Qualitative and quantitative analysis of quercetin and phyllanthin

1. A SEMINAR ON
QUALITATIVE AND QUANTITATIVE ANALYSIS
OF QUERCETIN & PHYLLANTHIN
By
Sri Krishna

2. 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.

3. DIETARY SOURCES
Dietary 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

4. 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

5. EXTRACTION
 Plant source : Dried bark of Quercus tinctoria
 Solvent: Alcoholic solvent
 Removal of impurities by
lead acetate solution
o Crystallization: Cold water

6. 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

7. 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

8. 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

9. 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

10. 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

11. HPTLC ANALYSIS
Standard 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.

12. QUERCETIN ESTIMATION BY HPTLC
HPTLC
Stationary 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

13. QUERCETIN HPTLC GRAPH

14. 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

15. HPLC ANALYSIS GRAPH

16. 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

17. UV SPECTRA OF ISOLATED COMPOUND

18. IR SPECTRA OF ISOLATED COMPOUND

19. IR SPECTRAL DATA FOR ISOLATED
COMPOUND
Wave Length Group
3411 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-1
In 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.

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

21. HERBAL PRODUCTS OF QUERCETIN

24. 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

25. PROPERTIES
Colour Mostly colourless
Shape Crystals
Melting point 96 °C+
Molecular Formula C24H34O6
Molecular Weight 418.52 g/mol
pH value

26. 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 .

27. 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.

28. 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)

29. 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

30. HPLC GRAPHS
 Std Phyllanthin  Extraction Phyllanthin

31. IR ANALYSIS
Wave length Functional group
2917.93 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

33. HPTLC
HPTLC
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

34. USES OF PHYLLANTHIN
 Anti Oxidant
 Hepatoprotective (hepatitis B)
 Anti viral
 Immunomodulatory
 Hepolipidemic
 Antimalarial
 Anidiabetic

35. HERBAL PRODUCTS OF PHYLLANTHIN

36. 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.