This document discusses the isolation and estimation of two phytoconstituents - glycerrhizin from liquorice root and phyllanthin from Phyllanthus amarus. It describes the biological sources, chemical constituents, isolation methods including acid precipitation, alcohol extraction and ammonia extraction for glycerrhizin. For phyllanthin, the isolation involves extraction with petroleum ether followed by column chromatography. Estimation methods discussed are TLC densitometry, colorimetric and HPLC methods for glycerrhizin and TLC and HPLC for identifying and estimating phyllanthin.
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INTRODUCTION TO
GLYCYRRHIZA
Common name: Liquorice, Sweet Wood, Regolizia
(Italian), Jeshtamadh, Yastimadhu.
Biological sources:
It is obtained from the dried, unpeeled, roots and
stolons of Glycyrrhiza glabra.
Family: Leguminosae
Part used: Roots & Stolons
Geographical Source: commercially cultivated in
Spain, and England.
Allied species: Glycyrrhiza uralensis
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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CHEMICAL CONSTITUENTS
Chief constituent of Liquorice- Glycyrrhizin.
Glycyrrhizin:
A triterpenoidal saponin glycoside.
Also called Glycyrrhizic acid or Glycyrrhizinic
acid.
Present as potassium and calcium salt of
Glycyrrhizinic acid.
Being a glycoside, Glycyrrhizic acid, on hydrolysis
gives an aglycone and a glycone portions:
Glycyrrhizinic acid Glycrrhetinic acid
(Glycyrrhetic acid), which is an aglycone of
triterpenoidal structure + 2 moles of Glucuronic
acid.
Upon hydrolysis, the glycoside loses its sweet taste.AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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INTRODUCTION TOINTRODUCTION TO
GLYCYRRIZINGLYCYRRIZINGlycyrrhizin is the main sweet tasting compound from liquorice
root.
It is 30–50 times as sweet as sucrose.
Pure glycyrrhizin is odorless.
Glycyrrhizin and other products with this component have a
long history of medicinal value and are used in the
treatment of peptic ulcer, especially glycyrrhetinic acid
(triterpene derivative).
IUPAC Name:
(3β,18α)-30-hydroxy-11,30-dioxoolean-12-en-3-yl 2-O-β-D-
glucopyranuronosyl-β-D-glucopyranosiduronic acid
Molecular formula: C42H62O16
Molecular weight: 822.94gm/mol. NASEEFA. JSSCP ,MYSORE
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ISOLATION OF GLYCYRRHIZINISOLATION OF GLYCYRRHIZIN
The isolation of glycerrhizin from glycerrhiza is based on
its solubility. Glycerrhizin is soluble in hot water,
alcohol, but slightly soluble in cold water and
insoluble in ether.
The three methods of isolation are:
1. Acid precipitating method
2. Alcohol extraction method
3. Ammonia extraction method
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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1. Acid precipitating method:
Procedure:
1. wiegh 50g liquorice powder add 300ml water in the beaker and
boil with stirring
2. decant the supernant liquid
3. filter the remaining residue and collect the filterate
4. Adjust pH 2.8 by the addition of acid, Glycerrhizin precipitates out
5. Filter and collect the ppt
6. Wash the ppt with cold water to make it free from acid
7. Transfer the ppt to china dish and heat gently to remove the
water content, shiny brown mass of glycerrhizin is seen.
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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2. Alcohol extraction method
Procedure:
1. wiegh 50g of powdered drug into 500ml beaker & add 100ml
methanol mix it .
2. Add another 100ml methnol & left for 24hrs
3. Filter and collect the filterate
4. Extract this methnolic extract with 3 portions of petroleum
ether, subsequently with benzene, ethylacetate chloroium and
solvent ether.
5. Transfer methnolic layer into china dish & evaporate on water
bath to get glycerrhizin.
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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3. Ammonia extraction method
Procedure:
1. Glycerrhiza is extracted with hot water and filtered.
2. Filtrate is acidified with con H2SO4 to pH2.8
3. Ppt is dissolved in dil.NH4OH and poured into
acetone to ppt ammonium glycerrhizinate.
4. The ppt is dissolved in hot water and evaporated to
get ammonium glycerrhizinate.
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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1.TLC- densitometry:
TLC plates- kieselgel 60HF 254
Solvent system: n Butanol: acetic acid:water 7:1:2
Detection:1% cerric sulfate in 10% H2SO4 heating at 105oC for
5min
Std solution: A known conc. Of glycerrhizin (0.5-2.0mg/ml)
Sample solution: Reflux 1gm of samplewith 50ml of 50%ethanol
cool and filter. Reflux the marc with 50ml of 50% ethanol.
Evaporate and dissolve the residues in 50% ethanol(10ml).
Development and chromatograms: 13-14cm.distance application
and 10µl std and sample
Visualisation of spots: visualise and scan in densitometer. Integrate
the area of spots corresponding to glycerrhetic acid. Calculate
the % of it in the sample.
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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2. Colorimetric method:
Glycerrhizin gives purple color with anisaldehyde in the
presence of H2SO4 . this forms the basis of
estimation. The intensity of color developed is
proportional to the amount of glycerrhizin, which is
measured at 556nm.
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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3.Spectrophotometric method
1g extract+50ml 3%
solution of
trichloroacetic
acid in acetone
stand for few
minutes &
decant the
liquid
repeat the extraction 2
more times by
adding 25ml of
trichloroacetic
acid in acetone
filter after the
third
extraction
wash the residue with
25ml of acetone .
combine all the
extracts
Add drop by drop
con.ammonia until
abandunt cheesy ppt
is formed. fliter in
buchner funnel
wash residue with
50ml of
acetone 3-
4times
dissolve the
residue in
50ml of
water
make up the volume
upto 250ml
(solutionA)
take 10ml from above
solution and dilute
upto 500ml this is
solution B
measure optical
density of
solution B at
258nm using
water as blank
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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4. HPTLC METHOD:
Sample Preparation:
Sample Application: Bandwise with linomat 2, 3, 4 μl of standard & sample
solution. Band length remains 6mm; distance between bands 4mm.
Chromatography: 2 step chromatogram development: 1st run: chloroform-
acetone (9:1). 2nd run: Chloroform- diethyl ether- formaldehyde
(80:15:1)
Quantitative evaluation: Done at 254nm using Mercury lamp as
monochromator.
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USES:
• Demulcent
• Anti-inflammatory
• Flavoring agent and sweetner
• Anti-tussive and expectorant
• Anti-bacterial
• Useful in gastric and duodenal ulcers
• Anti-spasmolytic activity
Phyllanthin
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Biological source : It consists of the aerial parts of the plant Phyllanthus
amarus belong to the Family – Euphorbiaceae
common names: Bhuiamla, Bhumyamalaki
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.
Phyllanthin Hypophyllanthin
PHYLLANTHIN
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PROCEDURE :
1.Mix the powder drug with lime water , It is allowed to stand for
overnight .
2.The mass is then transferred to a Soxhlet apparatus and extraction is
carried out with petroleum ether
3.Collect the petroleum ether extract and concentrate under reduced
pressure.
4. Mix the extract with methanol and boil , collect the dewaxed methanol
extract , evaporate to dryness, collect the residue.
5. Dissolve the residue in petroleum ether , concentrate and allow to
stand (Yellow oil gets separated)
6. The residue is subjected to column chromatography on Alumina and
elute with n-hexane: ethyl acetate (99:1)
7. 99- 108 fractions corresponds to hypophyllanthin and 109- 137
fractions corresponds to phyllanthin Subject them to
chromatography further to yield pure phyllanthin and hypophyllanthin
ISOLATION:
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
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IDENTIFICATION
BY T.LC :
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)
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By 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.
ESTIMATION :
AZEEZ. MYMOONA. NASEEFA. JSSCP ,MYSORE
