PHARCHEM MEMBERS: TIU, Chrisanthydel R. UY, Philene C. VICTA, Ma. Nerissa Dianne U. VILLAFLOR, Ruby Anne Mae S.

YSIT, Raymond Ivan M. 3DPH

FLAVONOIDS Flavonoids have antioxidant properties, which help protect people against various diseases. It influences archidonic acid metabolism which causes its anti-inflammatory, anti-allergic or antihistamine, antithrombotic and vasoprotective effects. Quercetin, the main flavonoid constituent of Zea mays or corn is found in fruits, flowers, and vegetables that, among other functions, give them their color.

QUERCETIN I. EXTRACTION PROCEDURE 1) Air-dried plant materials (or dried at room temperature) were ground into a fine powder using a commercial blender. 2) The material that passed through an 80-mesh sieve was used for extraction purposes. 3) Powdered material (20g) for each sample was extracted with 200 mL of 80% methanol or ethanol-water (1:1), overnight at room temperature using an orbital shaker or using ordinary maceration methods. 4) The extracts were separated from the residue by filtering through Whatman No. 1 filter paper. 5) The residues were extracted twice with the same solvent and extracts pooled. 6) The pooled extracts were concentrated to dryness under reduced pressure at 45 C, using a rotary evaporator. 7) The dry extracts were weighed to calculate the yield and stored in a refrigerator (-4 oC), until used for further analyses. II. GENERAL AND SPECIFIC TESTS A. GENERAL First Test for Flavonoids 1) 2 grams of lyophilizes sample re-dissolved in water for 5 mins. then filtered 2) Collect filtrate in a test tube then put 5 gtts. of 5% NaOH followed by addition of 2mL 10% HCl

3) Yellow solution that turns colorless upon addition of HCl indicates presence of flavonoid Second Test for Flavonoids 1) A few drops of 1% aluminium solution were added to a portion of the filtrate. 2) A yellow coloration indicates the presence of flavonoids. Third Test for Flavonoids 1) A portion of the extract was heated with 10 ml of ethyl acetate over a steam bath for 3 min. 2) The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow coloration indicates the presence of flavonoids. Fourth Test for Flavonoids 1) Aliquot of 4 ml of aqueous NaOH was added to 2ml of each of ethanol extract. 2) If a yellow precipitate was observed, it indicates the presence of flavonoids in the extracts. Otherwise, it indicates the absence of flavonoids. Shinoda Test 1) A little amount of magnesium powder and 3 drops of concentrated HCl were added to 4ml of each of the ethanol extract. 2) If a red color was observed, it indicates the presence of flavonoids. Otherwise, it shows the absence of flavonoids. OTHERS: Determination of total flavonoids / TF Dewantos Procedure 1) One milliliter of aqueous extract containing 0.01g/mL of dry matter was placed in a 10 mL volumetric flask. 2) Five mL of distilled water were added followed by 0.3 mL of 5% NaNO2. 3) After 5 min, 0.6 mL of 10% AlCl3 were added. 4) After another 5 min 2 mL of 1M NaOH were added and the volume made up with distilled water. 5) The solution was mixed and absorbance was measured at 510 nm using a spectrophotometer. 6) TF amounts were expressed as (+-) catechin equivalents g/100g of dry matter. All samples were analyzed thrice and results averaged. Colorimetric Aluminum Chloride Method 1) CS extract (0.5 ml of 1:10g ml-1) in methanol was separately mixed with 1.5 ml of Methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate, and 2.8 ml of distilled water. 2) The extract remained at room temperature for 30 min 3) The absorbance of the reaction mixture was measured at 415 nm with a double beam Perkin Elmer UV/Visible spectrophotometer (USA). 4) The calibration curve was prepared by preparing quercetin solutions at concentrations 12.5 to 100 mg ml-1 in methanol.

B.

SPECIFIC Tests for Quercetin

Flame Test 1.) Place 3-5 gtts. of the liquid sample in a small evaporating dish and apply a lighted match. If the sample is solid, use a pinch amount. 2.) Observe if the sample is flammable or not. Note the color of flame produced. 3.) A luminous or yellow flame and the production of soot or smoke indicates the presence of carbon atoms due to the three benzene rings that quercetin contains. Chromic Acid Test or Jones Test 1.) Dissolve 1 gtt. or a small amount of the solid sample in 1 mL of acetone in a small vial. 2.) Add 2 gtts. of 10% aqueous K2Cr2O7 solution and 5 gtts 6M H2SO4 . 3.) Primary and secondary alcohols are rapidly oxidized by chromium trioxide in acidic, aqueous acetone, whereas tertiary alcohols are stable to oxidation. 4.) Oxidation is readily detected by the appearance of the green Cr3+ ion or a blue-green solution. Oxidation is a qualitative analytical test for the presence of primary or secondary alcohols in the sample. Lucas Test Lucas Reagent: Dissolve 16 grams of anhydrous zinc chloride in 10 mL of conc. HCl with cooling. 1.) Add about 50 mg (2-3 gtts) of the sample to 1 mL of the reagent in a small vial. Cap the vial and shake vigorously for a few seconds. 2.) Allow to stand at room temperature. 3.) Coordination of the zinc chloride with the hydroxyl results in the formation of a sufficiently good leaving group. The carbon-oxygen cleavage can occur when reasonably stable carbocation is produced. 4.) Turbidity indicates the presence of an alcohol compounds in the sample. Tertiary alcohols react immediately with Lucas reagent to produce turbidity while secondary alcohols do so in five minutes. Primary alcohols do not react appreciably with Lucas reagent at room temperature. Baeyers Test The Baeyer test for unsaturation is for determining the presence of carbon-carbon double bonded compounds, called alkenes or carbon-carbon trible bonded compounds, called alkyne bonds. 1.) Place 5 gtts. of sample in a dry test tube. 2.) Add 2 gtts. of 2% KMnO4 solution. Shake the test tube vigorously and observe the rate and extent by which the reagent is decolorized. 3.) Note the formation of a brown suspension. Decolorization of the reagent is immediate if it occurs within 1 minute. 4.) The reaction is important because it doesnt work on alkanes (compounds with carboncarbon single bonds) or aromatic compounds. A negative reaction for this test indicates that the sample is either and alkane or an aromatic compound.

Test for Aromaticity : Nitration 1.) Place 2 mL of conc. HNO3 in an Erlenmeyer flask. Immerse the flask in a water bath and gradually add 2 ml conc. H2SO4. Cool the resulting mixture to room temp. This will serve as the nitrating mixture. 2.) Place 5 gtts. of the sample in a dry test tube. Add 8 gtts of the nitrating mixture and shake the test tube to ensure complete mixing. Note the formation of a yellow oily layer or droplet. Dilute with 20 gtts of water. 3.) The presence of a yellow oily layer or oil droplets indicates aromaticity of the sample. Xanthoproteic Test Aromatic groups can undergo reactions that are characteristics of benzene and benzene derivatives. One such reaction is the nitration of a benzene ring with nitric acid. Those containing activated benzene rings can readily undergo nitration such as the amino acids tyrosine and tryptophan. However, those containing inactivated benzene rings like phenylalanine soesnt readily undergo nitration. 1) Slowly add 10 gtts of conc. HNO3 to the diluted samples. Mix and note the color of the solution. 2) Slowly add 10 gtts conc. NaOH then mix. A yellow solution is produced with a sample containing activated benzene. Ferric Chloride Test The ferric chloride test is used to determine the presence or absence of phenols in a given sample. Enols give positive results as well. The bromine test is useful to confirm the result, although modern spectroscopic techniques (e.g. NMR and IR spectroscopy) are far superior in determining the identity of the unknown. 1) The sample is dissolved in water, or a mixture of water and ethanol 2) A few drops of dilute ferric chloride solution is added. 3) The formation of a red, blue, green, or purple coloration indicates the presence of phenols. Where the sample is insoluble in water, it may be dissolved in dichloromethane with a small amount of pyridine. Bromine Test The bromine test is a qualitative test for the presence of unsaturated CC bonds and phenols. 1. The sample is treated with a small amount of elemental bromine either as an aqueous solution, or as a solution in dichloromethane or carbon tetrachloride. 2. A positive test for the presence of unsaturation and/or phenol is indicated by the disappearance of the deep brown coloration of bromine, which happens because the bromine has been consumed by reaction with the unknown sample.

3. The formation of a white precipitate indicates the formation of a brominated phenol. III. USES a) Allergies, asthma, and hives Quercetin may inhibit histamine release from basophils (a type of white blood cell) and mast cells (large cells in connective tissue). b) Cancer Quercetin may be beneficial in the treatment of skin cancer, and may have anti-tumor effects in other cancers, such as ovarian cancer. c) Canker sores Quercetin may reduce the frequency of mouth sores and produce mild symptomatic relief. d) Diabetes mellitus Quercetin may help prevent cataracts, retinal disorders, nerve diseases, and other complications of diabetes. Flavonoids, including quercetin, also promote insulin secretion, increase vitamin C levels, protect blood vessels, prevent easy bruising, and support the immune systemall of which are beneficial to individuals with diabetes. e) Heart disease: Individuals with very low intakes of flavonoids are at higher risk for heart disease. f) Infection :Quercetin may control the spread of certain viruses within the body. g) Rheumatoid arthritis: Quercetin may help reduce tissue destruction. h) Quercetin may also be beneficial in the treatment of dysentery (an intestinal infection causing severe diarrhea), gout (a disease where crystals of uric acid, a component of urine, are deposited in the joints and cause swelling), and psoriasis (a chronic skin disease). IV. FOLKLORE Diuretic: Take decoction of hairs or cobs as tea. Decoction of pith of cob as tea is used for stomach complaints. Decoction of roots, leaves, and corn silk used for dysuria, bladder complaints, and bedwetting. The water in which unhusked corn is boiled is a pleasant tasting remedy for urinary tract infection. The corn silk decoction is also thought to be diuretic. Poultice used for ulcers, rheumatic pains and swellings. Decoction of parched corn (buned or roasted) taken as tea for nausea and vomiting. Kidney stones: Infusion of corn hair in hot water, 3x daily. Poultice of corn silk for wounds and sores. In China, corn silk is used for fluid retention and jaundice. Corn was removed through cutting done on the first Friday after a full moon. Corns have been subjected to some of the same cures used for warts, including dome of the quasi-magical ones.