Phytosterols,triterpene alcohols,and phospholipids in seed oil from white lupin

This study was conducted to determine effects of genotypes and growing environment on phytosterols, triterpene alcohols, and phospholipids (PL) in lupin (Lupinus albus L.) oil from seven genotypes grown in Maine and Virginia. The unsaponifiable lipid (UNSAP) and phospholipid (PL) fractions ranged from 2.1 to 2.8% and from 2.6 to 2.8% of oil, respectively. UNSAP in lupin oil contained 19.9 to 28.7% sterols and 17.3 to 22.0% triterpene alcohols. Growing location significantly affected contents of total PL, PS, phosphatidylglycerol, β-sitosterol, campesterol, and β-amyrin. Genotypic effects were significant for stigmasterol. PC (32.6 to 46.3% of PL), PE (21.6 to 32% of PL), and PS (11.2 to 17.9% of PL) were the major PL in lupin oil. The concentration of PL classes in lupin oil were in the following descending order: PC>PE>PS>PI>phosphatidic acid > lysophosphatidylcholine > phosphatidylglycerol > diphosphatidylglycerol. In descending order of abundance, the sterols present in lupin oil were: β-sitosterol > campesterol > stigmasterol > Δ⁵-avenasterol > Δ⁷-stigmastenol Lupeol was the most prominent triterpene alcohol in lupin seed oil. In general, growing environment had a much greater influence on lupin oil characteristics than the genotypes.     

Characterization of Total and Individual Sterols in Canola Sprouts

In this study, the contents of total and individual phytosterols in sprouts made from seeds of seven canola (Brassica napus L.) lines (Acropolis, Banjo, Jetton, KS-7740, KSM3-1-124, Mussette and Virginia), grown at three locations in Virginia (Orange, Petersburg and Suffolk), were determined. Canola sprouts contained, on an average, 36.3 g sterols in 100 g of unsaponifiable matter (UNSAP), 10.7 mg sterols in 1 g of oil and 2.4 mg sterols in 1 g of dry sprouts. The contents of individual phytosterols (μg per g of oil) in canola sprouts were 1,162 brassicasterol, 3,799 campesterol, 34 stigmasterol, 5,359 β-sitosterol, 201 Δ⁵-avenasterol and 97 Δ⁷-stigmastenol. Canola lines had significant effects on the contents of oil, brassicasterol and campesterol. Locations had significant effects on the oil, UNSAP, total sterols, brassicasterol, stigmasterol and β-sitosterol. The oil content in canola sprouts was positively correlated with total sterols and Δ⁵-avenasterol, whereas oil content was negatively correlated with brassicasterol content. In general, the contents of campesterol and β-sitosterol increased with an increase in total sterol content. The concentrations of sterols were in the following decreasing order: β-sitosterol > campesterol > brassicasterol > Δ⁵-avenasterol > Δ⁷-stigmastenol > stigmasterol. These results indicate that canola sprouts may have the potential as a natural source of dietary sterols and might be desirable for human nutrition.     

Further Lipid Profiling of the Oil from the Mophane Caterpillar, <Emphasis Type="Italic">Imbrasia belina</Emphasis>

A comprehensive lipid profiling of the oil from the edible mophane caterpillar, Imbrasia belina, has been carried out as part of the study of the nutritional value of the caterpillar. GC-MS analysis revealed the composition of the major FA classes as 18:3 (29.98%), 16:0 (25.64%), 18:1 (17.97%), 18:0 (12.49%) and 18:2 (11.81%), which was in agreement with reported GC-FID analysis of the phane oil. ESI-FTICR mass spectrometric analysis showed phane oil to contain 20 TAG classes, with C54:4 (14.59%), C52:3 (14.71%) and C52:2 (10.49%) being the dominant classes, whilst ¹³C-NMR studies of the TAGs regiochemistry showed that occupancy of the sn-2 position was dominated by linolenyl and linoleoyl groups whereas the sn-1/3 positions were dominated by saturated groups. Normal-phase HPLC analysis of the unsaponifiable matter showed the presence of α-tocopherol (71.39 μg/g) and γ-tocopherol (1.66 μg/g) as the only tocol content in phane oil. GC-MS analysis of the total acetylated unsaponifiable matter gave the relative composition of the major sterols as cholesterol (53.77%), β-sitosterol (24.16%), 22-dehydrocholesterol (14.58%) and campesterol (6.26%), whilst GC-MS analysis of an SPE pre-fractionated unsaponifiable matter gave the absolute 4-desmethylsterol content (μg/g) as cholesterol (4482.44), β-sitosterol (1861.95), 22-dehydrocholesterol (1274.53), campesterol (503.83) and stigmasterol (21.78). Perhaps the adverse effect of such high dietary cholesterol content on humans could be mitigated by the presence of the substantial amounts of β-sitosterol and campesterol which are known to be blood plasma cholesterol lowering phytosterols.     

Physicochemical Characteristics and Composition of Indian Soybean Oil Deodorizer Distillate and the Recovery of Phytosterols

The deodoriser distillate (DOD) of Indian soybean oil obtained from two industries processing soybean oil was investigated for its physicochemical characteristics, its composition of tocopherols, phytosterols, fatty acids and recovery of phytosterols for use in nutraceutical products. It was found that the two DOD samples studied were dark in color and had higher amounts of free fatty acids (22.7 and 49.9%), unsaponifiable matter (11.8 and 21.9%) (5–10 times found in soybean oil), total tocopherols (1957–2256 mg/100 g) (20 times the amount in soybean oil), and 6–10% of phytosterols (12–20 times the soybean oil). The fatty acids found were palmitic (23.2–25.5%), stearic (1.4–2.4%), oleic (23.8–26.1%), linoleic (40.4–41.1%) and linolenic (2.7–3.2%) acids. The unsaponifiable matter (21.9%) and phytosterols (8.7%) content of DOD-2 were higher than in DOD-1 and hence was more suited for isolation of phytosterols. Using hexane and water for crystallisation, the DOD-2 yielded a phytosterol fraction with lower recovery of 13.2–17.8% while treatment with alkali to remove FFA and the glycerides followed by organic solvent extraction yielded unsaponifiable matter containing phytosterols with a recovery of 74.6%. Later the unsaponifiable matter was purified by double crystallisation into a mixture of phytosterols of 87% purity containing β-sitosterol (34.3%), stigmasterol (3.1%) and campesterol (50.1%). The product may find use in foods, pharmaceuticals, cosmetics and allied industries probably as a nutraceutical.     

Improvement of Canola Oil Frying Stability by Bene Kernel Oil’s Unsaponifiable Matter

The anti-rancidity effect of the unsaponifiable matter fraction of bene kernel (UFB) oil on canola oil (CAO) during frying was compared to that of tert-butyl hydroquinone (TBHQ). The UFB was separated into hydrocarbons (12.9%), carotenes (9.6%), tocopherols and tocotrienols (65.8%, mainly γ-tocopherol), linear and triterpenic alcohols (3.8%), methyl sterols (2.8%), sterols (3.0%, mainly β-sitosterol, stigmasterol, Δ⁵-avenasterol, and Δ⁷-avenasterol, respectively), and triterpenic dialcohols (2.2%). The results obtained from the measurements of the total polar compounds, the conjugated diene value, the carbonyl value, and total tocopherols showed that the stability of CAO improves similarly in the presence of UFB or TBHQ, and even more in the presence of UFB in some cases (especially inhibition of oxidized triglyceride monomers and triglyceride dimers). The analysis of polar components showed that the antioxidative additives were more effective to resist the formation of thermo-oxidative than hydrolytic products during the frying of CAO.     

Phytosterol accumulation in canola, sunflower, and soybean oils: Effects of genetics, planting location, and temperature

To assess the potential of traditional selection breeding to develop varieties with increased phytosterol content, we determined concentrations of those sterols in canola, sunflower, and soybean seed oils produced from breeding lines of diverse genetic backgrounds. Seed oils were extracted and saponified, and the nonsaponifiable fractions were subjected to silylation. The major phytosterols brassicasterol, campesterol, stigmasterol and β-sitosterol, were quantified by capillary gas chromatography with flame-ionization detection. Canola contained approximately twice the amount of total phytosterols (4590–8070 μg g⁻¹) as sunflower (2100–4540 μg g⁻¹) or soybean (2340–4660 μg g⁻¹) oils. Phytosterol composition varied among crops as expected, as well as within a crop. Both genetic background and planting location significantly affected total phytosterol concentrations. Soybean plants were maintained from flower initiation to seed maturity under three temperature regimes in growth chambers to determine the effect of temperature during this period on seed oil phytosterol levels. A 2.5-fold variability in total phytosterol content was measured in these oils (3210–7920 μg g⁻¹). Total phytosterol levels increased with higher temperatures. Composition also changed, with greater percent campesterol and lower percent stigmasterol and β-sitosterol at higher temperatures. In these soybean oils, total phytosterol accumulation was correlated inversely with total tocopherol levels. Owing to the relatively limited variability in phytosterol levels in seed oils produced under field conditions, it is unlikely that a traditional breeding approach would lead to a dramatic increase in phytosterol content or modified phytosterol composition.     

Varietal and crop year effects on lipid composition of walnut (Juglans regia) genotypes

The FA, unsaponifiable, and volatile constituents of oil from three walnut varieties from two consecutive crop years were studied. The walnut oils (WO) were rich in PUFA and low in saturated FA. The tocopherol fraction consisted mainly of γ-tocopherol. High contents of β-sitosterol were found, together with campesterol and Δ⁵-avenasterol in similar amounts. Methylsterols present in WO were identified as cycloartenol, cyclolaudenol, cycloeucalenol, and 24-methylenecycloartanol. The hydrocarbon fraction was characterized by the predominance of C₁₄–C₂₀ n-alkanes. The major volatiles were aldehydes produced through the linoleic acid oxidative pathway. FA, methylsterols, and some hydrocarbons presented statistically significant differences among varieties. Most of this variation was due to the genotype. The Franquette variety was noteworthy by its higher oil and oleic acid contents. In contrast, tocopherols and volatile compounds showed minor differences among varieties; they were strongly influenced by the crop year. Chemical data were subjected to principal component analysis. The parameters that gave the greatest discrimination between the walnut varieties were oleic and linolenic acids, tetradecane, eicosane, tetracosane, cycloartenol, and 24-methylenecycloartanol. These components presented the major varietal influences and could be useful to determine the identity of walnut genotypes.     

The Spectrum of Plant and Animal Sterols in Different Oil-Derived Intravenous Emulsions

Intravenous lipid constituents have different effects on various biological processes. Some of these effects are protective, while others are potentially adverse. Phytosterols, in particular, seem to be implicated with parenteral nutrition-associated cholestasis. The aim of this study is to determine the amount of plant and animal sterols present in lipid formulations derived from different oil sources. To this end, animal (cholesterol) and plant (β-sitosterol, campesterol, and stigmasterol) sterols in seven different commercially available intravenous lipid emulsions (ILEs) were quantified by capillary gas chromatography after performing a lipid extraction procedure. The two major constituents of the lipid emulsions were cholesterol (range 14–57% of total lipids) and β-sitosterol (range 24–55%), followed by campesterol (range 8–18%) and stigmasterol (range 5–16%). The fish oil-derived formulation was an exception, as it contained only cholesterol. The mean values of the different sterols were statistically different across ILEs (P = 0.0000). A large percentage of pairwise comparisons were also statistically significant (P = 0.000), most notably for cholesterol and stigmasterol (14 out of 21 for both), followed by campesterol (12 out 21) and β-sitosterol (11 out 21). In conclusion, most ILEs combined significant amounts of phytosterols and cholesterol. However, their phytosterols:cholesterol ratios were reversed compared to the normal human diet.     

Profiling of Phytosterols, Tocopherols and Tocotrienols in Selected Seed Oils from Botswana by GC–MS and HPLC

The phytosterol, tocopherol, and tocotrienol profiles for mkukubuyo, Sterculia africana, manketti, Ricinodendron rautanenni, mokolwane, Hyphaene petersiana, morama, Tylosema esculentum, and moretologa-kgomo, Ximenia caffra, seed oils from Botswana have been determined. Normal-phase HPLC analysis of the unsaponifiable matter showed that among the selected oils, the most abundant tocopherol and tocotrienol were γ-tocopherol (2232.99 μg/g) and γ-tocotrienol (246.19 μg/g), detected in manketti and mkukubuyo, respectively. Mokolwane oil, however, contained the largest total tocotrienol (258.47 μg/g). Total tocol contents found in manketti, mokolwane, mkukubuyo, morama, and moretologa-kgomo oils were 2238.60, 262.40, 246.20, 199.10, and 128.0 μg/g, respectively. GC–MS determination of the relative percentage composition of phytosterols showed 4-desmethylsterols as the most abundant phytosterols in the oils, by occurring up to 90% in moretologa-kgomo, mkukubuyo, and manketti seed oils, with β-sitosterol being the most abundant. Mokolwane seed oil contained the largest percentage composition of 4,4-dimethylsterols (45.93%). Besides 4-desmethylsterols (75%), morama oil also contained significant amounts of 4,4-dimethylsterols and 4-monomethylsterols (15.72% total). GC–MS determination of the absolute amounts of 4-desmethylsterols, after SPE fractionation of the unsaponifiable matter, confirmed that β-sitosterol was the most abundant phytosterol in the test seed oils, with manketti seed oil being the richest source (1326.74 μg/g). The analysis showed total 4-desmethylsterols content as 1617.41, 1291.88, 861.47, 149.15, and 109.11 μg/g for manketti, mokolwane, mkukubuyo, morama, and moretologa-kgomo seed oils, respectively.     

Variation in lipid composition of niger seed (Guizotia abyssinica Cass.) Samples collected from different regions in ethiopia

Niger seed samples were collected from different regions in Ethiopia for determination of oil content, and of fatty acid, tocopherol and sterol composition in the seed oil by gas-liquid chromatography and high-performance liquid chromatography methods. There was a large variation in oil content, ranging from 29 to 39%. More than 70% of the fatty acids was linoleic acid (18∶2) in all samples analyzed. The other predominant fatty acids were palmitic (16∶0), stearic (18∶0) and oleic (19∶1) at a range of 6 to 11% each. Total polar lipids recovered after preparative thin-layer chromatography comprised a small fraction of the total lipids. They had higher 16∶0 and lower 18∶2 contents than the triacylglycerols.α-Tocopherol was the predominant tocopherol in all samples, 94–96% of the total amounting to 630–800 μg/g oil. More than 40% of the total sterols wasβ-sitosterol,ca. 2000μg/g oil. The other major sterols were campesterol and stigmasterol, ranging from 11 to 14%. The Δ5- and Δ7-avenasterols were in the range of 4 to 7%. From the samples studied, no conclusion could be drawn regarding the influence of altitude or location on oil content, tocopherol and/or sterol contents. The results of the present study on niger seed oil are discussed in comparison with known data for common oils from Compositae,viz, safflower and sunflower.     

Effect of extraction system, stage of ripeness, and kneading temperature on the sterol composition of virgin olive oils

Comparative extraction trials were carried out among a classical pressing, a dual-, and a three-phase centrifugation system using olive crops of Koroneiki variety. Two different kneading temperatures, 30 and 45°C, were tested at three stages of ripeness for two consecutive years of harvest, 1995–1996 and 1996–1997. Composition of the sterol fraction was determined in the resulting olive oil samples (n=72). Stigmasterol was found to be affected by the extraction system; it was obtained in the highest amount in the pressing system. The ratio campesterol/stigmasterol was significantly higher in oils extracted by dual- and three-phase centrifugation. Sterols were significantly affected by the ripening stage of the fruit. During December, the ratio campesterol/stigmasterol reached the maximal and β-sitosterol the minimal values; this appears to be the optimal period for harvesting the olives. Comparison of the different kneading temperatures showed that at 30°C, Δ⁵-avenasterol and campesterol/stigmasterol ratio reached higher values than at 45°C.     

Analysis of tocopherols and phytosterols in vegetable oils by HPLC with evaporative light-scattering detection

Methods were developed for the separation, detection, and quantification of tocopherols and phytosterols by high-performance liquid chromatography with an evaporative light-scattering detector. Four tocopherols— α, β, γ and δ—and four phytosterols—campesterol, β-sitosterol, brassicasterol, and stigmasterol—were analyzed in soybean, sunflower, low-erucic acid rapeseed (LEAR) and corn oils. The use of an evaporative light-scattering detector, in conjunction with modification of methods from the literature to prepare and analyze tocopherols and phytosterols by HPLC, showed consistent results between trials and levels of these minor constituents. Presented at the Annual American Oil Chemists' Society Meeting, May 3–7, 1989, Cincinnati, OH.     

Camelina oil and its unusual cholesterol content

The oil in Camelina sativa L. Crantz has a combined linolenic and linoleic acid content that is greater than 50% and a relatively low saturated FA content (∼10%). Although the FA composition has been reported, no information is available on the sterol composition of camelina oil. The derivatized plant sterols were separated and quantified with capillary GC and their identity confirmed with GC-MS. The refined camelina oil sample contained approximately 0.54 wt% unsaponifiables, and over 80% of the unsaponifiables were desmethylsterols. Perhaps the most unusual characteristic of camelina oil is its relatively high content of cholesterol, particularly for a vegetable oil, since it contains several times the cholesterol found in other “high-cholesterol” vegetable oils. Camelina oil also contains relatively large amounts of another unusual sterol, brassicasterol. The major sterols identified in the camelina oil included cholesterol (188 ppm), brassicasterol (133 ppm), campesterol (893 ppm), stigmasterol (103 ppm), sitosterol (1,884 ppm), and Δ⁵-avenasterol (393 ppm).     

Effect of genetic modification on the distribution of minor constituents in canola oil

Oil derived from different lines of genetically modified canola varieties was analyzed for phospholipids, tocopherols, and phytosterols by various chromatographic techniques. As observed previously in genetically modified soybean oils, there was a decrease in the content and composition of phosphatidic acid in three of the modified canola oils derived from the 12 varieties investigated. Normal-phase high-performance liquid chromatographic (HPLC) analyses showed small variations in the phospholipid content of major classes, despite few differences in their composition. Reversed-phase HPLC data indicated that the molecular species distribution of phosphatidylethanolamine was significantly altered by genetic modification when compared to phosphatidylcholine. Impact of oilseed modification on the tocopherol content was variable, with greater variation in the concentration of α- and γ-tocopherols than δ-tocopherol. Phytosterol composition was markedly affected by genetic modification. Brassicasterol, campesterol, and β-sitosterol levels were consistently lowered in one genotype, whereas increased brassicasterol content was observed in the other variety. In general, genetic modification of canola seeds led to changes in the distribution of phospholipids, tocopherols, and phytosterols. Presented in part at the 89th AOCS Annual Meeting & Expo, Chicago, Illinois, May 10–13, 1998.     

Grapefruit seed oil sterols

Grapefruit seed oil sterols separated from other lipids by Florisil column chromatography were characterized by gas liquid chromatography. The presence of stigmasterol, campesterol and β-sitosterol is indicated. Expressed in terms of peak area, the three sterols are present in proportions of 2.5%, 7.4% and 90.1% of the total, respectively.     

Characterization of the Composition of <Emphasis Type="Italic">Caesalpinia bonducella</Emphasis> Seed Grown in Temperate Regions of Pakistan

Caesalpinia bonducella is an oilseed that is indigenous to Pakistan. The hexane-extracted oil content from the seed kernel was 17.3 ± 1.0% DM (dry matter). The proximate analysis of C. bonducella seed estimated protein, fiber and ash contents to be 20.8 ± 1.4, 5.3 ± 1.0 and 4.6 ± 0.8%, respectively. Trace metals were determined comparable to commonly consumed legume seeds. α-Tocopherol was the predominant tocopherol ranging from 345.10 to 460.21 mg/kg of oil, followed by γ- and δ-tocopherol. The major sterols were β-sitosterol, stigmasterol, campesterol, Δ5-avenasterol, Δ7-stigmastenol and Δ7 avenasterol. The kernel oil was found to contain a high level of linoleic acid (72.7 ± 1.0%) followed by oleic, stearic and palmitic acids. The high percentage of linoleic acid revealed that this oil is a potential source for the manufacture of cosmetics, paints, varnishes, soaps, liquid soaps and other products including biodiesel. These investigations suggest that C. bonducella oil is potentially an important dietary source of essential fatty acids and protein which could be employed for edible and commercial applications in various industries of Pakistan.     

Studies on phytosterol oxides. II: Content in some vegetable oils and in French fries prepared in these oils

Hydrogenated rapeseed oil/palm oil blend, sunflower oil and high-oleic sunflower oil, and French fries fried in these oils were assessed for contents of sterol oxidation products. Different oxidation products of phytosterols (7α- and 7β-hydroxy-sito-and campesterol, 7-ketosito- and 7-ketocampesterol, 5α,6α-epoxy-sito- and campesterol, 5β,6β-epoxy-sito-and campesterol, dihydroxysitosterol and dihydroxycampesterol) were identified and quantiated by gas chromatography (GC) and GC-mass spectroscopy. Rapeseed oil/palm oil blend contained 41 ppm total sterol oxides before frying operations. After two days of frying, this level was increased to 60 ppm. Sunflower oil and high-oleic sunflower oil had 40 and 46 ppm sterol oxides, respectively, before frying operations. After two days of frying operations, these levels increased to 57 and 56 ppm, respectively. In addition to campesterol and sitosterol oxidation products, small amounts of 7α- and 7β-hydroxystigmasterol were detected in the oil samples. Total sterol oxides in the lipids of French fries fried at 200°C in rapeseed oil/palm oil blend, sunflower oil, and high-oleic sunflower oil were 32, 37, and 54 ppm, respectively. The levels of total oxidized sterols, calculated per g sample, ranged from 2.4 to 4.0 ppm. In addition to the content of phytosterol oxides, full scan mass spectra of several oxidation products of stigmasterol are reported for the first time. Part of these results were presented at the 86th Annual Meeting of the AOCS, May 7–11, 1995, San Antonio, TX.     

Assessment and Implications of Intraspecific and Phenological Variability in Monoterpenes of Scots Pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis>) Foliage

Scots pine populations contain individuals with widely differing amounts and composition of monoterpenes and exist as one of two chemotypes: with or without Δ³-carene. We investigated the significance for ecological studies of two types of variation in monoterpenes: (1) the inherent variability in the concentration of monoterpenes or their relative amounts in needles of seedlings, saplings, and mature trees; and (2) phenological variation in developing needles. The relative composition of needle monoterpenes in 5-year-old saplings changed during the needle development period until the final composition was reached upon needle maturity. Changes in composition depended on chemotype. Needles of the “no-Δ³-carene” chemotype had higher absolute concentrations of α-pinene, β-pinene, camphene, and total monoterpenes than “Δ³-carene” chemotype. For the “Δ³-carene” chemotype, the relative concentration of Δ³-carene during the needle growing season and immediately after emergence of seedlings was higher compared to that reached at needle maturity. Repeated removal of single needles (at weekly intervals during growth) from 5-year-old saplings did not influence the composition of monoterpenes. Within a natural Scots pine dominated woodland, 18% of mature Scots pines (N = 574) belonged to the “no-Δ³-carene” chemotype. Chemotypic variation within populations means that the statistical power with which differences in monoterpene concentrations can be detected is lower when sampling from the whole population compared to sampling within chemotypes. Reduction of this background variation and accounting for chiral variation if present, would significantly aid efficiency, interpretation, and understanding of processes in chemical and ecological research. One method for achieving this is the screening of plants for chemotypes before the establishment of experiments or field sampling regimes. We present a summary of suitable analytical methods for needle tissue that facilitates this prior screening.     

Structuring of edible oils by mixtures of γ-oryzanol with β-sitosterol or related phytosterols

The relation between molecular structure of oil-structuring agents and their gel-forming capability was investigated for mixtures of the phytosterol ester γ-oryzanol with a series of phytosterols. Dihydrocholesterol, cholesterol, β-sitosterol, and stigmasterol were found to form firm transparent gels with γ-oryzanol in sunflower oil under the conditions used in this work. The mixture of β-sitosterol with γ-oryzanol in sunflower oil does not gel immediately on cooling, but mechanical agitation such as shear promotes gelling. Gels that are formed immediately after cooling show a higher modulus than gels for which there is a time delay between cooling and agitation (150 vs. 100 kPa). The effect of oscillatory shear parameters (amplitude, frequency) is small, as long as the yield stress of the gel is not exceeded. The gels withstand compression very well (up to deformations of 10%), but yield at very small deformations. The enthalpy of melting of the solid phase is estimated to be 26±4 kJ/mol, putting it in the same range as for certain fibrillar steroid-derived organogels.     

4-Demethylsterols and triterpene alcohols from two Vanilla bean species: Vanilla fragrans and V. tahitensis

4-Demethylsterol and triterpene alcohol compositions of two Vanilla bean species (V. fragrans and V. tahitensis) were investigated. From retention times and gas chromatography-mass spectrometry, nine 4-demethylsterols were identified in V. fragrans and seven in V. tahitensis. The 4-demethylsterol fraction of V. fragrans was characterized by a high content of 24-methylene cholesterol (27–40%) and of β-sitosterol (35–46%). The 4-demethylsterol fraction of V. tahitensis was characterized by a high content of stigmasterol (27%) and of β-sitosterol (57.5%), and a lower amount of 24-methylene cholesterol (5%). Vanilla tahitensis was also characterized by the presence of ergosta-5,25-dien-3β-ol (2%) and the absence of campesterol, stigmasta-5,22,25-trien-3β-ol, and ergosta-7,24(28)-dien-3β-ol. The beans’ age modified the ratio 24-methylene cholesterol/β-sitosterol in V. fragrans. Combining liquid chromatography and gas chromatography allowed the identification of four other demethylsterols in V. fragrans (brassicasterol, 0.02%; stigmasta-5,23-dien-3β-ol, 1.43%; stigmasten-22-ol, 0.1%; and fucosterol, 0.5%) from the 4-demethylsterol fraction. 24-Methylene cholesterol and β-sitosterol were isolated, and their structures were confirmed by ¹H and ¹³C nuclear magnetic resonance. Four triterpene alcohols were identified in V. fragrans, including cycloartenol (0.9–1.6%) from the triterpene alcohol fraction, 24-dihydrotirucallol (17–23%) from the triterpene alcohol fraction, tirucall-7-en-3β-ol (6–7.5%) from the triterpene alcohol fraction, and in a higher content cyclosadol (66–69%) from the triterpene alcohol fraction. The content ranges were studied as a function of the beans’ age. Demethylsterol and triterpene alcohols profile could be used for origin differentiation.