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(AND BIOLOGICAL)
SCREENING
AN ILLUSTRATION OF
PHYTOCHEMICAL SCREENING FIELD TRIP
TO WEST SUMATRA
PHYTOCHEMICAL
SCREENING
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
Cannell, 356-
EXTRACTION
Method: Continuous extraction using Soxhlet apparatus
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
Isoquinoline
Purine
IN ALKALINE MEDIUM
POWDERED DRUG
base (NH4OH, Na2CO3, etc)
ORGANIC PHASE
MARC
DIFFERENTIAL
SOLUBILITY OF
THE SALTS AND
BASES IN WATER
AND ORGANIC
SOLVENTS
Bruneton, 795.
AQUEOUS PHASE
alkaloid salts
dilute acid
(HCl, H2SO4, etc.)
ORGANIC PHASE
base
organic solvent
neutral alkaloids
ORGANIC PHASE
AQUEOUS PHASE
quarternary alkaloids
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
MAYER 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.
Farnsworth, 250
FILTRATE
in basic condition,
extract with chloroform
Aqueous phase
Organic phase
evaporate
Residue
+ acid
Dragendorff
reagent
Mayer
reagent
Orange precipitate
White precipitate
Alkaloid (+)
Alkaloid (+)
FILTRATE
Acidic solution
Mayer
reagent
Orange spot
White precipitate
Alkaloid (+)
Alkaloid (+)
SAPONINS
SAPONINS
STEROID SAPOGENIN
TRITERPENOID SAPOGENIN
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.
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.
2.
Sample + NaCl
3.
4.
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
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
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
SKELETONS
AND NUMBERING
SCHEMES
OF FLAVONOIDS
Marbry et al., 1970.
DETECTION OF FLAVONOID
cyanidin reaction of Willstatter gamma-benzopyrone nucleus
FILTRATE**
+ Mg powder +HCl
+ amylalcohol
shake
** 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.
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
-- applying the extract to a filter paper disk then placing it on the surface of a bacterial or yeast culture.
2 days
BRINE SHRIMP
LETHALITY TEST
Evaporate
+ ASW 5 mL
10 nauplii
1 day
LD50
KUSMARDIYANI
URL: http://www.iupac.org/symposia/proceedings/phuket97/hostettmann.html
1999 IUPAC
combine TLC with a bioassay in situ and allow localization of active constituents in a
complex matrix.
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.
-- 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
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