Algae from marine source are potential renewable resource and has been widely used as antioxidants, anti-mutagens, anti-coagulants etc. Considering the various medicinal potentials of marine algae, the present study was carried out on the preliminary phytochemical screening, free radical scavenging activity, anti-Î±-glucosidase activity and anti-Î±-amylase activity of various extracts prepared from the marine sample, Chlorodesmis sp, collected from the coastal areas of Mahabalipuram, Tamil Nadu, India. Phytochemicals were extracted using various solvents such as methanol, ethanol, acetone and ethyl acetate and tested for bioactivities. The seaweed showed potential antioxidant activity, along with alpha amylase and alpha-glucosidase inhibitory activity. Out of which methanolic extract has shown significant activity, with IC50 value of anti-DPPH assay found upto 0.075mg/ml. IC50 value of inhibiting Î±-glucosidase and Î±-amylase were 0.28mg/ml and 0.18mg/ml respectively. Phytochemical screening has shown the presence of alkaloids, carbohydrates, phenolic compounds, proteins and amino acids, confirming that the seaweed Chlorodesmis sp can be used as a possible source of various anti-oxidant compounds, alpha amylase inhibitory compounds etc in future studies.
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Keywords: antioxidant, phytochemical screening, free radical scavenging activity, anti-Î±-amylase, anti-Î±-glucosidase, seaweed, IC50.
Any large marine benthic algae, which can be differentiated from algae of microscopic size with naked eye can be referred to as seaweeds. Algae of such types are usually multicellular, thus macroscopic along with macrothallic body. From early times, seaweeds have been used as potential medicinal sources for human use. Phytochemicals isolated from marine algae have been of immense use in various pharmaceutical industries, dealing with the production of medicines for the treatment of various diseases. Seaweeds have also been considered as extraordinary sustainable resources in the marine ecosystem which have been used as a source of food, feed and medicines. Hydrocolloids like alginate, agar, carrageenan and other gelatinous substances, extracted from marine algae , attain significant commercial role as food additives. The food industry have exploited their gelling, water-retention, emulsifying and other physical properties. The mineral nutrients present are diverseand, of which iodine component have been potentially used to prevent goitre. Other compounds have been implicated in the treatment of diseases like tuberculosis, arthritis, colds and influenza, worm infestations and even tumors in some instances. Among other uses, seaweeds have also been considered for the production of bioethanol.
Materials and Methods
Collection and Identification of the sample:- The sample was collected from the coastal areas of Mahabalipuram, situated in the state of Tamil Nadu (12.63Â°N, 80.17Â°E). Marine algae sample was collected manually, by hand picking, wearing gloves from the coastal rocks, along the seashores and was transferred to the Marine biotechnology and Biomedicine Laboratory of VIT University, Vellore, Tamil Nadu. The algal sample was identified to be Clorodesmis sp. and the bioactive assays were also conducted.
Sample Preparation:- The marine algae that were collected from the seashore, were immediately washed with seawater, a number of times to irradicate any epiphytes and extraneous, unwanted matter. After seawater wash, the sample washed again, this time with freshwater to remove other contaminations, Once the washing was done, the algae was transferred to sterile zip-locked polyethene bags and transported to the laboratory at a temperature of 4Â°C. In the laboratory, the sample was washed again, first using distilled water and then ethanol, just to ensure that no superficial contaminations remain. Once washed, the algae was sun-dried, cut into small pieces and powdered in a mixer grinder. The dried sample can also be subjected to ultrasonication for the betterment of sample preparation in any further studies that is conducted in near future.
Aqueous extraction of potential bioactive compounds from the algae:- Sterile 100ml conical flasks were taken, into which 1gm of the powdered sample was transferred and 30ml of sterile deionised water was added to it. The conical was then kept for heating at the water bath for 1hr, temperature being set to 100Â°C. This was allowed to soak for 24hours at room temperature. The soaked sample mixture was taken into 50ml sterile centrifuge tubes and the sample was centrifuges at 2500rpm for 8mins at 4Â°C, using a cooling centrifuge. The supernatant was transferred to another 50ml sterile conical flask, filtering it using a sterile Whatmann filter paper no.1. As an extra precaution, the filtrate was also filtered using a cellulose-acetate membrane filter in a 5ml sterile syringe.
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Solvent extraction of potential bioactive compounds from the algae:- For solvent extraction, 0.5gm of the powdered sample was taken into 50ml sterile conical flasks, to which each of the solvents like methanol, ethanol, ethyl acetate, n-butanol and acetone were added gradually in each conical. The dried sample were kept for soaking in each solvent for 48hrs. Each of these mixtures were taken into 50ml sterile centrifuge tubes, and following to which, centrifugation at 2500rpm for 8mins at 4Â°C was performed. The supernatants were transferred to 50ml sterile conical flasks after filtering through sterile Whatmann filter paper no.1. Filtration using cellulose-acetate membrane and 5ml sterile syringe was also performed in this case.
Detection of alkaloids: 500Âµl of each solvent extract, was taken in a test tube; to it 2ml of dilute hydrochloric acid was added and mixed well. Now to this mixture 2-3 drops of Mayer's reagent was added by the side of the tube and then observed for white coloured creamy precipitate which indicate the positive result.
[Mayer's Reagent: It was prepared as follows- 1.358g of Mercuric Chloride was dissolved in 60ml of water while 5g of potassium iodine was dissolved in 10ml of water. The two solutions were mixed properly and the volume was adjusted and made up to 100ml with water].
Detection of carbohydrates: 500Âµl of each solvent extract, was taken in a test tube and to it two drops of alcoholic solution of Î±-naphthal were added and shaken well and mixed properly. To this mixture 1ml of conc. Sulphuric acid was added and was allowed to stand for sometime and was then observed for the formation of violet ring which indicate the presence of carbohydrate. This test is commonly known as Molisch's test.
Detection of saponins: For this particular test instead of the aqueous sample we directly opt for the powdered sample which was initially prepared. 2g of the powdered sample was boiled in 10ml of distilled water in the water bath and then filtered. 5ml of the filtrate was then mixed with 2,5ml of distilled water and shaken vigorously for the stable persistent from the formation. The frothing was now subjected to mix with 3-4 drops of olive oil and this mixture was again shaken vigorously and then observed for the formation of emission.
Detection of proteins and amino acids: 500ÂµL of extract solution was mixed with 2ml of acetic anhydride in a test tube. To this few drops of Million's reagent was added. A white precipitate indicates the presence of proteins.
[Million's reagent was prepared by dissolving 1g of mercury in 9ml of fuming nitric acid. When the reaction is complete equal volume of distilled water was added.]
Another test was also done for this analysis:
Biuret tests: 500Âµl of extract solution was treated with one drop of 2 % copper sulphate solution. To this 1ml of methanol was added followed by excess potassium hydroxide pellets, pink colour in the methanolic layer indicates the presence of protein.
Detection of phytosterols: 250Âµl of extract solution was taken in a test tube and to it few drops of neutral ferric chloride solution was added and observed for an array of colours which indicates phytosterols.
Detection of flavonoids: 3ml of dilute ammonium solution, a part of aqueous extract followed by addition of conc. sulphuric acid. Then it was observed for yellow colour which disappears on standing.
Detection of terpenoids: 3ml of each extract was mixed with 0.5ml of chloroform and conc. Sulphuric acid was added along with the sides of the tubes to form a layer and then was observed for reddish brown colour of the interface which indicates the presence of terpenoids.
Free radical scavenging activity (antioxidant):- Any molecule that has the capacity to inhibit the oxidation of other molecules, can be called as an antioxidant. Here, we utilise the stable free radical compound DPPH (1,1-diphenyl-2-picryl hydrazyl) in order to measure the anti-oxidant free radical scavenging activity of the algal sample. Different concentrations i.e. 10Âµg-50Âµg of each of the solvent extract of the sample was prepared and taken into test tubes, properly labelled. The volume of the extract in each test tube was made upto 1ml. To this, 1ml of DPPH-methanolic solution was added and kept for incubation in dark for atleast half an hour. Since DPPH is light sensitive, preparation and addition of DPPH was performed with utmost care in dark. Two controls were also prepared; the positive control consisting of DPPH-methanolic solution and the methanol solution alone acts as the negative control. If the sample contains any anti-oxidant properties, it will react with DPPH to reduce the compound. This reduction, hence results in change of colouration from deep violet to light yellow. The absorbance was measured at 517nm, followed by calculation of percentage of anti-oxidant activity, using the formula,
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% scavenging activity = [(A517 Extract- A517 Control)] Ã- 100
Î±-glucosidase inhibition assay:- Various concentrations of the extract, ranging from 50-300Âµg/ml were prepared. A volume of 100Âµl of the extract solution was taken in individual sterile 2ml eppendorf tubes. 200Âµl of enzyme solution containing Î±-glucosidase(1.0U/ml), dissolved in 0.1M phosphate buffer was added to each of the eppendorf tubes and kept for 10mins incubation at room temperature. Once the pre-incubation is completed, 100Âµl of the substrate i.e. 5mM p-nitrophenyl-Î±-D-glucopyranoside, dissolved in 0.1M phosphate buffer(pH 6.9) was added to each tube individually. All the tubes were kept for 5mins incubation at 25Â°C. a control was also prepared, containing 100Âµl buffer solution instead of the extract. After incubation, absorbance were recorded at 405nm using a UV-visible spectrophotometer. The readings were compared to the absorbance reading of the control. Henceforth, the percentage inhibition activity was calculated using the formula
% inhibitory activity= [(A405 Extract- A405 Control)] Ã- 100
Î±-amylase inhibition assay:- Î±-amylase inhibitors are those, which bind to the enzyme molecule, inhibiting its activity of breaking down starch molecules into glucose and maltose moieties. Different concentrations like 50Âµg-250Âµg of each of the solvent extract of the sample was prepared. 500Âµl of each extract concentration were taken into test tubes, to which 500Âµl of freshly prepared enzyme solution was added (100ml of enzyme solution required equal parts by volume of 20mM disodium hydrogen phosphate, 20mM of sodium dihydrogen phosphate and 6.7mM of sodium hydroxide, with 0.001gm of powdered enzyme added to it). The extracts, with enzyme solution added to it, were incubated for 15mins, followed by addition of 500Âµl of 1% starch solution was then added to each aliquot. This was kept for another short period of incubation for 15mins. The reaction was made to terminate by addition of 1ml of 3,5-dinitrosalicylic acid(DNSA) to the assay mixture (DNSA was prepared using 0.0299gm of Na-K tartarate and 1.6gm of NaOH along with 0.4379g of DNS was added to 40ml of dH2O). After incubation, the volume of each assay mixture was increased upto 10ml with distilled water, followed by recording of the absorbance at 540nm. If the sample contains Î±-amylase inhibitory compounds, it will bind to it, not allowing the enxyme to breakdown the substrate starch.
% inhibition= [(A540 Control - A540 Extract)] Ã-100
Phytochemical Analysis:- Chlorodesmis sp was tested upon the presence of various phytochemicals like alkaloids, carbohydrates, saponins, proteins and amino acids, phytosterols, flavinoids and terpenoids. Methanolic extract showed the presence of the highest number of phytochemicals. It possessed carbohydrates, proteins and amino acids, flavinoids. Presence of alkaloids in the extract were not convincing and requires further investigation. Table 1. shows the results obtained in phytochemical screening.
Table 1. Phytochemical analysis
Name of the tests
Proteins and amino acids
(Magnesium+ HCl reduction)
(Ferric chloride test)
(+++) indicates positive result; (---) indicates negative result; (++-/+-+/-++) indicates
moderate positive result; (+--/-+-/--+) indicates slight positive result.
Anti-scavenging activity:- Solvents extracts prepared from the marine algae were subjected to free radicals scavenging assay. Methanolic extract possessed the highest anti-DPPH activity followed by ethanol and ethyl acetate. IC50 value (figure 2.) for the methanol extract was calculated upto 0.075mg/ml. Acetone showed the least activity. Figure 1. Shows a comparative analysis of the various extracts in possessing anti-oxidant properties.
Figure 1. In vitro assay of free radical scavenging activity
Figure 2. IC50 value = 0.075mg/ml of methanol extract in antioxidant activity
Anti-Î±-glucosidase assay:- Various solvent extracts of the marine algae, Chlorodesmis sp showed potential Î±-glucosidase inhibition activity. The methanol extract showed the highest activity with an IC50 value (figure 4.) of 0.28mg/ml followed by ethanol and ethyl acetate extracts. Acetone extract of the sample showed the least inhibitory activity. Figure 3. Shows a comparative interpretation of the various extracts involved in inhibiting the alpha-glucosidase enzyme.
Figure 3. In vitro assay of anti-Î±-glucosidase activity
Figure 4. IC50 value =0.28mg/ml in inhibiting alpha-glucosidase
Anti-Î±-amylase assay:- Four solvents extracts of the seaweed was subjected to Î±-amylase inhibition activity. Methanol extracts possessed the highest inhibitory potential with an IC50 value (figure 6.) of 0.18mg/ml. Acetone extract showed least anti Î±-amylase property. On the other hand intermediate activities were observed in case of both ethyl acetate and ethanol extracts, ethanol possessing second best effectiveness after methanol. A comparative interpretation of the various extracts of Chlorodesmis sp. In inhibiting alpha-amylase enzyme is shown in the figure 5. that follows.
Figure 5. In vitro assay of anti-Î±-amylase activity
Figure 6. IC50 value =0.18mg/ml of methanol extract in inhibiting alpha-amylase
Discussions:- Various oxidation reactions occurring in the environment, produce free radicals. These radicals are responsible for stating chain reaction in the cell. The role of anti-oxidents is thus to terminate this chain reaction by removing the free radicals intermediates and inhibits other oxidation reaction. In the present study, extracts of marine algae Chlorodesmis has found to possess anti-DPPH or free radical scavenging activity. Antioxidents from such natural resources can be widely used in dietary supplements for the prevention of oxygen stress and particularly finds importance in the treatment of disease such as cancer, coronary heart disease and even altitude sickness along with the treatment for various forms of brain injury.
An entirely different aspects of bioactivity of Chlorodesmis sp lies with the prevention of diabetes mellitus. The disease falls among the group of metabolic disorders in association with a person having high blood sugar levels, either because the body does not produce enough insulin or due to the fact that the cells does not respond to the insulin being produced properly. The initial referred to as "insulin dependent diabetes mellitus (IDDM)" and the latter being called "non-insulin dependent diabetes mellitus (NIDDM)". While IDDM requires dietary management, integrated with insulin treatment, NIDDM follows an entirely different therapeutic approach. Inhibition of enzymes involved in the metabolism of carbohydrate is being frequently used nowadays to reduce early postprandial hyperglycaemia and postprandial hypoglycaemia, as an early treatment.
The objective of this present study was to find such enzyme inhibition from marine algae Chlorodesmis sp . Clinically, various oral antihyperglycemic drugs have been used to reduce the activity of Î±-glucosidase enzyme in carbohydrate digestion to release Î±-glucose, promoting in the increase of blood glucose level in the meal. However, these often cause severe gastrointestinal side effects. Therefore, search for new Î±-glucosidase inhibitors from natural resources has become a prospective approach for the treatment of hyperglycaemia.
Î±-amylase, similarly, is another enzyme that hydrolyse alpha bonds of large alpha linked polysaccharides, such as starch, glycogen yielding glucose and maltose. Thus finding Î±-amylase inhibitory activity from natural resources has also been employed as one of the approaches to treat diabetes.
From early times many natural resources have been reported for their ant diabetic properties. However, such resources have not gained much temperature in the medical fields due to the lack of proper scientific evidence. Î±-glucosidase and Î±-amylase inhibitory effects of brown and red algae have also been reported earlier.
In the present study, Chlorodesmis sp were subjected to anti Î±-glucosidase and anti Î±-amylase assay. Methanolic extracts of seaweed showed potential enzyme inhibitory activity, followed by ethanol and ethyl acetate.
In conclusion, results obtained from the present study, supports the fact that Chlorodesmus can be used as dietary supplements for its anti diabetic and anti oxidant properties. Further investigation in future may shed light on using the green algae as medical sources in detail.
Acknowledgements:- The authors would like to convey gratitude towards the authorities of Vellore Institute of Technology, Tamil Nadu, India, for providing facilities with constant support and encouragement.