Jainal, Rasheedkhan A.

BSPh 2C

PHARMACOGNOSY AND PLANT CHEMISTRY

(LECTURE)

GLYCOSIDES

Assignment 3: (60 pts)

Rubric:
Criteria 20 pts

Content 14

Mechanics 6

1. Trace the biosynthetic pathways in the formation of the different constituents of

Glycosides? (20 pts)
a) Cardioactive (Steroidal)
 The biosynthesis of cardiac glycosides starts at the dehydrogenation of
pregnenolone to isoprogesterone which are catalyzed by 3β-hydroxysteroid
dehydrogenase followed by the conversion to progesterone from its isomer using
the enzyme ketosteroid isomerase. Progesterone 5β-reductase, now catalyzes
the conversion of progesterone to 5β-pregnane-3,20-dione which is then reduced
by the enzyme 3β-hydroxysteroid dehydrogenases to 5β-pregnane3β-ol-20-one.
The synthesis then proceeds with the enzyme pregnane 14β-hydroxylase
oxidizing 5β-pregnane3β-ol-20-one forming 5β-pregnane3β,14β-diol-20-one and
is further oxidized by 14β-hydroxypregnane 21β-hydroxylase to 5β-pregnane-
3β,14β-21-triiol-20-one. Malonyl coenzyme A:21-hydroxypregnane 21-O-
malonyltransferase, which is required for the synthesis of the cardenolide lactone
ring to form 21-O-malonlyl5β-pregnane-3β-14βdiol-20-dione and is then
catalyzed by an unknown enzyme to proceed the reaction forming cardiac
glucosides.

b) Anthraquinone

Anthraquinones can be biosynthesized from the polyketide (a) or shikimate (b)

pathway as described in the image above. They can be formed either by the
cyclization of linear octa--ketoacyl CoA intermediates from the addition of one
acetyl CoA to three malonyl CoA or by the addition of succinoylbenzoic acid,
resulting from shikimic acid andα-ketoglutaric acid, to mevalonic acid.
c) Saponin

The synthesis of triterpenoids exclusively occurs in the cytosol utilizing IPP and
its isomer DMAPP derived from acetyl coA via cytosolic mevalonic acid pathway. One
molecule of IPP (5C) condensed with its isomer DMAPP (5C) to form a monoterpene
called Geranyl pyrophosphate (GPP, 10C) by the enzyme prenyl transferase. GPP
condensed with one more IPP to form a sesquiterpene called Farnesyl pyrophosphate
(FPP, 15C) by the enzyme prenyl transferase. In the both of the above reactions prenyl
transferases catalyses the head to tail condensations. Each condensation reaction
involves a carbocation formed as ppi and it is eliminated. Then two molecules of FPP
condensed to form a triterpene Squalene (30C) by the enzyme squalene synthase. This
 condensation is a dimerisation reaction in which one molecule of NADPH is involved and
it is eliminated as NADP+ along with two pyrophosphate molecules. Squalene is then
converted to 2,3- oxidosqualene by squalene epoxygenase, involves one molecule of
NADPH and O2 molecule. It was reported that from acetyl coA to 2,3- oxidosqualene all
the steps are same for the biosynthesis of steroid saponins and phytosterols. 2,3-
oxidosqualene was the last common precursor and the major branch point as well as its
cyclization is the first committed step in the biosynthesis of triterpenoid saponins, steroid
saponins and phytosterols.

d) Cyanophore

The initial step in the synthesis of a cyanogenic glucoside is the

conversion of tyrosine to (E)-p-hydroxyphenylacetaldoxime. This step, catalyzed
by CYP79A1, is rate limiting in the biosynthesis of dhurrin, a cyanophore, in
young sorghum seedlings. The (E)-p-hydroxyphenylacetaldoxime is then
converted to p-hydroxymandelonitrile in a CYP71E1-catalyzed reactio.
Glucosylation of p-hydroxymandelonitrile catalyzed by the soluble UGT85B1
enzyme generates dhurrin.

e) Isothiocyanate

The general method for the formation of isothiocyanates proceeds through

the reaction between a primary amine (e.g. aniline) and carbon disulfide in
aqueous ammonia. This results in precipitation of the ammonium
dithiocarbamate salt, which is then treated with lead nitrate to yield the
corresponding isothiocyanate.

f) Flavonol
The two enzyme activities responsible for formation of the pollen-unique
class of flavonol
glycosides were
F3GalTase, which used
UDP-galactose as a
substrate to convert
flavonols to the flavonol 3-
O-galactosides, and a
flavonol 3-O-galactoside-
20-O-glucosyltransferase
(F20GT) which transfers
glucose from UDP-glucose
to the 20 position of
galactose to form the
flavonol
glucosylgalactoside. Both
glycosyltransferase
activities were
simultaneously extracted
from pollen and catalyzed
the formation of the
flavonol diglycosides.

g) Alcohol

The biosynthesis for

different alcohol glycosides
follow the same manner. In
this example, the cinnamyl
alcohol glycoside rosin will
 be traced. The biosynthesis starts from l-phenylalanine, which is converted into
cinnamic acid by PAL. From cinnamic acid, cinnamyl-CoA ester is formed by the
activity of hydroxycinnamate: CoA ligase. This CoA ester is further reduced to
cinnamaldehyde by cinnamyl-CoA reductase. The cinnamaldehyde is further
reduced by cinnamyl alcohol dehydrogenase to produce cinnamyl alcohol. The
enzymes that take part in the formation of the glycosides of cinnamyl alcohol are
not yet described. The binding of glucose to cinnamyl alcohol would result in
rosin formation, which is the simplest cinnamyl alcohol glycoside. Finally, rosavin
and rosarin can be formed from rosin by the connection of an arabinopyranose
and arabinofuranose unit, respectively.

h) Aldehyde
 In this particular example, an aldehyde glucoside vanillin is illustrated. Its
biosynthesis is generally agreed to be part of the phenylpropanoid pathway
starting with l-phenylalanine, which is deaminated by phenylalanine ammonia
lyase (PAL) to form t-cinnamic acid. The para position of the ring is then
hydroxylated by the cytochrome P450 enzyme cinnamate 4-hydroxylase
(C4H/P450) to create p-coumaric acid. Then, in the proposed ferulate pathway,
4-hydroxycinnamoyl-CoA ligase (4CL) attaches p-coumaric acid to coenzyme A
(CoA) to create p-coumaroyl CoA. Hydroxycinnamoyl transferase (HCT) then
converts p-coumaroyl CoA to 4-coumaroyl shikimate/quinate. This subsequently
undergoes oxidation by the P450 enzyme coumaroyl ester 3’-hydroxylase
(C3’H/P450) to give caffeoyl shikimate/quinate. HCT then exchanges the
shikimate/quinate for CoA to create caffeoyl CoA, and 4CL removes CoA to
afford caffeic acid. Caffeic acid then undergoes methylation by caffeic acid O-
methyltransferase (COMT) to give ferulic acid. Finally, vanillin synthase
hydratase/lyase (vp/VAN) catalyzes hydration of the double bond in ferulic acid
followed by a retro-aldol elimination to afford vanillin. Vanillin can also be
 produced from vanilla glycoside with the additional final step of deglycosylation.
In the past p-hydroxybenzaldehyde was speculated to be a precursor for vanillin
biosynthesis. However, a 2014 study using radiolabelled precursor indicated that
p-hydroxybenzaldehyde do not synthesise vanillin or vanillin glucoside in the
vanilla orchids.

i) Lactone
 Simple coumarins, coumarin (1), umbelliferone (2), esculetin (3), and
scopoletin (4) have modifications in their benzene ring. They are biosynthesized
from the phenylpropanoid pathway via ortho-hydroxylation of cinnamate (10), p-
coumarate (11), caffeate (12), and ferulate (13), respectively. The ortho-positions
are shown by red arrows. Oxygen atoms introduced by ortho-hydroxylation are
also highlighted in red. The ortho-hydroxylases from Arabidopsis (AtF6′H1), Ruta
graveolens (RgC2′H), and Ipomoea batatas (Ib1 and Ib2) were functionally
analyzed. AtF6′H1 and Ib1 catalyze ortho-hydroxylation of feruloyl-CoA (15),
whereas RgC2′H and Ib2 were capable of reacting to both feruloyl-CoA (15) and
p-coumaroyl-CoA (14) as the substrates. After hydroxylation, trans/cis
isomerization and lactonization occur, resulting in the production of their
respective coumarins. Umbelliferone (2) is a key intermediate of prenylcoumarin
biosynthesis, from which furanocoumarins and pyranocoumarins (examples:
psoralen and xanthyletin, respectively) are derived.

j) Phenol

The biosynthesis of Phenol Glycosides arbutin takes place from the

shikimic acid via phenylalanine (an amino acid)—cinnamic acid—hydroquinone
and finally to the desired glycoside.
2. Identify and describe the different methods of extraction and purification of glycosides
constituents? (20 pts)
a) Cardioactive (Steroidal)

One of the most common methods of extraction and purification of cardiac

glycosides is the prior protection of plant material by its maceration in toluene
and allowing it to stand for many days at 25–37°C to avoid the enzymatic
hydrolysis. Then, it is followed by exhaustive extraction with water-alcohol
mixture. The aqueous extract could be evaporated to a small volume under
vacuum at 50°C. Fats could then be removed by extraction with petroleum ether
and the aqueous syrup of glycosides is diluted with an equal volume of water.
Tannic acid and other polyphenolic and acidic products are precipitated with
freshly prepared lead hydroxide and the mixture is filtered through Hyflo-Super
Gel. The clear filtrate is adjusted to pH 6, concentrated under vacuum and
subjected to fractional extraction: first with ether, then chloroform, and finally with
chloroform-alcohol, 2:1 and 3:2. For isolation of glycosides of high solubility in
water, the residual aqueous phase is half saturated with sodium sulfate and then
extracted with chloroform-alcohol. The less polar fractions are separated by
chromatography on neutral alumina. The more polar fractions are usually
chromatographed after acetylation or benzoylation and the free glycosides
recovered by hydrolysis with bicarbonate.

b) Anthraquinone

The extraction and purification procedure for anthraquinone glycosides

consisted of macerating the ground drug for 24 hr. with 5% acetic acid. The
purpose of this step is to liberate the free anthraquinones and their
corresponding glycosides from their magnesium, potassium, or sodium salts, in
which form some of them are present in the plant. The maceration mixture is then
dried and extracted with chloroform until free of anthracene derivatives. The drug
is again air dried and then extracted with warm 95% ethanol. The alcoholic
extract is then treated with 5% alcoholic potassium hydroxide solution until
precipitation of the anthraquinone glycosides is complete. Following this step, the
residue is filtered and washed with 95% ethanol to remove the last traces of
potassium hydroxide and is then sucked dry on a Buchner funnel. The
anthraquinone glycoside salt residue is suspended in warm ethanol, and the free
glycosides are liberated by adding glacial acetic acid dropwise with stirring until
the color change is complete. The solution is then filtered hot, and the residue is
further extracted with warm ethanol. The combined filtrates are then evaporated
to dryness under reduced pressure, and the residue is recrystallized from
isopropyl alcohol to produce the crude drug.

c) Saponin

The first step in the processing of saponins involves their extraction from
the plant matrix. The extraction solvent, extraction conditions (e.g., temperature,
 pH, and solvent to feed ratio), and the properties of the feed material (e.g.,
composition and particle size) are the main factors that determine process
efficiency. Most saponins extractions are performed on powdered plant material
(various parts) using MeOH, ethanol (EtOH), water, or aqueous alcohol as
extracting solvents. This is followed by a defatting (to remove lipophilic
substances) step (generally with petroleum ether or n-hexane) which is carried
out before the extraction step or on the extract itself. The extracts are then
dissolved or suspended in water and shaken with n-butanol saturated with water.
The n-butanol aliquots are then combined and the liquid removed to give crude
saponin extract to work with.

d) Cyanophore

Extraction and purification of cyanogenic glycosides are done by first

blending the ground seed with 500 ml of 95% or absolute alkanol (methanol,
ethanol or isopropanol) with 10% (w/w) ammonia for 2 min at 2000 rpm. After a
15-min quiescent period, 500 ml of hexanes is added to the mixture and the
slurry is again blended for 2 min at 2000 rpm. Grounded seed is then recovered
on filter paper by vacuum filtration. Samples are rinsed three times with a total of
125 ml of alkanol and then dried overnight at 40°C in vacuum oven. The
grounded seed is further defatted with hexanes using a Soxhlet apparatus and
dried. The hexane and alkanol phases are separated and the polar fraction is re-
extracted three times with hexanes to recover any residual oil. The hexane and
alkanol phases were evaporated to recover the oil and the solid residues
(cyanogenic glycoside), respectively.

e) Isothiocyanate

The extraction and purification of glucosinolates are carried out by

applying an aqueous solution containing the pure glucosinolates at a
concentration of 1.6 mM to 10 g of soil in a polypropylene 50-mL centrifuge tube.
The tubes are shaken gently for 5 min. The extractions are done at ambient
temperature. After a contact time of approximately 30 min, 70% methanol is
applied and the tubes are shaken vigorously (manually) and left to stand for 45
min. The contact time of 30 min is chosen to allow time for any chemical
reactions of the glucosinolates with the soils without their microbial degradation
or hydrolysis playing a significant role. The tubes are then centrifuged and the
supernatant was filtered through a 0.22 µm syringe-mounted nylon filter.

f) Flavonol

Flavonoids are extracted and purified using the Soxhlet method. In this
method. shade dried leaves are powdered and is soxhlet extracted with 70%
(v/v) ethanol and vacuum concentrated to dryness under reduced pressure at 60°
± 1 °C. After drying in hot air oven (40–45 °C), it is stored in an air tight container
in refrigerator at 5 °C. The residue was designated as hydro-ethanolic extract of
 the plant. The residue are then extracted successively with pet-ether, benzene,
chloroform, ethyl acetate, and ethanol and finally macerated with distilled water
(non-polar to polar) to get respective flavonoid extracts.

g) Alcohol

The Stas-Otto method is the conventional technique used to extract and

purify alcoholic glycosides. The drug containing alcohol glycoside is finely
powdered and the powder is extracted by continuous hot percolation using
soxhlet apparatus with alcohol as solvent. During this process, various enzymes
present in plant parts are also deactivated due to heating. The thermolabile
glycosides, however, should be extracted at temperature preferably below 45°C.
The extract is treated with lead acetate to pre-cipitate tannins and thus eliminate
nonglycosidal impurities. The excess of lead acetate is precipitated as lead
sulphide by passing hydrogen sulphide gas through solution. The extract is
filtered, concentrated to get crude glycosides. From the crude extract, the
glycosides are obtained in pure form by making use of processes like fractional
solubility, fractional crystallization and chromatographic techniques such as
preparative thin layer and column chromatography.

h) Aldehyde

In aldehyde glycosides extraction and purification, the dried plant material

is rendered into a moderately coarse powder. The powder is then extracted in a
Soxhlet apparatus with aqueous ethanol. The non-glycosidal impurities which get
extracted along with glycosides are removed by precipitating them with lead
acetate solution. The excess of lead acetate is then removed by passing
hydrogen sulphide gas through the extract. Lead gets precipitated as lead
sulphide, which is filtered out. The filtrate contains the glycosides. The glycoside
can be obtained by removal of the solvent under reduced pressure or any other
suitable procedure. Further purification of the isolated glycosides is done by
column chromatography.

i) Lactone

Simple lactone glycosides are extracted by polar solvents such as simple

phenols and phenolic acids. They are generally lipid-soluble and can be isolated
from dried plant material by petroleum ether, light petroleum, or dichloromethane.
In many cases crude lactone glycoside is obtained as a semicrystalline sediment
when the concentrated petroleum ether extract is stored at low temperatures,
and is next purified by use of column chromatographic techniques, preparative
TLC, or by crystallization from different solvents.

j) Phenol
 Ultrasound-assisted extraction (UAE) can be utilized to extract and purify
phenol glycosides. It is based on the action of ultrasonic vibrations directed
toward an extracted sample, which enhance efficacy of penetration of a sample
by solvent. This method is characterized by high speed, simplicity, and usually
takes several minutes. Apart from the type of a solvent, sample size, pH of
extract, temperature and pressure, factors such as particle size, duration of
sonification and its amplitude, also have an impact on the efficacy of the
extraction process. Regardless of these factors, the method is considered as the
simplest one possible to perform in a laboratory. One of its advantages is the
possibility to carry out the extraction of several samples simultaneously within a
relatively short time. However, it is necessary to decant the extract or to filter it
through appropriate paper filters.

3. Identify and describe the different qualitative methods of analysis of glycosides

constituents? (20 pts)

a) Cardioactive (Steroidal)

 Keller-kiliani test— To the alcoholic extract of drug equal volume of water

and 0.5 ml of strong lead acetate solution was added, shaked and filtered.
Filtrate was extracted with equal volume of chloroform. Chloroform extract
was evaporated to dryness and residue was dissolved in 3 ml of glacial
acetic acid followed by addition of few drops of FeCl3 solution. The
resultant solution was transferred to a test tube contain-ing 2 ml of conc.
H2SO4. Reddish brown layer is formed, which turns bluish green after
standing due to presence of digitoxose.

 Legal test— To the alcoholic extract of drug equal volume of water and 0.5
ml of strong lead acetate solution was added, shaked and filtered. Filtrate
was extracted with equal volume of chloroform and the chloroform extract
was evaporated to dryness. The residue was dissolved in 2 ml of pyridine
and sodium nitropruside 2 ml was added followed by addition of NaOH
solution to make alkaline. Formation of pink colour in presence of
glycosides or aglycon moiety.

 Baljet test— Thick section of leaf of digitalis or the part of drug con-taining
cardiac glycoside, when dipped in sodium picrate solution, it forms yellow
to orange colour in presence of aglycones or glycosides.
  3,5-dinitro benzoic acid test— To the alcoholic solution of drug few drops
of NaOH followed by 2% solution of 3,5-dinitro benzoic acid was added.
Formation of pink colour indicates presence of cardiac glycosides.

b) Anthraquinone

 Borntrager’s test— To 1 gm of drug add 5–10 ml of dilute HCl boil on

water bath for 10 min and filter. Filtrate was extracted with CCl4/ benzene
and add equal amount of ammonia solution to fil trate and shake.
Formation of pink or red colour in ammoni-cal layer due to presence of
anthraquinone moiety.

 Modified borntrager’s test— To 1 gm of drug, add 5 ml dilute HCl followed

by 5 ml ferric Chloride (5% w/v). Boil for 10 min on water bath, cool and
filter, filtrate was extracted with carbon tetra-chloride or benzene and add
equal volume of ammonia solution, formation of pink to red colour due to
presence of anthraquinone moiety. This is used C-type of anthraqui-none
glycosides.

c) Saponin

 Haemolysis test— A drop blood on slide was mixed with few drops of aq.
Saponin solution, RBC’s becomes ruptured in presence of saponins.

 Foam test— To 1 gm of drug add 10–20 ml of water, shake for few

minutes, formation frothing which persists for 60–120 s in presence of
saponins.

d) Cyanophore

 Sodium picrate test— Powdered drug was moistened with water in a

conical flask and few drops of conc. Sulphuric acid was added. Filter
paper impregnated with sodium picrate solution followed by sodium
carbonate solution was trapped on the neck of flask using cork. Formation
of brick red colour due to volatile HCN in presence of cynophoric
glycosides takes place.

e) Isothiocyanate

 Thymol-test HPLC— the isothiocyanate-containing sample is treated with

1% thymol in ethanol and 77% sulfuric acid and is then analyzed using
High-performance liquid chromatography (HPLC).s

f) Flavonol
  Ammonia test— Filter paper dipped in alcoholic solution of drug was
exposed to ammonia vapor. Formation of yellow spot on filter paper.

 Shinoda test— To the alcoholic extract of drug magnesium turning and dil.
HCl was added, formation of red colour indicates the presence of
flavonoids. To the alcoholic extract of drug zinc turning and dil. HCl was
added, formation of deep red to magenta colour indicates the presence of
dihydro flavonoids.

 Vanillin HCl test— Vanillin HCl was added to the alcoholic solution of drug,
formation of pink colour due to presence of flavonoids.

g) Alcohol

 Antimony trichloride test— To a solution of glycoside add a solution of

antimony tri-chloride and tri-chloroacetic acid, and then heat the mixture.
Appearance of blue or violet colour show presence of alcoholic glycosides.

h) Aldehyde

 Kedde test— A solution of glycosides is treated with a small amount of

Kedde reagent (Mix equal volumes of a 2% solution of 3, 5 dinitrobenzoic
acid in menthol and a 7.5% aqueous solution of KOH). Development of a
blue or violet colour that faded out in l to 2 hrs shows it presence of
aldehyde glycosides.

i) Lactone

 FeCl3 test— To the concentrated alcoholic extract of drug few drops of

alcoholic FeCl3 solution was added. Formation of deep green colour,
which turned yellow on addition of conc. HNO3, indicates presence of
coumarins.

 Fluorescence test— The alcoholic extract of drug was mixed with 1N

NaOH solution (one ml each). Development of blue-green fluo-rescence
indicates presence of coumarins.

j) Phenol

 Xanthydrol test— The crude is heated with 0.1 to 5% solution of

Xanthydrol in glacial acetic acid containing 1% hydrochloric acid. A red
colour is produced due to the phenol ring.