Elucidation and quantification health-promoting phenolic compounds, antioxidant properties and sugar levels of ultrasound assisted extraction, aroma compositions and amino acids profiles of macroalgae, Laurencia papillosa

Graphical abstract

Highlights

• •Ultrasound was used as alternative technology to convectional method (CE) for macroalgae.
• •Ultrasonic assisted extraction(UAE) enabled increased extraction of bioactive compounds from L. papillosa.
• •UAE was faster, boost efficiency, reduction of time, least expensive compared with CE.

Abstract

Currently, sustainability is one of the most critical issues confronting society today. The growing of macroalgae in ocean farms appears more sustainable than agriculture on land due to it does not require any fresh water, chemical fertiliser, or soil. Macroalgae have been shown to be a sustainable marine source of amino acids, novel bioactive phenolic and aroma compounds that can be exploitation in food, cosmetic, nutraceuticals, pharmacological applications. Despite starting the huge cultivation of macroalgae in world, bioactive compounds in the edible macroalgae have not been well characterized. Ultrasound assisted extraction (UAE) and conventional extraction (CE) techniques were compared and red macroalgae, L. papillosa extracts were characterized. The highest amount of amino acid was glutamic acid (GLU) and composed of 35% was essential amino acids. UAE at 10% amplitude for 15 min showed significantly highest (p < 0.05) phenolic (212.03±3.03 mg gallic acid equivalent/100 g) as well as antioxidant activity determined by DPPH (105.69±3.02 µmol Trolox/100 g), ABTS (238.69±2.23 µmol Trolox/100 g) radical assay and FRAP value (72.47±3.13 µmol Trolox/100 g) when in comparison with CE. Furthermore, bioactive compounds in extracts were indicated as phlorotannins, flavonoids, phenolic acids and other polyphenols using liquid chromatography coupled to diode array detection and electrospray ionisation tandem mass spectrometry (LC-DAD-ESI-MS/MS). This result confirmed higher antioxidant capacity detected with the UAE. A total of 46 volatile organic compounds were identified and quantified by GC-FID/MS with HS-SPME system. This study emerges as first report to novel extraction method used and deeply characterization of L papillosa. The results seem that significant potential application in the functional food, active packaging and nutraceuticals industry.

1. Introduction

Seaweed or Macroalgae is a promising natural resource for the functional food, feed, cosmeceutical and pharmaceutical industries since it includes novel compounds with a diverse range of biological activity [1]. The pursuit of new physiologically bioactive compounds in seaweed is presented an almost limitless field because of the enormous taxonomic diversity of these organisms [2]. Adapting quickly to new environmental conditions is critical for the survival of seaweeds, which live in complex territory and are subjected to harsh circumstances. To do so, they produce a wide range of secondary metabolites that are unique to them and cannot be found in any other creature. Phenolic, carotenoids, alginate, glucan, fucoidan, polyunsaturated fatty acids, proteins, amino acids, vitamins, minerals and volatile organic compounds (VOCs) are just the bioactive compounds in seaweed that have been associated with beneficial health effects [3]. Generally, the protein content and amino acid profile of seaweeds differs according to species, grown regions, salt content in sea and seasonal periods etc. The protein concentration of brown seaweeds is low (3–15%) when compared with that of the green or red seaweeds (10–47%) [4]. Moreover, the seaweeds exhibit a large number of VOCs are released for a variety of reasons, including: increasing tolerance to abiotic stresses; communicating stress to homogeneous algae to induce defence; playing allelopathic roles on heterogeneous algae and aquatic macrophytes for competing nutrients; and protecting against predators. The majority of research on VOCs in macroalgae has concentrated on their allelopathy effects, whereas their contributions in other areas have received far less attention [5]. Additionally, polyphenols including one or more aromatic rings bearing hydroxyl groups are perhaps the category of phytochemicals substances in seaweed that have garnered the most interest. Polyphenols isolated from seaweeds have antioxidant, antibacterial, antiviral, antifungal, anti-inflammatory, antidiabetic and anticancer properties, and have been confirmed these findings in in vivo and in vitro studies, and are divided into subclasses of phenolic acids, flavanols, flavones, phlorotannin, lignans and stilbenes based on the molecular structure [6]. Polyphenolic compounds present a formidable extraction challenge since they are deeply embedded in the seaweed. Conventional extraction (CE) techniques such as maceration and soxhlet extraction for obtaining polyphenols from seaweeds have relied on the use of potentially hazardous chemical solvents, as well as consuming much more of energy and time while providing low yields of polyphenols [1], [7], [8]. However, there has always been worry about toxicity, limited extract yield, and the presence of residues in the target molecule. To solve these shortcomings and apprehension and accelerate the transition to more sustainably and environmental friendly extraction technologies, novel, reliable, efficient, and cheap extraction technologies are needed to maximise yield, minimise residues, and achieve clean label compliance [6], [9]. Novel cold extraction technology, such as ultrasound assisted extraction (UAE), have minimal impact on the stability of bioactive compounds, have significant potential to minimise the utilization of hazardous solvents, boost efficiency, reduction of time, least expensive and improve quality and yield of bioactive compounds [10]. Effective extraction of phytochemical components as penetrate complicated tough cell wall in seaweed is facilitated by ultrasound technology, which can be employed pre-treatment or in combination with solvent to rupture cell membranes for enhanced extractability [11], [12]. Recently, some researchers reported that UAE is preferable for recovering bioactive compounds from seaweeds because it allows for extraction to occur at atmospheric pressure and not need the temperature. Ultrasound utilizes sound waves with a frequency that exceeds what the human ear could detect. The microporous particles of the matrix or cell wall are agitated by the cavitation caused by the ultrasonic waves, allowing the solvent to effectively enter the cell and begin the extraction process [3], [8], [9], [13].

Furthermore, bioactive components in the solvent can be efficiently extracted thanks to the increased contact area between the liquid and the solid surface caused by the ultrasonic jet. It has only been reported in a few number of studies that ultrasound-assisted extraction has been utilised to extract phytochemicals from seaweed [7], [14]. Kumar et al. [1] found that UAE and CE techniques were compared and characterized by extracting three edible macroalgae, viz., Sargassum wightii, Ulva rigida and Gracilaria edulis in methanol. Ummat et al. [9] investigated that UAE process conditions (frequency, time and solvent) to acquire high yields of flavonoids, total phenolics and antioxidant activities from 11 brown seaweed species. Apart from these reports, there is not much literature available about UAE of bioactive in the seaweed. The genus Laurencia of the red algae is the most chemically complex seaweed genus in the world due to the vast quantity and variety of secondary metabolites it generates [15].

The aim of study, therefore to compare the effect of CE and UAE techniques on the antioxidant activity, total phenolic content, amino acid profiles, volatile compounds and phenolic constituents’ characterization of Laurencia papillosa grown in Meditarrean sea. Identification and quantitative analysis of phenolic constituents, amino acids compositions and volatile compounds in extracts prepared by both methods was performed using liquid chromatography-tandem mass spectrometry, HPLC, and GC-FID/MS respectively. This is the first study determining both qualitative and quantitative characterisation of extracts to compare the effects of these extraction methods on the red algae, Laurencia papillosa. These results will shed light on their availability utilization as phytochemicals and functional ingredients that can be exploited in the food and pharmaceutical industries.

2. Materials and methods

Laurencia papillosa were collected by scuba diving on the Mersin, Çamlıbel coast, Turkey (36.7910° N, 34.6267° E) in May 2020. They were first washed in seawater to get rid of any organisms or sediments that might have been present, and then rinsed briefly in tap water to get rid of any salt and removed excess water. Samples were dried at 40 °C during 72 h. Dried samples were milled via grinder (Fakir Roxy, Turkey) to get Laurencia papillosa powder (LPP). The LPP in this way was stored in a ziplock plastic bags and was used for the extraction and further analysis. All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). Standard solutions and mobile phases utilized in HPLC were prepared daily.

2.1. Ultrasound-Assisted extraction (UAE) and Conventional extraction (CE) techniques

The algal extracts were performed by ultrasound-assisted extraction (Branson Digital Sonifier SFX 250, USA) using different process parameters such as amplitude (A) (10, 30, and 50%) and time (5, 10, and 15 min). Ethanol was chosen as solvent according to preliminary trials. Briefly, 5 g LPP was weighed and added to 50 mL of ethanol/water solution (80% v/v). The suspensions were mixed homogeneously at 10.000 rpm for 2 min by using Ultraturrax (IKA, Germany). The suspensions were placed into an ice bath during the ultrasound-assisted extraction of compounds, to prevent a temperature increase. Then, the sonification process was applied according to determined parameters, following which the suspensions were centrifuged (4 °C, 20 min, 6500 rpm, Nuve, Turkey).

Conventional extraction (CE) method was conducted as prescribed by Kumar et al. [1] slightly modification that was stirred at 50 °C for 24 h. The same sample-to-solvent ratio was utilized as for both UAE and CE. Before and after extraction process, ultraturrax mixing and centrifugation and filtration, respectively was performed same as UAE.

Algal extracts were filtered through a 0.45 μm syringe filter (Millipore) after centrifugation and collected in amber glass bottles and then stored at −20 °C to avoid the oxidative reactions until analysis.

2.2. Determination of antioxidant properties and total phenolic content

The antioxidant properties were determined by the DPPH (1,1-diphenyl-2-picryl-hydrazyl), ABTS (2,20-azino-bis-3-ethyl-benzothiazoline-6-sulfonic acid) assays and FRAP (ferric reducing antioxidant power) assays. DPPH radical scavenging assay was performed according to described by Brand-Williams, Cuvelier, and Berset (1995) and ABTS radical assay was carried out using the auto-bleaching method as reported by Re et al. (1999), with the modifications [16]. In addition, the total antioxidant reducing power of extracts was measured using FRAP method as described by Benzie and Strain (1999), with slight modifications [9]. The antioxidant activity of samples was expressed in μmol Trolox equivalent per 100 g.

Total phenolic content (TPC) was determined using previously described by Singleton and Rossi (1965) with slightly modifications [16]. To summarise, 200 µL of extract was mixed with 0.5 mL of Folin-Ciocalteu reagent for 3 min, then added 2 mL of 20% (w/v) sodium carbonate. After allowed to 30 min at room temperature, the mixture was measured for absorbance at 765 nm. The TPC results were expressed as milligrams of gallic acid equivalent (GAE) per 100 g.

2.3. Sugar analysis

In the HPLC system, sugars analysis (sucrose, glucose, and fructose) were performed by using a refractive index detector (G1362A RID) and Aminex HPX-87H column (300×7.8 mm, Bio-Rad). Flow was set at 0.5 mL/min, and eluent was composed of 0.09 mol/L H₂SO₄ and 6% acetonitrile (v/v). The correlation between peak area and concentration was calculated using a calibration curve based on known standards [16].

2.4. LC-DAD-ESI-MS/MS analysis of phenolic compounds

Algal extracts were filtered through a syringe filter (0.45 μm) before injection and analysis was carried out according to Kelebek [17]. The Agilent 1100 HPLC system (Agilent Technologies) was employed with Windows NT-based ChemStation software. The HPLC equipment consists of, a binary pump, an auto sampler a degasser, and a diode array detector. A Phenomenex Luna reverse phase C-18 column (4.6 mm × 250 mm, 5 μm) was utilized. The parameters of the analysis were as described in the following: Solvent A (water/formic acid, 99/1; v/v) and Solvent B (methanol/formic acid, 60/40; v/v) as a mobile phase, 0.5 mL/min flow rate and the temperature adjusted at 25 °C.

Analysis conditions were as follows: water/formic acid (99:1; v/v, Solvent A) and methanol/formic acid A (60:40; v/v, Solvent B) as a mobile phase, and at a temperature of 25 °C, respectively. Peak intensities were monitored in real time by setting the DAD at 280, 320, and 360 nm, and the full spectra (190–650 nm) were continually recorded for component identification of algal extracts.

The phenolic compounds were determined through the comparison of a retention index and UV spectrum to pure standards and the verification was also obtained by an Agilent 6430 LC–MS/MS spectrometer operated with an electrospray ionization source. Positive and negative ionisation modes were used concurrently to acquire mass spectra (from m/z 100 to 2000).

Identification and quantification of phenolic compounds were detected by Kelebek [17]. Mass spectrum data was gathered in negative ion and MRM mode to identify and quantify phenolic compounds. Analysis was performed in triplicate. Over all cases, the regression coefficient value of the standards was given to being above 0.995 (R²). Due to a lack of reference compounds, a calibration based on structurally related substances was employed, with the molecular weight correction factor properly considered. Under current chromatographic procedures, the limits of detection (LOD) and quantification (LOQ) were calculated at a signal-to-noise ratio (S/N) of approximately 3 and 10, respectively.

2.5. Determination of amino acid compositions by HPLC

Amino acid profiling of algal samples was determined using HPLC-20AD Nexera XR (Shimadzu Co., Kyoto, Japan). Samples (0.5 g) was hydrolized with 0.1 N aqueous solution of hydrochloric acid (HCl) under reflux condenser at 110 °C for 24 h. Extracts were filtered using syringe filter (0.45 µm) and filtrate was derivatized with 3-mercaptopropionic acid (3-MPA)/o-phthalaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC-Cl) reagent and then 2 µL of the extract was injected into Shimpack XR-ODS (100–4.6 mm) column (Shimadzu Co., Kyoto, Japan) whose temperature was set to 40 °C. The mobile phases were following: Potassium phosphate buffer (A) (20 mM; pH: 6.9) and a mixture of acetonitrile/methanol/water (B) (45/40/15). Amino acids were quantified with this external standard method according to peak areas of the standards and the retention time [18], [19]. The amino acid profiling analysis were executed in triplicates.

2.6. Headspace solid phase microextraction (HS-SPME) and analysis of volatile compounds

Headspace solid phase microextraction (HS-SPME) method was performed [20]. Briefly, the amount of 1 g of powdered sample together with a 2 mL of saturated NaCl solution in a headspace vial were homogenized in ultrasound bath for 10 min. Then an internal standard was added into vial and was capped. DVB/Carboxen/PDMS fiber (Sigma-Aldrich, USA) was utilized for extraction of volatile compounds with these following conditions for headspace sampling: incubated at 60 °C for 15 min and then extracted at 60 °C for 45 min. And trapping the volatile compounds at 250 °C for 2 min in injection port and then was flushed into the column.

Quantification of aroma constituents was analysed with the gas chromatography (GC) system [20]. It was consisted of Agilent 6890 chromatograph equipped with flame ionization detector (FID) and Network-mass selective detector (MSD) (Agilent 5973, DE, USA). A FID signal for quantification and an MS signal for identification were all obtained simultaneously using this system. Each of the two modes FID and MSD, received an equal amount of GC effluent. Volatile compounds were distinguished on DB-Wax capillary column (60 m × 0.25 mm × 0.4 µm). The temperature of the column was raised from 50 °C to 250 °C at a rate of 4 °C per minute, with a final standby at 250 °C for 10 min. The mass-selective detector employed the same set of oven temperature programme. Three microliters of extract were injected and helium was adjusted as the flow rate of carrier gas was 1.5 mL/min¹. Injector and detector were set at 250 °C. The MS (electronic impact ionisation) criteria were described in the following: mass range m/z of 30– 300, MS ionization energy of 70 eV, interface temperature of 250 °C, quadrupole temperature of 180 °C. The peaks were recognized by injecting the standard solutions for volatile compounds. The constituents were assessed with comparing their retention index and their mass spectra on the DB-Wax column using commercial aroma spectra databases on the computer (Wiley 7.0, NIST-98 and Flavor.2L). It was utilized 2 methyl-3 heptanone as internal standard to determine the quantification. Some of the identifications were verified after injection of the chemical standards into the GC-FID/MS systems. Each compound's retention index was determined using the n-alkane series.

2.7. Statistical analysis

The data were stated with analysis of variance (ANOVA) in SPSS (v.16.0, SPSS Inc., Chicago, IL, USA). Significance of the differences between means were carried out by utilizing Tukey's comparison test at 95% confidence interval (p < 0.05).

3. Results and discussion

3.1. Amino acid composition of L. Papillosa

The amino acid composition (mg/100 g of dried weight) and representative chromatogram are illustrated in Table 1 and Fig. 1, respectively. Protein is the most substantial nutrient for growth, so it is an essential factor of figuring out, food might be potential health beneficial. Not only is the amount of protein important, but also is the protein amino acid profile so essential amino acids (EAAs) are evaluated to decide the protein quality of a food. The EAAs, HIS, THR, VAL, TRP, PHE, ISO and LEU and non-EAAs, GLU, ASP, SER, GLY, ARG, ALA, TYR, CYS were present in L. papillosa. The major amino acid composition included GLU, 47.24 mg/100 g. Amino acids levels ranged from 3.62% to 31.1%. The ratio of essential amino acids to the total amino acid were 0.35. Therefore, the 35% of the amino acids are EAA and the results also point out a decent ratio of EAA to non-EAA was 0.53.

Table 1.

Amino acid composition of L. papillosa (mg/100 g).

Peak	Ret. Time	Amino acids	Encoded	Amount	FAO recommendation1	Soy meal2	Rice meal2	Ovalbumin3
1	1.76	Glutamic acid	GLU	47.24±2.14		51.8	8.2	9.9
2	4.48	Aspartic acid	ASP	9.47±0.01		22.9	3.1	6.2
3	4.81	Serine	SER	8.42±0.04		15.2	2.4	6.8
4	5.77	Histidine*	HIS	5.95±0.32		6.7	1.0	4.1
5	6.02	Glycine	GLY	6.50±0.02		11.2	2.1	3.4
6	6.22	Threonine*	THR	8.90±0.23	4.0	11.5	1.7	3.0
7	7.40	Arginine	ARG	8.14±0.02		23.7	3.8	11.7
8	7.65	Alanine	ALA	6.34±0.24		12.2	2.7	6.7
9	9.48	Tyrosine	TYR	7.11±0.00		10.0	0.5	1.8
10	10.67	Cysteine	CYS	6.29±1.04	3.5 (MET + CYS)	3.1	0.3	n.a.
11	11.33	Valine*	VAL	7.12±0.18	5.0	14.5	3.0	5.4
12	12.15	Tryptophan*	TRP	12.63±0.11		n.a.	n.a.	1.0
13	13.14	Phenylalanine*	PHE	6.15±0.65	6.0 (PHR + TYR)	16.5	2.7	4.1
14	13.17	Isoleucine*	ISO	6.36±0.16	4.0	14.0	2.0	4.8
15	13.72	Leucine*	LEU	5.50±0.29	7.0	24.6	4.3	6.2
	Total AA		152.11±1.25
	EAA		52.61
	Non-EAA		99.95
	EAA/Total AA		0.35

Values are expressed as mean ± STD, ¹ FAO [35], ²[23], ³[4], * meaning is EAA, n.a., not analysed.

AA: Amino acid.

EAA: Essential amino acid.

Fig. 1.

Representative chromatogram of amino acid composition from L. papillosa.

Among the two Laurencia species studied, the GLU fractions show as major AA, similarity. The amount of AA in L. filiformis was much higher than L. intricata described by Gressler et al. [21], but lower than our data. This great difference among species is explained in the literature due to grow up region, varieties, stress conditions, collection time, climate state [22].

With respect to the high quantity of EEAs in L. papillosa, the comparison with those of other food proteins such as soybean, cereal, eggs indicate the similar composition and unsurpassed nutritional value quality [4], [23].

3.2. Antioxidant properties and total phenolic content of L. Papillosa UAE and CE extracts

The impact of ultrasound assisted extraction process parameters (time and ultrasound amplitude) on total phenolic contents (TPC) and antioxidant capacities of L. papillosa was specified. Extracts of algae was obtained with UAE conditions in different time (5, 10 and 15 min) and amplitude (10, 30 and 50%) and compared with CE. The influence of the extraction times and amplitude levels on the antioxidant capacities and amount of TPC was determined. TPC of both UAE and CE were analysed and indicated in Table 2. Increasing extraction time in each amplitude levels had more polyphenols was exhibited. The same cannot be mentioned for increasing in amplitude level. TPC of the extracts varied with the extraction time and amplitude levels applied and results ranged between 71.56 (5 min and 10% A) and 212.03 mg GAE/100 g (15 min and 10% A). While extractions prepared with each amplitude levels for 5 min compared, it was detected the amount of TPC increased with increasing amplitude levels. But extracts were obtained in each amplitude levels during 10 min and 15 min, TPC decreased with increasing amplitude levels was emerged. Considering 5 min extraction time, higher TPC values were found in extracts prepared with 50% A which detected 137.19 mg GAE/100 g. However, extraction times performed with 10 and 15 min for 30% and 50% amplitude levels, amount of TPC showed declining which varied between 82.79 and 144.54 mg GAE/100 g. Rodrigues et al. [3], who found UAE results in significantly higher TPC in red (Osmundea pinnatifida) macroalgae of Portuguese coast compared the CE. Dang et al. [8] reported that parameters effect extracting phenolic compounds from Saccharina japonica, especially while excessive of the ultrasonic time and power could lead to a decrease in TPC level due to degradation of bioactive compounds by ultrasonic wave. Variations in total polyphenols content may be caused by the non-specific nature of extraction solvents, ultrasound amplitudes, extraction time and spectrophotometric analysis for TPC. During extraction type and process, nonphenolic compounds (vitamin C and minerals) and other reductive polar compounds (such as soluble polysaccharides, amino acids and peptides) may be released along with polyphenols which may be detected and quantified. This can reduce the effect of the Folin-Ciocalteu reagent due to response of radical scavenging could be affected by those molecules [3], [9], [11].

Table 2.

Effect of ultrasound assisted extraction process parameters (time and ultrasound amplitude) and CE on antioxidant activities and total phenolic content (TPC) of L. papillosa.

	UAE parameters		Biological activities
	Amplitude	Time (min)	DPPH	ABTS	FRAP	TPC
		5	50.35±1.87a	106.85±1.92a	23.61±3.46a	71.56±12.14a
	10%	10	96.76±3.18b	202.05±1.57b	63.91±4.19b	196.82±4.48b
		15	105.69±3.02c	238.69±2.23c	72.47±3.13c	212.03±3.03b
		5	67.33±0.44df	138.71±0.53d	38.18±2.17d	103.77±3.96c
UAE	30%	10	57.36±0.89e	116.80±2.29e	30.25±0.79e	82.79±4.79a
		15	66.96±4.34df	132.79±1.56f	37.87±2.16d	100.68±0.70c
		5	68.18±2.27df	145.30±1.56 g	41.30±3.71d	137.19±0.70d
	50%	10	72.10±2.27f	155.17±0.47 h	42.44±2.62d	144.54±0.08d
		15	63.22±0.30de	148.08±1.10 g	42.89±2.72d	102.29±6.90c
CE			54.73±0.58ae	160.73±1.37ı	20.50±0.78a	169.21±7.48e

Data are expressed as average±standard deviation (n = 3); The different letters in each row indicate statistical differences (p < 0.05). Antioxidant properties (DPPH, ABTS and FRAP) and total phenolic content (TPC) are expressed as μmol Trolox/100 g and mg GAE/100 g, respectively.

As presented in Table 2, the antioxidant properties of L. papillosa red algae extracts acquired from UAE and CE were emerged using DPPH, ABTS and FRAP assays. Results of the present study suggest that with increase in the extraction time at 10% level amplitude, antioxidant properties increases significantly. The results of antioxidant capacity in terms of DPPH and ABTS were found that were varied between 50.35 and 105.69 and 106.85 and 238.69 μmol Trolox/100 g sample, respectively. The findings of DPPH activity were confirmed with ABTS results. The extract obtained by UAE at 10% A for 15 min had the highest FRAP value is 72.47 μmol Trolox/100 g sample, while the extracted by CE had the lowest FRAP value is 20.50 μmol Trolox/100 g sample. No previous study of antioxidant activity in L. papillosa has been found so, results were evaluated according to other seaweed specifies. A study by Dang et al. [8] reported that DPPH, ABTS, and FRAP values using UAE were higher (154.6%, 166.8% and 150.6%, respectively) is comparison with CE employed. Ummat et al. [9] investigated 11 brown macroalgae species widely grown in Ireland, these DPPH free radical scavenging activities were found between 2.5 and 29.1 mg TE/g extract. The ferric reducing antioxidant power value of antioxidants in the extracts is correlative to the antioxidant potential [10]. Moreover, applications of UAE and CE protocols achieved extracts from red macroalgae, Gracilaria edulis done then these extracts were utilized for detecting antioxidant potentials. It was found that FRAP value and Radical scavenging activity in UAE protocol have higher than CE. So, It can been revealed that shown similar antioxidant activities to those in this study [1]. Algal derived compounds such as pigments, polyphenols, vitamins, fatty acids and polysaccharides have been associated with effect on antioxidant activity. Among the determined bioactive compounds in seaweed, pigments and polyphenolic compounds are probably the group that have indicated the most attention. However, polyphenolic compounds could be said that playing the predominant role in the antioxidant activity of this extract due to higher amount of phenolic compounds and antioxidant capacity compared to other components [2], [8].

When comparing UAE with CE, UAE was found to be significantly more effective. This may be owing to the efficient disruption of seaweed cell walls by UAE, which in turn may have allowed for the release of more polar bioactive. Similar findings were reported which investigated effects of ultrasound for extraction of phenolic compounds from red, green and brown seaweeds. They demonstrated that the extraction yield was increased and extraction time was shorted using ultrasound as a driving force for distributing the solvent into the solid matrix [1], [9], [12].

As 10% US amplitude exploits less energy compared to 30% and 50% in the same extraction time (15 min), the strongest antioxidant activities (DPPH, ABTS and FRAP) and highest TPC were exhibited, 10% A and 15 min were considered the optimum conditions. So, these parameters were utilized extract preparation for analysing of phenolic compounds.

3.3. Sugar contents of L. Papillosa

Three types of sugars amounts (sucrose, glucose and fructose) were assessed in the both U@10A_15 and CE extract of L. papillosa (Fig. 2). Fructose was quantified as the predominant sugar in both U@10A_15 and CE, for CE followed by sucrose (130.62 mg/100 g) and glucose (110.37 mg/100 g), while for UAE tracked by glucose (187.96 mg/100 g) and sucrose (174.70 mg/100 g). Total sugar content in U@10A_15 and CE were detected 2193.12 and 1307.37 mg/100 g, respectively. Rodrigues et al. [3] determined the glucose content of extract that hot water extraction and ultrasound-assisted extraction were applied on Sargassum muticum, Osmundea pinnatifida, and Codium tomentosum. They found that ranged between 37.8 and 83.2 mg glucose equivalent/g extract. Tropical red seaweed Hypnea musciformis hydrolysate was reported for total sugar content was 5.57 g/100 g [24]. Our results specified higher than compared with these findings. These findings suggest that the mechanical action of ultrasonic waves, when applied at the extraction conditions employed, facilitates the release of additional polysaccharides from the intracellular or wall contents of seaweeds.

Fig. 2.

The sugar contents of U@10A_15 and CE of L. papillosa extract.

3.4. Phenolic compounds of L. Papillosa

The evaluation of L. papillosa phenolic profile by HPLC-DAD-ESI-MS/MS (Fig. 3) revealed the presence of 12 peaks. Retention time (RT), mass spectral characteristics (MS), identity and compositions of phenolic compounds were presented in Table 3.

Fig. 3.

Representative chromatogram of phenolic compounds from extract of L. papillosa from UAE (a) and CE (b) obtained by liquid chromatography-diode array detection (LC-DAD).

Table 3.

Retention time, mass spectral characteristics, concentration of phenolic compounds in U@10A_15 and CE of L. papillosa.

No	RT (min)	Compounds	[M−H]−	MS2	U@10A_15	CE
1	4.89	Urolithin A	227	182	21.09±1.35	17.99±0.60
2	5.91	Phloroglucinol*	125	97	318.86±1.59	199.94±1.63
3	6.9	2-Hydroxy-2-phenylacetic acid	152	125	91.27±1.21	67.57±0.33
4	7.14	Quercetin caffeoyl-glucoside	625	463–301	447.43±0.70	363.34±0.43
5	15.91	p-Coumaric acid methyl ester	176	163–119	99.86±1.53	91.82±0.52
6	23.2	Vanillin	151	135	48.29±2.34	27.68±2.10
7	27.31	Hydroxytyrosol	370	267–192	40.70±0.75	21.82±1.35
8	29.51	p-Hydroxybenzoic acid*	137	93	238.00±0.61	154.20±1.17
9	37.84	p-Hydroxybenzaldehyde*	121	92.6	487.62±1.39	614.26±8.34
10	41.65	6.8-Dimethyl-4-hydroxycoumarin	189	145	16.33±0.64	9.89±0.06
11	45.24	6,7-Dihydroxycoumarin-6-glucoside	339	163–148.9	22.82±0.11	16.70±0.72
12	48.11	Sinapic acid	223	179	10.92±0.07	8.06±0.49
Total (µg/g)					1843.20±5.03	1593.28±11.04

Since extract of L. papillosa processed by UAE at 15 min and 10% amplitude showed higher phytochemical content and antioxidant activity than other UAE process parameters. The extract of U@10A_15 were carried out for the quantitative analysis of phenolic compounds mentioned above, and was subjected in comparison with CE. Qualification of the extracted compound was emerged out by comparing its retention time, negative ionization mode and ESI-MS spectrum with spectra of the reference standard.

Phlorotannins (Two compounds; Urolithin A and Phloroglucinol) and flavonoids (One compound; Quercetin caffeoyl-glucoside) were the phytochemical groups in both extracts of U@10A_15 and CE, followed by phenolic acids (Five compounds; 2-Hydroxy-2-phenylacetic acid, p-Coumaric acid methyl ester, p-Hydroxybenzoic acid, p-Hydroxybenzaldehyde, Sinapic acid) and other polyphenols (Four compounds; Hydroxytyrosol, Vanillin, 6,8-Dimethyl-4-hydroxycoumarin, 6,7-Dihydroxycoumarin-6-glucoside). The total content of phenolic compounds of U@10A_15 and CE extracts were found 1843.20 and 1593.28±11.04 mg/L, respectively. The phenolic compounds in U@10A_15 extract have more amount than CE, except p-hydroxybenzaldehyde. This could be regarding to the increment of the amount of phenolic compounds as a result of the disruption of the cell wall and matrix structure by ultrasound treatment.

The most major compound is phenolic acid, p-hydroxybenzaldehyde (with [M−H]⁻ at m/z 137) was presenting in both U@10A_5 and CE extracts. However, the compound in CE extract was detected higher quantity than U@10A_5. The MS² spectrum of p-hydroxybenzaldehyde indicated the product ions at m/z 93, emphasising the loss of CHO (29 Da). The presence of p-hydroxybenzaldehyde in brown and red macroalgae species was also previously demonstrated by [2], [25]. It was reported as potential compounds showing considerable free radical scavenging capacity [26].

The peak 4 data suggested a flavonoid identified quercetin caffeoyl-glucoside as probable compound due to the precursor ion of m/z 624 and fragments of m/z 463 and 301. The quercetin caffeoyl-glucoside in U@10A_15 and CE extracts was determined as one of the main phenolic compound calculated 447.43 and 363.34 mg/L, respectively. No results for this compounds in seaweeds analysed phenolic profile come across in the literature.

The other important compound is that phloroglucinol was detected with [M−H]⁻ at m/z 125 and the identity was confirmed by the MS² spectrum, which generated a major fragment ion at m/z 97, due to the loss of CO from the precursorion, data is in agreement with previous work that detected its presence in seaweeds [26]. The quantity of phloroglucinol in U@10A_5 and CE extracts were found 318.86 and 199.94 mg/L, respectively. Polymeric structures like phlorotannins, which can include as many as eight linked phloroglucinol units, are just one indication of the complexity and variety of macroalgal phenolic compounds [2]. Apart from accomplishing as antioxidants, phlorotannins, phloroglucinol, have been used effectively in preventing of neurological diseases, cancer, and intestinal disorders [27]. In addition, phloroglucinol has been demonstrated to have a wide range of biological actions, such as antibacterial activity, cytoprotective, anti-HIV-1, anti-inflammatory effect, anti-diabetic activity, wound healing property and decreasing the pain [28], [29].

The compound presented molecular ion of [M−H]⁻ at m/z 137 and fragments of m/z 93, there- fore this compound was tentatively suggested as a p-hydroxybenzoic acid. The presence of hydroxybenzoic acid and other phenolic acids in seaweeds was reported by [30], who appreciated the phenolic composition of three brown macroalgae species in Galicia coast, Spain. Our results showed including highest amount of hydroxybenzoic acid compared with the macroalgae.

Three hydroxycoumarins derivatives (Urolithin A, 6.8-Dimethyl-4-hydroxycoumarin and 6,7-Dihydroxycoumarin-6-glucoside) were determined. Urolithin A with [M − H]⁻ m/z at 227 was specified from L. papillosa MS/MS identification by product ions at m/z 182 (M – H − 45, loss of COOH) [26]. The amounts of Urolithin A compounds in U@10A_5 and CE extracts were measured 21.09 and 17.99, respectively. 6.8-Dimethyl-4-hydroxycoumarin with [M−H]⁻ m/z at 189 was proposed and was identified by the neutral loss of CO₂ (44 Da), resulting in precursor ion at m/z 145. The quantity of compound in extracts were determined as 16.33 mg/L for U@10A_15 and 9.89 mg/L for CE. The MS² spectrum of 6,7-Dihydroxycoumarin-6-glucoside displayed the product ions at m/z 163 and m/z 148.9, indicating precursor ion [M−H]⁻ at m/z 339 in negative mode.

p-Coumaric acid methyl ester was quantified with concentrations of 99.86 and 91.82 mg/L for U@10A_15 and CE, respectively. Following compound is vanillin, were present with a concentration of 48.29 mg/L for U@10A_15 and 27.68 mg/L for CE. The lowest amount of compound was measured as sinapic acid was 10.92 and 8.06 mg/L for U@10A_15 and CE, respectively.

Results of LC-MS/MS analysis strongly corroborated findings of TPC and all antioxidant assays (DPPH, ABTS and FRAP) for the L. papillosa.

Varieties, growth stage, size, age, reproductive status, depth, nutrient cycling, salinity, light intensity exposure, UV lights, predation severity, and harvest time are just some of the abiotic and biotic factors that can affect the content and diversification of seaweed metabolites. As a result, the biological activity and phenolic profile variability of macroalgae depends on the environmental effects and species [14].

As consumer have become more concerned about nutritionally food and their ingredients, the understanding of nutrients that people worldwide benefit from has changed. So they have shifted consuming the seconder metabolites due to their biological activities. The phenolic profiles of L. papillosa extract reflect several health-promoting compounds. The wide range of bioactive compounds (phlorotannins, flavonoids, phenolic acids and other polyphenols) found in L. papillosa extract and has included a different of nutraceutical properties such as antioxidant, anti-histamine, anti-hypertension, anti-photo damage, anti-osteoporosis, anti-diabetes, anti-angiogenesis, antiviral, antimicrobial, hepatoprotective, anti-brain damage, anti-inflammatory, anticancer activities as well as decreasing blood cholesterol, and prevention of cardiovascular disease [12], [14], [31].

Furthermore, the presence of these abundant polyphenols suggest evidence on behalf of macroalgae as a potential source of antioxidants for application in functional foods, packaging, cosmetics and pharmaceutical industries, while further toxicity, animal and clinical studies can be required for human use.

3.5. Volatile organic compounds (VoCs) of L. Papillosa

A total of 46 compounds were identified and quantified in L. papillosa had 169.07 mg/L volatile compounds, which included thirteen hydrocarbons (53.31%), ten ketones (10.81%), eight aldehydes (15.33%), eight aromatic hydrocarbons (7.88%), three alcohols (7.31%), one esters (3.63%), one phenol (1.09%) and one carboxylic acids (0.62%), shown in Table 4. An example of the chromatogram of VoCs is represented in Fig. S1. This is first report about determination VoCs in L. papillosa. Heptadecane is highest concentration of VoCs. Major compounds were found hydrocarbons including pentadecane, 1-pentadecene and heptadecane, alcohol was 1-Octen-3-ol, aldehyde was benzaldehyde, ester was 2(4H)-benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl. Berneira et al. [32] evaluated the volatile composition seven Antarctic macroalgae and concerning volatile compounds, between 28 and 55 considerable compounds could be detected and heptadecane were found in highest amounts, this is agreement with our data. However, it can be said that differences of quantitative and qualitative of VoCs due to species, grown region, harvest season, environment, different extraction method etc. Moreover, aldehydes are representative of the so-called “green odour” substances, which play an active role as repulsive and impressive in plant and insect interactions [33]. Wang et al. [5] analysed the VOCs of three red seaweeds (Grateloupia filicina, Polysiphonia senticulosa and Callithamnion corymbosum) from the Yellow Sea of China and found that the main VOCs were aldehydes, ketones, alkenes and alcohols. As previous reported by Gressler et al. [34], algae-derived compounds, only 46 metabolites were identified as terpenes, alcohols, acids, esters, ketones, and hydrocarbons detected.

Table 4.

Volatile organic compounds of L. papillosa.

Peak	LRI	RT	Groups	Compounds	L. papillosa
1	1078	7.592	Aldehydes	Hexanal	3.45±0.012
2	1126	8.854	Aromatic Hydrocarbons	Ethylbenzene	1.02±0.20
3		9.153	Hydrocarbons	β-Pyronene (1,3-Cyclohexadiene, 1,2,6,6-tetramethyl-)	0.72±0.07
4	1134	9.430	Aromatic Hydrocarbons	p-Xylene	1.38±0.35
5	1147	9.908	Hydrocarbons	1-Undecene	1.32±0.16
6	1108	10.737	Hydrocarbons	β-Pinene	0.64±0.09
7	1181	11.992	Aldehydes	Heptanal	3.21±0.26
8	1185	12.180	Hydrocarbons	D-Limonene	3.77±0.78
9	1121	14.535	Ketones	3-Penten-2-one	1.67±0.02
10		14.648	Aldehydes	(E,2R)-2,4-dimethyl-4-hexenal	2.98±0.48
11	1254	14.901	Aromatic Hydrocarbons	Styrene	1.14±0.19
12	1630	15.257	Hydrocarbons	1-Hexadecene (Cetene)	1.59±0.25
13	1364	15.625	Aromatic Hydrocarbons	2-ethyl-1,4-dimethyl-Benzene	0.81±0.05
14		17.015	Ketones	4,6-Dimethyl-2-heptanone	1.55±0.01
15	1282	18.074	Ketones	2,2,6-trimethyl-Cyclohexanone	1.13±0.05
16		18.346	Hydrocarbons	Tridecane	1.46±0.17
17	1314	18.742	Aldehydes	(E)-2-Heptenal	1.57±0.17
18		19.098	Ketones	2,3-Octandione	1.12±0.13
19	1339	19.699	Ketones	6-methyl-5-Hepten-2-one	1.92±0.14
20		20.409	Aromatic Hydrocarbons	p-Methylstyrene	1.44±0.08
21	1353	20.766	Hydrocarbons	1-Tridecene	2.02±0.15
22	1359	21.817	Alcohols	1-Hexanol	4.20±0.33
23	1595	23.491	Ketones	Isophorone	1.83±0.26
24		24.695	Hydrocarbons	Tetradecane	3.13±0.26
25		25.280	Aromatic Hydrocarbons	o-Allyltoluene	2.04±0.15
26	1452	26.469	Carboxylic acids	Acetic acid	1.02±0.01
27	1454	26.627	Hydrocarbons	1-Tetradecene	2.07±0.12
28	1456	26.964	Alcohols	1-Octen-3-ol	5.06±0.34
29	1468	27.310	Alcohols	1-Heptanol	2.79±0.16
30	1528	28.719	Aldehydes	Benzaldehyde	7.92±0.74
31		29.091	Hydrocarbons	Pentadecane	10.79±0.99
32	1546	29.754	Hydrocarbons	β-Cubebene	2.27±0.02
33		30.532	Hydrocarbons	1-Pentadecene	7.55±0.50
34		32.375	Ketones	3-Acetyl-2,6-heptanedione	2.84±0.18
35	1632	32.752	Aldehydes	β-Cyclocitral	2.04±0.75
36		32.858	Aldehydes	Decanal	1.61±0.06
38	1652	34.436	Ketones	Acetophenone	1.42±0.12
39		35.554	Hydrocarbons	Heptadecane	50.45±0.79
40	1789	37.490	Aldehydes	4-(1-methylethyl)-Benzaldehyde	2.46±0.08
41	1832	39.650	Aromatic Hydrocarbons	4-isopropyl-1,6-dimethyl-1,2,3,4-tetrahydronaphthalene	3.67±0.23
42	1954	42.732	Ketones	E)-β-Ionone	2.35±0.31
43	1987	43.753	Phenol	Phenol	1.80±0.21
44		46.043	Ketones	Hexahydrofornesylacetone (6,10,14-trimethyl-2-Pentadecanone)	1.98±0.03
45	2200	47.037	Aromatic Hydrocarbons	Cadalene	1.47±0.03
46	2354	48.745	Esters	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-	5.97±0.15
Total (mg/L)					167.09±16.79

4. Conclusions

This study is the first report for characterization of L. papillosa exceedingly: determination of amino acid profile, ultrasound assisted extraction, sugar content, biological activites, quantification of polyphenols and volatile organic compounds of extracts through LC-MS/MS and GC/MS system. Two methods of extraction were employed, it was chosen UAE parameters by evaluating the total phenolic content and antioxidant capacities. The optimised paramters of UAE (10 %A and 15 min) was emerged to result in significantly higher antioxidant activities (DPPH and ABTS) (p < 0.05), and significantly higher total phenolic compound (p < 0.05) than CE. This could be ascribed to the existence of unique phenolic compounds that are including phlorotannins, flavonoids, phenolic acids and other polyphenols with characterized by LC-MS/MS. The all phenolic compounds in U@10A_15 extract have more amount than CE, except p-hydroxybenzaldehyde. One of the major compounds was phloroglucinol that in U@10A_15 and CE is 318.86±1.59 and 199.94±1.63, respectively. Moreover, the essential amino acids, HIS, THR, VAL, TRP, PHE, ISO and LEU were detected. Tryptophan was presented highest EAA. Fructose was quantified as the predominant sugar in both extracts. Other important seconder metabolites, volatile organic compounds were explored by carrying out quantification and identification by GC–MS/MS in L. papillosa. Heptadecane is highest concentration of VoCs. In accordance with the results, it could be committed that utilization of UAE in macroalgae extraction appears to be effective extraction method due to energy and time saving in addition supporting the sustainability. In the future, as we believe the results of the present study to be of tremendous value to the functional foods, active food packaging and nutraceuticals industries due to prefer ultrasound method, utilization of macroalgae and detection of bioactive compounds. It could be suggested as model for contribution of circular economy due to as alternative sources about the different macroalgae metabolites and their possible industrial applications.

CRediT authorship contribution statement

Gulden Goksen: Conceptualization, Methodology, Formal analysis, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

No data was used for the research described in the article.