Introduction

Coconut is a tropical fruit of coconut palm, Cocos nucifera, and is largely consumed in many countries. Coconut oil is derived from the dried fruit (endosperm) which undergoes refining steps of alkali treatment, bleaching and deodorization while the virgin coconut oil is produced without any of these chemical processes [1]. It is known to impart many health benefits [1].

Coconut oil contains mainly saturated fatty acids (SFA) (≈93 %) with lauric acid (C12:0) (≈50–55 %) being the most prevalent fatty acid present [2]. It also contains medium chain fatty acids (MCFAs) consisting of caproic acid (C6:0), caprylic acid (C8:0), capric acid (C10:0) and lauric acid (C12:0) that can be easily burned for energy rather than being stored in the body [2]. Coconut oil possesses antiviral, antibacterial and antiprotozoal properties due to the presence of C12:0 and C10:0 fatty acids [3]. Monounsaturated and polyunsaturated fatty acids are less in coconut oil (≈7–8 %) and, hence, it is highly stable towards oxidation and eventually provides longer shelf-life for the food products prepared in it [4].

Coconut oil contains minor components like tocopherols, tocotrienols, phytosterols and phenolics which are the natural antioxidants. Polyphenols are ubiquitous in food and are found to act as antioxidants, free radical scavengers and peroxidation inhibitors [5]. It also acts as an anti-carcinogenic and reduces cardiovascular diseases [6–8]. Phytosterols are present predominantly in oilseed plants. In addition to nuts, they are found in legumes, vegetables and unrefined vegetable oils [9]. Tocopherols are fat soluble antioxidants having vitamin E activity. Tocotrienols are present in coconut oil, palm oil and oil from bran sources such as rice bran and wheat bran. No other vegetable oils contain tocotrienols in significant amounts. They act against the membrane lipid peroxidation of polyunsaturated fatty acids [10].

Testa is the brown part covering coconut kernel, i.e., brown skin. It is obtained from coconut processing industries as a by-product through paring of wet coconut during the preparation of products like desiccated coconut, coconut milk and virgin coconut oil. Testa is used as animal feed. Consumption of coconut kernel provides the beneficial effect on human health but the coconut testa is getting wasted.

Nevin and Rajamohan [11] have reported that the presence of phenolic compounds is mainly responsible for the antioxidant properties of virgin coconut oil. Seneviratne et al. [12] have reported that the final phenolics content of coconut oil depends on the components of the endosperm. Information on the components of coconut testa is rare. Hence, the study focuses on the evaluation of composition and minor components including tocopherols, tocotrienols, phytosterols and phenolics of oil from coconut testa in comparison with coconut whole and coconut white kernel.

Materials and Methods

Wet coconut whole (WCW) and copra whole (CW) were purchased from the local market. The white kernel and testa of wet coconut and copra were separated manually to get wet coconut white kernel (WCWK) and wet coconut testa (WCT); copra white kernel (CWK) and copra testa (CT). Standard gallic acid, cholesterol, FAME mix, tocopherols, hydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, coumaric acid, caffeic acid, ferulic acid and cinnamic acid were procured from Sigma Chemicals Co., St. Louis, USA. All reagents and chemicals used were of analytical grade.

Proximate Composition of Starting Materials

The moisture content of the coconut samples was determined according to the AOCS method Ac 2–41 [13]. Fat was extracted from dried coconut samples by using hexane in a Soxhlet apparatus according to AOCS O.M.No. Ac 3–44 [13]. The micro-Kjeldahl method was used to determine total proteins described by the AOAC Official Method 950.48 [14]. The method described by the AOAC Official Method 950.02 [14] was used for crude fiber determination. Ash content of the dried and defatted coconut samples was determined gravimetrically according to AOCS O.M.No. Ba 5a-49 [13]. The iron, zinc, sodium, potassium and calcium content of coconut samples were analyzed by atomic absorption spectroscopy (AAS) [15].

Acylglyerol Content of the Oil Samples by Column Chromatography

The TAG, DAG and MAG fractions from oil samples were separated according to the AOCS Official Method No. Cd 11c-93 [13].

Fatty Acid Composition of Oil Samples by GC

The fatty acid methyl esters of the fat extracted from the coconut kernels and coconut testa were prepared by transesterification according to the AOCS Method [13]. Analysis was carried out using a gas chromatograph (model-GC-15A, Shimadzu Corporation, Kyoto, Japan) equipped with a FID detector and a stainless steel column of 3 m length × 0.5 mm ID, coated with 15 % diethylene glycol succinate on 60–80 mesh chromosorb WAW. The operating conditions were as follows: nitrogen flow 40 ml/min, hydrogen flow 40 ml/min, air flow 300 ml/min, column temperature 180 °C, injector temperature 220 °C and detector temperature 230 °C. A reference standard FAME mix (Supelco Inc., Bellefonte, PA, USA) was analyzed under the same operating conditions to determine the peak identity. The FAMEs were expressed as relative area %.

Triacylglycerols Composition of Oil Samples by HPLC

Triacylglycerols composition was determined according to Swe et al. [16]. The HPLC system used was equipped with a Shimadzu LC-10A liquid chromatograph with a Refractive Index (RI) detector. The column used was a C18 Discovery column (Supelco, Bellefonte, USA) with a mobile phase acetone/acetonitrile (70:30, v/v) at the flow rate of 1 ml/min. The oil samples were dissolved in acetone. TAG peaks were identified based on the retention time of the TAG standards.

Phenolics Extraction from Oil Samples

The phenolics extraction from the samples was carried out according to Seneviratne and Dissanayake [17]. The phenolics were extracted using methanol: water (80:20 v/v). About 5 g of the oil sample was mixed with 1 ml of 80 % methanol and vortexed for 2 min (twice). The samples were centrifuged at 2,500 rpm for 10 min at room temperature. The methanol: water layer was collected in another tube. This step was repeated four times and the extracts pooled were made up to 4 ml with 80 % methanol.

Determination of Total Phenolics Content (TPC) of Oil Samples by the Colorimetric Method

The total phenolics content (TPC) was determined using the Folin–Ciocalteu reagent. Different aliquots were mixed with 0.2 ml of Folin–Ciocalteu reagent and were kept for 3 min. About 1 ml of 15 % Na2CO3 solution was added and made up to 7 ml with distilled water. The tubes were incubated for 45 min and centrifuged at 2,000 rpm for 10 min at room temperature. The absorbance was read at 765 nm using a UV–Visible spectrophotometer (Shimadzu corporation, Kyoto, Japan, model UV—1601). The TPC (mg/100 g) was calculated using gallic acid as standard compound [18].

Determination of Total Phytosterols Content of Oil Samples by the Colorimetric Method

Total phytosterols content was analyzed by using the Liebermann-Burchard method. About 0.3 g of oil sample was dissolved with 1.2 ml of chloroform and 2 ml of the Liebermann–Burchard reagent (0.5 ml of sulphuric acid dissolved in 10 ml of acetic anhydride, covered and kept in an ice bucket). Final volume was made up to 7 ml with chloroform and mixed well. The tubes were kept in the dark for 15 min and their absorbance was read at 640 nm using a UV–Visible spectrophotometer (Shimadzu corporation, Kyoto, Japan, model UV—1601). A blank was prepared without the sample. The total phytosterols content was calculated using standard cholesterol [19].

Analysis of Individual Phenolic Acids of Oil Samples by HPLC

The extraction of phenolic acids from coconut oils for HPLC analysis was done according to Brenes et al. [20]. The analysis of phenolic acids in the coconut oil was conducted using a Waters Atlantis C18 (250 mm × i.d. 4.6 mm, 5 μm) Bondapack column. Isocratic elution was carried out with a mobile phase consisting of water: methanol (82:18 v/v) containing 2 % (v/v) acetic acid, at a flow rate of 1 ml/min. A photo diode array (PDA) detector was used for detection of phenolic acids and the HPLC profiles were obtained at 280 and 320 nm. The injection volume for all samples was 10 μl. Identification of phenolic acids was based on retention times in comparison with standards [21].

Analysis of Tocopherols and Tocotrienols of Oil Samples by HPLC

Tocopherols and tocotrienols were estimated in the oil samples by HPLC according to the AOCS method O.M.No. Ce 8–89 [13]. The analysis of tocopherols and tocotrienols was achieved by normal phase HPLC separation on a silica column (Lichrosorb Si60 5 μm) employing Shimadzu HPLC system consisting of a LC-10A pump, injector fitted with a 20 μl loop and fluorescence detector (FLD). The mobile phase was hexane: isopropyl alcohol (99.5:0.5, v/v) at the flow rate of 1 ml/min. The excitation wavelength of 290 nm and an emission wavelength of 330 nm were kept for the fluorescence detection of all the peaks. The tocopherols and tocotrienols were identified using standard tocopherols and expressed as α-tocopherol.

Statistical Analysis

All the analyses were carried out in triplicate and the average values ± SD are reported. One-way ANOVA was used to calculate significant differences among the coconut and oil samples [22]. A two-tailed p value was determined to show the significant differences at a p value ≤0.001.

Results and Discussion

Proximate Composition of Starting Materials

The composition of CW, CWK, CT, WCW, WCWK and WCT were analyzed and are presented in Table 1. The composition included moisture content, fat content, protein content, carbohydrate content, crude fiber content and ash content. The moisture content was less in copra (3.8, 4.0 and 4.3 %) when compared to the wet coconut (43.5, 32.9 and 42.2 %). WCT contained a lower moisture content than WCW and WCWK. Solangi et al. [23] had reported that the moisture content of mature coconut ranges from 38 to 62 % and ash content 0.85–1.26 % for different coconut varieties. According to Obasi et al. [24], the moisture content of copra was 7.51 %, crude fiber was 7.70 % and fat content was 47.80 %. The chief constituent of coconut kernel was carbohydrate, followed by lipid. On the wet basis, the fat content of copra was high ranging from 59.8 to 63.4 % and the wet coconut was low ranging from 34.7 to 38.8 %. CT had the same amount of fat when compared to CW and CWK. The crude fiber content of copra was less (6.6–11.6 %) and wet coconut was more (11.7–17.2 %). Both CT (11.6 %) and WCT (17.2 %) had high crude fiber content. The crude fiber content of healthy coconut kernel was 13.13 % [25]. The ash content of copra was slightly higher than that of the wet coconut. CW had an ash content of 1.4 %, CWK was 2.1 % and CT was 1.4 %. WCW had an ash content of 1.0 % of which WCWK was 0.9 % and WCT was 0.7 %. WCT had less ash content when compared to WCW and WCWK. CWK had more ash content than in CW and CT. Santoso et al. [26] showed that the ash content of wet coconut and copra (dry matter) was 1.15 and 2.11 %, respectively. The protein content of CW, CWK, CT, WCW, WCWK and WCT were 10.2, 8.1, 9.3, 7.5, 6.2 and 7.1 % respectively [23, 24]. The carbohydrate content was calculated by difference. The carbohydrate content of WCT (24.6 %) and CT (26.3 %) was more than other coconut samples (10.6–24.3 %).

Table 1 Proximate composition of starting material
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Mineral Composition of Coconut Samples

The mineral composition of coconut samples is presented in Table 2. The coconut testa and kernel have substantially more potassium than sodium. CW, CWK, CT, WCW, WCWK and WCT had 120.3, 124.1, 120.3, 122.1, 123.8 and 107.8 mg of potassium/100 g, respectively. They had 15–29 mg/100 g of sodium, 14–18 mg/100 g of calcium, 1.5–7.9 mg/100 g of iron and 1.6–3 mg/100 g of zinc content. CT contained 29.8 mg/100 g of sodium content. CW, CT and WCW contained 7.9, 6.2 and 7.9 mg of iron content/100 g, respectively. CW (2.9 mg/100 g) and CT (3.0 mg/100 g) contained slightly high zinc content. CT (22.4 %) and WCT (29.8 %) had more sodium content than in other coconut samples. The results are in good agreement with that reported by Solangi et al. [23].

Table 2 Mineral composition of coconut kernel and testa
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Acylglycerol and Fatty Acid Compositions of Oil Samples

The acylglycerol and fatty acid compositions of oil samples are presented in Table 3. CW, CWK, CT, WCW, WCWK and WCT oils had a TAG content of 90–98.2 %, DAG of 1–8 % and MAG of 0.4–2 %. CW contain a high amount of DAG, i.e. 8.4 %, CT oil with 5.3 % and WCT oil with 3.2 %. WCWK oil contained a high amount of TAG (98.2 %) and CW oil was 90.00 %. Oils from wet coconut (96.4–97.7 %) contained a slightly high TAG content when compared to that of copra oils (90–94.1 %). Whereas for DAG content, it was more in CW oil (8.4 %) and less in WCWK oil (1.1 %). DAG content was higher in copra (4.6–8.4 %) than in wet coconut (1.1–3.2 %). MAG content was also slightly higher in copra (0.6–2.0 %) when compared to the wet coconut (0.4–0.6 %). DAG is used in small quantity in foods as an emulsifier [27, 28].

Table 3 Acylglycerol and fatty acid compositions of oils extracted from coconut kernel and testa
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The fatty acid compositions of coconut oil samples are reported in Table 3. Lauric acid is the major fatty acid of coconut oil [1]. The lauric acid content of CT and WCT oils was 40.94 and 32.4 %, respectively whereas CW, CWK, WCW and WCWK oils were 50–53 %. The caprylic acid (C8:0) and capric acid (C10:0) content of CT and WCT oils were slightly less when compared to other oil samples. Palmitic acid (C16:0) content of CT (11.31 %) and WCT (14.07 %) oils were higher than the other oil samples, which is varying from 6.8 % for WCW to 7.4 % for WCWK and CWK oils. MCW (2.3 %) had more linolenic acid (C18:0) followed by CWK (1.9 %), WCWK (1.9 %), CT (1.6 %), WCT (1.2 %) and CW (1.1 %). CT and WCT oils contained 12.19 % and 17.82 %, respectively, of oleic acid (C18:1) and 5.32 and 10.6 %, respectively, of linoleic acid (C18:2) content whereas CW, CWK, WCW and WCWK oils contained 4–6 % of C18:1 and 1–2 % of C18:2. The oils from CT and WCT were found to be having more C18:1 and C18:2 than other oil samples. The SFA of CT and WCT oils (82.51–71.59 %) was marginally less when compared to CW, CWK, WCW and WCWK oils (92–95 %); MUFA of CT was 12.19 %, WCT oils was 17.82 % and CW, CWK, WCW and WCWK oils were 4–6 %; PUFA of CT and WCT oils was 5.32 % and 10.6 %, respectively, and CW, CWK, WCW and WCWK oils were 1–2 %. MCFA was less in CT (48.25 %) and WCT (36.16 %) oils whereas CW, CWK, WCW and WCWK oils were 64–68 %. The CT and WCT oils have higher PUFA and MUFA as compared to other coconut oil samples. The low SFA and MCFA in CT and WCT are most probably due to the presence of a high amount of MUFA and PUFA than in other samples. Oils from CT and WCT were significantly different from other coconut oil samples in the fatty acids composition. The fatty acids composition of whole coconut kernel oil was in good agreement with that reported by Bhatnagar et al. [3].

Triacylglycerol Composition of Oil Samples

The major TAGs in the coconut oils were CCLa, CLaLa, LaLaLa and LaLaM; these counted for around 50–75 % of the total TAG composition (Table 4). The sum of these four TAGs in CW, CWK, CT, WCW, WCWK and WCT oils were 51.82, 64.96, 50.4, 74.61, 76.67 and 39.97 %, respectively. WCT oil showed comparatively slightly higher LaLaO (6.42 %), LaMO (8.04 %), LaMP (8.0 %), LaSO (3.46 %), MOO (3.48 %), MPO (2.79 %), OOO (3.35 %), POO (2.22 %) than CWK, CT, CW, WCWK and WCW oils. CCLa (4.64 %), CLaLa (8.56 %), LaLaLa (12.34 %) and LaLaM (14.43 %) were less in WCT oil as compared to other samples. The trilaurin (LaLaLa) is less in oils from WCT (12.34 %) and CT (14.30 %) whereas WCW, CW, CWK, WCWK showed 23.04, 16.23, 20.01 and 22.28 %, respectively. CT oil contained the least amount of dilaurin (LaLaM) (5.04 %) than CWK (16.38 %), CW (16.0 %), WCWK (15.93 %), WCT (14.43 %) and WCW (10.41 %) oils. WCT oil had a low amount of CCLa (4.64 %) and CLaLa (8.56 %). WCT oil (8.04 %) and CT oil (6.21 %) showed comparatively higher LaMO content than oils from CWK (2.93 %), CW (4.62 %), WCWK (1.33 %) and WCW (1.54 %). CT oil had 0.29 % and WCT oil had 8.00 % of LaMP content. MPL was marginally more in CT oil (1.92 %) and very less in CWK (0.33 %), CW (0.54 %), WCWK (0.20 %), MCT (0.18 %) and WCW (0.08 %) oils. Triolein content was more in WCT oil (3.35 %) whereas CT, CW, WCWK and WCW oils had 0.13, 1.53, 0.14 and 0.53 % respectively. The TAG composition of wet coconut kernel and copra were similar to that of what Gopala Krishna et al. [1] had reported. The TAG composition of oils from testa showed some differences when compared to other oils.

Table 4 Triacylglycerol composition of oils from coconut kernel and testa
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Composition of Oil Samples

In the coconut industry, the testa is removed during oil extraction as it gives a yellowish color to oil. The oils extracted along with the testa gave little higher phenolics content than the oil extracted without testa. Nutraceuticals content and compositions of oil samples are presented in Table 5. The total phytosterols content of CW, CWK, CT, WCW, WCWK and WCT oil were 49.89, 33.31, 42.52, 50.27, 30.66 and 50.97 mg/100 g respectively. The total phytosterol content was more in CW (49.89 mg/100 g) and WCW (50.27 mg/100 g) than in CWK (33.31 mg/100 g) and WCWK (30.66 mg/100 g). This was may be due to the presence of testa while extracting the oil. The total phytosterols content of oils from CT and WCT was almost similar to that of oils from CW and WCW whereas the oils from CWK and WCW were less when compared to other coconut oils. Raja Rajan et al. [29] had reported that the total phytosterols content of whole coconut oils was 87 mg %.

Table 5 Composition of oils extracted from kernel and testa
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Oil samples showed fewer amounts of phenolics. CW, CWK, CT, WCW, WCWK and WCT had 1.1, 1.9, 1.4, 0.2, 0.5 and 0.7 mg of TPC/100 g oil, respectively. TPC was more in the copra than in the wet coconut. Henna et al. [30] reported that virgin coconut oil contained 0.65 mg of total phenolics/100 g of oil. All the samples showed the presence of gallic acid, hydroxybenzoic acid, vanillic acid, syringic acid, coumaric acid, caffeic acid, ferulic acid and cinnamic acid in significant amounts. The total phenolic acids content of oils from WCT and CT (388.9 and 313.1 μg/100 g, respectively) were higher when compared to that of the WCW, WCWK, CWK and CW oils (291.1, 94.7, 141.8 and 131.1 μg/100 g of oil, respectively). Among the phenolic acids, coumaric acid content was found to be more in WCT oil (230.6 μg/100 g) and hydroxybenzoic acid content in CT oil (126.4 μg/100 g). Seneviratne et al. [31] had reported the same phenolic acids composition in coconut kernel. The results are in agreement with the reports of Seneviratne and Dissananyake [17]. Marina et al. [32] had reported that the phenolic acids composition of coconut kernel oil varies with different extraction methods and contained protocatechuic acid, vanillic acid, caffeic acid, syringic acid, ferulic acid and p-coumaric acid. Phenolic acids were found to be more in mature coconut than in copra. The oils from testa were found to have higher total phenolic acids than CW and WCW followed by CWK and WCWK.

The coconut kernel oils contained less tocopherols (T) and tocotrienols (T3) content when compared to oils from testa. The T and T3 content of CW, CWK, WCW and WCWK oils were only 2.9, 6.7, 4.4 and 2.5 mg/100 g of oil, respectively. However, CT and WCT oils had 22.3 and 100.1 mg/100 g oil of T and T3 content. CW (2.1 mg/100 g) and CWK (3.7 mg/100 g) oils had more of β + γ tocopherols content whereas WCW (3.6 mg/100 g) and WCWK (2.5 mg/100 g) oils had more of α-tocopherol content. Oils from CT and WCT were found to have higher tocotrienols concentration than other coconut oil samples. There was 90.2 mg/100 g for WCT oil followed by CT oil, 16.6 mg/100 g. WCT oil contained 2.9 mg/100 g of δ-tocopherol content. CT oil contained 0.7 mg/100 g of δ-tocotrienols content. Raja Rajan et al. [29] had reported that whole coconut oil contained 1.7–3.1 mg/100 g of total tocopherols and tocotrienols content.

Conclusion

Coconut testa and kernel samples are rich in potassium content. The CT and WCT oils have higher PUFA and MUFA as compared to other coconut oil samples. Oils extracted from coconut testa were found to be rich in minor components like tocopherols, phenolics and phytosterols than oil extracted from kernel or whole coconut. They contained ≈50 mg of total phytosterols/100 g of oil. CT and WCT oils contained slightly higher DAG content than WCWK, WCW and CWK oils. WCT oil contained more amounts of coumaric acid whereas CT oil was found to have more of hydroxybenzoic acid than CW, CWK, WCW and WCWK oils. The oil from WCT had less trilaurin content and slightly higher triolein content when compared with other coconut oil samples. To our knowledge, reports on composition and natural antioxidants/minor components of oil from coconut testa have not been reported so far. Dried CT and WCT yield same amount of fat like copra and dry coconut kernel; therefore, they can also be used as a good oil source. Oils extracted from WCT and CT could be used for regular consumption as they contain a similar fatty acid composition, acylglycerol and triacylglycerol profile, minor components like phenolics, phytosterols, tocopherols and tocotrienols for beneficial health effects.