HOW TO DETERMINE TOTAL PHENOLIC, FLAVONOID CONTENTS AND IN VITRO

ANTIOXIDANTACTIVITY OF SELECTED PLANTS

Vinars Dawane

Do 1. Screening of 3-4 plant species from forest region

having antioxidant properties.

2. Identify primary and secondary metabolite profile of the plant spices of interest.

3. Isolate and quantify the bioactive antioxidant compound and find out its medicinal values.

4. Determine the Comparative antioxidant profile between screened plants species.

Material and Methods:-Objective- 1

• Screen the Plant species from forest area.

Extract preparation• washing with tap water, • shade dried and powdering the sample.• then soxhelt apparatus with methanol as solvent (different

combinations of solvents) will be used for extraction.

Identification of primary and secondary metabolite.Objective - 2

• Phytochemical Analysis:• Phytochemical analysis to screen the plants for the presence of

alkaloids, glycosides, saponins tannins, flavonoids and carbohydrates, will be performed according to the method described by Sofowora (1993) and Evans (1998).

• Test for Carbohydrates:• Add few drops of Molisch’s reagent to 2ml of each of the water extract in

two tubes. A small quantity of concentrated sulphuric acid will then add and allowe to form a lower layer. A purple ring at the interface of the liquids indicates the presence of carbohydrates. Each mixture will be then shaken and allowed to stand for 2 minutes and diluted with 5ml of water. A purple precipitate also showed the presence of carbohydrates (Evans, 1989).

• Test for Alkanoids:• Five test tubes will be used for each of the different drug sample

(ethanolic extract). Few drops of the following reagents manager’s reagent, Drangendorff’s reagent. Wanger’s reagent, Hanger’s reagent and 10% tannic acid solution will be added respectively to each of the five test tubes. The presence of precipitate in at least 3 or all of the above reagents indicates the presence of alkaloids (Evans 1989).

• Test for Saponins:• A small quantity of the ethanolic extract wil be boiled. The mixture will

be filtered and 2.5ml of the filtrate will be added to 10ml of the distilled water in a test tube. The test tube will be corked and shaken vigorously for about 30 seconds, then it will be allowed to stand for half an hour. A honeycomb forth will an indicator of the presence of saponins (Sofowora, 1993).

• Test for Tannins (Ferric chloride test):

• A portion of the water extract will be diluted with distilled water in a ratio of 1:4 and few drops of 10% ferric chloride solution was added. A blue or green colour indicates the presence of tannins (Evans,1989).

• Test for Glycosides:• To a portion of the aqueous extract will be added fehlings reagent and

boiled for 2 minutes. A brick red colouration indicates the presence of glycosides.

Total Phenolic Contents (TPC) determination

• Folin – Ciocalteu reagent method –

• Method is used for the colorimetric in vitro assay of phenolic and poly-phenolic antioxidants.

• Reagent - a mixture of phospho-molybdate and phospho - tungstate.

• Method - Samples (100μL) + 2 ml of 2% Na2CO3 + 100 μL of Folin-Ciocalteu reagent + absorbance was measured at 743 nm against a blank.

Presence of flavonoid shinoda test –

• Four pieces of magnesium fillings (ribbon) are added to the ethanolic extract followed by few drops of concentrated hydrochloric acid.

If –

• A pink or red colour indicates the presence of flavonoid.• Colours varying from orange to red indicated flavones.• crimson to magenta indicated flavonones.

Total Flavonoid Content (TFC) Determination

• Aluminium Chloride Method –

• Steps –

• 0.5ml of sample (1mg/ml) mixed with• 0.1ml of 10% aluminium chloride and 0.1ml of

potassium acetate (1M).• Addition of 4.3ml of 80% methanol.• Absorbance measure at 415nm.

Antioxidant capacity DetectionObjective - 3

• DPPH: Quenching Activity method.

• ATBS Cation production.

• Hydroxyl Radical Scavenging Activity method.

• Potassium Ferricyanide Reduction Method for Reducing Capacity testing.

• In vitro hemolysis of methanolic extract method for Antihemolytic Activity testing.

Antioxidant capacity• DPPH: Quenching Activity method.

• α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method.• This method is based on the reduction of DPPH in methanol solution in the

presence of a hydrogen donating antioxidant due to the formation of the non radical form DPPH-H.

• 0.1mM solution of DPPH in methanol + added to 3 ml of extract + mix.• Absorbance measure at 517 nm. And Ascorbic acid was used as the

reference.• DPPH scavenging effect (% inhibition) = {(A0 – A1)/A0)*100}.

• Where, A0 is the absorbance of the control reaction. and A1 is the absorbance in presence of all of the extract samples and reference.

• ABTS – Cation Decolorizing Test.

• ATBS Cation production –

• 7mM ABTS solution + 2.45 mM potassium Per sulphate + mix in dark room.

• The ABTS + Solution were diluted with ethanol to an absorbance of 0.70+0.02 at 734 nm.

• 100μL of sample + to 3.9 mL of diluted ABTS+ Solution + absorbance at 734 nm.

• ABTS radical cation activity = {(A0 –A1)/A0)*100}.

where , A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference.

Hydroxyl Radical Scavenging Activity method.

• Deoxyribose assay was used to determine the hydroxyl radical scavenging activity in an aqueous medium.

• Extract + FeCl 3 (100 µM) + EDTA (104 µM) + H2O2 (1 mM) and 2-deoxy- D-ribose (2.8 mM) + mix them + phosphat buffer for makeup.

• Mixture + 95 0 C in water bath for 15 min + 1 ml each of TCA (2.8%) and TBA (0.5% TBA in 0.025 M NaOH containing 0.02% BHA).

• Mixture will be cooled on ice and centrifuged at 5000 rpm for 15 min.

• Absorbance of supernatant was measured at 532 nm.

Potassium Ferricyanide Reduction Method for Reducing Capacity testing.

• Various concentrations of the extracts and standards (50, 100, 250, 500, 750, 1000 mg/ml) .

• Then add to 2.5 ml of (0.2 M) sodium phosphate buffer (pH 6.6) and 2.5 ml of potassium ferricyanide [K 3Fe 3 (CN) 6 ] (1%) solution and vortex.

• Incubation at 50°C for 20 min + 2.5 ml of TCA  + centrifuged at Χ3000 g for 10 min.

•  Mix with deionized water (5 ml) and then 1 ml of FeCl 3 (1%).• Formation of Perl's Prussian color was measured at 700 nm.

Note - Increased absorbance of the reaction mixture indicated increasing reducing power. 

In vitro hemolysis of methanolic extract method for Antihemolytic Activity testing.

• Inhibition of H 202 induced red blood cell (RBC) haemolysis of methanolic extract  are examined by this method.

• Haemolytic activity of the extract can investigate by measuring the lysis of a 10% (v/v) human red blood cells suspension in a spectrophotometric assay.

• In this experiment, method will be used same as - Naim M, Gestetner B, Bondi A, Birk Y. Antioxidative and antihemolytic activities of soybean isoflavones. J Agric Food Chem 1976;24:1174-7.

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• N. Bunyapraphatsara et al. Pharmecological studies of plants in the mangrove forest. Thai Journal of Pharmacy; 2003, Vol.10 (2).

• Vagi E, Rapavi E, Hadolin M, Vasarhelyine Peredi K, Balazs A, Blazovics A, et al. Phenolic and triterpenoid antioxidants from Origanum majorana L. herb and extracts obtained with different solvents. J Agric Food Chem. 2005; 53:17–21. [PubMed: 15631502].

• Jonathan Yisa. Phytochemical Analysis and Antimicrobial Activity Of Scoparia Dulcis and Nymphaea Lotus. Australian Journal of Basic and Applied Sciences, 3(4): 3975-3979, 2009.

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