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Article

Comprehensive Phytochemical Profiling of Ulva lactuca from the Adriatic Sea

by
Zorana Mutavski
1,
Igor Jerković
2,
Nada Ćujić Nikolić
1,
Sanja Radman
2,*,
Ivana Flanjak
3,
Krunoslav Aladić
3,
Drago Šubarić
3,
Jelena Vulić
4 and
Stela Jokić
3,*
1
Institute for Medicinal Plants Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia
2
Faculty of Chemistry and Technology, University of Split, Ruđera Boškovića 35, 21000 Split, Croatia
3
Faculty of Food Technology Osijek, Josip Juraj Strossmayer University of Osijek, Franje Kuhača 18, 31000 Osijek, Croatia
4
Faculty of Technology Novi Sad, University of Novi Sad, Boulevard cara Lazara 1, 11000 Novi Sad, Serbia
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(21), 11711; https://doi.org/10.3390/ijms252111711
Submission received: 29 September 2024 / Revised: 26 October 2024 / Accepted: 29 October 2024 / Published: 31 October 2024
(This article belongs to the Special Issue Updates on Synthetic and Natural Antioxidants)
Review Reports Versions Notes

Abstract

:
The potential of the green macroalga Ulva lactuca is increasingly recognized, not only for its environmental benefits, but also for its applications in various industries, including food, pharmaceuticals, and cosmetics. Given this insight, a comprehensive analysis of the chemical profile of U. lactuca from the Adriatic Sea was carried out. The hydrodistillate, rich in (Z,Z,Z)-hexadeca-7,10,13-trienal and hexadecanoic acid, underlines its importance for health-related uses, particularly in lipid metabolism and cellular integrity. Fatty acid analysis showed a predominance of palmitic acid and a favorable n-6/n-3 polyunsaturated fatty acid ratio, suggesting that U. lactuca can make a valuable contribution to a balanced diet. In addition, essential amino acids, including leucine, valine, and isoleucine, support its use as a functional ingredient for muscle repair and metabolic health. The ethanol extract contained 56 compounds, including derivatives of fatty acids, phenolic acids, pigments, flavonoids, and steroids. Many of them, such as hexadecasphinganine, azelaic acid, 5-sulfosalicylic acid, and pheophytin a, have proven roles or potentials in promoting human health. These results confirm that U. lactuca is a rich source of bioactive compounds, emphasizing its potential in scientific research and its expanding industrial applications in health, nutrition, and cosmetics.

1. Introduction

Ulva lactuca, commonly known as sea lettuce, has enormous chemical potential. This widespread green macroalgae is an important producer in marine ecosystems and thrives in coastal waters in temperate and tropical regions worldwide. However, the importance of U. lactuca goes far beyond its ecological role. Recent studies have focused on the remarkable adaptability of this alga, discovering numerous important chemical components used in various industries [1,2].
The key to unlocking the full potential of U. lactuca lies in understanding its complex chemical structure. This knowledge can pave the way for the development of new and sustainable applications [3]. Further research into the potential health-promoting components of U. lactuca would also be in the interest of the dietary supplement industry. It can also be used to fortify functional food formulations and supplement our diet by providing specific nutrients as well as bioactive ingredients such as vitamins, minerals, and polysaccharides [3,4,5]. Finally, the potential of U. lactuca as a source for biofuel production offers a glimpse into a future energy-efficient landscape. The seaweed may be used as a renewable feedstock for biofuel production, as it contains lipids and carbohydrates [6,7]. In addition, the chemical composition of U. lactuca could offer a wealth of opportunities for the pharmaceutical and cosmetic industries.
The high content of fatty acids and amino acids in U. lactuca from the Adriatic Sea also increases its potential to promote human health and sustainable practice [8]. Further research into the specific health benefits and optimal use of these compounds is essential to maximize the value of this versatile marine resource. U. lactuca contains a diverse range of fatty acids, which contribute to its nutritional value. Its fatty acid profile is predominantly composed of polyunsaturated fatty acids (PUFAs), including omega-3 and omega-6 fatty acids. Key fatty acids found in U. lactuca include α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) [9,10]. The presence of these essential fatty acids highlights U. lactuca’s role in supporting cardiovascular health and reducing inflammation. Its balanced fatty acid composition also makes it a valuable addition to a diet aimed at maintaining overall health and well-being.
The bright green color of U. lactuca is produced by a combination of pigments, with chlorophylls a and b being the most important for the absorption of sunlight and photosynthesis [2]. In addition to the chlorophylls, auxiliary pigments such as carotenoids absorb additional wavelengths of light and protect the alga from harmful sunlight. U. lactuca thrives in its marine habitat thanks to this mixture of pigments [11].
This study focuses specifically on U. lactuca harvested in the Adriatic Sea and highlights the unique chemical profile of this marine environment. To date, there has been no comprehensive characterization of the overall chemical composition of U. lactuca from this region. By thoroughly investigating the diverse chemical landscape of U. lactuca from the Adriatic Sea, this research aims to fill a significant gap in the existing literature. The focus is on the characterization of the biomolecules, revealing the astonishing diversity of nature’s chemical ingenuity. Fatty acids, volatile compounds, amino acids, and pigments are all analyzed. By thoroughly investigating the unique chemical composition of U. lactuca from the Adriatic Sea, this research lays a solid foundation for future study and development. This work will not only improve our understanding of this marine resource but also contribute to its meaningful utilization. Ultimately, discovering the secrets hidden in the chemical composition of U. lactuca has the potential to improve human diet and environmental well-being while contributing to the development of a more sustainable future.

2. Results and Discussion

2.1. The Headspace Composition Obtained by HS-SPME/GC-MS

The comparative studies on the odor characteristics of five seaweeds indicated that Ulva sp. received higher scores for seaweed, marine, and seafood odor [12]. Ulva sp. chemical composition was found to vary depending on geographical distribution and seasons; the principal environmental factors were water temperature, salinity, light, nutrients, and mineral availability [13,14,15], and therefore, it was of interest to investigate U. lactuca from the Adriatic Sea. Two fibers of different polarities (PDMS/DVB and DVB/CAR/PDMS) were used for collecting U. lactuca, and a total of 28 compounds were extracted by HS-SPME and identified by GC-MS with relatively different compound abundance among the fibers (Table 1).
Unsaturated alkane heptadec-8-ene ranged in the headspace from 21.80% to 12.46%. It was previously detected in the headspace of dehydrated U. lactuca from Spain [12], fresh U. lactuca from China [16], or the extract of U. lactuca from Egypt. The amount of heptadec-8-ene in brown and green algae is generally very low but was enriched when these algae were damaged mechanically or physiologically [17,18,19]. In comparison, the percentages of heptadec-8-ene in red algae Pyropia yezoensis, Pyropia haitanensis, and Bangia sp. (Rhodophyta) were 30–50% of the headspace volatile compounds, much higher than in the green alga Ulva australis (formerly Ulva pertusa) (Chlorophyta), the brown alga Sargassum thunbergii, and the red alga Gracilariopsis lemaneiformis (formerly Gracilaria lemaneiformis) [20]. From the marine green alga, Bryopsis maxima in a phosphate buffer (Z)-heptadec-8-ene was released by mechanical wounding, but this process was inhibited by heat treatment [17]. Therefore, it was supposed that (Z)-heptadec-8-ene would be also formed enzymatically from a fatty acid and might act as a chemical signal or a semiochemical. There is likely an enzymatic system (a type of heme protein) in Pyropia (Rhodophyta), which catalyzes eicosapentaenoic acid to produce heptadec-8-ene [20] with the optimal conditions of pH 9 and 25 °C, but the enzyme was thermal-resistant with over 50% activity remaining at 60–100 °C. Although the enzyme inhibitors phenidone, phenanthroline, and L-cysteine had no effect, the enzyme was significantly inhibited by hemoprotein NaN3, which is consistent with the result obtained for Pyropia tenera and Pyropia sp. (Rhodophyta) [20]. (Z)-heptadec-8-ene exhibited the reduction of reproduction of Varroa destructor [21].
Other abundant compounds were lower aldehydes heptanal (20.17%; 16.35%) and nonanal (10.57%; 6.62%), followed by minor percentages of pentanal (0.86%; 0.97%), hexanal (2.05%; 2.04%), octanal (1.86%; 1.11%), and decanal (2.12%; 1.13%). The algal aldehydes are formed through the degradation of fatty acids via the oxidation or enzymatic action of lipoxygenases [22]. Hexanal and heptanal are mainly derived from linoleic acid [23]. Nonanal could originate from ω-9 monounsaturated fatty acids (MUFAs) and ω-6 polyunsaturated fatty acids (PUFAs), such as linoleic acid [24].
Benzyl alcohol (8.53%; 9.71%) and benzaldehyde (2.61%; 2.61%) were the main benzene derivatives in the headspace. It was previously reported that benzyl alcohol was more abundant in the dry algal samples [25], while benzaldehyde could be decreased in the dried alga due to evaporation. The volatile benzene derivatives can be formed from phenylalanine when the side chain of a carbon skeleton shortens by C2-unit (via the oxidative pathway) [26]. In addition, benzaldehyde was readily reduced to benzyl alcohol by five cultures of photosynthetic microalgae [27].
The main volatile compounds in U. lactuca headspace from Spain were different: dimethyl sulfide, ethyl acetate, heptadecane, α-ionone, 2-phenylethyl acetate, and dimethyl sulfoxide [28]. Therefore, the obtained results agree with Narain [29] concerning a greater variety of volatile organic compounds identified in green seaweeds compared to red and brown species.

Volatiles Obtained by Hydrodistillation (HD)

The composition of hydrodistillate (Table 1) was considerably different in comparison to the headspace composition as was expected. Only five common compounds were found (heptanal, benzaldehyde, (E,Z)-hepta-2,4-dienal, (E,E)-hepta-2,4-dienal, 2,6-dimethylcyclohexanol, and heptadec-8-ene) but with considerably lower abundance in the hydrodistillate. A total of 30 compounds were identified in the hydrodistillate with higher aliphatic compounds as dominant.
The major compounds of the hydrodistillate were (Z,Z,Z)-hexadeca-7,10,13-trienal (22.67%), followed by hexadecanoic acid (13.18%), pentadecanal (10.61%), and methyl hexadeca-4,7,10,13-tetraenoate (7.50%). It was previously reported that (Z,Z,Z)-hexadeca-7,10,13-trienal is the main volatile compound in the green algae, a typical component of the essential oil of marine green alga U. pertusa [30,31], together with the major characteristic compounds pentadecanal, (Z)-heptadec-8-enal, (Z,Z)-heptadeca-8,11-dienal, and (Z,Z,Z)-heptadeca-8,11,14-trienal, also characteristic for Ulvaceae essential oils [32]. They accounted for ca. 40% of the hydrodistillate and were considered to exhibit the characteristic flavor of seaweeds [31]. These aldehydes were shown to be produced enzymatically from unsaturated fatty acids in U. pertusa collected from the sea [32]. In addition, the long-chain aldehydes formed by the enzymes from unsaturated fatty acids of the thalli culture were the same as in the field fronds, i.e., (Z)-heptadec-8-enal from oleic acid, (Z,Z)-heptadeca-8,11-dienal from linoleic acid, (Z,Z,Z)-heptadeca-8,11,14-trienal from α-linolenic acid, and (Z,Z,Z)-heptadeca-5,8,11-trienal from γ-linolenic acid [31]. (Z,Z,Z)-hexadeca-7,10,13-trienal was reported as antioxidant [33].

2.2. Fatty Acid Composition and Nutritional Indices of Ulva lactuca

The fatty acid composition of a freeze-dried sample of green macroalgae U. lactuca is presented in Table 2.
Palmitic acid (C16:0) was the predominant fatty acid (45.16%) and together with oleic acid isomers (C18:1n9c+t) and arachidic acid (C20:0) comprises more than 70% of the total identified fatty acids. The total saturated fatty acids (SFAs) were 73.35%, while unsaturated fatty acid (UFA) content was 27.53%, with similar content of monounsaturated and polyunsaturated fatty acids. High SFA content with a predominance of palmitic acid in U. lactuca was reported by Mohy El-Din [34] and Peñalver et al. [35]. Although present in lower amounts than SFAs, the content of essential fatty acids, cis-linoleic acid (C18:2n6c) and α-linolenic acid (C18:3n3), was notable (4.69 ± 0.62% and 5.82 ± 0.51%, respectively). Based on the results of nutritional indices, U. lactuca is characterized by a low PUFA/SFA ratio and a high index of atherogenicity (IA) and thrombogenicity (IT) that is slightly less favorable compared to other green macroalgae but comparable or even lower than nutritional indices of food products of animal origin (meat and dairy products) [36]. A similar PUFA/SFA ratio was determined in our previous study for another green macroalgae, Codium adhaerens [37]. n-6 and n-3 fatty acids are very important in human and fish nutrition, and a low n-6/n-3 ratio (preferably lower than 10) is beneficial to health. The n-6/n-3 ratio for U. lactuca obtained in this study was 0.99 which is comparable to or even slightly lower than the literature data [8,38]. Due to the favorable nutritional composition of fatty acids, incorporating U. lactuca consumption has significant advantages in a healthy diet and health preservation and reveals the possibility of using macroalgae as a source of bioactive components in functional food products.

2.3. Pigment Content of Ulva lactuca

The total quantified carotenoid content of U. lactuca was reported as 3.46 mg/100 g. Specifically, the concentrations of β-carotene, lutein, α-carotene, and β-cryptoxanthin were 1926.62 ± 0.36 μg/100 g, 1389.30 ± 0.60 μg/100 g, 131.81 ± 0.11 μg/100 g, and 12.05 ± 0.11 µg/100 g, respectively (Table 3). The results show remarkably high levels of β-carotene and lutein, which are consistent with the improvements observed in aquaculture environments as reported by Eismann et al. [39], who also found significant carotenoid concentrations in U. lactuca. Although the exact amounts differ, the observations confirm the presence of high amounts of lutein and β-carotene. Nevertheless, the content of β-cryptoxanthin was relatively low. Abd El-Baky et al. [40] reported that U. lactuca had the lowest concentration of β-cryptoxanthin, while the content of β-carotene was low compared to β-carotene in the present research. Abd El-Baqi et al. [40] reported the presence of 34 bioactive compounds in U. lactuca, including carotenoids such as α-carotene, all-trans β-carotene, 9-cis β-carotene, vioxanthin, astaxanthin, lutein, zeaxanthin, and cryptoxanthin.
The carotenoid composition of U. lactuca can vary due to factors such as environmental factors, growth, post-harvest storage, and season [41,42,43]. Recently, attention has focused on Ulva species rich in carotenoids with high neoxanthin or vioxanthin content, as these carotenoids increase free radical scavenging activity and oxidative stability. In addition, marine antioxidants are considered promising for the prevention of many modern diseases [44]. Antioxidant properties include quenching of singlet and triplet oxygen, scavenging of superoxide and hydroxyl radicals, and proven reducing power in phenols, carotenoids, terpenoids, and sulfated polysaccharides [45]. β-cryptoxanthin is considered a provitamin A, as it is converted into vitamin A (retinol) by the body. Similar to other carotenoids, β-cryptoxanthin acts as an antioxidant, potentially averting free radical damage to cells and DNA and helping to restore oxidative DNA damage [46].

2.4. Amino Acids in U. lactuca

The amino acid composition of macroalgae U. lactuca is presented in Table 4. Amino acid content was expressed as mg of amino acid per 100 g of proteins. The protein content in the U. lactuca freeze-dried sample was 17.66 ± 0.54%. The obtained protein content complied with results reported by Debbarma et al. [47] and Peñalver et al. [35], but lower and higher results are available in the literature [1,8,33,38]. The differences in protein content are attributed to different geographical origins, seasons, and environmental conditions (like sea temperature and salinity). The major amino acids determined in this study were glycine, threonine, aspartic, and glutamic acid (Table 4). U. lactuca contains all essential amino acids (EAA) in considerable amounts, except histidine and leucine content, which accounted for almost one-third of the total amino acid content. The content of threonine which accounted for about 50% of total EAA in U. lactuca was considerably higher compared to the threonine content in other edible algae (Undaria pinnatifida, Arthrospira platensis, Himanthalia elongate, and Porphyra umbilicales) from Spain where threonine content was about 1/10 of total EAA [35]. Threonine is an important amino acid for the formation of collagen, elastin, and tooth enamel and aids liver and lipotropic function when combined with aspartic acid and methionine. A low content of leucine was also found in sea lettuce from Spain [35], but in the literature, the limiting amino acids reported are also lysine, methionine, and isoleucine [8,35,38]. Partially, the difference can be attributed to the extraction procedure.
Namely, most of the studies perform acid hidrosis before derivatization, but according to Ummat et al. [48], the extraction using green solvents (water and citric acid) results in greater extraction yield, but additional parameters (temperature, time, and algae species) also have an impact on the amino acid profile. Aspartic and glutamic acid together with asparagine and glutamine contribute to the specific flavor and taste of the seaweeds, the umami taste. As presented in Table 4, aspartic and glutamic acid are present in large amounts in U. lactuca (123.77 ± 0.92 and 97.15 ± 1.70, respectively). Having in mind the obtained results, U. lactuca is a great source of proteins as well as amino acids that contribute to the specific umami taste and therefore can be added to enrich the taste of food products and possibly replace the umami additives that are increasingly used in food preparation.

2.5. Non-Volatile Compounds in Ethanol Extract

A non-targeted analysis of the ethanol extract from U. lactuca led to the tentative identification of 56 compounds. These include twenty-three derivatives of fatty acids, eleven phenolic acids, eight pigments and their derivatives, three flavonoids, and three steroids and their derivatives. Phenolic acids, flavonoids, and three fatty acid derivatives were found using ESI− mode, while the rest were found using ESI+ mode (Table 5).
Hexadecasphinganine (compound 11) was identified as the most abundant compound (Table 5). It belongs to the complex class of lipids known as sphingolipids, whose spingoid bases serve as building blocks of cell membranes. They also play roles in intra- and extracellular signaling [49]. In marine organisms, they are found as secondary metabolites. Researchers have shown interest in them due to their antiviral, antibacterial, anti-inflammatory, antitumor, and immunostimulatory activities [50,51]. The presence of sphingosine derivatives has been reported in the ethanol extract of Ulva lactuca (formerly Ulva fasciata) [52,53], methanol extract of the sponge Spheciospongia inconstans (formerly Spirastrella inconstans) [54], and methanol–dichloromethane extracts of brown algae Ericaria crinita and E. amentacea [55].
Azelaic acid (compound 24) was the second most abundant fatty acid derivative (Table 5). It has been recognized as an efficient therapeutic against some skin disorders such as acne and hyperpigmentation. It also has cytotoxic and antiproliferative activities in the case of human malignant melanocytes and has antibacterial properties [56]. The antibacterial behavior of diatom Asterionellopsis glacialis was probably due to the presence of azelaic acid [57]. Kalasariya et al. [58] detected it in methanolic extract of Ulva lactuca and proposed its antiviral activity against SARS-CoV-2 [58].
Among phenolics, 5-sulfosalicylic acid (compound 8) was the most abundant, followed by 4-hydroxybenzoic acid-4-O-sulphate (compound 13), vanillic acid 4-sulfate (compound 4), and 4-hydroxybenzoic acid (compound 7) (Table 5). They were all detected in [M-H]- mode. 5-sulfosalicylic acid, a derivative of salicylic acid that contains a sulfonate group, possesses antioxidant properties [59]. It has shown effectiveness against breast cancer cell lines (MCF-7) and HUVEC cells, with low toxicity [60]. 4-hydroxybenzoic acid-4-O-sulphate, also known as 4-(sulfooxy) benzoic acid, was found in the methanol extract of green alga Dasycladus vermicularis [61]. Welling et al. highlighted the importance of sulfated phenolic metabolites, which function as storage forms for more active metabolites [62]. Although limited data exist on vanillic acid 4-sulfate, this storage role may also apply to it. 4-hydroxybenzoic acid, which has antibacterial, antifungal, anti-inflammatory, and antioxidant properties, was the highest phenolic compound detected in methanol–hexane extract U. fasciata [63].
Pheophytin a (compound 47), hydroxypheophytin a (compound 45), and pheophorbide a (compound 29) were the most abundant among pigment derivatives (Table 5). Chlorophylls and pheophytins are used in the food industry as antioxidants [64]. Pheophytin a [65] and pheophorbide a [66] detected in ethanol extract of Ulva prolifera (previously Enteromorpha prolifera) have been linked to antigenotoxic and antitumor-promoting activities and antioxidant properties, respectively.

3. Materials and Methods

3.1. Macroalga Sampling and Extraction

Macroalga Ulva lactuca was collected in May through a single-site sampling from the Adriatic Sea. Geographical coordinates were 44°07′00″ N, 15°14′00″ E (Zadar). The collection occurred at the sea depth of 1 m, with a sea temperature of 16 °C. The collected algae and seawater were sealed in an airtight plastic bag and promptly transported to the laboratory after collection. The sample was kept in the dark at 4 °C for 48 h before further analysis.
The U. lactuca sample was washed with tap water (5×) and with deionized water (2×), then cut into 5–10 mm slices. For the analysis of volatiles, the slices were air-dried for 7 days at room temperature in the shadow. For all other analysis, the sample was stored in an ultra-low freezer (CoolSafe 55-9 PRO, Labogene, Allerød, Denmark) for 24 h at a temperature of −60 °C. After freezing, the sample underwent freeze-drying under a high vacuum (0.13–0.55 hPa) for 24 h. Primary and secondary drying temperatures were set to −30 °C and 20 °C, respectively.

3.2. The Headspace Analysis

The air-dried sample (1 g) was put in a 10 mL headspace vial, sealed with a PTFE-silicon septum, and extracted by a manual holder (Supelco, Bellefonte, PA, USA) using DVB/CAR/PDMS solid-phase microextraction (SPME) fiber (Supelco, Bellefonte, PA, USA) and PDMS/DVB fiber (Supelco, Bellefonte, PA, USA). The fibers were conditioned according to the manufacturer’s instructions. Equilibration of the sample was carried out for 15 min at 60 °C, after which the sample was extracted for 45 min. Thermal desorption of the fiber was performed directly to the GC column for 6 min at 250 °C. Three independent extractions were conducted, and the obtained results were reported as mean values. Hydrodistillation (HD) was performed according to a previously published procedure by Radman et al. [37].
A gas chromatograph (7890B Agilent Technologies, Palo Alto, Santa Clara, CA, USA) tandem mass-spectrometer detector (model 5977A MSD, Agilent Technologies) was used to analyze VOCs isolated from U. lactuca. The separation of VOCs was underrun on an HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness, 19091 S-433 UI-INT Agilent Technologies, Palo Alto, Santa Clara, CA, USA). The GC–MS analysis conditions and the identification procedure of the compounds were as specified by Jokić et al. [67].
The volatiles were identified by with their mass spectra comparing with the spectra from the NIST08 and Wiley275 libraries. The National Institute of Standards and Technology (NIST) Gas Chromatography Library (http://webbook.nist.gov/chemistry/, 10 June 2024) was used for the comparison with calculated retention indices (RIs) for each compound. RIs were calculated based on the retention times of C9-C20 n-alkanes under the same GC-MS working conditions.

3.3. Gas Chromatography Flame-Ionization Detection Analysis of Fatty Acids

Determination of fatty acids in a freeze-dried sample of U. lactuca was performed using the GC-FID method as described in our previous papers [37,68]. Briefly, macroalgae lipid fraction was extracted using the Folch method, after which the fatty acids were transesterified into volatile methyl esters (FAME) using methanolic potassium hydroxide. After filtration of separated FAMEs through a 0.45 μm membrane nylon filter, the sample was injected into the GC system.

3.4. Determination of Protein Content and Determination of Amino Acids Using HPLC-FLD Method

Protein content in U. lactuca was determined by the standard Kjeldahl method. Nitrogen content obtained by the Kjeldahl method was multiplied with a conversion factor (6.25) to calculate protein content in U. lactuca.
HPLC analysis was performed with the Shimadzu HPLC system consisting of a Shimadzu LC-20AD solvent delivery module, Shimadzu CTO-20AC column oven, Shimadzu SIL-10AF autosampler, and Shimadzu RF-20Axs fluorescence detector coupled with LabSolution Lite software (Release 5.52). The macroalgae sample (1 g) was dissolved in 25 mL of ultrapure water, vortexed, and placed into an ultrasonic bath for 15 min for incubation. After filtration through filter paper (21/N), an aliquot of filtrate (120 μL) was used for derivatization with OPA (o-phthalaldehyde) and FMOC (9-Fluorenylmethoxycarbonyl chloride). Filtrate aliquot was placed in a glass tube, 150 µL of borate buffer (pH 10.2) was added and vortexed, and after 30 s, 30 µL of OPA reagent, 30 µL of FMOC reagent, and 1920 µL of ultrapure water were added. After the addition of each reagent, the mixture was vortexed. Before HPLC analysis, solutions were filtered through a 0.45 µm nylon membrane filter.
Separation of fluorescent amino acid derivates was performed on the Inertsil ODS-3V column (250 × 4.6 mm, 5 μm). Column temperature was set at 40 °C. The mobile phase was composed of 40 mM NaH2PO4 (pH 7.8) (solvent A), while solvent B was a solution of acetonitrile, HPLC grade methanol, and ultrapure water (45:45:10, v:v:v). Mobile phase flow was 1 mL/min with gradient conditions as follows: starting percentage of solvent B was 15%, linear increase in solvent B up to 55% at 40 min, hold up to 45 min, and at 65 min, the initial conditions were achieved. The detection of separated amino acids was at 340 nm (excitation wavelength) and 450 nm (emission wavelength) except for proline, where wavelengths were 266 nm and 305 nm, respectively. Identification was achieved by comparing the retention time of each amino acid in the sample with the retention time of the same amino acid in a standard solution (AA standard 1 nmol/μL, Agilent Technologies and L-tryptophan, Acros Organics, Geel, Belgium). Quantification of identified components was performed with an external calibration method, and the results were expressed as mg of amino acid/100 g protein.

3.5. Determination of Pigments Using High-Performance Liquid Chromatography (HPLC-DAD) Method

Quantification of individual carotenoid compounds was performed by high-resolution liquid chromatography (High-Performance Liquid Chromatography, HPLC) on Shimadzu Prominence equipment (Shimadzu, Kyoto, Japan), which contains an LC-20AT binary pump, CTO-20A thermostat, and SIL -20A automatic dispenser connected to a DAD detector. The separation was performed on a column GRACE-Vydac 201TP54 C18, 250 4.6 mm, 5 mm (Hesperia, CA, USA). A solvent used as the mobile phase was stabilized methanol: stabilized THF (95:5, v/v), flow rate 1 mL/min. Samples and solvents were filtered before analysis through 0.45 µm pore size membrane filters (Millipore, Bedford, MA, USA). Carotenoids were detected at 445 nm. The wavelength range used was 200–600 nm.

3.6. Ultra-High-Performance Liquid Chromatography–High-Resolution Mass Spectrometry (UHPLC-ESI-HRMS) of Ethanol Extract

A freeze-dried sample weighing 1 g was extracted with ethanol under sonication for 60 min at 40 °C. The sample was filtered through a 0.2 µm PVDF syringe filter (Agilent Technologies). The UHPLC-ESI-HRMS analyses were conducted with an ExionLC AD UHPLC system (AB Sciex, Concord, ON, Canada), coupled to a TripleTOF 6600+ quadrupole time-of-flight (Q-TOF) mass spectrometer (AB Sciex, Concord, ON, Canada) featuring a duospray ion source. Electrospray ionization was operated in positive (ESI+) and negative (ESI−) modes using collision-induced dissociation (CID) in information-dependent acquisition (IDA) mode to collect MS/MS mass spectra. Gases were set as follows: nebulizer gas (GS1) 40, heater gas (GS2) 15, and curtain gas (CUR) 30. The temperature for positive mode was set to 500 °C, while negative mode was 400 °C. Ion spray voltage was set at 5000 V (ESI+) and −4000 V (ESI−). Declustering potential (DP) was set to 80 V (ESI+) and −35 V (ESI−), while collision energy (CE) was set to 40 V (ESI+) and 30 V (ESI−) with the 20 V collision energy spread. ACD/Spectrus Processor 2021.1.0 software (ACD/Labs, Toronto, ON, Canada) was used for data processing. Compound identification was proposed based on mass spectra, reported elemental compositions, and database searches in MassBank, Lipid Maps, ChemSpider, and ChEBI.
Chromatographic separation of the polyphenolic compounds (ESI−) was achieved with an Acquity UPLC CSH Phenyl-hexyl analytical column (Waters, Milford, MA, USA), while for the compounds in ESI+, separation was achieved with an Acquity UPLC CSH Phenyl-Hexyl analytical column (Waters, Milford, MA, USA), both measuring 2.1 mm × 100 mm and featuring a particle size of 1.7 µm. The mobile phases consisted of water with 0.1% formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B). The analysis was carried out at a steady flow rate of 0.4 mL/min, with the column oven maintained at 30 °C. The injection volume was kept at 4 µL. Gradient elution was set as described in Table 6.

4. Conclusions

This extensive chemical profiling of Ulva lactuca from the Adriatic Sea revealed the presence of various compounds, some of which have bioactive properties. HS-SPME/GC-MS and analysis of the hydrodistillate revealed the presence of various volatile organic compounds such as various aldehydes, benzyl alcohol, (Z,Z,Z)-hexadeca-7,10,13-trienal, and hexadecanoic acid, all of which are used in the cosmetic industry. The predominance of saturated fatty acids, especially palmitic acid, together with a favorable ratio of n-6/n-3 polyunsaturated fatty acids, makes this alga a valuable component of a balanced diet. The analysis of amino acids revealed that it is an excellent source of proteins and amino acids such as leucine, valine, and isoleucine, which play an important role in muscle repair and metabolic health. The presence of carotenoids, especially β-carotene and lutein, enhances the value of this algae as a source of antioxidants. In addition, 56 compounds were identified when analyzing the ethanol extract, including derivatives of fatty acids, phenolic acids, pigments, flavonoids, and steroids. In particular, several compounds such as hexadecasphinganine; azelaic acid; phenolics like 5-sulfosalicylic acid, 4-hydroxybenzoic acid 4-O-sulphate, vanillic acid 4-sulphate, and 4-hydroxybenzoic acid; and pigment derivatives such as pheophytin a, hydroxypheophytin a, and pheophorbide a have shown various functions or potential health benefits, including antiviral, antibacterial, anti-inflammatory, antitumor, and antioxidant activities. Given its rich chemical composition and the proven presence of numerous bioactive compounds, Ulva lactuca holds significant potential for use in various industries, including environmental protection, food production, pharmaceuticals, and cosmetics.

Author Contributions

Conceptualization, D.Š., S.J. and Z.M.; methodology, Z.M., S.R. and K.A.; formal analysis, I.J., S.R., I.F., J.V. and K.A.; investigation, Z.M., I.J., J.V. and S.J.; resources, I.J. and S.J.; data curation, Z.M., I.J., S.J., S.R., I.F., K.A. and N.Ć.N.; writing—original draft preparation, Z.M., S.J., I.F., S.R. and D.Š.; writing—review and editing, Z.M., I.J., N.Ć.N. and S.J.; supervision, S.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Croatian Government and the European Union through the European Regional Development Fund—the Competitiveness and Cohesion Operational Programme (KK.01.1.1.01) through the project Bioprospecting of the Adriatic Sea (K.K.01,1.1.01.001) granted to the Scientific Centre of Excellence for Marine Bioprospecting—BioProCro.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from authors for a limited time.

Acknowledgments

We would like to thank the Croatian Government and the European Union (European Regional Development Fund—the Competitiveness and Cohesion Operational Programme—KK.01.1.1.01) granted to the Scientific Centre of Excellence for Marine Bioprospecting—BioProCro. We thank also Donat Petricioli for the sample collection and identification.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. The volatile compounds from Ulva lactuca isolated by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS): U. lactuca extracted by DVB/CAR/PDMS fiber and by PDMS/DVB fiber. HD, hydrodistillation.
Table 1. The volatile compounds from Ulva lactuca isolated by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS): U. lactuca extracted by DVB/CAR/PDMS fiber and by PDMS/DVB fiber. HD, hydrodistillation.
No.CompoundRtRIPDMS/DVBDVB/CAR/PDMSHD
1.Dimethyl sulfide1.565<9004.465.81-
2.(E)-But-2-enal1.837<9000.053.48-
3.Pent-1-en-3-ol1.943<9002.103.48-
4.Pentanal2.001<9000.860.97-
5.3-Methylbut-2-enal 2.364<9000.711.54-
6.Hexanal2.734<9002.052.04-
7.(E)-Hex-2-enal 3.387<9000.490.31-
8.Hexan-1-ol3.549<9001.401.11-
9.Heptanal4.15090420.1716.350.33
10.(E)-Hept-2-enal5.3439610.841.23-
11.Benzaldehyde5.5279682.612.610.44
12.2-Pentylfuran 6.197992--0.24
13.(E,Z)-Hepta-2,4-dienal6.4109990.110.351.45
14.Octanal6.55210041.861.11-
15.2-(2-Ethoxyethoxy)- ethanol (Carbitol)6.6211007-0.70-
16.(E,E)-Hepta-2,4-dienal6.81710143.731.890.50
17.Benzyl Alcohol7.66310438.539.71-
18.(E)-Oct-2-enal8.29110622.985.56-
19.Heptanoic acid9.21510870.050.74-
20.(E,E)-Octa-3,5-dien-2-one 9.56010950.110.66-
21.Nonanal9.914110510.576.62-
22.2,6-Dimethylcyclohexanol 10.11911111.580.760.18
23.(E,Z)-Nona-2,6-dienal11.82111570.220.80-
24.(E)-Non-2-enal 12.04611631.691.80-
25.Decanal13.90012062.121.13-
26.(E)-Dec-2-enal 16.22512650.050.54-
27.(E,Z)-Deca-2,4-dienal17.5781294--0.29
28.(E,E)-Deca-2,4-dienal 18.5291318--3.15
29.α-Ionone23.0911429--0.20
30.(E)-6,10-Dimethylundeca-5,9-dien-2-one24.1181455--0.15
31.Dodecan-1-ol25.1001478--1.01
32.trans-β-Ionone25.4381486--3.04
33.Pentadec-1-ene25.68814920.110.56-
34.Pentadecane26.00315002.694.24-
35.Tridecanal26.4281511--0.28
36.Tridecan-1-ol29.0681580--0.23
37.Tetradecanal30.3521613--0.99
38.Heptadec-8-ene32.764167921.8012.464.39
39.(Z)-Pentadec-11-enal33.3331693--1.66
40.Heptadecane33.5621700-0.49-
41.Pentadecanal34.0981715--10.61
42.Tetradecanoic acid36.1861775--3.93
43.(Z)-Hexadec-11-enal36.9381795--0.39
44.Hexahydrofarnesyl acetone38.6371846--0.79
45.Hexadecan-1-ol38.8391852--2.29
46.(Z,Z,Z)-Hexadeca-7,10,13-trienal 39.0971863--1.61
47.(Z)-Hexadec-9-en-1-ol 39.2081863--1.12
48.Methyl hexadeca-4,7,10,13-tetraenoate 39.7571879--7.50
49.Hexadecan-1-ol39.9131884--1.34
50.(Z,Z,Z)-Hexadeca-7,10,13-trienal40.2371893--22.67
51.Dibutyl phthalate42.4831963--1.13
52.Hexadecanoic acid42.9821978--13.18
53.Sulfur, mol. (S8)44.09820121.163.45-
54.Phytol47.4392114--4.15
RI—retention index; Rt—retention time.
Table 2. Fatty acid composition determined by GC-FID and nutritional indices of Ulva lactuca.
Table 2. Fatty acid composition determined by GC-FID and nutritional indices of Ulva lactuca.
No.Fatty AcidAv ± SD (%)
1.Dodecanoic acid (lauric acid) (C12:0)4.43 ± 0.19
2.Tetradecanoic acid (myristic acid) (C14:0)2.76 ± 0.58
3.Hexadecanoic acid (palmitic acid) (C16:0)45.16 ± 1.82
4.Octadecanoic acid (stearic acid) (C18:0)5.40 ± 0.55
5.Eicosanoic acid (arachidic acid) (C20:0)14.57 ± 0.84
6.Docosanoic acid (behenic acid) (C22:0)1.03 ± 0.08
Total saturated fatty acids (SFAs)73.35
7.Palmitoleic acid (C16:1)2.37 ± 0.17
8.Cis-oleic acid+trans-oleic acid (C18:1n9c+t)13.59 ± 1.47
Total monounsaturated fatty acids (MUFAs)15.96
9.Cis-linoleic acid (C18:2n6c)4.69 ± 0.62
10.α-linolenic acid (C18:3n3)5.82 ± 0.51
11.Docosadienoic acid (C22:2n6)1.06 ± 0.11
Total polyunsaturated fatty acids (PUFAs)11.57
Total n-3 fatty acids (n-3 PUFAs)5.82
Total n-6 fatty acids (n-6 PUFAs)5.75
Nutritional indices
PUFA/SFA0.16
Index of atherogenicity (IA)2.20
Index of thrombogenicity (IT)1.82
Hypocholesterolemic/hypercholesterolemic ratio (HH)0.48
Unsaturation index (UI)44.92
Av—average value of three replicates expressed in percentage (%) with standard deviation (SD).
Table 3. Carotenoid content in Ulva lactuca.
Table 3. Carotenoid content in Ulva lactuca.
CarotenoidContent (µg/100 g)
β-carotene1926.62 ± 0.36
lutein1389.30 ± 0.60
α-carotene131.81 ± 0.11
β-cryptoxanthin12.05 ± 0.11
Table 4. Amino acid composition of Ulva lactuca determined by HPLC-FLD.
Table 4. Amino acid composition of Ulva lactuca determined by HPLC-FLD.
Amino Acid Av ± SD (mg/100 g Protein)
Aspartic acid123.77 ± 0.92
Glutamic acid97.15 ± 1.70
Serine32.73 ± 0.15
Histidine3.19 ± 0.02
Glycine182.15 ± 1.59
Threonine135.11 ± 1.68
Arginine50.76 ± 4.03
Alanine53.80 ± 0.73
Tyrosine9.60 ± 0.09
Cystine56.98 ± 1.44
Valine14.73 ± 0.25
Methionine16.04 ± 0.12
Tryptophane15.41 ± 0.63
Phenylalanine21.86 ± 0.93
Isoleucine12.73 ± 0.21
Leucine9.85 ± 0.35
Lysine38.04 ± 1.99
Proline50.23 ± 3.47
Av—average value of duplicates expressed as mg amino acid/100 g protein with standard deviation (SD).
Table 5. Major non-volatile compounds in U. lactuca ethanol extract identified using high-performance liquid chromatography–high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI–HRMS) in both positive (ESI+) and negative (ESI−) mode.
Table 5. Major non-volatile compounds in U. lactuca ethanol extract identified using high-performance liquid chromatography–high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI–HRMS) in both positive (ESI+) and negative (ESI−) mode.
No.NameMass[M-H]−
or [M+H]+		Molecular FormulatR
(min)		Mass Difference (ppm)Peak Area (Arbitrary Units)
Phenolic acids
34-Hydroxybenzaldehyde122.037121.02950C7H6O21.550.01.42 × 104
74-Hydroxybenzoic acid138.032137.02442C7H6O35.6687.22.89 × 105
353-Hydroxyphenylacetic acid152.047151.04007C1H1O316.5540.02.46 × 103
18Hydroxytyrosol154.063153.05572C1H10O311.8211.74.15 × 104
485-(3,4-Dihydroxyphenyl)pentanoic acid210.089209.08193C11H14O420.1177.23.35 × 104
134-Hydroxybenzoic acid-4-O-sulphate217.989216.98123C7H6O6S8.6016.67.30 × 105
85-Sulfosalicylic acid217.989216.98123C7H6O6S5.6846.83.35 × 106
4Vanillic acid 4-sulfate247.999246.99180C1H1O7S3.1947.24.21 × 105
2Caffeic acid 4-O-sulfate259.999258.99180C9H1O7S1.4143.32.30 × 104
54-(β-D-Glucosyloxy)benzoic acid300.085299.07724C13H16O13.8040.72.17 × 103
10Caffeic acid 4-O-glucuronide356.074355.06707C15H16O107.0919.86.86 × 103
Flavonoids
1Sativanone300.1299.09250C17H16O50.8729.71.02 × 105
63-Hydroxyterphenyllin354.11353.10306C20H18O64.1936.28.76 × 104
153,3′′-Dihydroxyterphenyllin370.105369.09798C20H18O710.8721.65.07 × 104
Fatty acid derivatives
16Ethyl 3-oxohexanoate158.094157.08702C8H14O310.8724.33.28 × 106
17Ethyl 2-ethylacetoacetate158.094157.08702C8H14O311.1773.51.43 × 106
24Azelaic acid188.105187.09758C9H16O414.2965.44.09 × 106
9Loliolide196.11197.11722C11H16O36.2272.33.32 × 106
19Tetradecanamide227.225228.23219C14H29NO12.49211.67.57 × 104
12Palmitoleamide253.241254.24784C16H31NO8.1570.75.25 × 104
21Palmitamide255.256256.26349C16H33NO13.6882.55.27 × 105
11Hexadecasphinganine273.267274.27406C16H35NO27.9030.41.53 × 108
20Linoleamide279.256280.26349C18H33NO13.4152.26.35 × 105
239-Octadecenamide281.272282.27914C18H35NO14.0810.41.11 × 106
26Octadecanamide283.288284.29479C18H37NO14.7816.12.63 × 105
14Palmitoylethanolamide299.282300.28971C18H37NO210.760.01.40 × 104
2811-Eicosenamide309.303310.31044C20H39NO15.0544.62.16 × 105
33Erucamide337.334338.34174C22H43NO15.9610.33.01 × 106
22Glycerol palmitate330.277331.28429C19H38O413.9796.88.23 × 104
27Glycerol monostearate358.308359.31559C21H42O415.024.11.95 × 105
341-(9Z-octadecenoyl)-2-(9Z-tetradecenoyl)-glycero-3-phosphocholine729.531730.53813C40H76NO8P16.3887.69.24 × 103
361-(9Z-octadecenoyl)-2-(9Z-pentadecenoyl)-glycero-3-phosphocholine743.547744.55378C41H78NO8P16.6277.87.72 × 103
421-(9Z-octadecenoyl)-2-(9Z-nonadecenoyl)-glycero-3-phosphocholine799.609800.61638C45H86NO8P18.4253.35.21 × 103
411-(11Z,14Z-eicosadienoyl)-2-heptadecanoyl-glycero-3-phosphoserine801.552802.55926C43H80NO10P18.0666.03.33 × 104
431-(11Z,14Z-eicosadienoyl)-2-nonadecanoyl-glycero-3-phosphoserine829.583830.59056C45H84NO10P19.1611.36.87 × 103
373-{[6-O-(α-D-Galactopyranosyl)-β-D-galactopyranosyl]oxy}-2-[(9Z)-9-hexadecenoyloxy]propyl (9Z,12Z,15Z)-9,12,15-octadecatrienoate912.581913.58830C49H84O1516.9530.03.69 × 102
381-hexadecanoyl-2-(9Z,12Z,15Z-octadecatrienoyl)-3-O-(α-D-galactosyl-1-6-β-D-galactosyl)-sn-glycerol914.597915.60395C49H86O1517.0215.05.67 × 103
Pigments and derivatives
31(2E)-3-[21-(Methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-9,14-divinyl-3,4-didehydro-3-24,25-dihydrophorbinyl]acrylic acid586.222587.22890C35H30N4O515.4991.43.87 × 103
303-[(21R)-21-(Methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-9,14-divinyl-3,4-didehydro-3--24,25-dihydrophorbinyl]propanoic acid588.237589.24455C35H32N4O515.4652.97.37 × 103
29Pheophorbide a592.269593.27585C35H36N4O515.3622.52.95 × 104
25Fucoxanthin658.423659.43062C42H58O614.7474.56.10 × 103
44Divinyl pheophytin a868.55869.55755C55H72N4O519.9490.01.21 × 103
47Pheophytin a870.566871.57320C55H74N4O520.1031.02.87 × 105
453-Phorbinepropanoic acid, 9-acetyl-14-ethylidene-13,14-dihydro-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-, 3,7,11,15-tetramethyl-2-hexadecen-1-yl ester886.561887.56811C55H74N4O619.9495.17.19 × 104
46Methyl (3R,10Z,14Z,20Z,22S,23S)-12-ethyl-3-hydroxy-13,18,22,27-tetramethyl-5-oxo-23-(3-oxo-3-{[(2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecen-1-yl]oxy}propyl)-17-vinyl-4-oxa-8,24,25,26-tetraazahexacycl;o[19.2.1.16,9.111,14.116,19.02,7]heptacosa-1(24),2(7),6(27),8,10,12,14,16,18,20-decaene-3-carboxylate902.556903.56303C55H74N4O719.9664.22.11 × 104
Steroids and derivatives
40(3β,20R,22E,24S)-Stigmasta-5,22-dien-3-ol394.36395.36723C29H4617.5186.65.99 × 104
327-Dehydrocholesteryl acetate426.35427.35706C29H46O215.7393.85.66 × 104
39(3β)-3-Hydroxystigmast-5-en-7-one428.365429.37271C29H48O217.3812.09.12 × 103
Table 6. Gradient elution for ESI+ (Acquity UPLC CSH Phenyl-Hexyl) and ESI− mode (Acquity UPLC BEH C8).
Table 6. Gradient elution for ESI+ (Acquity UPLC CSH Phenyl-Hexyl) and ESI− mode (Acquity UPLC BEH C8).
ESI+ESI−
Time (min)A (%)B (%)Time (min)A (%)B (%)
0.09820.01000
0.69822.0955
18.5010025.05545
25.0010030.00100
35.00100
Note: starting conditions were set in 0.1 min, and column equilibration was achieved in 2 min.
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Mutavski, Z.; Jerković, I.; Nikolić, N.Ć.; Radman, S.; Flanjak, I.; Aladić, K.; Šubarić, D.; Vulić, J.; Jokić, S. Comprehensive Phytochemical Profiling of Ulva lactuca from the Adriatic Sea. Int. J. Mol. Sci. 2024, 25, 11711. https://doi.org/10.3390/ijms252111711

AMA Style

Mutavski Z, Jerković I, Nikolić NĆ, Radman S, Flanjak I, Aladić K, Šubarić D, Vulić J, Jokić S. Comprehensive Phytochemical Profiling of Ulva lactuca from the Adriatic Sea. International Journal of Molecular Sciences. 2024; 25(21):11711. https://doi.org/10.3390/ijms252111711

Chicago/Turabian Style

Mutavski, Zorana, Igor Jerković, Nada Ćujić Nikolić, Sanja Radman, Ivana Flanjak, Krunoslav Aladić, Drago Šubarić, Jelena Vulić, and Stela Jokić. 2024. "Comprehensive Phytochemical Profiling of Ulva lactuca from the Adriatic Sea" International Journal of Molecular Sciences 25, no. 21: 11711. https://doi.org/10.3390/ijms252111711

APA Style

Mutavski, Z., Jerković, I., Nikolić, N. Ć., Radman, S., Flanjak, I., Aladić, K., Šubarić, D., Vulić, J., & Jokić, S. (2024). Comprehensive Phytochemical Profiling of Ulva lactuca from the Adriatic Sea. International Journal of Molecular Sciences, 25(21), 11711. https://doi.org/10.3390/ijms252111711

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