5-_saponins_and_cardiac_glycosides.pptx

SAPONINS AND CARDIAC GLYCOSIDES
ASSOC. PROF. DR. MOSTAFA MAHMOUD HEGAZY

 content
 Saponins (Properties, Extraction and separation)
 Steroidal Saponins (Diosgenin and Hecogenin)
 Triterpenoid Saponins (Ginseng, Liquorice, Quillaia and senega)
 Cardiac Glycosides

Glycosides Dr. Mostafa Hegazy
 Saponins
 Saponins are a group of organic compounds that
foam or form persistent froth with water. They
are named from the Latin word sapo =soap like.
 The term saponin is applied to identify
glycosides, which have the ability to lower
surface tension of aqueous solutions, and to
bring about haemolysis of red blood corpuscles.

 Saponins are amorphous colloidal
glycosides, which are used technically
to decrease surface tension e.g. as
emulsifiers and in fire extinguishers.
 They also have some important
pharmacological and medicinal uses.
 Saponins are widely distributed in
several plant families e.g. Rosaceae,
Liliaceae, Scrophulariaceae and
Polygonaceae

Properties:
1-They form persistent froth upon
shaking with water, as they lower the
surface tension; their solutions form
stable emulsions with oils, fats and
resins (i.e. emulsifiers). They were
also used as detergents.
2- Saponins have haemolytic action
on red blood corpuscles, so they are
toxic when injected ???? into the
blood stream. However, they are
comparatively harmless when taken
orally, as they are absorbed by the
intestines to a small extent.

3- Saponins cause sneezing when snuffed and
causes subcutaneous inflammation when injected
hypodermally.
4- Saponins often have a particular affinity for
cholesterol. This property can be used for the
determination of cholesterol in blood (e.g.
digitonin).

5- Saponins are soluble in water or aqueous-
alcohol and can be precipitated addition of
organic solvents such as acetone or ether.
They can also precipitated by lead acetate as
lead-saponin complex and regenerated by
addition of dilute sulfuric acid.
6- On hydrolysis a saponin splits into
sugar(s) and an aglycone called sapogenin.
Saponins ® sapogenin + sugar

Extraction and separation of saponins:
contain up to 12 sugar residues (monosaccharides or uronic acids). Therefore, they
are of high molecular weights, highly polar, and hence difficult to be isolated in
a pure state. They can be hydrolyzed by acids to give sapogenins and various
sugars, sapogenins are easier to be isolated and purified.
1- Plant material + hot dilute alcohol, filter while hot, filtrate is cooled + excess
acetone or ether ® saponins precipitate, collect, repeat this process several times.
Individual saponins can be separated by chromatography (e.g. reversed-phase
techniques).

2-The plant material is extracted with water, the water extract is treated with
neutral lead acetate to precipitate acid saponins (e.g. quillajic acid) and filtered.
The filtrate is then treated with basic lead acetate, which precipitate neutral
saponins. Saponins can then be liberated from the resulting saponin lead salts by
passing H2S gas or adding dilute sulfuric acid.
3- Saponins can be extracted by aqueous-alcohol and then the extract is freed
from the alcohol by distillation under vacuum. The remaining aqueous extract is
treated with calcium hydroxide to precipitate saponins. The obtained precipitate
is collected by filtration and the saponins are liberated from the precipitated
complex by boiling with alcohol.

Classification of saponins:
According to the structure of the aglycone, saponins can
be either a triterpene or a steroid. So, there are two kinds
of saponins:
1- Steroidal (C-27) saponins that have a steroidal genins
they are triterpenes which are based on the cyclopentane
perhydrophenanthrene ring system. On dehydrogenation
with selenium they give Diel's hydrocarbon.
2- Triterpenoid (C-30) saponins that have a pentacyclic
triterpenoid genins. On dehydrogenation with selenium
they give naphthelene or picene derivative.
Steroidal saponins are less distributed than triterpenoid
type.

Steroidal Saponins
Steroidal saponins are of great importance and interest owing to their structural
relationship with the important steroidal hormones (sex hormones and
corticosteroids), cardiac glycosides and vitamin D.
The first synthesis of cortisone from desoxycholic acid from ox bile involved 32
steps. Other successful partial syntheses of sapogenins to steroidal hormones were
early achieved. These sapogenins are converted a number of sex hormones by
techniques of enzyme hydroxylation (especially at position 11) with
microorganisms. These techniques not only resulted in phenomenal increase in
availability and dramatic decrease in the cost of these hormones substances as
medicinal agents, but also led to deliberate search for mass screening of thousands
of plants for sapogenins. The most common starting materials for chemical
conversion to medicinal hormones are diosgenin and hecogenin.

Diosgenin:
Diosgenin can be obtained from the roots
(tubers) of Dioscorea genus, family
Dioscoreaceae. The tubers contain up to 8%
diosgenin, whichis obtained by acid hydrolysis
of thesaponin. Fermentation for 4-10 days gives
better yield. Diosgenin is also found in
fenugreek seeds.
Uses: Diosgenin has been the dominant raw
material for the synthesis of important steroids
e.g. sex hormones, cortisone, contraceptive pills
and other steroids; however, total synthesis is
now much used.

Hecogenin:
Hecogenin is a steroidal sapogenin obtained from
the sisal leaves Agave sisalana f. Agavaceae.
Uses:
Hecogenin has a keto group in ring C, which can
be moved to position 11 by chemical or
microbiological methods and becomes similar to
corticoids. Therefore, hecogenin can be used for
the synthesis of glucocorticoids and
mineralocorticoids.

Triterpenoid Saponins
This group of saponins is generally characterized
by the C-30, pentacyclic terpenoid aglycones.
Unlike the steroidal saponins, they are abundant in
dicotyledons and rare in monocotyledons. They are
found in many plant families such as:
Polygonaceae, Caryophyllaceae, Myrtaceae,
Zygophyllaceae, Rutaceae.. etc. They also occur in
some important drugs including Liquorice,
Quillaia and Senega. Triterpenoid glycosides are
often formed of a sapogenin to which a chain sugar
or uronic acid units or both attached at C-3.

The triterpenoid sapogenins can be
classified as derivatives of one of three
hydrocarbons:
A- Oleanane B-Ursane C-Lupane
Hydroxyl, ketone and carboxylic
functions are the most frequent of the
main triterpenoidal skeletons. Most
triterpenoid saponins contain at least
one carboxyl group and the oleanane
type is the most common one.
Replacing a methyl group of the
original triterpene with a carboxyl
group in positions 4, 17 or 20 forms the
triterpenoid acids.

Ginseng saponins:
The roots of Panax ginseng f. Araliaceae,
have been thoroughly studied and the
medicinal activity appears to be due to the
presence saponins, termed as ginsenosides
and panaxosides. Hydrolysis of the
panaxosides yields oleanolic acid, panaxdiol
and panaxtriol.

Quillaia Saponins
Quillaia bark (Quillaia extract (E 999))
is the dried inner bark of Quillaja
saponaria (family Rosaceae). It has an
acrid taste and causes sneezing when
snuffed. It contains about 10% of
saponins of the triterpenoid type.
Hydrolysis of the saponins yield the
principal sapogenin quillaic acid and
gypsogenin. Quillaia saponins is a
mixture of acylated triterpinoidal
oligoglycosides (acylated saponins).

Uses:
 Used as expectorant.
 It is also used as an emulsifying agent,
particularly for tars and volatile oils.
 Quillaia also finds technical application as
mild detergent for washing fine textiles.

Liquorice Saponins:
Liquorice consists of the dried unpeeled roots and
stolons (under ground stem) of Glycyrrhiza glabra
fam. Leguminosae. The drug is cultivated in Europe
and the United States and collected from wild
sources in the Middle East and Russia. Constituents:
The main triterpenoid saponin of liquorice is a
strongly sweet principal named glycyrrhizin (6-13
%), to which it owes its sweet taste. Glycyrrhizin is
the potassium and/or calcium salt of glycyrrhizinic
acid.

Uses:
- Liquorice extract, which contains 10-15%
glycyrrhizin, is used in cough syrups as demulcent
and expectorant.
- The aglycone of glycyrrhizin has a keto group at
position 11, a structure that shown by
adrenocortical hormone. This may explain the
reported deoxy corticosterone effects of liquorice
extract. Hence glycyrrhetinic acid is used for the
treatment of rheumatoid arthritis and other
inflammatory diseases (unlike cortisone Liquorice
may give symptomatic relief from peptic ulcer
pain).

Uses:
-Flavoring and sweetening agent to
disguise nauseous medicines e.g. quinine,
cascara extract … etc
- Because of its sweet taste, liquorice
extract is employed in the confectionery
industry.
- Recently, glycerhizin gel act as a useful
vehicle for various topically used drugs, as
glycerhizin enhance the skin penetration
by the drug.

Senega Saponins:
The dried roots of Polygala snega, F. Polygalacea,
contain up to 12% of triterpenoidal saponins. One
of these saponins is Senegin II, which contain
fucose (a
deoxy sugar) and cinnamic acid derivative forming
branched chain at C-28.
Uses:
- Senega used as stimulant expectorant in chronic
bronchitis.
- It is also prescribed with other expectorants as
ipeca and ammonium carbonate.

General Uses and Pharmacology of Saponins
1- Saponin glycosides are used to decrease surface tension, so they are excellent
emulsifiers and can be used in fire extinguishers.
2-Steroidal saponins are used for economic synthesis of sex hormones and
corticoids by chemical or microbiological means.
3-.Drugs containing triterpenoid saponins are generally used as expectorants.
Although they are not absorbed they irritate the mucous lining (gastric mucosa) of
stomach and by reflex action they increase in glandular secretion, especially
bronchioles. Hence, they facilitate the expectation and discharge of phlegm in
infections of the respiratory organs.
4- Biologically saponins cause haemolysis as they attack the walls of the red blood
corpuscles and liberation of the hemoglobin.
5- Saponins increase the rate of absorption of many active drugs as cardiac
glycosides.

Tests for identity:
1-Froth test: saponins give persistent froth or foam with shaking.
2- Haemolysis test. A suspension of red blood corpuscles in physiological saline is
cloudy, if treated with saponin it becomes transparent and bright red. Blood agar in
Petri dish can use for the test and the presence of saponins in drugs. This can be
done by placing pieces of the drug on the surface of the blood agar. Saponins are
present when the pieces of the drug show transparent zones.
The above test could be modified for quantitative determination of saponins by
measuring the hemolytic index which is the lowest concentration required for the
complete hydrolysis of a suspension with a known content of red blood corpuscles.

Cardiac Glycosides
C23 or C24 steroidal glycosides that act directly on
the failing heart muscle producing a slowing and
strengthing effect, so called Cardiac glycosides.
Occurrence: Medicinally important glycosides are
obtained from a limited number of plant families.
Cardenolides are also found in the Scrophulariaceae
(e.g. Digitalis), Apocyanaceae (e.g. Strophanthus) and
Ranunculaceae (e.g. Adonis). Bufadienolides are less
common and occur in some Liliaceae (e.g. Urginea)
and Ranunculaceae (e.g. Helleborus).
Cardiac Glycosides still are used by primitive people
for preparation of arrow poisons.

General Structure of Cardiac Glycosides The
aglycones of cardiac glycosides fall in two main
types according to whether there is a five- or six-
membered lactone ring these types are known as:
1- Cardenolides, which have a C-23 skeleton and
a five membered-unsaturated lactone ring at C-17.
2- Bufadienolides (scilladienolide) which have a
C-24 skeleton and a six membered doubly
unsaturated lactone ring at C-17. In each class
(Cardenolides or Bufadienolides), the various
aglycones differ mainly in the presence
substituents at different positions of the steroidal
nucleus.

The sugar residue which is usually
formed of 3 or 4 units attached to
the aglycone at C-3, through a β-
linkage. In addition to glucose and
rhamnose some rare deoxysugars
are commonly present in cardiac
glycosides. (Some sugars lack the
hydroxyl group at C-2 and called
2,6-deoxysugar e.g. digitoxose),
cymarose and sarmentose. Other
sugars have the C-3 hydroxyl
group methylated, e.g. cymarose,
digitalose and sarmentose.)

Structure Activity Relationships:
1- The unsaturation of the β-oriented lactone is essential for
the activity.
2-Opening of the lactone ring renders the glycosides
inactive and saturation result in substational decrease of the
activity.
3- β-Orientation of the C-3 OH and lactone at C-17 is
important, if it changed to a-
orientation led to weaker activity
4- The activity is stronger if A/B and C/D junctions are cis.
5- The β-hydroxyl group at C-14 is important to the activity,
Change to the α - position
leads to loss of the activity.

- Tests for Identity of Cardiac Glycosides:
1-Liebermann test for the steroidal part:
Drop of conc. H2SO4 added to the glycoside in glacial acetic acid ---- red color ----
violet
---- blue ------ green color.
2-Kedde’s test (Modified Raymond) (the active methylene group in cardenolides)
(C- 21 of the lactone ring) Kedde's reagent (3,5- dinitrobenzoic acid / methanol +
aq. KOH 1:1 proportion) + alc. ext. ----- blue or violet color. The colour fades in
one or two hours, the reagent is used as a spraying agent and the test is used as a
quantitative determination for cardinolides.

3- Legal test for unsaturated lactones Small amount of the glycoside dissolved in
few drops of pyridine + a drop 2% sodium nitroprusside + a drop of 20% NaOH-----
deep red color indicates the presence of cardiac glycosides (Scillaren glycosides
give negative result).
4- Keller-kiliani test for 2-deoxy sugars Glycoside dissolved in glacial acetic acid +
FeCl3 soln + H2SO4 (contain the same amount of FeCl3) added by pipette at the
bottom of the soln. ----- blue color is formed between the two layers in 2-5 mins.,
spreading into the acetic acid layer.
5-Antimony trichloride (cardenolides, scilladienolides)The glycoside is heated with
2ml of SbCl3/ trichloroacetic acid ------- blue or violet colour.
6- Baljet test for cardiac glycosides. Small volume of the glycoside + Baljet
reagent (1% aq. soln of picric + 10% NaOH 1:1)-------- orange or red color.

Extraction and isolation of Cardiac Glycosides:
Procedure:
1- The fresh plant + solid (NH4)2SO4 to precipitate the enzymes + hot ethanol
(paste).
2- Saturated solution of ethanolic (NH4)2SO4 is added and the mixture is blended
again, and then filtered through a piece of cotton then through a sintered glass filter.
3- The filtrate is diluted with water and purified by addition of a soluble lead salt
and filtration. The excess lead is precipitated as sulfate or sulfide (by addition of
H2SO4 or passing H2S gas).
4- Then the glycosides are extracted from the filtrate chloroform-methanol (1:1)
mixture and the individual glycosides are isolated by chromatographic methods.

Cardenolides
All medicinal preparations of cardiac glycosides are derived from natural sources,
particularly Digitalis leaves as the synthesis of these compounds was not
successful, but recently, some monoside have been prepared as storaphanthidin-
mannoside. Glycosides of Digitalis purpurea, D. Lanata and Strophanthus gratus
are most commonly used in medicine.

Digitalis Glycosides:
Digitalis cardenolides, have been isolated from D. purpurea alone, the most
common aglycones are derived from digitoxigenin, gitoxigenin and digoxigenin.
Digitalis purpurea cardiac glycosides
Digitalis consists of the dried leaves of Digitalis purpurea family Scrophulariaceae.
The leaves contain about 0.3% (on dry basis) of cardiac glycosides calculated as
digitoxin. D. Purpurea contains, also, saponins, anthraquinone derivatives and
flavonoids.

D. purpurea contains the following
important cardio-active
constituents:
1- Primary (tetra) glycosides
(purpurea glycoside A and purpurea
glycoside B and glucogitaloxin)
which are all possess a chain of three
digitoxose sugars and a terminal
glucose unit.

2- Secondary glycosides
(digitoxin, gitoxin and gitaloxin),
which are the main constituents of
the dried drug. They exist as a result
of enzymatic degradation (during
drying) which causes loss of the
terminal glucose units of the genuine
primary glycosides. Gitoxin is also
found in dried D. purpurea.

3- Aglycones such as digitoxigenin
and gitoxigenin occur in small
amounts. The aglycones can also be
produced by acid hydrolysis of their
respective glycosides.

D. lanata , is the main source of
digoxin and lanatoside C. It contains
the following cardenolides:
1- 1ry glycosides (lanatosides A, B, C,
D and E), as those of D. Purpurea but
the terminal digitoxose moiety is
acetylated. Drying and storage may
cause partial hydrolysis and
deacetylation of these glycosides to
give purpurea A and B and the
deacetyl-lanatoside C.

2- 2ry glycosides such as
acetyldigitoxin, acetyldigoxin , digitoxin
and digoxin may present as a result of
hydrolysis during drying or storage. D.
lanata is more valuable than D. purpurea
as it contains higher glycosidic content
up to 1%.
Hydrolysis of Digitalis cardiac
glycosides:
The primary glycosides in Digitalis can
be hydrolyzed by the hydrolytic enzymes
present in the plant itself, acids or alkali
to give different products, as shown on
the structure schemes.

Strophanthus Cardiac Glycosides
K-Strophanthoside
The seeds of S. kombe f. Apocyanaceae contain up to
10% of a mixture of glycosides known as K-
strophanthin. The principal glycosides are K-
Strophanthoside, and cymarin, all based on the genin
strophanthidin. K-strophanthin-B can also be
obtained from the seeds of S. hispidus and was
formerly named H-strophanthin.
Hydrolysis of k-strophanthoside:
It is hydrolyzed by α-glucosidase, present in the
seed, to yield K-strophanthin-B
which is then hydrolyzed by strophanthobiase to
cymarin. Complete acid hydrolysis
yields strophanthidin aglycon and the sugar residue.

G- Strophanthin (Ouabain)
The seeds of S. gratus contain 4-8% of ouabain,
a rhamnoside glycoside and more stable than
other cardiac glycosides. Used for the preparation
of ouabain injections (as it is poorly absorbed
orally).
Hydrolysis: Acid hydrolysis yields an aglycone
called G-strophanthidin (ouabagenin)
and rhamnose. It is available as Ouabain® amp.
and drops.

Tests for identity of strophanthus glycosides:
1- Glycoside is moistened + mixture of sulfuric acid-water (4:1) ------ emerald
green color indicates the presence of k-strophanthin while a pink color changing
to brownish-red showing at last a green fluorescence indicates ouabain (G-
strophanthin).
2- Glycoside + one drop of amm. molybdate (Froehde's reagent), evaporate to
dryness, cool + a drop of H2SO4 ------ blue color indicates the presence of G-
strophanthin (k-strophanthin
gives negative result).
3- In the above test, if ammonium molybdate is replaced by ammonium vanadate
(Mandalin's reagent) ----- green color indicates G-strophanthin (K-strophanthin
does not give positive result with this test).

Bufadienolides
Bufadienolides have a little therapeutic use as
cardio-tonic drugs; this is because they have
low therapeutic index and cause side effects.
They are less widely distributed in nature in
comparison with cardenolides.
Squill Cardiac Glycosides
Squill (white squill) consists of the dried sliced
bulbs of Urginea maritima f. Liliaceae.

Constituents: Squill contains a number of bufadienolides.
Glucoscillaren-A, is a genuine primary glycoside. Removal
of one glucose unit gives a secondary glycoside scillaren
A, which is a secondary glycoside present also in squill.
Removal of one glucose residue from scillaren A gives
Proscillaridin A. Removal of all the sugars yields the
aglycone Scillaridin A (or scillarenin).
Squill also contains an amorphous mixture of glycosides
called scillaren B. is freely soluble in water and alcohol. It
forms one third of the total squill glycosides and is more
active than scillaren A.
Hydrolysis: Scillaren, which is the major glycoside of
squill, can be hydrolyzed by, scillarenase enzyme or by
acids as shown on the structure scheme.

Uses:
The crude drug squill is used for the extraction of
scillaren glycosides which are normally hydrolyzed
to proscillandin A. Proscillaridin A has a rapid effect
with short duration, and is not cumulative. It lowers
the pulse less than other cardiac glycosides.
Red Squill: It is a red variety of Uriginea maritime.
It differs from white squill in containing red
anthocyanin pigments and the glycoside scilliroside.
It is used as a rodenticide, to eradicate rats without
endangering other animal life seriously. Other
animals vomit it right away but rats can not vomit.

5-_saponins_and_cardiac_glycosides.pptx

5-_saponins_and_cardiac_glycosides.pptx

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