TY - JOUR
AU - Apak, Reşat
AB - Abstract This study was carried out to determine the main pigments in some different selected seaweeds and to reveal their antioxidant potential regarding the ever-increasing demand for utilization of marine pigments in human health and nutrition. The individual amounts of algal pigments were found by reverse phase high-performance liquid chromatography (HPLC) and their total antioxidant capacities (TAC) by two spectrophotometric TAC assays, namely: CUPRAC (CUPric ion Reducing Antioxidant Capacity) and ABTS/TEAC (2,2′-azinobis [3-ethyl benzo thiazoline-6-sulfonate])/(trolox equivalent antioxidant capacity). These two tests gave the same rank order for TAC. The TAC of HPLC-quantified compounds accounted for a relatively much lower percentage of the observed CUPRAC capacities of seaweed extracts, namely ranging from 11 to 68% for brown, from 4 to 41% for red and from 3 to 100% for green species, i.e., some TAC originated from chromatographically unidentified compounds. Fucoxanthin, chlorophyll a, and pheophytin a compounds were major pigments in brown algae. The relative carotenoid contents in red marine algae were generally lower than those of chlorophylls. Overall total quantities were quite low compared with those of brown species. In general, chlorophyll a and chlorophyll b were chiefly present in green algae, but β-carotene, violaxanthin and siphonaxanthin were also detected substantially higher in some species of green algae such as Caulerpa racemosa var. cylindracea and Codium fragile. Introduction Marine algae commonly known as seaweeds are photosynthetic organisms like plants and they are generally classified into three groups according to their color: brown algae (Phaeophyceae), red algae (Rhodophyceae) and green algae (Chlorophyceae). They are found in branched or unbranched forms, ranging from microscopic scale that are structurally prokaryotic (simple structured cell type) to a few meters in length that are eukaryotic (advanced cell type) plants (1, 2). They grow up in all aquatic areas, from warm tropics to cold and icy polar regions (oceans, rivers, freshwater lakes, streams, pole lakes, etc.). Seaweeds and seaweed-based products, some of which are edible, have recently gained great importance as an alternative source of bioactive compounds having mainly antioxidant properties such as polyphenols, proteins, fatty acids, minerals, vitamins and pigments (3). Due to their richness of bioactive ingredients, marine algae are also well known to exhibit anti-inflammatory, anticoagulant, antimicrobial, antiviral, antifungal and antitumor activities in addition to their antioxidant effects (4). Most of the marine macroalgae are green in color because of chlorophyll a. However, many common types of algae appear to be brown or red as the color of chlorophylls is masked by carotenoids or phycobilins (5). Carotenoids, largely due to their superior antioxidant properties, play a key role in the protection of plants against photooxidative processes with the aid of their scavenging effects of singlet oxygen and peroxyl radicals (6). Carotenoids can be divided into two groups based on their chemical structure: (i) carotenes that contain only carbon and hydrogen and (ii) xanthophylls that contain carbon, hydrogen and additional oxygen. They can also be classified as primary or secondary depending on their function; primary carotenoids (β-carotene, violaxanthin and neoxanthin) are responsible for transmitting light energy absorbed from sunlight by chlorophyll (photosynthesis), whereas secondary ones (α-carotene, β-cryptoxanthin, zeaxanthin, antheraxanthin) do not exhibit any function directly during photosynthesis (7, 8). Although zeaxanthin and antheraxanthin are classified as secondary carotenoids, they are crucial pigments contributing to maintaining the xanthophyll cycle in many plants including algae. Typical products of the protective xanthophyll cycle are lutein and zeaxanthin that are synthesized from β-carotene by hydroxylation and epoxidation reactions (9). Carotenoids in algae serve mainly as photoprotective agents and secondarily as light-converting pigments through photosynthetic pathways against photodamage. Furthermore, the relative composition of photosynthetic and photoprotective pigments in marine algae can be indicative of providing a significant taxonomical and physiological information for algae classification. Thus, pigment distribution can demonstrate taxonomic composition, and the presence or absence of specific marker pigments can be used to determine the seaweed profile. For example, brown marine algae have been reported as the main source of fucoxanthin pigment, which is responsible for the color of brown seaweeds. Fucoxanthin, commonly known as a marine xanthophyll, is significantly effective in the treatment of many different cancer cells (10). It has photoprotective features in human fibroblast cells via inhibition of oxidative DNA damage and thus increases the antioxidant activity (11). The main carotenoids in red marine algae have been noted as β-carotene, α-carotene and their dihydroxy derivatives zeaxanthin and lutein, respectively. As for green algae, their carotenoid profile is similar to that of higher plants; β-carotene, lutein, violaxanthin, antheraxanthin, zeaxanthin and neoxanthin have been described as major carotenoids (12). On the other hand, there are widely incompatible reports in the literature about carotenoid content among red marine algae, particularly in the studies of Schubert et al. (13) and Terasaki et al. (14). Thus, it can be concluded that there is no complete correlation between the carotenoid content and the taxonomic classification of marine algae species. Chlorophylls and metal-free chlorophyll derivatives (pheophytins) are generally defined as cyclic tetrapyrroles that are functional in photon harvesting or responsible for separation in photosynthesis. The main pigments in green marine algae and higher plants are chlorophyll a and b. Red marine algae are known to contain chlorophyll a as the dominant pigment, whereas brown ones can additionally have chlorophyll c and pheophytin a particularly. It has been reported that chlorophylls and pheophytins possess antioxidant and antimutagenic activity that can break radical chain reactions in the prevention of autoxidation of edible oils during storage (15). Today, there is an increasing number of studies to reveal the antioxidant capacity of seaweeds (16–20). ABTS/TEAC (21), FRAP (ferric-reducing antioxidant power) (22) and DPPH (2,2-diphenyl-1-picrylhydrazyl) are among the most common spectrophotometric methods and reagents applied in the literature (23–26). In this study, the CUPRAC assay that was developed by Apak et al. (27) has been used for the first time for the determination of total antioxidant capacity (TAC) of different seaweed varieties. The CUPRAC assay is simple and diversely applicable to both hydrophilic and lipophilic antioxidants, and its reagents are stable and easily available at low cost. Besides, it responds favorably to small-molecule thiols and antioxidant proteins in algae. The chromogenic oxidizing reagent of the CUPRAC assay, bis (neocuproine: 2,9-dimethyl-1,10-phenanthroline) copper(II), was successfully applied for the determination of TACs of some plant foods derived from carotenoids and chlorophylls pigments previously (28). The TACs of seaweeds presented to this study were comparatively assayed with the CUPRAC and the widely used ABTS (21) methods. Some research groups dealing with antioxidant chemistry have either used or evaluated the CUPRAC method. For example, Prior et al. (29) classified CUPRAC as one of the electron transfer-based methods and summarized the superiorities of the CUPRAC method over other antioxidant assays. They indicated that because of the lower redox potential of the CUPRAC reagent, sugars and citric acid—which were not real antioxidants but oxidizable substrates in other similar assays—were not oxidized with the CUPRAC reagent, meaning that it selectively responds to antioxidant compounds. There are several studies conventionally carried out by high-performance liquid chromatography (HPLC) to specify and particularly quantify the pigment compounds (14, 30–32). Thus, the individual contribution of each pigment constituent to TAC can be determined. This work has been intended primarily to develop a simple, rapid and reliable HPLC method for the identification and quantification of carotenoids and chlorophylls. In the presented study, the theoretical TAC of various widespread marine algae was measured by HPLC involving the determination of their constituents, and the theoretical TAC values were calculated by spectrophotometric CUPRAC and ABTS assays. An additional target is to emphasize the potential of seaweeds due mainly to their manurial value or their micronutrient suites as a renewable bioresource in sustainable agricultural systems and animal feed industry. Experimental Seaweed samples The seaweeds in the full-grown period were collected from Çanakkale located on the western coast and Antalya on the southern coast in Turkey. The name of collected seaweed samples and their abbreviations: Cladostephus spongiosum f. verticillatum: C. spongiosum; Cystoseira foeniculacea: C. foeniculacea, Dictyota dichotoma: D. dichotoma; Stypopodium schimperi: S. schimperi; Colpomenia sinuosa: C. sinuosa; Petalonia fascia: P. fascia; Hypnea musciformis: H. musciformis; Jania rubens: J. rubens, Polysiphonia scopulorum: P. scopulorum, Caulerpa taxifolia var. distichophylla: C. taxifolia; Ulva rigida: U. rigida; Caulerpa racemosa var. cylindracea: C. racemosa, Codium fragile: C. fragile. The description of the seaweeds collection process was also listed in Table I. Also, the availability of algae and their potential cultivation were presented. The macroscopic epiphytes and other impurities on the collected seaweeds were removed by rinsing and brushing. Then the seaweeds were immersed in containers with liquid nitrogen. After transferring to the laboratory, they were lyophilized, ground and stored in polypropylene bottles in the dark at room temperature until needed. The identification of the materials was done with Olympus brand SZX16 stereo zoom and BX51 model binocular light microscopes. Table I The List of Seaweeds Collected From Brown, Red and Green Algae Groups and General Conditions of Supply Seaweed . Location . Latitude/longitude . Depth (m) . Date . Availability . Brown (Phaeophyceae) Cladostephus spongiosum f. verticillatum (Lightfoot) Prud'homme van Reine 1972 Çanakkale 40°13′6.47"N 26°31′27.30"E 0–1 19 July 2016 Can be easily cultureda Cystoseira foeniculacea (Linnaeus) Greville 1830 Antalya 36°49′59.84"N 31°7′28.67″E 1–2 13 August 2016 Can be easily cultured Dictyota dichotoma (Hudson) J.V. Lamouroux 1809 Antalya 36°53′1.14″N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Stypopodium schimperi (Kützing) M. Verlaque & Boudouresque 1991 Antalya 36°53′1.14"N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier in Castagne 1851 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Petalonia fascia (O.F. Müller) Kuntze 1898 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Red (Rhodophyceae) Hypnea musciformis (Wulfen) J.V. Lamouroux 1813 Çanakkale 40°13′6.47″N 26°31′27.30″E 0–1 19 July 2016 Found in abundance/can be easily cultured Jania rubens (Linnaeus) J.V. Lamouroux 1816 Antalya 36°49′59.84″N 31°7′28.67″E 1–2 6 September 2016 Can be easily cultured Polysiphonia scopulorum Harvey Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Green (Chlorophyceae) Caulerpa taxifolia var. distichophylla (Sonder) Verlaque, Huisman & Procaccini in Jongma et al. 2013 Antalya 36°27′36.42″N 30°33′0.14″E 15–20 10 September 2016 Can be easily cultured Ulva rigida C. Agardh 1823 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman & Boudouresque 2003 Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Codium fragile (Suringar) Hariot 1889 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured Seaweed . Location . Latitude/longitude . Depth (m) . Date . Availability . Brown (Phaeophyceae) Cladostephus spongiosum f. verticillatum (Lightfoot) Prud'homme van Reine 1972 Çanakkale 40°13′6.47"N 26°31′27.30"E 0–1 19 July 2016 Can be easily cultureda Cystoseira foeniculacea (Linnaeus) Greville 1830 Antalya 36°49′59.84"N 31°7′28.67″E 1–2 13 August 2016 Can be easily cultured Dictyota dichotoma (Hudson) J.V. Lamouroux 1809 Antalya 36°53′1.14″N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Stypopodium schimperi (Kützing) M. Verlaque & Boudouresque 1991 Antalya 36°53′1.14"N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier in Castagne 1851 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Petalonia fascia (O.F. Müller) Kuntze 1898 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Red (Rhodophyceae) Hypnea musciformis (Wulfen) J.V. Lamouroux 1813 Çanakkale 40°13′6.47″N 26°31′27.30″E 0–1 19 July 2016 Found in abundance/can be easily cultured Jania rubens (Linnaeus) J.V. Lamouroux 1816 Antalya 36°49′59.84″N 31°7′28.67″E 1–2 6 September 2016 Can be easily cultured Polysiphonia scopulorum Harvey Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Green (Chlorophyceae) Caulerpa taxifolia var. distichophylla (Sonder) Verlaque, Huisman & Procaccini in Jongma et al. 2013 Antalya 36°27′36.42″N 30°33′0.14″E 15–20 10 September 2016 Can be easily cultured Ulva rigida C. Agardh 1823 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman & Boudouresque 2003 Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Codium fragile (Suringar) Hariot 1889 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured aA culture can be defined as an artificial environment in which the algae grow. Open in new tab Table I The List of Seaweeds Collected From Brown, Red and Green Algae Groups and General Conditions of Supply Seaweed . Location . Latitude/longitude . Depth (m) . Date . Availability . Brown (Phaeophyceae) Cladostephus spongiosum f. verticillatum (Lightfoot) Prud'homme van Reine 1972 Çanakkale 40°13′6.47"N 26°31′27.30"E 0–1 19 July 2016 Can be easily cultureda Cystoseira foeniculacea (Linnaeus) Greville 1830 Antalya 36°49′59.84"N 31°7′28.67″E 1–2 13 August 2016 Can be easily cultured Dictyota dichotoma (Hudson) J.V. Lamouroux 1809 Antalya 36°53′1.14″N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Stypopodium schimperi (Kützing) M. Verlaque & Boudouresque 1991 Antalya 36°53′1.14"N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier in Castagne 1851 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Petalonia fascia (O.F. Müller) Kuntze 1898 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Red (Rhodophyceae) Hypnea musciformis (Wulfen) J.V. Lamouroux 1813 Çanakkale 40°13′6.47″N 26°31′27.30″E 0–1 19 July 2016 Found in abundance/can be easily cultured Jania rubens (Linnaeus) J.V. Lamouroux 1816 Antalya 36°49′59.84″N 31°7′28.67″E 1–2 6 September 2016 Can be easily cultured Polysiphonia scopulorum Harvey Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Green (Chlorophyceae) Caulerpa taxifolia var. distichophylla (Sonder) Verlaque, Huisman & Procaccini in Jongma et al. 2013 Antalya 36°27′36.42″N 30°33′0.14″E 15–20 10 September 2016 Can be easily cultured Ulva rigida C. Agardh 1823 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman & Boudouresque 2003 Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Codium fragile (Suringar) Hariot 1889 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured Seaweed . Location . Latitude/longitude . Depth (m) . Date . Availability . Brown (Phaeophyceae) Cladostephus spongiosum f. verticillatum (Lightfoot) Prud'homme van Reine 1972 Çanakkale 40°13′6.47"N 26°31′27.30"E 0–1 19 July 2016 Can be easily cultureda Cystoseira foeniculacea (Linnaeus) Greville 1830 Antalya 36°49′59.84"N 31°7′28.67″E 1–2 13 August 2016 Can be easily cultured Dictyota dichotoma (Hudson) J.V. Lamouroux 1809 Antalya 36°53′1.14″N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Stypopodium schimperi (Kützing) M. Verlaque & Boudouresque 1991 Antalya 36°53′1.14"N 30°41′59.89″E 18–20 8 September 2016 Found in abundance/can be easily cultured Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier in Castagne 1851 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Petalonia fascia (O.F. Müller) Kuntze 1898 Çanakkale 40°6′43.19″N 26°24′8.20″E 0–1 27 February 2017 Can be easily cultured Red (Rhodophyceae) Hypnea musciformis (Wulfen) J.V. Lamouroux 1813 Çanakkale 40°13′6.47″N 26°31′27.30″E 0–1 19 July 2016 Found in abundance/can be easily cultured Jania rubens (Linnaeus) J.V. Lamouroux 1816 Antalya 36°49′59.84″N 31°7′28.67″E 1–2 6 September 2016 Can be easily cultured Polysiphonia scopulorum Harvey Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Green (Chlorophyceae) Caulerpa taxifolia var. distichophylla (Sonder) Verlaque, Huisman & Procaccini in Jongma et al. 2013 Antalya 36°27′36.42″N 30°33′0.14″E 15–20 10 September 2016 Can be easily cultured Ulva rigida C. Agardh 1823 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman & Boudouresque 2003 Çanakkale 40°4′26.86″N 26°21′28.08″E 22 25 August 2017 Can be easily cultured Codium fragile (Suringar) Hariot 1889 Çanakkale 40°4′26.86″N 26°21′28.08″E 0–1 25 August 2017 Found in abundance/can be easily cultured aA culture can be defined as an artificial environment in which the algae grow. Open in new tab Chemicals and standards β-Carotene, α-carotene, lutein, zeaxanthin, fucoxanthin, violaxanthin, astaxanthin, triethylamine (TEA), potassium persulfate, neocuproine (2,9-dimethyl-1,10-phenanthroline), ammonium acetate, (Sigma-Aldrich), ABTS (2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonate]) chlorophyll a, chlorophyll b (Sigma), CuCl2·2H2O (copper (II) chloride dihydrate), hydrogen peroxide (Merck), HPLC-grade solvents (acetone, methanol [MeOH], ethanol [EtOH], acetonitrile [ACN]) (Riedel-de Haën) were supplied from the indicated sources. Instrumentation A Metrohm Herisau E512 pH meter was used for pH measurements (Herisau, Switzerland). The extraction operations were made using a Bransonic 221 ultrasonic bath, an HB4 basic KIKA-WERKE water bath, and an Elektro-mag vortex mixer (Shelton, USA). Seaweed samples were lyophilized using a Telstar Cryodos freeze dryer (Terrassa, Spain). Spectrophotometric measurements were made with a Cary 1E UV–Vis spectrophotometer (Varian Instruments) (Sydney, Australia), and chromatographic separation and identification of pigment constituents were performed using a Perkin Elmer HPLC system (comprised of series 200 UV–Vis detector, binary gradient pump and vacuum degasser) (Massachusetts, USA). Pure distilled water was used throughout, as obtained from Millipore Simpak1 Synergy 185 ultra-pure water system. Sample preparation The extraction procedure for carotenoids and chlorophylls was applied as follows: (i) 2 g of the lyophilized sample powder of seaweeds was weighed in flasks and (ii) extracted for 10 min with 5 mL of acetone in an ultrasonic bath at room temperature protected from light; (iii) the upper phase was decanted and (ii) and (iii) were repeated five times. The extracts were combined and completed to 25 mL with acetone and centrifuged at 4000 rpm for 5 min; the supernatants were filtered with glass fiber/polyethylene terephthalate 1.0/0.45-μm microfilters. The extracts were generally used freshly but, if necessary, were stored at −20°C and checked for their absorbances at maximum absorption wavelength before use (28). Reagents and solutions β-Carotene (β-car), α-carotene (α-car), lutein (Lt), zeaxanthin (Zx), fucoxanthin (Fx), violaxanthin (Viox), neoxanthin (Neox), astaxanthin (Astx), chlorophyll a (Chl a) and chlorophyll b (Chl b) solutions were prepared as stock solutions in HPLC-grade acetone and were used freshly; if necessary, they were stored at −20°C for <1 week and checked by absorbance at maximum absorption wavelength before use (28). For the CUPRAC test of TAC, the following solutions were prepared: CuCl2 solution, 1.0 × 10−2 M, prepared by dissolving CuCl2 2H2O in water; ammonium acetate (NH4Ac) buffer at pH 7.0, 1.0 M, prepared from NH4Ac in water; neocuproine (Nc) solution, 7.5 × 10−3 M, prepared daily by dissolving Nc in 96% EtOH (27). For the ABTS test of TAC determination, the chromogenic radical reagent ABTS, at 7.0 mM concentration, was prepared by dissolving this compound in water and adding K2S2O8 to this solution such that the final persulfate concentration in the mixture to be 2.45 mM. The resulting ABTS radical cation solution was left to mature at room temperature in the dark for 12–16 h and then used for ABTS/TEAC assays. The reagent solution was diluted with acetone at a volume ratio of 1:10 before use. Methods used in antioxidant assays Spectrophotometric methods CUPRAC method The CUPRAC method, as described above (27), was applied as follows: a mixture comprised of 1 mL of 1.0 × 10−2 M copper(II) chloride, 1 mL of 1 M ammonium acetate buffer at pH 7.0 and 1 mL of 7.5 × 10−3 M neocuproine solution was prepared, x mL sample solution and (1.0 − x) mL acetone were added, and well mixed (total volume: 4.0 mL). This final mixture in a stoppered test tube was let to stand at room temperature for 30 min. At the end of this period, the absorbance at 450 nm was measured against a reagent blank. This method was applied to the pigment standards and seaweed extracts. ABTS method The ABTS/persulfate method (21) was followed. Briefly, 1 mL acetone was added to 3 mL of the radical cation (ABTS•+) solution produced by the reaction between ABTS and potassium persulfate, and the absorbances were recorded at 734 nm at the end of the sixth minute (i.e., this optimal time was experimentally determined for β-carotene and other pigments). The procedure was repeated for the antioxidant pigment by adding 3 mL of the radical cation solution to x mL of the sample solution and (1.0 − x) mL of acetone and recording the absorbance readings at the fifth minute. The absorbance of the reagent blank (A0) diminished in the presence of antioxidant pigment, the absorbance decrease (ΔA) being proportional to antioxidant pigment concentration. Linear regression equations were obtained using both trolox and β-carotene as ΔA versus molar concentration. Chromatographic analysis The analyses were performed using a Waters YMC C30 (250 mm × 4.6 mm, 5-μm particle size) chromatographic column. Detection was carried out within the wavelength range of 200–800 nm with a photodiode array (PDA) detector. The elution rate was 1.5 mL min−1, and the column temperature was 35°C. For pigment compounds, two different solutions of the mobile phase, i.e., MeOH/ACN (50:50, v/v) with 0.1% (v/v) TEA (A) and acetone (B), were used in gradient elution. The following working mode was adopted for gradient elution (28): 15 min 100% A (slope 0); 10 min from 100% to 30% A (slope, 4); 15 min from 30% to 0% A (slope, 4) (using Empower 2 Software, Waters Corporation). Using the above working mode, the calibration curves and linear equations of peak area versus concentration were determined for the pigment antioxidants of interest with three repetitive injections. Thus, they were quantified from their peak areas of the respective reference standards. Each peak was identified by HPLC retention times, absorption spectra of the standards and the data from PDA detection. To confirm quantitative identification, the change in the peak areas of certain chromatographic bands with fixed retention times was followed with the technique of standard additions. A coelution of the standard pigment with a sample was done to assay more precisely, if necessary (28). With the aid of these calibration curves, plant extracts in acetone and synthetic mixtures of pigments were analyzed. Qualitative and quantitative analyses of sample solutions were conducted with at least three injections. Injection volume was set at 20 μL. The chromatographic column was washed for 10 min with acetone before each injection and equilibrated for 10 min with a solvent mixture containing (A). Statistical analysis Spectrophotometric measurements were performed by triplicate for each sample and standard. The results of statistical analyses were obtained using Excel software (Microsoft Office 2010) for determining the means and the standard error of the mean. Results were presented as mean values ± standard deviation. HPLC data were obtained as the average of three values for each standard and the mixture of standard. Results Spectrophotometric results of the tested carotenoid and chlorophyll standards The TAC values derived from carotenoid and chlorophyll pigments expected to be present in the tested seaweeds was calculated as trolox equivalents and for this purpose, the linear calibration equations of trolox were calculated as y = 1.634 × 104 c + 0.0011 (R2 = 0.9992) for CUPRAC method and as y = 1.473 × 104c−0.0605 (R2 = 0.9998) for ABTS/persulfate spectrophotometric reference method. The absorbance values measured for carotenoid and chlorophyll pigments were recorded against their tested concentrations (absorbance measurements were realized in quartz cuvettes having 1-cm light path), and linear calibration equations were established between concentration and absorbance values. The molar absorption coefficients (ε) (i.e., the slope of the calibration line) and linear working ranges conforming to Beer’s law were given in Table II. The TAC values were expressed as trolox and β-carotene equivalents (trolox and β-carotene antioxidant capacity equivalents were given as TEAC and CEAC, respectively). To find these values, the molar absorptivity coefficient of the tested pigment was divided by that of trolox and β-carotene under the same conditions (TEACCUPRAC; εPigment/εTR) and (CEACCUPRAC; εPigment/εβ-carotene); β-carotene was chosen to evaluate the TAC of seaweed samples since it is one the most common carotenoid and often considered the general representative of pigments. Table II Linear Regression Data and the TEAC Coefficients of the Tested Pigments With Respect to the Spectrophotometric CUPRAC Method Pigment/abbreviation . Calibration equationsa . R 2 . Linear range (mol/L) . β-CEACCUPRAC (εPigment/εβ-carotene) . TEACCUPRAC (εPigment/εTrolox) . Fx y = 0.35 × 105 c − 0.035 0.997 3.85 × 10−6 − 2.96 × 10−5 0.35/0.52 = 0.67 2.19 Viox y = 0.14 × 105 c + 0.0001 0.998 6.88 × 10−6 − 6.86 × 10−5 0.14/0.52 = 0.27 0.88 Neox y = 0.25 × 105 c + 0.003 0.998 3.90 × 10−6 − 4.01 × 10−5 0.25/0.52 = 0.47 1.56 Astx y = 0.34 × 105 c − 0.05 0.997 3.00 × 10−6 − 3.00 × 10−5 0.34/0.52 = 0.65 2.13 Lt y = 0.77 × 105 c − 0.01 0.9996 1.40 × 10−6 − 1.25 × 10−5 0.77/0.52 = 1.48 4.81 Zx y = 0.33 × 105 c − 0.05 0.999 3.20 × 10−6 − 3.00 × 10−5 0.33/0.52 = 0.63 2.06 α-car y = 0.81 × 105 c − 0.04 0.999 1.25 × 10−6 − 1.20 × 10−5 0.81/0.52 = 1.55 5.06 β-car y = 0.52 × 105 c − 0.01 0.9992 2.50 × 10−6 − 2.00 × 10−5 0.52/0.52 = 1.00 3.25 Chl a y = 1.20 × 105 c − 0.01 0.9991 0.90 × 10−6 − 0.80 × 10−5 1.2/0.52 = 2.30 7.5 Chl b y = 1.04 × 105 c − 0.01 0.998 1.00 × 10−6 − 0.90 × 10−6 1.04/0.52 = 2.00 6.5 Pigment/abbreviation . Calibration equationsa . R 2 . Linear range (mol/L) . β-CEACCUPRAC (εPigment/εβ-carotene) . TEACCUPRAC (εPigment/εTrolox) . Fx y = 0.35 × 105 c − 0.035 0.997 3.85 × 10−6 − 2.96 × 10−5 0.35/0.52 = 0.67 2.19 Viox y = 0.14 × 105 c + 0.0001 0.998 6.88 × 10−6 − 6.86 × 10−5 0.14/0.52 = 0.27 0.88 Neox y = 0.25 × 105 c + 0.003 0.998 3.90 × 10−6 − 4.01 × 10−5 0.25/0.52 = 0.47 1.56 Astx y = 0.34 × 105 c − 0.05 0.997 3.00 × 10−6 − 3.00 × 10−5 0.34/0.52 = 0.65 2.13 Lt y = 0.77 × 105 c − 0.01 0.9996 1.40 × 10−6 − 1.25 × 10−5 0.77/0.52 = 1.48 4.81 Zx y = 0.33 × 105 c − 0.05 0.999 3.20 × 10−6 − 3.00 × 10−5 0.33/0.52 = 0.63 2.06 α-car y = 0.81 × 105 c − 0.04 0.999 1.25 × 10−6 − 1.20 × 10−5 0.81/0.52 = 1.55 5.06 β-car y = 0.52 × 105 c − 0.01 0.9992 2.50 × 10−6 − 2.00 × 10−5 0.52/0.52 = 1.00 3.25 Chl a y = 1.20 × 105 c − 0.01 0.9991 0.90 × 10−6 − 0.80 × 10−5 1.2/0.52 = 2.30 7.5 Chl b y = 1.04 × 105 c − 0.01 0.998 1.00 × 10−6 − 0.90 × 10−6 1.04/0.52 = 2.00 6.5 ay = stands for absorbance, c = molar concentration, R2 = determination coefficient, εTrolox = 0.16 × 105. Open in new tab Table II Linear Regression Data and the TEAC Coefficients of the Tested Pigments With Respect to the Spectrophotometric CUPRAC Method Pigment/abbreviation . Calibration equationsa . R 2 . Linear range (mol/L) . β-CEACCUPRAC (εPigment/εβ-carotene) . TEACCUPRAC (εPigment/εTrolox) . Fx y = 0.35 × 105 c − 0.035 0.997 3.85 × 10−6 − 2.96 × 10−5 0.35/0.52 = 0.67 2.19 Viox y = 0.14 × 105 c + 0.0001 0.998 6.88 × 10−6 − 6.86 × 10−5 0.14/0.52 = 0.27 0.88 Neox y = 0.25 × 105 c + 0.003 0.998 3.90 × 10−6 − 4.01 × 10−5 0.25/0.52 = 0.47 1.56 Astx y = 0.34 × 105 c − 0.05 0.997 3.00 × 10−6 − 3.00 × 10−5 0.34/0.52 = 0.65 2.13 Lt y = 0.77 × 105 c − 0.01 0.9996 1.40 × 10−6 − 1.25 × 10−5 0.77/0.52 = 1.48 4.81 Zx y = 0.33 × 105 c − 0.05 0.999 3.20 × 10−6 − 3.00 × 10−5 0.33/0.52 = 0.63 2.06 α-car y = 0.81 × 105 c − 0.04 0.999 1.25 × 10−6 − 1.20 × 10−5 0.81/0.52 = 1.55 5.06 β-car y = 0.52 × 105 c − 0.01 0.9992 2.50 × 10−6 − 2.00 × 10−5 0.52/0.52 = 1.00 3.25 Chl a y = 1.20 × 105 c − 0.01 0.9991 0.90 × 10−6 − 0.80 × 10−5 1.2/0.52 = 2.30 7.5 Chl b y = 1.04 × 105 c − 0.01 0.998 1.00 × 10−6 − 0.90 × 10−6 1.04/0.52 = 2.00 6.5 Pigment/abbreviation . Calibration equationsa . R 2 . Linear range (mol/L) . β-CEACCUPRAC (εPigment/εβ-carotene) . TEACCUPRAC (εPigment/εTrolox) . Fx y = 0.35 × 105 c − 0.035 0.997 3.85 × 10−6 − 2.96 × 10−5 0.35/0.52 = 0.67 2.19 Viox y = 0.14 × 105 c + 0.0001 0.998 6.88 × 10−6 − 6.86 × 10−5 0.14/0.52 = 0.27 0.88 Neox y = 0.25 × 105 c + 0.003 0.998 3.90 × 10−6 − 4.01 × 10−5 0.25/0.52 = 0.47 1.56 Astx y = 0.34 × 105 c − 0.05 0.997 3.00 × 10−6 − 3.00 × 10−5 0.34/0.52 = 0.65 2.13 Lt y = 0.77 × 105 c − 0.01 0.9996 1.40 × 10−6 − 1.25 × 10−5 0.77/0.52 = 1.48 4.81 Zx y = 0.33 × 105 c − 0.05 0.999 3.20 × 10−6 − 3.00 × 10−5 0.33/0.52 = 0.63 2.06 α-car y = 0.81 × 105 c − 0.04 0.999 1.25 × 10−6 − 1.20 × 10−5 0.81/0.52 = 1.55 5.06 β-car y = 0.52 × 105 c − 0.01 0.9992 2.50 × 10−6 − 2.00 × 10−5 0.52/0.52 = 1.00 3.25 Chl a y = 1.20 × 105 c − 0.01 0.9991 0.90 × 10−6 − 0.80 × 10−5 1.2/0.52 = 2.30 7.5 Chl b y = 1.04 × 105 c − 0.01 0.998 1.00 × 10−6 − 0.90 × 10−6 1.04/0.52 = 2.00 6.5 ay = stands for absorbance, c = molar concentration, R2 = determination coefficient, εTrolox = 0.16 × 105. Open in new tab HPLC results of the tested carotenoid and chlorophyll standards The analysis of pigments was performed by reverse phase high-performance liquid chromatography (RP-HPLC). The linear calibration graphs were drawn between concentration and chromatographic peak area and the calculated equations and R2 values were shown in Table III. The maximum absorption wavelengths (λmax) were determined by PDA spectrum of pigment standards and these values were also compared with literature findings. These experimental (Found λmax) and theoretical (Reported λmax) value and retention times were shown in Table III. Table III Linear Equations, Retention Times and Maximum Absorbance Wavelengths of Carotenoid (450 nm) and Chlorophyll Pigments (650 nm) Obtained by HPLC Pigment . RT(min) . Found λmax (nm) . Regression equations . R 2 . Reported λmax (nm) . References . Fx 8.29 448.9 y = 1 × 1011 c − 96319 0.999 447; 449 (30, 33) Viox 10.38 441.6, 472.0 y = 5 × 1010 c + 95665 0.992 440, 469; 441, 470 (30, 34) Neox 12.63 438.0, 467.1 y = 2 × 1011 c − 49600 0.999 438, 467; 440, 468 (32, 35) Astx 18.79 478.9 y = 2 × 1011 c − 68542 0.999 478 (32) Lt 19.50 447.7, 475.7 y = 2 × 1011 c − 78808 0.999 446, 474; 446, 476 (32, 36) Zx 20.95 453.8, 480.5 y = 2 × 1011 c − 48856 0.999 452, 480; 450, 476 (34, 35) α-car 26.17 448.9, 476.9 y = 2 × 1011 c − 27839 0.999 446, 475; 448, 476 (32, 35) β-car 28.19 455.0, 481.7 y = 4 × 109 c − 12478 1 455, 482; 453, 480 (30, 36) Chl a 21.80 430.7, 618.7, 665.4 y = 3 × 1010 c + 5671 0.999 431, 618, 665; 431, 618, 666 (34, 36) Chl b 19.43 465.9, 649.4 y = 5 × 1010 c + 57380 0.998 461, 649; 468, 652 (32, 35) Chl ca 4–5 447.7, 629.8 y = 1 × 1011 c + 96319 0.999 445, 633; 445, 629 (30, 34) Php aa 26–30 408.9, 503.7–506.1, 666.6–667.8 y = 3 × 1010 c + 5671 0.999 409, 505, 665; 408, 504, 666 (34, 36) Spxa 7.4 451 y = 1 × 1011 c − 96319 0.999 445; 463 (36, 37) Pigment . RT(min) . Found λmax (nm) . Regression equations . R 2 . Reported λmax (nm) . References . Fx 8.29 448.9 y = 1 × 1011 c − 96319 0.999 447; 449 (30, 33) Viox 10.38 441.6, 472.0 y = 5 × 1010 c + 95665 0.992 440, 469; 441, 470 (30, 34) Neox 12.63 438.0, 467.1 y = 2 × 1011 c − 49600 0.999 438, 467; 440, 468 (32, 35) Astx 18.79 478.9 y = 2 × 1011 c − 68542 0.999 478 (32) Lt 19.50 447.7, 475.7 y = 2 × 1011 c − 78808 0.999 446, 474; 446, 476 (32, 36) Zx 20.95 453.8, 480.5 y = 2 × 1011 c − 48856 0.999 452, 480; 450, 476 (34, 35) α-car 26.17 448.9, 476.9 y = 2 × 1011 c − 27839 0.999 446, 475; 448, 476 (32, 35) β-car 28.19 455.0, 481.7 y = 4 × 109 c − 12478 1 455, 482; 453, 480 (30, 36) Chl a 21.80 430.7, 618.7, 665.4 y = 3 × 1010 c + 5671 0.999 431, 618, 665; 431, 618, 666 (34, 36) Chl b 19.43 465.9, 649.4 y = 5 × 1010 c + 57380 0.998 461, 649; 468, 652 (32, 35) Chl ca 4–5 447.7, 629.8 y = 1 × 1011 c + 96319 0.999 445, 633; 445, 629 (30, 34) Php aa 26–30 408.9, 503.7–506.1, 666.6–667.8 y = 3 × 1010 c + 5671 0.999 409, 505, 665; 408, 504, 666 (34, 36) Spxa 7.4 451 y = 1 × 1011 c − 96319 0.999 445; 463 (36, 37) The data were measured with repeated injections (n = 3) of a mixture of pigment standards at a concentration of 10−5–10−6 M each. In the calibration equation, c represents concentration of the analyte (pigment) and y represents the peak area. aThe existence of these components was determined based on the literature. Open in new tab Table III Linear Equations, Retention Times and Maximum Absorbance Wavelengths of Carotenoid (450 nm) and Chlorophyll Pigments (650 nm) Obtained by HPLC Pigment . RT(min) . Found λmax (nm) . Regression equations . R 2 . Reported λmax (nm) . References . Fx 8.29 448.9 y = 1 × 1011 c − 96319 0.999 447; 449 (30, 33) Viox 10.38 441.6, 472.0 y = 5 × 1010 c + 95665 0.992 440, 469; 441, 470 (30, 34) Neox 12.63 438.0, 467.1 y = 2 × 1011 c − 49600 0.999 438, 467; 440, 468 (32, 35) Astx 18.79 478.9 y = 2 × 1011 c − 68542 0.999 478 (32) Lt 19.50 447.7, 475.7 y = 2 × 1011 c − 78808 0.999 446, 474; 446, 476 (32, 36) Zx 20.95 453.8, 480.5 y = 2 × 1011 c − 48856 0.999 452, 480; 450, 476 (34, 35) α-car 26.17 448.9, 476.9 y = 2 × 1011 c − 27839 0.999 446, 475; 448, 476 (32, 35) β-car 28.19 455.0, 481.7 y = 4 × 109 c − 12478 1 455, 482; 453, 480 (30, 36) Chl a 21.80 430.7, 618.7, 665.4 y = 3 × 1010 c + 5671 0.999 431, 618, 665; 431, 618, 666 (34, 36) Chl b 19.43 465.9, 649.4 y = 5 × 1010 c + 57380 0.998 461, 649; 468, 652 (32, 35) Chl ca 4–5 447.7, 629.8 y = 1 × 1011 c + 96319 0.999 445, 633; 445, 629 (30, 34) Php aa 26–30 408.9, 503.7–506.1, 666.6–667.8 y = 3 × 1010 c + 5671 0.999 409, 505, 665; 408, 504, 666 (34, 36) Spxa 7.4 451 y = 1 × 1011 c − 96319 0.999 445; 463 (36, 37) Pigment . RT(min) . Found λmax (nm) . Regression equations . R 2 . Reported λmax (nm) . References . Fx 8.29 448.9 y = 1 × 1011 c − 96319 0.999 447; 449 (30, 33) Viox 10.38 441.6, 472.0 y = 5 × 1010 c + 95665 0.992 440, 469; 441, 470 (30, 34) Neox 12.63 438.0, 467.1 y = 2 × 1011 c − 49600 0.999 438, 467; 440, 468 (32, 35) Astx 18.79 478.9 y = 2 × 1011 c − 68542 0.999 478 (32) Lt 19.50 447.7, 475.7 y = 2 × 1011 c − 78808 0.999 446, 474; 446, 476 (32, 36) Zx 20.95 453.8, 480.5 y = 2 × 1011 c − 48856 0.999 452, 480; 450, 476 (34, 35) α-car 26.17 448.9, 476.9 y = 2 × 1011 c − 27839 0.999 446, 475; 448, 476 (32, 35) β-car 28.19 455.0, 481.7 y = 4 × 109 c − 12478 1 455, 482; 453, 480 (30, 36) Chl a 21.80 430.7, 618.7, 665.4 y = 3 × 1010 c + 5671 0.999 431, 618, 665; 431, 618, 666 (34, 36) Chl b 19.43 465.9, 649.4 y = 5 × 1010 c + 57380 0.998 461, 649; 468, 652 (32, 35) Chl ca 4–5 447.7, 629.8 y = 1 × 1011 c + 96319 0.999 445, 633; 445, 629 (30, 34) Php aa 26–30 408.9, 503.7–506.1, 666.6–667.8 y = 3 × 1010 c + 5671 0.999 409, 505, 665; 408, 504, 666 (34, 36) Spxa 7.4 451 y = 1 × 1011 c − 96319 0.999 445; 463 (36, 37) The data were measured with repeated injections (n = 3) of a mixture of pigment standards at a concentration of 10−5–10−6 M each. In the calibration equation, c represents concentration of the analyte (pigment) and y represents the peak area. aThe existence of these components was determined based on the literature. Open in new tab As the parametric symbols used in the calibration equations given in Table III, y represents peak area, c pigment molar concentration and R2 linear correlation coefficient. Ten different pigments that are common for tested seaweed were mixed and the chromatographic separation was applied. The chromatograms were obtained with the optimized separation scheme at 450 and 650 nm for carotenoid and chlorophyll pigments, respectively. The results were shown in Figure 1. Figure 1 Open in new tabDownload slide Chromatogram of a synthetic mixture of (a) carotenoids (detected at 450 nm) and (b) chlorophylls (detected at 650 nm), together with their PDA spectra (inset). Peaks: (a) Fx, Viox, Neox, Astx, Lt, Zx, α-car, β-car; (b) Chl a, Chl b. Figure 1 Open in new tabDownload slide Chromatogram of a synthetic mixture of (a) carotenoids (detected at 450 nm) and (b) chlorophylls (detected at 650 nm), together with their PDA spectra (inset). Peaks: (a) Fx, Viox, Neox, Astx, Lt, Zx, α-car, β-car; (b) Chl a, Chl b. As seen from the Figure, all of these pigments could be separated within 30 min. The other pigments that can be found in seaweeds, namely chlorophyll c (Chl c), siphonaxanthin (Spx) and pheophytin a (Php a), were detected concerning the literature sources (33, 34, 36, 37). Chl c pigment experimentally determined particularly in brown algae extracts was confirmed by literature-based data that present the PDA spectrum and retention times (30, 33, 34). Since Chl c is much more polar than the other chlorophyll pigments, it is located in the chromatogram approximately at the fifth min (Table III). Furthermore, unlike the other Chl components analyzed at 650 nm (λmaks = 650 nm [Chl b], 665 nm [Chl a]), the wavelength at which the maximum absorption occurred is about 445 nm, and it can be analyzed at 450 nm, with carotenoids. As a result, the chlorophyll c component can easily be identified since there is no overlap problem between the peak of the Chl c component and the peaks of other chlorophyll components. As shown in Table III and Figure 1, it was observed that the more hydrophilic component left the column previously, i.e., the retention time decreased (38). HPLC analyses of seaweed samples In this study, the main pigment profile of 13 different marine algae was revealed. The chromatograms of six selected seaweed samples from brown, green and red algae groups were presented. All the pigments existing in various seaweed samples were identified and quantified with RP-HPLC. The chromatograms of a part of some brown, red and green algae extracts showing the profile of the carotenoid (450 nm) and chlorophyll (650 nm) pigments are presented in Figures 2–4. Since the color of the algae can vary depending on the composition and the amount of the pigments they contain, the chromatogram of each algae was evaluated by corresponding color classification. Figure 2 Open in new tabDownload slide Chromatograms and identified peaks of (a) C. sinuosa and (b) D. dichotoma algae samples (carotenoids and chlorophyll c at 450 nm, chlorophyll a, b and pheophytin a at 650 nm were determined): (a) Fx, Zx, Chl a, β-car, α-car, Php a; (b) Chl c, Chl a, Php a. Figure 2 Open in new tabDownload slide Chromatograms and identified peaks of (a) C. sinuosa and (b) D. dichotoma algae samples (carotenoids and chlorophyll c at 450 nm, chlorophyll a, b and pheophytin a at 650 nm were determined): (a) Fx, Zx, Chl a, β-car, α-car, Php a; (b) Chl c, Chl a, Php a. Figure 3 Open in new tabDownload slide Chromatograms and identified peaks of (a) H. musciformis and (b) J. rubens algae samples (carotenoids at 450 nm, chlorophyll a, b and pheophytin a at 650 nm were determined): (a) Fx, Chl b, Zx, Chl a, α-car, β-car, Php a; (b) Fx, Lt, Zx, Chl a, β-car, Php a. Figure 3 Open in new tabDownload slide Chromatograms and identified peaks of (a) H. musciformis and (b) J. rubens algae samples (carotenoids at 450 nm, chlorophyll a, b and pheophytin a at 650 nm were determined): (a) Fx, Chl b, Zx, Chl a, α-car, β-car, Php a; (b) Fx, Lt, Zx, Chl a, β-car, Php a. Figure 4 Open in new tabDownload slide Chromatograms and identified peaks of (a) C. racemosa and (b) C. fragile algae samples (carotenoids at 450 nm, chlorophyll a, b and at 650 nm were determined): (a) Spx , Chl b, Chl a, α car, β-car; (b) Spx , Viox, Chl b, Chl a, α-car. Figure 4 Open in new tabDownload slide Chromatograms and identified peaks of (a) C. racemosa and (b) C. fragile algae samples (carotenoids at 450 nm, chlorophyll a, b and at 650 nm were determined): (a) Spx , Chl b, Chl a, α car, β-car; (b) Spx , Viox, Chl b, Chl a, α-car. As can be seen from the chromatograms of the algae groups in Figures 2–4, the main pigments generally found in the brown group are Fx, Chl a and Php a, a derivative of Chl a. In addition to these major components, significant amounts of β-car and Chl c, frequent but small amounts of Zx, trace amounts of Lt and α-car compounds were detected in some types of brown algae. The total amounts of pigment compounds in seaweed samples were calculated as μg pigment/g dry sample. Accordingly, the pigment constituents were found as a percentage of the total amount: Thus, C. sinuosa (Figure. 2a) and D. dichotoma (Figure 2b) algae were revealed to contain approximately 21% Fx, 10% β-car, 1.4% Chl a, 67% Php a and a very small amount of Zx and α-car and 7.4% Chl a, 23.2% Chl c, 69.4% Php a, respectively. As shown by the chromatogram of D. dichotoma algae (Figure 2b), the peak observed at approximately fifth minute was identified as the Chl c pigment as described previously, based on a large number of studies (16, 30, 32–36). The identification of the Chl c component was readily accomplished because the PDA spectrum and the retention time of the tested pigment matched exactly with the literature data. The quantitative result of Chl c pigment was determined as the equivalence of Fx since Chl c had almost the same maximum absorption wavelength as Fx. Chl a component was identified as one of the major pigments of all the tested seaweeds except for U. rigida and C. fragile green algae. Since Php a is a derivative of Chl a and also the maximum absorption wavelength of both components is around at 665–666 nm, the quantitative result of Php a was obtained from the calibration curve generated for the Chl a pigment and calculated as the equivalence of Chl a. Another brown algae, S. schimperi was found to contain 45% Fx, 40% Chl a and 11% Php a. The pigment content of P. fascia algae was found to be approximately 24% Fx, 17% β-car and 59% Php a. According to the experimental results obtained in this study, the major carotenoid was fucoxanthin, a characteristic marine carotenoid, and the major chlorophyll was pheophytin a in brown marine algae. Thus, some carotenoids were specific to certain algae species and classes, and together with chlorophylls, may be used as references for chemotaxonomy of algae. The pigments found in the red algae groups were mainly Chl a, Fx and Php a, but the amounts were generally much less than those in brown algae. The composition and amounts of the pigments of H. musciformis algae were found to be about 18% Fx, 65% Chl a, 7% Chl b, 9% Php a and a very small amount of Zx (Figure 3a). Jania rubens was determined to have a major content of 76.8% Chl a, 13.5% Fx, a small amount of 4.2% Chl b, 3.7% Php a, 1.4% Lt and very small amount of Zx (Figure 3b). Another red algae P. scopulorum was found to have a very low total amount of pigments (8.7 μg/g algae) and was mainly composed of about 66% Php a, 21% Fx and 13% Chl a. These obtained results were associated with the studies performed for the analysis of pigments in macrostructural red marine algae. Caulerpa racemosa and C. fragile algae from green algae were found to have high total pigment amounts (Figure 4a and b). Codium fragile seaweed was determined to contain the most amount of Spx (17% of the total pigment amount corresponding to about 140 μg Spx/g algae). The quantitative amount of the siphonoxanthin pigment found in green seaweeds was calculated as Fx pigment equivalent having almost the same maximum absorption wavelength (~450 nm) with the Spx pigment. Thus, the amount of Spx was obtained from the calibration curve generated for Fx (36, 37). Other components were found to be composed of 4% Viox, 1.3% α-car, 31.2% Chl a and 46.5% Chl b. The amount of Viox was calculated as 32.45 μg/g algae corresponding to 4% of the total amount of pigments in C. fragile seaweed. Compared with other seaweeds, C. fragile contains a fairly high amount of Viox component, and thus Viox seems to be an important distinguishing pigment for this seaweed. As seen from the chromatogram of C. fragile (Figure 4b), the undefined peak observed at 12.78 min was presumably a derivative of Chl b, because it exhibited the same the maximum absorptions at 465.2 and 649.4 nm as that of Chl b. Zx and Lt components could not be detected in any species from the green algae group. Evaluation of TACs of the seaweed samples Seaweed extracts were spectrophotometrically analyzed with CUPRAC and ABTS methods to find out TAC values. The individual contribution of each carotenoid and chlorophyll pigment to the TAC of algae samples was determined with the help of calibration curves in HPLC (Table III). Taking advantage of the additivity property of TAC in a complex sample, the theoretical TACs of seaweed samples were calculated by multiplying the concentration with the TEAC value of each identified antioxidant (pigment) and summing up the products. Thus, the theoretical TAC values of the investigated seaweed sample could be estimated using the equation: $$\left(\mathrm{Theoretical}\ \mathrm{TAC}\right)={\sum}_{i=1}^n{C}_i{\left(\mathrm{TEAC}\right)}_i$$ where ci is the concentration of antioxidant component i found with the help of HPLC, and (TEAC)i is the trolox equivalent antioxidant capacity (TEAC) coefficient of component i for a given spectrophotometric method (either CUPRAC or ABTS). The theoretical TAC (thus calculated) and experimental TAC (directly measured with a given spectrophotometric method) values were compared as μmol trolox and μmol β-carotene equivalent per g dw (dry weight) seaweed samples in Table IV. Table IV The Theoretical and Experimental TAC Values of Seaweed Samples in the Units of mmol Trolox g−1 dw Algae and μmol β-Carotene g−1 dw Algae Algae sample . CUPRAC . ABTS . . TAC (TE) . TAC (β-CE) . HPLC–TAC (TE) . HPLC–TAC (β-CE) . TAC (TE) . Brown (Phaeophyceae) Cladostephus spongiosum f. v. 1.67 ± 0.01 0.48 ± 0.009 0.20 0.06 1.31 ± 0.01 Cystoseira foeniculacea 0.56 ± 0.01 0.15 ± 0.002 0.29 0.09 0.29 ± 0.01 Dictyota dichotoma 8.10 ± 0.02 2.54 ± 0.020 0.91 0.28 4.68 ± 0.01 Stypopodium schimperi 10.03 ± 0.01 3.03 ± 0.012 3.74 1.34 6.71 ± 0.02 Colpomenia sinuosa 2.67 ± 0.01 0.85 ± 0.010 1.24 0.38 1.44 ± 0.01 Petalonia fascia 0.98 ± 0.003 0.09 ± 0.003 0.67 0.06 0.31 ± 0.01 Red (Rhodophyceae) Hypnea musciformis 0.72 ± 0.01 0.2 ± 0.007 0.20 0.06 0.60 ± 0.01 Jania rubens 0.87 ± 0.01 0.25 ± 0.009 0.36 0.11 0.46 ± 0.01 Polysiphonia scopulorum 1.43 ± 0.01 0.45 ± 0.01 0.06 0.02 1.20 ± 0.01 Green (Chlorophyceae) Caulerpa taxifolia 1.16 ± 0.003 0.32 ± 0.003 0.04 0.02 0.46 ± 0.01 Ulva rigida 2.35 ± 0.014 0.84 ± 0.01 2.37 0.73 1.60 ± 0.02 Caulerpa racemosa var. cylindracea 1.31 ± 0.02 0.39 ± 0.007 1.53 0.47 0.59 ± 0.01 Codium fragile 7.80 ± 0.04 2.46 ± 0.02 5.49 1.68 2.85 ± 0.01 Algae sample . CUPRAC . ABTS . . TAC (TE) . TAC (β-CE) . HPLC–TAC (TE) . HPLC–TAC (β-CE) . TAC (TE) . Brown (Phaeophyceae) Cladostephus spongiosum f. v. 1.67 ± 0.01 0.48 ± 0.009 0.20 0.06 1.31 ± 0.01 Cystoseira foeniculacea 0.56 ± 0.01 0.15 ± 0.002 0.29 0.09 0.29 ± 0.01 Dictyota dichotoma 8.10 ± 0.02 2.54 ± 0.020 0.91 0.28 4.68 ± 0.01 Stypopodium schimperi 10.03 ± 0.01 3.03 ± 0.012 3.74 1.34 6.71 ± 0.02 Colpomenia sinuosa 2.67 ± 0.01 0.85 ± 0.010 1.24 0.38 1.44 ± 0.01 Petalonia fascia 0.98 ± 0.003 0.09 ± 0.003 0.67 0.06 0.31 ± 0.01 Red (Rhodophyceae) Hypnea musciformis 0.72 ± 0.01 0.2 ± 0.007 0.20 0.06 0.60 ± 0.01 Jania rubens 0.87 ± 0.01 0.25 ± 0.009 0.36 0.11 0.46 ± 0.01 Polysiphonia scopulorum 1.43 ± 0.01 0.45 ± 0.01 0.06 0.02 1.20 ± 0.01 Green (Chlorophyceae) Caulerpa taxifolia 1.16 ± 0.003 0.32 ± 0.003 0.04 0.02 0.46 ± 0.01 Ulva rigida 2.35 ± 0.014 0.84 ± 0.01 2.37 0.73 1.60 ± 0.02 Caulerpa racemosa var. cylindracea 1.31 ± 0.02 0.39 ± 0.007 1.53 0.47 0.59 ± 0.01 Codium fragile 7.80 ± 0.04 2.46 ± 0.02 5.49 1.68 2.85 ± 0.01 HPLC–spectrophotometric values represent the theoretically found TAC using the additivity principle. Open in new tab Table IV The Theoretical and Experimental TAC Values of Seaweed Samples in the Units of mmol Trolox g−1 dw Algae and μmol β-Carotene g−1 dw Algae Algae sample . CUPRAC . ABTS . . TAC (TE) . TAC (β-CE) . HPLC–TAC (TE) . HPLC–TAC (β-CE) . TAC (TE) . Brown (Phaeophyceae) Cladostephus spongiosum f. v. 1.67 ± 0.01 0.48 ± 0.009 0.20 0.06 1.31 ± 0.01 Cystoseira foeniculacea 0.56 ± 0.01 0.15 ± 0.002 0.29 0.09 0.29 ± 0.01 Dictyota dichotoma 8.10 ± 0.02 2.54 ± 0.020 0.91 0.28 4.68 ± 0.01 Stypopodium schimperi 10.03 ± 0.01 3.03 ± 0.012 3.74 1.34 6.71 ± 0.02 Colpomenia sinuosa 2.67 ± 0.01 0.85 ± 0.010 1.24 0.38 1.44 ± 0.01 Petalonia fascia 0.98 ± 0.003 0.09 ± 0.003 0.67 0.06 0.31 ± 0.01 Red (Rhodophyceae) Hypnea musciformis 0.72 ± 0.01 0.2 ± 0.007 0.20 0.06 0.60 ± 0.01 Jania rubens 0.87 ± 0.01 0.25 ± 0.009 0.36 0.11 0.46 ± 0.01 Polysiphonia scopulorum 1.43 ± 0.01 0.45 ± 0.01 0.06 0.02 1.20 ± 0.01 Green (Chlorophyceae) Caulerpa taxifolia 1.16 ± 0.003 0.32 ± 0.003 0.04 0.02 0.46 ± 0.01 Ulva rigida 2.35 ± 0.014 0.84 ± 0.01 2.37 0.73 1.60 ± 0.02 Caulerpa racemosa var. cylindracea 1.31 ± 0.02 0.39 ± 0.007 1.53 0.47 0.59 ± 0.01 Codium fragile 7.80 ± 0.04 2.46 ± 0.02 5.49 1.68 2.85 ± 0.01 Algae sample . CUPRAC . ABTS . . TAC (TE) . TAC (β-CE) . HPLC–TAC (TE) . HPLC–TAC (β-CE) . TAC (TE) . Brown (Phaeophyceae) Cladostephus spongiosum f. v. 1.67 ± 0.01 0.48 ± 0.009 0.20 0.06 1.31 ± 0.01 Cystoseira foeniculacea 0.56 ± 0.01 0.15 ± 0.002 0.29 0.09 0.29 ± 0.01 Dictyota dichotoma 8.10 ± 0.02 2.54 ± 0.020 0.91 0.28 4.68 ± 0.01 Stypopodium schimperi 10.03 ± 0.01 3.03 ± 0.012 3.74 1.34 6.71 ± 0.02 Colpomenia sinuosa 2.67 ± 0.01 0.85 ± 0.010 1.24 0.38 1.44 ± 0.01 Petalonia fascia 0.98 ± 0.003 0.09 ± 0.003 0.67 0.06 0.31 ± 0.01 Red (Rhodophyceae) Hypnea musciformis 0.72 ± 0.01 0.2 ± 0.007 0.20 0.06 0.60 ± 0.01 Jania rubens 0.87 ± 0.01 0.25 ± 0.009 0.36 0.11 0.46 ± 0.01 Polysiphonia scopulorum 1.43 ± 0.01 0.45 ± 0.01 0.06 0.02 1.20 ± 0.01 Green (Chlorophyceae) Caulerpa taxifolia 1.16 ± 0.003 0.32 ± 0.003 0.04 0.02 0.46 ± 0.01 Ulva rigida 2.35 ± 0.014 0.84 ± 0.01 2.37 0.73 1.60 ± 0.02 Caulerpa racemosa var. cylindracea 1.31 ± 0.02 0.39 ± 0.007 1.53 0.47 0.59 ± 0.01 Codium fragile 7.80 ± 0.04 2.46 ± 0.02 5.49 1.68 2.85 ± 0.01 HPLC–spectrophotometric values represent the theoretically found TAC using the additivity principle. Open in new tab As given in Table IV, the TAC values (in trolox and β-carotene equivalents, as TE and β-CE, respectively) were found with both spectrophotometric CUPRAC and ABTS methods and combined HPLC–spectrophotometric (i.e., HPLC–CUPRAC) method for seaweed samples. Inspecting the results presented in Table IV, TEAC values of D. dichotoma, S. schimperi ve C. sinuosa brown algae were significantly higher than those of the other algae in this group. Although CEAC values for the same algae were high enough, they were found to be much less than the trolox equivalent capacity values. This was because the absorption coefficient obtained for β-carotene was approximately 3.2 times greater than that of trolox (Table I). Since β-carotene is the general representative of carotenoids, it was selected as a reference constituent. The theoretical TAC values obtained by HPLC were compared with the TAC values found spectrophotometrically. Thus, it became possible to evaluate the HPLC–TAC values corresponding to the percentage of the TAC of constituents in seaweeds. The percentage ratios of 100 × HPLC–TEAC/TEAC and 100 × HPLC–Please change as CEAC/Please change as CEAC were found as about 11.2% and 11%, 37.3% and 44.2%, 46.4% and 44.7%, 12% and 12.5%, 52% and 60%, 68% and 67% for D. dichotoma, S. schimperi, C. sinuosa, C. spongiosum f. v., C. foeniculacea and P. fascia algae species, respectively. The ratios for the H. musciformis, J. rubens, P. scopulorum algae species from the red algae group were as follows: 28% and 30%, 41.4% and 44%, 4.2% and 4.4%, respectively. The same ratios were found as 3.44% and 6.25%, 101 and 87%, 117% and 121%, 70% and 69% for C. taxifolia, U. rigida, C. racemosa, C. fragile green algae species, respectively. Discussion Pheophytin a compound, the main derivative of the chlorophyll a pigment, occurs by the removal of the central Mg-atom from the chlorophyll molecule having a porphyrin ring structure, and it is often found in significant amounts in marine algae. Thus, pheophytin a is generally described simply as chlorophyll a without a central Mg-atom (33). Advantageously, the degradation of chlorophyll a that results in the formation of pheophytin a increases the antioxidant activity of the raw extract (39). Thus, the formation of Php a increases during the extraction process of plants. There are also environmental factors such as time and ambient temperature affecting this transformation. Php a component, which is more hydrophobic than chlorophyll a due to the absence of central metal Mg, was observed in the chromatogram later (29–30th min) in comparison to chlorophyll a component (21.8 min) since it was more strongly retained on the nonpolar C30 column. Although the polarity of an analyzed compound is an effective parameter determining the retention time observed in the chromatogram, quite different values are found in the literature depending on the composition of the mobile phase used, the type of column and also the method applied. However, the HPLC-chromatogram sequence of retention times of the chlorophyll components typically takes place as Php a > Chl a > Chl b. In this study, even though the retention times for all detected carotenoid pigments including chlorophylls slightly differ from literature, the retention time observed for Php a is 29–30 min, which is generally compatible with the literature independent of the mobile phase or method used. Besides, in the chromatograms of the algae extracts, the PDA spectrum of the component that is expected to be pigment Php a corresponded to the spectrum given in the literature (33). Thus, Php a was identified based on the literature providing sufficient data (34, 36). In a study conducted by Esteban et al. (40), it was reported that carotenoids found in 13 distinct red marine algae were mainly β-car, Lt, Zx, and some species also contained antheraxanthin in high amounts and α-car partially. However, the presence of the Fx component, which is likely to be found in algae, was not mentioned at all. In another study, carotenoids were analyzed in edible brown and red marine algae, and it was stated that brown marine algae contained Fx predominantly, whereas red algae contained Fx component in general with much lower amounts (14). In the same work, other carotenoid compounds present mainly in red algae were identified as Lt and Zx from the xanthophylls group. These findings were quite compatible with the results obtained for brown and red algae in this work. Although the β-carotene pigment has been reported to be found frequently in red algae by many other researchers (13, 40), it was not specified by Terasaki et al. (14). Thus, α- and β-carotene compounds were also not detected in the red algae samples presented in this work. Apart from the major components such as Fx, Chl a, Php a and β-car found in algae, it was revealed that both presence and the amount of some other minor xanthophylls subgroup components of the carotenoid group were important to distinguish the features of algae species (13). Among them, particularly Lt, Zx and partly Viox pigments were found as distinctive components. In addition to mentioned xanthophyll compounds, Esteban et al. (40) and Schubert et al. (13) reported that the antheraxanthin component from xanthophyll group was the major component commonly found in the red algae (Corallina elongata which is synonym of Ellisolandia elongata (Hind and Saunders 2013), J. rubens, and Bossiella orbigniana (Corallinales, Rhodophyta) P.C.Silva 1957). Based on the retention time (~10–12th min) and the PDA (λmax = 444 nm) spectrum data in mentioned above studies, the presence of an antheraxanthin component in the seaweeds was identified in our study. As can be seen from the chromatograms of C. sinuosa, D. dichotoma, H. musciformis, J. rubens algae shown in Figures 2 a, b and 3 a, b, the undefined peak (λmax = 442.8-444 nm) in about 12th minute could probably be assigned to the antheraxanthin component. Since the J. rubens seaweed presented in this study was the same as the seaweed given in the literature (40), the verification of the antheraxanthin component could easily be made. However, the carotenoid composition in seaweeds can vary depending on the species of algae, seasonal cycles and the location they are collected from as well as on the growth phase of algae. Besides, the most important thing that can be said for seaweeds is that all algae can show similar characteristics depending on the group they belong to, but they do not have a unique carotenoid and pigment profile. Furthermore, the xanthophyll group carotenoids starting from β-carotene transform into one another by the effect of β-carotene hydroxylase, zeaxanthin epoxidase and violaxanthin epoxidase enzymes (β-car → Zx ↔ antheraxanthin ↔ Viox) (40, 41). The species from the green algae group in this research were found to contain mainly Chl a, Chl b, partially Php a and Spx components. Particularly, the amount of Chl b component was remarkably higher in comparison with red algae. For example, the amount of Chl b was found to be 1.94, 1.97 and (not detected) μg Chl b/g-algae for the red algae, H. musciformis, J. rubens and P. scopulorum, respectively and, while it was recorded as 1.65, 98.29, 59.77 381 μg/g-algae for the green algae, C. taxifolia, U. rigida, C. racemosa and C. fragile, respectively. Considering the extensive studies examining the pigment composition of green marine algal groups, chlorophyll compounds and their derivatives are observed as the main components that are responsible for the green color of these algae groups (35, 38). In addition to chlorophyll pigments, some major carotenoids such as β-car, Viox, Neox, small amounts of Zx, rare antheraxanthin and small and variable amounts of Lt are also reported to be possibly present in green algae (41, 42). It was noted by Bianchi et al. (16) that some brown algae species from the Baltic Sea region contained a large amount of Fx as a major carotenoid though some red and green algae species did not have any. Takaichi (41) stated that the Fx component was not detected in five different green marine algae analyzed but a small amount in red algae. In the same study, it was also stated that the Lt component was generally a pigment specific to some red and green algae. According to this research, C. fragile green marine algae, an edible species, was determined to contain very high amounts of Spx pigment (140 μg/g-algae) (Figure 4b). This finding was also supported by Ganesan et al. (43) that siphonaxanthin, a marine carotenoid derived from green seaweeds, was extracted mainly from C. fragile green algae. Siphonaxanthin was also reported to effectively inhibit the viability of human leukemia HL-60 cells via induction of apoptosis. On the other hand, if the pigments found in the algal samples studied are compared relatively with other similar studies regarding their chromatograms, it appears that the chromatograms of the algal extracts may contain many intertwined peaks and possibly many shoulders of pigment isomers, and some undefined peaks. Some chromatograms from the studies on the analysis of algal pigments confirm this approach (19, 32, 44). It was clearly stated by Schmid and Stich (45) that there was no ideal method for the separation of algal pigments by HPLC. Besides, marine algae contain numerous different molecules in a highly complex structure, so we cannot expect the chromatograms of algae extracts to be as pure, sharp and clear as standards. Also, for example, isomers/epimers of chlorophylls (Chls’) are almost always present in the chlorophyll and its derivatives preparations: they are naturally present in small amounts in photosynthetic organisms; on the other hand, chlorophylls can be, in small amount, converted to the 132-epimers (Chls’) during the extraction processes (46). In the HPLC determination of chlorophylls and carotenoids, chemical changes of pigments during or before HPLC analysis (i.e. acid–base equilibria, isomerization, etc.) were shown to cause additional peaks, shoulders and tails in the HPLC elution diagram, limiting the accuracy of the quantification (47). According to Schmid and Stich (45), the use of an ion-pairing reagent (ipr) is often employed by researchers to suppress dissociation of compounds and enhance peak separation. Thus, they added ipr (tetraethylammonium acetate) to both the sample and the mobile phase. However, they reported that the use of ipr was only restricted to the extraction of samples. Because the successful chromatographic separation was obtained without the use of ipr during the combined C18 ODS with the MZ-PAH C18 column applications, but fucoxanthin was superposed on chlorophyll c. The effective separations feasible with C30 columns have proven useful for pigment isolation in our previous study. Pigment separation and resolution can also be tuned by the column temperature, mobile phase composition and the gradient program (28). For all algae species analyzed, the calculated HPLC–spectrophotometric TAC values corresponded to the percentage ratios ranging from about 3% to 100% of experimental TAC values, except for C. racemosa algae. This means that experimental and theoretical antioxidant capacity values are generally quite different from one another. These somewhat incompatible results, apart from the compounds determined by standards and literature data on the studied algae species, may stem from distinct pigment components in the chromatogram whose content and quantity cannot be assayed. Another reason is that the phenolic components, which are partially extracted with acetone but do not form a peak (i.e., do not exhibit absorption) in the range of 450–650 nm, probably increase the TAC value. On the other hand, that the above-mentioned ratios (117% and 121%) obtained for C. racemosa var. cylindracea algae are greater than 100% can be explained by the coincidence of the retention of some constituents without antioxidant properties with that of pigments having an antioxidant effect in the HPLC chromatogram, and thus increase the peak area values. As the composition and the number of constituents present in algae may change greatly depending on many factors such as seasonal changes, location, growth period and species of algae, the obtained variable results are expected. Among the seaweeds from the same color group, there may be great differences in terms of content and quantity, but even for the same algae, significant differences may be determined. In this study, the seaweed samples were particularly collected in the abundant periods in which they were considered to be more productive. As the results of HPLC–TEAC/TEAC and HPLC–CEAC/CEAC ratios were very compatible, in addition to trolox, β-carotene constituent was found suitable and necessary as a reference agent to assess the antioxidant potential of seaweeds. In the spectrophotometric TAC determination, the commonly used ABTS/TEAC method having partially a similar mechanism to that of the CUPRAC/TEAC method was applied as a reference method involving the use of trolox, a water-soluble synthetic derivative of vitamin E as standard reference material. The differences between the CUPRAC and ABTS results in Table III stem from the different mechanisms of the two methods (i.e., ABTS is a mixed-mode assay comprising both electron transfer and hydrogen atom transfer mechanisms), and the reagents used respond differently to antioxidant species. Conclusions In this study, HPLC–spectrophotometry combinations were carried out to estimate the theoretical TAC values as trolox and β-carotene equivalents of seaweed samples using the chromatographic standards of the most widely encountered antioxidant pigments. The TAC values of HPLC-quantified antioxidant pigments of seaweed extracts were found and compared for the first time with those measured by CUPRAC. The TAC of HPLC-quantified compounds accounted for a relatively much lower percentage of the observed CUPRAC capacities of seaweed extracts, namely ranging from 11 to 68% for brown, from 4 to 41% for red and from 3 to 100% for green species. The additivity of TAC with HPLC–CUPRAC is approximately valid for seaweed extracts because seaweeds contain pigments, some of the derivatives and additional configurational and conformational isomers, and it is almost impossible to find the exactly matching standards for their HPLC analysis. As a result, the chromatographic peaks of certain isomers may easily overlap and render their precise HPLC estimation and quantification almost impossible. 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TI - HPLC Detection and Antioxidant Capacity Determination of Brown, Red and Green Algal Pigments in Seaweed Extracts
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa107
DA - 2020-12-14
UR - https://www.deepdyve.com/lp/oxford-university-press/hplc-detection-and-antioxidant-capacity-determination-of-brown-red-and-XmbbTevrZb
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -