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Article

Domination of Tocotrienols in Passifloraceae Species’ Seeds and Recovery Using Ethanolic Extraction

by
Danija Lazdiņa
,
Inga Mišina
,
Krists Dukurs
and
Paweł Górnaś
*
Institute of Horticulture, Graudu 1, LV-3701 Dobele, Latvia
*
Author to whom correspondence should be addressed.
Separations 2026, 13(3), 78; https://doi.org/10.3390/separations13030078
Submission received: 5 January 2026 / Revised: 3 February 2026 / Accepted: 24 February 2026 / Published: 27 February 2026
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Abstract

Current industrial sources of tocotrienols are almost entirely composed of tropical monocots. However, recent reports have observed significant tocotrienol (T3) contents in eudicot families, including Passifloraceae. While passion fruits are also tropical, their cultivation is not strictly limited to rainforests, and seeds are often a by-product of fruit processing. To elucidate tocochromanol production in the Passifloraceae family, seeds (54 samples representing 18 species) were gathered from botanical gardens worldwide. Ultrasound-assisted extraction in ethanol (UAEE) was compared with the standard saponification protocol as a greener alternative. Tocotrienols constituted a major percentage (48–91%) of Passifloraceae species’ seed tocochromanols, and γ-T3 (12–53%) and δ-T3 (8–68%) were major contributors. Although a higher δ-T3 content was observed in some Passiflora species, it was less consistent than the γ-T3 content between and within species. The highest total tocochromanol content was observed in P. subpeltata (28.98 ± 5.83 mg 100 g−1 dry weight). The UAEE protocol recovered tocotrienols and tocopherols at degrees similar to those of saponification (100% and 93%, respectively). Therefore, UAEE could also be proposed for the effective recovery of these valuable phytochemicals from by-products of Passiflora fruits.

Graphical Abstract

1. Introduction

Tocochromanols are a class of prenylated lipids with relatively well-characterized antioxidant functions across various biological systems. Tocopherols (Ts) and tocotrienols (T3s) are the most common and most widely studied. Tocopherols, tocotrienols, and other tocochromanols are differentiated by their lipophilic side chains: tocopherols possess a saturated phytyl tail, whereas tocotrienol isoprenoid side chains have three unsaturated bonds. The chromanol ring structure is shared between tocochromanols, bearing a hydroxyl group (acting as a hydrogen donor) and one, two, or three methyl substituents, which distinguish individual homologues [1]. Tocochromanols are natural antioxidants and are often used in food, cosmetic, and pharmaceutical products [2]. Recently, tocotrienols have gained increasing attention as phytochemicals with potential adjunctive roles in cancer chemotherapy [3]. In nature, tocopherols, especially α-T and γ-T, are the most commonly occurring tocochromanols. While leaf tocochromanols are generally dominated by α-T, seeds usually primarily contain either γ-T or α-T alone or combined in similar amounts. In contrast, high concentrations of other homologues are relatively rare in nature [1].
The main dietary sources of tocotrienols are cereal products and cereal brans [4], which may have limited bioavailability due to their indigestible plant matrices. Currently, industry primarily utilizes palm oil and annatto seeds to produce tocopherol and tocotrienol extracts [5], which may be used in food as additives or supplements and in pharmaceutical or cosmetic products as antioxidants or active ingredients. However, the use of palm oil is becoming unpopular due to environmental and sustainability concerns, creating space for alternative sources in the market, such as Hypericum inflorescences [5]. Studies increasingly suggest that tocotrienols may be a more significant seed antioxidant than previously believed. While high tocotrienol content in cereal bran oils is well-documented, recent studies have shown consistent, relatively high tocotrienol content in dicotyledonous plant families as well, including Vitaceae [6], Hypericaceae and Clusiaceae [7], and Apiaceae [8,9,10]. Prior to the increased screening of dicot species, tocotrienols were believed to be essentially absent in them, seemingly as a result of sample selection and preparation, since early studies observed no tocotrienols in Apiaceae species [11]; however, several studies have observed consistently high tocotrienol content as early as the next year [8,9,10]. Subsequent studies on the relationship between tocochromanol composition and taxonomy have mostly focused on monocot species [12]. Even the spurge family, from which tocotrienols were first isolated [13], has not been widely analyzed.
Based on the available information and reports of no tocotrienol presence, studies have often omitted tocotrienols from analyses to save on standard costs. Currently, commercial tocotrienol standards are available at significantly more affordable prices. Moreover, time and labor required for sample preparation for tocochromanol determination can be substantially reduced by employing ultrasound-assisted extraction, and noxious solvents can be replaced with 96.2% (v/v) ethanol (UAEE). The streamlined UAEE protocol has been proven efficient and comparable to other protocols for tocochromanol extraction from cranberry [14] and grape seeds [6], as well as Hypericum inflorescences [15]. However, the broader application of the UAEE protocol requires validation across various plant parts and species, owing to the potential presence of tocochromanol derivatives (esters or glucosides), which are physically bonded tocochromanols [1,16,17]. The current UAEE protocol, combined with the RP-HPLC-FLD method, does not allow for the determination of the bonded tocochromanols [6].
Passifloraceae is a family of eudicot plants in the Malpighiales order. It contains about 27 genera and 750 tree, shrub, and liana species primarily growing in tropical and subtropical climates (www.gbif.org, Accessed: 31 December 2025). In the Passiflora genus, which is the most widely cultivated, between 50 and 60 species produce edible fruit, with P. edulis being the most economically relevant. Passiflora ligularis, P. quadrangularis, P. alata, and P. laurifolia are cultivated as fruit crops on a smaller scale [18]. Several other species of the Passiflora genus have been employed for their therapeutic potential, primarily owing to their recognized sedative and anxiolytic effects [18]. Some parts of the globally produced fruit are processed into juice, while the remaining seeds may be processed into oil. In the Turnera genus (Passifloraceae family), T. diffusa (damiana) is used as a medicinal plant and in the production of traditional liquors, tea, and cosmetics [19]; its seeds are not processed, but they could be processed for oil or phytochemical extraction.
Like many liquid plant oils [20,21], Passiflora seed oil predominantly contains three fatty acids—linoleic (68–74%), oleic (14–17%), and palmitic (9–11%) acid [22,23,24]—but, unlike most plant oils, it contains a significant amount of tocotrienols [23]. However, studies on Passiflora oil often only investigate a few species [22], mainly P. edulis [25,26,27]. Even in seemingly well-studied species, reports on the tocochromanol content and composition may be contradictory; tocotrienols may be omitted from analyses [28,29,30,31] or included in analyses, but their content and proportion differ significantly between studies [23,27,32]. In other cases, only a few tocotrienols are analyzed [22].
This study is aimed at filling the knowledge gaps on Passifloraceae species’ seed tocotrienols and providing a unified expectable tocotrienol content reference by assessing a large seed sample set and directly comparing tocochromanol extraction efficiency using UAEE. This method can be considered a greener alternative to the standard saponification protocol.

2. Materials and Methods

2.1. Reagents

Potassium hydroxide (purity 90%), pyrogallol (purity ≥ 99%), sodium chloride (purity ≥ 99%), n-hexane, methanol, ethyl acetate, and ethanol (HPLC grade) were obtained from Sigma-Aldrich (Steinheim, Germany). Then, 96.2% (v/v) ethanol was received from Kalsnavas Elevators (Jaunkalsnava, Latvia). Eight tocochromanol standards (purity > 95%, HPLC) were purchased from LGC Standards (Teddington, Middlesex, UK) and Merck (Darmstadt, Germany): α-, β-, γ-, and δ-tocopherols and tocotrienols.

2.2. Plant Material

Plant material was obtained from botanical gardens across the world, mainly Eurasia, in accordance with botanical garden seed exchange programs. Passifloraceae was one of over a hundred families investigated during the funding project. A full list of involved botanical gardens can be found in the Supplementary Materials. Species were verified by the staff of the donor botanical garden. To reduce the risks of species misidentification and crossbreeding and to account for natural variability between populations, origin (botanical garden) diversification was prioritized alongside species diversity. However, only one biological replication (n) could be obtained for some species during the project (2019–2024). In total, 54 samples from 18 species were investigated: Passiflora adenopoda (n = 2), P. amethystina (n = 4), P. apetala (n = 1), P. caerulea (n = 2), P. subpeltata (n = 5), P. capsularis (n = 2), P. coriacea (n = 6), P. edulis (n = 4), P. foetida (n = 4), P. gracilis (n = 3), P. incarnata (n = 1), P. maliformis (n = 1), P. morifolia (n = 5), P. quadrangularis (n = 1), P. rubra (n = 2), P. suberosa (n = 4), P. tenuifila (n = 3), and Turnera ulmifolia (n = 4). Species names and phylogenetic classifications were checked using online resources such as wikispecies.com (higher classifications), worldfloraonline.com (synonymic species), and passiflora.it (lower classifications) according to Feuillet and MacDougal [33]. Seeds were air dried and stored at ambient temperature in botanical gardens to retain viability. The seeds were sent via mail or a courier service. The plant material was catalogued, prepared for extraction (cleaned from other plant part residues if required), and analyzed as it was received within 1–3 months of receipt. Whole seeds were frozen at −80 °C for 1–3 h, freeze dried using a FreeZone freeze-dry system (Labconco, Kansas City, MO, USA) at −51 ± 1 °C and <0.01 mbar for 24–48 h, depending on the size and number of seeds tested experimentally, to obtain 3–7% seed moisture. The number of obtained seeds was generally insufficient to determine individual moisture content, so an average moisture content of 5% was assumed for all seeds during tocochromanol content calculation. Dry seeds (0.1−0.5 g) were powdered using an MM 400 mixer mill (Retsch, Haan, Germany) to obtain a powder of ~5 μm, and tocochromanols were extracted within the same day using ultrasound-assisted extraction and 96.2% (v/v) ethanol (UAEE), as described below in Section 2.3.2 for all samples and Section 2.3.1 for five randomly selected samples (recovery study).

2.3. Tocochromanol Extraction

2.3.1. Saponification

For the saponification protocol, 0.05–0.10 g of powdered seed sample was placed in a 15 mL conical tube with 2.5 mL of ethanol and 2% (w/v) pyrogallol (an antioxidant) at a sample:solvent ratio 1:50–1:25, w/v. Afterward, 0.25 mL of 60% (w/v) aqueous potassium hydroxide was added, and the samples were incubated in a water bath at 80 °C for 25 min. After saponification, the sample was cooled and tocochromanols were extracted three times using 2.5 mL n-hexane:ethyl acetate solution (9:1, v/v). The saponification and extraction protocol has been described in detail previously [14]. The n-hexane:ethyl acetate (3 × 2.5 mL = 7.5 mL) was evaporated, and the remaining sample was re-dissolved in 1 mL of ethanol. Finally, the sample was transferred to 2 mL glass vials and analyzed immediately as described in Section 2.4.

2.3.2. Ultrasound-Assisted Extraction in Ethanol (UAEE)

The UAEE method was adopted from a previously developed protocol [14]. Briefly, powdered seeds (0.05–0.10 g) were placed in 15 mL tubes and supplemented with 96.2% (v/v) ethanol to achieve a sample:solvent ratio of 1:100–1:50 (w/v) according to the seed mass, vortexed for 1 min, and extracted at 60 °C for 15 min in a Sonorex RK 510 H ultrasonic bath (Bandelin Electronic, Berlin, Germany) with a 160 W nominal ultrasonic power and 35 kHz ultrasound frequency. Afterward, the samples were mixed (1 min) as before and centrifuged at 11,000 × g at 21 °C for 5 min. The supernatant was transferred directly to a 2 mL glass vial and analyzed immediately as described in Section 2.4.

2.3.3. Tocochromanol Recovery

Owing to the inherent complexity of different plant matrices, together with the possibility that tocochromanols may occur in variable proportions as esterified or matrix-associated forms, newly proposed extraction methods should be validated against the conventional saponification protocol [34]. To this end, seeds from five randomly selected Passiflora species were analyzed using both UAEE and saponification, allowing a direct comparison of tocochromanol recovery achieved by UAEE relative to the established saponification protocol (5 species × 3 replications for UAEE vs. 5 species × 1 replications for saponification).

2.4. Tocochromanol Determination by Reversed-Phase High-Performance Liquid Chromatography with Fluorescent Detection (RP-HPLC-FLD)

This study follows a well-established chemotaxonomic framework developed in our earlier work on Aquifoliaceae [35], Celastraceae [36], Cornaceae [37], Rutaceae [38], and Vitaceae [34] families. In those studies, the separation method described below enabled robust profiling of tocopherols and tocotrienols. We now broaden its application to Passifloraceae, thereby expanding the comparative chemotaxonomic scope of tocochromanol analysis across plant families.
The four tocopherols and four tocotrienols were identified using authentic standards, and the contents were calculated using the calibration curves produced earlier [39]. Separation was performed on a Luna PFP column (3 µm, 150 × 4.6 mm) (Phenomenex, Torrance, CA, USA) using 93% (v/v) methanol as the mobile phase. The system operated under isocratic conditions with a 1 mL min−1 flow rate and 40 °C column-oven temperature. Measurements were done on a LC 10 series (Shimadzu, Kyoto, Japan) system equipped with a RF-10AXL fluorescence detector using excitation and emission wavelengths of λex = 295 nm and λem = 330 nm, respectively. Samples were not filtered following either extraction method; they were only centrifuged. Details of method validation are provided in Table S1 and Figure S1 (Supplementary Materials).

2.5. Statistical Analysis

Results are presented as means ± standard deviation (n = 2–6), unless only one sample was available for analysis (n = 1), in which case the measurement is provided for the one sample. R (version R 4.3.2) packages dplyr, ggplot2, ggtext, ggthemes, forcats, patchwork, scales, and tidyr were used for data structuring and visualization. R package GGally was used for Spearman correlation coefficient (ρ) calculation and visualization, and the factoextra package was used for principal component analysis (PCA) using unscaled data since the tocochromanol content is expressed on the same scale and scaling the data is likely to create data noise from minor compounds. R calculations were done in RStudio (version 4.4.1) using “Mariposa Orchid” release (f0b76cc0, 4 May 2025) for Windows.

3. Results and Discussion

3.1. Recovery–Saponification vs. UAEE

Saponification has the highest tocochromanol recovery [1] but is time-consuming, uses noxious solvents such as hexane, and does not distinguish between free and esterified tocochromanols [1]. The present study used a simplified UAEE method, which is more efficient for testing a large number of samples due to its reduced time, cost, and environmental impact. The method has already demonstrated similar recovery ability to saponification [6,14]. While quantified tocochromanol contents may be similar with or without saponification, they should not be compared directly, and a pilot study using both extraction methods is strongly advisable when investigating new plant material. The prevalence, function, and properties of esterified tocochromanols (except α-tocopheryl acetate) are not widely studied, and their proportions in plant material vary significantly [17]; saponification may release bonded tocochromanols [1]. Therefore, five species in the Passiflora genus (P. morifolia, P. subpeltata, P. coriacea, P. edulis, and P. maliformis) were selected, and the seeds were prepared using a saponification protocol. The recovery varied depending on the specific tocochromanol and plant species; however, certain patterns were consistently observed. Tocotrienols exhibited higher extractability (100%) compared to tocopherols (93%) with the UAEE protocol. When analyzed by homologues, the extractability followed the order δ ≥ γ > α (Figure 1, Table S2 in Supplementary Materials).
Saponification and UAEE protocol repeatability calculated for the five species’ seeds ranged from 1.22 to 14.52% for tocopherols and from 0.24 to 10.53% for tocotrienols (Table 1, Table S2 in Supplementary Materials).
The observed lower precision was for tocochromanols with low and very low concentrations, mainly for α-homologues. The two protocols were characterized by similar precision (Table 1). High tocochromanol—particularly tocotrienol—extractability using the UAEE protocol has previously been reported for cranberry [14] and grape [6] seeds. Key factors differentiating the saponification protocol from UAEE include potential losses of tocotrienols and the release of esterified, glycosylated, or physically matrix-bound tocochromanols during saponification [1], whereas the UAEE extraction does not release bound tocochromanols, and only free-form tocochromanol contents are determined.
In contrast, the higher extractability of tocotrienols relative to tocopherols, and of δ- and γ-homologues compared to α-homologues observed with UAEE, can be attributed to differences in physicochemical properties among tocochromanols, namely, the degree of saturation in the side chain and the number of methyl group substituents. This trend is clearly illustrated by their elution behaviors in reversed-phase liquid chromatography. Tocotrienols (unsaturated side chain) are slightly more polar than tocopherols (saturated side chain) and therefore elute earlier. Polarity is further modulated by the methylation pattern of the chromanol ring: as the number (and position) of methyl substituents changes, retention shifts accordingly—δ homologues elute first, whereas the more methylated α homologues elute last. The length of the side chain also contributes substantially to the overall polarity of tocochromanols. Tocopherols (three isoprenoid units) generally display higher extractability than plastochromanol-8 (eight isoprenoid units) by hydroethanolic solutions, which is consistent with the greater hydrophobicity conferred by a longer side chain. Collectively, these observations support a clear principle: increasing side-chain length and saturation, together with increasing chromanol-ring methylation, enhances non-polarity and thereby reduces extractability by hydroethanolic solutions [6]. The UAEE protocol leads to high tocochromanol recovery, especially for tocotrienols. This finding highlights its applicability for comparative studies of Passiflora species and its potential for industrial implementation.
In the present study, we show that the UAEE protocol can substantially streamline sample preparation by reducing processing time, operator workload, and the consumption of toxic organic solvents while preserving high tocochromanol recovery and robust analytical reproducibility. The efficiency of sample handling differs strikingly between the two approaches. UAEE requires less than 30 min per sample, whereas saponification extends preparation time to roughly twice that duration. The difference is even more pronounced in terms of hands-on labor: UAEE entails fewer than 5 min of active operator time per sample, while saponification demands four times more. Moreover, saponification consumes about 15% more energy and requires larger quantities of reagents and hazardous solvents, including hexane and ethyl acetate. Taken together, these metrics unequivocally underscore the advantages of UAEE and its strong alignment with the principles of green extraction.

3.2. Tocochromanol Profile

Passifloraceae species seed tocochromanols are generally tocotrienol-dominated (Figure 2), especially δ-T3 and γ-T3, followed by γ-T, δ-T, and α-T in different concentrations. The average values for the Passiflora genus are 7.51 (δ-T3), 4.88 (γ-T3), and 2.33 (γ-T) mg 100 g−1 dw (average of species means). Tocotrienols make up between 47% (in P. adenopoda seeds) and 91% (in P. morifolia seeds) of the total tocochromanols in Passiflora species. In T. ulmifolia, γ-T3 constitutes the tocochromanol portion (46%, 6.99 mg 100 g−1 dw), followed by γ-T (32%, 4.94 mg 100 g−1 dw). Some species are very rich in δ-T3 (P. subpeltata and P. tenuifila), while others contain very little δ-T3 (P. apetala, P. coriacea, P. morifolia, and P. suberosa)/sample variance is high (35.06), and γ-T3 content is less variable (3.15). This implies sub-groups within the genus, but the dataset is poorly clustered (Hopkins statistic 0.80 > 0.5), the contents of the two compounds are not related (R2 = 0.0025, Pearson correlation coefficient = 0.05), and the content of δ-T3 can be variable within a species. In P. subpeltata samples, the δ-T3 content varies in the range of 14.79–23.44 mg 100 g−1 dw. The three samples with significantly higher δ-T3 contents are from the same location, which suggests that variability is related to growing conditions. The systematic bias related to the assumed 5% moisture content can be considered low (0–2%). However, this error is smaller than the observed standard deviation for analysis of the same sample; therefore, it can be skipped. Our results indicate that the choice of extraction procedure can introduce a measurable, albeit relatively small, effect on the tocochromanol concentrations quantified in the analyzed seeds. Nonetheless, the prevailing evidence across the literature points to genetic background and environmental conditions as the main determinants of within-species variation in seed tocochromanol content, with genetic effects typically exerting the stronger influence [14,40,41,42].
To some extent, differences between tocochromanol profiles in Passiflora samples are also explained by phylogeny—the Bryonioides and Cieca supersection has higher tocopherol proportions. However, profiles are not as consistent in the Decaloba supersection—C. apetala in the Decaloba section has a higher tocopherol proportion than species in the Xerogena section. Unfortunately, C. apetala can only be procured in one biological replication, and it is possible that this is a biological outlier. Tocotrienol proportions are consistently very high in the Passiflora subgenus. Although the data pool is insufficient to analyze Turnera and Passiflora species separately, there are trends in the family regarding tocochromanol synthesis. Tocopherol and tocotrienol contents do not appear related (ρ = −0.027), however, there appear to be differences in their biosynthesis. While tocotrienol content correlates strongly with intermediary (δ- and γ-tocochromanols) synthesis products (ρ = 0.985), tocopherols are generally present as final (β- and γ-tocochromanols) biosynthesis products (ρ = 0.765). Additionally, tocotrienol content is tied to tocochromanols methylated at C7 of the chromane ring (ρ = 0.921), while tocopherol content is correlated with tocochromanols containing a hydryl group at chromane ring C7 (ρ = 0.747). The literature concerning the tocochromanol profile of passion fruit seeds and seed oil is inconsistent. Tocotrienol standards are not used in three studies, and the unusually high concentration of δ-T (about twice that of γ-T in the present study) reported in these studies raises additional concerns regarding analytical accuracy [28,29,30]. One of the other two studies reports all four tocopherol homologues, including a relatively high content of β-T [31]—the rarest tocopherol in nature [1]—along with trace amounts of α-T3. Three recent and methodologically sound studies consistently report a clear predominance of δ-T3, followed by γ-T3 and γ-T [23,27,32]. Our findings strongly support the accuracy of these latter reports regarding the correct identification of tocochromanols in passion fruit seed oil. The reliable identification of tocochromanols, particularly in less-studied plant species or in thermally processed materials such as roasted seeds, remains a significant challenge. Misidentification often arises due to incorrect analytical tools and methodologies and the lack of use of reference standards [1]. Tocochromanols have not previously been investigated in T. ulmifolia seeds or other species of the Turnera genus. Turnera ulmifolia tocochromanols are primarily constituted by γ-T3 and γ-T, while the δ-T3 content is minor. The mean free tocochromanol contents in Passifloraceae species are provided in Table 2.
While uncharacteristic for eudicot plants, significant tocotrienols contents have been observed in other Malpighiales order plants, such as rubber plants (Hevea brasiliensis), the sap of which also predominantly contains δ-T3 [11]. Tocotrienol derivatives—likely δ-T3 derivatives in G. paucinervis fruits, but not δ-T3 itself [43], and dimeric tocotrienol derivatives in G. oblongifolia fruits [44]—have been observed in the Garcinia genus (Clusiaceae family). Tocotrienols have been reported in other Clusiaceae species as well—several tocotrienol derivatives are in various Clusia minor plant parts [45], Z-δ-tocotrienoloic acid is in Clusia pernambucensis stem bark [46], and δ-T3 has been observed in Calophyllum inophyllum seeds [11], which belong to the closely related Calophyllaceae family. Recent studies observed tocotrienols in several Clusia species’ leaves [7] but did not analyze tocotrienol derivatives. While P. flavicarpa was not analyzed in the present study, P. edulis, the other most widely grown species of passion fruit, contains less δ-T3 or other tocotrienols than current industrial sources e.g., annatto seeds (Bixa orellana, Bixaceae family).

3.3. Tocochromanol Content Variability in Passifloraceae Genera

Principal component analysis (PCA) identifies δ-T3 as the main tocochromanol variable. Principal component (PC) 1 explains 84.73% of the variation, and PC 2 explains 10.40% of the variation in the dataset, for a total of 95.14% explained variance. PC 1 has a very high loading with δ-T3 (0.99), a slightly negative loading with γ-T3 (−0.097), and less than 0.1 loading with other tocochromanols, while PC 2 has high loadings with γ-T3 (0.86), γ-T (0.44), and α-T (0.17). Passiflora species and T. ulmifolia samples are plotted according to their PC1 and PC2 scores in Figure 3. Certain species have higher tocopherol (T. ulmifolia, P. amethystine, P. apetala, and P. caerulea) or δ-T3 (P. subpeltata and P. tenuifila) contents, but differences are not genus dependent. Datapoints do not form apparent clusters according to their PCA scores, and seed tocochromanol profiles within the Passiflora genus are not more similar to each other than they are to the tocochromanol profile in T. ulmifolia seeds.

3.4. Passiflora By-Products as Novel Sources of Tocotrienols

Passiflora is a widely cultivated genus in the tropics, a significant amount of its fruit is processed, and its by-products (seeds) are used to produce oil for cosmetic formulations. According to the presented results, the Passiflora seeds are also a valuable material due to uncommon tocochromanol profile-tocotrienols domination. Potential hurdles in the use of Passiflora seed oil and extracts as sources of T3s include the reported presence of cyanogenic glucosides in the fruit [47], but their presence, content, and intended topical application have not warranted regulatory discussion. While there is no official figure on passion fruit production and harvest area, the largest producer of passion fruit is Brazil, but there is substantial cultivation in South Asia, subtropical North America, and Australia as well, and the plant is cultivated in subtropical Europe for ornamental purposes. Like palm oil, passion fruit can be grown in warm, tropical, and semi-tropical climates, but their growing areas are not as narrow. According to a 2023 survey of US passion fruit growers, there is no distinct preference for yellow (P. edulis) or purple (P. flavicarpa) fruits as it differs by region, the mean yield ranges from 2405.5 to 9000 lb per acre (2696.3 to 10,088.1 kg per ha), and the plants may be grown in the ground, in containers, or in raised beds and may be propagated by seed, cutting, or tissue culture, depending on the region [48]. Passion fruit pulp may be processed into products with seeds, but the pulp is usually separated from the rind and seeds [49]. No official figures are available on global production and processing quantities; passion fruit seed oil is available on the market as a cosmetics ingredient from a variety of suppliers but is not currently marketed as a source of tocotrienols. The 8–29 mg 100 g−1 dw tocochromanol content is similar to fresh palm fruit tocochromanol content, which contains about 40% oil [50] and the typical 5–10 mg 100 g−1 range found in grape seeds, which contain 9–15% oil depending on the variety [51]. Passiflora seeds contain about 22–26% oil [28,52], but oil yield and tocochromanol content differ in terms of oil extracted using different extraction methods. In terms of the tocochromanol profile, passion fruit seed oil is closest to annatto (Bixa orellana), which has a significantly higher tocotrienol content—about 500 mg 100 g−1 seeds [5]. The production of passion fruit seed oil is largely limited by the availability and transportation of the raw material. Moreover, it differs significantly in terms of fatty acids and bioactive constituents like carotenoids, phenolic compounds and volatile compounds, which affect the nutritional, medicinal, and cosmetic value of the oil. Quantification of these and other compound classes is beyond the scope of this paper.

4. Conclusions

The results clearly show that tocotrienols constitute a significant part of Passiflora genus species and T. ulmifolia seed tocochromanols, particularly δ-T3 and γ-T3. Although the mean δ-T3 content in Passiflora species seeds is higher, it is more variable than γ-T3 between and within species. Based on tocochromanol composition, Passiflora species’ seed oils may most closely resemble annatto seed oil; however, this study did not evaluate their competitiveness or the tocochromanol content in the oil.
Considering the number of families in the Malpighiales order with preference for tocotrienol accumulation in the seeds and photosynthetic organs (Clusiaceae, Calophyllaceae, Hypericaceae, Euphorbiaceae, and Passifloraceae), re-examination is warranted in other branches of the plant kingdom as well, and it is increasingly clear that tocotrienol standards are a necessary analytical expense for producing reliable data on tocochromanol content and composition in the Passifloraceae family. For routine analyses, a streamlined ultrasound-assisted extraction in ethanol protocol can be used to analyze Passifloraceae fruit seed tocochromanol content with similar recovery to the saponification protocol. The use of ethanol can also be proposed as a solvent for the effective recovery of these valuable phytochemicals from by-products of Passiflora fruits as a more sustainable approach.
Considering the prevalence of tocochromanol derivatives in related plant families, mass spectrometry investigation of the Passifloraceae family may also be advised, as well as investigation of tocochromanol composition in other plant parts, such as the leaves, fruit flesh, and rind. Turnera is a genus of 139 accepted species across four continents, but only T. ulmifolia could can procured for this investigation—additional screening of the genus is highly recommended. Moreover, this study investigated only two of 31 accepted genera in the Passifloraceae family—expanded screening of other genera is also highly recommended, as conclusions on tocochromanol content and composition on a family level cannot be made based on the results of this study.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/separations13030078/s1, Figure S1: Chromatogram of the RP-HPLC-FLD separation of four tocopherol (T) and four tocotrienol (T3) homologue standards; Table S1: Linearity, limit of detection (LOD), limits of quantification (LOQ), and retention time (RT) of the RP-HPLC-FLD method for tocopherols and tocotrienols determination; Table S2: Recovery (%) and repeatability (%) for the determination of tocopherols and tocotrienols in the seeds of Passifloraceae family.

Author Contributions

D.L.: Conceptualization, Investigation, Resources, Data Curation, Validation, Software, Visualization, Writing—Original Draft, Writing—review and editing; I.M.: Resources, Formal analysis; K.D.: Resources, Formal analysis, Data Curation; P.G.: Conceptualization, Methodology, Investigation, Visualization, Supervision, Writing—Original Draft, Writing—review and editing, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Latvian Council of Science project “Dicotyledonous plant families and green tools as a promising alternative approach to increase the accessibility of tocotrienols from unconventional sources”, project No. lzp-2020/1-0422.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed toward the corresponding author.

Acknowledgments

We would like to recognize Georgijs Baškirovs for contribution to the sample analysis and data handling, and Arturs Stalažs for support in the collection of seeds. We were able to perform this research due to the generous support from over 150 botanical gardens around the world, in the form of seed donations. A list of botanical gardens that support this project is provided in the Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

RP-HPLC, reverse-phase liquid chromatography; dw, dry weight; T, tocopherol; T3, tocotrienol.

References

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Separations 13 00078 g001
Figure 1. Recovery (%) of tocochromanols—total tocopherols (Ts) and tocotrienols (T3s)—from seeds of five Passiflora species by using the UAEE protocol. Recovery (%) was calculated as an average value for three sample replications and assuming the saponification protocol as 100% recovery of tocochromanols. UAEE, ultrasound-assisted extraction in ethanol.
Figure 1. Recovery (%) of tocochromanols—total tocopherols (Ts) and tocotrienols (T3s)—from seeds of five Passiflora species by using the UAEE protocol. Recovery (%) was calculated as an average value for three sample replications and assuming the saponification protocol as 100% recovery of tocochromanols. UAEE, ultrasound-assisted extraction in ethanol.
Separations 13 00078 g001
Separations 13 00078 g002
Figure 2. Free tocochromanol proportion and content in Passiflora species and T. ulmifolia seeds. Data are presented as species mean tocochromanol proportion of total tocochromanols (left) and total content, including total tocochromanol standard deviation (right). α-, β-, γ-, δ-T, and T3 are homologues of tocopherol and tocotrienol.
Figure 2. Free tocochromanol proportion and content in Passiflora species and T. ulmifolia seeds. Data are presented as species mean tocochromanol proportion of total tocochromanols (left) and total content, including total tocochromanol standard deviation (right). α-, β-, γ-, δ-T, and T3 are homologues of tocopherol and tocotrienol.
Separations 13 00078 g002
Separations 13 00078 g003
Figure 3. PCA results of Passiflora species and T. ulmifolia, denoted by point shape and color. (A) Variable plot by contribution. (B) Individual points plotted by their PC1 And PC2 scores, color and point shape denote subgenus. (C) Individual points plotted by their PC1 And PC2 scores, color and point shape denote supersection.
Figure 3. PCA results of Passiflora species and T. ulmifolia, denoted by point shape and color. (A) Variable plot by contribution. (B) Individual points plotted by their PC1 And PC2 scores, color and point shape denote subgenus. (C) Individual points plotted by their PC1 And PC2 scores, color and point shape denote supersection.
Separations 13 00078 g003
Table 1. Precision (repeatability, %) for the determination of tocopherols (Ts) and tocotrienols (T3s) in the seeds of five Passiflora species using saponification (Sap.) or ultrasound-assisted extraction in ethanol (UAEE).
Table 1. Precision (repeatability, %) for the determination of tocopherols (Ts) and tocotrienols (T3s) in the seeds of five Passiflora species using saponification (Sap.) or ultrasound-assisted extraction in ethanol (UAEE).
TocochromanolP. morifoliaP. subpeltataP. coriaceaP. edulisP. maliformis
Sap.UAEESap.UAEESap.UAEESap.UAEESap.UAEE
α-T7.9914.525.364.742.185.5810.6713.7312.9612.27
γ-T1.531.332.994.031.852.810.971.313.484.12
δ-T––5.683.483.973.8611.778.211.221.77
α-T310.538.11––2.852.68––4.6810.28
γ-T31.441.122.601.091.560.771.420.473.832.30
δ-T36.354.361.320.901.091.272.162.320.240.90
Total Ts1.211.240.771.950.580.321.081.102.131.42
Total T3s1.741.171.601.050.560.600.791.490.991.06
Repeatability (coefficient of variation) refers to the results of independent determinations carried out on a sample by analyzing three replicates of the sample on the same day. The lower the value in the table, the higher the precision. –, not calculated. The β-homologue was not detected in any of the species.
Table 2. Mean free tocochromanol content in Passifloraceae species’, mg 100 g−1 dw of seeds.
Table 2. Mean free tocochromanol content in Passifloraceae species’, mg 100 g−1 dw of seeds.
Speciesδ-T3γ-T3α-T3δ-Tγ-Tα-TTotal
P. adenopoda (n = 2)0.70 ± 0.063.45 ± 0.18ndnd3.71 ± 0.280.88 ± 0.128.73 ± 0.63
P. amethystina (n = 4)5.75 ± 0.302.02 ± 0.58nd1.04 ± 0.151.03 ± 0.310.19 ± 0.0410.04 ± 1.23
P. apetala (n = 1)2.129.72ndnd4.082.8418.76
P. caerulea (n = 2)5.46 ± 0.662.27 ± 0.06nd2.34 ± 0.270.90 ± 0.151.40 ± 0.2712.44 ± 0.86
P. capsularis (n = 2)6.19 ± 0.216.19 ± 0.560.36 ± 0.111.83 ± 0.192.40 ± 0.430.15 ± 0.0817.37 ± 1.23
P. coriacea (n = 6)1.94 ± 0.685.15 ± 0.741.09 ± 0.430.30 ± 0.143.78 ± 0.661.77 ± 0.6314.03 ± 2.07
P. edulis (n = 4)8.17 ± 2.074.44 ± 2.10nd1.24 ± 0.632.95 ± 0.810.74 ± 0.8317.54 ± 4.22
P. foetida (n = 4)9.42 ± 1.055.32 ± 0.070.35 ± 0.081.60 ± 0.141.45 ± 0.070.20 ± 0.2018.41 ± 1.10
P. gracilis (n = 3)2.86 ± 0.084.24 ± 0.260.72 ± 0.070.43 ± 0.401.87 ± 0.070.05 ± 0.0210.17 ± 0.80
P. incarnata (n = 1)12.616.89ndnd1.800.5221.82
P. maliformis (n = 1)10.163.760.132.132.680.0718.93
P. morifolia (n = 5)0.86 ± 0.334.35 ± 1.090.55 ± 0.33nd3.12 ± 1.100.26 ± 0.129.14 ± 2.46
P. quadrangularis (n = 1)4.792.46ndnd0.710.058.01
P. rubra (n = 2)8.61 ± 0.155.59 ± 0.22nd0.37 ± 0.013.31 ± 0.151.45 ± 0.2219.65 ± 0.79
P. suberosa (n = 4)1.22 ± 0.336.69 ± 0.780.75 ± 0.680.25 ± 0.193.14 ± 0.442.28 ± 2.0814.34 ± 1.54
P. subpeltata (n = 5)19.64 ± 4.085.55 ± 1.25nd1.04 ± 0.322.12 ± 0.540.63 ± 0.4728.98 ± 5.83
P. tenuifila (n = 3)13.09 ± 1.622.50 ± 0.320.30 ± 0.041.74 ± 0.240.88 ± 0.030.85 ± 0.1019.99 ± 1.49
Turnera ulmifolia (n = 4)1.90 ± 0.606.99 ± 1.130.68 ± 0.980.06 ± 0.094.94 ± 0.510.74 ± 0.8315.32 ± 0.96
Results are presented as species means ± standard deviation unless only one sample was available for analysis (n = 1), in which case the measurement is provided for the one sample. n, number of analyzed samples of a species; T3, tocotrienol; T, tocopherol; nd, not detected. β-T3 was detected only in P. capsularis, P. rubra, and P. tenuifila (0.25 ± 0.04, 0.32 ± 0.05, and 0.22 ± 0.10 mg 100 g−1 dw, respectively), while β-T was detected in P. caerulea, P. foetida, and P. tenuifila (0.08 ± 0.11, 0.08 ± 0.04, and 0.42 ± 0.06 mg 100 g−1 dw, respectively).
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Lazdiņa, D.; Mišina, I.; Dukurs, K.; Górnaś, P. Domination of Tocotrienols in Passifloraceae Species’ Seeds and Recovery Using Ethanolic Extraction. Separations 2026, 13, 78. https://doi.org/10.3390/separations13030078

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Lazdiņa D, Mišina I, Dukurs K, Górnaś P. Domination of Tocotrienols in Passifloraceae Species’ Seeds and Recovery Using Ethanolic Extraction. Separations. 2026; 13(3):78. https://doi.org/10.3390/separations13030078

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Lazdiņa, Danija, Inga Mišina, Krists Dukurs, and Paweł Górnaś. 2026. "Domination of Tocotrienols in Passifloraceae Species’ Seeds and Recovery Using Ethanolic Extraction" Separations 13, no. 3: 78. https://doi.org/10.3390/separations13030078

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Lazdiņa, D., Mišina, I., Dukurs, K., & Górnaś, P. (2026). Domination of Tocotrienols in Passifloraceae Species’ Seeds and Recovery Using Ethanolic Extraction. Separations, 13(3), 78. https://doi.org/10.3390/separations13030078

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