Dr. Fatma M. Abdel Bar

Assoc. Professor of PharmacognosyPharmacognosy Department,

College of Pharmacy Sattam Bin Abdulaziz

University,

Al-Kharj. KSA.

Email: f.abdelbar

Pharmacognosy -2

PHG 322

Credits Type

15

Theoretical part(30):

Midterm I1

15Midterm II2

5Homework, Discussion, quizzes and activities3

40Final exam5

15Practical part (25):

Attendance, Continues evaluation and report1

10Practical exams3

100Total

Course Evaluation

• Definition and description of alkaloids

• Physical & chemical properties

• Pharmacological activity and uses of alkaloids

• Naming of alkaloids

• Extraction of alkaloids

• Classifications of alkaloids

Alkaloids

No definitive difference between an alkaloid and naturally

occurring complex amines.

Alkaloids: the term “alkaloid” (alkali-like) is commonly used to

designate basic heterocyclic nitrogenous compounds of plant

origin that are physiologically active.

Definition

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.

Alkaloids occur only exceptionally in bacteria (pyocyanine from Pseudomonas aeruginosa).

They are rarely found in fungi (psilocin from the hallucinogenic mushrooms of Central America, ergolines from Claviceps and other actinomycetes, sporidesmins, roquifortine, and others).

Pteridophytes rarely contain alkaloids, and among them the Lycopodiaceae represent the main exception (alkaloids derived from lysine).

The same comment applies to the Gymnosperms (alkaloids from Cephalotaxus).

Thus, alkaloids are compounds essentially found in Angiosperms, and some authors estimate that 10 to 15% of these synthesize those products.

Occurrence, distribution, Localization & Functionof alkaloids

Occurrence

Occurrence, distribution, Localization & Functionof alkaloids

Certain families have a marked tendency to produce alkaloids: this is true for the Monocotyledons (Amaryllidaceae, Liliaceae), as well as the Dicotyledons (Annonaceae, Apocynaceae, Fumariaceae, Lauraceae, Loganiaceae, etc).

Within these families, some genera produce alkaloids and others do not. Sometimes, they are found in all of the genera (Papaveraceae), although this is far less common.

Alkaloids concentrations have a wide range of variation: from ppm as in the case of the anticancer alkaloids of Catharanthus roseus to more that 15% in the bark of the trunk of Cinchona ledgeriana.

Alkaloid-containing plants rarely contain only one alkaloid: sometime they contain virtually only one constituent (e.g. hyoscyamine from the leaves of belladonnae), but, most often, they yield a complex mixture, which may be dominated by one major constituent.

It is not uncommon to find several dozen alkaloids in one drug.

Distribution

Occurrence, distribution, Localization & Functionof alkaloids

Alkaloids cont…

Localization

• In the plant, alkaloids occur as soluble salts (citrates, malates, tartrates, meconates, isobutyrates, benzoates), or in combination with tannins.

• Microchemistry shows that alkaloids are most often localized in the peripheral tissues: e.gexternal layers of the bark of the stems and roots, or seed tegument.

• Most of these compounds have a basic character and an antimetabolite activity which make their distribution necessary: they are normally stored in the cell vacuoles, which may be specialized (laticiferous) or not.

• Most often, alkaloid synthesis takes place at specific sites (growing root, laticiferous cells, and chloroplasts); the compounds are subsequently transported to the storage site.

Occurrence, distribution, Localization & Functionof alkaloids

Alkaloids cont…

Function

1. They may act as protective agents that protect the plants against insects, fungus,

predators due to their bitterness & toxicity.

2. They may be end products of nitrogen detoxification.

3. They may act as growth regulators.

4. They may act as reserve substances which can supply nitrogen when needed.

5. They may be utilized as a source of energy in case of deficiency in CO2 assimilation.

in several cases, they have been shown to act as intermediate metabolites.

<The question remains unanswered>

As in the case of many other secondary metabolites, almost nothing is known of the role of

alkaloids in plants. However, the following functions were suggested:

Naming of AlkaloidsAlkaloids cont…

Trivial names should end by "ine". These names may refer to:

1-The genus of the plant, such as Atropine from Atropa belladona.

2-The plant species, such as Cocaine from Erythroxylon coca.

3-The common name of the drug, such as Ergotamine from ergot.

4-The name of the discoverer, such as Pelletierine that was discovered by Pelletier.

5-The physiological action, such as Emetine that acts as emetic, Morphine acts as narcotic.

6-A prominent physical character, such as Hygrine that is hygroscopic.

7-Other such as morphine derived from ancient Greek mythology –Morpheus – god of dreams

Prefixes and suffixes:

Prefixes:

• "Nor-" designates N-demethylation or N-demethoxylation, e.g. nor-pseudoephedrine and nor-nicotine.

• "Apo-" designates dehydration e.g. apomorphine.

• "Iso-, pseudo-, neo-, and epi-" indicate different types of isomers.

Suffixes:

• "-dine" designates isomerism as quinidine and cinchonidine.

• "-inine" indicates, in case of ergot alkaloids, a lowerpharmacological activity e.g. ergotaminine is less potent thanergotamine.

Physical-Chemical Properties of Alkaloids

A) Physical Properties

II- Color:

The majority of alkaloids are colorless but some are colored e.g.: Colchicine and

berberine are yellow. Canadine is orange.

I- Condition:

Most of 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.

• 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 monosulphate.

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

Physical-Chemical Properties of Alkaloids

A) Physical Properties

Optically active isomers may show different physiological activities.

IV- Isomerization:

l-ephedrine is 3.5 times more

active than d-ephedrine.

l-ergotamine is 3-4 times more active than

d-ergotamine.

d- Tubocurarine is more

active than the

corresponding l- form.

Quinine (l-form) is antimalarial

and its d- isomer quinidine is

antiarrythmic.

The racemic (optically inactive) dl-

atropine is physiologically active.

Physical-Chemical Properties of Alkaloids

B) Chemical PropertiesI- Nitrogen:Primary amines R-NH2 e.g. Norephedrine

Secondary amines R2-NH e.g. Ephedrine

Tertiary amines R3-N e.g. Atropine

Quaternary ammonium salts R4-N e.g d-Tubocurarine

II- Basicity:R2-NH > R-NH2 > R3-N

Saturated hexacyclic amines is more basic than aromatic amines.

According to basicity alkaloids are classified into:

• Weak bases : e.g. Caffeine

• Strong bases: e.g. Atropine

• Amphoteric: Phenolic Alkaloids e.g. Morphine &

Alkaloids with Carboxylic groups e.g. Narceine

• Neutral alkaloids e.g. Colchicine

III- OxygenMost alkaloids contain Oxygen and are solid in nature e.g. Atropine.

Some alkaloids are free from Oxygen and are mostly liquids e.g.

Nicotine, Coniine

Physical-Chemical Properties of Alkaloids

Bitter tasting,

Most alkaloids are well-defined crystalline substances which form salts with acids.

In plants, they may exist

in the free state,

as salts or

as N-oxides

Alkaloids which do not contain O2 are liquid at room temperature (nicotine), while those that do are solids.

In rare cases they are coloured.

Most solid bases, rotate the plane of polarized light,

have high melting points.

Free bases are normally insoluble in water

(occasionally slightly soluble), soluble in a polar or

slightly polar organic solvents and soluble in

concentrated hydroalcoholic solutions.

Strychnine N-oxide

• Alkaloids are particularly interesting substances because of their multiple pharmacological activities:

• On the CNS, whether they are: depressants (morphine, scopolamine) or stimulants(strychnine, caffeine).

• On the autonomic nervous system: sympathomimetics (ephedrine) or sympatholytic(yohimbine, certain ergot alkaloids), parasympathomimetic (eserine, pilocarpine), anticholinergics (atropine, hyoscyamine), or ganglioplegics (sparteine, nicotine).

Pharmacological activity and uses of alkaloids

In addition they have the following uses: - alkaloids include curare, - local anesthetics (cocaine), - agents to treat cardiac fibrillation (quinidine),- antitumor agents (vinblastine, ellipticine),- antimalarials (quinine),- antibacterials (berberine), - amebicides (emetine).

>>>> Although some are only used as galenicals (belladonna, datura, henbane), many others are only used as starting materials for industrial extraction.

Tests for alkaloids

They give a precipitate (from neutral or slightly acidic) with heavy metal iodides. Caffeine, a purine derivative, does not precipitate like most alkaloids

Mayer's reagent (potasium mercuric

iodide solution) Cream coloured

precipitate

Wagner’s reagent (iodine in potassium

iodide) red-brown precipitate

Alkaloids cont…

Dragendorff's reagent (solution of potassium

bismuth iodide) orange coloured precipitate

Hager’s reagent (picric acid)

yellow precipitate

The specificity of these reagents is

not absolute:

proteins, α-pyrones, some

coumarins, hydroxyflavones,

lignans, and other compounds can

give false positive reactions with

Dragendorff's reagent

Extraction

Extraction method normally depends on the raw material, the purpose of extraction & the scale on which is to be performed.

For research purposes: chromatography allows for quick and reliable results.

If larger amounts of alkaloids need to be extracted, one of the following methods can be used:

Herbs often contain other materials which can interfere with

extraction such as large amounts of fat, waxes, terpenes,

pigments and other lipophilic substances (e.g. by forming

emulsions) – avoided by defatting the crushed herb (using

petroleum ether and hexane).

Alkaloids cont…

Extraction is based on the basicity of alkaloids and on the fact

that they normally occur in plants as salts (i.e. on the solubility

of bases and salts in water and organic solvents).

Column chromatography

General Method(Acid-Base extraction )

-Powdered, defatted herb is mixed with an alkalineaqueous solution.

-Free bases are then extracted with organic solvents(chloroform, ethyl acetate, alcohol).

-Organic solvent containing alkaloids (bases) is separatedfrom residue & concentrated by distillation underpressure if needed

Step I:

Alkaloids cont…

Step II:

-Solvent is stirred with an acidic aqueous solution:alkaloids go into the solution as salts. Impuritiesremain in the organic phase.

-Aqueous solution of alkaloid salts is washed with an apolar solvent (hexane)

-Alkalinize with a base, use an organic solvent which is not miscible with water.

-Alkaloids precipitate and dissolve in the organic phase.

-Extraction of aqueous phase continues till all alkaloids have moved into the organic phase (tested when Mayer’s reaction on the aqueous phase becomes negative).

-This purification step may be carried out in a separation funnel or in centrifugal extractors

Step II:

General MethodAlkaloids cont…

Step III:

Organic solvent containing alkaloid bases is decanted, freed from water traces (drying over anhydrous salt e.g. sodium sulphate) and evaporated under reduced pressure.

A dry residue remains: total basic alkaloids.

Acid-Base extraction procedure of alkaloids

Diagram for extraction of alkaloids

alcohol extraction

alkaloids

Sugars

Soluble peptides

Protein

Plant pigments

Cellulose

fibers

Extract

residue

Water layer

CHCl3 layerAll undesired materials

Alkaloids as salt

1. anhydrous sodium sulphatee

2. concentrate

Plant powder

1.Consetration

2.Desolve in dil. acid

Add Alkaline NH4OH

CHCl3 layer

Water layerQuaternary ammonium salt

Row alkaloids

Alkaloids cont…

Extraction of Alkaloids

• Process remained unchanged >1,000 years

Plant Material

Acid solution

EtOAc: neutral/weakly basic alkaloids

1) Methanol

2) Concentrate

3) Partition EtOAc / 2% acid

Petroleum ether extracts non-polar fats and waxes

Residue: polar material

Wash with petroleum ether

Basic aqueous solution of quaternary alkaloids

1) Ammonia

2) Partition with EtOAcEtOAc: basic alkaloids

their are two possible methods

1-Plant powder is extracted directly with acidified water.

2-Plant powder is extracted with acidified alcoholic or a hydroalcoholic solution. This is then followed by

distillation under vacuum (eliminates that alcohol, leaving behind and acidic aqueous solution of alkaloid salts)

Extraction of liquid alkaloids

Alkaloids cont… Specific Method

Test of Alkaloids

I-Qualitative test for Alkaloids:

They are used to:

1- Indicate the absence or presence of Alkaloids.

2- Test for complete extraction.

Disadvantages: The specificity of these reagents is not absolute:

proteins, α-pyrones, some coumarins, hydroxyflavones, lignans, and other

compounds can give false positive reactions with Dragendorff's reagent

Colour Reagents

1- Froehd’s reagent: Phosphomolybdic acid

2- Marqui’s reagent: Formaldehyde/ Conc. H2SO4

3- Mandalin’s reagent: Sulphovanidic acid

4- Erdmann’s reagent: Conc. HNO3/Conc. H2SO4

5- Conc. HNO3

Precipitation Reagents:

Mayer's reagent (potassiomercuric iodide

solution) Creamy precipitate

Hagers reagent (picric acid) yellow

precipitate

Dragendorff's reagent (solution of potassium

bismuth iodide) orange precipitate

Wagner’s reagent (iodine in potassium

iodide) red-brown precipitate

II-Quantative test for Alkaloids:

Volumetric methods:These are based on reaction of alkaloidal bases with acids (Acid-Base titration).

They include:• Aqueous titration: This is carried by either:

1- Direct titration of the alcoholic solution of the alkaloidal residue with standard acid, or2- Back titration by dissolving the residue in a known amount of standard acid and back titration of residual acid against standard alkali.

• Non-aqueous titration: This method is suitable for determination of weak bases e.g. Caffeine

Gravimetric methods:These methods are recommended for determination of:

1- Very weak bases which can not be determined by volumetric methods e.g. caffeine andcolchicine.2- Mixtures of alkaloids that are obtained from the same plant but differ greatly in theirmolecular weight e.g. Cinchona and Rawolfia alkaloids.

Colourimetric Method: e.g. Morphine + NaNO2/HCl

Ergot + p-dimethylaminobenzaldehyde

Spectrophotometric Methods.

Polarimetric Method.

Fluorimetric Method.

Chromatographic Methods.

Isolation of Alkaloids

All methods of alkaloid extraction yield impure compounds, so alkaloids therefore have to be separated.

TLC and HPLC are most commonly used.

Chromatography is used for complex alkaloids and if only small amounts of alkaloids are needed.

Large-scale extractions may be sent to a factory for purification & separation (for cinchona and cocoa alkaloids).

Separation and final purification may be done using fractional precipitation or fractional crystallization of salts.

Chromatography is used for complex alkaloids and if only small amounts of alkaloids are needed

Classification of Alkaloids

Alkaloids have a large variety in their botanical & biochemical origin, in chemical structure and pharmaceutical action. Therefore a range of different classification systems exist for grouping alkaloids.

They can therefore be classified according to:

1-General classification.

2-Biological uses.

3-Botanical Source (According to the plant source of alkaloids).

4-Type of Amines (Primary, Secondary, Tertiary alkaloids).

5-Biosynthetic or biogenetic pathway (Based on the biogenetic pathway that form the alkaloids)

6-Basic Chemical Skeleton ( 2 divisions)

• i. Atypical/non-heterocyclic alkaloids (proto-alkaloids or biological amines).

• ii. Typical/heterocyclic alkaloids (divided into 14 groups according to their ring structure).

Alkaloids cont…

Alkaloids Classification

Alkaloids cont…

1-Hagnauer system of classification

1) True (Typical) alkaloids: that are derived

from amino acids and have nitrogen in a

heterocyclic ring. e.g Atropine

2) Proto alkaloids: that are derived from

amino acids and do not have nitrogen in a

heterocyclic ring. e.g Ephedrine

3) Pseudo alkaloids: that are not derived from

amino acids but have nitrogen in a heterocyclic

ring. e.g terpenoidal alkaloids

Alkaloids Classification

2-According to pharmacological action:

Narcotics

3-Biosynthetic pathway

Alkaloids ClassificationAlkaloids cont…

Biosynthetic pathways to alkaloids

vary greatly depending on the

nitrogen source. Alkaloids are

biosynthesized from various kinds

of amino acids

1-Ornithine derived alkaloids

2-Tryptophan derived alkaloids

3-Lysine derived alkaloids

4-Phenylalanine derived alkaloids

5-Tyrosine derived alkaloids.

6-Histidine derived alkaloids

7-Alkaloids derived from

substance other than amino acids.

4-Chemical classification

i. Atypical/non- heterocyclic alkaloids (proto alkaloids or biological amines)

Alkaloids cont…

Alkaloids Classification

Terpenoids & Steroidal (Steroids, Aconite)

Tropolone(Colchicine)

phenylalkylamine(Mescaline, ephedrine, hordenine, capsaicinoids, cathinone )

ii. Typical/heterocyclic alkaloids (divided into 8 groups

according to their ring structure):

Alkaloids cont…

Alkaloids Classification

4-Chemical classification

Opium Alkaloids

Carboline

1-Imidazole: e.g. pilocarpine

2-Indole: e.g. ergometrine

3- Isoquinoline: e.g. Morphine

4-Quinoline : e.g. quinine and quinidine

5-Purine : e.g. caffeine

6-Pyridine & piperidine: e.g. lobeline, nicotine

7-pyrrolyzidine e.g. platyphylline, rosmarinine

8-Tropane e.g. Atropine.

Amino Acid Precursors

N

N

CH3

nicotine

from ornithine

NH2

OCH3

H3CO

H3CO

mescaline

fromtyrosine

N

H3C

CO2CH3

O Ph

Ococaine

from ornithine

N

O

N

O

strychnine

from tryptophan

O

HO

N

CH3

HO

morphine

fromtyrosine

N

NH

HO2C CH3

lysergic acid

from tryptophan

NH

N

HO

H3CO

H

from tryptophan

NH

N

HO

MeO2C

NMeO

N

OH

CO2CH3

O

O

R

R= -CH3 vinblastineR= -CHO vincristine

from tryptophan

N OH

H3C

Lycopodine

from lysine

Am

ino

aci

ds