A SEMINAR ON QUALITATIVE AND QUANTITATIVE ANALYSIS OF QUERCETIN & PHYLLANTHIN

By Sri Krishna

QUERCETIN Boilogical Sources : Quercetin occurs in the bark

of Quercus tinctoria belonging to family Hippocastanaceae.

Quercetin is a bioflavonoid (or flavonoid), which is a

type pigment found in almost all herbs, fruits, and

vegetables.

When acid hydrolysis it divided into two parts aglycone

and glycone part is rhamnose.

DIETARY SOURCESDietary sources Quantity mg/kg

Black and green tea (Camellia sinensis) 2000–2500 mg/kg

Onion, especially red onion 1910 mg/kg

Capers 1800 mg/kg

Red grapes, Citrus fruit, Tomato, broccoli and other leafy green vegetables, and Raspberry, Bog

Whortleberry

158 mg/kg, fresh weight

Cranberry cultivated 83 mg/kg, wild 121 mg/kg

Sweet rowan 85 mg/kg

Apples 44 mg/kg

PROPERTIES Properties

Color Mostly Yellow in color

Shape Like Crystals

Insoluble Cold Water and Ether

Molecular formula C15H10O7

Molecular weight 302.236 g/mol

Exact mass 302.042653

Density 1.799 g/cm3

Melting point 316 °C

EXTRACTION Plant source : Dried bark of Quercus

tinctoria

Solvent: Alcoholic solvent

Removal of impurities by lead acetate solution

o Crystallization: Cold water

FLOW CHART FOR EXTRACTION

Prepare a thimble with dried bark powder is subjected to taken into Soxhlet apparatus and extract under reduced pressure

Then take alcoholic extract add small amount of lead acetate while the brown colour ppt is changed into yellow – orange then centrifuge and the ppt is separated

Extraction process

Removal of impurities

Yellow residue is dissolved in water and allow to stand for some time .

That ppt is re-suspended in alcohol and decompose with a stream of hydrogen sulphide filtered to remove the lead sulphide and filtrate is evaporated to give yellow residue.

Removal of lead acetate

Re crystallization

QUALITATIVE ANALYSIS It exhibits a brown fluorescence under the UV-light.

Lead acetate solution test: sample +10% lead acetate solution yellow ppt

FeCl3 test: sample +FeCl3 change the colour from green to black

Shinoda test : Sample +with magnesium ribbon and conc. hydrochloric acid yellow or crimson red colour

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:-100-200mesh

Solvent System:-Chloroform: Acetic acid: Water (50: 45: 5 ml) Developing Reagent:-Iodine Vapours RF = 0.93

HPTLC ANALYSISStandard Quercetin:- 10 mg of standard quercetin was dissolved in 10 ml HPLC grade Methanol.1 ml of this solution was diluted with 10 ml of methanol (1:10). Sample Solution:-100 mg of extract powder was dissolved in 5ml (ethanol:water,4:1) solution using sonicator for 15 mins.

QUERCETIN ESTIMATION BY HPTLCHPTLCStationary Phase Silica gel F 254

Moblie Phase Ethyl acetate:Dichloromethane: Formic acid: Glacial acetic acid:Water (10: 2.5: 1: 1: 0.1)

Standard Quercetin 1 mg/ml (5μL)

Migration distance 80mm

Detection wavelength 254nm

Mode of scanning Absorption (Deuterium)

Rf value 0.93

Peak area 57858

QUERCETIN HPTLC GRAPH

HPLC ANALYSIS

Column(Column size) HiQ Sil C18HS(4.6 mm × 250 mm × 5µ)

Mobile phase Methanol: 0.1% ortho phosphoric acid (65:35%)

Flow rate 1 mL1min-1

Detectors UV detector, 369 nm

Std Preparation Accurately weighed 25 mg of standard quercetin was transferred to a 25 ml volumetric flask and dissolved in Methanol.

Concentration of Samples 10 pm (standard and isolated fraction of quercetin)

Retention time 8.4 min

HPLC ANALYSIS GRAPH

UV SPECTRA OF ISOLATED COMPOUND Preparation of standard stock solution:

100mgof drug dissolved in 100 ml methanol in 100 ml of volumetric flask 1000µg/ml

Then prepare 10µg/ml solution from stock and determined λ max (400-200nm)

The λ max of quercetin is 372nm, π → π∗ transition energy

UV SPECTRA OF ISOLATED COMPOUND

IR SPECTRA OF ISOLATED COMPOUND

IR SPECTRAL DATA FOR ISOLATED COMPOUNDWave Length Group3411 cm-1 O-H stretching vibration of Phenols

1663.1 cm-1 C=O Aryl Ketonic stretch

1608.8 cm-1, 1523.5cm -1, 1496cm-1 C ---C Aromatic ring stretch

1383.1 cm-1 In plane O-H bending of Phenols

1318.9 cm-1In plane bending of C-H bond in

Aromatic Hydrocarbon

1265 cm-1 C-O stretch of Aryl ether

1203 cm-1 C-O stretch of Phenol

1167 cm-1 C-CO-C stretch and bending in Ketone

940.6, 821.4, 677, 602.3 cm-1 Out of plane C-H bending of Aromatic

Hydrocarbon.

HEALTH BENEFITS Cancer Diarrhoea Allergies, Asthma, Hay Fever Heart Disease Hypertension Interstitial Cystitis Prostatitis Diabetes Rheumatoid Arthritis (RA) Athletic Endurance

HERBAL PRODUCTS OF QUERCETIN

PHYLLANTHIN

PHYLLANTHIN  Biological source : It consists of the aerial parts of the

plant Phyllanthus amarus belong to the Family – Euphorbiaceae

Common names: Bhuiamla, Bhumyamalak

 Chemical Constituents: Phyllanthin and Hypophyllanthin are the two major constituents of the drug phyllanthus. Chemically , Phyllanthin is a diaryl butane, where as hypophyllanthin is an aryl tetra-hydronaphthalein. other constituents are : phyllanirurin, niranthin, nirtetralin, tannins like amariniic acid , geraniic acid

PROPERTIESColour Mostly colourlessShape CrystalsMelting point 96 °C+Molecular Formula C24H34O6

Molecular Weight 418.52 g/molpH value

PROCEDURE Mix the powder drug with lime water , It is allowed to stand for

overnight . The mass is then transferred to a Soxhlet apparatus and extraction is

carried out with petroleum ether . Collect the petroleum ether extract and concentrate under reduced

pressure. Mix the extract with methanol and boil , collect the dewaxed methanol

extract , evaporate to dryness, collect the residue. Dissolve the residue in petroleum ether , concentrate and allow to stand

(Yellow oil gets separated) . The residue is subjected to column chromatography on slica gel and

elute with n-hexane: ethyl acetate (99:1) . 99- 108 fractions corresponds to hypophyllanthin and 109- 137

fractions corresponds to phyllanthin Subject them to chromatography further to yield pure phyllanthin .

QUALITATIVE ANALYSIS

Test for lignans: 0.5mL of aqueous solution of extract+2mL of 2% furfuraldehyde in a test tube Red color indicates the presence of flavonoids.

TLC  Adsorbent – Silica gel GF254

Solvent system – n- hexane: ethyl acetate (2:1)

Detection – Vanillin in sulphuric acid reagent

Rf value – Phyllanthin – 0.20 ( Blue spot) Hypophyllanthin – 0.25 ( Brown spot) 

HPLC Column : µ- Bondapak-18 ( 3.9mm X 30cm) Mobile phase :– Methanol – Water (66: 34) Flow rate – 1.8ml/Minute Detection: – UV at 230nm Standard – Known concentration

(0.05- 2µg) Sample: – Macerate 1gm of the drug powder with lime water

at room temperature for 18hrs .Reflux with 30ml methanol containing 3% KOH for 1hr .cool and filter .collect the filtrate Reflux mark with methanol containing 3% KOH . Filter and collect the filtrate . Combine the extracts and concentrate under reduced pressure. Make up to 50ml.

Sampling: – Apply 10µl of both standard and sample solutions

Determination: – Note the peak areas corresponding to phyllanthin and hypophyllanthin in both standard and samples and calculate their percentages accordingly

HPLC GRAPHS Std Phyllanthin

Extraction Phyllanthin

IR ANALYSISWave length Functional group2917.93 cm-1 , 2959.97 cm-1 , 2998.9 cm-1

C-H stretching (aromatic)

2850.37cm-1 C-H stretching (aliphatic)1088.34 cm-1 , 1102.36 cm-1 , 1127.37cm-1 , 1151.21 cm-1 , 1185.42 cm-1 , 1202.61cm-1

C-O-C stretching

1400 cm-1  C-N bending 1625 cm-1  N-H bending

HPTLCHPTLC

Stationary Phase Silica gel F 254

Mobile Phase n- hexane: ethyl acetate :acetone (24:8:12)

Standard Phyllanthin

Migration distance 80mm

Detection wave length 580nm

Reagent Vanillin in sulphuric acid reagent (BLUE)

Rf value 0.24

USES OF PHYLLANTHIN Anti Oxidant

Hepatoprotective (hepatitis B)

Anti viral

Immunomodulatory

Hepolipidemic

Antimalarial

Anidiabetic

HERBAL PRODUCTS OF PHYLLANTHIN

REFERENCES Vandana Patil et al ;J. Chem. Pharm. Res., 2015,

7(1):99-104. Yu Duan ; Ultraviolet-Visible spectrum

characterizations of Quercetin in aqueous ethanol solution, J. Chem. Pharm. Res., 2014, 6(9):236-240 .

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.

Kim YH. Lee YJ. TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation. Journal of Cellular Biochemistry.2007;100:998-1009.