PHYTOCHEMICAL

(AND BIOLOGICAL)
SCREENING

Farnsworth, N.R., Biological and Phytochemical Screening of Plants,

J. Pharm. Sci., 1966.
Cannell, R.J.P., Natural Product Isolation, 1998.

AN ILLUSTRATION OF
PHYTOCHEMICAL SCREENING FIELD TRIP
TO WEST SUMATRA

PHYTOCHEMICAL
SCREENING

PHYTOCHEMICAL SCREENING METHODS

- SIMPLE
- RAPID
- MINIMUM EQUIPMENT
- SELECTIVE
- ..
Get complete information from the journal.

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

monosaccharides
oligosaccharides
polysaccharides

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

fatty acids
oils (triacylglycerols)
waxes
alkamides (including
isobutylamides)

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

sulfur compounds
(Garlic)
glucosinolates
cyanogenic glycosides
5-HTP
amines
enzymes

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

simple phenols
phenolic acids
phenylpropanoids
coumarins
lignans
stilbenoids
xanthones
styrylpyrones
flavonoids
isoflavonoids
benzofurans
chromones
quinones
phloroglucinols

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

monoterpenes
sesquiterpenes
essential oils
diterpenes
triterpenes & saponins
tetraterpenes
(carotenoids)

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

steroidal saponins
phytosterols
cardiac glycosides

PHYTOCHEMICALS:
MAJOR CATEGORIES
Carbohydrates
Lipids
Amino acids &
derivatives
Phenolic
compounds
Terpenoids
Steroids
Alkaloids

betalain alkaloids

diterpenoid alkaloids
indole alkaloids
isoquinoline alkaloids
methylxanthines
monoterpenoid alkaloids
peptide alkaloids
pyrrolidine alkaloids
piperidine alkaloids
pyrrolizidine alkaloids
quinoline alkaloids
quinolizidine alkaloids
tropane alkaloids

GENERAL REAGENTS FOR THE DETECTION

OF VARIOUS PHYTOCHEMICAL GROUPS
None of these reactions is specific.
Structural similarities with compound
of completely different types
may result in false-positive reactions.
A compound may occur in too low
concentration that give a negative
reaction.
KUSMARDIYAN

Cannell, 356-

EXTRACTION
Method: Continuous extraction using Soxhlet apparatus

Powder of crude drug

Hexane Extract

Marc

Ethylacetate Extract

Methanol Extract

Marc

Marc

---------------------------------QUALITY ASPECTS OF BAWANG TIWAI [ELEUTHERINE AMERICANA (AUBL.) MERR.] BULB AS A NATURAL MEDICINE------------------------------

PHYTOCHEMICAL SCREENING
CRUDE
DRUG

HEXANE METHANOL
EXTRACT EXTRACT

Alkaloid

Flavonoid

Quinone

Tannin

Steroid/Triterpenoid

Saponin

-----------QUALITY ASPECTS OF BAWANG TIWAI [ELEUTHERINE AMERICANA (AUBL.) MERR.] BULB AS A NATURAL MEDICINE-----------

ALKALOIDS

ALKALOIDS
False-positive reactions
with:
coumarins, polyphenols,
purines, amino acids,
proteins, and other
nitrogenous compounds.

Pyridine

Piperidine

Imidazole

Pyrrolidine

Tropane
Quinoline

False-negative reactions
with:
quarternairy alkaloids,
amine oxides
Indole

Not all alkloids give a positive reaction

because of structural idiosyncracies.

Isoquinoline

Purine

IN ALKALINE MEDIUM

POWDERED DRUG
base (NH4OH, Na2CO3, etc)

organic solvent not missible with water

(CHCl3, CH2Cl2, Et2O, etc.)

ORGANIC PHASE

MARC
DIFFERENTIAL
SOLUBILITY OF
THE SALTS AND
BASES IN WATER
AND ORGANIC
SOLVENTS

Bruneton, 795.

alkaloids, lipids, pigments,

AQUEOUS PHASE
alkaloid salts

dilute acid
(HCl, H2SO4, etc.)

ORGANIC PHASE

base
organic solvent

neutral alkaloids

ORGANIC PHASE

AQUEOUS PHASE

total basic alkaloids

quarternary alkaloids

ALKALOID TEST METHODS

USED IN PHYTOCHEMICAL SCREENING
1. ACIDIC OR AQUEOUS EXTRACTS
2. ACIDIC OR AQUEOUS EXTRACT, FOLLOWED BY ALKALI
TREATMENT, IMMISCIBLE SOLVENT REACTION, AND
PARTITION WITH DILUTE ACID.
3. ALCOHOL EXTRACTION FOLLOWED BY CONCENTRATING
AND ADDITION OF ACID.
4. EXTRACTION OF ALKALINIZED SAMPLE WITH ORGANIC
SOLVENT.
5. . CHROMATOGRAPHY

CLASSIFICATION OF ALKALOID PRECIPITANTS

1. REACT WITH BASIC COMPOUNDS TO FORM INSOLUBLE SALTS
silicotungstic, phosphomolybdic, and phosphotungstic acids.
2. REACT WITH ALKALOIDS AS LOOSE COMPLEXES TO FORM
PRECIPITATES Wagners and Bouchardats reagents.
3. REACT TO FORM INSOLUBLE ADDITION PRODUCTS THROUGH
THE ALKALOID NITROGEN Mayers, Valsers, Marmes,
and Dragendorffs reagents
4. REACT THROUGH THE ATTRACTION OF ORGANIC ACIDS WITH
BASIC ALKALOIDS TO FORM INSOLUBLE SALTS
Hagers (picric acid)

Farnsworth

DRAGENDORFF REAGENT:
drop test reagent VS spray reagent
Check for this one:
- Dissolve 8.0 g Bi(NO3)3.H2O in 30%w/v HNO3 and 27.2 g KI in 50mL water.
- Combine the solution and stand for 24h.
- Filter and make up with water to 100 mL.
- Use only in acid solutions where an orange-brownish precipitate will
appear.
- The alkaloid may be recovered by treatment with sodium carbonate and
subsequent extraction with ethyl ether.
This reaction may also be performed on a filter paper or on a TLC plate by
adding a drop of the reagents onto a spot of the sample

Compare with these reagents:

Modifications:
Munier and Macheboeuf
Thies and Reuther

Stahl, E., 874

FALSE-POSITIVE REACTIONS WITH DRAGENDORFF

Lloydia, (1962) 25, 316-318.

MINIMUM STRUCTURAL FEATURES NEEDED

FOR A POSITIVE REACTION WITH DRAGENDORFF

Lloydia, (1962) 25, 318.

NEGATIVE REACTION WITH DRAGENDORFF

Lloydia, (1962) 25, 318.

FALSE-POSITIVE REACTIONS WITH DRAGENDORFF

CONYUGATED CARBONYL (KETONE OR ALDEHYDE) OR LACTONE FUNCTIONS ?

Lloydia, (1962) 25, 316-318.

Discuss the following reagents:

MAYER REAGENT

-Dissolve 1.36 g HgCl2 in 60 mL water and 5 g KI in 10 mL water.

-Combine both solution and make up with water to 100 mL.
-Add a few drops to an acidic solution (HCl or diluted H2SO4) containing the alk.
-If alkaloids are present, a white to yellowish precipitate will appear.
Notes:

the precipitate may be redissolved by AcOH or EtOH in the solution or

excess of reagent.

WAGNER REAGENT
- Dissolve 1.27 g I2 (sublimed) and 2 g KI in 20 mL water
- Make up with water to 100 mL.
- A brown precipitate in acidic solution will suggest the presence of alkaloids.

AMMONIUM REINECKATE
- Add 0.2 hydroxylamine to a saturated solution of 4% ammonium reineckate,
- Acidify with dilute HCl.
- A pink precipitate will appear if alkaloids are present.
- The precipitate is soluble in 50% acetone that may also be used to recrystallize it.

Structures of false-positive alkaloid reactions

Farnsworth, 250

DETECTION OF ALKALOIDS ----1

ACIDIC EXTRACT,
FOLLOWED BY ALKALI TREATMENT, IMMISCIBLE SOLVENT REACTION, AND PARTITION WITH DILUTE ACID.

POWDERED CRUDE DRUG

in acid solution,
filter

FILTRATE
in basic condition,
extract with chloroform

Aqueous phase

Organic phase
evaporate

Residue
+ acid
Dragendorff
reagent

Mayer
reagent

Orange precipitate

White precipitate

Alkaloid (+)

Alkaloid (+)

DETECTION OF ALKALOIDS ----2

EXTRACTION OF ALKALINIZED SAMPLE WITH ORGANIC SOLVENT.

POWDERED CRUDE DRUG

+ ammonia, grind in mortar
+ chloroform, grind, filter

FILTRATE

extract with acid

Spot on filter paper

Dragendorff
reagent

Acidic solution
Mayer
reagent

Orange spot

White precipitate

Alkaloid (+)

Alkaloid (+)

SAPONINS

SAPONINS

STEROID SAPOGENIN

TRITERPENOID SAPOGENIN

- hemolyze red blood cells

- produce persistent foam after vigorous shaking of aqueous solution
- toxic for fish
- produce characteristic color reaction with Liebermann-Burchard reagent

DETECTION OF SAPONINS

by shaking an aqueous solution of the sample and observing the production of foam.
POWDERED CRUDE DRUG
add water
filter.

FILTRATE**
vertically shake for 10 sec.

Persistent foam for 10 min.

Saponin +
+ HCl
Persistent foam
Saponin (+)
** A mixture of I gram powdered crude drug and 100mL hot water is boiled for 5 minutes
and filtered to get the test solution ( = filtrate)

TANNINS

CONDENSED TANNINS
or procyanidin or catechuic tannins

catechin

Example:
derivate of catechin

HYDROLYZABLE TANNINS
predominantly gallotannins and ellagitannins

gallic acid

Example:
derivate of gallic acid

TANNINS
HYDROLYZABLE TANNINS
CONDENSED TANNINS
1.

Sample + Fe Cl3 precipitate: green, blue , black

2.

Sample + NaCl

3.

Sample + gelatin 1% precipitate

4.

Sample + gelatin 1% + NaCl precipitate

precipitate

If : #3 positive tannin +
#3 + #4 positive tannin +
#2 positive false-positive reaction

Examples:
tannins from Punica granatum

CONDENSED or
HYDROLYZABLE
TANNINS ?

DETECTION OF TANNINS
POWDERED CRUDE DRUG
+ water,
filter

FILTRATE

+ FeCl3

gelatin

green, blue, black color

PRECIPITATE

Tannin +

Tannin +

FILTRATE
+ Na Ac

Blue ink
Gallotannins +

Steasny
[formaldehyde 30% - HCl (2:1) ]

RED PRECIPITATE
Catechuic tannins

PRECIPITATE
+ FeCl3

STEROIDS
TRITERPENOIDS

DETECTION OF STEROID/TRITERPENOID
POWDERED CRUDE DRUG
+ ether
filter

FILTRATE
evaporate

RESIDUE
+ Liebermann Burchard reagent

Blue - green steroid

Red - violet triterpenoid

- to be updated CLINICAL CHEMISTRY, Vol. 20, No.7, 1974, 794-801.

Mechanisms of the Liebermann-Burchard and Zak Color Reactions for Cholesterol

R. W. Burke, B. I. Diamondstone, R. A. Velapoldi, and 0. Menis

Cholesterol reacts with various strong acids of the Bronsted and Lewis
types to give colored products. Although these reactions have been used
empirically for many years for the qualitative and quantitative
determination of cholesterol, their mechanisms still are not clearly
understood.
Among the many color reactions for cholesterol, the Liebermann-Burchard
probedure is perhaps the most widely used. This reaction was described
initially by Liebermann in 1885 and applied to cholesterol analysis shortly
after by Burchard. Chloroform was used as a solvent in the early studies,
but the Liebermann-Burchard (L-B) reaction, as performed today, is carried
out in an acetic acid-sulfuric acid-acetic anhydride medium.

-- DETECTION OF STEROIDS/TRITERPENOIDS --

LIEBERMANN-BUCHARD TEST:
- Combine 1 mL anhydrous acetic acid and 1 mL chloroform and cool to 0oC
- Add one drop concentrated sulfuric acid.
- When the sample is added, either in the solid form or in solution in
chloroform: blue, green, red, or orange colors that change with time will
indicate a positive reaction,
- A blue-greyish color in particular is observed for 5-sterol, with maximum
intensity at 30 min.
(See previous slide)

SALKOWSKI REACTION:
- Dissolve 1-2 mg of the sample in 1 mL chloroform
- Add 1 mL concentrated sulfuric acid, forming two phases, with a red or
yellow color indicating the presence of sterol and methylated sterols.

QUINONES

Anthraquinones

Farnsworth, 266

-- DETECTION OF QUINONES --

How if
tannin
(+) ?
Farnsworth, 266

DETECTION OF QUINONES
POWDERED CRUDE DRUG
+ water
filter.
FILTRATE**
+NaOH

Red color
Quinone (+)

** A mixture of I gram powdered crude drug and 100mL hot water is boiled for 5 minutes
and filtered to get the test solution ( = filtrate)

FLAVONOIDS

FLAVONOIDS

I = flavones, II = flavonols, III = isoflavones, IV = catechins, V = flavanones,

VI = leucoanthocyanins, VII = anthocyanins, VIII = aurones, IX = chalcones.
Farnsworth, 263

SKELETONS
AND NUMBERING
SCHEMES
OF FLAVONOIDS
Marbry et al., 1970.

DETECTION OF FLAVONOID
cyanidin reaction of Willstatter gamma-benzopyrone nucleus

POWDERED CRUDE DRUG

+ water
filter.

FILTRATE**
+ Mg powder +HCl
+ amylalcohol
shake

red, yellow, orange in the amylalcohol layer

Flavonoid (+)

** A mixture of I gram powdered crude drug and 100mL hot water is boiled for 5 minutes
and filtered to get the test solution ( = filtrate)

FLAVONOIDS
The reagents may give a false positive reaction with other polyphenols.
SHINODA TEST:
- To an alcoholic solution of the sample, add magnesium powder and a few
drops of concentrated HCl.
- Orange, pink, red to purple colors will appear when flavones, flavonols, the
corresponding 2,3-dihydro derivatives, and/or xanthones are present.
- It is advisable to add t-butyl alcohol before adding the acid to avoid
accidents from a violent reaction.
- The colored compounds will dissolve into the upper phase.
- By using zinc instead of magnesium, only flavonols give a deep-red to
magenta colors or no color at all.

SULFURIC ACID:
- Flavones and flavonols dissolve into concentrated sulfuric acid giving a
deep yellow solution.
- Chalcones and aurones produce red or red-bluish solutions
- Flavanones give orange to red colors.

COUMARINS

COUMARINS

Farnsworth, 265

-- DETECTION OF COUMARINS --

.
..
Farnsworth, 265

CARBOHYDRATES

CARBOHYDRATES
MOLISCH REAGENT:
- Solution I: 1% alpha-nafphtol in 80% ethanol.
- Solution II: concentrated H2SO4.
- Add 2 to 3 drops of solution I to a sample solution (crude aqueous
extracts) and acid, without mixing, to form an upper phase.
- A purple ring will appear in the interphase as a result of the reaction
between alpha-naphtol and furfural and 5-hydroxymethyl furfural
aldehydes produced by dehydration of saccharides.
- A red color will appear if alpha-naphthol is replaced by 5 % thymol.

ANILINE ACETATE REACTION:

- The solid sample is heated over a flame
- The vapors are allowed to react with aniline acetate impregnated on
a filter paper placed on the vapors.
- A red color will appear in presence of heterocyclic aldehydes
produced from carbohydrate dehydration.

OTHERS

SESQUITERPENE LACTONES
AND CARDIAC GLYCOSIDES
Positive reactions with compounds containing alpha, betha- unsaturated
lactones

KEDDE REAGENT:
- Solution I: 25 of 3,5-dinitrobenzoic acid in MeOH.
- Solution II: 5.7% KOH in water.
- Add one drop of each solution to 0.2-0.4 mL of the sample solution, and a
bluish to purple color will appear within 5 min.
- The solution should not contain acetone, which give a deep bluish color.

Others:
BALJET REAGENT
LEGAL REAGENT

BIOLOGICAL
SCREENING

BIOLOGICAL SCREENING

- SCREENING FOR BIOACTIVE COMPOUNDS FROM PLANTS

- USING LIVING SYSTEMS

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY TEST

-- applying the extract to a filter paper disk then placing it on the surface of a bacterial or yeast culture.

THE AMES TEST

-- using bacteria to screen for cancer causing agent.

BRINE SHRIMP TEST

POTATO DISC ASSAY
etc.

Artificial sea water (ASW)

38 gram sea salt/L

EXTRACTS / FRACTIONS / ISOLATES

Brine shrimp eggs

2 days

BRINE SHRIMP
LETHALITY TEST

Evaporate
+ ASW 5 mL

10 nauplii

A brine shrimp hatchery made from a plastic soap

dish and placed in the light. The shrimp should hatch
within 24-48h. Significant if LD50 <30 ug/mL.

Get information and draw the schemes

for other biological screenings.

1 day

Count the number of dead nauplii,

use Program Finney

LD50

KUSMARDIYANI

THE COMBINED USE OF

CHEMICAL AND BIOASSAY

URL: http://www.iupac.org/symposia/proceedings/phuket97/hostettmann.html
1999 IUPAC

Strategy for the Biological and Chemical Evaluation of Plant Extracts

Kurt Hostettmann

TLC autographic assays

combine TLC with a bioassay in situ and allow localization of active constituents in a
complex matrix.

TLC screening for radical scavengers and antioxidants

These can be detected on a TLC plate by spraying with 2,2-diphenyl-1picrylhydrazyl (DPPH) radical. Antioxidants reduce the radical, producing white spots on a
purple background. Alternatively, the bleaching of crocin (which normally gives a yellow
colour on the plate) can be used to distinguish components of plant extracts with potential
antioxidant or radical-scavenging properties.

TLC bioautography (C. cucumerinum) and LC/UV/MS analysis of Swertia calycina

(Gentianaceae) whole plant dichloromethane extract.

-- the combined use of TLC and HPLC in the search for new antifungal metabolites --

URL: http://www.iupac.org/symposia/proceedings/phuket97/hostettmann.html
1999 IUPAC

Strategy for the Biological and Chemical Evaluation of Plant Extracts

Kurt Hostettmann

An antifungal naphthoquinone from Swertia calycina (Gentianaceae)

Among the examples of natural products isolated in our laboratory using the TLC bioautographic
approach is a 2-methoxynaphthoquinone from Swertia calycina (Gentianaceae), a small plant
found in Rwanda. This example illustrates well the combined use of TLC and HPLC in the search
for new antifungal metabolites (Fig. 1). TLC bioautography of the dichloromethane extract of S.
calycina showed a compound which strongly inhibited the growth of C. cucumerinum. HPLCUV
and HPLC-MS analyses of the extract revealed the presence of three main compounds: a
bitter principle, a xanthone and a naphthoquinone derivative with a MW of 188. Comparison of
on-line UV and MS data with a data bank allowed identification of the bitter principle as
sweroside and the xanthone as decussatin. As these have no antifungal properties, the strong
activity of the dichloromethane extract was attributed to the naphthoquinone, a class of
compounds which is known to have strong antimicrobial properties.

ASSIGNMENTS:
Find and write all mechanism of reactions (mentioned in
this chapter) for the detection of major phytochemical
groups in plants.
Get information and draw the schemes for other examples
of biological screening.
KUSMARDIYANI
SCHOOL OF PHARMACY ITB