SEMINAR ON

PHYTOCHEMICAL STUDY OF
GLYCOSIDE
 

BY INDER MAKHIJA , MCOPS MANIPAL UNIVERSITY

EMAIL: indermakhija5@gmail.com
 CONTENTS

 INTRODUCTION  CARDIAC GLYCOSIDES

 CLASSIFICATION  SAPONIN GLYCOSIDES
 TYPES OF SUGARS IN  CYANOGENETIC
GLYCOSIDES GLYCOSIDES
 PROPERTIES  FLAVONOIDAL GLYCOSIDES
 METHODS OF ISOLATION  COUMARIN GLYCOSIDES
 QUALITATIVE TESTING  SULPHUR CONTAINING
 ESTIMATION OF GLYCOSIDE GLYCOSIDES
 MEDICINAL IMPORTANCE  ALDEHYDE GLYCOSIDES
OF GLYCOSIDES  ALCOHOLIC AND PHENOLIC
 ANTHRAQUINONE GLYCOSIDES
GLYCOSIDES  BIOSYNTHETIC PATHWAYS
FOR DIFFERENT
GLYCOSIDES
 INTRODUCTION :


 Phytochemical investigation of a plant involves the

following steps:-
 Authentication and extraction of plant material
 Separation and isolation of constituents of interest
 Characterization of isolated constituents
 Investigation of biosynthetic pathways to particular
compounds
 Quantitative evaluations.


 DEFINITION :
 Glycosides may be defined as, the organic compounds
of plant or animal sources which on enzymatic or acid
hydrolysis gives one or more sugar moieties along with
non-sugar moiety.
 OR
 They are acetals or sugar ethers, formed by the
  CLASSIFICATION:


 Based On Aglycone Moiety:


 Glycosides


 HOLOSIDES HETEROSIDES
 (Aglycone: sugar)


 Holosides are of two types, they are

 1. OLIGOSACCHARIDES
 2. POLYSACCHARIDES 



Based On The Chemical Nature Of The Aglycone


Moiety:

 Anthracene glycosides
 Steroidal/cardiac glycosides
 Saponin glycosides
 Cyanogenetic glycosides
 Isothiocyanate glycosides
 Flavonol glycosides
 Coumarin/furanocoumarin glycosides
 Aldehydic glycosides
 Phenolic glycosides
 Bitter glycosides

 Based On The Nature Of The Simple Sugar:
 Glucosides
 Galactosides
 Mannosides
 Arabinosides


 According To The Type Of Glycosidic Linkage:

 C-glycoside: linkage is through carbon. Ex: anthraquinone
glycosides like aloe.
 Glycone -OH+CH-aglycone glyconeC-
aglycone+H2O
 O-glycoside: Interaction of nucleotide glycoside such as UDP-
glycone with alcoholic/ phenolic group of second compound
aglycone. Such glycosides are called as O-glycosides.
 Glycone-OH+OH- Aglycone glycone-O-
aglycone
 Ex: Rhein -8-glucoside obtained from rhubarb.In senna ,
rhubarb and frangula.
 N- Glycosides:Best example of it is Nucleosides. Amino group of
 TYPES OF SUGARS IN GLYCOSIDES:
 Monosaccharide (glucose in salicin and rhamnose in
ouabain)
 Disaccharide(gentiobiose in amygdalin)
 Trisaccharide (strophanthotriose)
 Tetrasaccharide (purpurea glycoside)
 Rare sugars (deoxy sugars)


 PROPERTIES:
 Diversity in structure makes it difficult to find general
physical and chemical properties.
 They are crystalline or amorphous substances.
 Most of them are water soluble or soluble in alcohols.
 They are either insoluble or less soluble in non-polar
organic solvents.
 Aglycone moiety is soluble in organic solvents.
 More sugar units in a glycoside lead to more solubility in
polar solvents.
 ENZYME HYDROLYSIS:
 It is specific for each glycoside.
 The same enzyme is capable to hydrolyze different
glycosides, but α and β stereoisomers of the
same glycoside are usually not hydrolyzed by the
same enzyme.
 Emulsin is a β- glycosidase.
 Maltase and invertase are α- glycosidases.
 

 ACID HYDROLYSIS:
 It is non-specific
 Except C-glycosides all glycosides are hydrolysable by
acids non-specific.
 Glycosides containing 2-deoxy sugars are more unstable
towards acid hydrolysis even at room temperature.

 METHOD OF ISOLATION
 Stas-Otto Method:
 Powdered drug


Extracted by soxhlet method using alcohol

Extract

 Treated with lead acetate to precipitate

tannins

 PPT of lead subacetate


Pure Glycoside Can Be Obtained By,
 Fractional solubility
 Fractional crystallization
 Chromatographic techniques
 

Characterization is Done By,



 IR , UV, visible, NMR, mass

spectrometry.
  QUALITATIVE TESTING OF
GLYCOSIDES:


 Test depending on the chemical nature of the genin:

 Steroidal or cardiac glycosides:
 Give positive Liebermann's test (steroidal structure)
 Anthraquinone glycosides:
 Give positive Borntrager’s test, characteristic reddish
coloration with alkalis.
 Flavanoidal glycosides:
 Characteristic color with NH4OH, AlCl3 , FeCl3.
 Cyanogenetic glycosides : give upon hydrolysis
hydrocyanic acid which can change color of
sodium picrate paper from yellow to red.
 Sulphur containing glycosides : give black ppt of
silver sulphate upon treatment with AgNO3.
 METHOD OF ESTIMATION:
 Generally estimated in terms of ether-soluble extractive

 crude drug

 extracted with water/alcohol

 Extract is hydrolyzed with HCL

 aglycone portion

 extracted with ether/ other non-polar solvents

 small portion of this ether extract is evaporated to dryness.

 residue of aglycone part is dissolved in suitable solvent.

 examined


 By TLC
 BY UV /Visible Spectrophotometry at particular wavelength
 Some glycosides analyzed by UV spectroscopy are:
 Cardiac glycosides…………… 217 nm

Vanillin…………………………….301nm  
 Some are analyzed by VISIBLE spectroscopic range . They
are
 Cardiac glycosides……………….590nm by Keller- kiliani
reaction.
 Anthraquinone …………………….505 after treatment with
alkali.
 IR spectroscopy
 Steroidal sapogenins at 11.11
 Fluorimetric analysis
 Rheum emodium…… brown fluorescence

 MEDICINAL IMPORTANCE OF
GLYCOSIDES
 Cardiac drugs: cardiotonic glycosides
 Ex: digitalis glycosides, strophanthus, squill.
 Laxatives:
 Ex: anthraquinone glycosides of senna, aloes, rhubarb,
cascara , frangula.
 Counter-irritants:
 Ex: thioglycosides and their hydrolytic products
 Analgesics:
 Ex: methyl salicylate a hydrolytic product of gaultherin.
 Anti-rheumatics:
 Ex: salicin
 Anti-inflammatory:
 Ex: glycyrrhizin has demulcent, expectorant and anti-spasmodic
action.
 Glycosides that reduce the capillary fragility:
 Ex: flavanoidal glycosides, rutin, hesperidin.
 ANTHRAQUINONE GLYCOSIDE

  CHEMISTRY:-
 Parent molecule for all these is Anthraquinone. It is present in different
forms along with methyl, hydroxymethyl, carboxyl, dihydroxy phenol,
carboxylic acid group.
 In reduced form anthraquinone is present as anthranol or anthrone which
are isomeric with each other.
 Oxanthrone is intermediate between anthraquinone and anthrone.
 The anthrone molecule orients in bimeric form called dianthrone which is
therapeutically more important.
 The reduced anthraquinone are more active.
 Anthraquinones with different groups like,
 Methyl ( chrysophanol )
 Carboxyl( rhein )
 Hydroxymethyl (aloe-emodin)

ISOLATION METHOD OF ANTHRACENE
GLYCOSIDE
 Sample+ methanol+ water
 ↓
 FeCl3 + HCl reflux for 30 min.
 ↓
 Cool and shake with chloroform


 Chloroform phase aqueous phase

 (contains all glycosides) (rejected)


 Add NaHCO3 and shake



 CHCl3 phase acidify with HCL &shake CHCl3


(neutral and acidic aglycone)


 CHCl Phase Aqu.Phase

CHEMICAL TEST
 Borntrager’s test:
 This test is given by free anthraquinones only.
 Powdered drug- boil with dil. sulphuric acid.
 Filter
 Add benzene
 Shake- pink, red or violet color- positive for anthraquinone derivatives.
 Borntrager’s reaction can distinguish anthraquinones from anthrones
and anthranols which do not give the test unless they are converted
to anthraquinone by ↓oxidation with mild oxidants such as hydrogen
peroxide or ferric chloride.
 MODIFIED BORNTRAGER’S TEST:
 If the anthraquinones are reduced or stable test will be negative. These
are treated with fuming nitric acid. All the anthranols are converted
in to anthraquinones. A drop of ammonia is allowed to mix gradually
with acid, liquid produces violet color.
 ESTIMATION:
 By Spectrophotometry
 The method ensures the complete elimination of other minor non
carboxylic anthracene derivatives, as well as flavanoidal
contamination. The method quantitates the actual total sennosides
content, through the elimination of these contaminates and
 CARDIAC GLYCOSIDES
 INTRODUCTION:
 These are steroidal in nature, which act as cardiotonic
agents.
 Glycosides having the action on cardiac muscle are known
as cardiac glycosides
 In 1785, a Scottish physician William withering first
introduced the use of digitalis in heart therapy that is why
these are called as digitalis glycoside.
 PHYSICAL AND CHEMICAL PROPERTIES OF CARDIAC
GLYCOSIDE:
 The different cardiac glycosides show different solubilities in
aqueous and organic solvents. They are usually soluble in
water or aqueous alcohol and insoluble in the fat solvents
with exception of chloroform and ethyl acetate.
 The higher the number of sugar units in the molecule, the
greater will be the solubility in water but lower solubility
in chloroform.
 Alcohols are good solvents for both the glycosides and the
CHEMISTRY
 All cardio-active glycosides are characterized by the following structural features:
 β-OH at position 3, which is always involved in a glycosidic linkage to a mono, di, tri,
tetra saccharide.
 β-OH group at C-14.
 Presence of unsaturated 5 or 6 membered lactone ring at position C-17, also in the β
configuration.
 The A/B ring junction is usually (cis), while the B/C ring junction is always (trans) and
the C/D ring junction is always (cis).
 Additional OH groups may be present at C-5, C-11 and C-16.
 Cardiac glycosides that have α-β unsaturated 5-membered lactone ring in position C-
17 are known as cardenolides. These are represented by the digitalis and
strophanthus group.
 Cardiac agents that have doubly unsaturated 6-membered lactone ring in position-
17 are referred to as bufadienolides.
 This group includes the squill glycosides and the toad venom, bufatoxin.
 The aglycone portion at position C-3 of cardiac glycosides may contain four
monosaccharide molecules linked in series. Thus, from a single genin one may
have a monoside, bioside, a trioside or a tetroside.
 With the exception of D-glucose and L-rhamnose, all the other sugars that are found
in cardiac glycosides are uncommon de-oxy sugars.
 Ex: Digitoxose, cymarose, Thevetose.
ISOLATION
 Procedure :
 10 gm of coarsely powdered dry drug
 Macerate with 50ml of 70% alcohol for 1 hr, filter & retain the
filterate.
 Add lead sub acetate until precipitation is completed and
centrifuge
 supernatant
residue(rejected)
 Slowly add sodium sulphate to ppt excess lead and filter.
 Filterate
residue(rejected)
 Evaporate to dryness to recover cardiac glycosides.


  Major difficulty lies in the isolation of 1ry glycosides
from the crude glycoside, because 1ry glycosides
are converted in to 2ry glycosides by hydrolysable
enzymes.
 Other difficulty is the existence of several closely
related glycosides in the same drug, which are
extremely difficult to separate and purify.
 

 CHEMICAL TESTS: 
Keller-kiliani tests for digitoxose(sugar moiety)


 Boil 1gm of drug in 70% alcohol for 2-3 mins

 Filter+10ml water + 0.5ml strong lead acetate
solution
 Filter+ shaken chloroform layer is separated and
evaporated
 Residue is dissolved in 3ml glacial acetic acid +
2drops Fecl3
 Transfer this solution to 2ml con.H2so4
  CGs +ve Liebermann’s and salkowski’s test.
 Cardenolides are distinguished from scillarens by
a group of colour reagents, which are all
alkaline solutions of aromatic nitro compounds
namely
 Kedde’s reagent- 3,5 dinitro benzoic acid
 Raymond’s reagents- meta dinitrobenzene
 Baljet’s reagent- picric acid,
 Legal’s reagent- alkaline solution of sodium
nitrprusside.
 All these nitro compounds react with the active
methylene of five membered lactone ring to
give characteristic colours.
  

  ESTIMATION:
By enzymatic assay:


 Inhibition of Na+, k+ ATP ase can be applied to estimate the

total cardenolide content in Digitalis lanata. 


By HPTLC analysis:


 Leaf powder is accurately weighed and extracted with 50%

methanol containing des acetyl lanatoside B as an internal
standard.
 Ultrasonication for 1hr and the extract is filtered and
evaporated to dryness.
 Residue is dissolved in MeCN water (2:8)(28ml) and MeCN
water (3:7)(10ml) are successively passed through the
cartridge.
 After evaporation the residue obtained is dissolved in MeCN –
methanol-water (20:1:50)(1ml) and the sample solution is
submitted to HPTLC .
 Lanatoside C and digoxin are determined by the internal
standard method.
 Caliberation graphs are constructed by plotting the ratio of
peak area of Lanatoside C or digoxin to the peak area of the
internal standard.
 SAPONIN GLYCOSIDES
 INTRODUCTION:
 Saponins are a group of amorphous colloidal glycosides which rae widely distributed
in higher plants
 Soap wort plant (saponaria), root used as soap(isapo soap)
 Referred to as natural detergents
 Have ability to form long lasting foam when shaken in aqueous solution
 PROPERTIES:
 Amorphous powder with high molecular weight
 Soluble in and form colloidal solution
 Bitter in taste, non alkaline in nature
 Saponins are colorless and have haemolytic properties.
 They are excellent emulsifying agents.
 CHEMISTRY:
 Aglycone called as sapogenin.
 Saponins are classified according to the genin part in to two types:
 Steroidal type(C-25)
 Triterpenoidal type(C-30).
 Both types of saponins have the glycosidic linkage at positon-3
 Steroidal saponins:
 Used as raw material for the synthesis of various medicinally useful steroids like
 ISOLATION:
 Procedure is as follows:
 Reflux 5g of the drug with 100 ml 2N HCl for 2 hrs. cool and
filter.
 Neutralize the filterate by passing dil.ammonia through the
filter.
 Pack the residue in to a soxhlet thimble and extract with
pet.ether in a soxhlet apparatus and reduced the volume
and filter off the crystals.


 ANOTHER METHOD OF ISOLATION:

 Take powdered drug
 extract with pet.ether
 Defatted extract is obtained
 mix of alc+water(diff ratio)
 Alcoholic extract is obtained
 water saturated with n-butanol
CHEMICAL TEST:


 pink red (triterpenoid)

Saponin glycoside + acetic anhydride--


 Blue green(steroidal)
 
ESTIMATION:

By TLC

Detection:

 Without chemical treatment: number of saponins are detected

by exposure to UV 254 nm or UV 356nm.
Spray reagents:

 Blood reagent:
 Identification of haemolytic saponins . White spots are
formed on red background. Spot formation is some times fast
and some times it takes long time , or after treating with hot
air the spot is formed.
 Vanillin sulphuric acid reagent:
 Blue or blue violet zones are observed.

 CYANOGENETIC GLYCOSIDES
 These are the group of glycosides which yield HCN as one of the products
of hydrolysis.
 More commonly found in rosaceous plants.
 Common cyanophore glycosides are derivatives of mandelonitrile
 These are hydrolyzed by the enzyme emulsin.
 Emulsin contain at least 4 enzymes out of which two are amygdalase and
prunase.
 These are O-glycosides containing nitrogen heteroatom in the form of C=N
functional group.
 The aglycone part is cyanohydrin of a carbonyl compound (condensation
product of HCN with an aldehyde or ketone.
 These are widely employed as flavouring agents.
 Anti-cancer claims have also been made for an amygdalin containing
preparation known as laetrile or vitamin B-17.
 CHEMICAL TESTS:
 SODIUM –PICRATE TEST: Moistened drug+ dil H2SO4. Sodiumpicrate
paper turns from yellow to red. Benzidine / cupric acetate paper turns to
blue colour.
 MERCUROUS ACETATE TEST: Drug solution with 3% mercurous acetate
 FLAVANOIDAL GLYCOSIDES
INTRODUCTION:


 Largest group of naturally occurring phenols.

 Flavanoids constitute the majority of the yellow colored plant
pigments. (yellow colour of derivatives increases with rise in
number of –OH groups and pH )
 Many flavanoidal compounds present as glycosidic or as free
forms.
 They are formed from three acetate units and a phenyl
propane unit.
 All are derived from the same parent nucleus, 2-phenyl
benzopyran (flavan), thus they have a basic C-15 skeleton.
 Occurs free or as O and C glycoside.
 Responsible for the colour of fruit and flowers.
 This group is known for its anti-inflammatory, and anti-allergic
effects, for anti-thrombotic and vasoprotective properties,
for inhibition of tumour promotion and as a protective for
the gastric mucosa.
 CHEMISTRY

ü Shows a 15 carbon skeleton which consists of 2 phenyl

rings connected by a 3 carbon bridge.
ü They are “ phenyl- benzo –gamma -pyrone” derivatives.
ü Chalcone and dihydrochalcones represents the two classes
of flavanoids in which three- carbon bridge is
ü open where as in the other group 3-carbon bridge is a
part of heterocyclic ring, involving a phenolic group in
adjacent ring.
ü Most of the flavanoids have a carbonyl functional group
situated at one end of the bridge.
PROPERTIES OF FLAVANOIDS:
 Flavanoids dissolve in alkalies and give intense yellow
colour solution, on the addition of acid it becomes
colourless.
 Flavanoids exhibit strong fluorescence under UV light.
 Flavanoidal glycosides are soluble in water and alcohol.
Ethyl acetate is the choice of solvent for the extraction
of flavanoids from aqueous solution.
 Flavanoid compounds may be characterized through the
investigation of their UV spectra.
 CLASSIFICATION:
 Based on the oxidation level of central pyran ring they are
classified in to:
 Flavones
 Isoflavones
 Flavanols
 Flavanones
 Anthocyanidins (true flavanoids)
 COUMARIN GLYCOSIDES
 These are benzo-α-pyrone derivatives.
 Exhibit aromatic smell.
 Alcoholic solution if made alkaline it shows either
blue/green fluorescence.
 Coumarin bear oxygen atom either as(-OH), alkoxy(-
OCH3) at C-7 position.
 Ex: umbelliferone- OH,
 Herniarin-OCH3 


 FURANOCOUMARIN GLYCOSIDES
 These are obtained by the fusion of ‘ furan ring’ at C-6
and C-7 or C-7 and C-8 OF coumarin.

SULPHUR CONTAINING
GLYCOSIDES


 THIOGLYCOSIDES
 A number of plants of the family cruciferae yield glycosides
containing sulphur. They are also called glucosinolate
compounds.
 Hydrolysis of these yield volatile genins of thiocyanate structure
 Ex: mustard oil.
 The best known compounds are sinigrin and sinalbin,two
glycosides occurring in black mustard and white mustard
respectively.
 Many of the plants belonging to such families contain myrosinase
enzyme.
 The glycosides and their specific enzymes are found in different
cells in the seeds. They donot interact until they are brought
together by the destruction of the cell walls.
 The anion is called glucosinolate ion, R may be aliphatic or
aromatic . The cation (X) may be a simple metal ion or a
complex organic cation.
 Ex: sinalpine ion of sinalbin.
 ALDEHYDE GLYCOSIDES

 Glucovanillin is a glycosidal constituent of green

vanilla pods.
 The fruits of the plant (pods) are collected and
carefully cured. To permit enzymatic action on the
glycoside with the liberation of vanillin (the
aglycone) which is the principal flavouring
constituent of the pods.
 Vanillin is widely used as a flavouring agent .It may
be obtained from vanilla pod or prepared from the
glycoside coniferin, lignin or from the phenolic
volatile oil constituent eugenol.
 The bulk of vanillin which is produced commercially is
prepared from lignin, which gives upon hydrolysis
coniferyl alcohol.
 lignin →→→→→→→coniferyl alcohol
 ALCOHOLIC GLYCOSIDES
 Salicin:
 It is classified both as alcoholic glycoside, as it contains free
primary alcoholic group & as a phenolic glycoside, as its
aglycone is phenolic in nature.
PROPERTIES:


 Salicin is obtained from different species of salix, the principle

commercial source is Salix fragilis.
 Salicin is used for many years as a remedy in the treatment of
fever and rheumatism.
 It is now used as an analgesic-anti-pyretic in case of periodic fever.
It is better tolerated in the stomach than sodium salicylate,
aspirin, and other anti-pyretics and anti-inflammatory agents,
which have largely displaced in medical practice.
 Salicin is hydrolyzed by the enzyme emulsin into saligenin(salicyl
alcohol) and glucose.
 Acid hydrolysis of salicin gives glucose and a phenolic ether called
saliretin which is a condensation product of two molecules of
saligenin.
PHENOLIC GLYCOSIDES

 Arbutin is a phenolic glycoside that

occurs in bearberry leaves
Arctostaphylous uva-ursi.
 When hydrolyzed with acids or with
emulsin it yields glucose and
hydroquinone.
 It is used as a diuretic and also has
bactericidal action. This activity is
due to the hydroquinone given by
hydrolysis.

 GLYCOSIDES FOUND IN DIFFERENT
PARTS OF PLANT

ROOT RHIZOMES STEM BARK LEAVES FRUITS SEEDS

Liquorice Rhubarb Brahmi Cascara Senna Gokhru strophanthus

Ginseng Digitalis Quillia Aloe Bitter almond

Shatavari Dioscorea Wild cherry bark Shatavari , gingko Mustard

 E
C
N
A
R
U
C
C
O
GLYCOSIDE DICOT MONOCOT OTHER

Anthracene
 Euphorbiaceae,Ericaceae,Lythraceae Lilliaceae Fungi and
Polygonaceae,Rubiaceae,Rhamnaceae, lichens
Leguminosae,

2. Sterol / Angiosperms(broadly restricted) Lilliaceae,

Cardiac Leguminosae,Sterculiaceae,Cruciferae, Ranunculacea
Scrophulariaceae,Euphorbiaceae e

3. Saponin Lilliaceae,
a) Steroidal Leguminosae,Solanaceae,Apocyanaceae Dioscoreaceae
saponin
BIBILIOGRAPHY

 Text book of pharmacognosy :- Trease

& Evans 16th edition pg no:165-178.
 Text book of pharmacognosy :- Taylor
10th edition
 Text book of pharmacognosy :- kokate
16th edition.
