Isolation & Identification of Quercetin

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Evaluation Seminar onIsolation & identification of Quercetin : 

Evaluation Seminar onIsolation & identification of Quercetin Presented By Alok Pratap Singhlokratap Singh M.Pharm-II Dept. of Pharmacognosy K.L.E.U College of Pharmacy Hubli- 31.

Contents : 

Contents Introduction Dietary sources of quercetin Biosynthesis of Quercetin Health benefits of Quercetin Isolation of Quercetin Identification of Quercetin References

Introduction : 

Introduction Flavonoid Glycosides Flavonoid constitute one of the largest class of naturally occuring plant products mostly phenols either in the Free State or as their respective glycosides. They are usually yellow-coloured compounds (flavous is a latin word yellow colour). Interestingly, more than 5000 different chemical compounds have been isolated, identified and reported from plant sources. In fact, their chemical structures are solely based upon a C6—C3—C6 carbon skeleton having a pyran or chroman ring bearing a second benzene (aromatic) ring strategically positioned at C—2, C—3 or C—4. Pyran Benzopyran

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In nature they are available as: flavones, flavanones, flavonols, isoflavones, and anthocyanidins. The structures of a few typical flavonoids are represented here as follows: Flavones Flavanones Flavonols Isoflavones Anthocyanidins

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The flavonoid glycosides mostly occur as O-glycosides or C-glycosides contained in the cell sap of relatively younger tissues of higher plants belonging to several families, such as: Compositae, Leguminosae, Polygonaceae, Rutaceae and Umbelliferae . It has been observed that a host of natural plant products containing flavonoid glycosides exert a variety of therapeutic effects, namely: antiasthmatic, antispasmodic, diuretic, fungicidal and oestrogenic activities. A few typical flavonoid glycosides: (a) Flavone Glycosides, (b) Flavonol Glycosides, (c) Flavanone Glycosides, (d) Chalcone Glycosides, (e) Isoflavonoid Glycosides, and (f ) Anthocyanidin Glycosides.

Flavonol Glycosides : 

Flavonol Glycosides The two well known glycosides belonging to this class are namely: Rutin and Quercetin, whereas the less important ones are—galangin, gossypin, hibiscitrin, kaempferin and avecularin. Quercetin Quercetin is a bioflavonoid (or flavonoid), which is a type pigment found in almost all herbs, fruits, and vegetables. Quercetin is the aglycone form of other flavonoid glycosides, such as rutin and quercitrin, found in citrus fruit, buckwheat and onions. Quercetin forms the glycosides, quercitrin and rutin, together with rhamnose and rutinose, respectively.

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Synonyms :- Quercitroside; Quercimelin; Quercitin – 3-L-rhamnoside; Thujin; Quercitin; Quercetin, Sophoretin, Meletin, Quercetine, Xanthaurine, Quercetol, Quertine, Flavin meletin. Biological Sources Quercetin occurs in the bark of Quercus tinctoria and some other species of Quercus. It is also obtained from Alsculus hippocastarum L., horse chest nut, belonging to family Hippocastanaceae. It is also found in Thuja occidentalis L., Morus alba L., Humulus lupulus L., Fraxinus excelsior L., Vitis vinifera and the other plants.

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Description The crystals are yellow in colour when obtained from ethanol or methanol. It is practically insoluble in cold water and ether. Flavine yellow shade obtained from the quercitron bark by extraction under high pressure steam which is used exclusively in dyeing fabrics. The structure of quercetin IUPAC name 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one Quercitin on hydrolysis in an acidic medium gives rise to rhamnose and quercetin (i.e., 5, 7, 3’, 4’-tetrahyroxy flavonol).

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Chemical Tests It exhibits a brown fluorescence under the UV-light. It gives a distinct yellow precipitate initially with a solution of basic lead acetate, but it gets dissolved on further addition of the reagent in excess. It reduces Tollen’s reagent to give a silver mirror. It gives Shinoda test to form yellow or crimson red colour when it mix with magnesium ribbon and conc. hydrochloric acid.

Biosynthesis of Quercetin : 

Biosynthesis of Quercetin Phenylalanine is converted to 4-coumaroyl-CoA in a series of steps known as the general phenylpropanoid pathway using phenyl ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumaroylCoA-ligase. 4-coumaroyl-CoA is added to three molecules of malonyl-CoA to form tetrahydroxychalcone using 7,2’-dihydroxy, 4’-methoxyisoflavanol synthase. Tetrahydroxychalcone is then converted into naringenin using chalcone isomerase. Naringenin is then converted into eriodictyol using flavanoid 3’ hydroxylase. Eriodictyol is then converted into dihydroquercetin with flavanone 3-hydroxylase, which is then converted into quercetin using flavanol synthase.

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+ Phenylalanine 4-coumaroyl-CoA malonyl-CoA tetrahydroxychalcone naringenin eriodictyol dihydroquercetin Quercetin

Health benefits : 

Health benefits Cancer Diarrhea Allergies, Asthma, Hay Fever Heart Disease Hypertension Interstitial Cystitis Prostatitis Diabetes Rheumatoid Arthritis (RA) Athletic Endurance

Quercetin Side Effects : 

Quercetin Side Effects Quercetin Cause Cancer On the other hand, a handful of studies from reputable sources claim high doses of quercetin cause cancer. Researchers found that the high concentrations of the element would actually bind with and damage chromosomes and DNA structures resulting in a cancerous mutation. High concentrations of the flavonoid also disrupted the activity of enzymes and particularly interfered with estrogen and thyroid hormones. These studies claim that there is a documented risk to young children consuming high doses of it and getting sick with a rare form of leukemia. Birth Defects Scientists theorize that birth defects could occur in the unborn children of women who consumed high doses of quercetin at the time of conception and throughout pregnancy.

Extraction : 

Extraction Neem Leaves Air-dried Powder (40-60 mesh) (300 gm) Petroleum ether 60-800C Benzene Acetone Methanol Hot Water

Isolation of Quercetin Methanolic Extract of Azadirachta indica leaves : 

Isolation of Quercetin Methanolic Extract of Azadirachta indica leaves Methanolic extract of Neem leaves was subjected to fractionation by Column Chromatography. The Column 72 cm in length and 5 cm in diameter was used. Slurry of silica gel (400gm; 60-120 mesh size) prepared in petroleum ether (600-800C) was added to the column. Methanolic extract (3 gm) was loaded on the column and the column was than eluted with various solvents i.e. Petroleum ether (600-800C), Benzene, CHCl3, CHCl3: Acetone (1:1), Acetone, Acetone: Ethanol (1:1), Ethanol, Methanol and distilled Water in the order of their increasing polarity.

Slide 17: 

The acetone fraction resulted in the precipitation of white crystalline compound. The methanolic fraction, kept in refrigerator resulted in the precipitation of yellow powder which was further purified. Qualitative analysis of the isolated compound was done. The isolated compound was subjected to TLC to ascertain its nature and its m.p. was determined. The isolated compound was subjected to U.V. (U.V.-Visible Spectrophotometer, Elico-make), I.R, Mass, 1H and13C NMR Spectroscopy (C.D.R.I., Lucknow) and the structure of isolated compound was determined.

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Azadirachta indicaLeaves Methanolic Extract (3gm) Column Chramatography Eluted with Petroleum ether (60-80 0C), (1:1), Acetone, Acetone: Ethanol (1:1), Benzene, CHCl3, CHCl3: Acetone Ethanol, Methanol, Water Methanolic Yellow powder (Flavonoid in nature; m.p. 3160C) Acetone White Crystalline Compound (Phenolic in nature; m.p. 61-640C)

Thin Layer Chromatography of Crude Methanol extract : 

Thin Layer Chromatography of Crude Methanol extract Plate Size:-20 X 5.5 cm Silica Gel:-100-200mesh Solvent System:- Chloroform: Acetic acid: Water (50: 45: 5 ml) Developing Reagent:-Iodine Vapours

Thin Layer Chromatography of Isolated Compound : 

Thin Layer Chromatography of Isolated Compound Plate Size:-20 X 5.5 cm Silica Gel:-100-200 mesh Solvent System:- Chloroform: Acetic acid: Water (50: 45: 5 ml) Developing Reagent:-Iodine Vapours Rf= 0.93

HPLC Analysis of Quercetin : 

HPLC Analysis of Quercetin The chromatographic analyses were performed on a 250 mm × 4.6 mm i.d., C18 (ODS), Shimadzu, Japan with 0.5% aqueous solution of Orthophosphoric acid and Methanol (HPLC Grade) as mobile phase at a flow rate of 1 mL min-1. The HPLC equipment comprised Hewlett-Packard (HP) 1050 ChemStation Software, an HP model 35900 interface units, an HP 9000 Series 300 computer, and an HP DeskJet 500 Printer. A Waters 486 tuna-ble absorbance detector was operated at 254 nm; detector sensitivity was 0.05 AUFS and the column oven temperature was 30°C. Determinations were performed after three separate extractions of each sample, and each extract was injected in triplicate (n = 3).

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HPLC graph for Quercetin

Estimated by HPTLC Technique : 

Estimated by HPTLC Technique The samples were spotted in the form of bands with Camag microlitre syringe on a precoated silica gel plates F 254 (10 cm X 10 cm with 0.2 mm thickness, E.Merck) using Camag linomat V. Automatic sample spotter of band width 7 mm. The plates were developed in a solvent system in CAMAG glass twin through chamber previously saturated with the solvent for 30 min. the distance was 8 cm. subsequent to the scanning, TLC plates were air dried and scanning was performed on a Camag TLC Scanner in absorbance at 254 nm and operated by wincats software 4.03 version (Sethi 1996).

Quercetin estimation by HPTLC : 

Quercetin estimation by HPTLC

Quercetin HPTLC graph : 

Quercetin HPTLC graph

UV Spectra of Isolated Compound : 

UV Spectra of Isolated Compound 257nm, 307nm 432nm

IR Spectra of Isolated Compound : 

IR Spectra of Isolated Compound

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IR Spectral data for Isolated Compound

Mass Spectroscopy : 

Mass Spectroscopy

Nuclear Magnetic Resonance : 

Nuclear Magnetic Resonance 1H NMR Spectra of Isolated Compound

13Carbon- NMR spectroscopy (13C-NMR) : 

13Carbon- NMR spectroscopy (13C-NMR)

Slide 38: 

Solaray - Mega Quercetin, 60 capsules Innate Response Formulas Quercetin - 90 Tablets Nutricology/ Allergy Research Group Quercetin Nature's Life - Quercetin Citrus-Free, 400 mg, Quercetin Bioflavonoids 100 caps Quercetin 500 Plus

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Healthy Aging Nutraceuticals Quercetin Complex Quercetin Complex Super Quercetin 100 – Capsule Quercetin 500mg Citrus fruits Apples are an excellent source of quercetin

References : 

References Ashutosh kar.Pharmacognosy and pharmacobiotechnology. new age international (p) limited, publishers New delhi. 2003; 2:161-2. Harborne JB. The Flavonoids. Chapman and Hall, London (1988) 622-645 Adebamowo CA, Cho E, Sampson L, et al. Dietary flavonols and flavonol-rich foods intake and the risk of breast cancer. Int J Cancer. 2005;114:628-33. Cruz-Correa M, Shoskes DA, Sanchez P, et al. Combination treatment with curcumin and quercetin of adenomas in familial adenomatous polyposis. Clinical Gastroenterology & Hepatology.2006;4:1035-38. Hubbard GP, Wolffram S, de Vos R, Bovy A, Gibbins JM, Lovegrove JA. Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study. British Journal of Nutrition 2006;96:482-8. Kim YH. Lee YJ. TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation. Journal of Cellular Biochemistry.2007;100:998-1009.

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7. Schabath MB, Hernandez LM, Wu X, Pillow PC, Spitz MR. Dietary phytoestrogens and lung cancer risk. JAMA. 2005;294:1493-04. 8. Shoskes DA, Zeitlin SI, Shahed A, Rajfer J. Quercetin in men with category III chronic prostatitis: A preliminary prospective double-blind, placebo-controlled trial. Urology. 1999; 54:960-63. 9. Swati D, Sadhna T. Quercetin-a potential compound of Azadirachta indicaa.juss.(neem) leaves exhibiting activity against wood decaying fungi and termites. J. Serb. Chem. Soc. 2007; 72: 921–39. 10. Rajalakshmi PV, Kalaiselvi KS. Direct HPLC analysis of quercetin in exudates of Abutilon indicum (linn). Malvaceae. Journal of Pharmaceutical Science and Technology. 2009; 1 (2):80-3. 11. Sarunya C, Sukon P. Method Development and Determination of Phenolic Compounds in Broccoli Seeds Samples. Chiang Mai J. Sci. 2006; 33(1):103-7. 12. Fathiazada F, Delazara A, Amiria R, Satyajit DS. Extraction of Flavonoids and Quantification of Rutin from waste Tobacco Leaves. Iranian Journal of Pharmaceutical Research. 2006; 3:222-7.