INTRODUCTION:

Marine macroalgae are important ecologically and commercially to many regions of the world, especially in Asian countries such as China, Japan and Korea ^[1]. They are a valuable food resource which contains low calories, and they are rich in vitamins, minerals, proteins, polysaccharides, steroids and dietary fibers^[2-4]. Seaweeds have been one of the richest and most promising sources of bioactive primary and secondary metabolites^[5] and their discovery has significantly expanded in the past three decades^[1,6,7].

The algae synthesize a variety of compounds such as carotenoids, terpenoids, xanthophylls, chlorophyll, vitamins, saturated and polyunsaturated fatty acids, amino acids, acetogenins, and antioxidants such as polyphenols, alkaloids, halogenated compounds and polysaccharides such as agar, carrageenan, proteoglycans, alginate, laminaran, rharrman sulfate, galactosyl glycerol and fucoidan ^[8-17].

These compounds probably have diverse simultaneous functions for the seaweeds and can act as allelopathic, antimicrobial, antifouling, and herbivore deterrents, or as ultraviolet-screening agents ^[18]. They are also used by the pharmaceutical industry in drug development to treat diseases like cancer, acquired immune-deficiency syndrome (AIDS), inflammation, pain, arthritis, infection for virus, bacteria and fungus ^[19]. As compared to terrestrial plants these marine plants is embryonic stage because it is lack of an analogous etho medical history. Marine floras have been used for medicinal purposes in India, China and coastal countries, since ancient times.

These seaweeds especially brown seaweeds are rich in proteins,iodine, sterols, poly phenols, poly saccharides and dietary fibers are perform various functions which are applicable for antioxidant and anticancer activities ^[20].

MARINE BROWN ALGAE:

Brown algae Phaeophyceae^[21] are a class of algae consisting mainly of complex, macroscopic seaweeds whose brown colour come from a carotenoid pigment, fucoxan thin, and in some species ,various phaephycean tannins .The marine brown algae are macroalgae hence they are macroscopic and multicellular. The seaweeds have made its place in the field of research and development as they are widely used as food, medicine ,fertilizer etc. Inhabiting great array of habitats. Critical primary producers in pelagic and coastal environments and in both temperate and tropical regions around the world. Brown algae, which comprise the Class Phaeophyceae ,are unique in being very far removed phylogenetically from all other eukaryotic macrophytes^[22].

PHYTOCHEMICALS:

Phytochemicals are responsible for medicinal activity of plants ^[23]. These are non-nutritive chemicals that have protected human from various diseases. So, phytochemical analysis of the seaweeds will be a good preliminary approach to reveal its secondary metabolite constituents and the resultant medicinal values. Macroalgae produce a wide variety of chemically active metabolites including alkaloids, polyketides, cyclic peptide, polysaccharide, phlorotannins, diterpenoids, quinones, lipids and glycerols that have a broad range of biological activities.

Preliminary phytochemical screening is a part of chemical evaluation. The two main methods used are qualitative and quantitative analysis. The qualitative tests are used to identify the constituents. The quantitative tests are used to identify or determine the amount of active constituents present.

MATERIALS AND METHODS:

COLLECTION OF THE SAMPLE:

Study area Collection site was along coast of Mandapam, Rameshwaram it is one of the most important places of interest for algal growth in Tamil Nadu. The selected sample of Turbinaria ornata is a known species of brown seaweed. It was collected by hand, washed with seawater at the sampling site to remove the adhered sediments and impurities and then packed in polyethylene bags and brought to the laboratory for further analyses.

PREPARATION OF THE SAMPLE:

The collected sample was washed successively with tap water and distilled water to remove all the salt on the surface. The water was drained off and the seaweed was spread on blotting paper to remove excess water. The clean seaweed was shade dried for one week under room temperature and was milled to obtain the powdered form. The dried algal material was ground to 2 mm or smaller particle size. The powdered sample was stored for further use.

PREPARATION OF ALGAL EXTRACTS:

The seaweed powder was successively extracted using solvents of increasing polarity^[24] , with some modifications. 10 g powdered algal sample was initially soaked in 100 ml of methanol and sterile double distilled water in air tight flask. The flask was periodically subjected to shaking on an electronic shaker for 24 hours at 37˚ C and then it was first filtered through double layered muslin cloth and then filtered through Whatmann No.1 filter paper and filtrate was collected into sterile air tight bottle. The extract was stored in refrigerator for further use.

QUALITATIVE ANALYSIS:

The phytochemical screening of different algal extracts was assessed by standard method^[25]. Phytochemical screening was carried out to identify the major natural chemical groups such as alkaloids, terpenoids,steroids, tannins, saponins, flavonoids, phenols, quinones and glycosides. General reactions in these analyses revealed the presence or absence of these compounds in the algal extracts tested.

TESTS FOR ALKALOIDS:

A small quantity of the extracts were separately treated with few drops of dilute hydrochloric acid and filtered. The filtrates were used for the following tests.

Wagner's test:

0.5 ml of each plant extract was treated with Wager's reagent gives a brown or reddish brown precipitate.

TEST FOR CARBOHYDRATE:

The minimum amount of extracts were dissolved in 5m1 of distilled water and filtered. The filtrate was subjected to the following test to detect the presence of carbohydrates.

Fehling's Test:

0.5 nil of plant extract was mixed add equal qty of Fehling's sol. A & B. After heating brick red precipitate is obtained.

TEST FOR GLYCOSIDE:

Borntrager's Test:

To the test tubes containing 2 ml of extract 2 ml of dilute sulphuric acid was added, boil for 5 min and filtered. To the filtrates, equal volumes of chloroform was added and mixed well. organic layers were separated and ammonia was added to this. Pinkish red colour of the ammonia layer indicates the presence of anthraquinone glycosides

Test For Saponins:

Foam Test: The extract (2g) was shaken vigorously with 20 nil of water and observed for persistent foam, which indicates the presence of saponins.

Test for Proteins:

Biuret's Test: To 3 ml of extract 1 ml of 4% w/v sodium hydroxide and 1 ml of 1% w/v copper sulphate were added. The change in colour of the solution to violet or pink indicates the presence of proteins.

NINHYDRIN TEST:

Two drops of ninhydrin solution (10 mg ninhydrin in 200 ml of acetone) was added to 2 ml of aqueous filtrate. A characteristic purple colour indicates the presence of aminoacids.

Test for Phenolic compounds:

Ferric chloride test:

The extract was dissolved in 5 ml of distilled water. To this, few drops of neutral 5 % ferric chloride solution was added. A dark green colour indicates the presence of phenolic compounds.

Test for terpenoids (Salkowski test):

To 0.5 g each of the extract was added 2 ml of chloroform. Concentrated H₂SO₄ (3 ml) was carefully added to form a layer. A reddish brown colouration of the interface indicates the presence of terpenoids.

Test for steroids (Libermann test):

To 0.5 ml of the extract of plant was mixed with 2 ml of acetic anhydride, 2 ml of chloroform followed by 2 ml of sulphuric acid. The colour changed from violet to blue or green in some samples indicated the presence of steroids.

Test for flavonoids:

The stock solution (1 Intl was taken in a test tube and added few drop of dilute NaOH solution. An intense yellow colour was appeared in the test tube. It became colourless when on addition of a few drop of dilute acid that indicated the presence of flavonoids.

Test for tannins:

The stock solution (3 mL) was taken in a test tube and diluted with chloroform and added acetic anhydride (1 mL). Finally, sulphuric acid (1 mL) was added carefully by the side of test tube to the solution. A green colour was formed which showed the presence of tannins.

Quantitative analysis:

Quantitative analysis of phytochemical substances in algal extracts

1. Estimation of phenols:

Phenols react with phosphomolybdic acid in Folin-Ciocalteau"s reagent in alkaline medium to produce a blue-colored complex (molybdenum blue) which can be estimated spectrophotometrically at 760 nm.

Procedure:

Total phenols in different algal extracts were determined according to the protocol described by Chandler and Dodds.

One millimetre of sample was mixed in a test tube containing 1 ml of 95% ethano1, 5m1 of distilled water, and 0.5 ml of Folin-Ciocalteau reagent.

Mixture was allowed to react for 5 min, after which 1 ml of 5% Na2CO3 was mixed thoroughly and placed in the dark for one hour.

Absorbance was measured at 720nm using a UV-VIS spectrometer.

Gallic acid in the range of 20-200 mg/L was used to construct a calibration curve.

Estimation of the total phenols was carried out in triplicate.

The results were mean values ± standard deviations and expressed as mg Gallic acid equivalent (GAE)/g dried weight.

Estimation of flavonoids:

Principle:

Total flavonoids content in alcoholic and aqueous extracts of was measured by aluminum chloride colorimeter assay. Flavonoids react with aluminum chloride reagent to produce a coloured product which can be measured spectrophotometrically at 510nm.

Procedure:

Total flavonoids in different algal extracts were determined by aluminium chloride calorimetric method described by Brighente et. al., (2007).

_{0.5 ml of 2% aluminium chloride in methanol was mixed
with the same volume of algal} extract.

After 1 hour incubation at room temperature, the absorbance of the mixtures was measured at 415 nm using UV/Vis spectrophotometer.

Rutin in the range of 20-200 mg/L was used to construct a calibration curve.Estimation of the total flavonoids was carried out in triplicate.

The results were mean values + standard deviations and expressed as mg rutin equivalents (RU)/g dried weight.

RESULT:

COLLECTION OF SAMPLE AND ITS PREPARATION:

Three kg marine macroalgae Turbinaria ornata was collected from the coast of Mandapam, TamilNadu. The fresh sample was in dark green in colour and washed with sterile distilled water and dried (Fig.1).The dried sample was very hard in nature.Then the dried algal sample was milled using the electric grinder to get the powdered form for the further extraction process (Fig.2.).

Fig 1: Fresh algal sample

Fig 2: Dried powder of the algal sample

Extract Characteristics:

1g of the dried powdered sample was mixed with 100m1 of two different solvents (Methanol,Aqueous solution) in two different flaks .The mixture of algae with solvent was kept in orbital shaker for 24 hours and then the solvent was filtered and then the solvent was used to determine the further activities.

PHYTOCHEMICAL ANALYSIS:

Two different extracts of the algae Turbinaria ornata was determine to check the phytochemical analysis of the sample.The powder sample of marine brown algae showed the presence of alkaloids, carbohydrates, saponins, glycosides, proteins and amino acids, steroids, phenolic compounds, flavonoids, terpenoids, tannins which added to its potentiality as a bioactive principle. Results of preliminary phytochemical analysis of various extracts of Turbinaria ornata are given in Table 1.

TABLE 1 Preliminary Phytochemical Analysis of Different Solvent Extracts

	Results
Test	Methanolic Extract	Aqueous Extract
ALKALOIDS	+	−
CARBOHYDRATES	−	−
GLYCOSIDES	−	+
SAPONINS	−	−
PROTEINS	−	+
AMINOACIDS	+	−
PHENOLS	+	+
TERPENOIDS	−	+
STEROIDS	+	−
FLAVANOIDS	+	+
TANNIN	+	−

QUANTITATIVE ANALYSIS:

The biochemical composition of seaweeds Turbinaria ornata were studied on dry weight basis. Macronutrients such as total phenols and total flavonoid content were quantified using spectrophotometric method. The total phenol contents of the crude extracts as determined by established method are reported as gallic acid equivalents. Among the two extracts, aqueous extract contained the highest amount of phenol and flavanoid (Fig 3) compounds of about 1.187 and 1.020. The methanol extract contained 0.264 of phenol and 0.215 of flavanoid.

Fig 3: Quantitative Estimation of phenolic and flavonoid content from Methanol and Aqueous extract.

The chemical constituents in the algal or crude extracts are known to be biologically active ingredients. The phytochemical screening of crude extracts from dry powder algal samples of T. ornata is used in this present study. This revealed that the crude extracts contained flavonoids, saponins and steroids. The screening of the methanol extracts showed the presence of active constituents like Alkaloids, amino acid, steroids, flavanoids, Tannins, phenols. The other groups of compounds were not present in methanol extracts. Phenol, Glycoside, Protein, Terpenoids and flavoinoids are present in the aqueous extract.

The total phenol contents of the crude extracts as determined by established method are reported as gallic acid equivalents. Among the two extracts, aqueous extract contained the highest amount of phenol compounds. The difference of results obtained might possibly be due to the different method of extraction and solvents polarities. During the samples processing and drying, may be some volatiles active compounds destroyed or evaporated from the samples.

CONCLUSION:

Medicinal plants are the best sources for chemical ingredients, antimicrobial and antioxidant agents for cure of different diseases. The aqueous and methanol crude extracts from T. ornata showed good amounts of total phenol and flavonoids contents and these crude extracts could be used as antibiotics or different aliments.

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Received on 26.08.2018 Modified on 29.09.2018

Research J. Pharm. and Tech 2019; 12(1): 108-112.

DOI: 10.5958/0974-360X.2019.00021.0