Alga from marine beginning are possible renewable resource and has been widely used as antioxidants, anti-mutagens, anti-coagulants etc. Sing the assorted medicative potencies of Marine algae, the present survey was carried out on the preliminary phytochemical showing, free extremist scavenging activity, anti-I±-glucosidase activity and anti-I±-amylase activity of assorted infusions prepared from the marine sample, Chlorodesmis sp, collected from the coastal countries of Mahabalipuram, Tamil Nadu, India. Phytochemicals were extracted utilizing assorted dissolvers such as methyl alcohol, ethyl alcohol, propanone and ethyl ethanoate and tested for bioactivities. The seaweed showed possible antioxidant activity, along with alpha amylase and alpha-glucosidase inhibitory activity. Out of which methanolic infusion has shown important activity, with IC50 value of anti-DPPH check found upto 0.075mg/ml. IC50 value of suppressing I±-glucosidase and I±-amylase were 0.28mg/ml and 0.18mg/ml severally. Phytochemical showing has shown the presence of alkaloids, saccharides, phenolic compounds, proteins and aminic acids, corroborating that the seaweed Chlorodesmis sp can be used as a possible beginning of assorted anti-oxidant compounds, alpha amylase inhibitory compounds etc in future surveies.
Keywords: antioxidant, phytochemical showing, free extremist scavenging activity, anti-I±-amylase, anti-I±-glucosidase, seaweed, IC50.
Any big Marine benthal algae, which can be differentiated from algae of microscopic size with bare oculus can be referred to as seaweeds. Algae of such types are normally multicellular, therefore macroscopic along with macrothallic organic structure. From early times, seaweeds have been used as possible medicative beginnings for human usage. Phytochemicals isolated from marine algae have been of immense usage in assorted pharmaceutical industries, covering with the production of medical specialties for the intervention of assorted diseases. Seaweeds have besides been considered as extraordinary sustainable resources in the Marine ecosystem which have been used as a beginning of nutrient, provender and medical specialties. Hydrocolloids like alginate, agar, carrageenin and other gelatinlike substances, extracted from Marine algae, attain important commercial function as nutrient additives. The nutrient industry have exploited their gelling, water-retention, emulsifying and other physical belongingss. The mineral foods present are diverseand, of which iodine constituent have been potentially used to forestall goiter. Other compounds have been implicated in the intervention of diseases like TB, arthritis, colds and grippe, worm infestations and even tumours in some cases. Among other utilizations, seaweeds have besides been considered for the production of bioethanol.
Materials and Methods
Collection and Identification of the sample: – The sample was collected from the coastal countries of Mahabalipuram, situated in the province of Tamil Nadu ( 12.63A°N, 80.17A°E ) . Marine algae sample was collected manually, by manus picking, have oning baseball mitts from the coastal stones, along the coasts 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 checks were besides conducted.
Sample Preparation: – The marine algae that were collected from the coast, were instantly washed with saltwater, a figure of times to irradicate any air plants and immaterial, unwanted affair. After saltwater wash, the sample washed once more, this clip with fresh water to take other taints, Once the lavation was done, the algae was transferred to sterile zip-locked polyethene bags and transported to the research lab at a temperature of 4A°C. In the research lab, the sample was washed once more, foremost utilizing distilled H2O and so ethanol, merely to guarantee that no superficial taints remain. Once washed, the algae was sundried, cut into little pieces and powdered in a sociable bomber. The dried sample can besides be subjected to ultrasonication for the improvement of sample readying in any farther surveies that is conducted in close hereafter.
Aqueous extraction of possible bioactive compounds from the algae: – Sterile 100ml conelike flasks were taken, into which 1gm of the powdery sample was transferred and 30ml of sterile deionised H2O was added to it. The conical was so kept for heating at the H2O bath for 1hr, temperature being set to 100A°C. This was allowed to soak for 24hours at room temperature. The besotted sample mixture was taken into 50ml unfertile extractor tubings and the sample was extractors at 2500rpm for 8mins at 4A°C, utilizing a chilling extractor. The supernatant was transferred to another 50ml unfertile conelike flask, filtrating it utilizing a unfertile Whatmann filter paper no.1. As an excess safeguard, the filtrate was besides filtered utilizing a cellulose-acetate membrane filter in a 5ml unfertile syringe.
Solvent extraction of possible bioactive compounds from the algae: – For solvent extraction, 0.5gm of the powdery sample was taken into 50ml unfertile conelike flasks, to which each of the dissolvers like methyl alcohol, ethyl alcohol, ethyl ethanoate, n-butanol and propanone were added bit by bit in each conical. The dried sample were kept for soaking in each dissolver for 48hrs. Each of these mixtures were taken into 50ml unfertile extractor tubings, and following to which, centrifugation at 2500rpm for 8mins at 4A°C was performed. The supernatants were transferred to 50ml unfertile conelike flasks after filtrating through unfertile Whatmann filter paper no.1. Filtration utilizing cellulose-acetate membrane and 5ml unfertile syringe was besides performed in this instance.
Detection of alkaloids: 500Aµl of each dissolver infusion, was taken in a trial tubing ; to it 2ml of dilute hydrochloric acid was added and assorted good. Now to this mixture 2-3 beads of Mayer ‘s reagent was added by the side of the tubing and so observed for white colored creamy precipitate which indicate the positive consequence.
[ Mayer ‘s Reagent: It was prepared as follows- 1.358g of Mercuric Chloride was dissolved in 60ml of H2O while 5g of K I was dissolved in 10ml of H2O. The two solutions were assorted decently and the volume was adjusted and made up to 100ml with H2O ] .
Detection of saccharides: 500Aµl of each dissolver infusion, was taken in a trial tubing and to it two beads of alcoholic solution of I±-naphthal were added and shaken good and assorted decently. To this mixture 1ml of conc. Sulphuric acid was added and was allowed to stand for sometime and was so observed for the formation of violet pealing which indicate the presence of saccharide. This trial is normally known as Molisch ‘s trial.
Detection of saponins: For this peculiar trial alternatively of the aqueous sample we straight opt for the powdery sample which was ab initio prepared. 2g of the powdery sample was boiled in 10ml of distilled H2O in the H2O bath and so filtered. 5ml of the filtrate was so assorted with 2,5ml of distilled H2O and shaken smartly for the stable persistent from the formation. The frothing was now subjected to blend with 3-4 beads of olive oil and this mixture was once more shaken smartly and so observed for the formation of emanation.
Detection of proteins and aminic acids: 500AµL of extract solution was assorted with 2ml of acetic anhydride in a trial tubing. To this few beads of Million ‘s reagent was added. A white precipitate indicates the presence of proteins.
[ Million ‘s reagent was prepared by fade outing 1g of quicksilver in 9ml of fuming azotic acid. When the reaction is complete equal volume of distilled H2O was added. ]
Another trial was besides done for this analysis:
Biuret trials: 500Aµl of extract solution was treated with one bead of 2 % Cu sulfate solution. To this 1ml of methyl alcohol was added followed by extra K hydrated oxide pellets, pink coloring material in the methanolic bed indicates the presence of protein.
Detection of phytosterols: 250Aµl of extract solution was taken in a trial tubing and to it few beads of impersonal ferrous chloride solution was added and observed for an array of colors which indicates phytosterols.
Detection of flavonoids: 3ml of dilute ammonium solution, a portion of aqueous infusion followed by add-on of conc. sulfuric acid. Then it was observed for xanthous coloring material which disappears on standing.
Detection of terpenoids: 3ml of each infusion was assorted with 0.5ml of trichloromethane and conc. Sulphuric acid was added along with the sides of the tubings to organize a bed and so was observed for ruddy brown coloring material of the interface which indicates the presence of terpenoids.
Free extremist scavenging activity ( antioxidant ) : – Any molecule that has the capacity to suppress the oxidization of other molecules, can be called as an antioxidant. Here, we utilise the stable free extremist compound DPPH ( 1,1-diphenyl-2-picryl hydrazyl ) in order to mensurate the anti-oxidant free extremist scavenging activity of the algal sample. Different concentrations i.e. 10Aµg-50Aµg of each of the solvent infusion of the sample was prepared and taken into trial tubings, decently labelled. The volume of the infusion in each trial tubing was made upto 1ml. To this, 1ml of DPPH-methanolic solution was added and kept for incubation in dark for atleast half an hr. Since DPPH is light sensitive, readying and add-on of DPPH was performed with extreme attention in dark. Two controls were besides prepared ; the positive control dwelling of DPPH-methanolic solution and the methanol solution entirely acts as the negative control. If the sample contains any anti-oxidant belongingss, it will respond with DPPH to cut down the compound. This decrease, hence consequences in alteration of color from deep violet to light yellow. The optical density was measured at 517nm, followed by computation of per centum of anti-oxidant activity, utilizing the expression,
% scavenging activity = [ ( A517 Extract- A517 Control ) ] A- 100
I±-glucosidase suppression check: – Assorted concentrations of the infusion, runing from 50-300Aµg/ml were prepared. A volume of 100Aµl of the extract solution was taken in single unfertile 2ml eppendorf tubings. 200Aµl of enzyme solution incorporating I±-glucosidase ( 1.0U/ml ) , dissolved in 0.1M phosphate buffer was added to each of the eppendorf tubing and kept for 10mins incubation at room temperature. Once the pre-incubation is completed, 100Aµl of the substrate i.e. 5mM p-nitrophenyl-I±-D-glucopyranoside, dissolved in 0.1M phosphate buffer ( pH 6.9 ) was added to each tubing separately. All the tubings were kept for 5mins incubation at 25A°C. a control was besides prepared, incorporating 100Aµl buffer solution alternatively of the infusion. After incubation, optical density were recorded at 405nm utilizing a UV-visible spectrophotometer. The readings were compared to the optical density reading of the control. Henceforth, the per centum suppression activity was calculated utilizing the expression
% repressive activity= [ ( A405 Extract- A405 Control ) ] A- 100
I±-amylase suppression check: – I±-amylase inhibitors are those, which bind to the enzyme molecule, suppressing its activity of interrupting down amylum molecules into glucose and maltose medieties. Different concentrations like 50Aµg-250Aµg of each of the solvent infusion of the sample was prepared. 500Aµl of each extract concentration were taken into trial tubings, to which 500Aµl of newly prepared enzyme solution was added ( 100ml of enzyme solution required equal parts by volume of 20mM disodium H phosphate, 20mM of Na dihydrogen phosphate and 6.7mM of Na hydrated oxide, with 0.001gm of powdered enzyme added to it ) . The infusions, with enzyme solution added to it, were incubated for 15mins, followed by add-on of 500Aµl of 1 % starch solution was so added to each aliquot. This was kept for another short period of incubation for 15mins. The reaction was made to end by add-on of 1ml of 3,5-dinitrosalicylic acid ( DNSA ) to the assay mixture ( DNSA was prepared utilizing 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 check mixture was increased upto 10ml with distilled H2O, followed by entering of the optical density at 540nm. If the sample contains I±-amylase inhibitory compounds, it will adhere to it, non leting the enxyme to breakdown the substrate amylum.
% inhibition= [ ( A540 Control – A540 Extract ) ] A-100
Phytochemical Analysis: – Chlorodesmis sp was tested upon the presence of assorted phytochemicals like alkaloids, saccharides, saponins, proteins and aminic acids, phytosterols, flavinoids and terpenoids. Methanolic infusion showed the presence of the highest figure of phytochemicals. It possessed saccharides, proteins and aminic acids, flavinoids. Presence of alkaloids in the infusion were non converting and requires farther probe. Table 1. shows the consequences obtained in phytochemical showing.
Table 1. Phytochemical analysis
Name of the trials
( Mayer ‘s trial )
( Molisch ‘s trial )
( Foam trial )
Proteins and amino acids
( million ‘s trial )
( biuret trial )
( Liebermann-Burchard ‘s trial )
( Magnesium+ HCl decrease )
( Ferric chloride trial )
( +++ ) indicates positive consequence ; ( — – ) indicates negative consequence ; ( ++-/+-+/-++ ) indicates
moderate positive consequence ; ( + — /-+-/ — + ) indicates little positive consequence.
Anti-scavenging activity: – Solvents infusions prepared from the marine algae were subjected to free groups scavenging check. Methanolic infusion possessed the highest anti-DPPH activity followed by ethyl alcohol and ethyl ethanoate. IC50 value ( figure 2. ) for the methyl alcohol infusion was calculated upto 0.075mg/ml. Acetone showed the least activity. Figure 1. Shows a comparative analysis of the assorted infusions in possessing anti-oxidant belongingss.
Figure 1. In vitro check of free extremist scavenging activity
Figure 2. IC50 value = 0.075mg/ml of methyl alcohol infusion in antioxidant activity
Anti-I±-glucosidase check: – Assorted solvent infusions of the Marine algae, Chlorodesmis sp showed possible I±-glucosidase suppression activity. The methyl alcohol infusion showed the highest activity with an IC50 value ( figure 4. ) of 0.28mg/ml followed by ethyl alcohol and ethyl ethanoate infusions. Acetone infusion of the sample showed the least repressive activity. Figure 3. Shows a comparative reading of the assorted infusions involved in suppressing the alpha-glucosidase enzyme.
Figure 3. In vitro check of anti-I±-glucosidase activity
Figure 4. IC50 value =0.28mg/ml in suppressing alpha-glucosidase
Anti-I±-amylase check: – Four dissolvers infusions of the seaweed was subjected to I±-amylase suppression activity. Methanol extracts possessed the highest repressive potency with an IC50 value ( figure 6. ) of 0.18mg/ml. Acetone infusion showed least anti I±-amylase belongings. On the other manus intermediate activities were observed in instance of both ethyl ethanoate and ethyl alcohol infusions, ethanol possessing 2nd best effectivity after methyl alcohol. A comparative reading of the assorted infusions of Chlorodesmis sp. In suppressing alpha-amylase enzyme is shown in the figure 5. that follows.
Figure 5. In vitro check of anti-I±-amylase activity
Figure 6. IC50 value =0.18mg/ml of methyl alcohol infusion in suppressing alpha-amylase
Discussions: – Assorted oxidization reactions happening in the environment, produce free groups. These groups are responsible for saying concatenation reaction in the cell. The function of anti-oxidents is therefore to end this concatenation reaction by taking the free groups intermediates and inhibits other oxidization reaction. In the present survey, infusions of Marine algae Chlorodesmis has found to possess anti-DPPH or free extremist scavenging activity. Antioxidents from such natural resources can be widely used in dietetic addendums for the bar of O emphasis and peculiarly discoveries importance in the intervention of disease such as malignant neoplastic disease, coronary bosom disease and even altitude illness along with the intervention for assorted signifiers of encephalon hurt.
An wholly different facets of bioactivity of Chlorodesmis sp lies with the bar of diabetes mellitus. The disease falls among the group of metabolic upsets in association with a individual holding high blood sugar degrees, either because the organic structure does non bring forth adequate insulin or due to the fact that the cells does non react to the insulin being produced decently. The initial referred to as “ insulin dependant diabetes mellitus ( IDDM ) ” and the latter being called “ non-insulin dependant diabetes mellitus ( NIDDM ) ” . While IDDM requires dietetic direction, integrated with insulin intervention, NIDDM follows an wholly different curative attack. Inhibition of enzymes involved in the metamorphosis of saccharide is being often used presents to cut down early postprandial hyperglycemia and postprandial hypoglycemia, as an early intervention.
The aim of this present survey was to happen such enzyme suppression from marine algae Chlorodesmis sp. Clinically, assorted unwritten antihyperglycemic drugs have been used to cut down the activity of I±-glucosidase enzyme in carbohydrate digestion to let go of I±-glucose, advancing in the addition of blood glucose degree in the repast. However, these frequently cause terrible GI side effects. Therefore, hunt for new I±-glucosidase inhibitors from natural resources has become a prospective attack for the intervention of hyperglycemia.
I±-amylase, likewise, is another enzyme that hydrolyse alpha bonds of big alpha linked polyoses, such as amylum, animal starch giving glucose and maltose. Therefore happening I±-amylase inhibitory activity from natural resources has besides been employed as one of the attacks to handle diabetes.
From early times many natural resources have been reported for their ant diabetic belongingss. However, such resources have non gained much temperature in the medical Fieldss due to the deficiency of proper scientific grounds. I±-glucosidase and I±-amylase repressive effects of brown and ruddy algae have besides been reported earlier.
In the present survey, Chlorodesmis sp were subjected to anti I±-glucosidase and anti I±-amylase check. Methanolic extracts of seaweed showed possible enzyme inhibitory activity, followed by ethyl alcohol and ethyl ethanoate.
In decision, consequences obtained from the present survey, supports the fact that Chlorodesmus can be used as dietetic addendums for its anti diabetic and anti oxidizer belongingss. Further probe in future may cast visible radiation on utilizing the green algae as medical beginnings in item.
Recognitions: – The writers would wish to convey gratitude towards the governments of Vellore Institute of Technology, Tamil Nadu, India, for supplying installations with changeless support and encouragement.