Alkaloids

The term alkaloid was introduced by W. Meissner at the beginning of the 19th century to designate natural substances reacting like base, in other words like alkalis (from the Arabic alkaly = soda and the Greek word eiods = appearance).

The definition of alkaloids is not simple and precise and sometimes it is difficult to distinguish the thin line between alkaloids and other natural nitrogen-containing metabolites.

Natural nitrogen-containing compounds are:

• Amino acids
• Amines
• Alkaloids
• Indole
• Purines, pyrimidines, and cytokinins
• Cyanogenetic glycosides
• Chlorophylls

An alkaloid is an organic compound of natural origin that contains a nitrogen atom that is more or less basic, is of limited distribution, and has at low doses marked pharmacological properties.

This grouping has a sound basis which is confirmed by the fact that these compounds have in common some reactions of precipitation with the general reagent for alkaloid. Their nitrogen atom is a part of the heterocyclic system and causes significant pharmacological activity.

According to some authors, they occur only in the vegetable kingdom. They are found as salt and it can also say that they are formed biosynthetically from an amino acid.

Pseudo alkaloid most often has all of the characteristics of the true alkaloid but they are not derived from amino acids. Some of the most known examples are Isoprenoid, Aconite, Aconine, Hypoaconitine, etc.

Steroidal alkaloids like Conessine, Solanidine are a few examples. They are also known as heterocyclic nitrogen-containing substances arising from the metabolism of Acetate. An example is a coniine, the toxic principle of Hemlock.

Proto alkaloids are simple amines in which the nitrogen atom is not part of a heterocyclic ring. They are basic and derived from amino acids. A few examples of these are Colchicine, Ephedrine, and Pseudoephedrine.

Distribution of Alkaloids:

Alkaloids occur exceptionally in bacteria (Pseudomonas aeruginosa) and rather rarely in fungi (Psilocin from the hallucinogenic mushroom).

The Pteridophyta rarely contains alkaloids. Thus alkaloid are compounds essentially found in the angiosperm.

Alkaloid concentrations have a wide range of variation from a few ppm (for example Catharanthus roseus contain 3 gm alkaloid in one metric ton leaves) to more than 15% in the bark of the trunk of Cinchona ledgeriana.

Sometimes they do contain only one constituent (for example Hyoscyamine from the leaves of belladonna) but must often yield a complex mixture that may be dominated by one major constituent.

As a general rule, all of the alkaloids of a given plant have a common biogenetic origin even if their structure may at first be seen quite differently.

The conc. of alkaloid can vary from part to part and some parts may contain none. For example, Quinine accumulates in the trunk bark of cinchona but is completely absent from leaves.

For a long time alkaloid used to be considered products of the metabolism of the plant only. Alkaloid also occur in animals (Arthropods who secrete them in very small quantities in their exocrine glands).

Sometimes they are volatile enough to act as chemical signals defense compounds known as allomones or communication compounds called pheromones.

Localization

In the plant, alkaloid occurs as a soluble salt (citrate, Malate, tartarate, malonates, benzoates, isobutyrates) or in combination with tannins.

They are often localized in the peripheral tissue external layer of the bark and root or seed tegument. Alkaloids synthesis takes place at specific sites (growing root, chloroplast, laticiferous cell).

Functions of Alkaloids:

Alkaloids are poisonous but when used in small quantities they exert useful physiological effects on animals and human beings.

Their exact role in the plant is still a topic of research. Some of the predicted roles are:

1. They are reserve substances that can supply nitrogen.
2. They might be the defensive mechanism for plant growth in the dry region to protect from grazing animals, herbivores, and insects.
3. They may be the end product of the detoxification mechanism in the plant and by this way check the formation of substances that may prove harmful to the plant.
4. The possible role as a growth regulatory factor in the plant.
5. They are present normally in conjugation with plant acid-like mercuric acid, cinchotannic acid, etc. Therefore alkaloids could be acting as carriers within the plant for the transportation of such acids.

Physicochemical Properties of Alkaloids:

• Alkaloid have a molecular weight of 100-900.
• Although most of the bases that do not contain oxygen atoms are liquid at ordinary temperature (examples: nicotine, coniine). Those that do contain oxygen atoms are normally crystallisable solids and in rare cases, they are colored compounds (for example berberine).
• Almost all of the crystallized bases rotate the plane of polarized light and have melting points without decomposition especially below 200o C.
• As general rule alkaloids as bases are not soluble in water. They are soluble in polar or slightly polar organic solvents.
• The basicity of alkaloids varies greatly. Since this property depends entirely on the availability of the lone pair of electrons on the nitrogen atom.
• The electron-withdrawing group near the nitrogen atom decreases the basicity, whereas the electron-donating group enhances it.
• The basic character allows the formation of salts with mineral acids (i.e. hydrochloride, sulfates, and nitrates) and organic acids (i.e. tartarate, sulfonate).
• Alkaloidal salts are generally soluble in water and dilute alcohols and they are except in rare cases not soluble in an organic solvent.
• Pseudo alkaloids and proto alkaloids show higher solubility in the water while free bases of alkaloids are soluble in an organic nonpolar immiscible solvent.

Detection And Characterization of Alkaloids:

The different reagents used for the detection of alkaloids are:

• Mayer reagent (Potassium mercuric iodide solution) gives cream-colored precipitate.
• Dragendorff reagent (Potassium bismuth iodide solution) shows reddish-brown precipitate.
• Wagner reagent (Iodine Potassium Iodide solution) yields reddish-brown precipitate.
• Hager’s reagent (saturated Picric acid solution) gives yellow color precipitate.
• ρ-dimethyl amino benzaldehyde uses for ergot alkaloids and pyrrolizidine alkaloids.
• Cerium and ammonium sulfate for different indole (yellow), dihydro indole (red), and β-amino acelates (blue).
• Caffeine, purine derivatives do not precipitate like most alkaloids. It is detected by mixing with a very small amount of potassium chlorate and a drop of HCl, evaporating to dryness and exposing the residue to ammonia vapor. Purple color is produced with caffeine and other purine derivatives (Murexide test).

Care must be taken in the application of these alkaloidal tests, as the reagent also gives a precipitate with proteins. During the extraction of alkaloids from the plant and subsequent evaporation, some proteins will not be extracted and others will be made insoluble (denatured) by the evaporation process and may be filtered out. If the original extract has been concentrated to low bulk and the alkaloids extracted from an alkaline solution using an organic solvent and then transferred into dilute acid (e.g. Tartaric acid) the latter solution should be protein-free and ready to test for alkaloids.

Extraction of Alkaloids:

The extraction of alkaloids is based as a general rule on the fact that they normally occur in the plant as salts and on their basicity, in other words on the differential solubility of the bases and salt in water and organic solvent.

[A] Solvent extraction in alkaline medium

Step 1:

• The powdered defatted drug is mixed with an alkaline aqueous solution which displaces the alkaloids with acids tannins and other phenolic substances, the free base is then extracted with an organic solvent.
• Alkalization is very often achieved with aqueous ammonia. If the structure of the alkaloids to be extracted contains a fragile element for example ester or lactone fraction aqueous ammonia must be replaced by an alkaline carbonate solution.
• In some cases, a mixture of calcium hydroxide and sodium hydroxide is used i.e. in cinchona bark in which the alkaloids are combined with tannins.
• When using sodium hydroxide, it will turn phenolic alkaloids into phenolates which will remain in an aqueous solution, if necessary this can be used to fractionate the total alkaloids.
• The organic solvent can be chlorinated (dichloromethane or chloroform) depending on cost, toxicity, safety, and recovery.

Step 2:

• The organic solvent containing the alkaloids as bases is separated from the residue and if necessary partially concentrated by distillation under reduced pressure.
• The solvent is then stirred with an acidic aqueous solution. The alkaloids go into the solution in the aqueous phase as salts, whereas neutral impurities remain in the organic phase. Repeat the process till the organic phase no longer contain any alkaloids. Acids may be HCl, H₂SO₄, sulfonic, citric, and tartaric acid.

Step 3:

• The aqueous solution of the alkaloidal salt combines and if necessary, washed with non-polar solvent (hexane or diethyl ether), are alkalinized with a base in the presence of an organic solvent not miscible with water.
• The alkaloids as bases precipitate and dissolve in the organic phase.
• The extraction of the organic phase continues until the totality of the alkaloids has gone into the organic phase.
• Finally, the organic solvent containing the alkaloid, as bases are decanted, is freed from possible traces of water by drying over an anhydrous salt and evaporated under reduced pressure.

[B] Extraction in Acidic Medium:

• Two approaches are possible, in the first one the pulverized drug is extracted directly with acidified water.
• In the second case, it is extracted with an acidified alcoholic or hydroalcoholic solution.
• In the latter case, the extraction is followed by a distillation under vacuum which eliminates the alcohol and leaves behind the as acidic aqueous solution of the alkaloidal salts.
• In both cases, the result is an aqueous solution of an alkaloidal salt requiring purification. This can be accompanied by:
• Alkalizing the solution and extracting the bases with an immiscible organic solvent leads back to the above steps.
• Selectively absorbing the alkaloids in the solution of an ion exchange resin, then eluting with a strong acid.
• Precipitating the alkaloids are iodomercurals. The resulting complex is recovered by filtration dissolved in a mixture of water-alcohol and acetone and decomposed by passing it through an ion exchange resin. This technique can be used to extract quaternary ammonium salts.

Isolation of Alkaloids:

By extraction, we cannot yield pure compounds but total alkaloids which are a complex mixture of bases that must be separated. The method for isolation is following:

(a) Direct crystallization from solvent: This method is very simple and cannot be used for the isolation of complex mixtures. One of the alkaloids should be in major constituent and can be obtained by direct crystallization. For example- quinine is crystallized as basic sulfate by simply neutralizing the acidic extraction medium with sodium carbonate to pH 6.

(b) Steam distillation: This method is specially employed for volatile liquid alkaloids like coniine, spartine, and nicotine but otherwise this process is not suitable for alkaloids of high molecular weights.

(c) Chromatographic techniques: Sometimes it is necessary to follow the classical methods of isolation for complex mixtures, mainly chromatographic techniques. In research and development labs this technique, as well as high-performance liquid chromatography and preparative thin-layer chromatography, are most often used.

(d) Gradient pH technique: Generally it is known that alkaloids are basic and the extent of basicity variation exists among various alkaloids of the same plant. For this case, the crude alkaloidal mixture is dissolved in 2% tartaric acid solution and then extracted with benzene. The first fraction contains neutral or very weakly basic alkaloid. the pH of the aqueous solution is increased gradually by 0.5 increments up to pH 9 and extraction can be done with organic solvents. In this way, the alkaloid of different basicity is extracted at the end.

Classification of Alkaloids:

The classification is mainly based on pharmacological activity, taxonomical distribution, biogenetic origination, and the presence of chemical entities.

Pharmacological Classification

Depending on the physiological response the alkaloids are classified under various pharmacological categories like CNS stimulants or depressants, sympathomimetics, analgesics, purgatives, etc. The main drawback of this system is that it does not take into consideration about chemical nature of the crude drug. Within the same drug, the individual alkaloids may exhibit different actions. Example- 1) Morphine is narcotic and analgesia while codeine is mainly antitussive. 2) Cinchona quinine is antimalarial whereas quinidine is a cardiac depressant.

Taxonomic Classification

This method classifies the vast number of alkaloid based on their distribution in various plant families like solanaceous alkaloids in the Solanaceae family or papilionaceous alkaloids in the papillionaceae family. The grouping of alkaloids is done as per the name of the genus in which they occur e.g. Ephedra, cinchona, etc. The chemotaxonomic classification has been further derived from this classification.

Biosynthetic Classification

This method gives significance to the precursor from which the alkaloids are biosynthesized in the plant. Hence the variety of alkaloid with different taxonomic distribution and physiological activities can be brought under some groups if they are derived from the same precursor i.e. all indole alkaloids from tryptophan are grouped. Alkaloidal drugs are categorized on the fact whether they are derived from amino acids precursors as ornithine, lysine, phenylalanine, tryptophan, etc.

Chemical Classification

This is the most accepted way of classification of alkaloids which depends on the ring structure present in the alkaloid. The alkaloidal drugs are broadly categorized into two divisions:

• True alkaloids (subdivided into 12 groups).
• Proto alkaloids or biological amines and pseudo alkaloids.

(a) True alkaloids

1. Pyrrole and pyrrolidine: Hygrine, coca species.
2. Pyridine and piperidine: Arecoline, Anabasine, coniine, trigonelline.
3. Pyrrolizidine: Echimidine, symphitine.
4. Tropane (piperidine/N-methyl pyrrolidine): Atropine, hyoscine.
5. Quinoline: Quinine, Quinidine, Chinchonine.
6. Isoquinoline: Morphine, codeine.
7. Aporphine (reduced isoquinoline – Naphthalene): boldine.
8. Indole (Benzpyrole): Vincristine, Ergometrine, Reserpine.
9. Imidazole: Pilocarpine, Isopilocarpine.
10. Norlupiname: Cytisine, Spartine.
11. Purine (pyrimidine/ imidazole): Caffine/ theofronine, theophylline.
12. Steroidal (cyclo pentano per hydro phenathrene ring): Solanidine, Conessine.

(b) Pseudo alkaloids

Diterpenes- Aconitine, Aconine

(c) Proto alkaloids

Alkylamines (amino alkaloids) – Ephedrine, colchicine

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