Introduction

Reactive oxygen species (ROS) are produced naturally throughout the body as a by-product of cellular metabolism and also by exposure to radiation, metals, pollutants, toxic chemicals, smoking, wrong diets, etc. At low to moderate concentrations, they function in physiological cell processes, but at high levels, they produce harmful modifications to cell components, such as lipids, proteins, and DNA (Marnett 2002). Thus, the imbalance between the ROS production and biological system’s ability to detoxify the reactive intermediates or to repair the resulting damage is called “oxidative stress.” The oxidative stress can lead to many pathological conditions, such as cancer, cardiovascular diseases, neurological disorders, diabetes, reduced resistance, and accelerated aging. (Essick and Sam 2010). Unfortunately, we cannot avoid ROS and the risk of oxidative stress, but we can counteract oxidative stress with a balanced and healthy diet rich in antioxidants (Serra-Majem et al. 2006).

In the past decade, lifestyle has changed with a shift to the use of processed foods. Oxidation of lipid is one of the critical quality deterioration in processed foods. Lipid oxidation in foods not only reduces the nutritional value but also leads to various noncommunicable diseases as mentioned above (Essick and Sam 2010). Synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, and tertiary butyl hydroquinone (TBHQ) available in the markets, are very efficient in controlling lipid oxidation in foods. However, the use of synthetic antioxidants in foods has been limited due to the toxicity and their adverse health effects (Fukushima et al. 1987; Kahl and Kappus 1993). Moreover, consumer preference on more “natural” products has increased the use of natural ingredients instead of synthetic antioxidants. Several studies are available on natural antioxidants from terrestrial plants, and a number of herbs, fruits, vegetables, and spices have been reported to delay or prevent lipid oxidation efficiently in foods (Maqsood et al. 2014).

From time immemorial, seaweed has been used in traditional folk medicine in Asian countries (Moi 1987). Recently, their importance as a source of novel bioactive compounds is increasing, and studies have shown that algal-derived compounds exhibit various biological activities of potential medicinal value (Holdt and Kraan 2011; Alghazwi et al. 2016). Various antioxidant compounds have been isolated from different types of seaweeds, which include phlorotannins, proteins, sulfated polysaccharides, sterols, and carotenoids (Holdt and Kraan 2011).

The coastline of Kuwait has abundant seaweed resources with broad species diversity (113 species) (John and Al-Thani 2014). However, not much work has been done to explore the bioactive compounds present in these seaweeds and their antioxidant potential. Being a unique environment with high temperature (36 °C), salinity (34–45 ppt), and environmental stress (Al-Yamani et al. 2004), we hypothesize that the algae in this regions have some mechanism to combat the oxidative stress. Hence, the present investigation aims to study the phytochemicals, phenolics, and antioxidant properties of a range of seaweeds from Kuwait coast of Arabian Gulf. The antioxidant activity was evaluated by in vitro assays, such as radical scavenging activity, metal chelating activity, reducing power, and prevention of lipid oxidation in liposome model system, which will give insight into the possible antioxidant mechanisms. Phytochemical screening and the phenolic composition of the extracts were also determined to find out the nature of antioxidant compounds. This study will be a basis to selectively identify the most suitable species for further characterization.

Materials and methods

Chemicals

1,1-Diphenyl-2-picrylhydrazyl (DPPH), ferrozine, and phenolic standards, such as gallic acid, hydroquinone, protocatechuic acid, catechin, epicatechin, caffeic acid, vanillic acid, syringic acid, rutin hydrate, p-coumaric acid, t-ferulic acid, coumarin, salicylic acid, and quercetin, were from Sigma-Aldrich (Germany). All the other chemicals were of analytical grade unless otherwise specified.

Algal materials

Twenty-six species of seaweeds consisting of 11 brown, five green, and ten red seaweeds (Table 1) were collected from the Kuwait coast of Arabian Gulf (Fig. 1) between August 2015 and April 2016. Feldmannia irregularis, Colpomenia sinuosa, Iyengaria stellata, Polysiphonia platycarpa, and Cladophora sp. were collected from the sandy beaches near the Kuwait tower in December 2015. Canistrocarpus cervicornis, Polysiphonia brodiei, Ulva flexosa, Spyridia filamentosa, and Laurencia obtusa were collected from Ahua Island in February 2016. Hypnea cornuta, Ulva lactuca, and Bryopsis pulmosa were collected from Um-Al-Namil Island in February 2016. Sargassum aquifolium, Chondria cornuta, and Chondria dasyphylla were collected from Miskan Island in March 2016. Murrayella periclados and Hypnea valentiae were collected from Julaia beach in March 2016. Grateloupia sp. and Sargassum angustifolium were collected from Salmiya marina and Green Island respectively, in January 2016. Sargassum boveanum, Feldmannia mitchelliae, and Codium sp. were collected from Fintas in February 2016 and Padina gymnospora in August 2015. Sargassum asperifolium and Sargassum oligocystum were collected from Al-Khiran in January 2016. The collected seaweed samples were washed with seawater and cleaned off epiphytes. The cleaned seaweeds were immediately frozen, freeze-dried, powdered, and stored at − 80 °C under vacuum packing until further use.

Table 1 The extraction yield and total phenolic content (TPC) of different seaweeds of Kuwait coast
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Fig. 1
figure 1

Map of Kuwait coast of Arabian Gulf indicating the sampling locations

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Production of algal extract

Due to the safety concern in using organic solvent extracts in foods, we used approved food-grade solvents, such as water and ethanol for the extraction of seaweeds. Absolute ethanol, 50% ethanol, and water was used as extraction solvent. Water extract was prepared by overnight extraction of 5 g seaweed powder with 100 mL distilled water. The slurry was centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The residue was re-extracted three times under the same conditions, and the pooled extracts were freeze-dried. Ethanolic extracts were prepared by overnight extraction of 5 g seaweed powder with 50 mL of absolute or 50% ethanol. After centrifugation for 10 min at 3500 rpm, the supernatant was collected. The residue was re-extracted three times under the same conditions, and the pooled filtrate was concentrated at 40 °C in a rotary evaporator (BUCHI Labortechnik AG, Switzerland). In the case of 50% ethanol extracts, the extracts obtained after the evaporation of the solvent were freeze-dried. The absolute ethanol extracts (AE), 50% ethanolic extracts (50E), and water extracts (WE) were kept at − 80 °C until analysis.

Determination of total phenolics

The total phenolic content (TPC) was determined by the method of Farvin and Jacobsen (2013). The absorbance at 725 nm was measured using a microplate reader (Variscan Lux, Thermo Scientific, Finland). The total phenolics were calculated from the standard curve made from various concentrations of gallic acid and were expressed as milligram gallic acid equivalent per gram of dried extract.

Quantification of individual phenolic compounds using UHPLC

The individual phenolic compounds were identified and quantified with Agilent 1290 Infinity II Series UHPLC (Agilent Technologies, Germany), equipped with an Agilent G7117B diode array detector. The column used was ZORBAX SB-C18 analytical column (250 mm × 4.6 mm) with a 5-μm packing material. Before injecting into UHPLC, the extracts were filtered through a 0.45 μm filter (Merk Millipore, Ireland). Elution was performed with a gradient elution with phosphoric acid in deionized water (pH = 3) as mobile phase A and methanol-acetonitrile (50:50) as mobile phase B at a flow of 0.8 mL min−1. The injection volume was 5 μL, and the temperature was 40 °C. The detection of the phenolic compounds was done by a diode array detector at 280, 235, 255, 210, 320, 340, 360, and 520 with reference wavelength of 450 nm. Peak areas and retention times (RT) were computed automatically by an HP 3396A integrator. The identification of the phenolic compounds was made by comparing the RT of sample chromatographic peaks with that of standards under the identical UHPLC conditions.

Phytochemical screening

The different seaweed extracts were subjected to phytochemical screening as per the method of Sadasivam and Manickam (1996). The qualitative results are expressed as (+) for the presence and (−) for the absence of phytochemicals.

Libermann Buchard test for steroids and terpenoids

About 0.5 mL of the crude extract was mixed with few drops of acetic anhydride in a test tube. The mixture was boiled in a water bath and rapidly cooled in ice. Then concentrated sulfuric acid was added through the sides of the test tube. The development of a brown ring at the junction of two layers and turning the upper layer to green show the presence of steroids, while the development of deep red color in the lower layer indicated the presence of triterpenoids.

Test for cardiac glycosides

Four milliliters of the extract was extracted with 8 mL of chloroform. The chloroform layer was used for the following tests for the presence of glycosides.

  1. a)

    Kedde’s test for the presence of β-unsaturated-o lactones in the aglycone

About 1 mL of the chloroform extract was evaporated to dryness. Then, 1 mL of 2% 3,5-dinitro benzoic acid was added followed by few drops of 10% NaOH. The formation of the purple color indicates the presence of glycosides.

  1. b)

    Keller-Killiani test for 2-deoxy sugars

The chloroform extracts were mixed with few drops of glacial acetic acid and ferric chloride solution. Then, concentrated sulfuric acid was added through the sides of the test tubes, and the formation of two layers was observed. Lower reddish-brown layer and an upper acetic acid layer that turns bluish green would indicate a positive test for glycosides.

  1. c)

    Reaction due to steroidal nucleus

To one milliliter of the chloroform extract in a glass test tube was added 1 mL of glacial acetic acid. To this mixture, 1 mL of concentrated sulfuric acid was added through the sides of the test tube, and the formation of green color indicates a positive reaction.

Foam test for saponins

About 1 mL of the extract was mixed with 10 mL of water in a test tube and shaken. The formation of froth, which is stable for 15 min, indicates a positive test for saponins.

Dragendroffs test for alkaloids

About 5 mL of the extract was extracted with 3 mL of chloroform. The chloroform extract of the crude extract was evaporated and acidified by adding few drops of Dragendroffs reagent (potassium bismuth iodide). The appearance of orange-red precipitate indicates the presence of alkaloids.

Tests for flavonoids

Three tests were used.

  1. (a)

    Ferric chloride test: The development of blackish red color when the extracts are treated with few drops of ferric chloride solution indicates the presence of flavonoids.

  2. (b)

    Alkaline reagent test: The extracts when treated with sodium hydroxide solution show an increase in the intensity of yellow color. If flavonoids are present, the solution becomes colorless upon the addition of few drops dilute hydrochloric acid.

  3. (c)

    Lead acetate test: The development of yellow precipitate upon the addition of few drops of 10% lead acetate solution to the extracts indicates the presence of flavonoids.

Tests for tannins

Two tests were used.

  1. (a)

    Ferric chloride test: In the ferric chloride test, adjacent phenolic OHs will be evident. When the crude extracts were mixed with few drops of 15% ferric chloride solution, the black blue color indicates adjacent 3-OH phenolics and therefore suggests hydrolyzable tannins. The greenish gray color indicates 2-OH phenolics suggesting condensed tannins, and the yellow suggests that phenolic 1-OH is isolated.

  2. (b)

    Gelatin test: The extracts were treated with gelatin solution, and the formation of white precipitate indicates the presence of tannins.

Determination of antioxidant activity of extracts

The absolute ethanol (AE), 50% ethanolic (50E), and water extracts (WE) were subjected to antioxidant screening by in vitro assays, such as radical scavenging activity, metal chelating ability, reducing power, and the inhibition of lipid oxidation in liposomes.

Free radical scavenging activity

α,α-Diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging effect was measured according to the method of Farvin et al. (2014) in a microplate reader (Varioscan Lux, Thermo Scientific, Finland). The concentration of the extracts used for the assay was 0.1, 1, 5, and 10 mg mL−1. A blank with distilled water instead of sample and a sample control with sample and 95% ethanol were also made. BHT at the concentration of 1–0.05 mg mL−1 was used for comparison. The effective concentration EC50 (concentration of extracts required to scavenge 50% of DPPH radicals) was calculated. The results are expressed as antiradical power, which is 1/EC50.

Metal chelating property

The iron chelating property of the extracts was determined according to method of Farvin et al. (2014) in a microplate reader (Variscan Lux, Thermo Scientific). The concentrations of the extract used were 0.1, 1, 5, or 10 mg mL−1. EDTA at 1–0.04 mg mL−1 was used for comparison. The results are expressed as % iron chelating capacity.

Reducing power

The reducing power of the extracts was measured according to the method of Farvin et al. (2014) in a microplate reader (Varioscan Lux, Thermo Scientific). The concentrations of the extract used were 0.1, 1, 5, or 10 mg mL−1. About 200 μg mL−1 of ascorbic acid was used for comparison. The reducing power was expressed as absorbance at 700 nm.

Inhibition of lipid oxidation in liposome model system

Liposomes assay was performed according to the method of Farvin et al. (2014). The liposome system for the oxidation study constitutes extracts at 1 mg mL−1 concentration and liposomes at 0.1 mg mL−1 phosphatidylcholine concentration. The assay solution with buffer and liposome alone serves as a control. BHT at a concentration of 0.2 mg mL−1 was used for comparison. Oxidation was initiated by iron-ascorbate redox reaction. The thiobarbituric acid reactive substance (TBARS) formation was quantified, and the results are expressed as % inhibition of TBARS formation.

Statistical analysis

The results are given as mean ± SD. The data on TPC and different antioxidant assays are subjected to two-way analysis of variance (ANOVA) using the statistical program GraphPad Prism 7.04 (GraphPad Software Inc., USA). Comparison of the group means is done with Tukey’s multiple comparison test. A P value < 0.001 was used as statistically significant.

Results and discussion

Extraction yield

The yields of different seaweed extracts were shown in Table 1. The yield of the different extracts showed considerable variation with species as well as the extractant. In general, water extracts of most of the seaweeds showed higher yield than ethanolic extracts. The yield of water extracts ranged from 11.4 to 90.9%. Absolute ethanol extracts showed the lowest extraction yield, and it varies from 2.3 to 46.8%, while the 50% ethanolic extracts showed intermediate levels of yield, which ranged from 8.2 to 72.9% (Table 1). In water extracts and 50% ethanolic extracts, the highest yield was for Codium sp., which was followed by L. obtusa and C. cornuta. The lowest yield for water and 50% ethanol extract was for P. platycarpa. In absolute ethanolic extracts, the highest yield was for S. filamentosa and the lowest for P. platycarpa. A higher yield of water extracts when compared with other organic solvents was reported elsewhere (Wang et al. 2009; Farvin and Jacobsen 2013). The polarity of solvents plays an essential role in the extraction of antioxidant compounds (Alothman et al. 2009). A higher yield of water extracts might be due to the extraction of more polar, water-soluble components like proteins and polysaccharides. Ethanol precipitates proteins and polysaccharides, which results in a lower yield of absolute ethanolic extracts. As the polarity of 50% ethanol (polarity index 7.1) is intermediate between water (polarity index 9) and ethanol (polarity index 5.2), the extraction yield was intermediate by extracting compounds within that range of polarity.

Total phenolic content

The TPC of the different seaweeds analyzed is shown in Table 1. TPC showed wide variation depending upon the species and the extraction solvents. In general, TPC was higher for absolute ethanolic extracts than those of the other extracts. However, some species like Cladophora sp. and H. cornuta showed significantly (P < 0.001) high levels of TPC in 50% ethanolic and water extracts, whereas P. brodiei showed significantly (P < 0.001) high levels of TPC in 50% ethanol extracts than other extracts. The TPC in the absolute ethanol extract ranged from 8.0 ± 2.0 to 175.5 ± 53.4 mg GAE g−1 extract. In the absolute ethanolic extracts, S. boveanum, S. oligocystum, and S. angustifolium showed significantly (P < 0.001) high amount of TPC, which was followed by S. asperifolium, F. irregularis, P. gymnospora, and C. cervicornis. The TPC of 50% ethanolic extracts ranges from 6.5 ± 1.5 to 95.9 ± 17.1 mg GAE g−1 extract. In 50% ethanolic extracts, all the Sargassum sp., P. gymnospora, C. dasyphylla, and Cladophora sp. showed significantly (P < 0.001) high TPC than the other species. The TPC of water extracts ranged from 4.4 ± 0.9 to 42.3 ± 9.1 mg GAE g−1 extract. In water extract, Cladophora sp. showed significantly (P < 0.001) high phenolic content than other extracts.

In contrast to yield, the TPC showed an opposite trend and was high in ethanolic extracts than water extracts. This might be because of the release of bound phenolics into the solution when ethanol precipitates most of proteins and polysaccharides. This result is in accordance with earlier studies (Wang et al. 2009; Farvin and Jacobsen 2013) where they also report that the water was inferior to ethanol and other organic solvents in phenolic extraction. Interestingly, Cladophora sp. and H. cornuta showed higher TPC in water and 50% ethanolic extract when compared with those in absolute ethanolic extracts, indicating the presence of more polar phenolic derivatives. As the phenolic content varies with species, geographical locations, and season (Pavia and Åberg 1996; Fairhead et al. 2005; Paiva et al. 2018), it will be ideal to compare and discuss the phenolic content from Arabian Gulf. There are a few research papers available in the literature, which deal with the TPC of the seaweeds collected from the Iranian coast. However, the direct comparison of the results of the present study with these studies is difficult due to the difference in solvents, conditions used for the extraction, units, and species used for the research. Zahra et al. (2007) reported a TPC of 17 and 0.9 mg CE g−1 dry sample for water and ethanolic extracts of S. boveanum collected from the Iranian coast. In this study, the authors do not mention the strength of the ethanol used for the extraction. Similarly, the studies on five species of Ulva from Iranian coast showed a TPC range of 1.25 ± 0.12 to 5.08 ± 0.65 mg GAE g−1 extract, with U. flexosa being 2.67 ± 0.22 mg GAE g−1 extract (Farasat et al. 2014). Sadati et al. (2011) reported a TPC range of 0.92 ± 0.14 to 5.3 ± 0.77 mg GAE (100 g)−1 dried algae for n-hexane, chloroform, ethyl acetate, and methanolic extracts of C. sinuosa collected from the Iranian coast. However, in the present study, TPC of the S. boveanum, U. flexosa, and C. sinuosa from Kuwait coast was far higher than the one reported from the Iranian coast. This might be an adaptation to survive the higher salinity prevailing in Kuwait coast when compared with the Iranian coast.

Individual phenolic composition

The composition of different phenolic compounds was determined in HPLC and is shown in Table 2. In the present study, phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids), flavonoids (flavan-3-ols or flavanols and flavonols), coumarin, and hydroquinone were quantified. The hydroxybenzoic acids include gallic, protocatechuic, vanillic, syringic, and salicylic acid. The hydroxycinnamic acids include caffeic, p-coumaric acid, and t-ferulic acid. Flavan-3-ol (flavanols) includes catechin and epicatechin, whereas flavonols include quercetin and rutin. Seaweeds showed different phenolic profile in water and ethanolic extracts. Although there are exceptions, in general, ethanol was good in extracting rutin, quercetin, and salicylic acid, while water was good in extracting gallic acid. The influence of solvent polarity in the extraction of phenolic and flavonoids has been reported elsewhere (López et al. 2011; Stankovic et al. 2011). Considerable variation in the phenolic profile of different extracts in the present study might be due to the difference in polarity of the compounds present in the seaweeds and also the extractability of the solvents.

Table 2 The individual phenolic compounds (μg g-1 of dried extract) in ethanolic and water extracts of different seaweeds determined by UHPLC. The values are the mean of two injections from the same extract
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In general, hydroquinone is the most common phenolic compounds present in all the seaweeds except Codium sp. collected from Kuwait coast of Arabian Gulf. The highest content of hydroquinone was found in P. platycarpa followed by F. mitchelliae, F. irregularis, and P. gymnospora (Table 2). Hydroquinone has been used as a depigmenting agent in creams and for treatment of melasma (Garcia and Fulton 1996). So, these seaweed species can be a potential source of hydroquinone, which have application in the pharmaceutical and cosmetic industry. In addition to hydroquinone, a number of other phenolic compounds are also present in these seaweeds.

In green algae, in addition to minor quantities of various phenolic compounds, B. pulmosa showed high levels of rutin, and Cladophora sp. contained high levels of catechin and t-ferulic acid. U. flexosa and U. lactuca contained high levels of quercetin in the absolute ethanol extracts, and U. flexosa was found to contain more phenolic compounds than U. lactuca. Codium sp. contained only gallic acid out of the different phenolics analyzed.

In red algae, in addition to minor quantities of various phenolic compounds, C. dasyphylla contained high levels of rutin and protocatechuic acid in absolute ethanol extract and vanillic acid in water extracts, whereas C. cornuta showed high levels of coumarin and quercetin in absolute ethanolic extract. P. platycarpa and H. cornuta contained more number of phenolic compounds when compared with P. brodiei and H. valentiae, respectively. M. periclados showed high levels of protocatechuic acid in the absolute ethanolic extract and high levels of protocatechuic acid, syringic acid, rutin, and quercetin in water extract. S. filamentosa contained mainly gallic and protocatechuic acids, whereas Grateloupia sp. contained mostly hydroquinone along with other minor phenolics.

In brown algae, along with minor quantities of various phenolic compounds, ethanolic extracts of S. asperifolium and S. boveanum showed high levels of protocatechuic acid and rutin, respectively, whereas S. angustifolium showed high levels of catechin, coumarin, and quercetin than the other Sargassum species. F. irregularis showed high levels of protocatechuic acid in all the extracts and salicylic acid in absolute ethanol extracts, whereas F. mitchelliae contained high levels of salicylic acid, quercetin, and protocatechuic acid in absolute, 50% ethanol, and water extracts, respectively. In addition to high hydroquinone and other minor components, P. gymnospora contained a high level of quercetin in absolute ethanol extracts. Hydroquinone was the main phenolic compound in I. stellata, C. sinuosa, and C. cervicornis. Other than this, C. cervicornins also showed high levels of coumarin and salicylic acid in the absolute ethanol extract.

In the present study, significant inter- and intra-species variations were found for total and individual phenolics. The pronounced variation observed in the phenolic composition of these seaweeds may stem from environmental factors, such as high salinity, temperature, and high UV index prevailing in the Arabian Gulf. Factors, such as salinity, temperature, nutrients, seasonality, herbivory, maturity, age, and type of tissues, are reported to influence the composition of seaweed (Amsler and Fairhead 2006). All these factors can influence the spatiotemporal regulation of the phenolic metabolism, which can lead to quantitative and qualitative variations of phenolics among seaweeds, together with intra-individual variations (Amsler and Fairhead 2006; Lann et al. 2012). In addition to this, type of solvents and conditions during the extraction also play an essential role on the total and individual phenolic composition, which is evident in Tables 1 and 2 and many other studies as well (Santoso et al. 2002; Farvin and Jacobsen 2015). Various polyphenolic compounds have been identified from brown, red, and green algae, which include simple phenolics, catechins, flavonols, and flavonol glycosides (Santoso et al. 2002; Farvin and Jacobsen 2015). In the current study, we have identified only some of the phenolic acids and flavonoids. Because of the difference in species studied, extraction methods used, and lack of information about the seaweed phenolics of Arabian Gulf in the literature, our results on phenolic constituents cannot be compared with other studies from the Arabian Gulf. Further detailed studies on the phenolic components are in progress in our laboratory.

Phytochemical screening

Phytochemical screening of the seaweed extracts revealed the presence of secondary metabolites, such as alkaloids, glycosides, saponins, flavonoids, steroids, terpenoids, and phenolics/tannins (Table 3). The presence or absence of the different constituents varied with solvents used and species tested.

Table 3 The phytochemical composition of ethanolic and water extracts of different seaweeds. The symbol “+” indicates the presence, and the symbol “−” indicates the absence of the compounds
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Out of the 26 species screened, only nine species contained alkaloids. Alkaloids are naturally occurring organic nitrogen-containing bases, which include numerous biological amines and halogenated cyclic nitrogen-containing substances. The green algae B. pulmosa and U. lactuca, red algae C. dasyphylla, and brown algae S. boveanum, S. oligosystum, C. sinuosa, and C. cervicornis showed the presence of alkaloids in their ethanolic extracts, while water extracts of F. mitchelliae and P. gymnospora were also positive for alkaloids.

Glycosides are compounds containing carbohydrate and a noncarbohydrate residue in the same molecule. Cardiac glycosides are glycosides that consist of a steroid molecule attached to a sugar (glycoside) and lactone group. In the present study, cardiac glycosides were found to be restricted in ethanolic extracts of the seaweeds tested (Table 2). Of the 26 species of seaweeds screened, ethanolic extracts of all the brown algae except C. sinuosa were positive for cardiac glycosides. In addition to this, B. pulmosa from green algae and P. brodiei from red algae also showed the presence of cardiac glycosides in the ethanolic extracts. Saponins are glycosides with foaming characteristics, and the foaming ability of saponins is caused by the combination of a hydrophobic (fat-soluble) sapogenin and a hydrophilic (water-soluble) sugar part. In the present study, except for H. valentiae, L. obtusa, Grateloupia sp., and C. sinuosa, all the other seaweeds tested showed the presence of saponins in ethanolic or water extracts. Saponins were mainly present in 50% ethanolic extracts, though some of the species contained saponins in the absolute ethanol or water extracts.

The tannins were found only in some of the tested seaweed species (Table 3) and were present mainly in the absolute ethanolic extracts, and only a few species contained it in 50% ethanolic and water extracts. All these species were positive for condensed tannins than hydrolyzable tannins. All the seaweeds screened were found to be positive for flavonoids in any of the extracts, especially in their 50% ethanolic and water extracts (Table 3). HPLC analysis of phenolic compounds also confirms this by the presence of more flavonoids. This indicates that the seaweeds in this part of the oceans are producing more flavonoids rather than more complex phlorotannins. This might be a mechanism to cope with the high UV index prevailing in these regions. Flavonoids are reported to have UV protective properties (Saewan and Jimtaisong 2013).

The presence of steroids was observed in some of the seaweeds species and was found mainly in absolute and 50% ethanolic extracts except B. pulmosa, which also showed the presence of steroids in water extracts. Steroids were found to be absent in Codium sp., Cladophora sp., C. dasyphylla, H. valentiae, L. obtusa, C. cornuta, and M. periclados. Out of the 26 species studied, the presence of terpenoids was found only in the water extracts of M. periclados and C. cervicornis.

The phytochemical studies on the three Sargassum species from Iranian coast, viz., S. angustifolium, S. oligocystum, and S. boveanum, reported the presence of tannins and absence of glycosides in these seaweeds except in S. oligocystum (Mehdinezhad et al. 2016). However, contradictory to Mehdinezhad et al. (2016), in the present study, we observed the presence of glycosides and absence of tannins in these seaweeds except S. oligocystum. Similar to our study, tannins were rare or less abundant in the phytochemical screening of seaweeds from Qatar waters (Heiba et al. 1990). The antioxidant activity of seaweed polyphenols, tannins, sulfated polysaccharides, protein, and peptides is well known (Farvin et al. 2014; Farvin and Jacobsen 2015). However, the relationship between other chemical compounds, such as alkaloids and saponins, on antioxidant activity needs further investigation.

Antioxidant activity of the seaweed extracts

DPPH radical scavenging activity

DPPH radical scavenging activity of the different extracts is expressed as antiradical power (ARP = 1 / EC50) (Table 4). In general, ethanolic extracts showed high antiradical power when compared with water extracts. In absolute ethanol extracts, the ARP ranged from 0.4 ± 0.01 to 100.0 ± 5.1, and all the Sargassum sp. except S. aquifolium showed high antiradical power. S. boveanum showed the highest antiradical power in absolute ethanol extract, which was followed by S. angustifolium, S. asperifolium, S. oligocystum, and C. cornuta. All the other species showed < 10 antiradical power in absolute ethanolic extracts. In the case of 50% ethanolic extract, the antiradical power ranged from 0.4 ± 0.01 to 40.0 ± 0.1. The highest antiradical power in 50% ethanolic extract was shown by S. asperifolium, which was followed by C. cervicornis, S. aquifolium, S. boveanum, M. periclados, S. oligocystum, C. cornuta, C. dasyphylla, and S. angustifolium. In water extracts, the antiradical power was found to be low and ranged from 0.4 ± 0.01 to 20 ± 0.01. The highest antiradical power in water extract was shown by M. periclados, which was followed by Cladophora sp. and C. cervicornis. The synthetic antioxidant BHT used for the comparison showed an ARP of 153.8 ± 10.8, which was higher than the ARP of different seaweed extracts tested. In the present study, the extracts, which showed high ARP, also contained high TPC, indicating that the phenolic compounds play an essential role in the radical scavenging activity. However, extracts of some species with low phenolic content also showed high radical scavenging ability suggesting that the co-extracted compounds also contributed to radical scavenging activity. For example, in M. periclados, the water and 50% ethanolic extracts, which contain less TPC than absolute ethanolic extracts, showed higher ARP (Table 4). These extracts in addition to phenolic compounds contained saponins and terpenoids (Table 3). Similarly, 50% ethanolic extracts of S. asperifolium and C. cervicornis, which contained less phenolic content than absolute ethanolic extract, showed higher ARP. These extracts also contained saponins, flavonoids, steroids, and terpenoids, which also might have contributed to the overall radical scavenging activity. Besides this, individual phenolics present in the extracts also play an essential role in radical scavenging property.

Table 4 Antiradical power, ferrous iron chelating activity, reducing power, and % inhibition of liposome oxidation by the extracts from selected seaweeds of Kuwait coast
Full size table

Depending upon the structure, number, and location of the hydroxyl groups, the radical scavenging activity of phenolic compounds will vary (Cirillo et al. 2012). Phenolic compounds with two hydroxyl groups, such as caffeic acid, were reported to be more efficient than caumaric acid with one hydroxyl group. Also, trihydroxy phenol, such as gallic acid, is more potent than its dihydroxyl counterparts, such as gentisic and protocatechuic acid (Cirillo et al. 2012). Similarly, phenolics with their second OH− in the ortho- or para-position are more active than the one in the meta-position (Bendary et al. 2013). The flavonoids, such as quercetin, were reported to have better radical scavenging activity than rutin due to the extra OH group located in the C ring of quercetin (Aliaga and Lissi 2004). In the present study, different extracts contained a different combination of these phenolic/flavonoids, which might be the reason for the difference in the radical scavenging activity.

In the present study, all the Sargassum species studied exhibited high radical scavenging activity. The ARP of S. boveanum was comparable to the ARP reported for acetone extracts of Fucus vesiculosus (ARP = 93.9) and Fucus serratus (90.8) from Iceland (Wang et al. 2009). Similar to our study, several authors reported high antioxidant and phenolic content for various Sargassum species collected from Arabian Gulf (Zahra et al. 2007; Sadati et al. 2011). Zahra et al. (2007) and Jassbi et al. (2013) also reported a high DPPH radical scavenging activity of S. boveanum water extracts collected from the Iranian coast. However, in the present study, both TPC and radical scavenging activity were higher for ethanolic extracts than for water extracts. Due to the difference in extraction solvents, units used, species, and collection site, the comparison of our results with other studies in Arabian Gulf is not feasible.

Metal chelating property

The Fe2+ chelating activity of the different seaweed extracts at 1 mg concentration is shown in Table 4. In general, water and 50% ethanol extracts showed significantly (P < 0.001) high ferrous ion chelating activity than absolute ethanol extract (Table 4). In the case of absolute ethanol extract, the iron chelating activity ranged from 0 to 68.7 ± 2.2%. The highest iron chelating activity was shown by P. gymnospora, which is followed by C. cervicornis, I. stellata, L. obtusa, and S. angustifolium. Absolute ethanolic extract of Codium sp. showed no iron chelating activity (Table 4). In the case of 50% ethanolic extract, the iron chelating activity ranged from 1 to 87.2 ± 2.1%. The highest iron chelating activity was shown by P. gymnospora followed by P. platycarpa, U. flexosa, F. mitchelliae, M. periclados, F. irregularis, Cladophora sp., and C. cervicornis. In water extracts, the iron chelating activity ranged from 0 to 98.3 ± 0.1%. The highest iron chelating activity was shown by P. platycarpa, which was followed by P. gymnospora, F. irregularis, S. Angustifolium, Cladophora sp., S. boveanum, Grateloupia sp., F. mitchelliae, S. aquifolium, I. stellata, and S. oligocystum. The known chelator EDTA was used for comparison, and it showed an iron chelating activity of 97.0 ± 0.2% even at the concentration of 60 μg/mL. The water extracts of P. platycarpa, P. gymnospora, and F. irregularis at 1 mg concentration were comparable to EDTA at this concentration.

Similar to the present study, the higher iron chelating activity of water and 50% ethanolic extracts of seaweeds was reported by Wang et al. (2009) and Farvin and Jacobsen (2015). The higher iron chelating activity of water and 50% ethanol extracts indicates the importance of polysaccharide and other co-extracted components, such as proteins and peptides in iron chelating activity. The polysaccharide components, such as sulfated polysaccharides and oligosaccharides in seaweeds, are known for their metal chelation (Wang et al. 2008). In addition to this, some peptides and proteins from seaweeds also reported having metal chelating properties (Toyosaki and Iwabuchi 2009). The low iron chelating activity of the absolute ethanol extracts might be due to the absence of polysaccharides and proteins, as absolute ethanol precipitates most of the polysaccharides and proteins.

Interestingly, some species, such as Codium sp., U. flexosa, and S. filamentosa, showed very low iron chelating activity in water and absolute ethanol extract, but showed a high iron chelating activity only in 50% ethanolic extracts. The main difference of these extracts over the other extracts is the presence of saponins in 50% ethanol extracts (Table 3). This might be the reason for the difference in iron chelating activity of these 50% ethanolic extracts. Saponins are reported to have good iron chelating properties (Guelcin et al. 2004). In contrast, certain species like L. obtusa and C. cervicornis showed high iron chelating activity in ethanolic extracts and showed less iron chelating activity in water extracts. The absolute ethanolic extracts of L. obtusa contain epicatechin and salicylic acid, and 50% ethanolic extracts contained salicylic acid. In C. cervicornis, coumarin, salicylic, alkaloids, glycosides, and steroids were present in absolute ethanolic extracts, and salicylic, saponins, and steroids were present in 50% ethanolic extracts. Salicylic acid, alkaloids, and saponins are reported to have good metal chelating properties (Raskin 1992; Jang et al. 2009). The comparatively high metal chelating activity of the absolute and 50% ethanolic extracts of these seaweeds might be due to the presence of these metal chelating compounds. The reports on the metal chelating activity of seaweed polyphenols are controversial. Some studies reported a high metal chelating ability of polyphenols extracted from brown seaweeds (Senevirathne et al. 2006; Chew et al. 2008), and others showed that polyphenols play a minor role in metal chelation (Rice-Evans et al. 1996). An earlier study by Farvin and Jacobsen (2015) on the fractionation of P. fucoides extract into different components, such as polyphenol, protein, polysaccharide, and low molecular weight fraction, also showed a low iron chelating activity and high radical scavenging and reducing power for the polyphenol-rich fractions (Farvin and Jacobsen 2015).

Reducing power

The reducing property of the extracts indicates the presence of reductants, the compounds that have lower reduction potentials. These compounds act as antioxidants either by breaking the free radical chain reaction by donating an electron or preventing peroxide formation by reacting with certain precursors of peroxides. The reducing power of different seaweed extracts is presented in Table 4. In general, ethanolic extracts showed high reducing power than the other extracts. The reducing power of absolute ethanolic extracts ranges from 0.2 ± 0.1 to 1.5 ± 0.01, and S. boveanum showed the highest reducing power. The order of the reducing power in absolute ethanol extract was S. boveanum > C. cervicornis > P. gymnospora > S. oligocystum > S. asperifolium = S. angustifolium > F. irregularis = B. pulmosa > P. platycarpa. The reducing power of 50% ethanolic extracts ranged from 0.2 ± 0.0 to 1.1 ± 0.01, and reducing power was highest for S. boveanum. The reducing power of water extract was less when compared with those of other extracts, and it ranged from 0.1 ± 0.01 to 0.6 ± 0.05. In water extracts also S. boveanum showed the highest reducing power, which was followed by C. cervicornis. Ascorbic acid showed the highest reducing power (2.2 ± 0.1), and none of the extracts were as effective as ascorbic acid. The absolute ethanolic extracts of all the Sargassum sp. and C. cervicornis, which showed high reducing power, also have high TPC (Table 1), suggesting the vital role of phenolic compounds in reducing property.

Inhibition of lipid oxidation in liposomes

In the present study, the effect of the seaweed extracts on phosphatidylcholine liposome oxidation was evaluated in an assay model system where the oxidation was induced by ferric/ascorbic acid. The % inhibition of liposome lipid oxidation for each seaweed extract at 1 mg mL-1 concentration is shown in Table 4. In general, the % inhibition of lipid oxidation was high for ethanolic extracts when compared with those for water extracts with few exceptions. In absolute ethanolic extracts, the % inhibition ranged from − 23.5 ± 8.4 to 75.3 ± 1.3%. The highest prevention of lipid oxidation was shown by P. platicarpa and C. cervicornis. Most of the absolute ethanolic extracts showed > 50% inhibition of liposome oxidation, while P. gymnospora showed a pro-oxidant effect. In 50% ethanolic extract, the % inhibition ranged from − 12.7 ± 2.0 to 46.7 ± 2.3%. The highest inhibition of lipid oxidation was shown by C. cervicornis, and certain species, such as F. irregularis, C. dasyphylla, and P. brodiei showed pro-oxidative effects. In the case of water extracts, the % inhibition of lipid oxidation ranged from − 0.7 ± 2.0 to 60.7 ± 2.3%. P. gymnospora showed the highest inhibition of lipid oxidation in water extract, while F. mitchelliae, C. cervicornis, Grateloupia sp., M. periclados, H. valentiae, and H. cornuta showed a pro-oxidant activity. BHT at 0.2 mg mL−1 (the maximum concentration allowed in food) showed a 30.8% inhibition of lipid oxidation, and some of the seaweed extracts were better than BHT at 1 mg mL−1 concentration.

Though most of the extracts show high radical scavenging, chelating, and reducing power, their effectiveness in the prevention of oxidation in the real lipid-containing system varies. The antioxidant and pro-oxidant activity of the extracts in liposome depends on a number of factors, such as the partition of antioxidant components in the aqueous or lipid phase, the oxidation inducers, the property (reducing power, metal chelating, and radical scavenging), and the amount of the different antioxidant compounds in the extracts (Budilarto and Kamal-Eldin 2015). The absolute ethanolic extracts of most of the seaweeds prevented oxidation of liposomes, indicating the importance of polyphenolic compounds and other co-extracted compounds in antioxidant activity. The absolute ethanol extracts in addition to phenolic compounds may contain tocopherol, a known antioxidant, which also contributes to antioxidant activity (Farvin and Jacobsen 2013). The water extracts of P. gymnospora showed high inhibition of lipid oxidation in liposomes, while absolute ethanol extracts showed a pro-oxidative activity. Though the water extracts of this species contained less phenolic content and low radical scavenging activity when compared with ethanolic extracts, its iron chelating activity was high in water extracts. Thus, these extracts might have chelated the iron added for the induction of oxidation and prevented oxidation. The combination of low iron chelating, high reducing property, and other co-extracted pro-oxidative compounds in the absolute ethanol extract might have contributed to the pro-oxidative nature of this extract. The antioxidant/pro-oxidant ratio is an important determinant of the antioxidant activity of the plant extracts (Ling et al. 2010).

Several studies have shown that the regular consumption of foods high in antioxidants could reduce the risk of heart disease, cancer, and other aging-related diseases (Serra-Majem et al. 2006; Nakayama et al. 2011). Namvar et al. (2012) have shown that the phenolic-rich methanolic extracts of Eucheuma cottoni had breast tumor suppressive effect on rats. The polyphenolic compounds identified in a methanol extract of E. cottonii were catechin, rutin, and quercetin. The crude extracts of Porphyra dentata containing catechol and rutin were reported to have anti-inflammatory property in macrophages (Kazlowska et al. 2010). Seven labdane diterpenoids with potential antibacterial activity have been isolated from Ulva fasciata (Chakraborty et al. 2010). Caulerpin, a bisindole alkaloid isolated from seaweeds of the genus Caulerpa, had anti-inflammatory and antinociceptive properties (De Souza et al. 2009). In addition to this, phenolic-rich seaweed extracts were successfully used as antioxidant in skin care emulsions and in food products (Hermund et al. 2016; Poyato et al. 2017). In the present study, algae from Kuwait coast are also rich in some of the above-mentioned phenolics and phytochemicals, which may be useful for food and pharmaceutical applications. Further studies on the characterization and bioactive potential these seaweed extracts are being carried out in our laboratory.

Conclusions

The study presented here is the first comprehensive report on the antioxidant activities of macroalgae from Kuwait coast of Arabian Gulf, in which phenolic compounds and flavonoids were identified and quantified from a range of seaweeds. This study also reveals the chemical diversity of different seaweeds of this part of the ocean. The result of this study shows that different seaweed extracts varied in their chemical constituents, total, and individual phenolic content and possessed diverse antioxidant properties. The type of solvent played an essential role in the extraction of various components and hence the antioxidant activity. Ethanol was more efficient in the extraction of polyphenols, tannins, steroids, saponins, and glycosides, whereas water was efficient in the extraction of polysaccharides and proteins. A good correlation was shown between TPC, radical scavenging, reducing power, and inhibition of liposome oxidation, indicating that the polyphenols play an essential role in these properties. Iron chelating activity showed no correlation to TPC. Conversely, few species with low TPC showed good antioxidant activity in some of the assays, demonstrating the importance of the co-extracted compounds. In general, the seaweeds from this area with extreme climatic conditions possess more flavonoids than phlorotannins compared with the temperate counterparts, indicating their role in UV protection.

Interestingly, almost all seaweeds contained hydroquinone, a known skin whitening agent, and could be of interest for the cosmetic industry. All the Sargassum sp. and some species, such as C. cervicornis, F. irregularis, P. platycarpa, Chondria sp., and Cladophora sp., showed high antioxidant activity in different antioxidant tests and are rich in polyphenolic compounds. Theses seaweeds could be a potentially rich source of antioxidants. Though the antioxidant activity of synthetic antioxidant, such as BHT, was superior to the extracts, purification of active components of the extracts may improve their activity. Further characterizations of the extracts are going on at our laboratory.