3.2. Fatty Acid Composition of Seed Oils and Nutritional Quality Index
Total FA% compositions, corresponding to each FA component of oil and TAG fraction, are reported in
Table 2. SFA with carbon chains shorter than 14 carbon atoms, called short- and medium-chain FA, was not found in pumpkin seed oil, as confirmed in other papers [
14,
20,
37,
38]. SFA was represented especially by palmitic (C16:0) and stearic (C18:0) acids, at 14.2% and 5.8%, respectively. It is reported that oils rich in myristic (C14:0) and palmitic acids affect the ratio of total to high-density lipoprotein (HDL) cholesterol only a little, and stearic acid slightly reduces this ratio [
35]. PUFA and MUFA fractions were the most abundant (37.2% and 41.7%, respectively, for oil; 37.8% and 43.0%, respectively, for TAG); in fact, the main FA were oleic (C18:1
n-9) and linoleic (C18:2
n-6) acids. Berrettina pumpkin seed oil showed a higher content of oleic acid than linoleic acid (41.4% vs. 37.0% for oil); on the contrary, Procida et al. [
20] reported a higher content of linoleic acid (44.30–51.58%) than oleic acid (34.16–42.59%) for three Italian samples of pumpkin (Crudigno, Pepo, and Winter). It has been reported by some authors [
14] that oleic acid is the predominant FA (41–46%), followed by linoleic acid (33.4–34.3%), in pumpkin seed oil from Italy and Libya. Siano et al. [
16] found that the main FA of southern Italian pumpkin (
C. maxima) seed oil were linoleic acid (47.45%), followed by oleic (25.54%) and palmitic (17.58%) acids. Habib et al. [
17] found that pumpkin (
C. maxima, known as “Misti Kumra”) seed oil contained a high amount of oleic acid, 40.58%, while linoleic acid was 14.97%.
It was reported by Orsavova et al. [
38] that MUFA may reduce low-density lipoprotein (LDL) cholesterol, while it may possibly increase HDL cholesterol, and that oleic acid (C18:1
n-9) may promote insulin resistance contrary to PUFA, with protection against insulin resistance. The high content of linoleic acid is an important nutritional aspect, because it is an essential FA (EFA), together with linolenic acid (C18:3
n-3), and a lack of either of the two leads to ill health and causes deficiency symptoms. In addition, several studies [
39] have positively correlated EFA intake with reduction of numerous disorders (cardiovascular, neurological, visual, and cancerous).
Minor FA (contents lower than 0.5%) of Berrettina pumpkin seed oil were myristic, palmitoleic (C16:1
n-7), margaric (C17:0), heptadecenoic (C17:1
n-7), arachidic (C20:0), behenic (C22:0), and lignoceric (C24:0) acids. These data are in good agreement with similar studies [
14,
20,
37,
38].
The extreme variability of FA composition of pumpkin seeds, and consequently of the corresponding pumpkin seed oils, is affected not only by the variety of the cultivar, but also by the growth conditions and degree of ripeness [
40].
In addition, the nutritional quality of pumpkin (
C. maxima, var. Berrettina) cultivated in central Italy was evaluated, using different indices, based on the FA composition of the oils. It is known that some FA can help to prevent or promote coronary thrombosis and atherosclerosis based on their effects on LDL concentration and serum cholesterol [
41]. The equations proposed by Ulbricht and Southgate [
41] for the atherogenic index (AI) and thrombogenic index (TI) showed that C12:0, C14:0, and C16:0 FA are atherogenic, while C14:0, C16:0, and C18:0 are thrombogenic. PUFA
n-3, PUFA
n-6, and MUFA are antiatherogenic and antithrombogenic. Atherogenic indices have been described as powerful indicators of the risk of cardiovascular disease; the higher the value, the higher the risk of developing the disease, and vice versa. The AI of pumpkin (
C. maxima) seed oil was lower than that reported by Siano et al. [
16] (0.19 for Berrettina vs. 0.34), while the TI was comparable (0.50 for Berrettina vs. 0.65).
3.3. Stereospecific Analysis Data
The indirect method of analyzing TAG was based on a chemical–enzymatic–instrumental (stereospecific analysis) procedure and allowed us to carry out qualitative and quantitative analysis of all molecular TAG species, including enantiomeric ones. In fact, it is known that in TAG molecules the positions esterified by FA are numbered relative to their stereospecific numbering (
sn) as
sn-1,
sn-2, and
sn-3. The procedure allowed us to evaluate the FA % composition of each of the three
sn-positions of TAG (% intrapositional composition). These data could be used to obtain the distribution of FA among the three
sn-positions of TAG [
24,
25,
42,
43].
Initially, the lipid fraction was isolated by Soxhlet extraction, and then the TAG fraction was purified by TLC. As shown in
Figure 1, several steps were carried out. Initially, the total FA% composition (A
t) was determined by HRGC.
Figure 2a shows the characteristic HRGC profile of the FAME of the TAG fraction of pumpkin samples. Then, enzymatic hydrolysis of TAG with pancreatic lipase was used to obtain
sn-2-MAG, and finally, after HRGC analysis of the FAME (
Figure 2b), the acidic composition of
sn-2-position (A
2) of the glycerol backbone of TAG was obtained. TAG was also subjected to chemical hydrolysis with Grignard reagent, then separation of enantiomeric
sn-1,2(2,3)-DAG, realized by enzymatic synthesis of
sn-1,2-PA, allowed us to obtain the acidic composition of the
sn-1,2-positions (A
1,2) of the glycerol backbone of TAG, after HRGC analysis of the FAME (
Figure 2c).
The stereospecific analysis represents a potent analytical-investigative procedure to give the fingerprint of TAG fraction for each botanical variety or animal species. The results of the stereospecific analysis procedure are shown in
Table 2. It was observed that Berrettina pumpkin seed oil had a high percentage of UFA (98.5%) in
sn-2 position, represented by MUFA (36.2%) and PUFA (62.4%). In
sn-2 position, the main FA was linoleic acid (62.1%), followed by oleic acid (36.0%). SFA are preferentially esterified in
sn-1 position (44.7%), represented essentially by palmitic and stearic acids (28.6% and 13.7%, respectively). Regarding the two primary positions, oleic acid was equally distributed between the
sn-1 and
sn-3 positions, while linoleic acid prefers the
sn-3 position (42.2%).
3.4. Unsaponifiable Fraction
Another part of the research was analyzing the main components of unsaponifiable fractions, i.e., sterols and alcohols. Alkaline hydrolysis was carried out on pumpkin seed oils to obtain data relative to the qualitative composition of sterol and alcohol fractions. Phytosterols have been studied for their role in lowering cholesterol levels. In addition to this property, plant sterols have antiatherogenic, anti-inflammatory, anticancer, and antioxidation activities [
44]. Together with the high content of linoleic acid, sterols can help in the treatment of lipid-associated disorders such as atherosclerosis.
In contrast to the other vegetable oils with Δ
5-sterols (β-sitosterol, campesterol, and stigmasterol) as the major components, Wenzl et al. [
45] showed that pumpkin seed oil contains specific Δ
7-phytosterols, typical of only a few plant families (e.g.,
Cucurbitaceae), that provide a fingerprint for detection of adulteration. These Δ
7-sterols are supposed to give the pumpkin seed oil a beneficial effect in the treatment and prophylaxis of disorders of the prostate gland and the urinary bladder [
46].
In this paper, sterol identification was carried out by HRGC-MS. Each peak was analyzed via detection of the parent molecular ion and the fragmentation pattern of the TMSE derivative. In addition to the presence of specific ion fragments, the relative intensity of the ion fragments was considered. Some TMSE sterols were identified by comparison with the NIST mass spectra library; typical fragmentation is reported in
Table 3, together with sterol composition (% and mg/100 g). TMSE sterols give a molecular ion that is not abundant, while the first significant ion observed in the high mass range was usually equivalent to [M-15]
+, due to the loss of the methyl terminal group. Other main fragment ions useful for identifying the single sterol compounds are [M-90]
+, [M-105]
+, and [M-129]
+. They correspond to the loss of the trimethylsilanol, methyl group with trimethylsilanol, and fragmentation of the 1,2-cyclopenthanophenanthrene structure, respectively. The predominant sterols of Berrettina pumpkin seed oil are Δ
7-sterols, in particular Δ
7,22,25-stigmastatrienol, Δ
7,25-stigmastadienol, and spinasterol, which accounted for about 76.8% of the total sterols, followed by Δ
7-avenasterol and Δ
7-stigmastenol. The Δ
5-sterols were represented by campesterol, stigmasterol, and only a little cholesterol, as in other foodstuff [
47]. Differences between the contents of Δ
5- and Δ
7-sterols could be attributed to the maturity stage of seeds or to the solvent used in the extraction procedure [
15]. A total sterol content of 295 mg/100 g oil was measured in
C. maxima seed oil; 15.7 mg/100 g was represented by Δ
5-sterols and 279.3 mg/100 g by Δ
7-sterols. The total content was in agreement with other studies [
9,
48], even if a wide range of variability is reported [
9]. Hence, more detailed examinations of the composition of the sterol fraction of this oil could be of special interest. For example, analysis of a more extensive sampling is required for better characterization of pumpkin seed oils and for their authentication.
The alcoholic fraction (aliphatic and triterpenic classes) was also studied, after derivatization as TMSE and analysis by HRGC-MS; the typical fragmentation is reported in
Table 4, together with the alcohol composition (% and mg/100 g). Some aliphatic alcohols (from C16 to C25 members of the 1-alkanol homologous series) with odd and even numbers of carbon atoms of the aliphatic chain were identified. The TMSE alcohols were identified by comparison with the NIST mass spectra library. TMSE alcohols give a molecular ion that is not abundant, while the first significant ion observed in the high mass range was equivalent to [M-15]
+, due to the loss of the methyl terminal group, and [M-117]
+, equivalent to the loss of [(CH3)
3-Si-O]
+, i.e., the OTMSi group. Moreover, HRGC-MS analysis of alcohols after BSTFA derivatization resulted in various peaks with MS fragments
m/z 73, 75, 103, and 117 characteristics for OTMSi groups. The peak at
m/
z 129, corresponding to [(CH3)
3-Si-O
+=CH-CH=CH
2]
+, has been identified as the fragment originating from the breakdown of ring A along with the TMS moiety. Four main triterpenic alcohols were identified: butyrospermol, obtusifoliol, β-amyrine, and cycloartenol. The key fragmentation ions were molecular ion [M]
+, [M-15]
+, [M-90]
+, and [M-105]
+. Aliphatic alcohol content was 36.8%, of which hexadecanol and octadecanol were the most abundant (each about 4.4 mg/100 g oil), while triterpenic alcohol content was around 63.2%, of which obtusifoliol was the most abundant (11.9 mg/100 g oil).
These minor compounds are also important constituents of edible oils and could be useful in distinguishing different pumpkin oil varieties.