CHAPTER – 1

ALKALOIDS, AN OVERVIEW

INTRODUCTION
DISTRIBUTION/ OCCURRENCE
CLASSIFICATION OF ALKALOIDS
CHEMICAL TESTS FOR ALKALOIDS
PHARMACOLOGICAL ACTION OF ALKALOIDS
EXTRACTION AND ISOLATION
ROLE OF ALKALOIDS IN PLANTS
BIOSYNTHESIS/ BIOGENESIS OF ALKALOIDS
CHARACTERIZATION OF ALKALOIDS
REFERENCES

8
 ALKALOIDS, AN OVERVIEW

Alkaloids are naturally occurring organic substances, predominantly found in plant

sources including marine algae and rarely in animals (e.g. in the toxic secretions of fire ants,
ladybugs and toads). They occur mostly in seed-bearing plants mainly in berries, bark, fruits,
roots and leaves. Alkaloids often contain at least one nitrogen atom in heterocyclic ring (Fig. 1).
These are basic in nature and so referred the term alkaloid (alkali-like). Alkaloids possess
remarkable physiological action on human and other animals. These are the active components
of numerous medicinal plants or plant-derived drugs. Their structural diversity and different
physiological activities are unique to any other group of natural products. Many drugs used by
man for both medical and non medical purposes are produced in nature in the form of alkaloids
e.g. atropine, strychnine, caffeine, nicotine, morphine, codeine, cocaine etc. Naturally occurring
receptors for many alkaloids have also been identified in human and other animals, suggesting an
evolutionary role for the alkaloids in physiological processes. Alkaloids are relatively stable
compounds that accumulate as end products of different biosynthetic pathways, mostly starting
from common amino acids such as lysine, ornithine, tyrosine, tryptophan, and others. These
substances are usually colourless but several coloured alkaloids are also reported e.g. berberine is
yellow, sanguinarine salt is copper-red and betanidin is red (Kokate et al., 2005). These are
crystalline solids, having ring structure, definite melting points and bitter in taste. In plants they
may exists in free state, in the form of salt or as N-oxides, rarely found in the form of glycosides
(Biswas and Sharia, 1978; Tanahashi et al., 2000; Kashiwaba et al., 2000). In addition to the
elements carbon, hydrogen and nitrogen, most alkaloids contain oxygen. A few such as coniine
from hemlock and nicotine from tobacco are oxygen free. The free bases are sparingly soluble in
water but readily soluble in organic solvents, however with their salts, the reverse is often the
case, e.g. strychnine hydrochloride is more soluble in water than in organic solvents. Most of the
alkaloids are optically active, generally due to the presence of tertiary nitrogen in their structures.
This results the various isomeric forms having different physical, chemical and pharmacological
properties e.g. (+)-tubocurarine isolated from Chondrodendron tomentosum (Bisset, 1992), have
muscle relaxant activity, whereas its leavo isomer is less active. The structural formula of some
common alkaloids from plant origin are given in Fig. 1 and 2.

9
 CH3
O N
N

N HO N CH3
NCH3 O
H3CN
N CH3
CH3
N Tropine
Hygrine
O Nicotine
Caffeine

OH
OH O
H
CH3 N
N
COOCH3
CH3
OH
N CH3
CH3 H
OCO.C6H5
H H
N Coniine
Cocaine Ephedrin

Ecgonine

H3CO HO
H3CO

NH.COCH3 N
H3CO O
H3CO
OCH3
OCH3 NCH3
H
HO
O
OCH3 Morphine
Papaverine
OCH3
Colchicine
HOOC
NCH3

O H

N
N HO
HN H
R

N
O N H
H
N
H
Xanthine
Quinine - R= OCH3 Lysergic acid
Cinchonine - R= H

N O N
H3CO N
H OCH3 H

OC OCH3

O Solasodine
H3COOC
OCH3
OCH3
Reserpine

Fig. 1

10
 H3CO
O
O
N H3C
OH
N
NH
O
N O NH2
N
N N
NCH3 H
O O H3C H
HO
Theophylline Tryptophane
Strychnine Codeine

H3CO
OCH3

N
H3CO H3C N+
H H OH H
H3C O CH3
H
O N+
H
H
H
OCH3
H3CO
HN OH

(+)-Tubocurarine
Emetine OCH3

OH

O
N
OH
OCH3
O N CH3

C2H5N OCH3
H3CO
OR1 Atropine
H
OH

H3CO
HO OH N
OR2
Quinine
CH2OCH3
N+
OCH3
H3CO
Aconine - R1=H, R2=H
Aconitine - R1=COPh, R2=COMe OCH3
R1

H3CO
Palmatine
OCH3

R2 N
O
N
N+
O

H3CO OCH3

OCH3

Solanidine Tylophorine R1=OCH3 R2=H OCH3

Tylocrebine R1=H R2=OCH3 Berberine

Fig. 2

11
DISTRIBUTION/ OCCURRENCE
Alkaloids are generally occur in all parts of the plant but some times accumulated only in
particular organ, whereas at the same time other organs are free from alkaloids e.g. the edible
tubers of potato plant are devoid of alkaloids, whereas the green parts contain the poisonous
alkaloid solanine. The organ in which alkaloids accumulated is not always the site of their
synthesis, e.g. in tobacco, nicotine is produced in the roots and translocated to the leaves where it
accumulates (Harborne and Herbert, 1995).
After the isolation of first alkaloid narcotine by French apothecary Derosne in 1803 and
morphine by Hanoverian apothecary Serturner in 1806, more than ten thousand alkaloids have
been discovered from different sources (Evans, 2006). Alkaloids are commonly found in the
orders Centrospermae, Magnoliales, Ranunculales, Papaverales, Rosales, Rutales, Gentiales,
Tubiflorae and Campanulales. True alkaloids are rarely occur in lower plants. Among the
Pteridophytes and Gymnosperms, the bioactive alkaloids lycopodium, ephedra and taxus are well
known. Lysergic acid and sulphur containing alkaloid gliotoxin are best known examples
isolated from fungi.
Nearly 300 alkaloids belonging to more than 24 classes are found in the skin of
amphibians along with other toxins (Evans, 2006). The poisonous neurotoxic alkaloids were
isolated from the skin of frogs belonging to genus Phyllobates. Daly, (1993) isolated various
antimicrobial alkaloids from the skin of reptilian. Some indole and isoquinoline alkaloids were
isolated from mammals including mammalian morphine.

CLASSIFICATION OF ALKALOIDS
1. Taxonomical classification: This classification is based on the distribution of alkaloids in
various plant families, like solanaceous or papilionaceous alkaloids. Some times they are
grouped as per the name of grouped genus in which they occur, e.g. ephedra, cinchona, etc.

2. Biosynthetic classification: This method gives significance to the precursor from which the
alkaloids are biosynthesized in the plant. Hence the variety of alkaloids with different
taxonomic distribution and physiological activities can be brought under same group, if they
are derived from same precursor, e.g. all indole alkaloids from tryptophan are grouped
together. Alkaloids derived from amino acid precursor are grouped in same class such as
ornithine, lysine, tyrosine, phenylalanine, tryptophan, etc.

12
3. Pharmacological classification: This classification is based on the physiological action or
biological activity of alkaloids on animals like CNS stimulants or depressants,
sympathomimetics, analgesics, purgatives, etc. This method does not take account of
chemical nature of alkaloids. Within the same chemical structure the alkaloids can exhibits
more than one physiological action e.g. morphine is narcotic-analgesic, while quinidine is
cardiac depressant.

4. Chemical classification: this classification is most accepted way to specify the alkaloids.
The alkaloids are categorised into three divisions.

a. True alkaloids: These have heterocyclic ring with nitrogen and derived from amino acids.

b. Proto alkaloids: These does not have heterocyclic ring with nitrogen and derive from amino
acids, e.g. colchicine.

c. Pseudo alkaloids: These have heterocyclic ring with nitrogen and derived from terpenoids or
purines but not derived from amino acids.

The chemical classification of alkaloids is summarized in Table 1 and a general scheme for
classification of alkaloids is shown in Fig. 3.

13
Table 1. Classification of alkaloids along with some common examples and sources

S.No. Class Basic ring Example Biological sources References

1. Pyrrole and Hygrine, nicotine, Erythroxylum coca, (Moor, 1994;
N
Pyrrolidine N
H
H cuscohygrine, coca alkaloids Erythroxylum truxillense Evans, 1981)

NCH3 O

Hygrine

2. Pyridine and Piperine, coniine, trigonelline, Piper nigrum, (Parmar et al., 1997;
Piperidine N
N
H
arecaidine, guvacine, Areca catechu, Ravindran et al., 2000)
pilocarpine, cytisine, nicotine, Lobelia nicotianefolia
N sparteine, pelletierine, lobeline,
N
Nicotine arecoline, anabasine
3. Pyrrolizidine Echimidine, senecionine, Castanospermum (Molyneux, 1988;
N
senesiphylline, symphitine australe, Senecio sps. Hartmann and Witte,
CHCH3 OH
1995)
O H2 CH3 O
C
O H H CH O
3

N
Cenecionine

4. Tropane Atropine, cocaine, ecgonine, Atropa belladonna, (Munoz and Cortes,

NCH3

scopolamine, hyoscine, Dhatura stramonium, 1998; Leete, 1988;

OH

O meteloidine hyoscyamine, Erythroxylon coca, Drager and Schaal,

O N pseudo-pelletierine, Schizanthus porrigens 1993)
Atropine

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5. Quinoline Quinine, quinidine, brucine, Cinchona officinalis, (Giroud, 1991;
N

veratrine, dihydroquinine, Cinchona calisaya, Phillipson et al., 1981;

N

dihydroquinidine, strychnine, Almeidea rubra Santos et al., 1998;

O
H
N
OH
cevadine, cinchonine, cupreine, Scherlach and
Quinine
cinchonidine, prenylated Hertweck, 2006)
OCH3
HO HO
O
OCH3
quinolin-2-one
H
N O
H
Prenylated quinolin-2-one

6. Isoquinoline Morphine, codeine, thebaine, Papaver somniferum, (Wu et al., 2000;

N
papaverine, narcotine, Cephaelis ipecacuanha, Bisset, 1992; Schiff,
sanguinarine, narceine, Berberis aristata, 1991; Guinaudeau and
O
N+ hydrastine, berberine, d- Aristolochia elegans, Brunetonin, 1993;
O
OCH3
tubocurarine, emetine, Cocculus pendulus Bhakuni et al., 1976;
OCH3
Berberine
cephaeline, narcotine Uprety and Bhakuni,
1975)
7. Aporphine NCH3
Boldine, phoebine, Stephania venosa, (Pharadai, 1985; Castro
laurodionine, noraporphine, Phoebe valeriana et al., 1986)

H3CO
OCH3 norpurpureine, nordelporphine
NCH3
H3CO

O
O
Phoebine

15
8. Indole Psilocybin, serotonin, Strychnos nuxvomica, (Dembitsky et al.,
melatonin, reserpine, ergine, Claviceps purpurea, 2005; Lin, 1996)
N
H
vincristine, vinblastine, Rauwolfia serpentine,
strychnine, brucine, emetine, Catharanthus roseus,
HOOC
NCH3
physostigmine, harmine, Gelsemium elegans
ergotamine, ergometrine,
N
H lysergic acid, yohimbine,
Lysergic acid

hydroxygelsamydine
9. Imidazole N
Pilocarpine, pilosine Pilocarpus jaborandi, (Negri, 1998)
N
H Maranham jaborandi,
R NCH3
Ceara jaborandi
O O N
Pilocarpine R= C2H5
Pilosine R= PhCHOH

10. Norlupinane Cytisine, laburnine, lupanine, Punica granatum, (Yusuph and Mann,
N
sparteine Lobelia inflata 1997)
H
N

N
H
Sparteine

11. Purine N NH
Caffeine, theobromine, Theobroma cacao, (Shervington, 1998)
N

CH3
N
theophylline Coffea arabica
O N
N

H3CN
N
CH3

O
Caffeine

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12. Steroidal Solasodine, solanine, Solanum tuberosum, (Kumar and Ali, 2000;
solanidine, samandarin, Veratrum album, Shakirov, 1990;
protoveratrine, conessine, Whithania somniferum, Rahman and
ON
H
funtumine Holarrhena Chaudhari, 1995;
Solasodine antidysenterica Rahman and
Chaudhari, 1996)
13. Diterpene Aconitine, aconine, Aconitum napellus, (Rahman and
N

OH
OCH3
hypoaconitine Aconitum japonicum Chaudhari, 1995; Wang
C2H5N
H3CO
OR1
et al., 1992)
HO OH
OR2

CH2OCH3 OCH3

Aconine - R1=H, R2 =H
Aconitine - R1=COPh, R2=COMe

14. Alkylamine/ Ephedrine, pseudoephedrine, Ephedra gerardiana, (He et al., 1999)

Amino colchicine, demecolcine Ephedra sinica,
alkaloid CHOH Colchicum autumnale
CHCH3

NHCH3

17
 ALKALOIDS

Pyridine Pyrrolidine Tropane Quinoline Isoquinoline Phenethylamine Indole Purine Terpenoid Pyrazole Betaines
group group group group group group group group group group

Tryptamines
Quaternary
Hygrine, ammonium
Xanthines compounds
Cuscohygrine, Opium alkaloids Psilocybin,
Nicotine Serotonin,
Melatonin

Morphine, Ergolines
Aconite alkaloids
Codeine,
Thebaine,
Papaverine, Ergot alkaloids
Atropine, (Ergine, Ergotamine,
Narcotine,
Cocaine,
Sanguinarine, Lysergic Acid)
Ecgonine, Aconitine
Narceine,
Scopolamine Hydrastine,
Berberine Pyrazole,
Beta-carbolines
Fomepizole

Harmine,
Yohimbine, Steroids
Quinine, Reserpine,
Quinidine, Emetine
Dihydroquinine,
Dihydroquinidine,
Strychnine,
Brucine, Mescaline, Solanine,
Veratrine, Ephedrine, Samandarin
Piperine, Cevadine Amphetamine,
Coniine, Methamphetamine
Trigonelline, Muscarine,
Arecaidine, Choline,
Guvacine, Neurine
Pilocarpine, Caffeine,
Cytisine, Theobromine,
Nicotine, Theophylline
Sparteine,
Pelletierine

Fig. 3. General scheme for classification of alkaloids

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CHEMICAL TESTS FOR ALKALOIDS
The chemical tests used for detection of alkaloids depend on their character to precipitate
with organic acids in the form of their salts. These are also precipitated by the reaction of
compounds of heavy metals like mercury, gold, platinum etc. Caffeine and some other alkaloids
which are highly water soluble, do not give the tests with usual reagents. Some common reagents,
used to the detection of alkaloids are summarized in Table 2.

Table 2. Reagents used for the detection of alkaloids

S.No Name of reagent Chemical composition Colour obtained Example

1 Mayer’s reagent Potassium mercuric iodide Cream Common
solution
2 Wagner’s reagent Solution of iodine in potassium Reddish-brown Common
iodide
3 Dragendorff’s Potassium bismuth iodide Reddish-brown Common
reagent solution
4 Hager’s reagent Saturated solution of picric acid Yellow Common
5 Picrolonic acid Solution of picrolonic acid Yellow Common
6 Tannic acid Solution of tannic acid Common
7 Murexide test *Potassium chlorate+HCl+NH3 Purple Caffeine
8 Mineral acids Phosphotungstic acid, Yellow Colchicine
phosphomolybdic acid
9 Acidic p-methyl- p-Methyl-aminobenzaldehyde Bluish-violet to Indole
aminobenzaldehyde and sulphuric acid red
10 Nitric acid Dilute nitric acid Orange-red Morphine
*a very small amount of potassium chlorate and a drop of conc. HCl, evaporating to dryness and exposing the residue to NH vapour
3

PHARMACOLOGICAL ACTION OF ALKALOIDS

Alkaloids have traditionally been of great interest to humans because of their pronounced
physiological and medicinal properties. From the beginning of civilization, alkaloid-containing plant
extracts have been used in all cultures as medicines and poisons. Greek philosopher Socrates died in
399 B.C. by consumption of coniine containing hemlock (Conium maculatum) and Egyptian queen
19
Cleopatra (69-30 B.C.) used atropine containing plant extracts (such as belladonna) to dilate her
pupils. The physiological effects of alkaloids have made them important compounds in medicine.
They are used as a remedy for painkillers, stimulants, muscle relaxants, tranquilizers, anaesthetics,
antimalarial, antimicrobial, antidiabetic, anticancerous, anti-HIV, antioxidants etc. Proaporphines
and crotsparine isolated from Cocculus sparciflorus (Bhakuni et al., 1968), showed significant
hypotensive and anticancer activity (Bhakuni et al., 1969). Homoerythrine derived alkaloids isolated
from stem of Galipea bracteata (Vielra and Kubo, 1990) showed molluscicidal activity. In modern
times, the stimulants caffeine in coffee, tea and cacao and nicotine in cigarettes are consumed
worldwide. Alkaloids with hallucinogenic, narcotic or analgesic properties have found applications
in medicine e.g. morphine, atropine and quinine. Some alkaloids served as model compounds for
modern synthetic drugs whereas several are abused as illegal drugs e.g. cocaine. A number of
alkaloids are too toxic for any therapeutic use e.g. coniine and strychnine. Moreover the plant
constituents are still screened for new biologically active compounds. Taxol (Fig. 4) considered as
cytostatic drug, applied for the treatment of lung cancer, breast cancer, neck cancer and ovarian
cancer (Heinstein and Chang, 1994). Coniine, tropine, vendoline, morphine and tyrosine showed
significant anticancer activity (Hartwell and Abbot, 1969). Nicotine isolated from Nicotiana
tabacum of family Solanaceae is a highly addictive stimulant of the nervous system in small doses,
such as those obtained by smoking tobacco in cigarettes, whereas higher doses can be extremely
toxic. Nicotine is also used as an insecticide in the form of nicotine sulfate. Various alkaloids like
codeine, heroin, morphine and opium, derived from the sap of Papaver somniferum are widely used
as medicines or highly addictive and recreational drugs. An addictive narcotic drug cocaine, derived
from the foliage of Erythroxylon coca used as a stimulant and local anaesthetic (Novak et al., 1984).
Quinine, a bitter tasting alkaloid extracted from Cinchona ledgeriana is well known for its
antimalarial activity. Morphine is a powerful painkiller, often given to terminally ill patients.
Codeine having similar pharmacological functions to morphine, but is less potent. Heroin is a
synthetic derivative of morphine that is highly addictive. Papaverine isolated from Papaver
somniferum (Pictet and Gums, 1909) has relaxant effects on smooth muscle of the intestinal and
bronchial tract and the blood vessels. Strychnine is a powerful CNS stimulant and lysergic acid
produced by a fungus grows on rye in the form of lysergic acid diethylamide (LSD), is a powerful
hallucinogen. Atropine (Mann et al., 1994) acts as a smooth muscle relaxant and used to dilate the
pupils of the eye. Roots of Rauwolfia serpentine used in North India as a sedative, led to the
isolation of ajmaline, ajmalinine and serpentine (Siddhiqui and Siddhiqui., 1931). Tylophorine,

20
tylophoridine and tylocrebine (Hartwell and Abbot, 1969) isolated from different genus of
Tylophora showed anticancer activity. The bark of Holarrhena antidysentrica containing couessine,
conessimine and conssidine (Siddhiqui and Pillay, 1932; 1934) is widely used in the treatment of
dysentery. Ergotamine (Fig. 5) in the form of ergotamine tartrate combined with caffeine is used as a
specific analgesic in the treatment of migraine.

O
CH3

O O OH CH3
O
NH OH
O NH O O
N
H
O N
N O O
OH H
HO H O CH3
H

H3COCO HN

O O Fig. 5. Ergotamine
Fig. 4. Taxol

EXTRACTION AND ISOLATION

The extraction of alkaloids is based on their basic character and solubility pattern. The
general scheme for extraction is shown in Fig. 6. Extraction is usually served by one of the following
general methods;
1. The plants are defatted with petroleum ether, especially in case of seeds and leaves to remove the
fat soluble constituents and then with polar solvents. The extract is concentrated under reduced
pressure and treated with alkali so that the free bases convert in their salts and separated with
organic solvents. This process is known as Stas-Otto process. This method is frequently used in
the extraction of ergotamine (Kokate et al., 2005) from ergot.
2. The powdered material is moistened with water and mixed with lime, which combines with
acids, tannins and other phenolic substances and sets free the alkaloid salts. Extraction is then
carried out with organic solvents such as ether or petroleum sprit. The concentrated organic
liquid is then shaken with aqueous acid and allowed to separate. Alkaloid salts are now in
aqueous liquid, while many impurities remain behind in the organic liquid.

21
3. The powdered material is extracted with polar solvents such as water or aqueous alcohol
containing dilute acid. Pigments and other unwanted materials are removed by shaking with
chloroform or other organic solvents. The free alkaloids are then precipitated by the addition of
excess sodium bicarbonate or ammonia and then separated by filtration or by extraction with
organic solvents.
4. The extract is treated with ammonia so as to convert the alkaloid salts into their free bases. Such
liberated alkaloids in free base form are conveniently extracted with organic solvents like ether,
benzene, chloroform etc. This method is not useful for the isolation of alkaloids of quaternary
nitrogen.
5. The alkaloids present in the extract are converted into their reineckates by treating with
H[Cr(NH3)2(SCN)4] (Reinecke’s solution). The product is then dissolves in acetone and then
passed this solution through an ion exchange column which afforded the alkaloids in a high state
of purity.

POWDERED PLANT MATERIAL

(Containing alkaloidal salts like oxalates,
tannates and defatted if necessary)
Moisten and render alkaline with sodium
carbonate, ammonia, calcium hydroxide
ALKALOIDS FREED AS BASES
Exhaust with organic solvent e.g. chloroform,
ether or methylene dichloride

TOTAL EXTRACT
Concentrate and shake with successive quantities
of inorganic acids

RESIDUAL ORGANIC FRACTION AQUEOUS ACID SOLUTION

(Like pigments, fats, very weak bases or (alkaloidal salts), make alkaline
chloroform soluble alkaloids sulphates) and extract alkaloids with
immiscible solvents

ORGANIC SOLVENT OF ALKALOID BASES

Remove solvent
RESIDUAL AQUEOUS FRACTION
CRUDE ALKALOID MIXTURE
Purification by fractional crystallization, chromatographic separation, etc.

Structure identification by modern analytical techniques such as UV, IR, NMR, MS, etc.

Fig. 6. General scheme for extraction of alkaloids

22
 Further purification of crude extract of alkaloids is done by following ways, which may,
however vary for individual alkaloid.

1. Direct crystallization from solvent: It is a simple method of isolation in which the alkaloids
crystallise directly by fractionation process and may not be useful in case of complex mixtures.

2. Steam distillation: This method is specially employed for volatile liquid alkaloids like coniine,
sparteine and nicotine. However this method is not suitable for alkaloids of high molecular weights.
In this method, the aqueous extract is made alkaline with caustic soda or sodium carbonate and then
alkaloids are distilled off in steam.

3. Gradient pH technique: There is variation in the extent of basicity of various alkaloids of same
plant. On the basis of this property, the crude alkaloid mixture is dissolved in 2% tartaric acid
solution and extracted with benzene so that the first fraction contains neutral and/or very weakly
basic alkaloids. The pH value of the aqueous solution is increased gradually by 0.5 increments to pH
9 and extraction is carried out at each pH with organic solvents. By this way, the alkaloids of
different basicity are extracted and strongly basic alkaloids extracted at the end.

4. Chromatographic techniques: Chromatography is an ideal method for separation of a vast

number of alkaloids. The separation of alkaloids carried out by using stationary and mobile phase of
different organic solvents. The different techniques of chromatography used for separation of
individual alkaloid from complex mixture are as following;

a. Paper chromatography (PC): This technique is simplest and most widely used among other
chromatographic techniques because of its applicability to isolation, identification and some times
quantitative determination of all type of natural products. It is a partition as well as an absorption
type technique, in which the mobile phase is either individual or mixture of organic solvents and the
stationary phase is hydrophilic surface of paper. The choice of the solvent used to run a
chromatogram depends upon the nature of alkaloid. Usually non polar solvents like benzene/
acetone/ water are commonly used for separation of non polar alkaloids and polar solvents like
butanol/ acetic acid/ water used for separation of polar alkaloids. Both one and two dimensional
ascending and descending, some times horizontal chromatographic techniques applied using
Whatman filter paper sheet of various types i.e. No.1, 2, 3 etc. The paper sheet is washed with 5%
2N HCl to remove the impurities before starts the separation. The paper is visualised under UV light
23
of long wavelength or spread with iodoplatinate reagent in the form of coloured spots. The Rf values
and colour appearance of spots on paper sheet are useful in preliminary identification of alkaloids by
this technique.

b. Thin layer chromatography (TLC): TLC is an important tool extensively used for
identification, separation and determination of the purity of isolated alkaloid. Although TLC method
is used in qualitative analysis but it having a great importance in quantitative analysis also (Oswald
and Fluck, 1964; 1964). The TLC separation of alkaloids can be performed on silica gel, alumina,
cellulose powder or kieselguhr. Silica gel is the most active stationary phase and good separation is
achieved even when several milligrams substance applied. This method is also used for preparative
separation of alkaloids (Tschesche et al., 1963). In TLC, the substances are identified preliminary on
the basis of their mobility in suitable solvent system and moreover on the basis of their reaction with
selective or specific detection reagents or on the basis of their absorption or fluorescence in UV light
(Macek, 1972; Hais, 1970; Neher, 1970). Some typical solvent systems (Stahl, 1969) are as
following;

C6H6/ EtOAc/ (C2H5)2NH (5:4:1) + 8% MeOH C6H6/ EtOAc/ (C2H5)2NH (5:4:1, 7:2:1)
CHCl3/ (C2H5)2CO/ (C2H5)2NH (7:2:1) + 8% MeOH CHCl3/ (C2H5)2CO/ (C2H5)2NH (7:2:1)
HCON(CH3)2/ (C2H5)2NH/ EtOH/ EtOAc (1:1:6:12) CHCl3/ MeOH/ AcOH (6:1:3)
C6H12/ (C2H5)2NH (9:1 or 1:1) C6H6/ CHCl3/ (C2H5)2CO (14:3:3)
CHCl3/ EtOH (9:1 or 8:2) CHCl3/ (C2H5)2O/ H2O (7:1:2)
Xylene/ BuOH/ MeOH/ (C2H5)2NH (5:5:7:3) C6H14/ CCl4/ (C2H5)2NH (5:4:1)

Developed TLC plate is sprayed by several visualizing reagents for detection of alkaloids.
Many alkaloids can be seen on the chromatogram even in daylight and a large number yield typical
fluorescence colours in UV light (365 nm). The reagents most commonly used for detecting
alkaloids are Dragendorff reagent, acidified iodine- iodine solution, iodoplatinate, antimony-
(III)chloride, cerium sulphate in sulphuric acid and cerium sulphate in phosphoric acid.
Papaverrubine type alkaloids give a red spot with hydrogen chloride vapour, Rauwolfia alkaloids
give colour with mixture of ferric chloride and perchloric acid and also with iodine vapour,
phenylalkylamines are visualised with ninhydrin. Beside these reagents, cinnamaldehyde
hydrochloric acid for indole alkaloids, sulphuric acid for purine bases and betaines and the van Urk
reagent for ergot alkaloids are often used.
24
 Preparative TLC: In this method, comparatively thicker layer of adsorbent are employed for
preparative work and the separated bands of compounds are scraped from the plate and subjected to
solvent extraction. This technique generates sufficient quantity of material for complete spectral
analysis. Preparative TLC was performed for cryptopleurine and piperidylacetophenone (Al-
Shamma et al., 1982) on 1 mm thick commercial plates precoated with Al2O3 GF-254 developing
with ammoniated CHCl3 and determined the Rf values.

c. Column chromatography (CC): This method is very popular and used extensively for
quantitative separation and purification of different natural products from crude plant extract or
mixture. Column of different size and dimensions are used for separation. Choice of stationary phase
(adsorbents) and mobile phase (organic/ inorganic solvents) is based on the nature of components
present in the crude extract. A large number of adsorbents are available which can be used in the
column chromatography, some being better for one type of components and some for others. For the
separation of alkaloid, silica gel (Yinggang et al., 2003; Roumy et al., 2006) and neutral alumina
(Hart et al., 1968) are extensively used. Beside these, some more adsorbents are kieselguhr, silicates
(magnesium/calcium silicate), tricalcium phosphate, calcium sulphate, glass power, salts of
heteropoly- tungstic, molybdic and tetraboric acids, ferric and chromic oxides, zinc carbonate/
ferrocyanide, charcoal, zirconium phosphate, hydrous zirconium oxide, lanthanum oxide, bentonites,
cellulose powder (including acetylated, ion exchanger), starch, sucrose, mannitol, dextran gels
(sephadex), polyamides etc. Eluotropic solvents used in column chromatography, in order of
increasing polarity are petroleum ether, n-hexane, cyclohexane, carbon tetrachloride, trichloro-
ethylene, benzene, dichloromethane, chloroform, diethyl ether, diethyl formamide, ethyl acetate,
pyridine, n-propanol, ethanol, methanol, formamide and water. Polarity and composition of solvents
is depending upon the nature of compound to be eluted i.e. non-polar compounds elutes with non-
polar solvents whereas polar compounds elutes with polar solvents. Polarity is changed by observing
the mobility of the compounds to be elutes. Some solvent systems are commonly used for the
separation of alkaloids are as follows;
Petroleum ether- chloroform (1-100%), n-hexane- chloroform (1-100%), benzene-
chloroform (1-100%), methanol- chloroform (1-100%), methanol- ethyl acetate (1-100%), pet. ether-
ethyl acetate- diethylamide (5:1:0.3-20:1:0.3)

d. Gas liquid chromatography (GLC): Gas chromatography separates volatile substances by

percolating a gas stream over a stationary phase. It can be applied to almost any type of compound
25
which has reasonably volatility and stable under the chromatographic conditions. GLC is the most
selective and versatile form of gas chromatography, since there exists a wide range of liquid phases
usable upto 4500C. This method is applicable for the examination of alkaloids of opium, tobacco,
coniine and belladonna. Modification of the sample structure may be accomplished by derivatisation
and the most widely applied general derivatisation is silylation. The compound containing labile
functional groups viz. alcohols, amines, carboxylic acids and thiols are converted into corresponding
silylamines, esters and thioesters. The common derivatising agents used in GLC are
trimethylchlorosilane, hexamethyldisilazone and N, O-bis-trimethylacetamide.

e. High performance thin layer chromatography (HPTLC): This method is very useful in
both qualitative and quantitative analysis. It is a major advancement of TLC, which requires shorter
time, saving reagents and better resolution. The basic difference between conventional TLC and
HPTLC is only in particle and pore size of the sorbents. The linear development method is most
familiar technique in HPTLC. The analytical profiles for tropine alkaloids and several other natural
products i.e. flavonoids and steroids have been developed using this technique. HPTLC is
conveniently applied for compilation of profiles pertaining to varied range of bioactive constituents
such as berberine, quinine, opium alkaloids, colchicines, chelidonine, sanguinarine, serpentine,
raubasine, asarone, elemicine, eugenol, thymole coumarine, pulegon, flavones, diosgenin, silymarin,
catechin, anthraquinone derivatives, gibberellins, antibiotics and number of other compounds of
natural origin. Using external standards, the detection limits of bulaquine, chloroquine, and
primaquine by HPTLC (Saxena et al., 2003) on silica plates were 0.25, 0.59 and 0.53 µg respectively
while the lower limits of quantification were 0.52, 1.21 and 1.07 µg respectively with detection
wavelength at 254 nm. The separation was achieved on precoated Silica gel 60 F254 (20×20 cm, 0.2
mm) plates using n-hexane/ diethyl ether/ methanol/ diethylamine (37.5:37.5:25:0.5, v/v) as solvent
system. HPTLC is a sophisticated and automated form of TLC.

f. High performance liquid chromatography (HPLC): In HPLC method, the separation of

one component from another is based upon their absorption of UV light and the functional group
interaction with the analytical column substrate. The amount of caffeine in a sample of coffee may
be determined to a high degree of accuracy using HPLC techniques. It has become the most
versatile, safest, dependable, fastest and sensitive chromatographic technique for the quantitative and
qualitative analysis of natural as well as synthetic compounds. The alkaloids like morphine,
papaverine, codeine, emetine, antibiotics, ergot alkaloids etc. can be analysed by HPLC. Since the
26
time of its invention in 1966 by Horvath and Lipsky, several natural products including several
antibiotics (Cooper et al., 1973; Buhs et al., 1974; Wold et al., 1977) has been determined
qualitatively as well as quantitatively successfully. Chloroquine, primaquine and bulaquine has
resolved (Saxena et al., 2003) with the help of HPLC without no interference with retention times of
4.22, 5.72 and 17.26 min respectively by using RP C8 lichrospher 25 x 0.4 cm, 5µ column and the
solvent was acetonitrile: sodium acetate buffer (pH 5.6) in ratio of 55:45 with detection wavelength
265 nm. The profile of berberine bridge enzyme catalyzed transformation of (S)-reticulin to (-)-
scoulerine (Winkler et al., 2006) was carried out at four different time to determine the reaction
progress. The series of common solvents used in this technique with relative decreasing order of
polarity is acetic acid, ethylene glycol, glycerol, formamide, acetic anhydride, furfural, acetonitrile,
methanol, ethanol, dimethyl formamide, propanol, iso-propanol, methyl ethyl ketone, butanol,
cyclohexanol, pyridine, diethyl sulfoxide, methyl acetate, ethyl acetate, dioxane, tetrahydrofuran and
chloroform.

ROLE OF ALKALOIDS IN PLANTS

The functions of alkaloids in plants are mostly unknown and their importance in plant
metabolism has been debated. A single plant species may contain over one hundred different
alkaloids and their concentration can vary from a small fraction to as much as 10% of the dry
weight. Most alkaloids are very toxic and therefore, have the potential function in the chemical
defence arsenal of plants against attack by herbivores and micro-organisms (Levin, 1976; Levin and
York, 1978). For example, nicotine present in tobacco leaves inhibits the growth of tobacco
hornworm larvae. Nicotine in pure form is also applied as an effective insecticide in greenhouses. In
addition, alkaloids have been suggested to serve as a storage form of nitrogen or as protectants
against damage by ultraviolet light. Some Phytochemists suggested that alkaloids are by-products of
normal plant metabolism. It has been suggested that alkaloids may have a role in the defence of the
plant against singlet oxygen (1O2), which is damaging to all living organisms and is produced in
plant tissues (Larson and Marley, 1984; Larson, 1988) in presence of light. It is also thought that
alkaloids may provide a means of defence against insects and animals. Alkaloids may also be a
reservoir (Wink and Michael, 1999) for molecules that plants often use.
BIOSYNTHESIS/ BIOGENESIS OF ALKALOIDS
The molecular structure of the alkaloids along with other substances occurring with them led
to some formulations by which the biosynthesis of most of the alkaloids is studied. Biosynthesis of

27
different group of alkaloids has now been investigated to some extent using precursors labeled with
radioactive atoms. Ornithine is precursor or starting compound of the synthesis of tropine alkaloids
(Lounasmaa and Tamminen, 1993; Patron, 2000) and the intermediate produced during this
synthesis is methylornithine (Fig. 7). Tryptophan is starting compound for the biosynthesis of
quinoline alkaloids (Fig. 8) (Isaac, 1987). Phenylalanine is stating product in most of the amino acid
derived alkaloids like ephedrine (Fig. 9). Tyrosine (Fig. 10) is the starting product of a large family
of alkaloids including isoquinoline (Guinaudeau and Brunetonium, 1993; Hansine, 1984). The first
important intermediate is dopamine which is the starting product of the biosynthesis of colchicine
and related alkaloids (Fig. 11). Tryptophan and mevalonic acid are starting precursors in the
biosynthesis of indole alkaloids (Fig. 12). The biosynthetic pathways for pharmacognostically
important alkaloids are given in Fig. 7-12.

COOH COOH COOH

CHO
Methylation Decarboxylation NH2 Oxidation
NH2 NH2 NH2

NHCH3 NHCH3
NH2 NHCH3 NH2
Ornithine N-methylputrescine 4-Methylaminobutanal
δ -N-methylornithine N-methylputrescine

COOH
Acetoacetic acid COOH
Dehydrogenation
NCH3 CO N+CH3X-
N+CH3 CO
H3C
H3C NH2
∋
Hygrine N-methyl- ∆ -pyrroliniumsalt
NH2

Putrescine
Aldole condensation

Reduction
NCH3 O NCH3 OH

Tropinone Tropine

Fig. 7. Biogenesis of tropine and pseudotropine alkaloids

28
 COOH

O HO
N
NH2 N
N
N H
H N
H R
Tryptophan

N
N
OH Cinchoninone
Corynantheal CHO R=OCH3=Quinidine
R=H=Cinchonine

Geraniol

N H
HO N
HO H

R=OCH3=Quinine N
R=H=Cinchonidine
Cinchonidinone

Fig. 8. Biogenetic pathway of quinoline alkaloids

CH3
CH3COCOOH CO2 + H2O

1-phenylpropan-1,2-dione
CH2
CHO COOH

CHNH2

COOH H OH
H OH CH3
Phenylalanine
CH3
NH2
H O
NHCH3
H
CH3
(+)-Norephedrin
(+)-Pseudoephedrin
NH2
H

HO H HO H
Cathinone
CH3 CH3

NHCH3 NH2
H H

(-)-Ephedrin (-)-Norephedrin

Fig. 9. Biogenesis of amino alkaloid (ephedrine and related alkaloids)

29
 HO
CH2CH2NH2
HO HO
H3CO

HO NH NH
Dihydroxyphenylethylamine HO N
HO
COOH H3CO
HOOC
CH2 COCOOH HO HO
H3CO
NH2
HO

Tyrosine HO HO
H3CO
Norlaudanosoline carboxylic acid Norlaudanosoline
HO Papaverine

OH
Dihydroxyphenyl pyruvic acid

O
HO H
H3 CO
H3CO O
H3CO

NCH3 NCH3
NCH3 HO O
NCH3
H
O
H
H CO
H HO OR OCH3
HO
HO

H3 CO R=CH3=Narcotine
H3CO R=H=Narcotoline OCH3
H3CO Reticuline
Salutaridinol I
Salutaridine

HO
HO
H3 CO
O

NCH3
NCH3
NCH3
NCH3
O H
O H
O H
O H

HO
H3 CO
H3 CO
H3 CO Morphine
Codeine
Thebaine
Codeinone

Fig. 10. Biosynthesis of isoquinoline alkaloids

H3CO
OH

NH.CO.CH3
HO HO
NCH3
HO H3CO

OCH3
H3CO
NH2
CH2 HO OCH3 O
Dopamine H3CO OH
CHNH2 OCH3
Autumnaline
Colchicine
COOH
Dihydroxyphenylalanine

Fig. 11. Biosynthesis of colchicine and related alkaloids

30
 N
H3CO N
H

OCH3

H3COOC OCO OCH3

OCH3
Reserpine OCH3

COOH

NH2
H3C OH
N
H
N+ N
Tryptophan
N
H
COOH
CH3 O
OH O
Mevalonic acid
Strychnine
NH2
O
N
H3COOC H

Tryptamine
Serpentine

Ergot alkaloids

Dimethyl tryptophan Dimethyl allylpyrophosphate

HOOC
NH

Chanoclavine I Agroclavine Elymoclavine N

CH3 Lysergic acid

Fig.12. Biosynthesis of indole alkaloids

CHARACTERIZATION OF ALKALOIDS
Spectroscopic studies have revealed important informations regarding the structure of
alkaloids. UV, IR, NMR, MS including X-ray crystallography and ORD methods played a
significant role in the structure elucidation of natural products. Various modifications of NMR and
MS experiments have become of growing importance for the structural characterization of alkaloids.

UV-Visible spectroscopy
This valuable tool is most frequently used in the identification of structure, especially the
chromophore groups including conjugated double bonds present in the molecule. This analytical
method is based upon measurement of light absorption by substance in the wavelength of 190-
800nm. The region from 190 to 380 nm is known as UV region whereas upto 800 nm (some times
900 nm) is known as visible region. A variety of natural products of pharmaceutical significance

31
have been analysed and studied by UV-VIS spectroscopic technique. Some of the typical examples
are summarized in Table 3.

Table 3. Absorption maxima of some common alkaloids

S. No. Alkaloid Wavelength (λmax) in nm

1 Lobeline 249
2 Reserpine 268, 390 (with sodium nitrite in dilute acid)
3 Vinblastine 267
4 Vincristine 267
5 Tubocurarine chloride 280
6 Morphine 286, 442 (after nitroso reaction)
7 Colchicine 350
8 Ergot (total alkaloids) 550 (with p-dimethylaminobenzaldehyde
reagent), 532 (with vanillin in concentrate HCl)
9 Tropic acid, esters of 555 (with fuming nitric acid in methanolic KOH)
hydroxytropanes
10 Cardioactive glycosides 217, 590 (after Keller-Kiliani reaction)
11 Vaniline 301
12 Anthraquinone 505 (after treatment with alkali)
13 Indole chromophore 224, 277 (Mukhtar et al., 1997)
14 Aporphine 282, 312 (Castro et al., 1986)

Infra-red spectroscopy
IR spectrometric studies have proved a very important analytical tool for establishing the
presence of certain functional groups in alkaloids. IR spectroscopy is the study of the reflected,
absorbed or transmitted radiant energy in region from 0.8 to 500 µm. A more commonly used
measurement for IR spectroscopy is the frequency, expressed in wave number. The quantitative
analysis of alkaloids, quinine and strychnine (6.20 and 6.06 µ) is also possible. FTIR is a new
method of spectroscopy which has come into use more recently. It is especially useful for examining
small samples and for taking the spectrum of compounds produced in the out flow of a
chromatograph. The regions in which functional groups absorb are summarized in Table 4.
32
 Table 4. IR absorption regions of some groups

S. No. Functional Appearance of S. No. Functional groups Appearance of

groups band (cm-1) band (cm-1)
*
1 C-H (str.) 3300-2800 6 C-N (aliphatic) 1210-1200
C-N (aromatic) 1350-1250
2 O-H (str.) 3700-3350 7 C≡C, C≡N (str.) 2350-2000
3 C-O (str.) 1280-1000 8 C=C, C=N, N=O (str.) 1640-1500
and N-H (bend.)
4 C=O (str.) 1950-1640 9 Double bond (bend.) 950-750
#
5 N-H (str.) 3500-3300 10 Other str., bend. and 1400-400 (finger
combination bands print region)
*Saturated acyclic and cyclic hydrocarbons C-H (str.) = 2960-2850, C-H (in plane bend.) = 1470-1360; Unsaturated olefinic C-H (str.) =
3090-3000; Unsaturated acetylenic C-H (str.) = 3300-3270; Aromatic C-H (str.) = 3100-3000, C-H (out of plane bend.) = 900-650.
#NH ≈ 3200; -NH ≈ 2700; >NH ≈ 2000
3 2

Fluorescence spectroscopy
All the molecules absorb light usually over a specific range of wavelength and many of them
re-emit such radiations. This phenomenon is called as luminescence. When the re-emission of
absorbed light lasts only whilst the substance is receiving the exciting rays, the phenomenon is called
as fluorescence. The leaf and root of belladonna, wild cherry bark, gambier catechu aloes, jalap, etc.
show fluorescence in visible range. The UV light obtained from mercury vapour lamp or tungsten
produce fluorescence in many natural products, which do not visibly fluoresce in day light.
Fluorescence produced by a compound in UV light for quantitative evaluation is possible with the
help of an instrument spectrofluorimeter. Sennoside, extracts of rhubarb, berberis, and cinchona
alkaloids may be evaluated by this technique. Under fluorescent light alkaloids or source of
alkaloids are evaluated qualitatively and shows fluorescence is summarized in Table 5.

Table 5. Color of fluorescence shown by some common alkaloids and other sources

S. No. Alkaloid or source of alkaloids Color of fluorescence

1 Cinchona bark Luminous yellow and light blue
2 Calumba Yellow, dark green (phloem and cambium)

33
 3 Hydrastis rhizome Golden yellow
4 Rheum emodi Brown
5 Rheum rhaponticum Violet
6 Ergot powder Red
7 Olive oil Deep golden yellow
8 Arachis and sesame oils Blue
9 Berberis Yellow
10 Gentian Light blue
11 Aconitine Light blue
12 Emetine Orange
13 Precipitated chalk Greenish-violet

Nuclear Magnetic Resonance Spectroscopy

NMR spectroscopy was discovered in 1946 and after a number of modifications, it has been
routinely applied in organic chemistry since 1960. It is an extremely important tool for elucidation of
molecular structure, especially the stereochemistry and configuration of chemical compounds. This
technique reveals the position and environment of proton and carbon (some times other nuclei i.e.
phosphorus, fluorine etc.) in a complex molecule. It is an excellent choice for qualitative and
quantitative analysis of new compounds isolated from natural sources. The advent of 1D and 2D
techniques as DEPT, COSY, NOESY, 2D J-resolved, one-bond and long range hetero-
COSY/COLOC, 2D-INADEQUATE, HOHAHA etc. (Rahman, 1986; Rahman, 1989) have provided
the organic chemist with new powerful tools for structure elucidation of complex natural products.
Inverse measurement techniques such as HMBC and HMQC have allowed substantial enhancements
of sensitivity in the heteronuclear shift correlation experiment. A number of homonuclear and
heteronuclear 2D NMR techniques have been developed for identification of alkaloids, for example
anopterine alkaloid (John et al., 1985) was identified on the basis of modern 2D NMR analysis.
Rahman and Qureshi, (1990) summarized a review on the application of modern 2D experiments for
structure elucidation of various new alkaloids. The δ values for any particular compound show a
discrepancy with deuterated solvent to solvent (Breitmaier and Voelter, 1987; Derome, 1988;
Williams and Fleming, 2004). 1H and 13
C NMR chemical shifts (δ values) of some alkaloids and
heterocyclic compounds are given in Fig. 13.

34
 7.2, 122.3 N
N 6.1, 107.7
N 7.1, 121.4
7.7, 136.2 6.6, 118.0 8.5, 155.4
8.6, 156.4
N N 9.2, 158.0
H H N N

135.5
128.0
7.7, 126.2 7.5, 120.2
7.5, 135.7 7.7, 127.6 8.0, 135.7
7.6, 130.1 8.5, 142.7
7.1, 123.6 7.3, 120.8
7.4, 126.3 7.5, 127.0 N

8.5, 149.8 7.6, 129.2 8.8, 150.0 7.9, 127.3 9.1, 152.5
N N
8.0, 130.2 128.5

6.29
HO 113.0
6.40
127.6 139.8 120.9 7.42
2.27 7.88 123.5
142.5 134.0 129.3 36.1 136.9
7.5, 120.5
2.91 8.59
34.1 N 3.53
6.45, 102.1 1.75 64.8 148.8
6.9, 121.7 O 2.24 2.25
40.6 27.8 48.2 51.4 138.1 N
7.1, 119.6 7.26, 124.1
2.76 N 1.88 8.75
N 4.30 2.49 1.59 149.8
7.4, 111.0 H 60.0 30.0
88.1 38.3 20.6

135.5 5.59 2.27

HO 4.19 131.7 37.2
71.6 5.59
131.7

3.67 O 176.0 O
50.7
7.34 1.84 28.8 2.25 7.79
44.9 2.91
126.9 2.44 66.8 4.60 41.3
H
N O 167.0 O 63.6 2.87
7.16 136.7 1.50
5.76 142.2
123.8 46.7 28.6 59.4 1.55 22.3 52.8 H 100.3
3.90 3.03 1.88 1.55 8.0
130.5 NH
7.11 46.2 31.2 25.1 N
125.9 142.8 2.27
139.0 N 2.11 7.99 7.99 164.4 151.5
2.40 20.2 2.24 34.0
7.02 129.7 129.7
120.1 35.9 3.05 H 5.51 55.5 O N O
169.9 118.3 7.37 7.37 H
73.9
128.4 128.4 10.0
O O 4.04 7.46
2.36
37.2 63.8 132.8

Fig. 13. 1H and 13C NMR values for some common nucleus

35
One Dimensional NMR Spectroscopy

1
H NMR Spectroscopy
1
H NMR technique gives the information about the position, nature and numbers of protons
present in molecules. 1H NMR spectrum can distinguish protons of different chemical environments.
The spectrum shows direct proportionality between peak area and the number of nuclei responsible
for the peak which is important for quantitative determination of any sample. This technique can also
be employed for the determination of total concentration of a given kind of magnetic nuclei in a
sample. It is widely used for determination of aspirin, phenacetin and caffeine for commercial
analgesic preparations. In the structure determination of alkaloids, NMR spectrum shows clear
appearance for aromatic hydroxyl (δ 8.0-10.5), aliphatic hydroxyl (δ 4.4-5.2), methoxyl (δ 3.5-4.3),
N-methyl (δ 2.3-3.4), imine (δ 7.5-10.0), methylenedioxy (δ 5.8-6.2), aliphatic cyclic proton in close
proximity to heteroatom (δ 2.5-3.2) and anomeric protons in case of alkaloid glycosides (δ 4.2-5.1)
(Tanahashi et al., 2000; Kshiwaba et al., 2000).

13
C NMR Spectroscopy
13
C nucleus is magnetically active like to 1H nucleus having a spin number of ½. Since the
natural abundance of 13C is 1.1% to that of 12C and its sensitivity is only about 1.6% to that of 1H.
13
Therefore this method is very sensitive for structure elucidation. C NMR spectrum with its wide
chemical shift range about δ 220 ppm (down field to TMS) has proved to be distinctly advantageous
in structure elucidation of natural products (Wehrli and Wirthlin, 1976; Wehrli et al., 1989).
Different alkaloids (Ramirez et al., 2003; Chen et al., 1997; Crabb, 1978; Levy et al., 1980)
13
furnished characteristic peaks in C NMR spectrum for carbonyl (δ 162-178), methoxyl (δ 51.5-
58.2), oxygenation substituted aromatic carbon (δ 145-160), methylenedioxy (δ 98-104) and N-
methyl (δ 25-45).

Distortionless Enhancement by Polarization Transfer (DEPT)

This method is most efficient for the peaks assignments, whereby the peaks can be classified
as representing methyl (CH3), methylene (CH2), methine (CH) or quaternary carbon (C). The novel
feature in the DEPT sequence is a variable proton pulse that is set at 450, 900 or 1350 in three
separate experiments. The signal intensity at a particular time for each of the three different pulses
depends on the number of protons attached to a particular carbon atom. A separate sub-spectrum can

36
be recorded for each of the CH3, CH2 and CH groups. In the DEPT spectrum, the multiplet
components are not distorted, therefore leading itself to decoupling. This technique is therefore
provides an alternative means for multiplicity determination (Doddrell et al., 1982; Bendall et al.,
1982; Bendall et al., 1981). DEPT is usually being the method of choice when polarization transfer
is required.

Insensitive Nuclei Enhanced by Polarization Transfer (INEPT)

1D INEPT technique has proved to be a powerful tool for unambiguous carbon spectral
assignment and also provides the information about critical carbon framework. Morris and Freeman,
(1979); Cordell, (1991) used Selective INEPT spectroscopy (2D) for the multiplicity selection,
spectral assignment and structure elucidation in some alkaloids. Bolcskei et al., (1994) applied the
INEPT for structure elucidation of biologically active alkaloid, Catharanthine along with other 2D
NMR technique. INEPT spectral analysis was used for determination of multiplicity in all carbon
signals of dihydrochelerythrine, (±)-evodiamine and zanthobungeanine alkaloids (Joshi et al., 1991)
for structure elucidation.

Two Dimensional NMR Spectroscopy

1
H-1H Correlation Spectroscopy (COSY)
2D NMR spectra provide more information about the chemical compounds particularly that
are too complicated to work with using one-dimensional NMR. COSY was first discovery in the
field of 2D experiments. It was proposed by Jean Jeener, Professor at Université Libre de Bruxelles,
in 1971. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and Richard R.
Ernst, who published their work in 1976 (Martin and Zekter, 1988). A 2D experiment which
indicates all the spin-spin coupled protons in one spectrum is called 1H-1H Correlation or COSY
spectrum. In the general case, COSY spectra are interpreted as giving rise to off-diagonal or cross
peaks for all protons that have significant J-J coupling. The cross peaks correlate coupled protons. In
other words the experiment can be thought of as simultaneously running all pertinent decoupling
experiments to see which protons are coupled to which other protons. In this method two identical
chemical shift axes are plotted orthogonally. All peaks that are mutually spin-spin coupled detected
as cross peaks and symmetrically placed about the diagonal. Csupor et al., (2004) established the
structure of acovulparine a norditerpene alkaloid by using 1H-1H-COSY experiment. In (+)-

37
sebiferine (Fig. 18a) (Bartley et al., 1994), an aliphatic ABC coupling system was evident involving
the methylene hydrogens at δ 3.08, 3.38 and the methine hydrogen at δ 3.73; it was confirmed by
observation of the expected cross-peaks in the COSY spectrum. Hughes et al., (1989) elucidated the
structure of two lycopodium alkaloids, annotinine and annotine with the help of COSY experiments,
geminal and vicinal coupling between protons in their structures was also discussed.

Nuclear Overhauser Effect (NOE)

The interaction of one magnetic nucleus with another leading to spin-spin coupling takes
place through the bounds of the molecules. NOE is useful for detection of groups which are close in
space to each other and can provide the information about stereochemistry (Noggle and Schirmer,
1972) of molecule. The stereochemistry of cephamonine (Fig. 14a) and cephamuline (Fig. 14b) for
H-14 was confirmed by NOE correlation and observed that in previous case H is α-oriented while in
later case it is β-oriented (Kashiwaba et al., 1994). The α-, β-orientation of hydrogen in (-)-
xylopinine (Fig. 14c) (Patra et al., 1987) was also confirmed by this technique.

H H H
H H
H3CO H
H3CO H H3CO H H
H N H
H H H3CO
H HO H H H H
HO H H H H H H
H H H H
H N CH3 H
N CH3 H HH
H HH H OCH3
O OCH3 O OCH3
OCH3
OCH3 OCH3

b. Cephamuline c. (-)-Xylopinine
a. Cephamonine

Fig. 14. NOE crrelations

Nuclear Overhauser Enhancement Spectroscopy (NOESY)

A 2D spectrum which records all the 1H-1H NOE occurring in a molecule in a single
experiment is called NOESY spectrum. Superficially, it appears like a 1H-1H COSY spectrum.
NOESY spectrum provide crucial information about the geometry of the molecules. Joshi et al.,
(1994) established the structure of septatisine, a diterpenoid alkaloid, isolated from the roots of
Aconitum septentrionale with the help of NOESY experiments. The actual configuration (α-
orientation) of OH group in isoprostephabyssine (Fig. 15b) (Zhang and You, 2005) has been
38
significantly determined by NOESY spectrum. This compound is stereoisomer of prostephabyssine
(Fig. 15a) that is different only in the orientation of OH group (β-orientation). These compounds
verified the configuration of H-10, correlated with the N-methyl in prostephabyssine while no such
correlation was observed in another case because of α-orientation of OH group. The structure of
azaanthracene alkaloid (Fig. 15c) (Roumy et al., 2006) was confirmed with the help of NOESY
spectrum which showed all 1H-1H correlation present in its structure.

Heteronuclear Overhauser Enhancement Spectroscopy (HOESY)

This is an important method utilized for structure elucidation and conformational analysis of
small organic molecules (Yu and Levy, 1984; Rinaldi, 1983) as well as large bioactive compounds
such as alkaloids and other natural products (Niccolai et al., 1985; Niccolai et al., 1984). In order to
ensure that the magnetization transfer occurs via a dipolar mechanism, the HOESY pulse sequence
effectively eliminates scalar coupling between 1H and 13
C spins. A study of dipolar interactions
between quaternary carbons and protons was carried out significantly for several natural compounds
such as camphor (Yu and Levy, 1984), rifamycin S (Niccolai et al., 1985; Niccolai et al., 1984) and
valinomycine (Khaled and Watkins, 1983) with the help of 2D HOESY.

H H
H3CO H H3CO H H OCH3 CH3
OH H H H
HO HO
10 H OH
CH3 N CH3 H3CO N O
N
OCH3 OCH3 H
H O H O
OCH3 OCH3

c. Azaanthracene
a. Prostephabyssine b. Isoprostephabyssine

Fig. 15. NOESY correlations

39
Heteronuclear Correlation Spectroscopy (HETCOR)
HETCOR or 1H-13C COSY experiment correlates 13C nuclei with differently coupled protons
which is also known as one-bond (1JCH) coupling (Derome, 1988; Williams and Fleming 2004).
Joshi et al., (1997) elucidated the structures of various partially demethylated products from some
aconitine-type norditerpenoid alkaloids named aconitine, cammaconine, delphinine, falconerine,
lappaconitine, and talatizamine on the basis of HETCOR experiments. Demethylation process was
carried out with trimethylsilyl iodide and HBr in glacial acetic acid.

Incredible Natural Abundance Double Quantum Transfer Experiment (INADEQUATE)

13
This is useful technique to show direct C-13C correlations or connections in a compound
through a 2D spectrum. This technique is highly sensitive since the natural abundance of 13C is only
13
1%, the probability of finding one C attached to another is only 1 in 104. This technique is finds
applications only where either extremely concentrated solution of sample or the molecule is enriched
in the 13C isotope.

Abundance Double Quantum Transfer Experiment (ADEQUATE)

This technique is used for the determination of direct correlations between 1H and 13C nuclei
in an organic compound. The structure of azaanthracene alkaloid (Fig. 16) (Roumy et al., 2006) was
confirmed with the help of ADEQUATE spectrum which showed all 1H-13C correlation present in its
structure.

H OCH3 CH3 OCH3

H H
HO

H O
H3CO
H3CO N O H
N
OCH3 OCH3 H H3CO

Fig. 16. ADEQUATE correlations in azaanthracene Fig. 17. Incartine

40
Heteronuclear Single Quantum Coherence (HSQC)
HSQC experiments showed 1H-13C correlation using pulse sequence in spinning nuclei. The
structure of four alkaloids 16,17-dihydro-17β-hydroxy isomitraphylline; 16,17-dihydro-17β-hydroxy
mitraphylline; isomitraphylline and mitraphylline (Pandey et al., 2006) were elucidated with the help
of HSQC along with other 2D techniques. The structure and stereochemistry of lymphostin alkaloid
(Aotani et al., 1997) was determined by this method an elucidated as pyrrolo[4,3,2-de]quinoline.

Heteronuclear Multiple Quantum Coherence (HMQC)

13
In this technique the correlation is achieved between C and 1H (Martin and Zekter, 1988).
Zellagui et al., (2004) have successfully elucidated the structures of lupanine and S-calycotomine by
using HMQC experiments. The 1H-1H-COSY, HMQC and HMBC experiments established geminal
and vicinal hydrogen interactions as well as direct (1JCH) and two and three bond correlations
between carbon and hydrogen atoms in the structure of a natural quaternary alkaloid, N-
methylvoachalotine (Medeirosa et al., 2001). HMQC experiments of incartine alkaloid (Fig. 17)
isolated from Galanthus elwesii (Berkov et al., 2007) showed clear multiplicity for each carbons and
correlation of these carbons to their respective proton. In its spectrum, three quartate signals at δ
59.2, 56.1, 56.2 for the methoxy groups, three triplets at 56.7, 30.1 and 53.0 corresponding to the
methylene carbons, two doublets at 110.7 and 107.6 assigned to the aromatic carbons, five singlets at
66.9, 128.5, 147.8, 147.9 and 127.9 assigned to the quaternary carbons and five doublets at 67.7,
78.2, 59.0, 61.3, and 40.1 corresponding to the remaining carbons in the structure.

Heteronuclear Multiple Bond Correlation (HMBC)

HMBC is also known as long-range 1H-13C COSY in which a time delay in the pulse
1/2
sequence is set to correspond to J where J is in the region of 10 Hz. Since many two bond and
13
three bond coupling constant are rather similar in value and lie in the range of 2-20 Hz, then C
chemical shifts correlated with the chemical shifts of those protons separated from them by two and
three bonds. The relative positions of the methoxy substituents and the isolated unsaturated
hydrogens in the structure of sebiferine (Fig. 18a) were confirmed by multiple bond correlation
spectroscopy using the HMBC sequence (Bartley et al., 1994). Wansi et al., (2006) furnished the
exact structure of oriciacridone A (Fig. 18b) by using long-range 1H-13C correlation. Chemical
structure and the relative stereochemistry of alkylated piperazine alkaloid named 3,5-diisobutyryl-6-
isopropyl-piperazine-2-one (El-Desouky et al., 2007) was elucidated on the basis of HMBC

41
technique. The unambiguous structure of the side-chain in furoquinoline oxogeranyl ether (Fig. 18C)
(Inada et al., 2008) was established by HMBC experiments to indicate significant correlation peaks
between H-1’ / C-2’, C-3’, C-7; between H-2’ / C-1’, C-3’, C-4’, C-10’; between H-4’/ C-2’, C-5’,
C-10’ and between H-6’/ C-5’, C-8’, C-9’. The connection of the 5-oxogeranyl side-chain to C-7 on
the furoquinoline ring was revealed by the HMBC correlation peak between H-1’ / C-7. Li et al.,
(2001) were noted the usefulness of the gradient 1H–15N HMBC NMR spectroscopy in the structure
elucidation of 3-hydroxyrutaecarpine.

H O OH

H H HH OCH3
5
H3CO N CH3 N O 8' 6' 4' 10' 6 4a 4 3a 3
H 5' 3'
7' 7 2
H OH OH N 2'
H3CO H O 1' O O
8a N 9a 1
OH 9' H 8 9
H
H O H
OCH3 O O H
a. Sebiferine H OH c. Furoquinoline-oxogeranyl ether

b. Oriciacridone A

Fig. 18. HMBC correlations

Mass Spectrometry
The mass spectrometry is an essential tool for identification of organic compound. It provides
accurate molecular weights, elemental information and fragmentation pattern, correct interpretation
of which helps to determine the structure of unknown compound. The structure elucidation of
alkaloids by mass fragmentation pattern (Biemann, 1962; Budzikiewicz et al., 1964) is easier than
the other natural products because of complexity in their structure. Qian and Zhan, (2007) elucidated
the structures of various alkaloids such as sessilifolines A, B, tuberstemonine, sessilifoliamide A and
stemoninoamide, isolated from the stems of Stemona sessilifolia with the help of simple mass
fragmentation pattern. Various techniques used under mass spectrometry for structure elucidation are
as follows;

42
Electron Impact Mass Spectrometry (EIMS)
The volatile compounds introduced by vaporization or by GC through a very small orifice
followed by ionization by electron impact, determined under this procedure. The structures of
acovulparine (Csupor et al., 2004) a norditerpene alkaloid was established by HR-EIMS. EIMS
spectrum of parvineostemonine (Ke et al., 2003; Pilli and Oliveria, 2000) suggest that it gave
molecular ion peak at m/z 275, the other intense peaks were assign at 246 (Loss of ethyl group from
M+) and 137 (perhydroazaazulene skeleton). Jones et al., (1999) proposed the fragmentation for
pyridine alkaloid 2-[3-(6-propyl-2-pyridyl)-propyl]-1,3-dioxolane (Fig. 19). Santos et al., (2003)
described the mass fragmentation in lignoaporphine alkaloid (Fig. 20) using EIMS method at 70 eV.

O
O

N N N
O N O
m/z 206 O
m/z 190 m/z 164 m/z 134 100%

O
O

O N
O N
m/z 73 N
m/z 234
m/z 162
m/z 106

Fig. 19. Mass fragmentation in pyridine alkaloids

O
O O
O
O N O
N OH O N
O N O O

H H

H3CO OCH3 m/z 286 (26%)

m/z 316 (96%) m/z 316
OH
m/z 469

Fig. 20. Mass fragmentation of lignoaporphine alkaloid

Chemical Ionization Mass Spectrometry (CIMS)

The vaporized sample is introduced into the mass spectrometer with an excess of a reagent
gas (commonly methane) at a pressure of about 1 torr. In these spectral experiment the [M+H]+ ions
(quasimolecular ions) are often prominent (Silverstein and Webster, 1998) Mass spectra of several
Erythrina alkaloids (Boar and Widdowson, 1970) and their derivatives, arundamine and arundanine
(Khuzhaev, 2004) were recorded by using CIMS method. Jones et al., (1999) determined the

43
structure and chemistry of several quinoline and dihydroquinoline of animal origin and proposed a
mechanism of mass fragmentations in qinolizidine (Fig. 21).

CIMS
N N N
N NH3
H H H

m/z 195 m/z 196 m/z 98 m/z 126

retro Diels-Alder

N N

α−cleavage

m/z 152 (100%) m/z 124

N McLafferty
m/z 195 N

H m/z 138

m/z 180 (5%)

Fig. 21. Mass fragmentations of quinolizidine

Field Desorption Mass Spectrometry (FDMS)

This method is used for non or low volatile and thermally labile substances. Usually the
major peak represents the [M+H]+ ion. For molecular weight determination of large and polar
substances, a small amount of cation donor reagents (Prome and Puzo, 1977) is added to the sample.
Some times due to inorganic impurities in sample produce most intense [M+Na]+ and [M+K]+ peaks.
Ahmad et al., (2002), reported the fragmentation pattern in piperidine alkaloids isolated from
Andrachne aspera with the help of EI/FD/HRMS experiments. They proposed a suitable rout for
fragmentations in aspertin B is shown in Fig. 22.

HO H H OH
HO H H OH
N
N
H
H m/z 212.16
m/z 255.22

HO H
HO H
N
N
N H
H
H m/z 182.15
m/z 168.14
m/z 79.04

Fig. 22. Fragment ions in FDMS/HRMS of aspertin B

44
Fast Atomic Bombardment Mass Spectrometry (FABMS)
Polar molecules such as peptide and several others with molecular weights upto 10,000 Da
can be analyzed by this soft ionization technique. The bombarding beam consists of xenon or argon
atoms of high translational energy. This beam is produces by first ionizing xenon atoms with
electrons to give xenon radical cations. The sample compound is dissolved in a high boiling viscous
solvent such as glycerol, a drop is placed on a thin metal sheet and the compound is ionized by the
high energy beam of xenon atoms. Although the molecular ion itself is usually not seen but adduct
ions such as [M+H]+ are prominent. Other adduct ions can be formed from salt impurities or upon
addition of salts such as NaCl or KCl, which give [M+Na]+ and [M+K]+ additions. FABMS requires
a more simple instrumentation (Fenselan, 1978) which is easy to operate and has been used more
frequently. Belferdi and Belattar, (2001) suggested a suitable rout for mass fragmentation pattern in
oxoaspidospermidine-N-oxide (Fig. 23) in which all the rearrangements in the molecule are shown.

N
N

N N

N
N
H O m/z 124
H m/z 138 (100%)
m/z 296 m/z 268

N
N HN

H
N
N m/z 110 m/z 130
H
H
m/z 239
m/z 268

Fig. 23. Mass fragmentation ions of oxoaspidospermidine-N-oxide

Electrospray Ionization Mass Spectrometry (ESIMS)

This method involves placing an ionizing voltage of several kilovolts across the nebulizer
needle attached to the outlet from a HPLC. This technique is widely used on water soluble molecules
such as carbohydrates, protein and peptide. Ergot alkaloids (Lehner et al., 2004) ergovaline (m/z
534), ergotamine (m/z 582), ergocornine (m/z 562), ergocryptine (m/z 576) and ergocrystine (m/z
610) exhibited a consistent loss of water (-18 u) from the C-12' α-hydroxy functionality. Of this
group, ergovaline and ergotamine generated an m/z 320 fragment deriving from cleavage of ring E

45
amide and ether functions with retention of the peptide ring system methyl group. Ergocornine,
ergocryptine and ergocrystine similarly formed an m/z 348 fragment with retention of isopropyl.
Fragmentations of aporphine alkaloids (Caroline et al., 2004) by electrospray ionization with
multistage mass spectrometry (ESI-MSn) in positive mode showed the loss of the amino group and
its substituents in first step. Further steps display the loss of the peripheral groups, losses of
methanol and CO are observed if an OH is vicinal to an OCH3 on the aromatic ring. Otherwise the
spectra show radical losses of CH3· or CH3O· as the main fragmentations. If a methylenedioxy group
is present losses of formaldehyde followed by CO are observed. These fragmentations provide
important information on the structures of aporphine alkaloids. A typical ESI spectrum of aconitine
alkaloid (Sun et al., 2004) showed protonated molecular [M+H]+ ion and major fragment ion by the
loss of neutral molecular species of m/z 60 (CH3COOH), 122 (PhCOOH), 152, 279 etc.

Matrix Assisted Laser Desorption Mass Spectrometry (MALDIMS)

This method has been used recently in several variations to determine the molecular weight
of large molecules upto several hundreds kDa. The successful use of six beta-carbolines, harmane,
harmine, harmol, harmaline and harmalol (Nonami et al., 1997) is reported as matrices in matrix-
assisted MALDI/TOF-MS (λmax 337 nm) for proteins and sulfated oligosaccharides, using stainless-
steel probes and membranes to prepare the samples. The usefulness of the new matrices in
MALDI/TOP-MS of proteins and sulfated oligosaccharides is compared with those of classical
MALDI matrices such as alpha-cyano-4-hydroxycinnamic acid, gentisic acid, sinapinic acid, 6-aza-
2-thiothymine and 3-indole- trans-beta-acrylic acid. Direct quantitative analysis using TLC coupled
with MALDIMS has been demonstrated, an internal standard and a data collection protocol were
used for analysis directly from TLC plates to compensate for shot-to-shot signal degradation, as well
as deviations of analyte and internal standard spatial distributions within the TLC spot. Cocaine
hydrochloride (Nicola et al., 1996) was used as a model compound for this study, and cocaine itself
used as the internal standard.

46
Gas Chromatography-Mass Spectrometry (GCMS)
Gas chromatography coupled with mass spectrometry (GC-MS) has been used successfully
for the identification of tropine alkaloids (Christen et al., 1993). The capillary gas chromatography
with mass spectrometry (GC/MS) is convenient and very often used method for analysis of a
complex mixture containing pyrrolizidine alkaloids (Witte et al., 1993). GCMS spectrum of 3-α-
cinnamoyloxytropane (Fig. 24) (Munoza and Casaleb, 2003) having molecular formula C17H21NO2
exhibited the fragmentation pattern similar to 3-substituted tropane (Christen et al., 1995). The
molecular ion peak at m/z 271, a base peak at m/z 124 [M- PhCH=CHCOOH]+ along with other
strong peaks at m/z 148 [PhCH=CHCOOH]+, 140 [M-PhCH=CHCO]+, 131 [PhCH=CHCO]+, 103
[PhCH=CH]+, and 77 [C6H5]+. The similar fragmentation was found in 3-(3’-formyloxytropoyloxy)
tropane (Berkov, 2003) which showed molecular ion at m/z 317 (C18H23NO4) and most abundant ion
at m/z 124 (tropane group) together with ion at m/z 140 (M+-177), 103. The chemical profiling of
alkaloids from Bulgarian species of genus Senecio (Christova and Evstatievab, 2003) was
successfully carried out by the GC/MS analysis.

HO

O
O m/z 131 m/z 103 m/z 77
m/z 148
H3CN

H H3CN H3CN

O H

O OH m/z 124 100%

m/z 271
m/z 140
Fig. 24. Mass frgments of 3-α-cinnamoyloxytropane in GCMS

Liquid Chromatography-Mass Spectrometry (LCMS)

LCMS of any component facilitate quick characterization. The sample is dissolve in a
suitable solvent and separated its components with the help of LC and finally determined the
molecular weight and mass fragmentation in a particular component. Solid-phase extraction (cation-
exchange) of pyrrolizidine alkaloids and their N-oxides from honey samples (Kerrie et al., 2004;
Betteridge et al., 2005) was carried out by high-performance liquid chromatography-atmospheric
pressure chemical ionization mass spectrometry. Enantio-enriched building blocks (Feng et al.,

47
2006), several ephedrine type alkaloid (Gay et al., 2001; Thomas et al., 2007; Sharpless et al., 2006;
Joshi and Khan, 2005; Gray et al., 2004; Trujillo and Sorenson, 2003) were determined with the help
of LCMS using atmospheric pressure chemical ionization. Aconitum alkaloid (Hajime et al., 2001)
was analysed significantly from aconite roots with the help of LCMS technique. A semi-quantitative
LC-MS method (Beike et al., 2003) was developed for the detection of the pseudo alkaloids of Taxus
baccata. This method was used to examine the cause of death of a 43 year old man who died several
hours after he drank a decoction of taxus leaves.

X-Ray Diffraction
Many compounds are capable of crystallizing in more than one type of crystal lattice. At a
particular temperature and pressure, only one crystalline form is thermodynamically stable. It is used
to find several polymorphs of crystalline alkaloids existing under normal handling conditions.
Solubility, melting point, density, hardness, crystal shape, optical and electrical properties, vapour
pressure, etc. vary with polymorphic forms. Powder X-ray diffraction analysis is employed for the
characterization of crystalline structure. It has been used to determine the existence of polymorphic
forms of many substances such as carbon in graphite or diamond and drugs. Various other
techniques such as diffraction topography, double crystal diffractometry, interferometry, strain
measurements and texture analysis are very important techniques used for the study of crystal
perfection, structure defect, grain structure and orientation. The structure of altaconitine alkaloid
(Batbayar et al., 1993) isolated from the epigeal part of Aconitum altaicum was established on the
basis of x-ray structural method along with IR, 1H NMR, 13C NMR and MS studies.

48
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