2. Skrining Fitokimia (2)
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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.
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AN ILLUSTRATION OF PHYTOCHEMICAL SCREENING FIELD TRIP
TO WEST SUMATRA
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PHYTOCHEMICAL SCREENING
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- SIMPLE
- RAPID
- MINIMUM EQUIPMENT
- SELECTIVE
- ……..
PHYTOCHEMICAL SCREENING METHODS
Get complete information from the journal.
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PHYTOCHEMICALS: MAJOR CATEGORIES
• Carbohydrates• Lipids• Amino acids &
derivatives• Phenolic
compounds• Terpenoids• Steroids• Alkaloids
• monosaccharides• oligosaccharides• polysaccharides
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PHYTOCHEMICALS: MAJOR CATEGORIES
• Carbohydrates• Lipids• Amino acids &
derivatives• Phenolic
compounds• Terpenoids• Steroids• Alkaloids
• fatty acids• oils (triacylglycerols)• waxes• alkamides (including
isobutylamides)
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PHYTOCHEMICALS: MAJOR CATEGORIES
• Carbohydrates• Lipids• Amino acids &
derivatives• Phenolic
compounds• Terpenoids• Steroids• Alkaloids
• sulfur compounds (Garlic)
• glucosinolates• cyanogenic glycosides• 5-HTP• amines• enzymes
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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
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PHYTOCHEMICALS: MAJOR CATEGORIES
• Carbohydrates• Lipids• Amino acids &
derivatives• Phenolic
compounds• Terpenoids• Steroids• Alkaloids
• monoterpenes• sesquiterpenes• essential oils• diterpenes• triterpenes & saponins• tetraterpenes(carotenoids)
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PHYTOCHEMICALS: MAJOR CATEGORIES
• Carbohydrates• Lipids• Amino acids &
derivatives• Phenolic
compounds• Terpenoids• Steroids• Alkaloids
• steroidal saponins • phytosterols• cardiac glycosides
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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
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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
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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------------------------------
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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-----------
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1
ALKALOIDS
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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
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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
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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
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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)
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Farnsworth
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- 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:
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Stahl, E., 874
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FALSE-POSITIVE REACTIONS WITH DRAGENDORFF
Lloydia, (1962) 25, 316-318.
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MINIMUM STRUCTURAL FEATURES NEEDED FOR A POSITIVE REACTION WITH DRAGENDORFF
Lloydia, (1962) 25, 318.
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NEGATIVE REACTION WITH DRAGENDORFF
Lloydia, (1962) 25, 318.
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FALSE-POSITIVE REACTIONS WITH DRAGENDORFF
Lloydia, (1962) 25, 316-318.
CONYUGATED CARBONYL (KETONE OR ALDEHYDE) OR LACTONE FUNCTIONS ?
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-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:
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Farnsworth, 250
Structures of false-positive alkaloid reactions
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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
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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.
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2
SAPONINS
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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
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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)
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3
TANNINS
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CONDENSED TANNINS HYDROLYZABLE TANNINScatechin gallic acid
Example: derivate of catechin
Example: derivate of gallic acid
or procyanidin or catechuic tannins predominantly gallotannins and ellagitannins
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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
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Examples: tannins from Punica granatum
CONDENSED or HYDROLYZABLE
TANNINS ?
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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
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4
STEROIDS TRITERPENOIDS
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DETECTION OF STEROID/TRITERPENOID
POWDERED CRUDE DRUG
FILTRATE
RESIDUE
Blue - green steroid
Red - violet triterpenoid
+ ether filter
evaporate
+ Liebermann –Burchard reagent
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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 -
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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 --
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5
QUINONES
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Anthraquinones
Farnsworth, 266
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Farnsworth, 266
-- DETECTION OF QUINONES --
How if tannin (+) ?
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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)
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6
FLAVONOIDS
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Farnsworth, 263
I = flavones, II = flavonols, III = isoflavones, IV = catechins, V = flavanones, VI = leucoanthocyanins, VII = anthocyanins, VIII = aurones, IX = chalcones.
FLAVONOIDS
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SKELETONS AND NUMBERING SCHEMES OF FLAVONOIDS
Marbry et al., 1970.
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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
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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.
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7
COUMARINS
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Farnsworth, 265
COUMARINS
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Farnsworth, 265
………………………………… …….……………… ……………………………………..
-- DETECTION OF COUMARINS --
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8
CARBOHYDRATES
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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:
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9
OTHERS
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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
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BIOLOGICAL SCREENING
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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….
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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.
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THE COMBINED USE OF CHEMICAL AND BIOASSAY
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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.
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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 --
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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
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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.