Evaluation Seminar on Isolation & identification of Quercetin

Presented By Bijay kumarSingh

M.Pharm-II

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

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 C6C3C6 carbon skeleton having a pyran or chroman ring bearing a second benzene (aromatic) ring strategically positioned at C2, C3 or C 4
2' 1' 6' 3'

8 7

4' 5'

6 5 4

Pyran Benzopyran

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

O H H

O OH O

Flavones

Flavanones

Flavonols

Isoflavones

Anthocyanidins

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
The two well known glycosides belonging to this class are namely: Rutin and Quercetin, whereas the less important ones aregalangin, 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.

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.

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).

Properties
Molecular formula Molecular weight Exact mass Density Melting point Chemical Tests 1. It exhibits a brown fluorescence under the UV-light. 2. 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. 3. It reduces Tollens reagent to give a silver mirror. 4. It gives Shinoda test to form yellow or crimson red colour when it mix with magnesium ribbon and conc. hydrochloric acid. C15H10O7 302.236 g/mol 302.042653 1.799 g/cm3 316 C

Dietary sources Black and green tea (Camellia sinensis) Capers Lovage Apples Onion, especially red onion Red grapes, Citrus fruit, Tomato, broccoli and other leafy green vegetables, and Raspberry, Bog Whortleberry Lingonberry Cranberry Chokeberry Sweet rowan Rowanberry

Quantity mg/kg 20002500 mg/kg 1800 mg/kg 1700 mg/kg 44 mg/kg 1910 mg/kg 158 mg/kg, fresh weight Cultivated 74 mg/kg, wild 146 mg/kg. cultivated 83 mg/kg, wild 121 mg/kg 89 mg/kg 85 mg/kg 63 mg/kg

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.

OH

COOH

+
HO2C NH2
HO2C

X H2C COSCoA
OH

Phenylalanine
HO O OH

4-coumaroyl-CoA
HO

malonyl-CoA

CH3 O

OH

O
OH OH

naringenin
OH OH

tetrahydroxychalcone
HO O

HO

OH OH O

dihydroquercetin

eriodictyol

Quercetin

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

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
Neem Leaves

Air-dried Powder (40-60 mesh) (300 gm)

Petroleum ether 60-800C Benzene

Acetone

Hot Water Methanol

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 (600800C) 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.

 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.

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

Acetone

Methanolic

White Crystalline Compound (Phenolic in nature; m.p. 61-640C)

Yellow powder (Flavonoid in nature; m.p. 3160C)

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 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

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 30C. Determinations were performed after three separate extractions of each sample, and each extract was injected in triplicate (n = 3).

HPLC graph for Quercetin

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

Stationary Phase Mobile phase Silica gel F 254 plates Ethyl acetate:Dichloromethane: Formic acid: Glacial acetic acid:Water (10: 2.5: 1: 1: 0.1) Quarcetin 1 mg/ml (5 L) 80 mm 254 nm Absorption (Deuterium) 0.93 57858

Standard Migration distance Detection wavelength Mode of scanning Rf value Peak area

Quercetin HPTLC graph

UV Spectra of Isolated Compound

257nm, 307nm 432nm

UV-Vis Spectra of Four Flavonoids

Rutin
HHO O ORutinos e OH O OH 3' OH

Quercetin glycoside OH
OHH HHO O OGlu OH O

Ploridzin
HHO OH

OH

Anthocyanidin
OH OHH HHO + O OH OH

O
O--D-Glucose

Adapted from FEBS Letters, 401 (1997) 78Paganga and Rice -82, -Evans.

IR Spectra of Isolated Compound

IR Spectral data for Isolated Compound

1 3411 cm-1 2 1663.1 cm-1 3 1608.8 cm-1, 1523.5cm -1, 1496cm-1 4 1383.1 cm-1 5 1318.9 cm-1 In plane O-H bending of Phenols In plane bending of C-H bond in Aromatic Hydrocarbon 6 1265 cm-1 7 1203 cm-1 8 1167 cm-1 9 940.6, 821.4, 677, 602.3 cm-1 C-O stretch of Aryl ether C-O stretch of Phenol C-CO-C stretch and bending in Ketone Out of plane C-H bending of Aromatic Hydrocarbon O-H stretching vibration of Phenols C=O Aryl Ketonic stretch C ---C Aromatic ring stretch

Mass Spectroscopy
Identification of flavonoids by MS: spectrum of quercetin
100

179:

- C7H6O2

OH OH HO O

80

OH
60

151: - C7H6O2 CO

OH

C15H10 O7 Mw=302 40

c d n u b A v i t a l e R

273: - CO
20 107: C 7H6O2 - CO - CO2 0
193 229

257: - CO2
239

301 (M - H+)

100

140

180

220 m/z

260

300

340

Mass Spectrometer Results

Intensity
x10 1.25 1.00 0.75 0.50 0.25 0.00 100 200 300 400 500 600 700 5 303.3

Peak 1
633.0

Peak 1 m/z [M + H]+ 303, 633

331.3 405.3 465.1 487.2 591.2 779.0 800 900

Intensity
x10 4 5 303.3

Peak 2
487.1

Peak 2 m/z [M + H]+ 303, 487

325.2 628.9 400 500 600 700 800 900 100 200 300

Intensity
x10 3.0 2.5 2.0 1.5 1.0 0.5 0.0 325.2 5 303.2

Peak 3
471.1

Peak 3 m/z [M + H]+ 303, 471

642.9 400 500 600 700 800 900 100 200 300

Mass Spectrometer Results

Glycoside Flavonol

Aglycone

Glycone Sugar (e.g. glucose, galactose)

Ions Na+

Flavonol Aglycone

633

303

308

22

Identified first

MS/MS Compound Identification

The presence of quercetin was suspected based on: The compound mass of 302 (mass = positive ion 1) was present in all three major peaks in mimosa HPLC analysis showed that the compounds were flavonols
Intens. x10 4 1.5 0 1.2 5 1.0 0 0.7 5 0.5 0 0.2 5 110.9 100 228.9 164.9 136.9 152.9 150 285.0 201.0 213.0 200 247.0 250 274.0 0 300 m/z 110.9 100 136.9 152.9 150 201.0 213.0 200 247.0 25 0 274.0 30 0 m/z 257.0 1 164.9 228.9 3 All, 48.6-49.2min (#411-#416) 302.9 Inten s. 5 x1 0 4 All, 48.7-49.2min (#412-#418) 302.9

2 257.0 285.0

Common ion in mimosa foliage extract

Quercetin

Nuclear Magnetic Resonance

1H NMR Spectra of Isolated Compound

Position of signal ()
6.2 6.5 6.9 7.7 7.8

No. of Protons
1 1 1 1 1
OH 3'
4' 6' 5'

Multiplicity Assignment
Doublet Doublet Doublet Doublet Doublet H-6 H-8 H-5 H-6 H-2

2'

OH

HO
7 6

' 2 1
3

OH

OH

13

Carbon- NMR spectroscopy (13C-NMR)

Signal ( ) 155.59 134.57 174.39 160.11 96.99 162.88 92.28 Position 9 10 1 2 3 4 5 Signal ( ) 146.18 101.93 121.57 113.56 143.68 144.82 114.01

Position 2 3 4 5 6 7 8

Nutricology/ Allergy Research Group Quercetin

Innate Response Formulas Quercetin - 90 Tablets

Solaray - Mega Quercetin 60 capsules

Nature's Life - Quercetin Citrus-Free, 400 mg,

Quercetin Bioflavonoids 100 caps

Quercetin 500 Plus

Healthy Aging Nutraceuticals Quercetin Complex

Quercetin Complex

Super Quercetin 100 Capsule

Quercetin 500mg

Citrus fruits

Apples are an excellent source of quercetin

References
1. Ashutosh kar.Pharmacognosy and pharmacobiotechnology. new age international (p) limited, publishers New delhi. 2003; 2:161-2. 2. Harborne JB. The Flavonoids. Chapman and Hall, London (1988) 622-645 3. 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. 4. 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. 5. 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 collagenstimulated platelet activation pathway in man: a pilot study. British Journal of Nutrition 2006;96:482-8. 6. Kim YH. Lee YJ. TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation. Journal of Cellular Biochemistry.2007;100:998-1009.

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: 92139.

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.