Natural product and chemical analysis methods

NATURAL PRODUCTS
AND
CHEMICAL ANALYSIS METHODS
14.01.13EFG 702
By Omer Bayazeid
Prof. L.ÖMÜR DEMİREZER

Natural Products:
• A natural product is a chemical compound or substance produced
by a living organism. They may be extracted from tissues of
plants, marine organism or micro - organism fermentation.
• In that respect any biological molecule is a natural product, but
in general the term is reserved for secondary metabolites
(carotinoids, phytosterines, saponines, phenolic compounds,
alkaloids, glycosinates, terpenes etc).
• The extracts from plant tissue are a rich source of lead
compounds for pharmaceutical applications.

Extraction / Aim of Extraction:
• Is to separation medicinally active portions of
plant from the inactive or inert components by
using selective solvents in standard extraction
procedures.
• The products so obtained from plants are
relatively impure liquids, semisolid. These
include classes of preparations known as
decoctions, infusions, fluid extracts, tinctures
extracts and powdered extracts.

General Methods of Extraction of Medicinal Plants
1.Maceration:
In this process, the whole or powdered crude drug
is placed in a container with the solvent and
allowed to stand at room temperature for a period
of at least 3 days with frequent shaking until the
soluble matter has dissolved.
The mixture then is filtered, the marc (solid
material) is pressed.

2.Infusion:
Fresh infusions are prepared by macerating the
crude drug for a short period of time with cold
or boiling water. These are dilute solutions of
the readily soluble constituents of crude drugs.
3.Decoction:
In this process, the crude drug is boiled in a
specified volume of water for a defined time; it is
then cooled and filtered.
This procedure is suitable for extracting water-
soluble, heat-stable constituents.

Infusion & Decoction:
Sr.
No.
Infusion Decoction
1. Cold or boiling water is used as
menstruum.
Drug is boiled in water.
2. Drug having soft tissue is used. Drug having hard tissue is used.
3. Drug constituents may be
volatile.
Drug constituents should be non
volatile.
4. Final volume is adjusted. Final volume is not adjusted.
5. When boiling water is used as
menstruum, precaution are taken
to prevent the escape of heat
by covering the vessel with a
cloth .
No such precaution is required.
6MANJUL P. SINGH

4.Digestion:
This is a form of maceration in which gentle
heat is used during the process of extraction.
It is used when moderately elevated
temperature is not objectionable. The solvent
efficiency increased.

5.Percolation:
This is the procedure used most frequently to
extract active ingredients in the preparation of
tinctures and fluid extracts.
A percolator is generally used. The solid
ingredients are moistened with an appropriate
amount of the specified menstruum and allowed
to stand for approximately 4 hours in a well
closed container, Additional menstruum is added
and stand for 24 in the closed percolator.

6 . Hot Continuous Extraction (Soxhlet):
In this method, the finely ground crude drug is placed in a
porous bag made of strong filter paper, which is placed in
chamber E of the Soxhlet apparatus. The extracting
solvent in flask A is heated and its vapors condense in
condenser D.
The condensed extractant drips into the thimble containing
the crude drug, and extracts it by contact. When the level
of liquid in chamber E rises to the top of siphon tube C,
the liquid contents of chamber E siphon into fl ask A. This
process is continuous and is carried out until a drop of
solvent from the siphon tube does not leave residue when
evaporated.
The advantage of this method, compared to previously
described methods, is that large amounts of drug can be
extracted with a much smaller quantity of solvent.

SEPARATION & ISOLATION OF CONSTITUENTS
The most difficult operation in phytochemical
research is to isolate & purify plant constituents.
TECHNIQUES OF SEPARATION & ISOLATION
- Sublimation.
- Distillation.
- Fractional liberation.
- Fractional crystallization.
- Chromatography.

Collection of medicinal plants:
• Drugs may be collected from wild or cultivated
plants.
• It is known that the active constituents of
medicinal plants are affected by many factors
and may vary during the course of plant
growth.
• Proper time of collection is very important to
obtain a drug of a good quality.

Factors affecting collection:
1.Time of the year:
The plant may contain a substance in winter that is not
present in summer, or its amount varies markedly e.g.
Rhubarb contains no anthraquinone in winter, instead it
contains anthranols, which in summer, are oxidized to
anthraquinones.
Colchicum corm is free from bitterness and is devoid of
the alkaloid colchicine in autumn.Bitterness starts to
appear in spring and early summer when it is used as a
drug.

2.Time of the day:
Some drugs, like Digitalis, contain different amounts of active
constituents in different times of the day. Being highest in the
afternoon.
3.Stage of maturity and age:
The value and content of active constituents of many drugs
depends on the stage of maturity and age.
Conium fruits contain coniin when fruits are mature and
unripe.
Santonica flowers are rich in santonin, when unexpanded,
when it starts to open, the santonin content decreases.

Plant Identification:
Identification is a basic activity and one of the primary objectives
of systematics. Although identification is a separate activity or
process, in practice it involves both classification and nomenclature.
Identification is simply the determination of the similarities or
differences between two elements.
The comparison of an unknown plant with a named specimen and the
determination that the two elements are the same also involves
classification.
Both processes--identification and classification--involve comparison
and judgment and require a definition of criteria of similarities.
Identification is, therefore, a basic process in classification with
nomenclature playing an essential role in the retrieval of information
and as a means of communication.

Drying of crude drugs:
Reasons for drying:
1. To help in their preservation.
2. To fix their constituents, by preventing
reactions that may occur in presence of water.
3. To prevent the growth of micro-organisms such
as bacteria and fungi.
4. To facilitate their grinding.
5. To reduce their size and weight.

Methods of drying:
Drying is carried out either by natural or artificial
methods.
1.Natural drying: this is accomplished by natural
air in sun or shade.
2.Artificial drying: this is a rapid method done at
well-controlled temperature and is accomplished
by:

a) direct fire.
b) Use of heated stones.
c) Use of stoves.
d) Lyophilization (Freeze drying):
Frozen material is placed in an evacuated apparatus which has
a cold surface maintained at -60 to -80 °C. Water vapour
from the frozen material passes rapidly to the cold surface.
It is used for drying heat-sensitive substances e.g. antibiotics
and proteins.

Choice of solvent:
The ideal solvent for a certain pharmacologically active
constituent should:
1.Be highly selective for the compound to be extracted.
2.Have a high capacity for extraction in terms of
coefficient of saturation of the compound in the
medium.
3.Not react with the extracted compound or with other
compounds in the plant material.
4.Have a low price.
5.Be harmless to man and to the environment.
6.Be completely volatile.

• Aliphatic alcohols with up to three carbon atoms, or
mixtures of the alcohols with water, are the solvents
with the greatest extractive power for almost all
natural substances of low molecular weight like
alkaloids, saponins and flavonoids.
• According to the pharmacopoeias, ethyl alcohol is the
solvent of choice for obtaining classic extracts such as
tinctures and fluid, soft and dry extracts.

Puriﬁcation:
The puriﬁcation methods relay mainly on chromatography and the final
product is then obtained by crystallization.
Physical techniques are also used for separating and purifying the plant
constituents.
a) Fractional crystallization.
b) Fractional liberation.
c) Steam distillation.
d) Fractional distillation.
e) Sublimation.

a) Fractional crystallization:
Crystallization is an important method for the purification
of compounds from the mixture.
Crystallization mostly depends upon the inherent character
of the compound which form the crystals at the point of
super- saturation in solvent in which it is soluble.

Methods of crystallization:
1. Concentration.
2. Slow evaporation.
3. Refrigeration.
Based on differences in solubility of the components of a
mixture in a particular solvents Valuable.

b) Fractional liberation:
A mixture of alkaloid salts in aqueous solution, when
treated with aliquots of alkali gives first the weakest
base in the free state followed by base liberation in
ascending order of basicity.
If the mixture is shaken with organic solvent after each
addition of aliquot of a alkali, a fractional series of
bases shall be obtained.

c) Steam distillation:
Used for the extraction of volatile oils and hydrocyanic acid
from plant material.
d) Fractional distillation:
Used for the separation of components of volatile oils.
e) Sublimation:
We use Sublimation to isolate caffeine from tea and to
Purified materials present in the crude drug.

Herbarium:
• A herbarium is a collection of
preserved plant specimens. These
specimens may be whole plants or
plant parts: these will usually be in
a dried form mounted on a sheet
but, depending upon the material,
may also be kept in alcohol or other
preservative.
HERBARIUM SAMPLE
Senna

NAPRALERT:
NAPRALERT is a relational database of all natural
products, including ethno medical information,
pharmacological / biochemical information of
extracts of organisms in vitro, in vivo,in humans and
clinical studies. Similar information is available for
secondary metabolites from natural sources.
To date more than 200,000 scientific papers and
reviews are included, representing organisms from
all countries of the world, including marine
organisms, including the geographic origin from
where the organisms were obtained.

Coverage:
• The coverage of the literature in the following areas
is quite comprehensive:
• Clinical studies of natural products (including safety).
• Natural products that affect sugar metabolism.
• Natural products that affect mammalian reproduction.
• Extracts and compounds that affect cancer growth.
• Natural products and antiviral (including HIV) activity.
• Natural products and antitubercular activity.
• Natural products and tropical diseases.

General Reactions
for
Identification of Different groups
in
phytochemistry

(1) Tests for Alkaloids:
1. Dragendroff’s test:
1 ml of extract, add 1 ml of Dragendroff’s reagent (potassium
bismuth iodide solution). An orange-red precipitate indicates
the presence of alkaloids.
2.Mayer’s test:
1 ml of extract, add 1 ml of Mayer’s reagent (potassium
mercuric iodide solution). Whitish or cream colored precipitate
indicates the presence of alkaloids.

3.Hager’s test:
1 ml of extract, add 3 ml of Hager’s reagent (saturated
aqueous solution of picric acid). Yellow colored precipitate
4. Wagner’s test:
1 ml of extract, add 2 ml of Wagner’s reagent (iodine in
potassium iodide). Reddish brown colored precipitate

Tests for Glycosides:
Tests for free sugars:
The extract is hydrolyzed with mineral acid and then
tested for the glycone and aglycone moieties.
• Raymond’s test:
Test solution when treated with dinitrobenzene in hot
methanolic alkali, gives violet color.
• Legal’s test:
Treat the extract with pyridine and add alkaline sodium
nitroprusside solution, blood red color appears.
• Bromine water test
Test solution when treated with bromine water gives yellow
precipitate.

Test for Saponin Glycosides:
• Froth Test:
Place 1ml solution of drug in water in a semi-micro
tube and shaken well and noted for a stable froth.
• Hemolysis test:
Add 0.2ml solution of saponin (prepared in 1%
normal saline) to 0.2ml of v/v blood in normal saline
and mix well, centrifuge and note the red
supernatant compare with control tube containing
0.2ml of 10% blood in normal saline diluted with
0.2ml of normal saline.

Test for Anthraquinone Glycosides:
Borntrager's test:
Boil the test material with 1ml of dilute sulphuric acid in a test
tube for 5min (anthracene glycosides are hydrolyzed to aglycone
and sugars by boiling with acids) centrifuge or filter while hot,
filtrate, cool and shake with an equal volume of dichloromethane
(the aglycones will dissolve preferably in dichloromethane)
separate the lower dichloromethane layer and shake with half its
volume with dilute ammonia.
A rose pink to red color is produced in the ammonical layer
(aglycones based on anthroquinones give red color in the presence
of alkali).

Test for Cardiac Glycosides:
Kedde’s test:
Extract the drug with chloroform, evaporate to
dryness, add one drop of 90% alcohol and 2 drops
of 2% 3,5-dinitro benzoic acid(3,5-dinitro benzene
carboxylic acid -Kedde's reagent) in 90% alcohol.
Make alkaline with 20% sodium hydroxide solution. A
purple color is produced.
The color reaction with 3, 5-diinitrobenzoic acids
depends upon the presence of an β- unsaturated-o
lactones in the aglycone.

Keller killiani test [test for Deoxy sugars]:
Extract the drug with chloroform and
evaporate it to dryness. Add 0.4ml of glacial
acetic acid containing a trace amount of
ferric chloride. Transfer to a small test
tube; add carefully 0.5ml of concentrated
sulphuric acid by the side of the test tube,
blue color appears in the acetic acid layer.

Tests for Flavanoids:
Shinoda test:
Dry powder/extract + 5ml 95% ethanol + few
drops conc. HCl + 0.5 g magnesium turnings Pink
colour.
Lead acetate test:
Small quantity of extract + lead acetate solution
Yellow colour precipitated.

Sodium hydroxide test:
Plant extract + NaOH Yellow colour which
decolorize after addition of glacial acetic acid.
Ferric chloride test:
2-3 ml of alcoholic extract + 5% Fecl3 Deep
blue – black colour Geletin test : 2-3 ml of
alcoholic extract + Geletin 10% + NaOH (10%)
white ppt at lower level formed.

Test of Triterpenoids:
Liebermann -Burchard’s test:
2 mg of dry extract was dissolved in acetic
anhydride, heated to boiling, cooled and then 1
ml of concentrated sulphuric acid was added
along the sides of the test tube.
Formation of a pink colour indicates the
presence of triterpenoids.

Tests of Steroids:
(a) Liebermann-Burchard’s test:
2 mg of dry extract was dissolved in acetic anhydride, heated to
boiling, cooled and then 1 ml of concentrated sulphuric acid was added
along the sides of the test tube.
Formation of green colour indicates the presence of steroids.
(b) Salkowski reaction:
2 mg of dry extract was shaken with chloroform, to the chloroform
layer sulphuric acid was added slowly by the sides of test tube.
Formation of red colour indicated the presence of steroids.

Test of Tannins:
To 1-2 ml of the ethanolic extract, few
drops of 5% w/v FeCl3 solution was added.
A green colour indicated the presence of
gallotannins, while brown colour indicates
the presence of pseudotannins.

Detection
of Different groups
by
Thin Layer Chromatography

(1) TLC of Alkaloid:
Solvent system:
Toluene-ethyl acetate-diethylatnirre (70:2O: 10),
is suitable for the major alkaloids of most drugs.
Stationary phase:
The principal alkaloids OF the most common alkaloid
drugs can be identified by Silica gel 60 F254
precoated TLC plates Adsorbent.

Detection of Alkaloid:
UV-254nm some alkaloid types such as
indoles, quinolines, isoquinolines, purines.
UV-365 nm Blue, blue-green or violet
fluorescence ofalkaloids, e.g: Boldo folium.
Yellow fluorescence, e.g. colchicine.

(2) TLC of Flavanoids:
Solvent System:
Different solvent system can be used, ethyl acetate-formic
acid-glacial acetic acid-water(100-11-11-26 v/v) or
formic acid - water – ethyl acetate mixed in different
proportion with or without ethyl methyl ketone are suitable
for the TLC screening of polar flavonoids glycosides.
For less polar flavonoids aglycones we would use a mobile
phase composed of Toluene-ethyl formiate -formic acid
(50-40-10 v/v) or Toluene- dioxane - glacial acetic
acid(90-25-4 v/v).
Stationary phase: Silica gel,polyamide

Detection of Flavanoids:
The solvent must be thoroughly removed from silica gel layer before
detection UV-254nm
All flavonoids cause fluorescence. UV-365nm,Depending on the
structure type, flavonoids shows dark yellow, green or blue
fluorescence, which is intensified and changed by the use of various
spray reagent.
Spray Reagents Fast blue salt reagent (FBS)-Detection of phenolic
compounds.
Natural products reagents (NP/PEG) - The plate is sprayed with 1%
methanolic diphenylboric acid, β - ethylamino ester (=
diphenylboryloxyethylamine , NP), followed by 5% ethanolic
polyethylene glycone-4000spray.

(3) TLC of Anthracene Derivatives:
Solvent System:
Aloin, frangulin A/B, glucofrangulin A/B, rhein, aloe-
emodin and rliaponticoside are applied as 0.1%
methanolic solutions. Solutions Sennasides A and B are
prepared as a 0.1% solution in methanol-water (1: 1).
A total of 10 111 of each reference solution is used
for TLC.
Stationary Phase:
Chromatography is performed on silica gel 60 F254
precoated

Detection Anthracene Derivatives:
• UV 254 nm All anthracene derivatives quench
fluorescence.
• UV 365 nm All anthracene derivatives give yellow or
red-brown fluorescence.
Spray reagents:
Potassium hydroxide After spraying with 5% or 10%
ethanolic KOH, anthraquinones appear red in the
visible and show red fluorescence in UV-365 nm.

(4) TLC of Cardiac Glycoside:
Solvent System:
Ethyl acetate-methanol-water (100:13.5:10)
solvents. A generally applicable solvent system for
cardiac glycosides Ethyl acetate-methanol-ethanol-
water (81 : 11 :4: 8). The addition of ethanol
increases the Rf values of strongly polar compounds.
Stationary System:
• Adsorbent Silica gel 60 F254 precoated.

Detection of Cardiac Glycoside :
Without chemical treatment UV-254 nm very weak
fluorescence quenching of all cardiac glycosides UV-365 nm
no fluorescence at all.
Spray reagents:
Specific detection of the y-lactone ring of cardenolides:
Kedde reagent Immediately on spraying, cardenolides
generate a pink or blue-violet (vis) colour. The colour fades
after a few minutes, but can be regained by repeated
spraying. Raymond reagent also give red, red-orange or
violet (vis) cardenolide-specifics colors.

(5) TLC of Coumarin:
Solvent System:
For coumarin Aglycones solvent Toluene-ether (l:l,
saturated with 10% acetic acid) For glycosides Ethyl
acetate-fortnic acid-glacial acetic acid-water
(100:11:11:26).
Stationary Phase:
Adsorbent Silica gel 60 F254 precoated TLC plates.

Detection of Coumarin :
• UV-254 nm distinct fluorescence quenching of all coumarins.
• UV-365 nm run intense blue or blue-green fluorescence (simple
coumarins) yellow, brown, blue or blue-green fluorescence (furano-
and pyranocoumarins).
• The non-substituted coumarin fluoresces yellow-green in UV-365
nm only after trearment with KOH- reagent or ammonia vapour.
Spray reagents:
The fluorescence of the coumarins are intensified by spraying with
5%-10% ethanolic KOH. Concentrated ammonia vapour has the
same effect.

(6) TLC of Saponin:
Solvent System:
The solvent which is suitable for separation of the
saponin mixtures Chloroform-glacial acetic acid-
methanol-water( 64:32:12:8) solvents.
Chloroform-methanol-water (70:30:4) ginsenosides
(Ginseng radix).
Stationary Phase:
Adsorbent Silica gel 60 F254 precoated TLC plates.

Detection of Saponin:
• Without chemical treatment With the exception of
glycyrrhizin and glycyrrhetic acid (Liquiritiae radix),
no saponins are detectable by exposure to UV-254 or
UV-365 nm.
Spray reagents:
Hemolytically active saponins are detected as white
zones on a reddish background. Hemolysis may occur
immediately, after allowing the TLC plate to stand or
after drying the plate in a warm airstream.

(7) TLC of Triterpenes:
Solvent System:
Ethyl formiate-toluene-formic acid (50:50: 15)
Toluene-chloroform-ethanol (40:40:10)
Stationary Phase:
Silica gel 60 F254 precoated plates

Detection of Triterpenes:
• UV-254 nm calfeic acid, its derivatives and isoflavones
show quenching.
• UV-365 nm caffeic acid, its derivatives and isoflavones
fluoresce blue.
Spray Reagents:
Anisaldehyde-sulphuric acid reagent Tile sprayed TLC is
heated for 6 min at 100°C; evaluation in vis.: triterpenes
blue-violet (Cimicifugae rhizoma) and red to red-violet
(Ononidis radix).

(8) TLC of Lignans:
Solvent System:
Chloroform-methanol-water (70:30:4)
Cloroform-metllanol( 90:lO)
Stationary Phase:
Silica gel 60 F254 -precoated plates

Detection of Lignans:
• UV-254 nm all lignans show prominent quenching.
• UV-365 nm e.g. eleutheroside E, gives blue
fluorescence.
Spray reagents:
50% ethanolic sulphuric acid for Cubebae fructus
Vanillin-phosphoric acid reagent for Eleutherococci
radix.

(9) TLC of essential oil:
Solvent System:
Toluene-ethyl acetate (93:7).This system is
suitable for the analysis and comparison of all
important essential oils.
Stationary Phase:
Silica gel 60 F254 precoated TLC plates.

Detection of essential oil:
• Without chemical treatment UV-254nm Compounds
containing at least two conjugated doulble bonds quench
fluorescence and appear as dark zones against the light-
green fluorescent background of the TLC plate.
• UV-365 nm No characteristic Ruorescence of terpenoids
and propylphenols is noticed.
Spray reagents:
Anisaldehyde-sulphuric acid 10 min/110°C; evaluation in
vis.: essential oil compounds show strong blue, green, red
and brown colouration. Most of the compounds develop
fluorescence under UV-365 nm.

CLASSIFICATION OF DETECTED BIOACTIVITIES
The study of medicinal plants and their chemical constituents
can be focused to their speciﬁc bioactivities.These
bioactivities can be classiﬁed according to several scientists as
follows:
Action on the autonomic nervous system:
(1) Acetylocholine-like drugs as pilocarpine.
(2) Antagonists of acetylocholine as tropane esters alkaloids in
Solanaceae.
(3)adrenaline-like drugs: ephedrine from Ephedra spp.
(4) antagonists of adrenaline as ergot alkaloids from Claviceps
purpurea .

Actiononthecentralnervoussystem:
(1)Drugs affecting mentalactivity
(1a) Hallucinogenics as cannabinoids.
(1b) stimulating mental activity as purine bases as caffeine.
(2) central depressants of motor function as tropane alkaloids, and
(3) possessing analgesic acvtivity as morphine from Papaver
somniferum .
Action on heart muscle: cardiac glycosides mostly from Digitalis spp.,
and Strophanthus sp.
Action on blood vessels:
(1) peripheral vasoconstrictors drugs as ephedrine, nicotine, etc.,
(2) central vasoconstrictors drugs as picrotoxin,
(3) vasodilators as papaverine, ergotamine.

Action on the respiratory system:
(1) Bronchodilators as ephedrine.
(2) Cough depressants as codeine.
Action on the gastrointestinal tract:
(1) Anticholinergic drugs.
(2) Emetics as ipecacuahna,
(3) Bitters such as Gentian, Cinchona.
Action on the liver:
(1) Hepatoprotective activity as Silibum marianum ﬂavolignans.
(2) Hepatotoxic activity as pyrrolizidine alkaloids from Boraginaceae Fammily.

Action on skin and mucous membranes:
(1) Astringents as tannins,
(2) Emollients and demulcents as olive.
Treatment of malignant diseases:
Anticancer activity with vinca alkaloids from
Catharanthus roseus, the famous taxol from
Taxus sp., and semi synthetic derivatives as
etoposide and teniposide from Podophyllum
peltatum, etc.

Natural product and chemical analysis methods

Natural product and chemical analysis methods

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