NATURAL PRODUCTS AND

CHEMICAL ANALYSIS METHODS

Compiled by _Charan Ingole

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 Decoction1. 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 phytochemicalresearch 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 indicates the presence of alkaloids.

4. Wagner’s test:

1 ml of extract, add 2 ml of Wagner’s reagent (iodine in potassium iodide). Reddish brown colored precipitate indicates the presence of alkaloids.

Tests for Glycosides:Tests for free sugars:

The extract is hydrolyzed with mineral acid and thentested for the glycone and aglycone moieties.

• Raymond’s test:Test solution when treated with dinitrobenzene in hotmethanolic alkali, gives violet color.

• Legal’s test:Treat the extract with pyridine and add alkaline sodiumnitroprusside solution, blood red color appears.

• Bromine water testTest solution when treated with bromine water gives yellowprecipitate.

Test for Saponin Glycosides:

• Froth Test:Place 1ml solution of drug in water in a semi-microtube 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 salineand mix well, centrifuge and note the redsupernatant compare with control tube containing0.2ml of 10% blood in normal saline diluted with0.2ml of normal saline.

Test for Anthraquinone Glycosides:Borntrager's test:

Boil the test material with 1ml of dilute sulphuric acid in a testtube for 5min (anthracene glycosides are hydrolyzed to aglyconeand 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 itsvolume with dilute ammonia.

A rose pink to red color is produced in the ammonical layer(aglycones based on anthroquinones give red color in the presenceof alkali).

Test for Cardiac Glycosides:

Kedde’s test:

Extract the drug with chloroform, evaporate todryness, add one drop of 90% alcohol and 2 dropsof 2% 3,5-dinitro benzoic acid(3,5-dinitro benzenecarboxylic acid -Kedde's reagent) in 90% alcohol.Make alkaline with 20% sodium hydroxide solution. Apurple color is produced.

The color reaction with 3, 5-diinitrobenzoic acidsdepends upon the presence of an β- unsaturated-olactones in the aglycone.

Keller killiani test [test for Deoxy sugars]:

Extract the drug with chloroform andevaporate it to dryness. Add 0.4ml of glacialacetic acid containing a trace amount offerric chloride. Transfer to a small testtube; add carefully 0.5ml of concentratedsulphuric 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 phenoliccompounds.

Natural products reagents (NP/PEG) - The plate is sprayed with 1% methanolic diphenylboric acid, β - ethylamino ester (= diphenylboryloxyethylamine , NP), followed by 5% ethanolicpolyethylene 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, cardenolidesgenerate 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 isoflavonesshow quenching.

• UV-365 nm caffeic acid, its derivatives and isoflavonesfluoresce blue.

Spray Reagents:

Anisaldehyde-sulphuric acid reagent Tile sprayed TLC is heated for 6 min at 100°C; evaluation in vis.: triterpenesblue-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 Eleutherococciradix.

(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 terpenoidsand 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.Thesebioactivities 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 Podophyllumpeltatum, etc.