Ultrasonically-extracted marine polysaccharides as potential green antioxidant alternatives

Marine-extracted sulfated polysaccharides (SPs) have been the subject of myriad research since they are considered an eco-friendly source of biologically active compounds. Meanwhile, food and pharmaceutical industries are urgently producing natural sugar substitutes and antioxidants as alternatives to synthetic ones which are associated with cytotoxicity and safety issues. This study assesses the potential of using marine SPs obtained via the ultrasonic-assisted extraction of different marine species, to utilize them as antioxidant sugar substitutes. The carbohydrate, total phenolic contents and antioxidant activities were measured for SP extracts of the algal species of Ulva lactuca, Jania rubens and the marine plant mangrove Avicennia marina. These SPs were structurally elucidated by Fourier Transform Infrared (FTIR) spectroscopic and high-performance liquid chromatography (HPLC) analyses. The results revealed that SPs’ highest yield percent was obtained from Ulva lactuca, 5.50 ± 0.25%. The SPs of Avicennia marina had the highest carbohydrate content, 44 ± 1% and antioxidant activity, 78.85 ± 0.06 at the 100 μg/mL concentration and 89.50 ± 0.21 at the 250 μg/mL concentration. Meanwhile, the highest phenolic content was exhibited by algal SPs obtained from Jania rubens, 132.60 ± 2.50 mgGa/g. Results also showed that all extracts have potent antioxidant activity, while the highest antioxidant activity belonged to the SPs of Avicennia marina owing possibly to their balanced glucose and galactose contents as measured by HPLC. This work emphasizes the need to consider sulfated polysaccharides from marine sources for their antioxidant activity and to correlate it with their monosaccharide content to determine the effect of reducing sugar concentration on the antioxidant activity.


Introduction
Marine organisms have recently gained traction owing to their rich content of bioactive compounds. These organisms are mainly classified into six kingdoms including bacteria, protozoans, algae (seaweeds), fungi, plants like mangroves, and animals [1]. Some marine organisms are edible like sea lettuce (Ulva spp.) [2] and fruits of mangroves [3], and can, hence, be used in nutraceuticals after considering food safety measures. The antioxidants extracted from mangroves, particularly in the form of herbal drugs, play an essential role in protecting from free radical-induced injury [4]. The same applies for the antioxidants extracted from green algae (Ulva lactuca) which could be used in food supplements [5].
The extracts of these marine species contain polyphenolic compounds and polysaccharides that contribute to the antioxidant activity exhibited by the extracts [6]. As such, sulfated polysaccharides, especially fucans extracted from brown seaweeds, could be used as sources of natural antioxidant alternatives with potential food industry applications [7]. The search for natural antioxidants as alternatives to synthetic ones is of great interest recently due to their potential as economic, safe and non-toxic compounds [8,9]. These species are also edible, and after appropriate processing, they could be used as antioxidant supplements.
In the current study, sulfated polysaccharides (SPs) were extracted from the different marine species of green algae, red algae, and mangrove leaves using ultrasonic (US) extraction. These extracts were tested for their carbohydrate and total phenolic contents (TPC) then examined for their structure by Fourier Transform Infrared (FTIR) Spectroscopy. The sugar content, which was analyzed by highperformance liquid chromatography (HPLC), was then correlated to antioxidant activity (AO), tested by 1-Diphenyl-2-picrylhydrazyl (DPPH) in order to assess the potential of these extracts as alternative natural antioxidants. The scheme of this work is outlined in Figure 1.

Materials and Methods
Ulva lactuca green algae and Jania rubens red algae were collected from Alexandria city coast (31°19′ N 30°03′ E) and were immediately brought to the laboratory in sterile plastic bags containing seawater in order to prevent evaporation. Fresh mangrove leaves (Avicennia marina (Forssk.) Vierh were collected in winter of 2017 from Ras Mohammed National Park at the Aqaba Gulf in Sinai. Dr. Mohamed Massed Hejazi recognized the plants' voucher samples at the Botany Herbarium Laboratory of the Department of Marine Science, Suez Canal University, Ismailia, Egypt. All marine samples were then washed with fresh water, air dried, ground into powder and stored before extraction at −20 °C.
Ten grams of algal powders were soaked in 200 mL of distilled water and placed for four hours in the ultrasonicator (VWR ® sonicator model 150HT) at 60 °C for Ulva lactuca and at room temperature (RT) for Jania rubens. Meanwhile, the same ratio of mangrove leaves powder and distilled water were mixed for 15 min at RT in an ultrasonicator (Branson 2510 sonicator, model 2510DTH, Hampton, NH, USA). As per the reported literature, red algae and mangroves contained thermolabile metabolites, hence extraction under high temperature was avoided. All marine suspensions were filtered through a cheese cloth. The filtrates were separately dialyzed, collected and added to ethanol in the proportion of 1:3/3.5 (v/v) as previously reported [10,11] and then the supernatants (SPs) were collected. Extraction was performed in triplicate.
The respective total carbohydrate and phenolic contents of the extracted marine SPs were determined using glucose and gallic acid as standards, as described in our previous studies [5,12].
FTIR spectroscopy was used to determine the functional groups of the SPs of each marine species. In this regard, the powdered SPs were mixed with potassium bromide to form 1-mm pellets. Analysis was carried out using a Nicolet 380 Thermogravimetric Analysis/Fourier Transform Infrared (TGA/FTIR) spectrometer for a range of 500 to 4000 cm −1 .
High-performance liquid chromatography (HPLC) was performed to estimate the monosaccharide content of these SPs as previously outlined [10,11].
The free radical scavenging activity of the marine SPs was measured using 1,1-Diphenyl-2picrylhydrazyl (DPPH) antioxidant assay following Blois's method [13] with minor modifications. The radical scavenging activity was determined using equation 1 in which AR is the absorbance of the pure DPPH while As is the absorbance of the sample SPs in the presence of DPPH.

Results and Discussion
The ultrasonic-assisted extraction of SPs from the green U. lactuca (GU-SPs), the red J. rubens (RJ-SPs) and the mangrove A. marina (M-SPs) produced SPs yields of 5.50 ± 0.25, 0.36 ± 0.04 and 3.52 ± 0.94%, respectively. As shown in Figure 2a, the green and red algal SPs of GU-SPs and RJ-SPs constituted 7.70 ± 0.10 and 2.60 ± 0.30% carbohydrates, respectively, while the mangrove SPs exhibited a much higher carbohydrate content of 44.0 ± 1.00%. Figure 2b depicts the phenolic contents in mgGA/g which amount to 60.60 ± 7.90 for GU-SPs, 132.60 ± 2.50 for RJ-SPs and 50.62 ± 0.10 for M-SPs. The red algae SPs possess the highest phenolic content, whereas those of the green algae have comparable content to that of the mangrove SPs. Table 1 shows the structural elucidation of the extracted SPs by FTIR. The FTIR spectra ( Figures  S1,S2) [14,15]. On the other hand, spectra of the red algal RJ-SPs ( Figure S3) exhibit all the previously mentioned bands with the exception of the sulfate ester band at 1260 cm −1 ; however, the presence of sulfate is confirmed by the appearance of the bands corresponding to the galactose sulfate groups at 850 cm −1 .
The sugar contents of the extracted marine SPs were measured by HPLC and their values were compiled in Table 2. Results reveal that GU-SPs has glucuronic acid as the most dominant sugar, possibly due to the high temperature (60 °C) used for its extraction and which could have led to the oxidation of glucose and galactose fractions to glucuronic acid. As for RJ-SPs, it is primarily composed of glucose (94.04%), as a result of the ultrasonic-aided conversion of galactose to glucose [10]. On the other hand, M-SPs consisted mainly of glucose and galactose with 24.51 and 17.46%, respectively, probably due to its shorter extraction duration and consequently less exposure time to the ultrasonic waves that can convert galactose to glucose [11]. Table 1. Functional groups of the red Jania rubens SPs (RJ-SPs), the green Ulva lactuca SPs (GU-SPs) and the mangrove A. marina (M-SPs).   To assess their potential as antioxidants, the marine SPs used in this study were tested for their antioxidant activities at two different concentrations-100 and 250 μg/mL. Ascorbic acid was used as a reference standard and its antioxidant profile is given in Figure S4. The results, depicted in Figure  2c, show that the highest % DPPH scavenging activity was exhibited by M-SPs of mangrove yielding 79.85 ± 0.06% and 89.50 ± 0.21% at the two applied concentrations, respectively. Glucose is the principal sugar constituent in RJ-SPs and M-SPs comprising about 94.04 and 24.51%, respectively; which might have contributed to the antioxidant activities of these extracts, while the balanced amounts of glucose and galactose in M-SPs might be responsible for the higher antioxidant activity of M-SPs relative to the other two extracts.

Conclusions
Sulfated polysaccharides of the marine organisms U. lactuca, J. rubens and A. marina, were extracted using ultrasonication. SPs of U. lactuca showed the highest yield-5.50 ± 0.25% of all marine species being studied. Meanwhile, the SPs of A. marina exhibited the highest carbohydrate content-44 ± 1%. The SPs of J. rubens were characterized by the highest phenolic content-132.6 ± 2.5 mg GA/g. However, SPs of A. marina showed the highest antioxidant activity among other extracts at the two applied concentrations indicating that its SPs could be utilized as antioxidant alternatives. As per the HPLC analysis, the SPs of J. rubens and A. marina have glucose as their major sugar constituent comprising 94.04% and 24.51%, respectively, while A. marina SPs contain balanced amounts of glucose and galactose which implies that these monosaccharides could be partially responsible for the relatively high antioxidant activities of these SPs. The current study suggests that sulfated polysaccharides extracted from marine organisms can be utilized as natural antioxidant alternatives in diet supplements or nutraceuticals.