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Phycochemical Characterization of Marine macroalgae, Sargassum tenerrimum Collected from Beyt Dwarka, Western Coast of Gujarat, India

Industries are moving towards finding a natural source of functionally active constituents which is better and safer materials to fulfill customers’ demand. Marine algae contain a huge variety of biologically active compounds and express a promising role in different applications. Hence, the present study was carried out to characterize different biochemicals from brown alga Sargassum tenerrimum by FTIR, GCMS, HRLCMS Q-TOF, and ICP AES technique. First, the macroalga was collected from the Beyt Dwarka sea site, the Western coast of Gujarat, India. In FTIR, different types of bioactive functional groups were characterized as accountable for different beneficial components whereas ethanolic and methanolic extracts of S. tenerrimum reported fourteen and nineteen different beneficial phyco compounds in GCMS analysis, respectively. In HRLCMS Q-TOF analysis, two compounds were found carbohydrate derivatives and fifty-nine different compounds were determined to be different types of phycocompounds. Additionally, in the ICP AES study, Silicon was found to be high up in amount whereas Copper remained the minimum among studied elements. Moreover, the HRLCMS-QTOF study of amino acids reported that Glutamic acid (132.13 nmol/mL) was found to be the highest whereas Isoleucine (0.70 nmol/mL) was found to be the least amidst measured twenty-one amino acids. Bioactive potentials of these compounds have been reported in many previous studies. The inclusion of macroalgae-derived constituents in different applications has been broadly rising due to their bioactive potentials.


INTRODUCTION
High energy requirements, natural, nutritious, harmless, and nontoxic compounds in different types of products such as food, cosmetics, dairy, pharma as well as aquaculture supplements promote the search for alternative natural renewable and sustainable materials 1 . These compounds are eco-friendly, sustainable, and economically cheaper. Mainly, great attention towards using biomass for overcoming the demerits of harmful and synthetic components. There are different types of natural resources utilized for different applications but among them, marine macroalgae are well known due to having high nutritional compositions as well as the presence of bioactive compounds than terrestrial plants and animals 2 . Marine macroalgae are similarly known as seaweeds, which are macroscopic, multicellular, eukaryotic, marine photosynthetic microorganisms. It is highly diversified in length and morphology 3 . They are mainly growing in brackish water as well as coastal zone (water close to the sea coast). It is found attached to rocky sea fronts, sand gravels, or floating freely 4 . Some species of macroalgae can reach up to 65 meters in length. Seaweed species occupy several ecological areas. Some algal species are found wet in seafoam whereas other algae can attach to substratum several meter deeps. Its growth expands for miles to the deeper sea 5 . Moreover, other species have become to live in tide pools and must tolerate hasty changes in environments such as temperature, drying, and salinity 6 . According to pigment composition, it can be divided into three types: red algae, brown algae, and green algae belong to Rhodophyta, Phaeophyta, and Chlorophyta, respectively. Among these three types, the Plantae kingdom comprises Chlorophyta and Rhodophyta whereas the Chromista kingdom comprises Phaeophyta [7][8][9][10] . Red algae mainly contain phycocyanin, phycoerythrin, chlorophyll a, and carotenoids such as lutein, zeaxanthin, and beta carotene. Brown algae are well known for chlorophyll (a & c), and carotenoids such as fucoxanthin whereas chlorophyll a, b, and carotenoids are found in green algae 11 . As a result of the diversified components, macroalgae are extensively useful in many regions of the world as an ingredient of human food and animal feed, pharmaceutical, cosmeceutical, agriculture, and many more 12 . Marine algae produce proteins, amino acids, carbohydrates, fatty acids (primary metabolites) which play their role in reproduction and normal growth as well as phenols, sterols, minerals, vitamins, and some other active constituents (secondary metabolites) found under stressful environmental factors such as Ultraviolet exposure, temperature, high salinity or pollutants [13][14][15][16] .
Many marine algae have been completely practiced by humans for different uses as food for humans, animal feed, fertilizers, dairy and food industries, etc. China and Europe have had old practices to collect marine algae for nutritional food purposes as long as 500 B.C. 17 Macroalgae can be used as an alternative to today's foods that we consume. It gives us high nutritional content, high caloric, and lesser fat. Macroalgae provide a good amount of carbohydrates, vitamins, minerals, and fibers that improve intestinal transition and reduce the cholesterol amount in the blood 18 . They also have an old use as an animal food but it depends on the algal species, variable composition, time of collection, and habitat as well as some other atmospheric factors such as light intensity, temperature, and nutrient availability 19 . Macroalgae have wide application in the agriculture sector to improve crop productivity. It contributes certain elements such as nitrogen, phosphorus, potassium, magnesium, iron, and calcium. Besides, it is utilized to regulate the pH of the soils and possesses plant growth-promoting components such as auxins, gibberellin, cytokinin, and others 20 . It is also utilized to combat different plant infections caused by different insects and fungal pathogens 21 . Marine algae-derived bioactive molecules are widely used in various industrial applications like, shows anticoagulant, antitumor, anti-inflammatory, antimicrobial, antithrombotic, and immunomodulatory 22,23 Seaweed based bioactive compounds are useful in making soft drinks, glass, soaps, shampoos, dyes as well as biopolymers [24][25][26] . The phycocolloids exhibit an important action in pharmaceutical applications as a stabilizer, a thickener, as well as an, impart antimicrobial component 27 . Macroalgae are widely advantageous in the cure and hair washing due to their biding ability with hair proteins 28 . Previous studies reported that it can be used to treat warms infection, wound healing, treatment of gastritis, diarrhea, menstrual disorders, hypertension, skin disorders, ulcers, and syphilis [29][30][31] . Moreover, it is also useful in the skin cosmeceutical applications such as antiwrinkle, photoprotection, skin whitening, anti-aging, moisturizer, antiacne, and other skin benefits 32,33 .
Brown macroalga Sargassum tenerrimum is very commonly found on the western coast of Gujarat, India. The thallus of S. tenerrimum is soft, slender, light brown to greening brown in the color of about 30-40 cm in height. It has a very short main axis, terete, and alternatively arranged primary cylindrical branches with branchlets. Vesicles of S. tenerrimum are stalked, spherical, pointed, and ovate about 3-4 mm broad. It is fusiform, slightly compressive, edged, and possess toothed at the margin. Its holdfast discoid and grow on rocks in subtidal regions 34,35 . The present study aims to carry out the characterization of brown alga S. tenerrimum derived bioactive components such as fatty acids, amino acids, carbohydrates derivatives, as well as mineral content analysis by using different characterization techniques to show its phycochemical profile.

MATERIALS AND METHODS
hot percolation in the Soxhlet procedure. Afterward, the extract was then filtered and dried at 40°C for 24 h in a Hot air oven (REMI, India) to remove the excessive ethanol solvent (Sigma-Aldrich, India). This obtained filtrate was fixated to dryness at 20°C in reduced pressure (150 mbar) by using a vacuum evaporator (Sigma scientific, India). The aboveconcentrated extract was isolated in the air-free vessel and kept at freezing temperature (-20°C, Esquire Biotech, India).

Sample collection
The sample of marine macroalga Sargassum tenerrimum was done in sterile plastic bags from Beyt Dwarka sea site (22°28'37.7"N 69°07'48.2"E), the West coast of Gujarat, India. (Fig. 2-a) After collection, the sample was transferred (10°C) to the laboratory. This sample collection was carried out by handpicking from the low tide zone and environmental surroundings as follows: 0% Precipitation, 55% Humidity, 12 km/h Wind, and 27°C temperature. Then, this sample was cleaned with deionized water to separate extraneous materials and debris and kept shed dry at room temperature for six to seven days. This dried material was reduced to a fine powder by a mechanical grinder and preserved at freezing temperature until the next analysis.

Identification of Sargassum tenerrimum
The sample identification was accomplished by taking the help of Dr. N. Joshi, at the Aquaculture department, Veraval, Gujarat-India. The authenticated image is illustrated in Figure.

Phycocompounds characterization study by GCMS technique Extract Preparation
0.5 kg of dry S. tenerrimum's dried powder was added in 80% ethanol for 24 h by the continuous

GCMS Characterization study
For the characterization study, the T100GCV GC model and EB 5 column were used for chromatographic separation. GC specifications such as Helium (He) used as a Carrier gas; 1 mL/ min flow rate; 200°C Injector operation; 50-250°C Column oven temp.; Injection mode: 10 °C/minute.
AccuTof Mass from the jeol model was used in mass spectrometry analysis. MS specifications are as follows: 70 eV Ionization voltage; 250°C ion source temperature; 250°C interface temperature; 50-600 mass units mass range. The obtained gas chromatogram and mass spectra of the screened compounds were put in comparison with the available data of known compounds in the National Institute of Standard and Technology (NIST) library ver. 2005.

Characterization of Fatty Acids
The methanol solvent (99.8%) was used to prepare an extract from the shed dried fine powder of S. tenerrimum. This extract was prepared 10% (ratio 1:10) in a flask for three days. The above mixture was filtered out in another sterile vessel. After filtrates collection, the surplus solvent was eliminated by vacuum evaporator using fresh methanol solvent. This process was carried out twice with the same sample residues using fresh methanol. And this extract was used for further study. The GCMS characterization study was carried out by fixing the HP-5 column. Other specifications of GC and MS are used the same as above. The obtained gas chromatogram and mass spectra of each phycocompounds were put in comparison with known data in the NIST library (ver. 2005). The chemical identity, mol. weight and chemical framework of all obtained phycocompounds were regained and the percentage peak area was also calculated.

Functional Groups characterization study by FTIR
Total 5 mg of shed dried S. tenerrimum's a fine powder added with FTIR grade KBr (Potassium bromide) and evenly mixed to get a homogenized texture. This mixture was then placed in the mechanical mold using a sterile spatula and pressed by mechanical support for 30 seconds. This pellet of the mixture was conditioned on the pan proceeded for IR study. In this FTIR study, 3000 Hyperion Microscope with Vertex 80 FTIR model (Bruker, Germany) and 400-4000 cm -1 scanning range were used. The peak ratio (the peak value in the IR spectrum) was used to separate the functional groups of components.

Elemental analysis by ICP AES procedure Digestion and extraction procedure
All standard grade reagents (purchased from Sigma-Aldrich, India) were used in this analysis. About 50 milligrams of S. tenerrimum's powder was added into the TFM (modify PTFE-PolyTetraFluoroEthylene-) vessels. Following this, the mixture of reagents (3 mL HCl + 1 mL HNO 3 + 1 mL HF + 1 mL H 2 O 2 ) was added and the vessel was closed immediately. The digestion of this mixture was carried out in a microwave digester (Titan Microwave system, India) based on the below specifications: 15 min hold time 10 min with 130 degrees, and following 190 degrees ramp time. After cooling this hot vessel at 70°C, the sample mixture was vented and opened. By using Milli-Q water, made total volume up to 25 mL and then shaken completely to dilute the rest of the particles adhered to the vessel's wall. Blank was also kept for hydrolysis without adding the sample. The ICP-AES (Inductive Couple Plasma-Atomic Absorption Emission Spectrometer) instrument was considered to measure the amount of different elements. Model of the instrument: ARCOS, Simultaneous ICP Spectrometer.

Amino acid analysis Acid digestion and detection
0.1 gram of S. tenerrimum's fine powder was weighed and added into the 12 mL of 6N hydrochloric acid in it and the mixture was tightly packed after adding pure N 2 gas. Put this test tube at 120°C temperature for16 h in a hot air oven (REMI, India) for digestion. After hydrolysis, filtration was carried out and flash evaporation was done to eliminate excess hydrochloric acid. 0.05 N HCl was used to make a definite amount. Its filtration was carried out by a Whatman filter (0.45µ size). The filtrate was used as a sample for analysis. Moreover, standard amino acids were also run to get a standard chromatogram. Specifications such as, model of the instrument: 6550 iFunnel QTOFs (Quadrupole Time of Flights), Agilent Technologies, USA; Column details: Poroshell HPH C18 (4.6 ×100 mm), 2.7 µ; 60°C temp. in the oven; analysis mode: non-switch flow.

Phyco compounds analysis by HRLCMS-QTOF Acid Hydrolysis and detection
The uniform fine powder was obtained by drying the algal sample at 40°C for 48 hours. A total of 0.1 g of the selected macroalgal sample was placed in a sterile airtight tube and add 10 mL of 2M HCl containing 1% phenol. Then, the tube was tightly closed in presence of N 2 gas and kept at 80°C for 3 h in a Hot air oven, allowing it to cool and Whatman no. 41 paper was used to carry out vacuum filtration. The obtained filtrate was diluted to make a final volume of 25 mL with ultrapure water which was again filtered to get the hydrolysate. Different phycocompounds were analyzed by the HRLCMS QTOF technique. Specifications include.

Characterization of fatty acids and derivatives
Different fatty acids and derivatives are identified in this characterization depending on RT (in min), % peak area, mol. formula, and mol. weight. The gas chromatogram for the methanolic extract is illustrated in Fig. 4 whereas nineteen different phycocompounds were identified which are tabulated in Table 2 with its chemical information. Among identified phycocompounds, cis-Vaccenic acid is found to be the considerable compound that had the large value of % peak area (52.96%) with 19.68 min retention time whereas 9-Octadecenamide compound reported the lowest percentage peak area (0.29%) with 27.41 min retention time. Octadecenamide; 17-Octadecynoic acid; Linoleic acid, phenylmethyl ester; Oleic acid benzyl ester and Oleic acid are fatty acyl compounds which belong to class fatty acids or fatty acid derivatives. In addition, n-Hexadecanoic acid; is a saturated fatty acid. Some other compounds such as 9,17-Octadecadienal, [Z]-and 1-(4-Bromobutyl)-2-piperidinone are aldehyde derivative and organonitrogen heterocyclic compounds, respectively. In addition, carboxylic acid derivative cyclooctane acetic acid,2-oxo-was found. Lastly, Phytol and Patchouli alcohol is found that belong to terpenes (Diterpenes) whereas hydrocarbon derivative methyl di-t-butyl hydroxyhydro cinnamate was also detected. The chemical framework of each compound is revealed in supplementary data Table S2.

FTIR characterization study
Characterization of the functional groups of an active constituent was done by FTIR depending on the value of peaks in the IR spectrum. The FTIR spectrum of S. tenerrimum is depicted in Fig. 5. Peak values, functional group, and compound class are revealed in Table 3

Determination of Elements
Among different mineral elements, Silicon, Potassium, Calcium, and Magnesium were found in large proportion as compared to Sodium, Iron, Boron, and Copper in water extract of S. tenerrimum in ICP AES analysis. The result reported that Silicon (13.67%) was found in the highest amount whereas Boron (0.01%) remained at the lowest. The mineral % follows Silicon > Potassium > Calcium > Magnesium > Sodium > Iron> Boron order. The amount of each element (%) is tabulated in Table 4.

Determination of Amino Acids
In this analysis, a total of twenty-one  Fig. 6. Among 21 amino acids, Glutamic acid, Alanine, Glycine, and Aspartic acid were determined to be higher than 100 nmol/mL whereas Leucine, Serine, Arginine, Threonine, Tyrosine, Lysine, Phenylalanine, valine, and Isoleucine were found to be lower than 100 nmol/ml. Content (nmol/mL) of these amino acids were detected in the below order: Glutamic acid > Alanine > Glycine > Aspartic acid > Leucine > Serine > Arginine > Threonine > Tyrosine > Lysine > Phenylalanine > Valine > Isoleucine. Concentrations of detected amino acids (in nmol/ mL) are presented in Table 5.

CONCLUSION
Oceans contain a huge diversified marine organism, comprise the majority area of the earth. Among them, marine macroalgae offer a wide variety of bioactive ingredients such as polysaccharides, amino acids, proteins, vitamins, fatty acids, bioactive peptides, etc. It confers a broader range of beneficial actions such as antimicrobial, anticancer, antioxidant, anti-inflammation, antiaging, anticancer as well as other benefits in food and dairy sectors, medicinal and pharmaceutical sectors, fuel and remediation, agricultural benefits, etc. Researchers from around the world have demonstrated the biological potential of different macroalgae and derived compounds. The present findings showed that the Phaeophyta S. tenerrimum is a valuable source of bioactive ingredients. In the finding, 1-O-butyl 2-O-(6-ethyloctan-3-yl) benzene-1,2-dicarboxylate; Pentadecanal-; 1-O-tetradecyl 4-O-(2,3,6-trichlorophenyl) butanedioate; 3-Hexanone, 2,5-dimethyl-; Propane, 1,3-bis(octadecyloxy)-; Pentanal; Benzyl icosanoate,1,1,3,3,5,5,7,7,9,9,11,11-Dodecamethylhexasiloxane, and t-Boc-sarcosine, etc. compounds in ethanolic extracts whereas cis-Vaccenic acid, Erucic acid, n-Hexadecanoic acid, 9,17-Octadecadienal, [Z]-, 1-(4-bromobutyl)-2piperidone, 4-Tridecene, (Z)-, 17-Octadecynoic acid and Oleic acid were majorly found in methanolic extract of S. tenerrimum. Likewise, Lipid and lipid-like molecules, Carboxylic acid derivatives, Carbohydrate derivatives, Terpenes like compounds, alkaloids as well as some other organic compounds were screened in the HRLCMS characterization study. In addition, Glutamic Acid, Alanine, Glycine, Aspartic acid, Leucine, Serine, Arginine, Threonine, Tyrosine, and Lysine amino acids were obtained in larger amounts whereas Silicon, Potassium, Calcium, Magnesium, and Sodium elements were measured higher in amount. This finding revealed a very good phycochemical profile and that compounds can be applied in different types of applications after successful experimentation further. These compounds are natural, less toxic, economical, almost inexhaustible, and safer than synthetic ingredients. By different characterization studies, selected marine alga reported a good type of potentially active components. S. tenerrimum or derived bioactive constituents can be utilized whole or part in various applications such as food, beauty enhancer, medicinal and pharmacological properties, antioxidant activities, antimicrobial, anti-inflammatory, anticancer, antidiabetic activity, antiviral activity, cellular growth, plant growth promotion, vermifuge activity, antitumor activity, antiulcer, wound healing treatment, Goitre treatment, industrial for fuel production, renewable energy suppliers, animal feed preparation, organic manure preparation, domestic sewage treatment, wastewater treatment, etc. Along with its large availability of biomass (particularly on the Beyt Dwarka sea site), no need to worry about its cultivation, it could be utilized in various applications or product preparations after successful In vitro and in vivo evaluation as well as clinical assessment.