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CHAPTER-II ALKALOIDS
2.1 INTRODUCTIO
The term natural products refers to those organic compounds which are found in nature and are associated with living organisms, either plants or animals. Natural products serve a variety of functions in the organisms in which they occur. These natural products are classified as carbohydrates, trepans, steroids, malefic acids, and alkaloids etc. [4] Alkaloids are derived from plant sources, they are basic in nature, contain one or more nitrogen atoms (usually in the form of a heterocyclic ring) and have usually a marked physiological action on man and other animals. In practice, those substances present in plants and giving the standard qualitative tests are termed as alkaloids, and frequently in plants surveys this evidence alone is used to classify a particular plant as alkaloid-containing. [5]

2.2

DEFINITION AND CHARACTRISTICS OF ALKALOIDS

An alkaloid is a nitrogenous molecule that has pharmacological effects on humans and

other animals. The name alkaloid derives from the word alkali, because their chemical properties are similar to it; originally, the term was used to describe any nitrogen containing base (an amine in modern terms). Alkaloids are found as secondary metabolites in plants (e.g. in potatoes and tomatoes), in animals (e.g. in shell fish), and in fungi, and can be extracted from their sources by treating them with acids (usually hydrochloric acid-HCl or sulfuric acid-H2SO4, or through organic acids such as Maleic acid-C4H4O4, and Citric acid-C6H8O7 are sometimes used). Usually, alkaloids are derivatives of amino acids. Alkaloids have the following main characteristics according to its definition. i. ii. They contain Nitrogen; usually derived from an amino acid. They are bitter in taste and generally exist as white solids except Nicotine-C10H14N2 which exist as a brown liquid. iii. They give a precipitate with heavy metallic iodides.

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iv. v.

They are basic in nature and form water soluble salts. They occur in a limited number of plants. Nucleic acid exists in all plants, whereas, morphine exists in only one plant species. [6]

2.3

HISTORY
Alkaloid containing plants were used by humans since ancient times for therapeutic and

recreational purposes. For example, medicinal plants have been known in the Mesopotamia at least around 2000 B.C.
[4]

The Odyssey of Homer referred to a gift given to Helen by the

Egyptian queen, a drug bringing oblivion. It is believed that the gift was an opium containing drug. A Chinese book on house plants written in 1 st 3rd centuries BC have mentioned the medicinal use of Ephedra and Opium poppies, while Coca leaves were used by South American Indians since ancient times also. Proper study of alkaloids began in the 19th century. In 1804, the German chemist Friedrich Serturner isolated from opium a soporific principal (Latin: principium somniferum), which he called Morphium in honour of Morpheus the Greek God of dreams; in German and some other Central-European languages this is still the name of the drug. The term Morphine used in English and French was given by the French physicist Joseph Louis Gay-Lussac. A significant contribution to the chemistry of alkaloids in the early years of its development was made by the French researchers-Pierre Joseph Pelletier and Joseph Bienne Cavetto who discovered Quinine-C20H24N2O2 (1820) and Strychnine-C21H22N2O2 (1818). Several other alkaloids were discovered around that time including Xanthine-C5H4N4O2 (1817), Atropine-C17H23NO3 (1819), Caffeine-C8H10N4O2 (1820), Coniine-C8H17N (1827), NicotineC10H14N2 (1828), Colchicine-C22H25NO6 (1833), Sparteine-C15H26N2 (1851), and Cocaine-

C17H21NO4 (1860). The first complete synthesis of an alkaloid was achieved in 1886 by the German chemist Albert Ladenburg. He produced coniine by reacting 2-methylpyridine with acetaldehyde and reducing the resulting 2-propenyl pyridine with sodium. The development of the chemistry of alkaloids was accelerated by the emergence of spectroscopic and chromatographic methods in the 20th century, so that by the year 2008, more than 12000 alkaloids had been identified. [7]

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2.4

OCCURRENCE
The major sources of alkaloids in the past have been the flowering plants- the

angiosperm. In recent years, however, there have been increasingly numerous examples of the occurrence of alkaloids in animals, insects, marine organisms, micro-organisms and the lower plants. Some other diverse sources of alkaloids are given in table 1.1. VARIOUS DIVERSE SOURCES OF ALKALOIDS Serial No. 1 2 3 4 5 6 7 Musk Deer Canadian Beaver Sex hormone of several insects Gonyaulax Catenella Pseudomonas Aeruginosa Calviceps Purpurea Lysopodium Table 1.1 Muscopyridine Castor amine Pyrrole derivatives Sexitoxin Pyocyanine Chanoclavine-1 Lysopodine Source of Alkaloid Alkaloid Isolated

2.5

NAMING
The name alkaloids (German: Alkaloide) was introduced in 1819 by the German

chemist Carl F. W. Meissner and is derived from the Late Latin word alkali. However, the term came into wide use only after the publication of a review article by O. Jacobsen in the chemical dictionary of Albert Ladenburg in the 1880s. [8]

2.6

CLASSIFICATION OF ALKALOIDS
Alkaloids are usually classified by their common molecular precursors, based on the

metabolic pathway used to construct the molecule. When much was not known about the biosynthesis of alkaloids, they were grouped under the names of known compounds, even some non-nitrogenous ones (since those molecular structures appear in the finished product; the opium alkaloids are sometimes called phenanthrenes for example), or by the plants or animals they were isolated from. When more is learned about certain alkaloids, the grouping is

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changed to reflect the new knowledge, usually taking the name of a biologically important amine that stands out in the synthesis process. Pyrrolidine Group: Tropane Group: Quinoline Group: Hygiene, Cuscohygrine, Nicotine. Atropine, Cocaine, Ecgonine, Scopolamine Quinine, Quinidine, Dihydroquinie, Dihydroquinidine, Strychinie, Brucine, Veratrine, Ceradine. Isoquinoline Group: The opium alkaloids (Morphine, Codien, Thebaine, Heroin, Papaverine, Nicotine, Narcien, Hydrastine, Berberine) Phenethylamine Group: Indol Group: a) b) MDMA, Methamphetamine, Mescaline, Ephedrine. Tryptamines: DMT, NMT, Psilocybin, Serotonin, Ergolines: The ERGOT ALKALOIDS (Engine, Ergotamine,

Lysergic, Acid, etc.) LSD c) Beta-Carbolines: Harmine, Yohimbine, Resepine,

Emitine Purin Group: Xanthines: Terphenoid Group: Aconite Alkaloids: Steroids: Pyrazole Group: Aconitine Solanine, Samandarin. Pyrazole, Fomepizole. [9] Caffeine, Theobromine, And Theophylline.

2.7

OTHER METHODS FOR CLASSIFICATION OF ALKALOIDS

According to Hegnaurer, the most widely accepted classification system groups the

alkaloids as depending upon the sources from which they are obtained. 1) True alkaloids

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2) Proto alkaloids 3) Pseudo alkaloids

True Alkaloids

Proto Alkaloids

Pseudo Alkaloids

Contains Nitrogen in not Does Heterocyclic ring System

Does not Contain Nitrogen in Heterocyclic ring System

Contains Nitrogen in Heterocyclic ring

Derived from Amino acid

Not Derived from Amino acid

2.7.1 TRUE ALKALOIDS

True alkaloids have the following important characteristics: i. ii. iii. iv. v. vi. vii. Contain nitrogen in heterocyclic ring system Wide range of physiological activity Derived from amino acids Limited taxonomic distribution Occurs in plants as the salt of an organic acid Invariably basic Toxic

Examples of true alkaloids are: Nicotine Morphine

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Codiene

Structure of Nicotine Structure of True Alkaloids

Structure of Codiene

2.7.2 PROTO ALKALOIDS

Proto alkaloids have the following main characteristics: i. ii. iii. iv. Simple amine Nitrogen is not in a heterocyclic ring Biosynthesis from amino acids Basic

Examples of proto alkaloids are: Mescaline Ephedrine

Structure of Mescaline

Structure of Ephedrine

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Structure of Proto Alkaloids

2.7.3 PSEUDO ALKALOIDS

Pseudo alkaloids have the following main characteristics: i. ii. iii. They are not derived from amino acids They are usually basic in nature Two important series of alkaloids in this class are: a) Steroidal alkaloids b) Purine Examples of pseudo alkaloids are: 1. Conessine 2. Caffeine

Structure of Conessine Structure of Pseudo Alkaloids

Structure of Caffeine

2.8

PROPERTIES OF ALKALOIDS
There are two types of properties: i. ii. Physical properties Chemical properties

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2.8.1 Physical Properties

1. Most of the alkaloids that have been isolated are crystalline solids with a definite melting point or decomposition range, while very few alkaloids are amorphous solids e.g. Emetine-C29H40O4N2. 2. Some are liquids that are either volatile e.g. Nicotine and Coniine, or non-volatile e.g. Pilocarpine and Hyosine. 3. Most alkaloids are colourless, but some of their complex higher species are coloured, e.g. Berberine are yellow and Betanin are red. 4. Both alkaloid bases and their salts are soluble in alcohol. In general, the free base of alkaloids is soluble only in organic solvents, although, some of the pseudo and proto alkaloids are substantially soluble in water. The salts of alkaloids and the quaternary alkaloids are highly water soluble.

2.8.2 Chemical Properties

1. The most distinct property of most alkaloids is that they are basic. This property is of course dependent on availability of the lone pair of electrons on nitrogen. If functional group adjacent to nitrogen are electron e.g. an alkyl group, availability of the electrons on nitrogen is increased and compound is more basic. Thus: R2NH > RNH2 > R3N 2. Structural Hexocyclic amines are more basic than aromatic amines. 3. Alternatively, if adjacent functional group is electron withdrawing e.g. carbonyl group, the availability of the lone pair is decreased and the effect is to make the alkaloid neutral or even slightly acidic. A typical example is the amide group of compounds. 4. The basicity of the alkaloids also depends upon the nitrogen heterocyclic system. The electron pair on nitrogen in case of Piperidine and Pyrolidine is more available than in case of Pyridine, Isoquinoline, Pyrrole, and Idol. So, the former are more basic than the later. 5. The basicity of alkaloids makes them extremely susceptible to decomposition, particularly by heat and light in the presence of oxygen. The products of such reactions

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(often N-Oxides and alkaloid N-oxides) are becoming of increasing biosynthetic importance. 6. Most alkaloids contain oxygen and are solid in nature e.g. Atropine. Some alkaloids are free from oxygen and are mostly liquids e.g. Nicotine and Coniine. [10]

2.9 EXTRACTION AND ISOLATION OF ALKALOIDS

GENERAL PROCEDURE:
The alcoholic extraction method was applied to all the selected plants. The plants material was chopped into small pieces and then soaked overnight in methanol. The methanol extract was filtered and the process was repeated four times to ensure no alkaloids were left in the residue. All the extracts obtained in each set were combined and the methanol was then removed by rotary evaporation. The dried methanol extract obtained from each plant was then packed in glass bottles. The residue from the ethanol extracts were air dried and stored with proper labeling. Sequential extraction and isolation were done following the schematic procedures outlined and shown in the following scheme. The solvents in the filtrates were evaporated under reduced pressure and the crude alkaloids were obtained. The crude alkaloids were then separated using preparative thin layer chromatography on silica gel.

MACERATED PLANT:
1. Extract with petroleum ether 2. Filter, evaporate, fats, filtrate

PLANT RESIDUE:
1. Extract with MeOH / EthOH 2. Evaporate Filtrate

CRUDE PLANT EXTRACT:

1. Dissolve in dilute inorganic acid 2. Extract with ether,

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3. Ether Solution (Non-Basic Material)

ACID SOLUTION OF ALKALOID SALTS:

1. NaOH Solution 2. Extract with ether 3. Aqueous Residue

Ether Solution Evaporate Alkaloids Procedure of Isolation of Alkaloids from Plants

2.10 STRUCTURAL ELUCIDATION OF ALKALOIDS

The structure of an alkaloid is elucidated by the application of various spectroscopic and physical organic techniques. These techniques have many advantages, such as: 1. The case and rapidity with which information can be accumulated that the data obtained are accurate compared to the old methods. 2. Most instrumental procedures require only minute amount of material, which need not necessarily be pure and which can usually be recovered unchanged. In alkaloidal chemistry, the most important physical methods are the following: 1. Ultraviolet Spectroscopy 2. Infrared Spectroscopy 3. Nuclear magnetic resonance spectroscopy 4. Mass spectroscopy 5. Optical rotatory dispersion and circular dichorism 6. Conformational analysis 7. X-ray diffraction
[11]

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2.11 DETECTION METHOD AND SPRAY REAGENTS

Dragendroffs reagent and Mayers reagent were used for the detection of alkaloids in the plants extracts. Mayer reagent was prepared by dissolving mercuric chloride (1.4grams) and potassium iodide (5grams) in 100ml of water. Dragendroffs reagent was prepared as: a) Bismuth sub-nitrate (2.5grams) was dissolved in a solution of 20ml water and glacial acetic acid (5ml). b) Potassium iodide (4grams) was dissolved in 10ml water. Solution a and b were then mixed together and stored as a stock solution. The spray reagent was prepared by mixing the stock solution (5ml) with glacial acetic acid (10ml) and water (85ml) on TCL cards alkaloid give a brown coloration with this spray reagent. [12]

2.12 THE BIOLOGICAL ROLE OF ALKALOIDS

The role of alkaloids for living organisms which produce them is still unclear. Initially it was assumed that alkaloids are the final products of nitrogen metabolism in plants, and as urea in mammals. Later it was shown that alkaloid concentration varies over time and this hypothesis was refuted. [13] Most of the known functions of alkaloids are related to protection, e.g. aporphin alkaloid liriodenine produced by the tulip plant protects it from parasitic mushrooms. In addition, presences of alkaloids in plants prevent insects and chordate animals from eating them. However, some animals are adapted to alkaloids and even use them in their own metabolism.
[14]

Such alkaloid-related substances as Serotonin, Dopamine and Histamine are

important neurotransmitters in animals. Alkaloids are also known to regulate plants growth. [15]

2.13 APPLICATION OF ALKALOIDS

Alkaloids are in use in a variety of fields. Some important applications of alkaloids are as follows:

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2.13.1

Use in Medicines

Medical use of alkaloid plants has a long history, and thus, when the first alkaloids were synthesized in the 19th century, they immediately found application in clinical practice. Many alkaloids are still used in medicine, usually in the form of salts including the following: Name of Alkaloid Ajmaline Atropine, Scopolamin Vinblastine, Vincristine Vincamine Codeine Cocaine Colchicine Morphine Reserpine Tubocurarine Quinidine Quinine Ergot Alkaloids Action Antiarrhythmic Anticholinergic Antitumor Vasodilation, Anti-hypertension Cough medicine Anesthetic Remedy for Gout Analgesic Anti-hypertensive Muscle relaxant Antiarrhythmic Antipyretics, Antimalarial Sympathomimetic, Vasodilator

Many synthetic and semisynthetic drugs are structural modifications of the alkaloids which were designed to enhance or change the primary effects of the drug and reduce unwanted side effects. For example, naloxone, an opioid receptor antagonist is a derivative of Thebaine which is present in opium. [16]

2.13.2

Use in Agriculture

Prior to the development of a wide range of relatively low-toxicity synthetic pesticides, some alkaloids, such as salt of nicotine and anabasine were used as insecticides. Their use was limited by their high toxicity to humans. [17]

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2.13.3

Use as Psychoactive Drugs

Preparation of plant containing alkaloids and their extracts and later pure alkaloids have long been used as psychoactive substances. Cocaine and Cathinone are stimulants of the central nervous system (CNS). Mescaline and many of indole alkaloids have hallucinogenic effects. Morphine and Codeine are strong narcotic pain killers. There are alkaloids that do not have strong psychoactive effect themselves, but are precursors for semi-synthetic psychoactive drugs e.g. ephedrine and pseudo-ephedrine are used to produce methcathinone and methamphetamine. [18]

2.14 FUNCTION OF ALKALOIDS IN PLANTS

The characteristic nature of alkaloids and their often very marked pharmacological effects when administered to animals naturally led scientists to speculate on their biological role in the plants in which they occurred. In spite of many suggestions over the years, however, little convincing evidence for their function has been forthcoming. The following points are noteworthy. 1) Being of such diverse nature, alkaloids as a group could not be expected to have a common role (if any) in the plant, except possibly in situations requiring a non-specific basic compound. In this respect the increase in putrescine in barley seedlings when grown in a medium deficient in potassium is of interest. 2) Alkaloids often occur in plants in association with characteristic acids, e.g. the tropane alkaloids of the Solanaceae and Erythroxylaceae are esters, the cinchona alkaloids occur with quinic and cinchotannic acids, opium alkaloids are associated with meconic acids. In some cases the alkaloids could provide either a means of storing or transporting in soluble form the particular acids. In the case of solanaceous plants it has been shown that tropane esters formed in the roots are translocated to the aerial parts, where hydrolysis of the alkaloids and breakdown of the liberated acids occur. 3) As the majority of alkaloids are biosynthesized from readily available units by a series of ubiquitous reactions, their presence in the plant may be purely chance, depending on the enzymes present and the availability of precursors. Being apparently harmless to the plant, they are not eliminated through necessity by natural selection.

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4) By the use of grafts, plants which have normally alkaloids in the aerial parts (e.g. Nicotiana and Datura) are produced free of alkaloids. The lack of alkaloids in the scion appears in no way to impair its development, which suggests the non-essential nature of the alkaloids. 5) Plants which do not normally contain alkaloids appear usually to suffer no adverse reaction when administered alkaloids (Colchicines is an exception). Some foreign alkaloids may be metabolized. 6) Current research constantly demonstrates not only, that alkaloids participate in plants metabolism over the long term, but also that daily variation in alkaloid content (qualitative and quantitative) is very common in some species. This implies that even if the presence of alkaloids is not vital to the plant, they do participate in metabolic sequences and are not solely the waste-end products of metabolism. Pertinent to the above, it has been suggested that alkaloids may have a role in the defense of the plant against singlet oxygen (1O2), which is damaging to all living organisms and is produced in plant tissues in the presence of light. Of 15 alkaloids tested, most showed a good ability to quench singlet oxygen, with brucine and strychnine being especially efficient. Circumstantial evidence quoted is the turnover of poppy alkaloids on a diurnal basis and the formation of oxidized serpentine at the expense of the reduced ajmaline when Catharanthus roseus tissue cultures are exposed to light. Further, to harmonize with the above hypothesis, one would expect plants inhabit more alkaloids and confirmatory examples quoted are berberine in Berberis and tomatidine in Lycopersicum. To these could be added quinine in Cinchona. [19]