Molecular Composition of and Potential Health Benefits Offered by Natural East African Virgin Sunflower Oil Products: A 400 MHz 1 H NMR Analysis Study

Objectives: Sunflower oil (SFO) is regularly employed for cosmetic, emollient and food frying purposes, the latter representing its foremost use globally. Therefore, full investigations of the molecular composition and quality of SFO products are a major requirement. In this study high - field 1 H NMR analysis was employed to explore the molecular composition and authenticities of East African virgin (EAV) SFO products, particularly their acylglycerol fatty acid contents, together with those of selected minor constituents. Results acquired were statistically compared to those obtained on commercially - available, EU - approved refined SFO products via NMR - linked multivariate chemometrics strategies. Methodology: High - field 1 H NMR spectra of EAV and refined SFOs (n = 55 and 4 respectively) were acquired at an operating frequency of 400 MHz. Their triacylglycerol fatty acid, triacylglycerol hydrolysis product, and sterol and stanol contents were determined via intelligent frequency bucketing and electronic integration of selected resonances. Univariate analysis - of - variance, and multivariate ROC curve evaluations were conducted to determine the magnitude and statistical significance of analyte concentration differences between these two sample classifications. Further multivariate NMR - linked chemometrics analyses such as principal component, random forest and support vector machine classification analyses were also utilised for this purpose. Key Results: Multicomponent 1 H NMR analysis demonstrated that EAV SFOs had significantly higher and lower contents of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), respectively, than those of refined SFOs. Furthermore, significantly higher concentrations of ‘ health - friendly ’, cholesterol - blocking sterols and stanols were also found in these virgin SFO products. and EU - purchased significantly lower component and component


Introduction
Sunflower oil (SFO) is commonly and regularly employed for food frying and cooking, cosmetic and emollient purposes, and in 2014 the total global production of it was close to 16 million tonnes. Crude Moreover, within the same vegetable/plant species, the abundance of such components in oils arising therefrom are critically dependent on a variety of factors, such as the nature and quality of seeds employed, agronomic strategies utilised, climate and temperature, and the oil extraction system, notably the temperature employed for this process [1][2][3].
SFO production has made significant contributions towards the socioeconomic development of East African nations since its establishment during the first half of the twentieth century, and currently agriculture accounts for >80% of total worker employment in Uganda, for example. Indeed, the sunflower sector in this nation has been of valuable interest to developmental communities following its original inception, although cultivation has generally been restricted to poorer Ugandan regions.
These SFO markets were further developed to combat the deleterious economic effects arising from the diminishing cotton sector in the 1990's, and since then sunflower and SFO production has considerably expanded, i.e. more than two-fold since 2000. Such global sunflower output has been facilitated by resilient demands from developing markets, reflected by population growth and modifications in edible oil consumption patterns linked to rising incomes.
Commercially, many East African SFO products classify as 'virgin' status ones, since they have not been exposed to harsh chemically-/physically-demanding 'clean-up' refinement processes. Any refining processes involved, which of course precludes the classification of culinary oils exposed to this process as 'virgin' status products, will also exert a major influence on oil compositions [4,5], particularly their colouration status, reductions in their contents of α-tocopherol (vitamin E) and alternative phenolic antioxidants, along with those of other 'health-friendly' molecules such as sterols and stanols. Therefore, validated and detailed analytical determinations of the fatty acid, antioxidant, and sterol and stanol contents of unrefined 'natural' EAV SFOs is of critical importance, both to manufacturers and consumers, the latter now expressing a high level of interest and concern regarding the health and nutritional properties of their diets [6]. Indeed, such assessments provide much valuable information concerning preservation of the nutritional qualities of such products, and also serve to reassure consumers of the absence of any adulteration, refinement or alteration of these oils, and hence their authenticity.
Of further concern are the toxicological properties and adverse health effects potentially exerted by dietary, predominantly polyunsaturated fatty acid (PUFA)-derived, lipid peroxidation products (LOPs), and when employed as frying media, or allowed to peroxidatively degrade during prolonged periods of storage and/or exposure to light, such toxins can be generated within PUFA-rich SFO products. [7,8].
Therefore, in this investigation we have developed and applied 400 MHz 1 H NMR analysis and linked chemometrics strategies in order to explore the total molecular (predominantly lipidic) composition of a large cohort of virgin (unrefined) SFO samples produced by a range of mill manufacturing sources located in East Africa, specifically at Ugandan and Tanzanian sites.
Indeed, these techniques were employed to provide valuable molecular information to support the quality and authenticities of these oils, and also to test the effectiveness and reliabilities of NMR-based chemometrics strategies for the purpose of distinguishing between authentic, East African-produced virgin SFOs and corresponding products commonly available for purchase at EU (UK-based) retail outlets, and which have been exposed to rigorous industrial refinement processes.

Experimental Design and Statistical Analysis
The experimental design for univariate analysis of the total culinary oil 1 H NMR ISB intensity dataset involved an analysis-of-variance (ANOVA) model, which incorporated 1 prime factor and 2 sources of variation: (

1 H NMR profiles of East African virgin and refined
EU-available refined SFOs, including their saturated, monounsaturated and polyunsaturated fatty acid contents Typical 1 H NMR spectra of EAV and EU-refined SFO samples are shown in Figure 1. Resonances visible in these spectral profiles are listed in Table 1 for both major and minor acylglycerol functions, and also for a range of further minor agents detectable within the expanded regions of these spectral profiles, including hydrolysis products, along with sterols and stanols.
Results arising from the determination of differing classes of fatty acids in EAV SFOs are shown in Table 2, which provides the saturated, monounsaturated and polyunsaturated fatty acid contents of a total of n = 55 EAV and 4 commercially-available EU-purchased refined SFO samples analysed.
Comparisons of mean±95% confidence intervals (CIs) % values for the SFAs, MUFAs and PUFA contents of EAV SFOs with known international CODEX standard ranges for such oils, together with reference values available for Ugandan SFO products [17], are also provided. The full spectra displayed in Figure  However, no significant differences were found between the mean SFA contents of these two oil classifications.
Sixteen out of 55 of the estimated % PUFA (predominantly linoleic acid) contents were found to be lower than the codex standard for these values for SFOs      Table 3 These data are clearly consistent with the higher PUFA contents of the EU-available refined SFO samples.

ROC Curve Analysis of Differences between EA Virgin and EU-Available Refined SFOs
Indeed, since all ISB variables were constant sum-normalised, lower levels of PUFAs present in the EAV SFO products will give rise to relatively higher resonance intensities for the non-PUFA-and more generally the non-olefinic function-associated  (Table 3 and Table 4). Successful

AHC Analysis
Agglomerative hierarchal clustering (AHC) analysis demonstrated a very high level of distinction between the two SFO sample classifications investigated ( Figure 6). This AHC classification model was based on dissimilarities between the grouped samples, and Figure   6 shows the progressive grouping of the dataset, and also the finalised clear distinction of the refined SFO group from the EAV one. A range of further clusterings and sub-clusterings within the EAV SFO sample group (i.e. major and minor respectively) are visible, and the sources of these will be further explored, and reported in detail elsewhere.  (Table 3).

Discussion
Results acquired from our high-field 1     water containing dissolved 'gum' is allowed to drain out) [4]. Hence, this process may be favourable to the generation of γ-stearolactone, since the phosphoric acid may directly activate the transformation of any free (unesterified) oleic acid in the medium to this product.   [3], and these researchers found that in conventional ones, the minimum climatic temperature exerted a major effect on the triacylglycerol-bonded oleic acid:linoleic acid concentration ratios of these oils. Indeed, the % oleic acid content of these conventional genotypes could rise to and exceed 70% (w/w) in such oils, and higher locational temperatures gave rise to enhancements of up to 35% for this FA.