Alkaloid Isomers and Analysis

• Phytochemistry II is course PG-605 for the Spring semester of 2024-2025.
• The lecturer is Shaza Hussiny Aly, PhD, from the Faculty of Pharmacy at Badr University in Cairo.

BUC Pharmacy Department Vision

• To be a leading college locally, regionally, and internationally in advancing human health.
• This will be done by using innovations in education, scientific research, and applied practices.

BUC Pharmacy Department Mission

• To provide optimal practices in education, learning, and training.
• Create ongoing opportunities for students and graduates through innovation, publishing, and applying modern knowledge.
• Emphasize research and applications in pharmaceutical, clinical, and social sciences and contribute to health promotion.

Course Aims

• Demonstrate understanding of chemical characteristics of secondary plant metabolites like alkaloids, volatile oils, tannins, and antioxidants.
• Understand techniques for isolation using various chromatographic methods.
• Apply methods for the qualitative analysis of active phytochemical ingredients.
• Provide counsel to patients and communities regarding the appropriate and safe use of herbal drugs.

Course Topics

• Alkaloids
• Volatile Oils
• Plant polyphenolics

Key Competencies and Learning Outcomes

• Use appropriate pharmaceutical terminology.
• Determine methods for separating biologically active principles from extracts.
• Identify the chemical properties and biological activity of alkaloids and tannins.
• Explain various methods for identifying alkaloids and tannins.
• Choose suitable chromatographic techniques for isolating biologically active compounds from natural sources.

Essential Reading

• "Trease and Evans' Pharmacognosy" by W.C. Evans, W.B. Saunders Company, 16th edition (2009).

Recommended Reading

• "Textbook of Pharmacognosy and Phytochemistry" by B. A Shah, ELSEVIER; 2nd edition (2014).
• “A Textbook of Pharmacognosy and Phytochemistry" by G.S. Kumar, K.N. Jayaveera, S. Chand Publishing; 1st edition (2014).

Office Hours

• Sundays: 11 AM - 1 PM
• Tuesdays: 2 PM - 3 PM

Alkaloids - Introduction

• Alkaloid-containing plants are diverse taxonomically and chemically.
• Their unifying feature is a basic nitrogen atom and other classifications.
• Questions regarding the physiological role of alkaloids, their taxonomic importance, and biogenesis are best discussed within specific alkaloid classes.
• The therapeutic and pharmacological activities and the toxicity levels also require consideration.
• Plants with a strong alkaloid make-up don't feature strongly in herbal medicine.
• Alkaloids have been important in allopathic medicine where dosage is controlled and homeopathy where the dosage is harmless.
• A precise definition of alkaloids is challenging due to overlaps with complex amines.
• Typical alkaloids are plant-derived, basic, contain one or more nitrogen atoms (usually in a heterocyclic ring), and exert a marked physiological action.
• Proto-alkaloids or amino-alkaloids lack some properties of typical alkaloids.
• Substances in plants that yield positive qualitative tests are classified as alkaloids.
• Often, this evidence classifies a plant as 'alkaloid-containing'.

Lectures Outline

• Introduction
  • Definition
  • Classification
  • Functions
  • Nomenclature
  • Physiochemical properties
  • Detection and isolation
• Non-Heterocyclic Alkaloids.
• Heterocyclic Alkaloids.

1 - Definition

• Alkaloids are like alkali: They are naturally occurring organic compounds with:
  • Basic nitrogen-containing secondary metabolites.
  • Usually, 1 to 5 nitrogen atoms.
  • Marked pharmacological or toxicological effects.
  • Nitrogen atoms of heterocyclic or non-heterocyclic types
  • A heterocyclic ring e.g. tropane, quinoline, and indole.
• All alkaloids contain nitrogen, but not all nitrogenous compounds are alkaloids.

Properties

• Alkaloids comprise the largest class, with approximately 21,000 secondary metabolites.
• They are biosynthetically derived from amino acids like L-Ornithine, L-tyrosine, and L-tryptophane.
• Alkaloids have potent physiological effects. Atropine, morphine, taxol, and quinine are alkaloid examples.

2 - Classification

• Alkaloids can be classified according to their pharmacological action (biological action), biochemical origin (biosynthetic pathway), taxonomical origin (plant families rich in alkaloids), and/or chemical structure.
• They can also be classified by the type of nitrogen.

Classification by Pharmacological Action

• Analgesic: Morphine
• CNS stimulant: Caffeine
• Mydriatic: Atropine
• Myotics: Pilocarpine
• Anti-asthmatic: Ephedrine
• Antitussive: Codeine
• Skeletal muscle relaxant: Tubocurarine
• Anthelmintics: Pelletierine
• Anticancer: Vincristine

Classification by Biochemical Origin

• Ornithine: Pyrrolidine, Tropane alkaloids
• Lysine: Piperidine and Quinolizidine alkaloids
• Nicotinic acid: Pyridine alkaloids
• Tyrosine: Phenylethylamine alkaloids
• Tryptophane: Simple indole alkaloids, Terpenoid indole, Quinoline, and Ergot alkaloids
• Anthranilic acid: Precursors for Quinoline alkaloids
• Histidine: Imidazole derivatives

Hegnauer's Classification

• Based on nitrogen type and biochemical origin, alkaloids are divided into:
  • True alkaloids.
  • Proto alkaloids.
  • Pseudo alkaloids.

A - True Alkaloids / Typical / Heterocyclic

• These compounds:
  • are derived from amino acids.
  • contain nitrogen atoms in a heterocyclic structure.

B - Proto Alkaloids / Simple amines / Non-heterocyclic alkaloids

• These compounds:
  • are derived from amino acids.
  • do NOT contain nitrogen atoms in a heterocyclic structure.

C - Pseudo Alkaloids

• These alkaloids:
  • are NOT derived from amino acids.
  • contain nitrogen atoms in a heterocyclic structure.
  • are weakly basic compounds.
  • insert the nitrogen atom at a late stage through a transamination reaction.

Occurance

• Plant sources include:
  • Alkaloid-bearing families like Solanaceae, Papaveraceae, Apocyanaceae, Fabaceae.
• Alkaloid-free families like Oleaceae, Labiatae.
• Animal sources include:
  • Ergotamine and ergometrine derived from Ergot fungus.
  • Indole alkaloids derived from marine snails.

3 - Functions of Alkaloids in Plants

• Protection against insects.
• End products of metabolism.
• Waste products.
• Source of energy and nitrogen reserve.
• Growth regulators.

4 - Nomenclature

• Alkaloid names usually terminate with the suffix "-ine."
• Names may be derived from:
  • Genus: Atropine, ephedrine.
  • Species: Cocaine, belladonnine.
  • Common name: Ergotamine from Ergot.
  • Physiological activity: Emetine.
  • Discoverer: Pelletierine from Pelletier.

Prefixes and Suffixes

• "Nor-" indicates N-demethylation e.g. Nornicotine.
• "Apo-" indicates dehydration e.g. Apomorphine.
• "Iso-", "Pseudo-", "Neo-", "Epi-" indicate different types of isomers.
• "-dine" indicates optical isomerism where the two chemicals both have the same formula but a different atomic arrangement e.g. Quinidine is an optical isomer of quinine.
• "-inine" indicates lower pharmacological activity e.g. Ergotaminine is less active than ergotamine.

5 - Physicochemical Properties

A) Physical Characters

• Condition

  • Most alkaloids are solid and crystalline.
  • Volatile liquids: Nicotine, Coniine.
  • Non-volatile liquids: Pilocarpine, Hyoscine.

• Color

  • Most alkaloids are colorless or white.
  • Exceptions - Yellow: Colchicine, Berberine, Psychoterine.

Solubility

• Both alkaloid bases and their salts are soluble in alcohol.
• Alkaloidal bases are generally soluble in organic solvents but insoluble in water.
• Exceptions
  • Bases soluble in water: Caffeine, Ephedrine, Colchicine, Quaternary Ammonium Alkaloids.
  • Bases insoluble in organic solvents: Morphine, Psychotrine (in ether), Theobromine, Theophylline (in benzene).
• Alkaloid salts are usually water-soluble and insoluble in organic solvents.
• Salts insoluble in water: Quinine monosulphate.
• Importance of solubility:
  • Administered in injection form.
  • Useful for isolation and purification.

Optical Activity

• Many alkaloids are optically active because they contain an asymmetric carbon atom (chiral) in their molecule.
• Optically active isomers show different physiological activities with Levo (-) isomers.
• Levo is usually more active than dextro (+) isomers:
  • L-ephedrine is 3x more active than d-ephedrine.
  • L-ergotamine is 3x more active than d-ergotamine.

Exceptions

• d-Tubocurarine is more active than l- form.
• Both Quinine (l-form) and Quinidine (d- form) active.
• Racemic dl-Atropine is physiologically active.

B) Chemical Character

• In aliphatic side chain (Proto-alkaloids)or heterocycle structures (True alkaloids).
• Usually present in a Tertiary structure.
• Less common in the Secondary structure e.g. Ephedrine.
• Rarely present in Primary structures e.g. Mescaline.
• May also be present in Quaternary forms e.g. Tubocurarine.

1. Basicity of Alkaloids

• Basicity is due to the unshared electron pairs on the nitrogen atom.
• Salts form with many acids like HCl, H2SO4, and meconic acid in opium.
• Oxides also form (N→O).
• Strong basic alkaloids form salts with very weak acids.
• Weak bases require a more acidic medium.
• Some alkaloids are Amphoteric due to acidic groups in their molecule.
• Alkaloids form salts with acids and bases and possess a Phenolic (Morphine) or Caroxylic group (Narceine).

2. Stability of Alkaloids

Effect of Heat

• Alkaloids generally decompose when heated with some substances able to sublime e.g. caffeine.
• Alkaloids transform into N oxides in the presence of light, oxygen and elevated temperatures.

Effect of Acids

• Dehydration (anhydro- / apo-alkaloids).
  • Morphine to apomorphine
  • Atropine to apoatropine.
• O-Demethylation
  • Codeine to morphine
• Hydrolysis
  • Atropine [Tropane alkaloids]

Effect of Alkalis

• Weak alkalis (NH3) liberate most alkaloids from their salts.
• These salts contain alkaloids with a carboxylic group e.g. [Narceine].
• Strong alkalis (NaOH, KOH) form salts with phenolic alkaloids e.g. [Morphine].
• Hot alkalis can hydrolyze ester alkaloids e.g Atropine, Cocaine, Physostigmine and cleave a lactone ring e.g. Pilocarpine.

6 - Detection and Isolation

I - Qualitative Tests for Detection

A) Precipitation Reactions

• Reactions occur in alkaloid solution in test tubes.
  • The sensitivity is impacted by the concentration.
  • The selectivity is impacted by the effect of False Alkaloids (proteins, tannins, and coumarins).
• The method involves the formation of double salts or a complex with M.Wt. (heavy) metals like mercury (Hg), platinum (Pt), bismuth (Bi), gold (Au), and iodine (I₂) in a neutral or acidic solution.

Precipitation Reagents

• Mayer's reagent: Potassium-mercuric iodide - Creamy white precipitate. Some alkaloids don't react at a low conc.

The following don't react at a low concentration to Mayer's Reagent

• Purine Alkaloids
• Ephedrine
• Colchicine
• Ricinine
• Wagner's reagent: Iodine in potassium iodide - Reddish brown precipitate.
• Hager's reagent: Saturated solution of picric acid - Yellow precipitate.
• Dragendorff's reagent: Potassium bismuth iodide - Orange-reddish brown precipitate.
• Marmé's reagent: Potassium cadmium iodide - Yellow precipitate.

B) Coloring Reactions

• A residue is placed on an alkaloid porcelain slab.
  • Sensitivity is impacted by the concentration.
  • Selectivity is impacted.
• Involves dehydration and oxidation of the alkaloid structures.
• Sulphuric acid is always present.

General Coloring Reagents

• Froehd's reagent: Ammonium molybdate/conc. H2SO4. Used in spectroscopic analysis of alkaloids.
• Mandalin's reagent: Ammonium vanadate/conc. H2SO4. Used in spectroscopic analysis of alkaloids.
• Marquis' reagent: Formaldehyde/conc. H2SO4. Used in spectroscopic analysis of alkaloids.
• Erdmann's reagent: Conc. Nitric acid/conc. H2SO4. Used in spectroscopic analysis of alkaloids.

Special Coloring Reagents

• Vitali-Morin reagent: Violet color with Tropane alkaloids.
• Thaleoquine reaction: Red color with Cinchona alkaloids.
• Murexide reaction: Pink color with Purine bases.
• Van-Urk reagent: Blue or grayish-green color with Ergot alkaloids.

II - Quantitative Tests for Detection

• Used to:
• Determine the genuineness of raw material?
• Evaluate the plant material for marketing?
• Determine the best stage for collection?
• Evaluate the stability and activity of a preparation?
• Prevent of overdose and intoxication?
• Determine the bioavailability in different organs and tissues?

Estimation Methods

• Proximate assays: determination of total alkaloids.
• Ultimate assays: determination of individual alkaloids.
• Techniques used:
  • Volumetric
  • Gravimetric
  • Colorimetric/spectrophotometric.

Isolation of Alkaloids

• Follow established procedures to separate alkaloids from the other constituents.
• Process steps:
• Preparation of plant sample
• Liberation of free alkaloid with a weak alkali.
• Selective extraction of alkaloids.
• Purification of crude alkaloid extract.
• Fractionation of total alkaloids.

Liberation of Free Alkaloids

• Alkaloids occur as these salts (HCl, H2SO4, tannates, oxalates...).
• The salt of a strong base requires a strong alkali for liberation.
• Ester alkaloid (Atropine and Cocaine), Lactone alkaloids (Pilocarpine),↑ fatty content alkaloid ext. → Weaker bases (Na2CO3, NaHCO3)
• To prevent hydrolysis or saponification.
• NH3 is most widely used as it’s sufficiently basic, volatile (xss removed by A), and doesn't react with amphoteric alkaloids.

Explore alkaloid isomers with prefixes like 'Apo-' and suffixes like '-dine.' Learn about quinidine's optical isomerism and qualitative alkaloid estimation. The content focuses on alkaloid analysis and identification.