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

- SIMPLE

- RAPID

- MINIMUM EQUIPMENT

- SELECTIVE

- ……..

PHYTOCHEMICAL SCREENING METHODS

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 negativereaction.

Cannell, 356-…

KUSMARDIYAN

EXTRACTIONMethod: Continuous extraction using Soxhlet apparatus

Powder of crude drug

Hexane Extract Marc

Ethylacetate Extract Marc

Methanol Extract Marc

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

PHYTOCHEMICAL SCREENING

Alkaloid + - +

Flavonoid + - +

Quinone + + -

Tannin + - +

Steroid/Triterpenoid + + -

Saponin - - -

CRUDEDRUG

HEXANEEXTRACT

METHANOL EXTRACT

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

1

ALKALOIDS

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

Not all alkloids give a positive reaction because of structural idiosyncracies.

False-negative reactions with: quarternairy alkaloids, amine oxides

Pyridine Piperidine PyrrolidineImidazole

Tropane

QuinolineIsoquinoline

PurineIndole

DIFFERENTIAL SOLUBILITY OF

THE SALTS AND BASES IN WATER

AND ORGANIC SOLVENTS

POWDERED DRUG

MARC ORGANIC PHASE

ORGANIC PHASEAQUEOUS PHASE

AQUEOUS PHASE ORGANIC PHASE

IN ALKALINE MEDIUM

base (NH4OH, Na2CO3, etc)

organic solvent not missible with water (CHCl3, CH2Cl2, Et2O, etc.)

1

dilute acid (HCl, H2SO4, etc.)

2

baseorganic solvent

3

Bruneton, 795.

alkaloids, lipids, pigments, …

alkaloid salts neutral alkaloids

quarternary alkaloidstotal basic 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

2. REACT WITH ALKALOIDS AS LOOSE COMPLEXES TO FORM PRECIPITATES

3. REACT TO FORM INSOLUBLE ADDITION PRODUCTS THROUGH THE ALKALOID NITROGEN

4. REACT THROUGH THE ATTRACTION OF ORGANIC ACIDS WITH BASIC ALKALOIDS TO FORM INSOLUBLE SALTS

silicotungstic, phosphomolybdic, and phosphotungstic acids.

Wagner’s and Bouchardat’s reagents.

Mayer’s, Valser’s, Marme’s, and Dragendorff’s reagents

Hager’s (picric acid)

Farnsworth

- 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

DRAGENDORFF REAGENT: drop test reagent VS spray reagent

Modifications:Munier and Macheboeuf Thies and Reuther

Check for this one:

Compare with these reagents:

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

Lloydia, (1962) 25, 316-318.

CONYUGATED CARBONYL (KETONE OR ALDEHYDE) OR LACTONE FUNCTIONS ?

-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.

MAYER 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.

WAGNER REAGENT

- 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.

AMMONIUM REINECKATE

Discuss the following reagents:

Farnsworth, 250

Structures of false-positive alkaloid reactions

DETECTION OF ALKALOIDS ----1

POWDERED CRUDE DRUG

FILTRATE

Aqueous phase Organic phase

Residue

Orange precipitate

Alkaloid (+)

White precipitate

Alkaloid (+)

Dragendorffreagent

Mayer reagent

in basic condition, extract with chloroform

evaporate

+ acid

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

in acid solution, filter

DETECTION OF ALKALOIDS ----2

POWDERED CRUDE DRUG

FILTRATE

Spot on filter paper Acidic solution

Orange spot

Alkaloid (+)White precipitate

Alkaloid (+)

Dragendorffreagent

Mayer reagent

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

extract with acid

EXTRACTION OF ALKALINIZED SAMPLE WITH ORGANIC SOLVENT.

2

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

POWDERED CRUDE DRUG

FILTRATE**

Persistent foam for 10 min.

Saponin +

add water filter.

vertically shake for 10 sec.

Persistent foam

Saponin (+)

+ HCl

DETECTION OF SAPONINSby shaking an aqueous solution of the sample and observing the production of foam.

** 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)

3

TANNINS

CONDENSED TANNINS HYDROLYZABLE TANNINScatechin gallic acid

Example: derivate of catechin

Example: derivate of gallic acid

or procyanidin or catechuic tannins predominantly gallotannins and ellagitannins

TANNINSHYDROLYZABLE TANNINS

CONDENSED TANNINS

1. Sample + Fe Cl3 precipitate: green, blue , black

2. Sample + NaCl precipitate

3. Sample + gelatin 1% precipitate

4. Sample + gelatin 1% + NaCl 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 TANNINSPOWDERED CRUDE DRUG

FILTRATE

PRECIPITATE

Tannin +

green, blue, black color

Tannin +

FILTRATE

Blue ink

Gallotannins +

+ FeCl3 gelatin Steasny[formaldehyde 30% - HCl (2:1) ]

RED PRECIPITATE

Catechuic tannins +

PRECIPITATE

+ water, filter

+ Na Ac + FeCl3

4

STEROIDS TRITERPENOIDS

DETECTION OF STEROID/TRITERPENOID

POWDERED CRUDE DRUG

FILTRATE

RESIDUE

Blue - green steroid

Red - violet triterpenoid

+ ether filter

evaporate

+ Liebermann –Burchard reagent

Among the many color reactions for cholesterol, the Liebermann-Burchardprobedure 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.

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.

CLINICAL CHEMISTRY, Vol. 20, No.7, 1974, 794-801.Mechanisms of the Liebermann-Burchard and Zak Color Reactions for CholesterolR. W. Burke, B. I. Diamondstone, R. A. Velapoldi, and 0. Menis

- to be updated -

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.

-- DETECTION OF STEROIDS/TRITERPENOIDS --

5

QUINONES

Anthraquinones

Farnsworth, 266

Farnsworth, 266

-- DETECTION OF QUINONES --

How if tannin (+) ?

DETECTION OF QUINONES

POWDERED CRUDE DRUG

FILTRATE**

Red color

Quinone (+)

+ water filter.

+NaOH

** 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)

6

FLAVONOIDS

Farnsworth, 263

I = flavones, II = flavonols, III = isoflavones, IV = catechins, V = flavanones, VI = leucoanthocyanins, VII = anthocyanins, VIII = aurones, IX = chalcones.

FLAVONOIDS

SKELETONS AND NUMBERING SCHEMES OF FLAVONOIDS

Marbry et al., 1970.

DETECTION OF FLAVONOID

POWDERED CRUDE DRUG

FILTRATE**

red, yellow, orange in the amylalcohol layer Flavonoid (+)

+ water filter.

+ 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)

cyanidin reaction of Willstatter gamma-benzopyrone nucleus

FLAVONOIDSThe 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.

7

COUMARINS

Farnsworth, 265

COUMARINS

Farnsworth, 265

………………………………… …….……………… ……………………………………..

-- DETECTION OF COUMARINS --

8

CARBOHYDRATES

CARBOHYDRATES

- 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.

MOLISCH REAGENT:

- 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.

ANILINE ACETATE REACTION:

9

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….

EXTRACTS / FRACTIONS / ISOLATES

2 days

1 day

LD50

10 naupliiEvaporate

+ ASW 5 mL

Artificial sea water (ASW) 38 gram sea salt/L

Brine shrimp eggs

Count the number of dead nauplii, use Program Finney

BRINE SHRIMP LETHALITY TESTA 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.

KUSMARDIYANI

Get information and draw the schemes for other biological screenings.

THE COMBINED USE OF CHEMICAL AND BIOASSAY

URL: http://www.iupac.org/symposia/proceedings/phuket97/hostettmann.html© 1999 IUPACStrategy for the Biological and Chemical Evaluation of Plant ExtractsKurt Hostettmann

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

TLC screening for radical scavengers and antioxidantsThese can be detected on a TLC plate by spraying with 2,2-diphenyl-1-picrylhydrazyl (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 --

An antifungal naphthoquinone from Swertia calycina (Gentianaceae)Among the examples of natural products isolated in our laboratory using the TLC bioautographicapproach is a 2-methoxynaphthoquinone from Swertia calycina (Gentianaceae), a small plantfound in Rwanda. This example illustrates well the combined use of TLC and HPLC in the searchfor 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. HPLCUVand HPLC-MS analyses of the extract revealed the presence of three main compounds: abitter principle, a xanthone and a naphthoquinone derivative with a MW of 188. Comparison ofon-line UV and MS data with a data bank allowed identification of the bitter principle assweroside and the xanthone as decussatin. As these have no antifungal properties, the strongactivity of the dichloromethane extract was attributed to the naphthoquinone, a class ofcompounds which is known to have strong antimicrobial properties.

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

Strategy for the Biological and Chemical Evaluation of Plant ExtractsKurt Hostettmann

KUSMARDIYANI SCHOOL OF PHARMACY ITB

ASSIGNMENTS:

Find and write all mechanism of reactions (mentioned in this chapter) for the detection of major phytochemicalgroups in plants.

Get information and draw the schemes for other examples of biological screening.