Physicochemical characterization and nutraceutical compounds of the selected spice fixed oils

Abstract

Spices and herbs are well appreciated for their medicinal properties since ancient times. Till date, spices are being explored for volatile oils (essential), flavour and for addressing many chronic diseases. In the present study, we investigated the physicochemical properties, fatty acid composition, differential scanning calorimetry (DSC), elemental composition and nutraceutical compounds of fixed oils (non-volatile) from five selected spices viz., Alpinia galanga, Cinnamomum zeylanicum, Trigonella foenum-graecum, Foeniculum vulgare, and Myristica fragrans. The fixed oil (FO) content of volatiles-free powders of the five selected spices ranged from 1.58% (C. zeylanicum) to 26.43% (M. fragrans). The studied FO showed a good quality index which was analysed by estimation of free fatty acids, iodine value and unsaponifiable matter. The fatty acid analysis showed high palmitic acid in the FO of A. galanga and C. zeylanicum. High linoleic, oleic, and myristic acid levels were observed in T. foenum-graecum, F. vulgare and M. fragrans FOs, respectively. The nutraceutical compounds such as total phenolics were high in C. zeylanicum FO (0.53%). Hence the studied FO could be an excellent alternative to oil nutraceutical compounds. It may be used as a functional ingredient in foods which needs further validation for value addition.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04813-8) contains supplementary material, which is available to authorized users.

Introduction

Spices are originated from plant parts such as flowers, fruits, seeds, bark, leaves, and roots. They not only enhance the taste of food from ordinary to extraordinary but also extend the shelf-life of the food (Pop et al. 2019). As per available literature, the spices are present on earth since 7500 years and since 3000 years, these spices are being used in many traditional medicine systems such as Ayurveda and Siddha. According to the scriptures, many of the spices have hypolipidemic, hypoglycaemic, antioxidative, anti-atherosclerotic, anti-thrombotic, anti-inflammatory, anti-carcinogenic and anti-arthritic properties (Fathivand et al. 2017). Several in vitro, in vivo and clinical studies have been performed to provide scientific evidence to establish the therapeutic properties of these spices.

So far the research on spices is mainly focused on essential oils (volatile oils) content and its medicinal properties. The spice essential oils mainly constitute of terpenes, monoterpenes and sesquiterpenes. These volatiles are the secondary products of plants metabolism that possess antibacterial, antiviral, antifungal, and also have insecticidal properties (Chouham et al. 2017).

However, the FO (non-volatile oil) from spices remain an unexplored area. By definition, FO are non-volatile at room temperature and are generally soluble in organic solvents. The quality indices of the oils (volatile or non-volatile) is affected by its physicochemical properties such as colour, free fatty acids, peroxide value, iodine value, saponification and unsaponifiable matter, lipid classes and elemental analysis. Also, knowledge on the content of nutraceutical compounds in the FO is very limited.

In the present study, we focused to explore the physicochemical properties, GC–MS characterization of fatty acids, and nutraceutical content of the FO of the selected Indian spices. The five selected spices are Alpinia galanga, Cinnamomum zeylanicum, Trigonella foenum-graecum L., Foeniculum vulgare L., and Myristica fragrans. To the best of our knowledge, this is the first report describing the quality indices of the selected spice FOs, fatty acid composition, and nutraceutical contents of these Indian spices that could be used as a functional ingredient in foods for value addition. Below are the brief descriptions of the selected spice samples.

Alpinia galanga (Greater galanga) belongs to the Zingiberaceae family and is widely distributed in China, India, East Indies, and Polynesia. The volatile oil and flavonoids present in it are associated with many biological activities. A. galanga possessed numerous pharmacological activities, including antibacterial, antifungal, antiviral, and antiprotozoal (Kiuchi et al. 2002). Cinnamomum zeylanicum (Cinnamon) belongs to the Lauraceae family. The inner bark of the plant has several medicinal properties due to the presence of polyphenolic compounds. The most important constituent of cinnamon is cinnamaldehyde and trans-cinnamaldehyde present in essential oils to give aroma to the various food products prepared (Yeh et al. 2013). Trigonella foenum-graecum (Methi) belongs to Leguminoceae family, cultivated in the Central and South-Eastern Europe, Western Asia, India and Northern Africa. Recently, the anti-hyperlipidemic activity of the ethanolic extract of the T. foenum-graecum has also been reported (Kumar and Jhajharia 2019). Foeniculum vulgare (Fennel) belongs to a family Umbelliferae. It is reported to be used as a anti-ageing, anti-allergic, anti-colitic, anti-hirsutism, anti-inflammatory, anti-microbial and anti-viral agent respectively (Tripathi et al. 2013). Myristica fragrans (Nutmeg) belongs to Myristicaceae family and is cultivated in many tropical countries. It possess stimulant, narcotic, carminative, astringent, aphrodisiac properties, anti-thrombotic and anti-platelet aggregation, antibacterial, antifungal, anti-dysenteric, anti-inflammatory and analgesic activities (Gupta et al. 2013).

Materials and methods

Source of the spice material

The authentic five (5) selected Indian spices such as Alpinia galanga, Cinnamomum zeylanicum, Trigonella foenum-graecum L., Foeniculum vulgare L. and Myristica fragrans were purchased from Vriksha Vijnan Private Limited, Bangalore, Karnataka. The authentication of spice powders was performed through microscopy (data not shown). The spices were screened to remove unwanted material if any and powdered using CFTRI developed Multi-Mill-grinder for 20 ± 2 min at high speed with a uniform size sieve and stored in polythene airtight bags until further use.

Chemicals and reagents

The chemicals and reagents used in the current study such as gallic acid, β-carotene, Trolox, aluminum chloride (AlCl₃), Folin-Ciocalteu’s (FC) and boron trifluoride (BF₃) were purchased from Sigma-Aldrich, Bangalore, India. Except stated above, all the other chemicals such as potassium hydroxide (KOH), sodium hydroxide, sodium thiosulphate, and solvents used were of analytical grade (Hi-Media, Bangalore, India).

Volatile oil (VO) extraction

The pre-weighed spice powders, 100 g each were placed in distillation flasks at 100 °C which were connected to a steam generator via a glass tube, and to a condenser to retrieve the volatile oil (VO) until complete removal of volatiles. GC–MS was performed at various intervals to confirm the complete extraction of volatiles. Finally, the obtained VO was separated using separating funnel, and the percentage of volatiles was estimated according to the standard method (AOCS 2003). The obtained residual powders, devoid of volatile compounds, were made moisture-free at 60 °C for 4 h.

Determination of FO content

The FO was extracted from the volatile-free spice powders with hexane in a Soxhlet apparatus at 68 ± 2 °C for 8 h. The obtained hexane fraction was evaporated and the difference was measured to determine the FO content (AOCS 2003).

Physicochemical properties

Estimation of moisture content

The moisture content of the powdered spice samples were determined by toluene co-distillation method using Dean and Stark apparatus as described standard method (AOCS 2003). The distillation process was carried out for 4 h until the moisture content is completely condensed.

Colour measurement

The colour of extracted FO was determined by colour measurement instrument (Konica Minolta CM-5, VA, USA) where L values represent the lightness or darkness, a* values refer to red-green colour, and b* values refers to the blue-yellow colour.

Free fatty acid (FFA) estimation

The FFA of FO samples was estimated according to the method of Ca 5a-40 (AOCS 2003). The oil sample (1 g) was titrated against 0.1 N sodium hydroxide in a neutralised alcohol medium with a phenolphthalein indicator, and the results were expressed as the FFA percentage.

Peroxide value (PV)

The PV of the extracted FO (5 g) samples was determined by titrating against 0.1 N sodium thiosulphate in the presence of saturated potassium iodide solution using starch as an indicator Ca 8-53 (AOCS 2003).

Para-anisidine value (p-AV)

The p-AV is a measurement of carbonyl content in the oils or fats and was determined according to AOCS (2003). The reaction is mainly based on the reactiveness of the aldehydes carbonyl bond of the p-anisidine amine group to form Schiff base which was measured at 350 nm against isooctane containing p-anisidine.

TOTOX value

As an indicator of both primary and secondary oxidation products, the TOTOX values of the spice FOs were calculated in accordance with the following equation (Wan et al. 2009).

$$\text{TOTOX}=2\text{PV}+p\text{-}\mathit{AV}$$

where, PV is peroxide value and p-AV indicates p-anisidine value.

Iodine value (IV)

The IV of spice FO (1 g) samples was estimated according to standard Wij’s method Cd 1d-92 (AOCS 2003). The oil sample in carbon tetrachloride was treated with 25 mL of Wijs solution. The excess iodine monochloride was treated with potassium iodide and the iodine liberated was titrated with 0.1 N of sodium thiosulphate solution using starch as an indicator.

Saponification value (SV)

The SV of the spice FO samples was determined using AOCS method Cd 3-25 (AOCS 2003). The 5 g of oil sample, was saponified using 50 mL of 5% ethanolic KOH solution in a conical flask with a connected air condenser and boiled until the oil was completely saponified, cooled and titrated with 0.5 N hydrochloric acid (HCl) using phenolphthalein as indicator.

Unsaponifiable matter (USM)

The USM of extracted spice FO samples was determined according to the standard method Ca 6a-40 (AOCS 2003). A 5 g FO sample was refluxed with 5 mL of 50% KOH solution in the presence of 30 mL of ethanol until the oil was saponified completely, and the unsaponifiable matter was extracted with petroleum ether, further washed with 70% ethanol and finally desolvated and its weight was estimated.

Fatty acid methyl esters (FAME)

FAME of the spice FO samples was prepared by trans-esterification, according to the method (AOCS Ce 1-62 2003). Briefly, for 100 mg of the oil sample 1 mL of BF₃ methanol was added and incubated for 30 min at 60 °C. The tubes were immediately transferred on to the ice bath for 5 min to impede the reaction, 1 mL hexane, distilled water was added and the tubes were vortexed. Finally, the undisturbed top layer of methyl ester was transferred to GC vials. The heptadecanoic acid (C17:0), 50 µg was added as the internal standard.

Determination of fatty acid profile

GC–MS analysis was performed by using an Agilent Technologies 7890B chromatograph connected directly to a 5977A inert mass spectrometer (Agilent Technologies, Milan, Italy), with GC column, VF-23 ms (60 m × 250 µm × 0.25 µm film thickness). The MS detector was operated in electron ionization (EI) (70 eV, 200 mA), in full-scan mode (m/z 40–500), and ions at m/z 127, 140 and 256 for heptadecanoic acid as the internal standard. The transfer line was set at 230 °C, and the solvent delay was set at 3 min.

Glycerolipid composition

The glycerolipid composition (TAG, DAG, and MAG) of extracted spice FO samples were determined by using standard procedures Cd 11c-93 (AOCS 2003). The standard column chromatographic method was carried out using a glass column (i.d. 1.8 cm and 30 cm length) packed with a silica gel bed (100–120 mesh size) by using petroleum ether. The individual glycerolipid fraction (TAG, DAG, and MAG) were eluted with the optimized solvent system, and the quantity of each fraction was estimated gravimetrically after evaporating the solvent.

Thin-layer chromatography

The spice FO were subjected to thin-layer chromatography (TLC) with DC Kieselgel 60 TLC silica gel 60 aluminium sheets. The TLC mobile phase consisted of petroleum ether:diethylether:acetic acid (85:15:0.1, v/v), and the bands were detected with iodine vapours. The rice bran oil was used as a reference for comparing the results.

Determination of fatty acid profile for glycerolipid fractions

For the above obtained glycerolipid fractions (TAG, DAG, and MAG), a known weight of the sample was used for FAME preparation. GC–MS analysis was performed by following the same procedure as described in earlier section.

Differential scanning calorimetry (DSC)

DSC of the FO samples was analysed according to the method reported (Lee and Foglia 2000). The melting and crystallization of oils were measured using DSC (DSC 8000, PerkinElmer DSC 8000) by placing aluminium pan as a reference with accurately weighed oils (8–10 mg). The samples were initially heated to 80 °C and held for 10 min. After that the temperature was decreased to 10 °C/min to -60 °C with holding time of 10 min, the melting curve was obtained by heating the samples to 80 °C at 5 °C/min.

Carbon, hydrogen, nitrogen and sulphur (CHNS) content

The extracted FO samples were analysed for the CHNS content using Elemental Analyser (Vario EL III) where in sulphanilic acid was used as a standard compound. Briefly, a tin capsule was filled with 5.0–10.0 mg of FO using an electronic balance (Sartorius Balance). The percentage of CHNS content was determined as per Sekhar et al. 2018.

Sample preparation for total phenolics and flavonoid estimations

The FO sample (1 g) was extracted with 0.1% HCl in 70% methanol by vortexing for 15 min and centrifuging at 10,000 rpm for 10 min at 10 °C. The obtained pellet was extracted thrice, and all the supernatants were pooled, filtered through 0.45 µm filter, and stored in amber tubes to avoid light interference (Arcan and Yemenicioglu 2009). All the extractives were stored at −20 °C until further use.

Determination of total phenolic content

The soluble phenolic compounds in the FO sample extractives were determined using the FC reagent as per the published method (Dande and Manchala 2011) with slight modifications. Briefly, different concentrations of sample extractives to fit into standard curve were taken, 1.0 mL of 10% FC reagent and 0.8 mL of 7.5% sodium carbonate (Na₂CO₃) were added, vortexed thoroughly, incubated at room temperature for 90 min, and the absorbance was read at 725 nm. The values were expressed as g/100 g gallic acid equivalent (GAEq.), which is one of the most commonly used standards for phenolic estimations.

Determination of total flavonoid content

For the determination of the total flavonoid content (TFC), the spice FO extractive (200 µL) was diluted with absolute ethanol to an appropriate concentration. 1 mL of the extracted sample was mixed with 1 mL of 2% (w/v) methanolic solution of AlCl₃ (Djeridane et al. 2006). After incubation at room temperature for 15 min, the absorbance of the reaction mixture was read at 430 nm with a double beam spectrophotometer (UV-1800 A, Shimadzu Corporation, Kyoto, Japan). The TFC was expressed as Rutin equivalent (REq.) in mg/100 g of FO.

Determination of lignans content

The lignans present in the spice FO were determined by spectrophotometric method. 10 mg of oil sample was dissolved in 10 mL of HPLC grade chloroform and hexane (7:3 v/v), and the absorbance was measured at 288 nm followed by calculation using ${\text{E}}_{1\text{cm}}^{1\%}$ value of 230 (Bhatnagar et al. 2015).

Determination of carotenoid content

Carotenoids content of the spice FO was determined by diluting 1 g of oil with 10 mL of acetone. From this solution, 1 mL aliquot was further diluted to 10 mL with hexane, and the absorbance was measured at 446 nm using a UV–Vis spectrophotometer (Shimadzu UV-1601, Shimadzu Corporation, Kyoto, Japan). The carotenoid content was calculated by using the diffusion coefficient of 383 and expressed as g/100 g of oil (Chandrasekaram et al. 2009).

Statistical analysis

All the determinations were made at least in triplicate, and each reported value represents the mean ± S.D of independently prepared samples.

Results and discussion

VO extraction

Most of the earlier studies on spices were on characterization of their VO, and the role of these VO in combating various disorders. However, the aim of the present study was to characterize the FO from the spices, to study the, antioxidant potential and nutraceutical composition. Hence, to avoid the interference of the VO, steam distillation was performed to remove and estimate the VO content (Table 1). M. fragrans and A. galanga showed the highest and lowest percentage of VO (5.95% and 0.15% respectively) with a steam distillation time of 8 h for both the spice powders. The earlier reported VO percentages of the M. fragrans, F. vulgare and A. galanga were 5–6%, 2% and 0.04–0.15%, respectively (Damayanti and Setyawan 2012).

Table 1.

Optimization of steam distillation time, volatile, FO content and physicochemical properties of the FOs extracted from the Indian spices

Parameter	A. galanga	C. zeylanicum	T. foenum-graecum	F. vulgare	M. fragrans
Moisture (%)	8.94 ± 0.11	10.83 ± 0.35	11.69 ± 0.36	8.96 ± 0.07	7.75 ± 0.54
Steam Distillation (h) $	8	24	4	8	8
Volatile oil (%)	0.15 ± 0.03	3.01 ± 0.21	ND	1.13 ± 0.09	5.95 ± 0.20
Fixed oil (%)	1.58 ± 0.13	1.87 ± 0.01	6.73 ± 0.17	12.92 ± 0.83	26.43 ± 1.89
Color
L	667.04 ± 24.71	260.34 ± 4.84	14.49 ± 0.43	68.35 ± 0.80	15.12 ± 0.43
a*	−44.54 ± 3.81	20.33 ± 2.35	−1.96 ± 0.14	91.46 ± 2.86	−1.81 ± 0.15
b*	145.12 ± 18.10	54.72 ± 1.64	7.14 ± 0.62	91.34 ± 1.38	3.84 ± 0.28
FFA (%)	13.33 ± 0.38	12.11 ± 2.29	4.65 ± 0.27	3.68 ± 0.04	19.72 ± 0.14
PV (meq O2/kg)	192.64 ± 1.33	43.05 ± 1.70	145.64 ± 0.79	68.42 ± 3.85	9.14 ± 0.47
p-Anisidine value	142.24 ± 1.22	ND	12.34 ± 0.92	2.89 ± 0.00	36.97 ± 0.00
TOTOX value	528.28 ± 4.48	ND	302.84 ± 0.21	135.99 ± 5.90	21.31 ± 1.24
IV (I2/100 g)	63.39 ± 1.97	67.05 ± 0.89	89.36 ± 1.17	149.27 ± 1.17	11.02 ± 0.62
SV (mg/100 g)	ND	ND	330.58 ± 2.84	327.26 ± 3.22	317.78 ± 3.16
USM (%)	7.96 ± 0.45	7.85 ± 0.49	5.89 ± 0.49	7.69 ± 0.14	4.63 ± 0.03
TAG (%)	28.63 ± 2.30	40.67 ± 3.80	74.71 ± 2.90	83.20 ± 2.20	70.65 ± 6.20
DAG (%)	3.48 ± 0.80	6.04 ± 1.40	13.80 ± 2.10	8.01 ± 1.30	0.47 ± 0.01
MAG (%)	54.55 ± 4.60	41.17 ± 4.20	6.85 ± 4.20	5.10 ± 0.90	9.15 ± 0.50

All the values are mean ± SD of three replicates. ND  Not detected. ^$Indicates time required for the removal of volatile oils

FO content

Table 1 shows that the highest FO content (26.43%) was recorded in M. fragrans, and the lowest in A. galanga (1.58%). The reported total oil content of the M. fragrans is 25–40%, F. vulgare is 12–14%, and T. foenum-graecum is 3–7%, respectively, that was comparable with the present data (Cosge et al. 2008; Gu et al. 2017).

Physicochemical characterisation

Estimation of moisture content

The moisture content of spice powders was analysed by toluene distillation method (Table 1) wherein, M. fragrans showed the lowest moisture (7.75%) content, and the highest moisture content was observed in T. foenum-graecum (11.69%), which was similar with the previous reports (Altuntas et al. 2005). The toluene distillation method is the most appropriate method for moisture determination for spices (Altuntas et al. 2005). However, the moisture content mainly depends on the area of cultivation, season, and also the processing conditions.

Colour measurement

The extracted FO were subjected to the colour measurement by colour measurement spectroscopy. The L, a* and b* values obtained are shown in Table 1. A dark brown colour was observed in A. galanga FO whereas, the mild yellow colour was noticed in M. fragrans FO. The similar dark colour oil was reported to be extracted from mustard seeds (Chakraborty et al. 2018). The variation in colour of the oils is mainly due to the presence of carotenoids and other pigments or exposing the oil to higher temperatures for a longer time. Similarly, the green and yellow colour of oils is due to the combination of aldehydes and peroxides in the oils (Chakraborty et al. 2018).

Free fatty acid estimation

FFA is one of the important quality indices to monitor the quality of raw material and degree of purity of the oils during processing and preservation. The FFA content in FO were 3.68, 4.65, 12.11, 13.33, and 19.72% for F. vulgare, T. foenum-graecum, C. zeylanicum, A. galanga, and M. fragrans FO respectively. The increase in FFA content of FO is mostly due to the hydrolytic rancidity and high heat treatment during steam distillation, and oven drying methods (Osawa et al. 2007).

Peroxide value (PV)

The PV indicates the quality of oil during storage studies. Usually, fresh oils and highly saturated fatty acid-containing oils show low PV as oxidation occurs at a slower rate. The lowest peroxide value was observed in M. fragrans FO (9.14 meq. O₂/kg), which indicate the presence of the high amount of saturated fatty acids whereas, A. galanga FO (192.64 meq. O₂/kg) showed the highest PV (Table 1). The increase in PV of A. galanga FO is probably due to extended heat treatment during the steam distillation process that in turn causes the oxidation of unsaturated fatty acids. The higher PV of the FOs may be due to formation of lipid oxidations, hydroperoxides of unsaturated fatty acids, colour interference, storage, light exposure, metal contents in oils and the partial pressure in the headspace of the oil (Choe and Min 2006).

Para-anisidine value (p-AV)

The p-anisidine value is a measure of secondary products of oils formed due to oxidation. During lipid-oxidation, hydroperoxides, and the primary reaction products decompose to produce secondary oxidation products which are more stable during the heating process and are responsible for off-flavour and off-odours of oils. The p-AV is a suitable method to measure the amount of secondary oxidation products. The F. vulgare FO showed a lowest (2.89 ± 0.00) p-AV and highest (142.24 ± 1.22) was recorded in A. galanga FO. However, the C. zeylanicum FO does not produced any secondary oxidation products as there was no detectable p-AV (Table 1). Sunil et al. (2015) reported an increasing (1.9 to 110) p-AV of sunflower oil at 120 °C during heating up to 24 h. They also reported that the addition of purified oryzanol, tocopherols and combination reduces the production of secondary oxidation products. Similarly, the changes in p-AV values of the rapeseed oil ranged between 1.2 and 235.4 with different oil layer height after heating (Kobyliński et al. 2016).

TOTOX value

The combination of p-AV with PV indicates the overall oxidation state of FOs. The calculated TOTOX values showed that M. fragrans FO had a low (21.31 ± 1.24) value and A. galanga FO showed the highest (528.28 ± 4.48) values (Table 1). Similarly, the changes in TOTOX values of the rapeseed oil ranged between 211.4 and 325.7 with different oil layer height after heating (Kobyliński et al. 2016).

Iodine value (IV)

The IV represents the degree of unsaturation of oils, high IV values indicate the presence of the high amount of double/triple bonds in the fatty acids present in oils. The current results suggest that F. vulgare FO (149.27 I₂/100 g) has highest IV due to the presence of a high amount of unsaturated fatty acids when compared with other studied FO (Table 1). The high unsaturation of these FO indicates that these are more prone to oxidation. However, the antioxidant/nutraceutical compounds present in these FO may prevent/reduce the level of oxidation during storage. The M. fragrans FO (11.02 I₂/100 g) has low IV due to the presence of low levels of unsaturated fatty acids. Hence these FO may have excellent shelf-life stability. The IV of A. galanga FO (63.39 I₂/100 g) is comparatively more than that of M. fragrans FO due to the higher unsaturated fatty acid composition. T. foenum-graecum FO showed IV (89.36 I₂/100 g), which is comparable to an earlier report (Al-sebaeai et al. 2017).

Saponification value

SV represents the average molecular weight and fatty acid chain length of the oils. The SV of T. foenum-graecum FO was found to be 330.58 mg KOH/100 g followed by F. vulgare FO (327.26 mg KOH/100 g) and M. fragrans FO (317.78 mg KOH/100 g) (Table 1). The high SV of M. fragrans FO was due to the presence of significant amounts of medium-chain fatty acids. The SV are comparable with the available commercial oils soybean (189–195), canola (182–193), and sunflower (188–194 mg KOH/g) oils as reported earlier (Goli et al. 2008). The high SV may be due to low level of impurities in the FOs and a similar observation was recorded in almond seed oil (Odoom and Edusei 2015).

Unsaponifiable matter

The unsaponifiable matter (USM) of oils mostly contains the compounds that cannot be converted into soap. The USM constitutes mainly antioxidant compounds, and phytosterols (Table 1). The highest level of the unsaponifiable matter was observed in A. galanga FO (7.96%), and C. zeylanicum FO (7.85%) whereas, the lowest USM value was observed in M. fragrans FO (4.63%). The USM values of F. vulgare, and T. foenum-graecum FO were 7.69 and 5.89% respectively. The refined oils possess lower USM levels than its crude oil form due to the loss of minor nutraceuticals during processing. Hence crude oils with good quality are preferred to deliver maximum nutraceutical benefits to the consumers (Prashanth Kumar et al. 2017).

Fatty acid profile

The GC–MS analysis was performed to study the fatty acid profile of the FO. Figure 1 shows the fatty acid composition of the FO extracted from the spices. It was observed that C. zeylanicum FO showed the presence of a high amount of C16:0 (29.95 µmol%) followed by C18:1 (21.85 µmol%), C18:2 (24.74 µmol%), low levels of C12:0 (1.11 µmol%), and C15:0 (0.67 µmol%). F. vulgare FO showed high levels of C18:1 (63.56 µmol%), and low levels of C14:0 (0.86 µmol%). In M. fragrans FO the highest level was recorded for C14:0 (76.99 µmol%), and lowest for C18:3 (0.40 µmol%). Cosge et al. (2008) reported high percentage of oleic acid in F. vulgare oil.

Fig. 1.

Fatty acid composition of the selected five Indian spice fixed oils. All the values are mean ± SD of three replicate analyses

Glycerolipid composition

Table 1 shows the glycerolipid composition of the extracted spice FO, which contributes a major part of oils, and contains monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG). Out of these three glycerolipids, TAG is the major glycerolipid present in all vegetable oils. Whereas, DAG and MAG contribute to only 10% of the oil (Prashanth Kumar et al. 2017). The glycerolipid composition of the extracted spice FO showed the highest TAG in F. vulgare FO (83.20%) and lowest in A. galanga FO (28.63%). Similarly, the highest DAG and MAG levels were observed in T. foenum-graecum (13.80%), and A. galanga FO (54.55%) respectively. However, the DAG and MAG levels were found to be lowest in M. fragrans, and F. vulgare FO (0.47 and 5.10%), respectively.

TLC is the primary separation technique used for the identification of glycerolipids present in the lipids. This rapid and reliable method was used to ascertain the glycerolipids (TAG, DAG and MAG) in the spice FO samples. The rice bran oil was used as reference oil for the TLC analysis. The results of TLC indicate that a similar band spots pattern of TAG, DAG and MAG were observed in the extracted spice FO with respect to the reference (Supplementary Fig. 1).

Fatty acid profile of glycerolipid fractions

The fatty acid composition of the glycerolipid fractions did not show any difference in fatty acid percentage (Fig. 2). TAG fractions of A. galanga and C. zeylanicum FO were high in C16:0 (22.83 µmol% and 29.96 µmol%), respectively. High C18:2 levels were observed in T. foenum-graecum FO TAG fraction (39.81 µmol%). F. vulgare FO TAG fraction was found to be rich in C18:1 (75.12 µmol%). Finally, high amount of C14 was observed in M. fragrans FO TAG fraction (68.01 µmol%). A similar trend in fatty acid composition was also noted in DAG and MAG fractions respectively. Kamal-Eldin and Allelqvist (1994) reported fatty acid composition of the different acyl lipids in sesamum seed oils. They reported a similar fatty acid profiles in TAG and total lipids characterization. Higher levels of saturated fatty acids were observed in MAG when compared to TAG whereas higher levels of unsaturated fatty acids were seen in TAG and DAG when compare to MAG. The same trend was reflected in the studied FO.

Fig. 2.

Fatty acid composition of the glycerolipid fractions (a TAG, b DAG, and c MAG) from extracted fixed oils. All the values are mean ± SD of three replicate analyses

Differential scanning calorimetry (DSC)

DSC is a fast, very sensitive technique used to measure the enthalpy due to changes in the physical and chemical properties of the oils as a function of temperature or time. Table 2 shows the endothermic crystallization and melting profiles of the FO extracted from spices. The C. zeylanicum (58.17 °C) and M. fragrans (46.45 °C) showed a high melting temperature points compare to the other two oils. Which indicates that the C. zeylanicum and M. fragrans contains high saturated fatty acids and high melting glycerolipids than rest of the oils extracted. Whereas in the case of crystallization point, the F. vulgare has the highest cooling point (−26.70 °C) followed by T. foenum-graceum (−17.43 °C). As these two FO contain high unsaturated fatty acids, they showed the highest crystallization points while cooling. Similar variation in melting and crystallization peaks of blends of butterfat with phytonutrients retained palmoleins has been reported (Prasanth Kumar et al. 2016).

Table 2.

DSC of the FOs extracted from the Indian spices

Sample	Onset	Offset	ΔH (J/g)	Peak	Peak height	Peak Area
Melting point
A. galanga	ND	ND	ND	ND	ND	ND
C. zeylanicum	54.86	61.62	1.72	58.17	0.17	4.32
T. foenum-graecum	−19.84	2.79	4.00	−11.05	0.098	8.81
F. vulgare	11.52	22.28	53.96	19.18	2.87	151.09
M. fragrans	37.44	48.80	193.50	46.45	4.50	290.26
Crystallisation point
A. galanga	ND	ND	ND	ND	ND	ND
C. zeylanicum	44.35	47.65	−5.06	40.10	−0.28	−12.66
T. foenum-graecum	−10.52	−34.15	−8.55	−17.43	−0.205	−18.81
F. vulgare	−25.20	−29.90	−38.27	−26.70	−5.10	−107.18
M. fragrans	20.61	16.52	−132.70	18.73	−7.62	−199.05

ND not detected

Carbon, hydrogen, nitrogen and sulphur (CHNS) content

Table 3 shows the elemental (CHNS) composition of the FO extracted from spices. In the present study, the carbon and hydrogen were major elements found in the FO samples ranging from 54–78% and 7.73–15.87% respectively. There was a significantly less content of sulphur quantified in all the FO samples. Similar high carbon (76%) and hydrogen content with no detection of sulphur in Pithecellobium dulce seed oil was reported recently (Sekhar et al. 2018).

Table 3.

CHNS analysis of the FOs extracted from the Indian spices

Parameter	A. galanga	C. zeylanicum	T. foenum-graecum	F. vulgare	M. fragrans
Carbon	78.77 ± 0.28	64.02 ± 0.14	54.68 ± 0.45	63.46 ± 0.44	74.49 ± 0.36
Hydrogen	11.94 ± 0.24	7.73 ± 0.22	10.59 ± 0.23	14.63 ± 0.21	15.87 ± 0.08
Nitrogen	0.78 ± 0.02	0.90 ± 0.01	0.70 ± 0.00	0.16 ± 0.00	0.59 ± 0.01
Sulphur	0.91 ± 0.01	0.31 ± 0.01	0.23 ± 0.01	0.23 ± 0.00	0.03 ± 0.00

All the values are mean ± SD of three replicates

Determination of total phenolics content

Phenolics are a class of nutraceutical compounds present in oils and provide antioxidant activities (radical scavenging activity) to oils. The content of phenolics may vary from oil to oil, and the final retention of phenolics depends on the processing conditions employed. Table 4 shows that the C. zeylanicum FO (0.53 g/100 g) displayed the highest phenolic content, followed by M. fragrans FO (0.50 g/100 g). However, F. vulgare FO (0.03 g/100 g) exhibited the lowest phenolic content. The phenolic content of the studied oil is 9–10 folds high compared to walnut oil (Gao et al. 2019).

Table 4.

Estimation of nutraceutical compounds of the FOs extracted from the Indian spices

Parameter	A. galanga	C. zeylanicum	T. foenum-graecum	F. vulgare	M. fragrans
Lignans (g/100 g of oil sesemol Eq)	1.77 ± 0.15	1.16 ± 0.10	0.21 ± 0.03	0.15 ± 0.01	0.51 ± 0.05
Carotenoids (g/100 g of oil)	0.22 ± 0.03	0.17 ± 0.00	0.11 ± 0.03	0.12 ± 0.01	0.05 ± 0.02
Total Phenols-FC method (g/100 g of oil Gallic acid Eq)	0.22 ± 0.01	0.53 ± 0.01	0.05 ± 0.00	0.03 ± 0.00	0.50 ± 0.00
Total Flavonoid Content (mg/100 g of oil Rutin Eq)	40.95 ± 1.60	17.98 ± 3.36	4.65 ± 0.28	6.99 ± 0.34	7.15 ± 1.19

All the values are mean ± SD of three replicates

Determination of total flavonoids content

Flavonoid contents were analysed using AlCl₃ method that could form stable complexes of flavanones and dihydroflavonols. The results showed that among the FO, A. galanga showed the highest flavonoid content (40.95 mg/100 g) than the other oils. However, T. foenum-graecum FO (4.65 mg/100 g) showed the least amount of flavonoid content (Table 4). Vinha et al. (2005) compared the phenolics and flavonoids compounds in 18 different Spanish, Italian and Portuguese olive cultivars with rutin and luteolin-7-O-glucoside as the two main flavonoid compounds.

Determination of lignans content

Lignans are a group of compounds, found initially in sesame oil. It comprises of sesaminol, sesamin, sesamolin, and sesamolinol. These lignans are unique in functional, physiological bioactivity and nutritional properties. The present results show that A. galanga FO has high lignans (1.77 g/100 g) content among the studied oils, followed by C. zeylanicum FO (1.16 g/100 g). The lowest level of lignans was observed in F. vulgare FO (0.15 g/100 g). Kochhar et al. (2006) reported that T. foenum-graecum contains 0.38 g lignans/100 g of oil, which is comparable with the present lignans percentage (0.21 g/100 g) of T. foenum-graecum FO (Table 4).

Determination of carotenoid content

In general, vegetable oil such as palm oil contains carotenoids with health benefits, which play a pivotal role in eye diseases. The carotenoids content of the extracted FO was 0.22, 0.17, 0.12, 0.11, and 0.05% of A. galanga, C. zeylanicum, F. vulgare, T. foenum-graecum, and M. fragrans FO respectively (Table 4). The carotenoid content was positively correlated with the colour (L, a* and b* values) observed in oils. As we analysed crude oil, the contents of carotenoids were relatively high than the total carotenoid content reported in refines mustard and rapeseed oil with 1.8 and 1.2 mg/100 g (Konuskan et al. 2019).

Conclusion

The spices examined in this study have been used since ancient days in small quantities in the preparation of foods. These spices have not only played a major role in enhancing the food taste and flavour but also have been used for medicinal purpose. Only volatile oils have been extracted from these spices, and used for various purposes. The residual mass of the spices after isolation of volatiles can also be further used for extraction of FO. Hence, there was a need to explore the physicochemical and nutraceutical content of the FO present in these spices. The physicochemical characterization of these FO showed a good quality index which was confirmed by analysing various oil quality parameters and nutraceuticals such as polyphenols, flavonoids, carotenoids, and lignans. The predominant fatty acids quantified by GC–MS were palmitic acid in the FO of A. galanga and C. zeylanicum and high linoleic, oleic, and myristic acid levels in T. foenum-graecum, F. vulgare and M. fragrans FOs respectively. Hence all these studied FOs could be an excellent alternative to oil nutraceutical compounds that could be used to prevent lifestyle disorders. It may be used as a functional ingredient in foods which needs further validation.

Electronic supplementary material

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Conflict of interest

The authors have declared that there is no conflict of interest.