1. B.PHARM. 5th SEMESTER
PHARMACOGNOSY ANDPHYTOCHEMISTRY-II
BP504T.
UNIT-III
Isolation, Identification and Analysis of Phytoconstituents
Alkaloids: Atropine, Quinine,
Reserpine, Caffeine

2. ALKALOIDS

3. HISTORY OFALKALOIDS:
The term ‘alkaloid’ was coined by MEISSNER, a German pharmacist,
in 1819.
The French chemist, Derosne in 1803, isolated narcotine. In the same
year, morphine from opium was isolated by Serturner.
Pelletier and Caventon isolated emetine in 1817 and colchicine in
1819.
From the beginning of 19th century till to date, it has to proved to be
a perpetual work to discover new alkaloids from plants and animals.
As per a Russian review in 1973, the number of known alkaloids had
reached up to 4959, amongst which, the structures of 3293 alkaloids
were elucidated.
At present, the number of alkaloids discovered has exceeded 6000.

4. What is alkaloid ?
The term “alkaloid” (alkali-like) is commonly used to designate basic
heterocyclic nitrogenous compounds of plant origin that are
physiologically active.
They resemble some of the characters of naturally occurring
complex amines.
It posses specific physiological actions on human or animal body
,when used in small quantities.
STRUCUTE OF ALKALOID

6. The term alkaloid also covers Proto alkaloid, Pseudoalkaloids and True
alkaloids.
TRUE ALKALOIDS:
• The true alkaloids are toxic in nature and contain heterocyclic
nitrogen which is derived from amino acids and always basic in
nature. True alkaloids are normally present in plants as salts of
organic acids.
PROTOALKAOIDS:
• The ‘protoalkaloids’ or ‘amino alkaloids’ are simple amines in
which the nitrogen is not in a heterocyclic ring. They are basic in
nature and prepared in plants from amino acids
PSEUDOALKALOIDS:
• It includes mainly steroidal and terpenoid alkaloids and purines.
They are not derived from amino acids.

7. OCCURENCE OF ALKALOIDS:
• Alkaloids are occurs mostly in angiosperms and rarely in gymnosperms.
• But their presence is also detected in micro organisms, marine
organisms, insects, animals and some of the lower plants.
• In the lower plants, although the alkaloids are found in less number,
some important sources are ergot fungus giving peptide alkaloids,
ergometrine, ergotamine etc., and also gymnosperms like ephedra
alkaloids.
• Out of 60 different orders in higher plants, 34 orders contain alkaloids.
DISTRIBUTION
• Families with alkaloidal content are Apocynaceae, Berberidaceae,
Euphorbiaceae, Leguminosae, Papaveraceae, Ranunculaceae, Rutaceae,
Rubiaceae and Solanaceae.
• In most of the plants alkaloids are highly localized and concentrated in
certain morphological parts only.

8. Examples
All Parts :e.g. Datura.
Barks :e.g. Cinchona,kurchi
Seeds :e.g. Nux vomica, areca, physostigma
Roots :e.g. Aconite , rauwolfia
Fruits :e.g. Black pepper, conicum
Leaves :e.g. Tobacco, coca, lobelia
Latex :e.g. Opium

9. PROPERTIES OFALKALOIDS:
Mostly all alkaloids are colourless, crystalline solids
Sharp melting point
Bitter taste
Some alkaloids are coloured in nature, e.g. Berberine is yellow
Soluble in organic non-polar, immiscible solvents.
The alkaloids containing quaternary bases are only water soluble.
Some of the pseudoalkaloids and proto-alkaloids shows higher solubility in water.
Some alkaloids are liquid because of lacking of oxygen in their molecules. (e.g.
coniine, nicotine, spartenine)
Alkaloids are decomposed by heat, except Strychnine and caffeine.
Give a precipitate with heavy metal iodides.

10. The various methods proposed for classification of alkaloids are
explained as follows,
Pharmacological classification
Taxonomic classification
Biosynthetic classification
Chemical classification
CLASSIFICATION:
TAXONOMIC CLASSIFICATION:
This method classifies vast number of alkaloids based on their
distribution in various plant Families, like solanaceous or
papillionaceous alkaloids.
From this classification, the chemotaxonomic classification has
been further derived.

11. PHARMACOLOGICAL CLASSIFICATION
Depending on the physiological response, the alkaloids are classified under various
pharmacological categories, like central nervous system stimulants or depressants,
analgesics, purgatives etc.
Some of the
examples are
explained as
follows,
• Narcotic analgesic e.g.Morphine
• Antimalarial e.g. Quinine
• Reflux excitability e.g.Strychnine
• Respiratory stimulant e.g.Lobeline
• Neuralgia e.g. Aconitine
• Oxytoxic e.g. Erogotometrine
• Bronchodilator e.g. Ephedrine, vasicine
• Anticholinergic e.g. Atropine
• CNS stimulant e.g. Caffeine
• Antitussive e.g. Codeine
• Antiarrythmic e.g.Quinidine
• Antihypertensive e.g. Reserpine
• Anticancer e.g. Vincristine
• Antiglucoma e.g. Pilocarpine

12. BIOSYNTHETIC CLASSIFICATION
This method give significance to the precursor from which the alkaloids are biosynthesized in
plants.
The alkaloidal drugs are categorised on the fact whether they are derived from amino acid
precursor as ornithine, lysine, tyrosine, tryptophan, phenylalanine etc.
1. Ornithine derived alkaloids
• e.g. Pyrrolidine alkaloid-Nicotine
• Tropane alkaloid - Atropine, hyosyamine, coacaine
2. Lysine derived alkaloids
• e.g. Piperidine and pyridine alkaloid - conine, lobaline, arecoline
• Quinazolidine alkaloid- lupinine
3. Tyrosine derived alkaloids
• e.g. Isoquinoline alkaloid-Morphine, codeine, emetine, cephaline, berberine,d- tubocurine
• Amino alkaloid- colchicine
4. Tryptophan derived alkaloids
• e.g. Indole alkaloid- ergot alkaloid, vincristine, vinblastine, reserpine, strychnine, physostigmine,
strychinine, brucine
• Quinoline alkaloid- cinchonine, quinine.
5. Histidine derived alkaloids
• e.g. Imidazole alkaloid - Pilocrpine
6. Phenylalanine derived alkaloids
• e.g. Amino alkaloid- Ephedrine

13. CHEMICAL CLASSIFICATION
The alkaloidal drug care broadly categorised into 2 divisions.
Heterocyclic alkaloids (True
alkaloids) are divided into 12
groups according to nature
of their heterocyclic ring.
Non- heterocyclic alkaloids
or protoalkaloids or
Biological amines or
pseudoalkaloids.
This is the most accepted way of classification of alkaloids. The main
criterion for chemical classification is the type of fundamental
(normal heterocyclic) ring structure present in alkaloid.

14. A) True alkaloids
Sr. no. Type Structure Examples
1. Pyrrole and
pyrrolidine
N
H
N
H
e.g. Hygrine, coca species
2. Pyiridine
and
piperidine N N
H
e.g. Arecoline, anabasine,
lobeline, conine, trigonelline
3. Pyrrolizidine
N
e.g. Echimidine, senecionine,
seneciphylline
4. Tropane
N
e.g. Atropine, hyoscine, hyoscyamine,
cocaine, pseudopelletirine
5. Quinoline
N
e.g. Quinine, quinidine, cinchonine,
cupreine, camptothecine
6. Isoquinoline
N
e.g.Morphine, codeine, emetine,
cephaline, narcotine, narceine, d-
tubocurarine
The following chart indicates types of alkaloids and their occurence in
various plants along with basic chemical ring.

15. 7 Indole
N
H
e.g. Erotamine, ergotametriene,
reserpine, vincristine, vinblastine,
strychnine, brucine
8 Imidazole N
N
H
e.g. Pilocrpine, isopilocarpine,
pilosine
9 Norlupinane
N
e.g. Cystisine, laburinine
10 Aporphine
(reduced
isoquinoline
napthalene)
N
e.g. Boldine

16. B) PROTO ALKALOID
1. Alkyalamine
Ephedrine,
Pseudoephedrine
HO
NH
1. Purine
N
N N
HN
e.g. Caffeine, thophylline,
theobromine
2. Steroidal e.g. Solanidine, conessine,
protoveratrine
3. Diterpene C20H32 e.g. Aconitine, aconine, hypoaconine
C) PSEUDO ALKALOID

17. ISOLATION OFALKALOID:
Plant material is dried at a temperature not exceeding 60˚c and
finely powdered.
Macerate the powdered material with sufficient quantity of ethanol
and set aside over night. Most alkaloids and their salts being alcohol
soluble will get dissolved in the solution.
Filter and concentrate the extract to ¼ the initial volume. complete
the evaporation of the remaining solvent at a temperature not
exceeding 50˚c.
Treat the residue with dil. sulphuric acid and filter to remove resins,
fatty matter and other unwanted substances.
Basify the solution by the addition of alkali such as ammonia as it
effectively precipitates most alkaloids and because of its volatility.

18. ISOLATION OFALKALOID:
Extract this solution with successive portion of chloroform
or till complete extraction of all the alkaloids is effected.
Concentrate the pooled organic layer to yield a crude
mixture of alkaloids of the plant material.
Dissolve the above residue in dil. sulphuric acid and filter if
necessary.
Basify the solution with ammonia and successively extract
with chloroform.
Run the pooled chloroform layers through a bed of
anhydrous sodium sulphate and evaporate to dryness.
Note the weight of the mixture of total alkaloids.

19. QUALITATIVE CHEMICAL TEST:
Wagner’s Test
• (+) Reddish brown precipitate Reagent used: Wagner’s Reagent [Solution of
iodine in potassium iodide]
Mayer’s Test
• (+) Cream color precipitates Reagent used: Mayer’s Reagent [Potassium
mercuric iodide solution]
Dragendorff’s test
• (+) Orange precipitate Reagent used: Dragendorff’s reagent [Potassium
bismuth iodide solution]
Hager's test
• (+) Yellow color precipitate Reagent used: Hager's reagent [saturated
solution of Picric acid]
Tannic acid test
• (+)buff color precipitate Reagent used: 10% Tannic acid solution

20. Atropine

21. Atropine
Biological source: Atropine is a tropane alkaloid obtained
from the fresh or dried leaves and flowering tops of
• Atropa belladonna,
• Datura stramonium (Not less than 0.25%) and
• Hyoscyamus niger (Not less than 0.05%).
Family – Solanaceae

22. Isolation Required quantity of coarse powder is taken and
moistens with sodium carbonate solution.
The blended mixture is extracted in petroleum ether
and filters it.
To the filtrate aqueous acetic acid is added and
further the aqueous fraction is extracted with ether.
Both fraction are separated by separating funnel and
discard solvent ether fraction.
Aqueous (Acidic fraction) is made alkaline with
sodium carbonate solution to obtain precipitates of
tropane alkaloids.
Atropine

23. Atropine
Isolation The precipitate is filtered and dry to obtain residue.
The residue is dissolved in diethyl ether, filtered it
and concentrated the filtrate.
Atropine crystals will be separated out.
The crystals are filtered and dissolve in alcohol
containing sodium hydroxide solution (Hyocyamine
is converted to atropine).
The atropine sulphate is recrystallized from acetone
and crystals of atropine are separated

24. Atropine
Properties Appearance :Colourless crystal or white
crystalline powder.
Odour :Odourless
Taste :Bitter taste
Solubility :Easily soluble in water,
soluble in ethanol, but insoluble in
ether and chloroform

25. Atropine
Identification by chemical test
• Vitali–Morin test: Small quantity of the solid
atropine is taken and added 2 drops of Conc.
nitric acid in an evaporating dish and evaporated
to dryness on water bath.
• Then the residue is dissolved in 1ml of acetone
and few drops of freshly prepared alcoholic
potassium hydroxide solution is added.
• Violet coloration takes place due to tropane
nucleus

26. Atropine
Analysis
by
TLC
Sample preparation :1mg of Atropine is dissolved in 1ml
of chloroform
Standard sample :Atropine
Stationary phase :Pre-coated Silica gel
Mobile phase :Toluene: Ethyl acetate: Diethyl
amine (70:20:10)
Detecting agent :Dragendorff’s reagent
RF Value :0.70
Color spot :Yellow orange spot
Utilization :It is used as antispasmodic,
mydriatic and antidote in opium poisoning.
Storage condition :It should be store in air-tight containers
protected from light and in cool place.

27. Quinine

28. Background & Historical Perspectives
▶Important medical discovery of 17th
century
▶Component of bark of cinchona trees.
▶In early 1600s it was referred as “Jesuits
bark” “cardinal’s bark” or “sacred bark.”
▶Quina-quina trees in Andean Jungle.
▶Introduction to Europe in 1638.
▶Use before 1820 and in 1820.

29. Quinine
Biological source: Quinine is a quinolone alkaloid obtained from the
dried bark of Cinchona calisaya, Cinchona officinalis, Cinchona
ledgeriana and Cinchona succirubra.
Family: Rubiaceae.
Quinine and quinidine are stereo-isomers.
Quinine is levorotatory and quinidine is dextrorotatory.

31. Effective cinchona alkaloids against malaria
EFFECTIVE
CINCHONA
Cinchonine
Quinidine
Cinchonidine
Quinine

32. Properties of quinine
▶ Aryl amino alcohol group of drugs.
▶ Basic compound.
▶ Rapid action against intra-erythrocytic malarial parasites.
▶ Gametocytocidal.
▶ Analgesic properties.
▶ White crystalline.
▶ Insoluble in water.
▶ Bitter taste and levorotatory.
▶ Anti inflammatory.

33. OVERVIEW OF QUININE
▶ Distribution in our body.
▶ Treatment for uncomplicated malaria.
▶ Lower cure rate.
▶ Higher cure rate (sulphadoxine-pyrimethamine, tetracycline or
clindamycin).
2010 World health Organization 2009 by 31 African countries
Quinine+ Doxycycline+
Tetracycline
(2nd line treatment)
Quinine
(2nd line treatment)

34. Isolation: Quinine
Required quantity of dry powder bark material is first well mixed with about 30% of its weight of
alcoholic calcium hydroxide or calcium oxide or calcium oxide (20%) and sufficient quantity of sodium
hydroxide solution (5%) to make a paste.
It is allowed to stand for few hours so that alkali can convert cinchona alkaloids to free bases.
The mass is then transferred to a Soxhlet apparatus and extraction is carried out with benzene for 6
hours.
After competition of extraction the benzene extract is shaken with successive portions of 5% sulphuric
acid in separating funnel.
The aqueous acid extract is separated from benzene layer and adjusted the pH 6.5 with dilute sodium
hydroxide, cooled.
Crystals of quinine sulphate are formed, filtered and recrystallized with hot water

35. Quinine
Properties:
• Appearance :Colourless crystal or white crystalline
powder
• Odour :Odourless
• Taste :Intensely bitter taste
• Solubility :Sparingly soluble in water, readily soluble
in chloroform, alcohol and ether
Identification by chemical test:
• Thalleoquin test: Bromine water and ammonia solution is
added in small quantity of powdered the sample. Emerald
green colour takes place which indicates the presence of
quinine

36. Quinine
Analysis
by
TLC
Sample preparation :1mg of Quinine is dissolved
in 1ml of methanol
Standard sample : Quinine
Stationary phase :Silica gel-G
Mobile phase :Chloroform: Diethyl amine (9:1)
Detecting agent :Dragendorff’s reagent
RF Value : 0.17

37. Analysis by HPLC
• Method : Isocratic
• Stationary phase : C18 Column
• Mobile phase :Methanol: Acetonitrle-0.1mol/L:
ammonia: acetone (45:15:40)
• Detection : Fluorescence at excitation 325nm.
• Emission : 375nm
Utilization:
• Quinine is antimalarial. Quinidine is a cardiac depressant
therefore used in cardiac arrhythmias.
Storage condition:
• It should be store in well closed and air-tight containers
protected from light and in cool place.
Quinine

38. Reserpine

39. Reserpine
Biological source: Reserpine is an indole alkaloid
obtained from the dried roots of Atropa
belladonna, Rauwolfia serpentine.
Family: Apocynaceae.
Sarpagandha contains not less than 0.15% of
reserpine and ajmalcine

40. Isolation
Rauwolfia root powder is exhaustively extracted with 90% alcohol in Soxhlet
apparatus.
The alcoholic extract is filtered, concentrated and dried under reduced pressure
below 60°C to yield dry extract.
The dry extract is extracted with ether-chloroform- 90% alcohol (20:8:2.5) and
filtered.
In filtrate dilute ammonia is added with intermittent shaking.
Then water is added to precipitate the crude alkaloids mixture and allowed the drug
to settle after vigorous shaking.

41. Isolation
The solution is filtered off and extracted the residue with 4 volumes of 0.5N
Ammonium sulphate in separating funnel and combined all the extracts.
The extract is made alkaline with dilute ammonia to liberate alkaloid.
Finally it is extracted with 3 portion of chloroform. Chloroform extract is
collected, concentrated and evaporated on water bath to yield total rauwolfia
alkaloids.
Residue is subjected to column chromatographic fraction for the separation of
reserpine

42. Reserpine
Properties:
• Appearance : White or pale buff to slightly yellow
crystalline powder, darkening slowly
on exposure to light.
• Odor : Odourless
• Taste : Bitter taste
• Solubility :Soluble in alcohol, chloroform and
acetone, partially soluble in water,
freely soluble in acetic acid

43. Reserpine
Identification by chemical test:
• When sample is treated with solution of vanillin in acetic acid, a violet
red colour is produced which indicates the presence of reserpine
Analysis by TLC
• Sample preparation : 1mg of Reserpine is dissolved in 1ml
of methanol
• Standard sample : Reserpine
• Stationary phase : Silica gel-G
• Mobile phase : Chloroform: Acetone: Diethyl ether
(50:40:10)
• Detecting agent : Dragendorff’s reagent
• RF Value : 0.72-0.35
• Color spot : Orange spot

44. Mode of Action
Reserpine acts by blocking the vasicular monoamine transporter
VMAT, which normally transports free norepinephrine, serotonin,
and dopamine from the cytoplasm of the presynaptic nerve into
vesicles for subsequent release into the synaptic cleft.
The unprotected neurotransmitters then metabolized by MAO and
therefore never reach the synapse.
By this mode of action this give mainly the antihypertensive action
and also use in the treatment of dykynesia mania etc.

45. Therapeutic application:
Essential hypertension
Mild anxiety
Dyskinesia
Psychosis symptoms
Maintenance dose: 100 to 250 mg (once a day)
Side effects:
Nasal congestion
Nausia
Vomiting
Gastric intolerance
Gastric ulceration
diarrhoeia

46. Reserpine
Storage condition:
• It should be store in well closed and air-tight containers
protected from light and in cool place.

47. Caffeine
• Thea sinensis (Tea leaves)

48. Caffeine
Biological source: Caffeine is a purine alkaloid
obtained from Tea leaves, Coffee seeds, cocoa, and
other species. It is chemically 1,3,7, trimethyl
xanthine which is isolated from tea and coffee seeds
during decaffeination process. It is obtained from the
prepared leaves and leaf buds of Thea sinensis.
Family:Theaceae and
Dried ripe seeds of Coffea Arabica, C. liberica,
Family: Rubiceae.
Tea leaves contains 1-4% of caffeine and coffee
contains 1- 2% of caffeine.

49. Isolation: Caffeine
The powder tea leaves is extracted with boiling water and the aqueous extract is filtered while hot.
The warm extract is treated with lead acetate to precipitate tannins and filtered.
The filtrate is treated with excess of dilute sulphuric acid to precipitate lead in the form of lead
sulphate.
The filtrate is boiled with activated charcoal to remove colouring matter, if any and filtered to
remove charcoal.
The filtered decolourized solution is extracted with chloroform successively.
Combined the chloroform extracts and evaporated on water bath to yield caffeine (white powder).
It is recrystallized with alcohol

50. Caffeine
Properties:
• Appearance : White powder or white glistering needles
• Odor : Odourless
• Taste : Bitter taste
• Solubility : Soluble in hot water
Identification by chemical test:
• Murexide test: Sample is taken in a Petri dish to which
hydrochloric acid and potassium chlorate are added and
heated to dryness. A purple color is obtained by exposing
the residue to vapors of dilute ammonia. The purple color is
lost on addition of fixed alkali.

51. Caffeine
• Analysis by TLC
• Sample preparation : 1mg of Caffeine is dissolved in 1ml
of methanol or chloroform
• Standard sample : Caffeine
• Stationary phase : Silica gel-G
• Mobile phase : Ethyl acetate: methanol: acetic acid
(80:10:10)
• Detecting agent : Expose to vapors of iodine
• RF Value : 0.41
• Color spot : Brown spot [4, 5]
• Analysis by HPLC
• Method : Isocratic
• Stationary phase : C18 column
• Mobile phase : Methanol: Water (25:75)
• Detection : UV-Visible detection 254nm

52. Caffeine
Utilization:
• Caffeine is a CNS stimulant and Diuretic. It is used in
beverage.
Storage condition:
• It should be store in well closed and air-tight containers
protected from light and in cool place.