3.1. Solid Phase Extraction (SPE) and HPLC Analysis
Solid phase extraction (SPE), generally recognized as a cost effective and versatile method, can represent a very selective and effective technique only if the aim is the separation of components quite different in polarity properties. It does not represent, on the contrary, the choice method in other cases [
14].
SPE has been performed according to two main strategies. Under the condition of “bind and elute strategy” (SPE-BES), valuable compounds are expected to bind the sorbent material, while unwanted matrix components are washed off. Then, eluting with specific solvents, which disrupt the analyte and adsorbent interaction, only chemicals of interest could be removed from stationary phase. Otherwise, according to the “fractionation strategy” (SPE-FS), different classes of compounds adsorbed on the stationary phase could sequentially eluted by modifying the eluent composition. In SPE-BES, two different solvents were used as eluent: a
n-hexane fraction (1) corresponding to about 36% of the loaded sample and an ethyl acetate fraction (2), which accounted for the remaining about 63%. The use of
n-hexane that resulted was not appropriate for the complete elution of triglycerides. Moreover, the composition of the two obtained fractions was quite similar, as shown by the NMR spectra (
Figure S1, Supplementary Materials), both fractions retaining high triglycerides amounts, which makes it difficult to apply further purification steps.
The expansion of
13C NMR spectrum of fraction 2 (
Figure S2, Supplementary Materials) shows the coelution of sterols and carotenoids within the triglycerides fraction. To confirm what was observed by NMR analysis, fraction 2 was subjected to HPLC analysis for the identification of bioactive molecules of interest. As expected, the HPLC analysis (
Figure S3, Supplementary Materials) revealed the presence of sterols and carotenoids. In addition, from the HPLC data (see tables below), in this fraction, β-sitosterol (31 mg/g) and phenolic acids (111 µg/g) were about 8 and 6 times more concentrated than in crude corn oil, respectively, together with a certain amount of lutein and zeaxanthin (70 and 64 µg/g, respectively).
Despite these promising preliminary data, this type of selective extraction did not allow us to obtain good results in terms of purification of bioactive compounds. Thus, even in this case, a selective extraction of the molecules of interest was not achieved. So, we have adopted a new fractionation strategy. As reported in
Section 2, three low-polarity (3, 4, 5), two medium-polarity (6, 7), and two high-polarity (8, 9) fractions were collected operating with a fractionation strategy, based on a gradient solvents system. Gravimetrical data of the obtained fractions in relation to the eluted solvents are reported in
Table 1. As shown, a quantitative recovery was obtained (about 99%).
Each obtained fraction was previously analyzed by NMR spectroscopy.
1H and
13C NMR analysis of fractions 4, 5, and 6 revealed the presence of triglycerides, as also confirmed by the gas-chromatographic analysis of fractions (
Table S1, Supplementary Materials). The fractions 3, 7, 8, and 9 were submitted to NMR, HPLC, and MS analyses in order to better highlight the presence and the nature of the target compounds.
So, although fraction 3 gave a yield of only 0.01%, the
1H HMR analysis revealed the prevalent presence of squalene (
Figure 3), as confirmed by comparison with the spectrum of the reference standard. Considering the interest shown by this chemical specimen, the purity of the obtained extract and the exceptionally high quantities of side stream material to be managed, the squalene yield, although so low, deserves to be taken in consideration. The presence of squalene in corn oil is also confirmed in the literature [
15], in which an amount of about 25 mg/100 g was observed. These data are lower compared to our results (80 mg squalene/100 g) due to the different origin of the corn oil from bioethanol production. Squalene is one of the main polyunsaturated lipids of the skin surface. It exhibits emollient and antioxidant activity and is preventive against skin cancers. It is also used as a carrier in preparations of lipid emulsions and nanostructured lipid carriers [
16]. These characteristics give squalene an excellent potential in cosmetics and nutraceuticals. Based on the annual volume of side stream corn oil from ENVIRAL, the obtained putative amount of squalene could afford to about 300 Kg. The levels of sterols, tocols, and squalene were evaluated by HPLC-DAD in the obtained fractions after SPE fractionation strategy. Data are reported in
Table 2,
Table 3 and
Table 4. Fraction 3 showed an enrichment factor of 60 with respect to crude corn oil for squalene (
Table 2).
Fraction 7 had a yield of 3%, and NMR analysis had highlighted the presence of phytosterol esters along with triglycerides, so a further purification step has been performed as described in
Section 2.
A white precipitate (7a) was acquired by the purification step, which analyzed by NMR and showed to be a mixture of β-sitosterol esters. More detailed evaluation on the obtained extracts revealed characteristic signals in the olefinic region of the spectrum, suggesting the presence of ferulic and sinapic esters at the position 3 of β-sitosterol. Deconvolution of the selected signals showed a ferulic/sinapic ratio of 80:20 (
Figure 4).
As shown in
Table 2, fraction 7 has a content of about 38 mg/g β-sitosterol with a concentration of about 10 times that of raw corn oil. Given a precipitate yield of 0.3%, one gram of corn oil side stream would yield 3 mg of an extract enriched in phytosterols as a potential food supplement. As reported in the literature, several lipid compounds such as omega-3 fatty acids and plant sterol esters have shown important efficacy in preventing cardiovascular disease [
17]. Since plant sterols are also susceptible to oxidation after heat treatment, contact with oxygen, or exposure to sunlight, forming oxidation products, the presence of esters with antioxidant compounds (such as ferulic acid and synapic acid) is able to delay lipid oxidation [
18]. This combination of the antioxidant and hypocholesterolemic effects of phytosterols, within this fraction, could be an interesting strategy to reduce the risk of cardiovascular disease.
Fraction 8 gave a yield of 3%. The NMR spectra of this fraction exhibit the typical signals of sterols. For this reason, a further purification step afforded a precipitate (8a) about 40 mg, whose
1H NMR analysis revealed the presence of β-sitosterol together with other compounds. The obtained precipitate was further purified by HPLC semipreparative and MS analysis. High purity sample of β-sitosterol, confirmed by comparison with the standard reference, was obtained as shown by
Figure S4. Fraction 8 was the fraction showing the highest concentration of free sterols, mostly represented by β-sitosterol (about 60 mg/g) that here attained an enrichment factor of 14 compared to crude corn oil, but also by avenasterol, stigmasterol, campesterol, and ergosterol (enrichment factor of about 7–10) (
Table 2). These results confirm
1H NMR analyses. No detectable amounts of carotenoids were present in this fraction. These results are in line with what reported in the literature. In fact, corn oil is considered one of the richest sources of phytosterols, especially β-sitosterol (about 300 mg/100 g of corn oil), among the most commonly used commercial oils (about 100 mg/100 g of olive oil and other vegetable oil) [
19]. So, considering that these compounds reduce the absorption of cholesterol at the intestinal level, leading to a reduction in LDL plasma levels, and that their assumption could positively influence the metabolism of cholesterol [
20], fraction 8 presents an excellent pharmacological potential.
Fraction 9 (yield of 0.6%), which showed an intense reddish orange color indicating the presence of carotenoid and/or polyphenolic components, was subjected to HPLC analysis, by which three regions of interest were revealed (
Figure 5).
Several classes of compounds of different polarity such as phenolic acids, among which ferulic acid, p-coumaric acid, vanillic acid, syringic acid and sinapic acid (A region, Panel A), sterols, among which ergosterol, Δ-5-avenasterol, stigmasterol, campesterol, β-sitosterol (B region, Panel A), and carotenoids, among which lutein and zeaxanthin (C region, Panel B) were identified in the mixture.
Fraction 9, the most polar fraction, showed a high concentration of sterols (enrichment factor for β-sitosterol = 10), carotenoids (enrichment factor = 5), and phenolic acids (enrichment factor = 83), confirming the indications given by qualitative HPLC analyses. The concentration of free sterols, carotenoids, and phenolics in this fraction are reported in
Table 2,
Table 3 and
Table 4. Due to the high concentration of plant sterols (67 mg/g), phenolic acids (1.51 mg/g), as well as of carotenoids (2.39 mg/g).
For a better understanding of the molecular profile, this fraction was subjected to a further purification step. A suitable procedure has been developed and optimized for the recovery of the phytosterols and carotenoids, the most precious classes of target compounds.
Fraction 9 has been chromatographed on a 10 g RP18 silica gel, and five fractions (9a–e) have been collected as described in
Section 2. These fractions were subjected to HPLC analysis, which revealed the presence of sterols and carotenoids.
The five fractions obtained after RP C18 chromatography of fraction 9 have been analyzed for their content in bioactive compounds. Results are shown in
Table 2 and
Table 3. Fractions 9a–e showed a high concentration of sterols in free form. Fraction 9d was the fraction with the highest concentration of β-sitosterol (enrichment factor with respect to crude corn oil of about 64), stigmasterol and campesterol (enrichment factor about 98), avenasterol (enrichment factor about 29), and ergosterol (enrichment factor about 44). Tocotrienols and tocopherols appeared not to be concentrated in these fractions (
Table 2). Carotenoids were highly concentrated in fraction 9c (
Table 3), where lutein and zeaxanthin in
trans form (2573 and 2151 μg/g, respectively) together with several different
cis-isomers attained a total carotenoid concentration of 7903 μg/g (enrichment factor about 18). These results are better than those reported in the literature (about 60–200 µg/g) [
21,
22] since the fractionation strategy adopted allows us to concentrate more carotenoids from the corn oil under examination.
So, fraction 9 and fraction 9c are highly rich in valuable bioactive compounds with interesting functional and antioxidant properties.