POSTLAB DISCUSSIONS

(PHYTOCHEMICAL SCREENING EXPERIMENTS)

PHA6115 LABORATORY
INTRODUCTION
PHYTOCHEMICAL SCREENING
• Refers to the extraction, screening and identification
of medicinally active substances found in plants
• Some of the bioactive substances that can be derived
from plants are flavonoids, alkaloids, carotenoids,
tannins, antioxidants and phenolic compounds
PHYTOCHEMICAL EXTRACTION
• Separation of medicinally active portions of plant (and
animal) tissues using selective solvents through standard
procedures.
• Purposes:
• To attain the therapeutically desired portions
• To eliminate unwanted material by treatment with a
selective solvent known as menstruum
FACTORS INFLUENCING THE QUALITY OF
EXTRACT
• Plant part used as starting material
• Solvent used for extraction
• Extraction Procedure
EFFECTS OF EXTRACTED PLANT
PHYTOCHEMICALS
• Nature of the plant material
• Its origin
• Degree of Processing
• Moisture Content
• Particle Size
FACTORS AFFECTING QUANTITY AND
SECONDARY METABOLITE COMPOSITION
• Type of extraction
• Time of extraction
• Temperature
• Nature of solvent
• Solvent concentration
• Polarity
VARIATION IN EXTRACTION
METHODS
• Length of extraction period
• Solvent used
• pH of the solvent
• Temperature
• Particle size of the plant tissues
• The solvent-to-sample ratio
METHODS OF EXTRACTION
• Maceration
• Infusion
• Percolation
• Digestion
• Soxhlet extraction
• Sonication
• Serial exhaustive extraction
• Plant tissue homogenization
GLYCOSIDES
GLYCOSIDES
• Glycosides are simply inactive sugars (glycone)
bonded to a non-sugar active portion
(aglycone/genin)
• Usually bonded by an ether (R-O-R’), although other
bonds such as (R-C-R’) and (R-N-R’) are possible
• Hydrolysable by mineral acids (not bases) or enzymes
such as myrosin or emulsin (only target the beta forms
of glycosides)
CLASSES TESTED IN THE SCREENING
• Saponins
• Flavonoids
• Tannins*
• Cyanophores
• Anthraquinones
• Cardiac Glycosides
* Tannins are sometimes discussed as a separate class of secondary metabolites
SAPONINS
3 Main Properties
• Foams/lathers in aqueous solution
• Hemolyzes red blood cells
• Lowers surface tension of solution
SAPONINS
Types based on aglycone (aka the
sapogenin):
• Neutral/ Steroidal – similar to
cholesterol; predominant in
monocots
• Acidic/ Triterpenoid – five six
membered rings; predominant in
dicots
TESTS FOR SAPONINS
NAME OF TEST FOR REAGENT POSITIVE RESULT
Froth Plant extract itself Honeycomb froth greater than
2cm for 10 minutes or more
Hemolysis Plant extract on Zones of hemolysis (aka
blood agar hemolytical halos)

Capillary Plant extract itself Reduction of at least ½ height of

in capillary tube water in capillary tube
Liebermann- Nucleus Sulfuric acid and Green/blue (steroidal),
Burchard of acetic anhydride red/pink/violet (triterpenoid)
saponins
FLAVONOID GLYCOSIDES
• “Flavonoids” applies to the glycosides, with their aglycone
being “flavonols”
• Formally defined as two rings separated by a three-carbon
bridge whether enclosed as ring or not, having the gamma-
benzopyrone as nucleus
• With tannins, are described as polyphenols with antioxidant
activity
CHEMISTRY AND TESTS

• Gamma-benzopyrone (left) is found in majority of flavonoids

• Example of an actual flavonoid molecule (right)
 TESTS FOR FLAVONOIDS
NAME OF TEST FOR REAGENT POSITIVE RESULT
Batesmith- Chalcone/ aurone HCl Immediate red (to verify
Metcalf chalcone/aurone)
• If no color, boil, and
red/violet color is seen (to
verify leucoanthocyanins)
Wilstatter Gamma- HCl, Orange -> red -> crimson ->
Cyanidin benzopyrone Magnesium, magenta, ocasionally green or
nucleus of all Octanol blue
flavonoids
Matchstick Catechins Matchstick Pink stain on wood
in HCl
CHEMISTRY OF TANNINS
• Tannins – polyphenol compounds of high molecular weight
(1000-5000 g/mol) with astringent property
• Pseudotannin - <1000 g/mol, cannot precipitate leather
• Combine with proteins and exert inhibitory effect on
putrefaction of animal skin, preserving them to form leather
• With flavonoids, are described as polyphenols with
antioxidant activity
• Often tested in vitro under as “total phenolic content”
(ex. through the Folin-Ciocalteu reagent)
CLASSIFICATION
1) Hydrolyzable
a) Gallotannin
b) Ellagitannin
2) Nonhydrolyzable
TESTS FOR TANNINS
NAME OF TEST FOR REAGENT POSITIVE RESULT
Goldbeater’s skin Membrane of ox Black/brown
True tannins,
intestine, HCl, FeSO4
in general
Gelatin Gelatin TS Gel-like precipitate
Ferric chloride Ferric chloride TS Bluish black (hydrolysable),
greenish brown
Distinguishing (condensed)
Dry distillation true tannins Heat Pyrogallol as product
(hydrolysable), pyrocatechin
as product (condensed)
CYANOPHORES/ CYANOGENIC GLYCOSIDES
• Aglycones are derived from mandelonitrile, containing a
cyanide group (CN)
• They give a characteristic bitter almond/peach kernel odor,
and are found in bitter almonds, apricots, cherries, peaches,
plums and other rosaceous seeds
• Potentially toxic due to their cyanide content
CHEMISTRY AND TESTS
TESTS FOR CYANOPHORES
NAME OF TEST REAGENT POSITIVE RESULT
Guignard Sodium picrate paper Brick red/ various shades of red on the
paper

Odor n/a Bitter almond/ peach kernel smell due

to HCN
ANTHRAQUINONES
• Aglycones are derived from anthracene, and have a
characteristic cathartic/laxative action
• anthraquinones (2 =O),
• oxanthrones (1 -OH and 1 =O)
• anthranols (1 -OH),
• anthrones (1 =O), and
• dianthrones (two anthrones bonded)
CHEMISTRY AND TESTS
TESTS FOR ANTHRAQUINONES
NAME OF TEST FOR REAGENT POSITIVE RESULT
Borntrager’s Anthraquinones NH4OH Red
Modified Anthraquinone NH4OH + FeCl3 and Pink
Borntrager’s glycosides HCl
CARDIAC GLYCOSIDES
• Aglycones are steroid-lactones where the steroid always has
a OH at carbon 14, which are further classified according to
the unsaturated lactone present:
• Cardenolide – five-member unsaturated lactone, usually
found in plants and generally more common in nature
• Bufadienolide – six-member unsaturated lactone, usually
found in animals (ex. Frogs)
• Often have positive inotropic activity (ex. digoxin)
CHEMISTRY AND TESTS
TESTS FOR CARDIAC GLYCOSIDES
NAME OF TEST FOR REAGENT POSITIVE RESULT
Salkowski Sterol Sulfuric acid Red/violet
Liebermann- Sterol Sulfuric acid and acetic Emerald green
Burchard anhydride (unsaturated), yellow
(saturated)
Kedde’s Unsaturated Dinitrobenzoic acid in Blue-violet (or
lactone NaOH amethyst) color
Keller-Kiliani Deoxysugar FeCl3 in acetic acid and
Reddish brown
H2SO4
ALKALOIDS
ALKALOIDS
• Alkaloids are formally defined nitrogenous compounds
derived from amino acids with a wide range of physiological
activity.
• All alkaloids are (majorly crystalline, some amorphous)
solids, while only a few exist in the liquid form (ex. nicotine)
• Can be classified based on its amine (primary, secondary,
tertiary; some are even quaternary)
• Most alkaloids are bitter, physiologically active substances
Examples of significant alkaloids:
• Nicotine
• Atropine
• Cocaine
• Quinine
• Tubocurarine
• Morphine
• Ergotammine
• Physostigmine
• Pilocarpine
• Caffeine
CLASSIFICATION
• Almost all compounds considered to be alkaloids have their
nitrogen atoms enclosed within a ring system, and are called
true alkaloids.
• Cyclic nitrogenous “alkaloids” that are not derived from
amino acids are almost exclusively xanthine alkaloids, and
are sometimes called pseudoalkaloids.
• Some nitrogenous compounds with physiological activity do
not have their nitrogen atoms in the ring, and are called
protoalkaloids or alkaloidal amines
METHOD OF EXTRACTION
• They are extracted in two steps:
• Conversion into alkaloidal salts by addition of acid to
become water soluble, then
• Liberation as free base by addition of alkali such as
sodium bicarbonate or ammonium hydroxide.
TESTS FOR ALKALOIDS (COMPREHENSIVE)
NAME OF TEST REAGENT POSITIVE RESULT
Wagner’s Iodine in KI Reddish brown ppt
Valser’s Mecuric Iodide White ppt
Mayer’s HgCl2 in KI White/ Cream ppt
Dragendorff’s Potassium bismuth iodide Orange ppt
Marme’s Potassium cadmium iodide
Hager’s Saturated picric acid
Scheibler’s Phosphotungstic acid Precipitation
Sonnenschein’s Phosphomolybdic acid
Reaction with metals Gold/silver/platinum salts