Comparative quantitative analysis of fruit oil from Hippophae rhamnoides (seabuckthorn) by qNMR, FTIR and GC–MS

Abstract

Objective

To develop a qNMR method for quantitative analysis of triacylglycerols in fruit oil of Hippophae rhamnoides (seabuckthorn, SBT) and analyze commercial samples of SBT oils using GC–MS and FTIR.

Methods

SBT fruit oil (IPHRFH) was extracted with hexane and the triglyceride (TAG) was isolated by vacuum liquid chromatography. Six different branded SBT oils purchased from e-commerce suppliers (Amazon) and in-house prepared SBT oil was analyzed by qNMR and fatty acyl composition of TAGs determined by using NMR. In-house oil was also analysed by GC–MS and FTIR spectroscopy.

Results

The qNMR results showed that the oil contained 80.3% of triacylglycerol (TAG). The SBT oil TAGs comprised of linolenate 6.6%, palmitoleate/oleate 65.4%, and total saturated fatty acyl chain including palmitate 28% as determined by qNMR. GC–MS analysis revealed that the major acyl functionalities present in the TAG were palmitoleic acid 36.5%, oleic acid 12.9%, palmitic acid 21.2%, and linoleic acid 18%. Of the six commercial samples analyzed, samples from only one supplier (SW) were fruit oil; All others were the seed oils or mix of fruit oil and seed oil. The labels for samples except for the SW did not indicate whether it was fruit oil or seed oil.

Conclusion

The results suggest that SBT oil should be analyzed by combination of GC–MS, FTIR and qNMR for factual content of free fatty acid or TAGs, which are chemically different in nature and affect the quality of oil. GC–MS showed the content of omega free fatty acids after hydrolysis, while qNMR and FTIR showed the content of TAGs. The major acyl functionalities found in SBT fruit oil TAGs are palmitoleate/palmitate/oleate, while linoleate and linonelate make up a minor fraction. Furthermore, analysis of commercial samples showed discrepancies between label claims and actual content.

1. Introduction

Seabuckthorn (Hippophae rhamnoides L.) from the Elaeagnaceae family is a shrub, mainly wild or cultivated at high altitude regions of Himalayan Mountain range in India (Korekar, Dolkar, Singh, Srivastava, & Stobdan, 2014). It is widely distributed in European and Asian countries such as Russia and China (Rousi, 1971). Seabuckthorn (SBT) has a long history of usage in traditional medicine in Asia and Europe (Dong, Binosha, Durham, Stockmann, & Jayasena, 2023). The health benefits are documented in various medicinal texts dating back to ancient Chinese and Tibetan dynasties. SBT has also been used in traditional Mongolian medicine for more than 800 years now (Suryakumar & Gupta, 2011). SBT has been used in traditional Chinese medicine for almost 1000 years dating back to Tang Dynasty (AD 618–907) (Dhyan, Maikhuri, Misra, & Rao, 2010). SBT was first admitted in Chinese Pharmacopoeia in 1977 (The State of Pharmacopoeia Commission of P.R. China, 1977). In traditional Chinese medicine, SBT is reported for use in different disease conditions like cardiovascular diseases, hepatic disorders, fever, cold, toxicity, inflammation, metabolic disorders, depressive disorder, cough and gynecological diseases (Olas, 2018, Zhao et al., 2017, Liu et al., 2021). SBT is commonly called as Chinese medicinal plant as it is widely mentioned in ancient and recent literature for the use of its different parts in traditional Chinese medicine (Wani et al., 2016). China is the largest producer of SBT in the world; more than 90% of the SBT habitats are located in China (Dong, Binosha, Durham, Stockmann, & Jayasena, 2023). These factors may have contributed to the extensive research on SBT in China. SBT has attracted global attention due to its high nutritional and medicinal value. Seven species and eight subspecies of SBT have been reported (Suryakumar and Gupta, 2011, Ma et al., 2022). Different parts of SBT, namely leaves, fruits, pulp and seeds have been reported for various classes of bioactive compounds, which are lipophilic to hydrophilic in nature. It is a rich source of flavonoids, carotenoids, pigments, minerals, amino acids, fatty acids and phytosterols (Yuzhen and Fuheng, 1997, Teleszko et al., 2015, Żuchowski, 2023). The patents on a number of pharmacological activities, like cardiovascular, antioxidant, anti-inflammatory, antiulcer, anticancer, antihypertensive, radioprotective, anti-ulcer, antibacterial and etc, have recently been reviewed (Singh et al., 2019, El-Sohaimy et al., 2022), highlighting the important health benefits of this plant. SBT oil is more popular worldwide because it comprises the essential fatty acids like omega 3, 6, 7, 9 (Yang and Kallio, 2001, Cenkowski et al., 2006). This fascinating combination of chemical composition provides the health promoting values to SBT. Seed and pulp oils of SBT were evaluated for activity against gastric ulcers in rats and both the oils showed preventive as well as curative effects (Xing et al., 2002). Both the oils also showed protection against intestinal injury induced by radiation (Shi et al., 2017). A placebo-controlled double bind study showed beneficial effects of seed and pulp oil on atopic dermatitis (Yang et al., 1999).

Triacylgycerol (TAG) molecules contained in edible oils consist of saturated and unsaturated fatty acids attached as acyl chains to glycerol molecule. Glycerol can be esterified by single or different fatty acid molecules (Barison et al., 2010, Matthäus, 2010). The composition of oils is traditionally determined by GC and GC–MS, where TAGs are hydrolysed to fatty acids and then esterified to their methyl esters (Singh & Sharma, 2022). SBT oils are available in the market with label claims as omega fatty acids for health benefits. This analysis is done by GC–MS which determines the released fatty acids from TAGs but does not quantify the TAGs (Teleszko et al., 2015, Dulf, 2012). While fatty analysis is suitable for edible oils as TAGs are known to be metabolized into fatty acids in the body, this method of reporting fatty acids as components of oils may not be suitable where oils are used for external applications. In such cases, it is important to analyze qualitatively and quantitatively the oils for TAGs. The TAG composition of SBT of different geographical origins was determined through hydrolysis and analysis of fatty acid composition (Vereshchagin & Tsydendambaev, 2010). Similarly, in another study, Yang and Kallio (2006) also determined TAGs in SBT through hydrolyzed fatty acids composition using mass spectrometry. However, there has been no direct analysis of TAGs in SBT oil. Quantitative Nuclear Magnetic Resonance (qNMR) is more suitable and simple method for analyzing the TAGs in oils compared with GC and GC–MS (Barison et al., 2010, Gottstein et al., 2019). We report here direct analysis of SBT oil TAGs by qNMR and a comparison of the results with GC–MS analysis.

Various analyses of SBT fruit oil are reported in literature for the determination of the presence of different saturated and unsaturated fatty acids totaling to almost 100% (Fatima et al., 2012). During our work on SBT fruit oil, we isolated large amount (> 70%) of a compound from the fruit oil; The ¹H NMR spectrum of the isolated compound clearly indicated that this was a TAG and not the free omega fatty acids. This prompted us to study the composition of marketed oils, many of which claim the presence of omega fatty acids and its comparison with in-house prepared SBT fruit oil (IPHRFH). Six different branded SBT oils were purchased from e-commerce suppliers (Amazon): Seabuckwonder (SW), Deveherbs (DV), Ryaal (RY), Grace SBT (GS), Health Diva (HD), and Omega 7 (O7). Analysis of marketed oils and IPHRFH was done by using qNMR. Actual isolation of TAG was also done from all samples of the marketed oils.

2. Materials and methods

2.1. Materials

Chemicals: Hexane, dichloromethane, ethyl acetate, methanol, analytical grade acetic acid, formic acid, potassium hydroxide (KOH) and silica gel were purchased from CDH Laboratory Reagents (New Delhi, India). Absolute ethanol was purchased from Changshu Hongsheng Fine Chemical Co., Ltd. (Changshu, China). Potassium Bromide (KBr), deuterated chloroform (CDCl₃) 1,3,5-trimethoxybenzene were purchased from Sigma Aldrich (St. Louis, Missouri, USA). The NMR data was recorded on Bruker Avance III 400 spectrometer (400 MHz) and JEOL 600 MHz NMR instrument.

Plant materials: Plant materials were collected from Lahaul and Spiti Valley of Himachal Pradesh, India in September 2017. These samples were morphologically validated and submitted for authentication at the institute’s plant authentication department (Department of Environmental Technology, CSIR-IHBT, Palampur, H.P. India). The voucher specimen IHBT No. 1047 Hippophe rhamnoides is deposited at CSIR - Institute of Himalayan Bioresource Technology (IHBT), Palampur, HP herbarium. The berries were stored at –20 °C until they were processed.

2.2. Sample preparation

Samples: Six different branded seabuckthorn oils were purchased from e-commerce suppliers (Amazon) 1: Seabuckwonder (SW; lot No.1203), 2: Deve herbs (DV; batch No. 0618), 3: Ryaal (RY), 4: Grace SBT (GS; batch No. SBT-050), 5: Omega 7 (O7; batch No. ASC 01), 6: Health Diva (HD; batch No. SE/DEL/2018).

Extraction of oil from fruits of H. rhamnoides: Two alternate routes were adopted for extraction of fruit oil. SBT fruits (10 g) were subjected to extraction with hexane 30 mL × 3 times to yield 1.1 g fruit oil. The extracted fruits were then further extracted with 70% ethanol three times for 72 h each at room temperature to yield 3 g of extract (IPHRFEW-E1; route 1). In the second method, SBT fruits were first extracted with 70% ethanol by maceration for 3 d and the process was repeated three times (IPHRFEW-E2). Extracted SBT fruits was macerated with hexane at room temperature for 24 h and repeated for three times (route 2). Both the methods resulted in similar extractive yields. The extraction process was repeated three times to obtain average extractive yields. Route 2 was used for subsequent extractions for preparation of fruit oil.

2.3. HPLC analysis of 70% ethanol extract prepared by route 1 and route 2

The HPLC analysis was performed on a Shimadzu HPLC system (Shimadzu Corp., Kyoto, Japan) LC-10ATVP model fitted with SIL-20AC autosampler and SPD-M10AVP photodiode array detector (PDA). The separations were achieved using a reversed phase C₁₈ column (250 mm × 4.6 mm i.d.; 5 µm) subjected to gradient elution. An aliquot of the dried 70% ethanolic extract (5 mg) was dissolved in methanol (1 mL) and filtered through a 0.45 μm polytetrafluoroethylene syringe filter. The separation of compounds was accomplished on a C₁₈ column using the mobile phase gradient (mobile phase: methanol and B, 0.1% formic acid in water) 5% to 100% increment of methanol in 50 min then same concentration kept for up to 60 min. Flow rate was 1 mL/min, column at 40 °C temperature, detection at 368 nm, with injection volume of 10 μL.

Isolation of triglyceride: Vacuum liquid chromatography was used for isolation of TAG using TLC grade Silica gel as adsorbent. Triglyceride was eluted with 100% hexane.

2.4. FTIR spectroscopy

Infrared (IR) spectra were recorded by using Perkin Elmer–Spectrum II (PerkinElmer,Inc. Waltham, Massachusetts, US). All spectra were recorded from 4000 to 500 cm⁻¹. A small amount of oil sample was dissolved in DCM and the sample deposited over a KBr disk. The assignment of frequency band specific to the functional group vibration was compared with the reported literature values for triglyceride (Guillén et al., 2003a, Guillén and Ruiz, 2003b).

2.5. Quantitative NMR analysis of fruit oil and marketed oils

The weighed amount of oils and 1,3,5-trimethoxybenzene (TMB) as an internal standard was dissolved in 0.6 mL of CDCl₃. ¹H NMR spectra were acquired in a non-spinning mode at a temperature of 293 K. The number of scans was 64/128 and spectral width was 16 ppm. The other acquisition parameters were: 32 K data points, pre-acquisition delay 6 µs, acquisition time 4.0 s, and relaxation delay of 5.0 s and flip angle of 30 °C (Choudhary, Sharma, & Singh, 2016). Preliminary data processing was carried out with the Bruker software TOPSPIN 3.2 and JEOL Delta 5.2.0.

TAG was calculated using the following formula,

Wx = (Ax/As) (Ns/Nx) (Mx/Ms) ws

Where, Wx: analyte weight, Ws: internal standard weight, Ax: analyte integral, As: internal standard integral, Nx: integrated proton of analyte, Ns: integrated protons of standard, Mx: analyte molecular weight (trilinolein was taken as reference molecular weight), Ms: standard molecular weight.

2.6. GC–MS analysis

GC–MS analysis of oil sample was performed by using Perkin Elmer Clarus 600/600C GC/MS PerkinElmer, Inc. Waltham, Massachusetts, US. Oil sample was methylated with 2 mol/L methanolic potassium hydroxide with stirring for 5 min at 35–40 °C. For GC–MS analysis, Elite-5MS capillary column (30 m × 25 mm i.d, 0.25 µm film thickness) was used. The oven temperature was held at 60 °C for 5 min, and then programmed from 60 to 220 °C at 5 °C/min using nitrogen as the carrier gas at 1.5 mL/min. Injection and detector temperatures were 250 °C; split ratio was 1:20; injection volume was 0.5 μL. Quantification of fatty acid methyl esters was obtained directly from GC peak area integrations using an electronic integrator (Peak Simple-II programme) and expressed as percentages (Cakir, 2004). The flow chart is shown in Fig. 1.

Fig. 1.

Flow chart of work plan.

3. Results and discussion

A perusal of literature on SBT indicated that the plant material is either used for preparation of oil or of alcoholic extracts from the fruits (Cenkowski et al., 2006, Cakir, 2004, Chen et al., 2013, Zielińska and Nowak, 2017, Velioglu et al., 1998). Further usage of exhausted material is not shown. We wished to develop method where the same material can be used to prepare both the alcoholic extract as well as the oil. If the fruits are first extracted with hexane to produce oil, the residue, if used for preparation of alcoholic extracts may contain hexanes as residual solvents. Therefore, residual fruits may not be suitable for further extraction. However, if fruits are first extracted with alcoholic solution, the marc can be used for preparation of hexane extracts/oil. We compared the extracts prepared by these two separate routes: route 1 - first extraction with hexane (H1) followed by extraction with 70% ethanol (E1) and route 2 - first extraction with 70% ethanol (E2) followed by hexane (H2). As shown in Table 1, similar extractive yields were obtained by both the routes. HPLC chromatograms of ethanolic extract prepared by both the routes were also very similar (Fig. 2). These results indicated that fruits can be first used to prepare 70% alcoholic extract. Thereafter, the marc can be used to prepare the oil, for optimal utilization of this natural resource.

Table 1.

Extractive yields from SBT fruits.

Sr. No.	Plant materials	70% Ethanolic extract (%)	Hexane extract (Oil, %)
1	Fruits (10 g)	29.00 ± 1.05 E1	11.23 ± 0.90 H1
2	Fruits (10 g)	32.23 ± 0.97 E2	9.63 ± 1.30 H2

Fig. 2.

HPLC chromatogram of 70% ethanolic extracts of fruits A (IPHRFEW-E1) and B (IPHRFEW-E2).

The process was repeated on a large scale. The marc obtained after extraction of SBT fruits (1 kg) with 70% ethanol–water was further macerated with hexane 3 L × 3 times for 24 h each and yielded 80 g fruit oil. The oil showed the following physicochemical properties: density 0.929 g/mL; saponification value 231.4 mg KOH/g; refractive index 1.402–1.430 (31.6 °C); viscosity 49.3 centipoise (25.8 °C).

Approximately 21 g of triacylglycerol was isolated by vacuum liquid chromatography (VLC) from 30 g of fruit oil in a single batch. VLC is fast and less time consuming preparative technique, therefore it was used for separating triglycerides. Percentage of TAG by isolated yield from all marketed and in house prepared SBT fruit oil is mentioned in Table 2. The presence of TAG in SBT oil was suggested by FTIR spectra (Fig. 3). The FTIR spectra showed a band at 3 005.86 cm⁻¹ for stretching vibration of cis olefinic bonds. The C = O group of TAG showed stretching vibration at 1 744.28 cm⁻¹. The bending vibrations at 1 465.06 cm⁻¹ could be assigned to the CH₂ and CH₃ aliphatic groups and the symmetrical bending vibrations at 1 377.52 cm⁻¹ to the CH₃ groups. The C–O ester group stretching and the CH₂ group bending vibrations were observed at 1 238.92 cm⁻¹ and 1 163.02 cm⁻¹. In the FTIR spectrum of hydrolyzed SBT oil (Fig. S7), a band was found at 1 709.80 cm⁻¹ instead of 1 744.28 cm⁻¹ which indicated absence of glyceride ester (stretching vibration of C = O group of free fatty acid) (Guillén & Ruiz, 2003a).

Table 2.

Percentage of TAG by qNMR and isolated yield from marketed oil and IPHRFH.

Sr. No.	Sample code	Samples (mg)	Internal standard (TMB) (mg)	TAG (%) qNMR	Isolated (%) yield of TAG
1	IPHRHF	11.4	1.5	80.3	77.2
2	SW berry oil (Imported)	14.6	1.6	85.8	72.8
3	DV (Indian)	13.2	1.5	84.0	74.7
4	RY (Indian)	14.1	1.1	82.5	72.3
5	GS (Indian)	19.2	1.52	92.9	73.7
6	HD (Indian)	19.9	1.0	95.2	78.9
7	O7 (Indian)	12.5	2.1	88.0	76.4

Fig. 3.

FTIR of SBT oil.

3.1. Analysis of oil by NMR

¹H NMR spectra of IPHRFH and marketed oils were recorded under the conditions mentioned in experimental section. ¹H NMR signal indicated the typical triglyceride signals at δ 4.12–4.30 (two doublets of doublets, marked α in Fig. 4) due to the presence of protons on carbon atoms 1 and 3 of the glyceryl group (—CH₂OCOR), while the signal at δ 5.33–5.36 (marked β in Fig. 4) is due to the proton on carbon atom 2 of the same glyceryl group (> CHOCOR) (Guillén and Ruiz, 2003b, Guillén and Ruiz, 2003c). In all the studied samples, the signals observed in ¹H NMR spectra were clearly indicative of the presence of TAGs, the samples varied only in the integral values of signals relative to the internal standard thereby indicating the presence of different amounts of TAGs in all samples. The total amount of TAG present in IPHRFH was found to be 80.3% by qNMR (Table 2). The GC–MS data (Table 3) showed the presence of 18 fatty acids, five MUFAs, five PUFAs, and two trans-fatty acids. However, five fatty acids contributed 94% of the fatty acid profile while the others are in minor quantity. Therefore, only major five fatty acids were determined by qNMR (Table 4, Fig. 4). The GC–MS analysis of oil showed palmitoleic acid 40.95%, oleic acid 10.25%, palmitic acid 31.63%, and linoleic acid 10.2% as the major acyl functionalities present in the oil. It showed total monounsaturated fatty acids at 54%, polyunsaturated fatty acids at 10% and saturated fatty acids at 34.3% (Table 3, Fig. 5). A typical qNMR quantification of TAG in IPHRFH was shown below. A total of 1.5 mg of internal standard TMB was added to 11.4 mg of oil and 600 mL CDCl₃ was added. The NMR was acquired under the conditions mentioned in the experimental section. The integral value of two methylene protons at δ 4.1 was taken for calculations and molecular weight of trilinolein 879.3 is taken as standard. The calculated value of TAG was 9.16 mg (according to the equation below) in 11.5 mg of oil (80.3%).

$$x=\frac{2.36\times 3\times 879.3\times 1.5}{3.01\times 2\times 168.15}=9.16\mathrm{m}\mathrm{g}$$

Fig. 4.

¹H NMR spectrum of SBT fruit oil (IPHRFH).

Table 3.

Free fatty acid profile of in-house SBT fruit oil (IPHRFH) and isolated TAG by GC–MS.

Sr. No.	Fatty acid profiling	SBT oil area (%)	Isolated TAG area (%)
	Saturated fatty acid
1	Butyric acid	0.14	0.05
2	Caproic acid	0.04	0.05
3	Caprylic acid	0.02	0.02
4	Undecanoic acid	0.03	0.03
5	Lauric acid	0.03	0.03
6	Myristic acid	0.35	0.31
11	Pentadecanoic acid	0.06	0.07
12	Palmitic acid	31.63	21.18
13	Heptadecanoic acid	0.05	0.05
14	Stearic acid	1.29	1.08
15	Arachidic acid	0.30	0.17
16	Heneicosanoic acid	0.10	0.02
17	Behenic acid	0.12	0.03
18	Lignoceric acid	0.11	0.05
	MUFA
1	Palmitoleic acid	40.95	36.48
2	cis-10-Heptadecanoic acid	1.01	1.20
3	Oleic acid	10.25	12.93
4	cis-11-Eicosanoic acid	2.39	7.71
5	Nervonic acid	0.09	0.02
	PUFA
1	Linoleic acid	10.20	18.00
2	Linolenic acid	0.24	0.17
3	Arachidonic acid	0.09	0.04
4	cis-11,14-Eicosadienoic acid	0.15	0.02
5	cis-5,8,11,14,17-Eicosapentaenoic acid	0.10	0.02
	Trans fatty acids
1	Elaidic acid	0.16	0.02
2	Linoelaidic acid	0.02	−
	Saturated fatty acids	34.30	28.20
	MUFA	54.70	58.40
	PUFA	10.90	18.40
	Trans fatty acids	0.20
	Total	100.00	100.00

Table 4.

Percentage of fatty acyl chains by ¹HNMR.

Samples	Linolenate (%)	Linoleate (%)	Palmitoleate + oleate (%)	Saturated fatty acyl chain + Palmitate (%)
SBT fruit oil (IPHRFH)	6.6	−	65.4	28.00
SW (Imported)	7.0	−	64.6	28.4
DV (Indian)	14.5	49.0	27.0	10.0
RY (Indian)	13.0	38.0	37.0	12.0
GS (Indian)	18.2	37.0	27.4	17.2
HD (Indian)	25.9	22.4	23.9	21.0
O7 (Indian)	54.0	16.0	22.0	10.0

Fig. 5.

GC–MS chromatogram of SBT oil.

The ¹H NMR spectra of all marketed oils are shown in supplementary information (Figs. S1–S6). The results suggested that the marketed oils consist mainly of triglycerides and not the free omega fatty acids as claimed on labels, which are produced from TAGs.

3.2. Characterization of TAG

The fatty acyl composition of TAGs can be determined by using NMR and several methods and have been employed (Barison et al., 2010, Guillén and Ruiz, 2003c, Korekar et al., 2014). Guillén and Ruiz (2003b) have explained the use of ratios of integrals for various characteristic signals of saturated and unsaturated acyl chains as well as the C-1 and C-3 of glyceride moiety to determine the percentage of linoleiate, linolenate, palmitoleiate/oleate and saturated acyl chains. The ¹H NMR spectrum (Fig. 4) showed signal at δ 0.86 to 0.89 for methyl protons of saturated, monounsaturated, and di-unsaturated oleiate, palmitoleiate and linoleiate acyl groups (Signal A). A signal at δ 0.945 to 1.045 (B) for methyl protons is typical of linolenate or other similar polyunsaturated fatty acyl groups. The signals at δ 4.12 and 4.27 appear due to the protons on carbon atoms sn-1 and sn-3 of the glyceryl group (-CH₂OCOR). Signal C is due to the methylene groups in allylic position and signal E is due to methylenes in bis-allylic position. Signal D is due to the methylenes alpha to carbonyl group. Calculation of fatty acyl chains in TAG was performed by integration of these acyl signals and using the equations given below (Guillén & Ruiz, 2003c).

$$\mathrm{L}\mathrm{n}=\frac{\mathrm{B}}{\mathrm{A}+\mathrm{B}}$$

$$\mathrm{L}=\left(\frac{\mathrm{E}}{\mathrm{D}}\right)-2+[\frac{\mathrm{B}}{(\mathrm{A}+\mathrm{B})}]$$

$$S=1-\left(\frac{C}{2D}\right)$$

$$\mathrm{O}=\left(\frac{\mathrm{C}}{2\mathrm{D}}\right)-\left(\mathrm{L}+\mathrm{L}\mathrm{n}\right)=\frac{C}{2D}-\left(\frac{\mathrm{E}}{\mathrm{D}}\right)+[\frac{\mathrm{B}}{(\mathrm{A}+\mathrm{B})}]$$

The qNMR analysis showed linolenate 6.6%, palmitoleate/oleate 65.4%, total saturated fatty acyl chain including palmitate 28%. The fatty acid composition of TAG was also determined by GC–MS (Fig. 5), which showed palmitoleic acid 36.5%, oleic acid 12.9%, palmitic acid 21.2%, and linoleic acid 18% as the major acyl functionalities present in the oil. It showed total monounsaturated fatty acids at 58.4%, polyunsaturated fatty acids at 18.4% and saturated fatty acids at 28.2%. Using the same qNMR method, the fatty acyl composition of six commercial samples was also determined. The NMR spectral data of commercial samples is shown in Supplementary Information Figs. S1−S6.

As per most of literature reports, the analysis of free omega fatty acid is mainly done by GC–MS where the oil is hydrolysed converting TAGs into free fatty acids and then they are converted into their methyl esters which are analyzed by GC–MS. The FTIR spectrum of neat SBT oil (before hydrolysis) showed an intense IR band at 1745 cm⁻¹ for the stretching vibration of C = O group of glyceride ester. It did not show any band in the region around 1700 cm⁻¹ that suggested the absence of free fatty acids. Only after hydrolysis, the oil showed IR band at 1710 cm⁻¹ (stretching vibration of C = O group) of free fatty acid (Fig. S7). GC–MS data of isolated TAG is shown in Fig. S8. These observations suggested that SBT oil consisted mainly of TAGs and not free omega fatty acids.

These qNMR results indicated that SBT fruit oil TAG is a mixture of triacylglycerols with different acyl chains as given in Table 4. The major SBT fruit oil TAG consisted of palmitoleate/palmitate/oleate acyl functionalities. A perusal of literature indicates that fruit oils have major proportion of oleate, palmitoleate and palmitate while the seed oil has major proportion of linoleate and linolenate as shown in Table 5 (Fatima et al., 2012). As shown in Table 3, Table 4, out of the six commercial samples analyzed, the data showed that only SW contained oleate, palmitoleate and palmitate as major constituents (comprising about 93%) suggesting that it was the fruit oil. The other oils contained larger percentage of linolenate and linoleate suggesting that these were either seed oils or mix of fruit oil and seed oil. The label on SW indicated it was fruit oil while the labels from any of the other six vendors did not mention if it was seed oil or fruit oil.

Table 5.

Reported percentage of fatty acyl chains by GC–MS from fruit oil and seed oil.

Samples	Linolenate	Linoleate	Palmitoleate	Oleate	Saturated fatty acyl chain/palmitate	References
Reported fruits	1.6–1.7	8–9	49–50	9–11	28–29	Dulf, 2012
Reported seed oil	30–36	33–36	<4	17–20	about7	Fatima et al., 2012

4. Conclusion

This investigation suggests that SBT oil should be analyzed by combination of GC–MS, FTIR and qNMR for factual content of free fatty acid or TAGs, which are chemically different in nature and affect the quality of oil. GC–MS showed the content of omega free fatty acids after hydrolysis, while NMR and FT-IR showed the content of TAGs. SBT fruit oil TAG contained palmitoleate/palmitate/oleate acyl as major acyl functionalities, while linoleate and linonelate comprised minor fraction as determined by qNMR. GC–MS showed different omega fatty acids that were formed from TAGs. Analysis of commercial samples highlights the issues associated with the label claims of marketed oils. Six marketed samples were also analyzed by qNMR; SW contained oleate, palmitoleate and palmitate as major constituents (comprising about 93%) suggesting it was fruit oil while the others contained linolenate and linoleate as major constituents suggesting that these were seed oil or a mix of fruit oil and seed oil. The marketed oils (except one) did not mention if it the oil was prepared from seed or fruit.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: