1H-Nuclear Magnetic Resonance Analysis of the Triacylglyceride Composition of Cold-Pressed Oil from Camellia japonica

Camellia japonica (CJ) has oil-rich seeds, but the study of these oils has received little attention and has mainly focused only on their health properties. In the present work the relative composition of the fatty acid (FA) components of the triglycerides in cold-pressed oil from CJ is studied by 1H-NMR. The results obtained were: 75.75%, 6.0%, 0.17% and 18.67%, for oleic, linoleic, linolenic and saturated FA respectively. Levels of C18 unsaturated FA found in CJ oil were similar to those reported for olive oils. We also checked the possibility of using 13C-NMR spectroscopy; however, the results confirmed the drawback of 13C over 1H-NMR for the study of FA components of CJ triglycerides due to its low gyromagnetic ratio and its very low natural abundance.

Nuclear magnetic resonance (NMR) has become one of the most promising methods to determine organic structures in complex matrices such as foods, and pharmaceutical and biological samples [32,33]. 1 H nuclear magnetic resonance ( 1 H-NMR) offers many advantages over alternative analytical methods to study edible oils because it allows the rapid, simultaneous, noninvasive, and nondestructive study of oil composition, and also provides information about the acyl distribution and the acyl positional distribution of TAGs [34][35][36][37].
Although Galicia is one of the most important Camellia japonica producing-regions in Europe, there is no information available on the composition of CJ oil from this region. This lack of knowledge commonly leads to the waste of this quite easily obtained product. Thus, most of the Galician producers are not able to maximize their profits. In this context, the characterization and standardization of CJ oil would allow for the increase of its use in industries, and as a consequence, the economic value of this natural resource. All of this would stimulate its production. At the same time, it is necessary to measure elemental composition in order to provide correct denominations that can establish the minimum marketing level of the product and provide adequate consumer protection. The aim of this work was to study the TAG composition of cold pressed oil obtained from C. japonica cultivated in NW Spain by 1 H-NMR analysis.

Results and Discussion
The structure of the major TAGs present in CJ oil highlight the most singular kinds of hydrogen from the NMR point of view, and is represented in Figure 1. Vinylic hydrogens (H v ) have a characteristic chemical shift, and could be used to determine the ratio of saturated to unsaturated esters. Bisallylic hydrogens (H d , H t ) could be used to differentiate the nature of the unsaturated components. Finally, the tertiary hydrogen in the glycerin moiety (H g ) could be used to quantify the ratio of saturated to unsaturated esters since there is only one hydrogen for each TAG molecule. The proton resonances of the TAG acyl chains were assigned according to the literature data [38,39] and are shown in Tables 1 and 2.

Signal
Functional group Multiplicity Chemical shift (ppm) 1 Signal multiplicity: s, single; d, doublet; t, triplet; m, multiplet. The signal number agrees with those in Figure 2.  Signal multiplicity: s, single; d, doublet; t, triplet; m, multiplet; dt, double of triplet; dd, doublet of doblet. The signal number agrees with those in Figure 3.
We studied the 300 MHz spectra for CJ oil ( Figure 2); however the spectra obtained did not allow for the accurate integration of the tertiary hydrogen H g of the glycerine moiety, and no difference was found between bisallylic hydrogens (δ = 2.80 ppm approx.).
The use of a 750 MHz spectrometer to analyze the samples rendered a higher resolution spectra that in turn allowed for the separated integration of the signal from the tertiary glycerin hydrogen, and the vinylic ones (Figure 3). For CJ oil, once the peak of H g was normalized to 100, the signal of vinyl hydrogens was integrated as 5.26 (5.37-5.50 ppm), the bisallylic hydrogens giving 0.36 (linoleic) and 0.02 (linolenic).
The measured integrals [40] can be summarized as: Hv = 568.98, H d = 39.88, H t = 3.45 and H g = 100. Thus the values of each kind of acid are: A 80.67%, B 6.65%, C 0.29% and D 12.39%, for oleic, linoleic, linolenic, and saturated fatty acids, respectively. Table 3 shows the typical levels of C 18 unsaturated and total saturated FA reported in literature for common oils. The values recorded for linoleic, linolenic and oleic FA were similar to those obtained for olive oils. Table 3. Typical levels (in %) of C 18 unsaturated fatty acids and total saturated fatty acids in common oils.  [42]; c [43]; * Oil from Camellia oleifera; ** Present work.

Oil
We also checked the possibility of using 13 C-NMR spectroscopy for the rapid analysis of CJ oil [44]. Although this technique has been used for the study of the composition of several oils, its main drawback is the low natural abundance of the 13 C isotope. The same sample used for the 1 H-NMR experiment was used for the 13  The characteristic vinylic hydrogen region ( Figure 5 bottom) showed peaks of oleic ester at 130.16, 130.14, 129.84, and 129.81, together with 130.35, 130.11, 120.20, and 128.02 from linoleic esters, but no peaks corresponding to linolenic could be detected. These results confirmed the well known handicap of 13 C for rapid analysis of TAGs due to its low gyromagnetic ratio and its very low natural abundance.

Oil Obtention
The extraction of Camellia japonica oil (CJO) essentially involved preconditioning of camellia fruits, mechanical pressing of seeds and filtering to remove oil impurities. Mature Camellia japonica fruits were obtained from healthy plants grown in the live germplasm camellia bank at the Estación Fitopatolóxica do Areeiro (Pontevedra, Galicia, Spain). The fruits are large apple-shaped woody capsules that can contain several seeds, which are dropped on to the ground in late summer. For the study, ripe fruits were collected as soon as the capsules began to split open, and allowed to dry at room temperature for a week. Then seeds were removed from capsules. After crushing, seeds were cold-pressed for oil. The oil often had suspended particles in it which had to be filtered out. Finally, the CJ oil obtained was stored in amber glass bottles at ambient temperature.

1 H-NMR Analysis
1 H-NMR analyses were performed on Varian Mercury 300 (300 MHz for 1 H) and Varian Inova 750 (750 MHz for 1 H) instruments (Agilent Technologies®, Palo Alto, CA, USA), equipped with a 5 mm probe. Each oil sample, weighing 0.2 g, was dissolved in 400 µL of deuterated chloroform CDCl 3 , (Sigma-Aldrich®, Madrid, Spain) shaken in a vortex mixer, and the resulting mixture was placed into a 5-mm diameter ultra-precision NMR sample tubes (Norell®, Landisville, PA, USA). The temperature of the sample in the probe was 30 °C. The chemical shifts are reported in ppm, using the solvent proton signal as standard. The area of the signals was determined by using the equipment software, and the integrations were carried out three times to obtain average values. All figures of the 1 H-NMR spectra and of the expanded 1 H-NMR spectrum regions were plotted at a fixed value of absolute intensity to be valid for comparative purposes. 13 C-NMR analysis was performed on a Varian Inova 750 spectrometer working at 189 MHz for 13 C. The same samples subjected to 1 H-NMR analyses were used for 13 C-NMR analysis.

Conclusions
In summary, 750 MHz 1 H-NMR spectroscopy has proven to be a useful tool for the direct analysis of the triacylglyceride composition of cold-pressed oil from the Camellia japonica.