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PHG-322

Copyright: This manual was written for undergraduate Pharmacy Programs for students enrolled in the

college of Pharmacy Program at the Sattam bin Abdulaziz university Al-Kharj.

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Objectives:

The goal of this course is to provide primary knowledge of natural

product drugs to the pharmacy student in professional program of study

and give some practical guidance in the process of phytochemical

screening of medicinal plants using different extraction techniques. The

course will also cover chromatographic principles and methodologies

specially planer chromatography as well as their applications in

evaluation of natural products.

Note: This manual will help guide you to learn about natural Alkaloids

and Glycosides.

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Practical course contents:

Contents LLaabb.. NNoo..

Theory of Alkaloids 11))

Colour Test of Alkaloids 22))

Microchemical Test Alkaloids -I 33))

Microchemical Test Alkaloids -II 44))

Isolation of Caffeine from Tea 55))

Isolation of Piperine from Black Pepper 66))

Mid-term Practical Exam 77))

Introduction to Glycosides 88))

Chemical Identification of Anthraquinone Glycosides in Senna Leaves 99))

TLC Characterization of Senna Powder and Tablets 1100))

Extraction & Chemical Identification of Cardiac Glycosides 1111))

TLC Identification of Cardiac Glycosides 1122))

Thin Layer Chromatography Characterization of Flavonoids in Different Herbal Products

1133))

Final Exam 1144))

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Assessment:

99 %% MMiiddtteerrmm eexxaammiinnaattiioonn

44 %% LLaabb.. aasssseessssmmeenntt

1122 %% FFiinnaall eexxaammiinnaattiioonn

2255 %% TToottaall

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GENERAL INSTRUCTIONS

Laboratory work in phytochemistry is an interesting part.

In the lab, you have the opportunity to learn.

To make the best use of each lab, you should observe the following

1. Attendance

Students should attained all lab periods on time

2. Lab Coat

All students should wear a full-length buttoned lab coat while in lab.

3. Preparation for the lab

Usually the first 15-20 minutes are used by the lab instructor to introduce the materials of the

lab. A small quiz will be given during this short period and it will be considered in the final mark, thus

student should be well prepared for the new lab and study the material each week.

4. For good work and safety

S.N DO NOT DO

1 Ingest any Reagent Always wear lab coat in lab

2 Pour solution directly from containers on

to slides or into tubes rather use dropper

Keep your work area neat, clean and organized

3 Use any solution without being sure of its

nature

Throw solid wastes in wastebaskets and liquid in

sinks

4 Taste any solid/liquid chemicals Return materials to their original benches, and

clean up your work area before leaving the lab.

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Lab:1.

Theory of Alkaloids

Definition: the term “alkaloid” (alkali-like) is commonly used to designate basic heterocyclic nitrogenous

compounds of plant origin that are physiologically active. At present approximately 5000 alkaloids are

isolated.

Theory:

Alkaloid is a rather broad and vague term which describes natural products which contain a basic

nitrogen atom. Alkaloids are a naturally occurring large group of pharmacologically active nitrogen-

containing secondary metabolites of plants, microbial or animal origin. In most alkaloids, the nitrogen

atom is a part of the ring. Alkaloids are biosynthetically derived from amino acids. The name ‘alkaloid’

derives from the word ‘alkaline’, which means a water soluble base. There are three main types of

alkaloids:

Deviation from Definition

BASICITY: Some alkaloids are not basic, e.g. Colchicine, Piperine, Quaternary alkaloids.

Nitrogen: The nitrogen in some alkaloids is not in a heterocyclic ring e.g. Ephedrine, Colchicine,

Mescaline.

Plant Origin: Some alkaloids are derived from Bacteria, Fungi, Insects, Frogs, Animals

Most alkaloids are well-defined crystalline substances which unite with acids to form salts. In fact, one

or more nitrogen atoms that are present in an alkaloid, typically as 1˚, 2˚or 3˚ amines, contribute to the

basicity of the alkaloid. The degree of basicity varies considerably, depending on the structure of the

molecule, and the presence and location of the functional groups. Most alkaloids are crystalline solids

and are bitter in taste. In plants, they may exist in the free state, as salts or as N-oxides.

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The important of solubility differences of alkaloids is to isolate the alkaloid. In general alkaloid bases are

soluble in organic solvent but not in water, but we found that morphine is sparingly soluble in organic

solvent; also there are alkaloid bases which are soluble in water such as ephedrine, caffeine, colchicine

and pilocarpine. On the other hand alkaloid salts are usually soluble in water and insoluble in organic

solvent, but quinine sulfate is insoluble in H2O, and apoatropine salt is soluble in organic solvent.

Alkaloids give a precipitate with heavy metal iodides:

Most alkaloids are precipitated from neutral or slightly acidic solution by Mayr’s reagent (potassium-

mercuric-iodide solution).Cream colored precipitate.

Dragendorff’s reagent (solution of potassium bismuth iodide) gives orange colored precipitate with

alkaloids.

Caffeine, purine derivative, does not precipitate like most alkaloids.

Isolation of Alkaloids from

Plant-Root, leaves, bark, fruits, seeds

Animals –frogs, centipedes, ladybugs,

Bird-Pitohui birds

Classification:

True (Typical) alkaloids that are derived from amino acids and have nitrogen in a heterocyclic ring.

Example- Atropine

Proto alkaloids that are derived from amino acids and do not have nitrogen in a heterocyclic ring.

Example- Ephedrine

Pseudo alkaloids that are not derived from amino acids, but have nitrogen in a heterocyclic ring.

Example- Caffeine

False alkaloids are non alkaloids give a false positive reaction with alkaloidal reagents.

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Most acceptable definitions:

Alkaloids are cyclic organic compounds containing nitrogen in a negative state of oxidation with limited

distribution among living organisms.

Distribution and occurrence:

Rare in lower plants.

Dicots are richer in alkaloids than Monocots.

Families rich in Alkaloids: Apocynaceae, Rubiaceae, Solanaceae and Papaveracea.

Families free from Alkaloids: Rosaceae, Labiatae

Distribution in Plant

All Parts e.g. Datura.

Barks e.g. Cinchona

Seeds e.g. Nux vomica

Roots e.g. Aconite

Fruits e.g. Black pepper

Leaves e.g. Tobacco

Latex e.g. Opium

Physical Properties

I- Condition

Most alkaloids are crystalline solids.

Few alkaloids are amorphous solids e.g. emetine.

Some are liquids that are either: Volatile e.g. nicotine and coniine, or Non-volatile e.g. pilocarpine

and hyoscine

II- Color:

The majority of alkaloids is colorless, but some are colored e.g.:

Colchicine and berberine are yellow.

Canadine is orange.

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The salts of sanguinarine are copper-red

III- Solubility

Both alkaloidal bases and their salts are soluble in alcohol.

Generally, the bases are soluble in organic solvents and insoluble in water

Exceptions:

Bases soluble in water: caffeine, ephedrine, codeine, colchicine, pilocarpine and quaternary

ammonium bases.

Bases insoluble or sparingly soluble in certain organic solvents: morphine in ether, theobromine and

theophylline in benzene.

Salts are usually soluble in water and, insoluble or sparingly soluble in organic solvents.

Exceptions:

Salts insoluble in water: quinine mono sulphate.

Salts soluble in organic solvents: lobeline and apoatropine hydrochlorides are soluble in chloroform.

Extraction, Purification and Isolation of Alkaloids from Powdered plants

Method I:

The powder is treated with alkalis to liberate the free bases that can then be extracted with water

immiscible organic solvents.

Method II:

The powdered material is extracted with water or aqueous alcohol containing dilute acid. Alkaloids are

extracted as their salts together with accompanying soluble impurities.

Method III:

The powder is extracted with water soluble organic solvents such as MeOH or EtOH which are good

solvents for both of salts and free bases.

Types: Three main types

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YES Derived from amino acid Having heterocyclic ring

with nitrogen

True alkaloid

YES Derived from amino acid

(Cholchicine)

Does not Have

heterocyclic ring with

nitrogen

Proto alkaloid

NOT Derived from amino acid

(terpenoids, Purine)

Having heterocyclic ring

with nitrogen

Pseudo alkaloid

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Importance of alkaloids to plants:

Alkaloids may be poisonous for the invaders

Side product of detoxification.

Act as Regulatory growth factors

Nitrogen supplies.

Nomenclature:

1) Generic name Atropine.

2) Specified name Cocaine.

3) Pharmacological effect emetine, morphine, narcotine.

4) Physical property hygrine (moist).

5) Discoverer name pelletierine.

6) Drug name ergotamine.

Note: Suffixes or prefixes can be used to distinguish between individual alkaloids found in the same

plants.

Extraction Method:

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Their basicity means that alkaloids are able to exist as free bases or acid salts. This property may be

exploited in their isolation by means of acid/base extraction

Take the plant material and make alkaline by addition of a few drops of ammonia solution

Extract with a suitable organic solvent (CH2Cl2)

Partition the extract with 2M HCL three times, retaining and bulking the acid extracts

Make the acid extract basic(pH 9) with ammonia which will convert the alkaloids back to the free

base from

Partition with a suitable organic solvent three times to move the alkaloids into the organic phase.

By this point the alkaloids will be largely free of other plant constituents.

General Alkaloid Reagents:

1. Mayer's Reagent (KHgI3) ppt.

2. Dragendroff's Reagent (KBiI4) ppt.

3. Wagner's Reagent (KI3, KI/I2) ppt.

4. Tannic acid solution ppt.

5. Mercuric chloride ppt.

6. Hanger's Reagent (Sat. Solution of picric acid) ppt.

7. Gold chloride ppt.

8. Chloroplatinic acid ppt.

9. Mandalin's Reagent (Sulfovamidic acid ) Color.

10. Froehd's Reagent (Sulfomolybdic acid ) Color.

11. Marqui's Reagent (HCHO/H2SO4) Color.

12. Erdmann's Reagent (HNO3/H2SO4) Color.

13. Conc. H2SO4 Color.

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14. Conc. HNO3 Color.

15. Marme's Reagent (KCl 13) ppt.

1 % Test solution and few drops of HCI should be used (except with tannic acid neutral solution is

recommended).

Tests for Acidic Radicals of Alkaloid Salt

TEST for SO4

(I) Add 1 ml of BaSO4 T.S. to 1 ml of alkaloid test solution white ppt. or turbidity is observed,

which insoluble in HCl or HNO3.

TEST for Cl:

Add 1 ml of AgNO3 T.S. to 1 ml of the alkaloids test solution white ppt or opalescence is

produced, which is soluble in NH4OH solution and insoluble in HNO3.

TEST for NO3:

Add few drops of FeSO4 T.S. to the alkaloids test solution and shake then add I ml conc.

H2SO4 on the wall of the test tube. A brown ring is produced at the junction of the two

liquids.

Test for Po4:

Add 1 ml of HNO3 to 1 ml of the test solution add 1 ml of amonium molybdate is soluble in

dilute NH4OH solution.

Test for Salicylate:

Dilute solution of salicylate give a violet color with FeCl3 T.S. Changed to white ppt. on addition of dil.

HCl.

Test for Citrate:

To 1 ml of citrate solution add 0.5 ml H2SO4 + 1 ml KMnO4 and warm on a water bath, a

decolorization and a PPT. is observed.

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REPORT- Lab:1.

Identification of Alkaloids by using Mayers Reagent

S.N Alkaloids Result

1. Atropine

2. Brucine

3. Caffeine

4. Emetine

5. Ephedrine

6. Nicotine

7. Papaverine

8. Quinidine

9. Quinine

10. Strychnine

11. Theobromine

12. Theophylline

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Lab: 2.

Identification of Atropine, Quinine/Quinidine, Emetine, Theophylline,

Ephedrine, Papaverine, strychnine and Brucine using chemical (colour) test

Procedure:

1. Identification of Atropine (Vitalis Test)

Take 1ml sample in a china dish and then evaporate, add 4 drops of HNO3 again

evaporate to dry and cool. Add 4 drops of 10% alcoholic KOH

Result: A violet is colour produced on the surface of a china dish

2. Identification of Quinine/Quinidine specific test)

Take 2ml each solution in a separate test tube

Result: A blue fluorescence produced by Quinine (not by Quinidine)

3. Identification of Emetine (Mandalis test)

Take 1ml sample in a china dish and then evaporate to try. Add a few crystals of

Ammonium molybdate and 4 drops of H2SO4.

Result: A green colour is produced on the surface of a china dish

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4. Identification of Theophylline

Take 2 ml sample in a test tube. Add 1 ml of Ag NO3 (mucilaginous precipitate form) and 1ml of HNO3.

Result: Mucilaginous precipitate converts into Granular precipitate

5. Identification of Ephedrine

Take 1 ml sample in a test tube. Add 0.3 ml of CuSO4 and 1ml of NaOH solution, a blue colour is

produced. Again add 5ml of CHCl3; two layers will be formed (organic/aqueous)

Result: organic layer-Purple and aqueous layer blue

6. Identification of Papaverine (Marquis Reagent)

Take 2ml sample in a test tube; add 1ml of KMNO4, and Marquis Reagent (10 ml of concentrated sulfuric

acid and 1 ml formaldehyde)

Result: Green- Blue colour produced

7. Identification of strychnine

Take 2 ml sample of strychnine in a china dish and then evaporate to try; add few K2Cr2O7 crystals and 4

drops conc. H2SO4.

Result: deep violet colour produced, (change yellow)

8. Identification of Brucine

Take 2ml sample in a test tube; and a few drops of HNO3

Result: A deep red color is produced

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Lab-3:

Microchemical tests of Alkaloids-I

Overview: Micro-chemical tests for identifying alkaloids are an accurate and

specific technique, being better than color tests which are rarely specific. They are

based on the characteristic crystalline shapes of the alkaloid derivatives produced

by the addition of certain specific reagents. The shapes of these crystals are, in

most, cases specific for most alkaloids. Besides, this technique is applicable for

very minute quantities of all alkaloids and alkaloid preparations. Hence, it would

be useful in identifying alkaloids in their pharmaceutical preparations, e.g. tablets,

injections etc.

The following are specific reagents for the identification of alkaloids:

1. Ammoniacal silver nitrate solution: 2% solution in 5% ammonium hydroxide. It

must be freshly prepared.

2. Disodium hydrogen phosphate solution: 5% aqueous solution.

3. Gold bromide solution: Dissolve 1 g of HAuC14.4H2O and 1.5 ml of 40% HBr in

18 ml of water. The saturated aqueous NaBr solution may be substituted for

the HBr.

4. Gold chloride solution: 5% aqueous solution.

5. Hydrochloric acid: 5% aqueous solution.

6. Kraut's solution: Dissolve 5 g of bismuth nitrate in 20 ml of HN03 (sp gr. 1.18).

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Dissolve 27.2 g of KI in 50 ml of water. Mix the two solutions and dilute with

water to 100 ml. It must be freshly prepared.

7. Lead Iodide solution: To 33% aqueous potassium acetate solution, add one

drop of methyl red indicator and acetic acid until yellow color changes to

orange, then while gentle warming, saturate with PbI2, cool and filter.

8. Marme solution: Dissolve 3 g of CdI2 in 18 ml of water containing 6 g of KI. It

must be freshly prepared.

9. Mercuric chloride solution: 5% aqueous solution.

10. Platinic chloride solution: 5% aqueous solution.

11. Potassium ferrocyanide solution: 5% aqueous solution

12. Sodium benzoate solution: 5% aqueous solution.

13. Sodium carbonate solution: 5% aqueous solution.

14. Wagner's solution (Iodine solution): Dissolve 1.27g of I2 and 2 g of KI in 5 ml

of water and dilute to 100 ml with water.

15. Zinc chloride: 5% aqueous solution.

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Lab: 3.

Micro-chemical tests for identifying alkaloids based on the characteristic

crystalline shapes produced by addition of certain specific reagents. (Atropine,

Caffeine, Nicotine and Papaverine)

Preparations of Reagent

1 Iodo-potasium iodide (Wagner's

reagent for alkaloids)

Dissolve 2g of iodine and 6g of KI in 100ml of water

2 Mercuric chloride 0.25 M, 0.5 N. Dissolve 68g of HgCl2 in water. Dilute to

1 liter

3 Zinc Chloride 5% aqueous solution

4 Disodium hydrogen phosphate 5% aqueous solution

1 Atropine-Wagner's

reagent

Small dark rods and

triangular plates form in

great number, singly in

groups

2 Caffeine-Mercuric

chloride

Clusters of long, radiating

needle-shaped crystal

3 Nicotine-Mercuric

chloride

Radiating transparent

blades

4 Papaverine-Zinc

Chloride

Thin rectangular plates in

the presence of excess

HCl.

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Report:

Draw pictures

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Lab: 4.

Micro-chemical tests for identifying alkaloids based on the characteristic

crystalline shapes produced by addition of certain specific reagents.

(Theophyllin, Ephedrin, Theobromin and Quinine)

Reagents

Ammoniacal silver nitrate 2% solution in 5% ammonium hydroxide, it must be freshly prepared

Kraut's Reagent (A and B) (add I drop from each)

A. 80g bismuth nitrate in 200 c.c. nitric acid of 1.18 sp. gr. in a little water B. Dissolve 272 grams potassium iodide in a little water

1 Theophylline-

Ammoniacal silver

nitrate

Gelatinous at first, dense

spheres of dark radiating

needles

2 Ephedrine-Kraut's

Brown radiating and

inter lacing needle

3 Theobromine-

Kraut's

Tufts of brown radiating

needles

4 Quinine-Disodium

hydrogen phosphate

Silvery, leaf like crystal

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Report:

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Lab: 5.

Isolation of caffeine from Tea

Requirements:

S.N Requirement Quantity

1 Tea 10g

2 Lead acetate solution 5 ml

3 10% Dilute H2SO4 20ml

4 Activated Charcoal 0.5 g

5 10% KOH For alkaline

6 Chloroform 50ml×3

7 Ammoniacal silver nitrate

2% solution in 5% ammonium hydroxide, it must be freshly prepared

8 Kraut's Reagent (A and B) (add I drop from each)

A. 80g bismuth nitrate in 200 c.c. nitric acid of 1.18 sp. gr. in a little water B. Dissolve 272 grams potassium iodide in a little water

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Procedure:

1. Take 5 g teas and add 25ml distilled water boil for 15 min and filter, repeat 3 times

2. Infiltrate add Lead acetate 5ml and again a filter

3. Add 10 ml dilute H2SO4 and activated Charcoal (0.2g) and filter

4. Filtrate alkalinized with KOH (Test uses litmus paper)

5. Using separating funnel extract the filtrate with CHCl3 (3times)

6. Collect the organic layer together and evaporate

7. Calculate the percentage yield

% yield= weight of caffeine/weight of tea×100

(Weight of caffeine= weight of beaker with caffeine-weight of clean beaker)

Result:

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Lab: 6.

ISOLATION OF PIPERINE FROM BLACK PEPPER

Requirement:

S.N Requirement Quantity

1 Black Pepper 5g

2 Alcohol 50 ml 3 KOH (10%)

Alkalinized 1ml

4 CHCI3 20ml

5 Hot alcohol 20ml

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Procedure:

1. Take 5 g Black Pepper and add 50 ml distilled water boil for 15 min and filter, repeat 3 times

2. In filtrate add Concentrate to 1/3 rd using water bath

3. Add 1 ml KOH and leave for 5 min and filter

4. Dissolve the residue in hot alcohol and set aside for crystallization

5. Concentrate the extract to about 10 ml and add-30 ml of H2O and transfer to a Separator

5. Using separating funnel extract the filtrate with CHCl3 (3times)

6. Collect the organic layer together and evaporate

7. Calculate the percentage yield

% yield= weight of caffeine/weight of tea×100 (weight of caffeine= weight of beaker with caffeine-

weight of clean beaker)

Result:

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Lab. 8

Introduction to secondary metabolites (Glycosides) :

Plants produce a variety of compounds that can be divided into primary metabolites and

secondary metabolites. Primary metabolites are essential for the survival of the plant and

include sugars, proteins and amino acids. Secondary metabolites were once believed to be

waste products. They are not essential to the plant’s survival, but the plant does suffer without

them. Secondary metabolites also have many uses for us, too. Some are beneficial, and others

can be toxic.

Alkaloids

Alkaloids are secondary metabolites. They are primarily composed of nitrogen and are

widely used in medicine. They can also be highly toxic (morphine, caffeine …etc).

Terpenoids

Terpenoids are made of isoprene units and are found in all plants. They are the largest

group of secondary metabolites and are very volatile, which means they evaporate easily.

Phenols

The final type of secondary metabolites has recently become very popular due to their

health benefits: the phenols.The phenols consist of a hydroxyl group (–OH) attached to an

aromatic ring. Phenols are found in nearly all parts of the plant and in nearly every plant on the

planet.

The first group of phenols is the flavonoids. Flavonoids are water-soluble pigments

found in the vacuoles of plant cells. Flavonoids can be further divided into three groups:

anthocyanins (range in color from red to blue and purple), flavones and flavnols.

The other type of phenol is important to the structure of the plant and is called lignin. It

adds stiffness and strength to cell walls of the plant cells. Lignin is crucial to terrestrial plants

because it supports the branches and size. It also allows the cell wall to be waterproof and

protects the plant from fungal attacks.

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Glycosides

Glycosides comprise a very wide range of compounds, that are of common and

ubiquitous occurrence in almost all plants. Glycosides are said to play an important role in the

life of the plant, involving its regulatory, protective and sanitary functions.

Glycosides are compounds containing a carbohydrate and a noncarbohydrate residue in the

same molecule. The carbohydrate residue is attached by an acetal linkage at carbon atom 1 to a

noncarbohydrate residue or AGLYCONE. The nonsugar component is known as the

AGLYCONE. The sugar component is called the GLYCONE. If the carbohydrate portion is

glucose, the resulting compound is a GLUCOSIDE.

CLASSIFICATION OF GLYCOSIDES:

The Classification according to the chemical nature of the aglycone, which is

considered by any authors to be the most convenient method of classification.

According to this method glycosides are classified into:

Alcoholic and phenolic glycosides; the aglycones are alcohols or phenols.

Aldehydic glycosides; the aglycones are aldehydes.

Cyanogenetic glycosides; or cyanophore glycosides; the aglycones are nitriles or

derivatives of hydrocyanic acid.

Anthracene or anthraquinone glycosides; the aglycones are anthracene derivatives.

Steroidal glycosides; the aglycones are steroidal in nature i.e.; derived from

cyclopentanoperhydrophenanthrene.

Coumarin glycosides; the aglycones are derivatives of benzo- -pyrone.

Chromone glycosides; the aglycones are derivatives of benzo- γ-pyrone.

Flavonoidal glycosides; the aglycones have 2-phenyl chromone structure.

Sulphur-containing or thio-glycosides; the aglycone molecule contains sulphur, usually in

the form of thiocyanate or isothiocyanate.

Alkaloidal glycosides; the aglycone is alkaloidal in nature. e.g. the glycoalkaloids of

Solanum species.

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Chemical tests for Identification:

Cardiac glycosides give color reactions with different reagents. These can be used for

qualitative and quantitative purposes, as well as, spray reagents on TLC.

Color reactions due to the aglycone moiety:

1) Reactions due to the steroidal nucleus:

These are positive with any compound containing a steroidal nucleus including cardenolides

and bufadienolides:

- Antimony trichloride test: Cardiac glycoside + SbCl3/CHCl3 blue or violet.

- Liebermann’s test: Cardiac glycosides in glacial acetic acid + H2SO4 red, violet, blue to

green.

2) Reactions due to the (-CH2-) group of the lactone ring:

These are characteristic for cardenolides having a 5-membered lactone ring.

- Legal’s test:

- Raymond’s test: Cardenolide in 50% EtOH+ m-dinitrobenzene + NaOH violet color

blue.

- Kedde’s test : Cardenolide+ 3,5-dinitrobenzoic acid + NaOH violet.

- Baljet’s test : Cardenolide + picric acid + NaOH orange or red.

3) Color reactions due to the 2-deoxy sugar moiety:

Keller-Killiani’s test: The glycoside is dissolved in glacial acetic acid that contain traces of

FeCl3, and then concentrated H2SO4 is carefully added on the side of the test tube to form a

lower layer a blue ring is formed in between the two layers.

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LAB. 9

Aim: Chemical Identification of anthraquinone glycosides in Senna Leaves

Introduction:

The sugar part of the glycoside is called (Glycone) while the non-sugar compound with

which the sugar has reacted is termed (Aglycone) or (Genin).

The most characteristic chemical property of glycosides is their susceptibility to hydrolysis,

to give the free sugar and the free genin. Hydrolysis of glycosides is affected in the laboratory by

digestion with dilute acids (acid hydrolysis).

Anthraquinone glycosides upon hydrolysis yield aglycone, which are di, tri or tetra

hydroxy anthraquinone or derivative of these compounds. The drugs having these glycosides

possess cathartic activity. The free anthraquinone aglycone exhibits little therapeutic activity.

The sugar residue facilitates translocation and absorption of aglycone at the site of action. These

compounds are stimulant, cathartic or purgation and they exert their action by increasing the tone

of smooth muscle in the wall of large intestine.

Major Anthraquinone Glycoside-Containing Herbs

• Aloe vera (aloe) latex

• Frangula purshiana (cascara sagrada)

• Rhamnus frangula (alder buckthorn)

• Rheum palmatum (Chinese rhubarb)

• Rumex crispus (yellow dock)*

• Senna alexandrina (senna)

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Biological source of Senna: Consists of the dried leaflets of Cassia

acutifolia (Alexandrian senna), or Cassia angustifolia (Indian senna).

Family: Leguminoseae.

Active constituents:

1- Anthraquinone glycosides: known as Sennosides A, B, C

and D.A & B are dianthrone of RheinC & D are dianthrone

of Rhein and Aloe emodin.

2- Free Anthraquinones ( aglycones): Rhein, Aloe emodin.

3- Flavanoids.

4- Mucilage.

5- Naphthalene glycosides which can be used to differentiate between the 2 types by TLC.

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Chemical color tests:

There are two chemical tests for anthraquinone glycosides after extraction step:

(i) When alkali (KOH) is added to the powdered drug or to the section of drug, red colour

produced serve to locate the anthraquinone derivative in the tissue.

(ii) Borntrager Test:

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LAB. 10

Aim: To carry out the TLC comparison of Senna powder and tablets

Pre-lab preparation: An introduction to TLC can be found at

www.chemguide.co.uk/analysis/chromatography/thinlayer.html

Intruduction to TLC:

Chromatography is used to separate mixtures of substances into their components. All

forms of chromatography work on the same principle.

Thin-layer chromatography (TLC) is an extremely valuable analytical technique in the

lab. It provides a rapid separation of compounds, and thereby gives an indication of the number

and nature of the components of a mixture. The following are some common uses of Thin-Layer

Chromatography:

1. To determine the number of components in a mixture.

2. To determine the identity of two substances.

3. To monitor the progress of a reaction.

4. To determine the effectiveness of a purification (check purity).

5. To determine the appropriate conditions for a column chromatographic separation.

6. To monitor column chromatography.

Principles of TLC:

- TLC is normally done on a small glass or plastic plate coated with a thin layer of a solid

— the most common are silica (SiO2) or alumina (Al2O3). This is the stationary phase.

The mobile phase is an organic solvent or solvent mixture.

- The sample mixture is applied near the bottom of the plate as a small spot, then placed in

a jar containing a few ml of solvent. The solvent climbs up the plate by capillary action,

carrying the sample mixture along with it.

- Each compound in the mixture moves at a different rate, depending on its solubility in the

mobile phase and the strength of its absorption to the stationary phase. When the solvent

gets near the top of the plate, it is allowed to evaporate, leaving behind the components of

the mixture at various distances from the point of origin.

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- The ratio of the distance a compound moves to the distance the solvent moves is the Rf

value (retention factor). This value is characteristic of the compound, the solvent, and

the stationary phase

- The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer

chromatography also often contains a substance which fluoresces in UV light - for

reasons you will see later. The mobile phase is a suitable liquid solvent or mixture of

solvents.

- A pencil line is drawn near the bottom of the plate and a small drop of a solution of the

dye mixture is placed on it. Any labeling on the plate to show the original position of the

drop must also be in pencil. If any of this was done in ink, dyes from the ink would also

move as the chromatogram developed.

- When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a

covered beaker. It is important that the solvent level is below the line with the spot on it.

- The reason for covering the beaker is to make sure that the atmosphere in the beaker is

saturated with solvent vapour. To help this, the beaker is often lined with some filter

paper soaked in solvent. Saturating the atmosphere in the beaker with vapour stops the

solvent from evaporating as it rises up the plate.

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- As the solvent slowly travels up the plate, the different components of the dye mixture

travel at different rates and the mixture is separated into different coloured spots.

What if the substances you are interested in are colourless?

There are two simple ways of getting around this problem (unless the chemical

compounds are colored):

1) Using fluorescence:

You may remember that I mentioned that the stationary phase on a thin layer plate often has a

substance added to it which will fluoresce when exposed to UV light. That means that if you

shine UV light on it, it will glow.

That glow is masked at the position where the spots are on the final chromatogram - even if those

spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow

apart from where the spots are. The spots show up as darker patches.

While the UV is still shining on the plate, you obviously have to mark the positions of the spots

by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will

disappear again.

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2) Showing the spots up chemically:

In some cases, it may be possible to make the spots visible by reacting them with

something which produces a coloured product. A good example of this is in

chromatograms produced from amino acid mixtures. The chromatogram is allowed to dry

and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to

give coloured compounds, mainly brown or purple.

In another method, the chromatogram is again allowed to dry and then placed in an

enclosed container (such as another beaker covered with a watch glass) along with a

few iodine crystals. The iodine vapour in the container may either react with the spots on

the chromatogram, or simply stick more to the spots than to the rest of the plate. Either

way, the substances you are interested in may show up as brownish spots.

by spraying the dry chromatogram of mixture on the TLC with general indicator like

vanillin sulfuric acid (few crystals in 10% H2SO4 of methanol)

Or by applying ammonia vapor to the plate to produce visible colored spots.

This experiment will introduce you to the mechanics of TLC, and the chemical principles

behind it. In the first part, you will separate the soluble components of senna extract and

tablets; in the second, you will analyze and compare the compounds you extracted from

plant and tablets on TLC chromatogram to confirm the presence of sennosides of senna in

available pharmaceutical product.

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Work procedure:

1. Weight about 10g of dry powdered Senna leaves, transfer into a stoppered flask and

add 100 ml of methanol

2. Shake gently, allow standing for 30 min. on water bath to enhance extraction then filter .

3. Grind few tablets of senna tablet product and extract using methanol

4. Preparation of standard solution: (if available)

5 mg of Aloe-emodin glycoside are dissolved in 5 ml of methanol

5. Solvent system:

Ethyl acetate: Methanol: Water (100:17:13)

6. Spraying reagent: 10 % alcoholic solution KOH or treatment with ammonia solution

7. Calculation of Rf value:

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Results and Calculations :

Determine the Rf values for all spots seen on the senna extract chromatogram:

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Lab. 11

Aim: Extraction & Chemical Identification of Cardiac Glycosides

The cardiac glycosides are an important class of naturally occurring drugs which actions

include both beneficial and toxic effects on the heart, and have played an outstanding role in the

therapy of congestive heart failures (CHF){What is CHF ??? Homework}. The terms ‘cardiac

glycoside’ or ‘digitalis’ are used throughout to refer to any of steroid or steroid glycoside

compounds that exert characteristic positively inotropic effect on the heart.

The cardiac glycosides are composed of two structural features; the sugar (glycoside) and

the non-sugar (aglycon) moieties.

Digitalis Purpurea, Digitalis lanata, Strrophanthiusgratusand Strophanthuskombeare the major

source of cardiac glycosides and digoxin,digitoxin, and ouabain (G-strophanthin) are well known

cardiac glycosides.

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Preparation of cardinolides from Digitalis )or any cardiac glycosides

containing drugs and plants):

1. Weight accurately about 10g of powdered leaves, transfer into a flask and add 100 ml of

70% ethanol. (gradual addition 20 ml each time with trituration in big mortar &pastle)

2. Transfer the mixture to flask in hot water bath, add 10 ml of diluted HCl, shake gently

many times, and allow standing for 30minutes then keep to be cooled.

3. Purify the tincture by adding 10 ml of (7.5 % lead subacetate solution) to the flask then

filter ( optional ) .The filtrate is shaken with 50 ml chloroform in 250 ml separatory

funnel.

4. Take 2-3 ml of chloroform layer into a clean test tube to perform the following chemical

tests:

Color reactions of cardinolides procedures:

A) Reaction due to (--CH2 (- group of the lactone ring

Kedde's test:

To the purified 2-3 ml chloroform fraction of extract, add 1 ml of Kedde's reagent (equal

volumes of 2 % 3,5-dinitrobenzoic acid in alcohol and 10 % NaOH )→ purple-violet color

Baljet's test:

To the purified 2-3 ml chloroform fraction of extract, add few drops of Baljet's reagent (95

ml of 1 % picric acid solution and 5 ml of 10 % sodium hydroxide solution freshly prepared just

before use) → orange or red will be found.

B) Reaction due to steroidal nucleus

Liebermann's test:

To the chloroform solution, add 1 ml of acetic acid followed by the addition of sulfuric acid

down the walls of the test tube to form a layer, the formation of a reddish-violet color at the

junction of the two layers, and a green color in the chloroform solution indicates the presence of

unsaturated sterols and/or triterpenes.

C) Reaction due to presence of 2-deoxy sugar

Keller killiani's test:

2ml of filtrate will be treated with 2ml of glacial acetic acid in a test tube and a drop of ferric

chloride solution (25g made up to 100ml + drops of Hcl) will be added to it. Then add 1 ml

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carefully of conc. sulfuric acid dropwise on the side of the test tube → a reddish brown ring

will be formed between two layers.

Results:

Describe your results and conclusions:

Kedde's test:

Baljet's test:

Liebermann's test:

Keller killiani's test:

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LAB. 12

Aim : To carry out the TLC of Cardiac glycosides

Continued notes of TLC:

- In TLC, a plastic, glass or aluminum sheet is coated with a thin layer of silica gel (stationary

phase)

regular silica gel, followed by end-capping

- A very small amount of a solution of the substance to be analyzed is applied in a small spot

with a capillary tube, ~1cm from the bottom of the TLC plate

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- The TLC is developed in a chamber which contains the developing solvent (the mobile

phase).

- Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the

developing chamber and the farthest extent of the solvent (the solvent front) is marked with a

pencil.

- The solvent is allowed to evaporate from the TLC sheet in the hood.

- The spots are visualized using a UV lamp.

- The separated chemicals may be colorless, so several methods used to view the spots:

• Visualization of spots under a UV254 lamp. The adsorbent layer will thus fluorescence

light green by itself, but spots of analyte quench this fluorescence.

• Iodine vapors are a general unspecific color.

• Specific color reagents exist into which the TLC plate is dipped or which are sprayed on

the plate.

• Once visible, the Rf value of each spot can be determined:

• The Rf is defined as the distance the center of the spot moved divided by the distance the

solvent front moved (both measured from the origin).

• Rfvalues can be used to aid in the identification of a substance by comparison to

standards.

• Two substances that have the same Rf value may be identical; those with different Rf

values are not identical.

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Work procedure of identification of cardinolides (Cardiac glycosides):

A) Sample preparation:

Weight accurately about 10g of powdered leaves, transfer into a stoppered flask and add

100 ml of 70 % ethanol. Add 10 ml of diluted HCl. Shake gently many times, and allow

standing for 30minutes then keep to be cooled then filter. The filtrate is shaken with

chloroform. The solvent is evaporated to concentrate.

B) Preparation of standard solution:

5mg of Digoxin (if available) dissolved in 2 ml of methanol

C) Solvent system:

Ethyl acetate: Methanol: Water (81:11: 8)

D) Spraying reagent:

Vanillin-Sulphoric acid reagent (few crystals of vanillin + 50 % sulphoric acid in methanol

solution). / OR /

Kadde’s reagent (3,5-dinitrobenzoic acid in methanol + 5% w/v solution of KOH).

E) How to run a TLC plate:

1. Draw a pencil line about a 1 cm from the bottom of the plate (along the short side). Mark places

along the line for each spot (pure reference spots and your mixture).

2. Dip the capillary into the solution and gently and quickly place a 1-2 millimeter spot on the plate

at the position you’ve marked. Keep the spots small!

3. Pour approx. 5 mL of solvent into a caped- jar, place a piece of filter paper in the jar and wet the

paper with the solvent to saturate the atmosphere. Make sure that the solvent is shallow enough

that it will be below the spot line on your plate.

4. Place the plate carefully in the chamber (use tongs or tweezers), being careful not to dunk the

spots under the solvent. Put the cap on the jar and let the plate develop.

5. When the solvent front has almost reached the top of the TLC plate, remove the plate and

immediately mark the solvent front with a pencil line.

6. Allow the solvent to evaporate and clarify the visible spots then look at the invisible spots under

UV lamb cabinet. /OR/ Spray the dried plate with suitable reagent, then heat the sprayed plate

using hotplate until the colors appear.

7. Circle the spots in pencil, break out a ruler and calculate Rf values.

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F) Calculation of Rfvalue:

Results and discussions:

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LAB. 13

Thin Layer Chromatography Characterization of

Flavonoids in Different herbal products

Introduction:

- Flavonoid is a class of plant secondary metabolites based around phenylbenzopyrone

structure.

- Flavonoids are most commonly known for their antioxidant activity.

- Flavonoids are also commonly referred to as bioflavonoids because all Flavonoids are

biological in origin.

- They have been referred to as nature's biological response modifiers because of strong

experimental evidence of their ability to modify the body's reaction to allergens, viruses

and carcinogens.

- They show anti-allergic, anti-inflammatory, anti-microbial and anticancer activity. In

addition, flavonoids act as powerful antioxidants, protecting against oxidative and free

radical damage.

- The beneficial effects of fruits, vegetables and tea have been attributed to flavonoid

compounds rather than to known nutrients and vitamins.

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- They are widely distributed in plants producing yellow or red/blue pigmentation in

flowers and protection from attack by microbes and insects.

Important Dietary Sources:

- All citrus fruits, berries, onions, green tea and dark chocolate are good sources of flavonoids.

- The citrus bioflavonoids include hesperidine, quercetin, rutin and tangeritin.

Aim of the experiment:

-In this experiment you will identify flavonoids by TLC in different herbs and pharmaceutical

products.

Work procedure of identification of flavonoids:

A) Sample preparation:

Weight accurately about 10g of powdered leaves and flavonoids containing capsules or

tablets, transfer into a stoppered flask and add 100 ml of 80% methanol. Shake gently many

times, and allow standing for 20 minutes then keep to be cooled then filter. The solvent is

evaporated to concentrate.

B) Preparation of standard solution:

5mg of Rutin (if available) dissolved in 2 ml of methanol

C) Solvent system:

ethyl acetate: formic acid: water (8:1:1).

D) Spraying reagent:

- Vanillin-Sulphoric acid reagent (few crystals of vanillin + 20 % sulphoric acid in methanol

solution). / OR /

- Spray the plate with the reagent consisting of (15ml 3% boric acid solution and 5ml 10%

oxalic acid) heat the plate and examine in UV light. Flavonoids treated with boric and oxalic

acids give compounds which after heating fluorescence yellowish green.

E) Calculation of Rfvalue:

F) Compare the results of the herbal products with standard to chick the availability of

flavonoids in them.

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Results and discussions:

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