Physico-Chemical Characterization of Oils Extracted from Noni, Spinach, Lady’s Finger, Bitter Gourd and Mustard Seeds, and Copra

Abstract

The physico-chemical properties of solvent-extracted oil from the seeds of noni (Morinda citrifolia L.), spinach (Spinacia oleracea L.), lady’s finger (Abelmoschus esculentus (L.) Moench), bitter gourd (Momordica charantia L.), mustard (Brassica nigra (L.) Koch), and the dried kernel (copra) of coconut (Cocos nucifera L.) were characterized. Among these sources, spinach seed had the lowest oil content (4.5 ± 0.4%) while coconut kernel had the highest oil content (63.1 ± 2.8%). Palmitic, oleic, and linoleic acids were the major fatty acids for spinach, lady’s finger and noni seed oils, while erucic, eleostearic, and lauric acids were the major fatty acids for mustard seed oil, bitter gourd seed oil, and coconut kernel oil, respectively. All of the oils possessed at least three major peaks in their triacylglycerol profiles except for bitter gourd seed oil which had only one major peak (1-stearoyl, 2,3-dieleostearoyl). The last endothermic peaks were –12.4, –6.0, 6.8, 57.7, 2.7, and 24.3ºC for noni, spinach, lady’s finger, bitter gourd and mustard seed oils, and coconut oil, respectively. Initially, the solid fat content of bitter gourd seed oil decreased gradually, but became rapidly after 50 until 60ºC. Coconut oil had its solid fat content reduced rapidly around 14 to 28ºC.

Keywords:

• Bitter gourd seed oil
• Lady’s finger seed oil
• Mustard seed oil
• Noni seed oil
• Physico-chemical properties
• Spinach seed oil

INTRODUCTION

According to the 8th Global Oils and Fats Forum 2013,^[1] the world’s commercially most important vegetable oils in 2012 (global production calculated in thousands metric tonnes) were palm oil (53,446), soybean oil (42,758), rapeseed oil (24,444), and sunflower oil (14,831). Global consumption of these oils has increased steadily due to growth of global population, energy crisis (used as biofuels), emergence of new functionality (applications in the oleo-chemical industry as surfactant, detergents, lubricants, flavor esters, and other purposes, such as blended with other oils for new functionality and utilization), and nutritional transition trend from saturated animals fat to unsaturated vegetable oils.^[2] This has led to either inadequate supply or increases in price, and, thus highlights the need for finding new plants as complimentary oil sources. In the end, it becomes more important to conduct further research on currently known plant oils that have incomplete properties information or varied characteristics due to different hybrids and cultivars. This is the case with the oils extracted from noni, spinach, lady’s finger, and bitter gourd seeds, and these sources of oils may have future applications both in the food and non-food industries. Noni seeds are a waste product of noni juice industry when the juice is commercialized worldwide.^[3,^4] Noni is a shrub that grows widely in Polynesia and is used as folk medicine, food, and fabric dye by the locals since thousand years ago.^[4] Other countries where noni is also grown include Vietnam,^[5] Malaysia,^[6] China,^[7] Costa Rica,^[8] and America.^[9,^10] The Noni seed contains 7% oil, 44% dietary fiber, 43% carbohydrate, 4% protein, and 2% ash.^[9] The major fatty acid of noni seed oil is linoleic acid while oleic acid and palmitic acid are the minor fatty acids.^[9]

TABLE 1 Oil content (%) of noni, spinach, lady’s finger, bitter gourd and mustard seed oils, and coconut oil

	Oil content mean value (%)
Source of oil	Value obtained^a	Literature value	Reference
Noni seed	15.1 ± 1.8	12.5	West et al.^[^9^]
		20.1	Bai et al.^[^37^]
Spinach seed	4.5 ± 0.4	3.0	Itoh et al.^[^11^]
Lady’s finger seed	16.4 ± 0.7	16.3	Andras et al.^[^39^]
		17.9	Rao^[^12^]
Bitter gourd seed	20.2 ± 0.8	19.3 ± 1.8	Nyam et al.^[^14^]
Mustard seed	36.7 ± 0.7	38.0	Tiwari et al.^[^42^]
		33.0	Fadhil & Abdulahad^[^41^]
Coconut kernel	63.1 ± 2.8	65-68	Langstraat^[^40^]
		68.5 ± 0.3	Nik Norulaini et al.^[^43^]

^aMean ± standard deviation.

Spinach seed contains approximately 3% oil.^[11] Known properties of spinach seed oil include iodine value (IV; 129.2 g of I₂/100 g), saponification value (SV; 190.8 mg of KOH/g) and unsaponifiable matter content (2.9%). The major compounds in the unsaponifiable matter are sterols from the category of α-spinasterol and stigmastenol. On the other hand, the lady’s finger seed contains a higher quantity of oil (17.9%) than the noni and spinach seeds.^[12] Rao^[12] reported that the lady’s finger seed contains 21.1% protein, 23.4% crude fiber, and 4% ash. The lady’s finger seed oil is highly unsaturated and is linoleic acid-rich (41.7%). Other fatty acids in the oil include 23.8% palmitic acid and 27.1% oleic acid. Anwar et al.^[13] reported that alcoholysis of the lady’s finger seed oil with methanol produces methyl esters that have fuel properties comparable with diesel. Bitter gourd seed contains 19.3% oil, 11.8% protein, and 34.8% crude fiber.^[14] The major fatty acid for bitter gourd seed oil is eleostearic acid^[14,^15] while the predominant triacylglycerol (TAG) is dieleosteroylstearin.^[15]

Mustard seed is oil-rich and has been reported to contain oil content in the range of 17–40%.^[16] The major fatty acid of mustard seed oil is erucic acid (0–62%) while eicosenoic acid, linolenic acid, linoleic acid, and oleic acid are the minor fatty acids.^[17] However, erucic acid is not suitable for human consumption as this fatty acid is detrimental to the function of heart^[18,^19] and liver,^[20] besides affecting fertility.^[21] Traditionally, coconut oil is obtained from the white edible flesh inside the kernel. The major fatty acids of coconut oil are lauric (51.0%) and myristic acids (23.0%) while stearic, palmitic, capric, and caprylic acids are present in trace amounts.^[22] Coconut oil can be formulated into an environment-friendly lubricant^[23] or used as feedstock of biodiesel.^[24] Coconut oil has also been found to possess antimicrobial properties against Candida species and is potentially for usage as an antimicrobial drug in the treatment of fungal infections.^[25] Thus, in this study, the physicohysic-chemical properties of oils that have been solvent-extracted from the seeds of the bitter gourd, spinach, lady’s finger, mustard, noni, and dried coconut kernel were characterized in order to determine their potential applications in food systems, and eventually as new edible oil.

MATERIALS AND METHODS

Sources and Extraction of Oil

Bitter gourd, lady’s finger, and spinach seeds were purchased from the Malaysian Agricultural Research and Development Institute (MARDI; Selangor, Malaysia). Mustard seeds, which are used as a spice by the local people, were bought from a local store in Serdang, Selangor, Malaysia. Noni seeds were extracted from fruits which were collected at the “hard-white” maturity stage^[26] from trees growing at the Universiti Farm, Universiti Putra Malaysia. These fruits were then stored five fruits per plastic bag (21 × 12 cm) at ambient temperature for four days until the fruits became soft. The seeds were separated from the fruit pulp manually. In order to remove residual pulp, the seeds were placed in a beaker containing 1 L of 0.5% (v/v) Pectinex Ultra SP-L (Novozymes, Bagsværd, Denmark) solution and incubated in a shaking water bath at 50ºC and 200 rpm for 5 h. The seeds were then cleansed under running tap water and oven-dried for 48 h at 90ºC^[27] and stored at room temperature until needed. Fresh coconut kernel was removed from eight mature coconut fruits obtained from a local market and then chopped into smaller pieces. The chopped kernel was then oven-dried at 90ºC for 48 h.^[28] The dried kernel was stored under cool, dried condition at room temperature and used for oil extraction within three days. Prior to solvent-extraction, the dried copra pieces were flaked manually into thin pieces and then ground finely using a home blender (Panasonic, Japan).

The oil from ground samples (for a total of three extractions per sample, with each extraction using approximately 500 g, except for noni seeds, 205.0–248.7 g) were extracted with 2 L of petroleum ether (boiling point 40–60ºC, from Merck, Darmstadt, Germany) in a 5 L Soxhlet apparatus for 8 h.^[29] Residual solvent was removed using a rotary evaporator (Model N-1, Eyela, Tokyo Rikakikal Co., Ltd., Japan) at 50ºC. The oils were placed in screw capped bottles, flushed with 99.9% nitrogen gas, capped, and then stored at –18ºC before analysis.

Chemical Analyses of Oil

Determination of SV, IV, and free fatty acids (FFAs) were done according to the official standard method.^[29] Samples were thawed in an oven at 60ºC for 90 min and mixed gently before the analyses were performed. All analytical determinations were done in triplicate, and the mean and standard deviation were calculated.

Determination of Color

The color of extracted oil was determined using a Lovibond tintometer (Model E, Salisbury, England).^[29] The melted sample was added to the tintometer cell up to three-quarters full. The color of each sample was determined at room temperature and by finding the best possible match with the standard color slides that were provided.

Determination of Fatty Acid Composition

The fatty acid composition of the oil samples was determined using the method described by Cocks and van Rede.^[30] Briefly, the oil was converted into fatty acid methyl ester (FAME) by dissolving 50 mg of sample in 0.9 mL of n-hexane and 0.1 mL of 30% sodium methoxide. After vortexing for 1 min and leaving to stand for 5 min, 1 µL of the upper layer was injected into a gas chromatograph (Perkin Elmer Clarus 500, Shelton, USA) which was fitted with a flame-ionization detector (FID) and a polar capillary column of BPX70 (0.32 mm internal diameter, 30 m length and 0.25 µm film thickness; SGE International Pty, Ltd., Victoria, Australia). The pressure of the column was set at 10 psi, while the initial temperature for the oven was set at 90ºC. The temperature was then programmed to increase to 220ºC at three different rates: 15ºC/min for the first 5 min, 2ºC/min for the following 20 min, and 15ºC/min for the remaining 1 min. The temperature of the injector and detector were maintained at 240ºC. The FAME peaks were identified by comparing their retention time with peaks of the standard (C6-C24, Even Carbon Number, Saturated FAMEs Kit, Sigma-Aldrich, St. Louis, USA) that was analyzed under the same conditions. Total percentage of saturated and unsaturated fatty acids was calculated by summing up the relative percentage of saturated and unsaturated fatty acids, respectively. Unsaturated and saturated fatty acids ratio was calculated by taking the total percentage of unsaturated fatty acids over the total percentage of saturated fatty acids.

Determination of TAG Profile

The TAG profile was determined using reversed phase high-performance liquid chromatography (HPLC) according to Ghazali et al.^[31] The instrument used was an Alliance Waters 2695 Separations Module HPLC system equipped with a Waters auto-injector, a Waters 2414 refractive index detector, column oven and two Waters 515 HPLC pumps (Waters Corp., Milford, USA). The chromatogram was processed using Water’s Empower Pro Chromatography software version 1.0. The separation column was an RP-18 column (250 × 4 mm) with a particle size of 5 µm (LichroCART 250-4, Merck, Darmstadt, Germany) placed in the column oven at 30ºC. The mobile phase for TAG separation was a mixture of acetone/acetonitrile in the ratio of 63.5:36.5, delivered into the HPLC column at a flow rate of 1 mL/min. An aliquot (10 µL) of oil sample (5% [v/v] in acetone) was injected into the system and the total run time was 45 min. Mean percentage for each peak were calculated by dividing the area of the respective TAG peaks over total area of all TAG peaks (for a sample) and times with 100%. Each sample was run in triplicate and the standard deviation for the respective mean percentage was calculated. In order to identify the TAG peaks, another oil sample, palm oil, which was freshly extracted from oil palm fruit using Soxhlet extraction, was included, as many of its TAG peaks have been identified. Instruments and method that used for running this extra oil sample was same as the other oils sample. The TAG peaks for the palm oil were identified based on the chromatogram of Chen et al.,^[32] Lida et al.,^[33] and Ghazali et al.^[31] The equivalent carbon number (ECN) was assigned for each identified TAG peaks according to the method of Wolff et al.^[34] for the palm oil TAG peaks. Graph of log α´ (log of relative retention time of each palm oil TAG peaks) against ECN (for each respective peaks) was plotted and linear equation obtained from this plotted graph was used to identify the ECN of other oil sample. ECN for the reference graph was calculated by the following formula:

where, CN is carbon number, D₁ = 2.6, D₂ = 2.35, D₃ = 2.17, N₁ is monounsaturated fatty acid (MUFA), N₂ is diunsaturated fatty acid, N₃ is triunsaturated fatty acid.^[34] The coefficient (D₁, D₂, and D₃) are empirical data values obtained from the study of Wolff et al.^[34] The relative retention time (α´) for each TAG peaks was calculated with respect to the relative retention time of tripalmitin by the following formula:

where, α´ = relative retention time, RT_n = retention time of TAG peaks – retention time of solvent, RT_p = retention time of tripalmitin peak – retention time of solvent. As coconut oil HPLC chromatograms contained some peaks with α´ that were not within the reference range α´, these were identified based on the chromatogram obtained by Adhikari et al.^[35] and Laureles et al.^[36] On the other hand, bitter gourd and spinach seed oil contained peaks with ECN that were not identifiable through comparison with the reference ECN and were identified based on the report by Chang et al.^[15] The HPLC chromatogram of mustard seed oil has never been reported in the literature, and thus, a compromised method was used to identify the peaks of mustard seed oil with unknown ECN. Three fatty acids that appear in the fatty acid profile of mustard seed oil were randomly chosen and the total CN of these fatty acids was assigned into the ECN formula and ECN for these three fatty acids were calculated. Such ECN calculation procedure was continuously carried on until the assigned CN (of the three-chosen fatty acids) gave ECN that was same as the unknown peak.

Thermal Properties

A differential scanning calorimeter (DSC; PYRISTM Diamond DSC, Perkin Elmer, Shelton, Washington, USA) was used to determine the thermal behavior and solid fat content (SFC) of the oils sample. The instrument was calibrated using indium and was connected to a laboratory Peak Scientific^® NG1000A nitrogen generator (Peak Scientific, Billerica, Massachusetts, USA) and a cooling accessory (Intracooler 2P, Perkin Elmer, Shelton, Washington, USA). A data processing software, PYRIS Instrument Managing Software, Version 10.1, was used to analyze the thermal property of oils. Before analysis, the oil samples were placed in an oven at 60ºC for 90 min to ensure complete melting. Approximately 4–9 mg of oil was placed into a volatile aluminum pan (Perkin Elmer, Shelton, Washington, USA) and crimp-sealed with a crimper (Perkin Elmer, Shelton, Washington, USA). The crimped volatile aluminum pan was then placed in the DSC’s sample furnace. An empty-crimped volatile aluminum pan that was used as reference was placed into the DSC’s reference furnace. The DSC furnaces were then covered up with two platinum furnace covers. All the oils sample were subjected to the following temperature programmed: heating from the load temperature (30ºC) to 60ºC and held for 5 min; cooling from 60 to –60ºC with a cooling rate of 10ºC/min and held for 2 min; then, heating from –60 to 60ºC with a heating rate of 10ºC/min and held for another 2 min. Nitrogen gas was purged into the furnaces at 20 mL/min. Peaks that appeared in thermogram can be exothermic or endothermic and their temperature was recorded. Due to the fact that these peaks represent the phase changes process (from solid to liquid or vice versa), temperature of these peaks was collectively known as transition temperature.

The solid fat content percentage (SFC%) for all oil samples were derived from the heating thermogram of the respective oil sample. By using the PYRIS data processing software, the partial peak areas (%) of the melting peaks against temperature were generated. Due to the fact that when the partial area of melting peak was 0%, the SFC% of the oil was 100% and vice versa, the SFC% of the oil sample at different temperature was obtained by reversing the percentage of partial area (from 0–100% to 100–0%).

Statistical Analysis

All measurements with replication were statistically analyzed by using Minitab 14 software. Test of one-way ANOVA (Analysis of variance) was carried out and the average values were compared for significance difference using Turkey’s test. Statistical significance differences were considered at the level of p < 0.05.

RESULTS AND DISCUSSION

Oil Content

Table 1 shows the oil contents of noni, spinach, lady’s finger, bitter gourd and mustard seeds, and coconut copra and it can be seen that the oil content (15.1 ± 1.8%) of the noni seeds used in this study was slightly higher than value (12.5%) reported by West et al.^[9] This slight difference can be due to factors such as cultivation region, temperature, or climate. On the other hand, Bai et al.^[37] recovered 20.13% of oil from noni seeds using a supercritical CO₂ extraction method. Apart from varietal differences, the greater extraction yield may be because supercritical fluid extraction method is more effective than solvent-extraction method as supercritical fluid possess diffusion coefficient that is similar to gases and solubility that is comparable to liquid.^[38]

Among the oil samples, spinach seed oil has the lowest oil content (4.5 ± 0.4%) and is similar to the value reported by Itoh et al.,^[11] making it least suitable as a source of industrial oil unless it has a special property such as having antimicrobial activity. On the other hand, the lady’s finger seed, or more commonly known as okra seed, has an oil content 3.6 times more than spinach seeds, making it a potential source of industrial oil. This value is similar to the oil contents reported previously, i.e., 16.3^[39] and 17.9%.^[12] The oil content of bitter gourd seed (20.2 ± 0.8%) is considered as high since it is comparable to the commercially important oil-bearing cotton seed (18–20%).^[40] This value is similar to the oil content (19.3 ± 1.8%) reported by Nyam et al.^[14] but different from the value (41–45%) reported by Chang et al.^[15] Higher oil content value reported by Chang et al.^[15] is probably due to the de-shelling of seeds process before they extracted the oil, thereby increasing the oil content significantly on a weight basis. Mustard seed has the second highest oil content value among oils sample used in this study (36.7 ± 0.7%). This value is within those reported by Fadhil and Abdulahad^[41] (33%) and Tiwari et al.^[42] (38%). The oil content for coconut copra is 63.1 ± 2.8%. This value is slightly less than the range reported in the literature (65–68%) for dry process extraction method for copra.^[40,^43] The slight difference could be due to a variety of factors and the most possible factor for this case is the different extraction methods that were used.

Chemical Analysis

The chemical properties of noni, spinach, lady’s finger, bitter gourd and mustard seeds, and coconut copra are shown in Table 2. FFA content is one of the parameters that used to judge the quality of oil where values obtained for all oils sample were below 2%, and hence, better than the set parameter (2.3%) for good quality of crude oil.^[44] Oil with high FFA (30–50%) is usually obtained from bruised, crushed, or fermented sources. Color is one of the food properties that can give out it impact in a variety of ways. Natural colorants that usually exist in food are compounds such as carotenoids, anthocynanins, and flavonoids.^[45] These colored compounds can act as an indicator of the quality of crude oil because oxidation, polymerization, or other chemical reactions can cause color changes in the original oil.^[45] In this study, results obtained for R/Y (red color over yellow color ratio) is below 0.3 for all oils sample indicating that all oils sample appear visually more yellow than red.

TABLE 2 Properties of noni, spinach, lady’s finger, bitter gourd and mustard seed oils, and coconut oil

	Source of oil
Property	Noni seed	Spinach seed	Lady’s finger seed	Bitter gourd seed	Mustard seed	Coconut kernel
FFA (%)	0.8 ± 0.1^ac	1.8 ± 0.0^b	1.4 ± 0.4^b	0.5 ± 0.1^a	1.7 ± 0.1^b	0.6 ± 0.1^a
Lovibond color (R/Y)	15.0R; 50.0Y	2.5R; 50.0Y	0.4R; 70.0Y	0.6R; 8.0Y	2.0R; 70.0Y	1.2R; 5.0Y
IV (g of I2/100 g oil)	136.7 ± 1.0^d	125.5 ± 3.0^f	92.7 ± 0.4^c	105.8 ± 0.9^a	110.9 ± 5.5^a	9.7 ± 1.1^b
SV (mg of KOH/g)	189.8 ± 0.8^a	173.6 ± 2.7^c	191.1 ± 2.1^a	191.1 ± 0.4^a	168.3 ± 2.5^c	249.5 ± 6.5^b

Means ± standard deviation in the same row with different letters are significantly different (p < 0.05).

Results obtained for IV for all the oils sample are also shown in Table 2. IV indicates the degree of unsaturation of the respective oils and noni seed oil is significantly higher (p < 0.05) than those of the other oil samples, due to its very high content of linoleic acid (72%; Table 3). Noni and spinach seed oils have an IV that are similar to that of safflower (136–148 g of I₂/100 g) and sunflower seed oils (118–141 g of I₂/100 g) which are both mainly used as edible oil and can also being utilized as industrial lubricant basestock.^[46,^47] The IV of lady’s finger seed oil is comparatively lower than noni seed oil due to its higher content of saturated fatty acids (Table 3) and its IV is slightly lower that the value reported by Anwar et al.^[13] probably due to varietal differences. Bitter gourd seed oil has a lower IV when compared with linseed oil (IV, 127.8–202.8 g of I₂/100 g) that has a similar percentage of tri-unsaturated fatty acids (56% of linolenic acid).^[48,^49] This is probably because of the presence of eleostearic acid which possesses conjugated double bonds, known to result in lower IV compared to oils with non-conjugated double bonds.^[50] The IV obtained is similar to the value reported by Nyam et al.^[14]

TABLE 3 Fatty acids relative percentage of noni, spinach, lady’s finger, bitter gourd and mustard seed oils, and coconut oil

	Source of oil
Fatty acids	Noni seed	Spinach seed	Lady’s finger seed	Bitter gourd seed	Mustard seed	Coconut kernel
Caprylic/Octanoic acid (C8:0)	ND	ND	ND	ND	ND	6.1 ± 1.5
Capric/Decanoic acid (C10:0)	ND	ND	ND	ND	ND	4.8 ± 1.5
Lauric/Dodecanoic acid (C12:0)	ND	ND	ND	ND	ND	55.4 ± 3.4
Myristic/Tetradecanoic acid (C14:0)	ND	ND	ND	ND	ND	18.2 ± 0.9^a
Palmitic/Hexadecanoic acid (C16:0)	10.9 ± 1.2^b	22.6 ± 0.3^a	36.1 ± 0.1^c	1.4 ± 0.2^f	1.8 ± 0.3^f	7.5 ± 1.1^d
Stearic/Octadecanoic acid (C18:0)	ND	1.7 ± 0.3^b	2.3 ± 0.6^b	41.2 ± 4.7^a	ND	1.6 ± 0.7^b
Oleic/Octadecenoic acid (C18:1)	17.1 ± 0.9^c,d	15.4 ± 2.2^d	19.4 ± 0.4^c	1.4 ± 0.3^a	15.7 ± 1.1^d	5.5 ± 0.9^b
Linoleic/Octadecadienoic acid (C18:2)	72.1 ± 1.8^d	49.3 ± 1.7^f	42.3 ± 0.3^b	2.6 ± 0.2^a	17.6 ± 1.1^c	0.7 ± 0.7^a
Linolenic/Octadecatrienoic acid (C18:3)	ND	ND	ND	ND	19.4 ± 1.4	ND
Eleostearic/Octadecatrienoic acid (C18:3n5)	ND	11.1 ± 4.4^b	ND	53.4 ± 4.4^a	ND	ND
Gadoleic/Eicosenoic acid (C20:1)	ND	ND	ND	ND	3.2 ± 0.6	ND
Erucic/Docosenoic acid (C22:1)	ND	ND	ND	ND	42.3 ± 3.0	ND
Total fatty acid (%)	100	100	100	100	100	100
Saturated fatty acid (SFA) (%)	10.9	24.3	38.4	42.6	1.8	93.6
Unsaturated fatty acid (USFA) (%)	89.1	75.7	61.6	57.4	98.2	6.4
Ratio of USFA/SFA	8.2	3.1	1.6	1.3	54.6	0.07

Means ± standard deviation in the same row with different letters are significantly different (p < 0.05); ND: Not detected.

The IV for mustard seed oil is within the IV range of commercial edible oils such as sesame oil (104–120 I₂/100 g) and cotton seed oil (99–119 I₂/100 g).^[46,^51] However, unlike sesame oil and cotton seed oil that have their degree of unsaturation contributed mainly by oleic and linoleic acids,^[46,^52] a large part of the degree of unsaturation of mustard seed oil is contributed by erucic acid (Table 3). Compared with other oil samples, the IV of coconut oil is the lowest, and statistically different (p < 0.05) from the other oil samples, largely due to an abundance of medium-chain saturated fatty acids (Table 3). The IV is within the value range (8.8–10.4 g of I₂/100 g) reported by Gopalakrishnan et al.^[53]

One-way ANOVA analysis shows that the SV of noni seed oil is not significantly different (p > 0.05) from those for lady’s finger and bitter gourd seed oils but was significantly different from the SV of the other oils used in this study. As SV is inversely related to the mean molecular weight of the oil, the data obtained indicates that noni, lady’s finger, and bitter gourd seed oils have relatively similar molecular weights. Such property can be deduced from their fatty acid profiles (Table 3) as they all contain fatty acids of similar chain lengths. The SV of these three seed oils are within the reported SV range of commercial edible oils such as sunflower (188–194 mg of KOH/g) and soybean (189–195 mg of KOH/g) seed oils.^[46,^54] The unsaponifiable matter in the three oils have been reported to be 1.2, 0.37, and 1.12–1.71% for noni,^[9] lady’s finger,^[55] and bitter gourd seed^[56] oils, respectively. The SV of spinach seed oil is found to be lower than the SV of the three oils despite the presence of a higher content of palmitic acid. Itoh et al.^[11] reported that spinach seed oil has a high percentage of unsaponifiable matter (2.9%) and oil without unsaponifiable materials has a SV of 190.8 mg of KOH/g. The difference is probably due varietal differences and possible high unsaponifiable content in the spinach seed oil under study. The SV of mustard seed oil is the lowest because of its high content of erucic acid (Table 3) and is within the value reported for Brassica family (159–179 mg of KOH/g).^[57] As expected, the SV of coconut oil is statistically the highest (p < 0.05) when compared with other oil samples due mainly to the presence of medium chain fatty acids such as lauric and myrtistic acids (Table 3). The SV of coconut oil is within the value range reported in the literature, 248–265 mg of KOH/g.^[52]

Fatty Acid Composition

The fatty acid composition (Table 3) shows that mustard seed oil is the most unsaturated (98.2%) of the oil samples, followed by noni and spinach seed oils. The high degree of unsaturation of mustard seed oil is largely due to the presence of erucic, oleic, linoleic, and linolenic acids where MUFA content made up 61.2% of the total. The MUFA content is close to those of other oils with high MUFA content such as olive, hazelnut, and almond oils^[58–60] and Moringa oleifera, Myrcianthes pungens, and Rollinia sylvatica seed oil.^[61,^62] High MUFA in mustard seed oil is largely due to erucic acid, which has been reported to cause nutritional deficiency and cardiac lesion in tested animals and, thus is prohibited from being used as an edible vegetable oil in the U.S., Canada, and Europe regions.^[63] The fatty acid profile of mustard seed oil is in accordance with the result reported by Velasco et al.^[64] Mustard seed oil together with noni seed oil (89% unsaturated fatty acid) would probably be the more susceptible to oxidative rancidity than other oils as mustard seed oil is high in linoleic and linolenic acids (total 37.0%) while noni seed oil is rich in linoleic acid (72.1%). In fact, the fatty acid composition of mustard seed oil is quite similar to that of rapeseed [Brassica napus, and Brassica rapa (campestris)] oil.^[17] However, through the effort of selective breeding, low erucic acid B. napus, and B. rapa have been successfully grown since 1968 and 1974, respectively. Subsequent research resulted in the release of low erucic acid and low glucosinolate cultivars of both species which are used to produce canola oil.^[65]

Both spinach and bitter gourd seed oils contain a non-typical fatty acid, namely eleostearic acid, in their fatty acids profile (Table 3). The presence of eleostearic acid makes these two oils suitable for food usage as conjugated double bonds have been found to be cytotoxic to human tumor cells.^[66] When fatty acid with conjugated double bonds was tested on human normal cells, no detrimental effects were exhibited and the cytotoxic effect is believed to act selectively on fast-growing human tumor cells.^[67] Furthermore, the use of bitter gourd seed in food is certainly not a modern discovery because traditional Indian culinary had long employed bitter gourd fruit together with its seeds in a dish which is more specially known as “Karela sabzi.”^[68] Tung oil which contains 90% eleostearic acid is used industrially in paints and coatings due to its “fast-drying” property.^[15] With its higher oil content compared to spinach seed, bitter gourd seed may be a potential source of oil that have useful applications in the food and non-food industries. Bitter gourd oil is especially interesting as it is the only seed oil under study with a saturated fatty acid to unsaturated fatty acid ratio of 43:57, while the other oils are largely more unsaturated.

Lady’s finger seed oil is rich in palmitic, oleic, and linoleic acids (Table 3). Therefore, this oil has the potential to be formulated into food products that can provide energy and essential fatty acids such as fat powder with high proportion of polyunsaturated fatty acids.^[12,⁶⁹,^70] The three most abundant fatty acids in the oil account for 97.8% of total fatty acid which is in agreement with the range (94.8–99%) reported in the literature.^[12,³⁹,^71] On the other hand, coconut oil is rich in medium chain fatty acids, namely lauric and myristic acids (Table 3), and is more suitable as cooking/frying oil when compared to the other seed oils because it contains a low level (0.7%) of polyunsaturated fatty acids making it less susceptible to oxidative deterioration. Besides, coconut oil also contains a high amount of short and medium chain fatty acids (84.5%) that makes it suitable as feedstock in the production of biodiesel,^[72] lubricant,^[23] flavor esters,^[73] and α-amylase.^[74]

TAG Profile

The TAG profiles of the extracted oils are shown in Fig. 1a–1f. Included is the TAG profile palm mesocarp oil (Fig. 1g) from which a plot of log α´ versus ECN was generated (Fig. 2). The equation derived from the linear regression plot was used to determine the ECN of the TAG peaks for the seed oils used in the study (Table 4). Due to linoleic acid being the dominant fatty acid in noni seed oil, the most abundant TAG in the oil was found to be trilinolein (LLL) (31.6 ± 0.03%), followed by PLL (21.3 ± 0.07%), and OLL (20.1 ± 0.05%), where P, L and O are palmitic, linoleic and oleic acids. For spinach seed oil, the major TAG were ones that contain eleostearic acid such as LEE (where E is eleostearic acid) and linoleic acid (e.g., PLL and POL/PLO) which were both present in nearly equal proportions. To date, there is as yet no report on the TAG profile and composition of noni seed and spinach seed oils.

TABLE 4 Triacylglycerol composition of noni, spinach, lady’s finger, bitter gourd and mustard seed oils, coconut oil, and palm olein

		Relative composition^a (%)
Triacylglycerol	ECN	Bitter gourd seed	Mustard seed	Spinach seed	Noni seed	Lady’s finger seed	Coconut kernel	Palm olein
CaCC	28	–	–	–	–	–	1.29 ± 0.08	–
CCC	30	–	–	–	–	–	4.33 ± 0.03	–
CaLaLa/CCLa	32	–	–	–	–	–	14.7 ± 0.05	–
CCM/CLaLa	34	–	–	–	–	–	19.2 ± 0.03	–
LaLaLa	36	–	–	–	–	–	22.5 ± 0.04	–
LaLaM	38	–	–	–	–	–	17.7 ± 0.19	–
LLL	39.9	–	–	9.98 ± 0.39	31.6 ± 0.03	5.38 ± 0.27	–	–
LEE	39.9	2.49 ± 0.04	–	14.5 ± 0.04	–	–	–	–
EEE	39.9	5.16 ± 0.02	–	–	–	–	–	–
LaMM	40	–	–	–	–	–	9.72 ± 0.23	–
LaMP/MMM	42	–	–	–	–	–	4.75 ± 0.25	–
OLL	42	–	–	3.41 ± 0.26	20.1 ± 0.05	5.76 ± 0.16	–	0.71 ± 0.04
OEE	42	5.05 ± 0.07	–	–	–	–	–	–
OOLn	42.3	–	5.18 ± 0.59	–	–	–	–	–
EaLnLn	42.4	–	4.14 ± 0.27	–	–	–	–	–
PLL	42.6	4.4 ± 0.04	–	15.1 ± 0.08	21.3 ± 0.07	19.4 ± 0.72	–	4.12 ± 0.19
PEE	42.6	3.36 ± 0.06	–	–	–	–	–	–
MPL	43.3	–	–	–	–	–	–	0.77 ± 0.01
OOL	44.1	–	–	6.93 ± 0.06	6.72 ± 0.07	4.48 ± 0.20	–	–
SEE	44.6	77.08 ± 0.22	–	–	–	–	–	–
PLO/POL	44.7	–	–	13.7 ± 0.68	16.3 ± 0.04	15.8 ± 0.83	–	15.0 ± 0.75
PPL	45.3	0.55 ± 0.00	–	10.5 ± 0.26	–	18.1 ± 0.16	–	12.2 ± 0.28
OOO	46.2	–	4.85 ± 1.84	4.90 ± 0.09	–	2.58 ± 0.05	–	–
POO	46.8	–	3.35 ± 0.35	4.91 ± 0.92	3.95 ± 0.04	6.71 ± 0.44	–	23.4 ± 1.00
PPO	47.4	–	–	6.74 ± 0.52	–	8.78 ± 0.67	–	29.0 ± 0.41
PPP	48	–	–	–	–	–	–	3.16 ± 1.95
EaOL	48.1	–	7.00 ± 2.70	–	–	–	–	–
EaLnG	48.3	–	1.93 ± 0.56	–	–	–	–	–
SOO	48.8	–	–	–	–	–	–	2.75 ± 0.16
PSO	49.4	–	–	–	–	–	–	5.39 ± 0.21
PPS	50	–	–	–	–	–	–	0.45 ± 0.52
EaLnEa	50.3	–	31.9 ± 2.03	–	–	–	–	–
SSO	51.4	–	–	–	–	–	–	0.34 ± 0.01
EaLEa	52.5	–	28.3 ± 1.86	–	–	–	–	–
EaOEa	54.2	–	9.59 ± 0.61	–	–	–	–	–
Others^b		1.9 ± 0.00	3.69 ± 1.27	9.34 ± 0.28	–	13.0 ± 0.49	5.84 ± 0.24	2.68 ± 0.15
Total		100	100	100	100	100	100	100

^aMean ± standard deviation; Standard deviation is calculated based on the triplicate of the mean; ^bTriacylglycerol peaks that are yet to be identified; Not identified for column with symbol.

FIGURE 1 HPLC chromatograms for (A) noni seed oil; (B) spinach seed oil; (C) lady’s finger seed oil; (D) bitter gourd seed oil; (E) mustard seed oil; (F) coconut oil; (G) palm olein.

FIGURE 2 Graph log α´ against equivalent carbon number for palm olein.

As lady’s finger seed oil is rich in palmitic and linoleic acids, its TAG profile is dominated by PLL, PLO/POL, and PPL in an almost equal proportion. Since different subspecies of lady’s finger seed produce oil with varying fatty acids composition and TAG profile,^[71] the TAG composition obtained is thus, slightly different from the values reported by Fiad.^[71] Being abundant in eleostearic and stearic acids, bitter gourd seed oil has SEE (where S is stearic acid) as its major TAG. LEE, which is the dominant TAG of tung oil^[15] and spinach seed oil, is <6% in bitter gourd seed oil. Other minor TAG in bitter gourd seed oil with similar quantities is EEE, PEE, and OEE. On the other hand, the TAG profile of mustard seed oil is dominated by EaLnEa (31.9 ± 2.03%) and EaLEa (28.3 ± 1.86%), where Ea and Ln represent erucic and linolenic acid, respectively. EaOEa is present in a lower amount, since O (oleic acid) appears to be used to form other TAG such as OOO and POO. For coconut oil, the major TAG are all composed of short and medium chain fatty acids such as LaLaLa, CLaLa/CCM, LaLaM, and CaLaLa/CCLa, where La, C, Ca, and M stand for lauric, caprylic, capric, and myristic acids, respectively. A total of 74.1% of coconut oil is composed by these short- to medium-chain TAGs. The TAG composition of coconut oil is approximately similar as the values reported in the literature.^[35]

Thermal Behavior

The heating and cooling thermograms of the oil samples are shown in Fig. 3a–3f and Fig. 4a–4f, respectively. Due to the presence of a greater variety of dominant TAG in noni and lady’s finger seed oils, the thermograms of these two oils exhibited more endothermic and exothermic peaks when compared with other oil samples. All endothermic peaks of noni seed oil are found at temperature below 0ºC (D₁ = –56.4ºC, D₂ = –38.8ºC, D₃ = –29.5ºC, D₄ = –20.4ºC, D₅ = –12.4 ºC; Fig. 3a). The endset temperature (ET, temperature where the last peak goes back to the baseline, and indicates complete melting) of noni seed oil is 4.6ºC. Compared with noni seed oil, lady’s finger seed oil which has more saturated fatty acids (Table 3), has its last endothermic peak above 0ºC (B₁ = –32.0ºC, B₂ = –19.6ºC, B₃ = –3.2ºC, B₄ = –0.3ºC, and B₅ = 6.8ºC) and also a higher ET of 13.3ºC (Fig. 3c). The cooling thermogram of noni seed oil contains two broad and blunt exothermic peaks that are approximately equal in size (D₆ = –14.7ºC and D₇ = –42.8ºC; Fig. 4a). Lady’s finger seed oil has two unequal size exothermic peaks (B₆ = –5.7ºC and B₇ = –34.3ºC) in its cooling thermogram, with B₆ larger and broader than B₇ peak (Fig. 4c).

FIGURE 3 DSC melting curves of for (A) noni seed oil; (B) spinach seed oil; (C) lady’s finger seed oil; (D) bitter gourd seed oil; (E) mustard seed oil; (F) coconut oil.

FIGURE 4 DSC cooling curves of for (A) noni seed oil; (B) spinach seed oil; (C) lady’s finger seed oil; (D) bitter gourd seed oil; (E) mustard seed oil; (E) coconut oil.

The heating thermogram of spinach seed oil (Fig. 3b) showed two endothermic peaks (F₁ = –34.4ºC and F₂ = –6.0ºC) with peak F₁ as a small shoulder peak while peak F₂ as a large and broad peak and an ET at 6.3ºC. In the cooling thermogram of spinach seed oil (Fig. 4b), two exothermic peaks (F₃ = –9.7ºC and F₄ = –40.4ºC) can be observed, with peak F₄ as the broad and large peak. On the other hand, bitter gourd seed oil has only one tall endothermic peak (E₁ = 57.7ºC) in its heating thermogram and an ET at 59.7 ºC (Fig. 3d). Such a high transition temperature may be largely due to the high content of stearic acid (41.2%). The cooling thermogram of the oil (Fig. 4d) shows a single, sharp exothermic peak (E₂ = –8.7ºC).

The heating thermogram of mustard seed oil (Fig. 3e) shows two endothermic peaks (A₁ = –15.4ºC and A₃ = 2.7ºC) and one exothermic peak (A₂ = –7.6ºC). The ET of mustard seed oil is at 7.9ºC. Existence of peak A₂ is most probably due to recrystallization of the melted fat crystal. Although further investigation is needed to confirm the true cause of peak A₂ existence, it can be deduced as an exothermic peak caused by recrystallization through crystallization principle.^[75] Cooling thermogram of mustard seed oil (Fig. 4e) shows a single, narrow and sharp exothermic peak (A₄ = –35.45ºC).

Coconut oil has only one large and broad endothermic peak (C₁ = 24.3ºC) in its heating thermogram (Fig. 3f) with ET at 28.1ºC, because it contains mostly of medium-chain saturated fatty acids. In the cooling thermogram (Fig. 4f), two not-completely-resolve peaks (C₂ = 1.8ºC and C₃ = –7.6ºC) were observed and this indicates that coconut oil contains TAGs that crystallized at very similar temperature.

SFC%

The SFC% of the oil samples were derived from their heating thermograms and are shown in Fig. 5. The SFC of noni (Fig. 5a) and lady’s finger (Fig. 5c) seed oils share two common characteristics: A gradual decrease in SFC% with an increase in temperature and the formation of a half-solid (50% solid) state at temperatures below 0ºC. The latter feature is shared by spinach seed oil (Fig. 5b) but it melts at a higher rate. This indicates that these three oils will be useful in any applications that require high liquidity as only extensive cooling can change these oils into total or half-solid state. Potential applications of such oils are as salad oil or whipped-soft margarine that does not solidify when stored at refrigerator temperature.

FIGURE 5 Solid fat content (%) for (A) noni seed oil; (B) spinach seed oil; (C) lady’s finger seed oil; (D) bitter gourd seed oil; (E) mustard seed oil; (F) coconut oil.

The SFC% of bitter gourd seed oil is shown in Fig. 5d. The half-solid state of the oil is 54.9ºC. Subsequent increase of 4.8ºC from this half-solid state liquefies the oil completely at 59.7ºC. This narrow melting temperature range from half-solid to liquid state is valuable in any applications that require immediate phase change from solid to liquid. The SCF% of mustard seed oil (shown in Fig. 5e) decreased rapidly between –1.3 to 3.8ºC. Its onset of melting temperature occurred at –3.7ºC. At ambient tropical temperature, this mustard seed oil would remain mainly in the liquid state. In contrast, coconut oil can be regarded as hard oil since the SFC% is 100% at 0ºC. The SFC% decreases gradually from 0 to 14ºC, then decreased more rapidly after 14ºC (Fig. 5f) to melt completely at 28.1ºC. This indicates that food substances that need sharp melting behavior may employ coconut oil as cocoa butter substitute and ice-cream fat which are supposed to melt rapidly within body temperature.^[76,77]

CONCLUSION

The study shows that, apart from spinach seed, the samples used have an oil content >15% and the physico-chemical characterization of the oils provides valuable information including their potential applications and utilization in food and nonfood industries. All the extracted oil samples have FFA (%) value that is <2% and thus, intensive neutralization will not be needed if being processed for food usage. The noni seed oil has the highest red color index while lady’s finger and mustard seed oils have the same highest yellow color index. The IV and SV of all extracted oil samples are in accordance with the values reported in the literature. It is interesting to note that the fatty acid compositions of the extracted oils, except bitter gourd seed oil, show high proportions of fatty acids with antibacterial activity.^[78–81] Fatty acids with antibacterial activities include linoleic acid in noni, spinach, and lady’s finger seed oils, linolenic acid in mustard seed oil, and lauric acid in coconut copra oil. Although bitter gourd seed oil does not contain any fatty acid that has antibacterial activity, it contains a high level of eleostearic acid, a conjugated trienoic acid that may have the ability to inhibit microbial growth. The SFC% profile of noni, lady’s finger, and spinach seed oils show potential application in food usage as salad oil or whipped-soft margarine. Bitter gourd seed oil may have an application that is similar as coconut oil used to its narrow phase-changing temperature range.

FUNDING

The authors would like to acknowledge the Ministry of Education, Malaysia, for the research grant (ERGS/1/2012/STG04/UPM/01/9 Vot. 5527111) awarded to H. M. Ghazali.

Additional information

Funding

The authors would like to acknowledge the Ministry of Education, Malaysia, for the research grant (ERGS/1/2012/STG04/UPM/01/9 Vot. 5527111) awarded to H. M. Ghazali.