PHYTOCHEMICAL INVESTIGATION OF DRUGS: A TEACHER’S COMPREHENSIVE GUIDE
Welcome, future pharmacognosists and phytochemists!
Phytochemistry involves the study of chemicals (mainly the secondary metabolites) the plants produce as a measure to protect themselves from insects, pests, pathogens, herbivores, UV exposure, and environmental hazards. Phytochemistry includes the structural compositions, the biosynthetic pathways, functions, mechanism of actions in the living systems, and the medicinal, industrial, and commercial applications of secondary metabolites.
As a pharmacognosy educator with years of experience teaching phytochemical investigation, I have observed that students often find the analytical techniques challenging. Let me tell you: Phytochemical investigation is the key to unlocking the therapeutic potential of medicinal plants.
In this comprehensive guide, I will walk you through the stages of phytochemical investigation, extraction methods, and isolation, purification, and identification techniques—including spectroscopy (UV-Visible, IR, NMR, Mass) and chromatography (Paper, TLC, HPLC). By the end of this article, you will have a thorough understanding of how medicinal plant constituents are investigated. Let us begin!
Dpharmguru’s exam insights:
Phytochemical investigation is frequently tested in pharmacognosy exams. Remember: Phytochemicals are biologically active compounds in plants. Extraction methods include Maceration, Digestion, Percolation, Soxhlet, Supercritical fluid extraction, Microwave-assisted, and Ultrasonic-assisted extraction. Spectroscopy includes UV-Visible (190-900nm), IR (functional groups), NMR (proton and carbon), and Mass Spectrometry (m/z ratio). Chromatography includes Paper (Rf value), TLC, and HPLC. These are classic exam questions!
STAGES OF PHYTOCHEMICAL INVESTIGATION OF DRUG
- Stage-I: Quality controls of raw material investigated on initial level
- Stage-II: Extraction, purification, characterisation, and quality control of interested pharmaceutical components
- Stage-III: Investigations of biosynthetic pathway leading to specific compounds
- Stage-IV: Quantitative and qualitative evaluations using spectroscopy, chromatography, etc.
12.1. EXTRACTION OF DRUGS
12.1.1. Introduction
The process of separating medicinally active constituents of plant and animal tissues with the help of selective solvents and standard procedures is termed extraction.
The extracted products include decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts, or powdered extracts—named as galenicals after Galen (a Greek physician of 2nd century).
The undissolved residue left behind is termed mare.
Properties of Menstruum (Solvent)
- Chemically and physically inert
- Non-toxic
- Inexpensive
- Selective—should dissolve desired active constituents with minimum inert material
Most commonly used solvents: Water, Ethanol, and their mixtures.
Steps of Drug Extraction
- Solvent penetrates the drug
- Drug constituents dissolve in the solvent
- Solution within the cells diffuses out
- Dissolved portion separates from the exhausted drug
12.1.2. Modern Methods of Extraction
- Maceration: Softening—simplest method; Official in I.P., 1966—used for tinctures, extracts, concentrated infusions
- Digestion: Modified maceration at higher temperature (not affecting active ingredients); reflux condenser used if menstruum volatilises easily
- Percolation: From Latin percolare (to pass through)—also termed lixivation; menstruum slowly passes through granulated/powdered drug
- Continuous Hot Extraction (Soxhlet Extraction): Uses Soxhlet apparatus with flask, extractor, and reflux condenser; limited menstruum required; repeated percolation until complete extraction
- Supercritical Fluid Extraction
- Counter Current Extraction
- Microwave Assisted Extraction
- Ultrasonic Assisted Extraction
- Infusion and Decoction
- Pressure Cooker Extraction
12.2. ISOLATION, PURIFICATION, AND IDENTIFICATION OF DRUGS
The progress in isolation and analysis techniques has led to the identification of many unknown compounds. Complete identification depends on measuring properties—melting point, boiling point, optical rotation, retention factor—and comparing data with standard literature using UV, IR, NMR, and Mass Spectrometry.
12.2.1. Spectroscopy
Spectroscopy involves the study of interaction of electromagnetic radiation with matter.
12.2.1.1. Ultraviolet (UV) and Visible Spectroscopy
Measures light absorption in wavelength region from 190-900nm.
- UV region: 190-400nm
- Visible region: 400-900nm
Absorption results from electronic transitions within the molecule. Widely used analytical procedure in pharmacy. Used as a detector in HPLC.
UV and Visible Range of Some Compounds
| Class of Compounds | UV Range (nm) | Visible Range (nm) |
|---|---|---|
| Anthocyanins | 275-330 | 475-555 |
| Betacyanins | 250-275 | 540-554 |
| Carotenes | 400-500 | 400-525 |
| Anthraquinone derivatives | 425-400 | 220-290 |
| Flavonoids | 250-275 | 350-390 |
| Chlorophylls | Very short range | 430-470 and 650-660 |
12.2.1.2. Infrared (IR) Spectroscopy
Used for identification of functional groups. Different functional groups have specific absorbance. Used for finger printing analysis for qualitative identification.
Three IR Regions:
- Near IR: 1250-4000 cm⁻¹
- Mid IR: 4000-400 cm⁻¹
- Far IR: 400-20 cm⁻¹
Methods of Measurement:
- Chloroform or carbon tetrachloride solution
- Potassium bromide pellets
IR Frequencies of Important Groups
| Group | Characteristic Bands (cm⁻¹) |
|---|---|
| Alkanes | 2850-2960 |
| Alkenes | 1600-1700 |
| Alkynes | 2100-2250 |
| Alcohols | 3000-3700 |
| Phenols | 3200-3550 |
| Aldehydes | 1710-1720 |
| Ketones | 1715-1730 |
| Esters | 3300 |
| Carboxylic acids | 3200-3600 |
| Amines | 3400 |
12.2.1.3. Nuclear Magnetic Resonance Spectroscopy (NMR)
Involves absorption of radioactive constituents at limited radio frequency in a particular magnetic field.
- ¹H NMR (Proton NMR): Measures magnetic moments of different hydrogen atoms—determines number of hydrogen atoms and establishes structure
- ¹³C NMR (Carbon Spectroscopy): Measures nature of carbon skeleton
Standard solvent: TMS (Trimethylsilane). Used for elucidation of molecular structure, stereochemistry, and configuration.
12.2.1.4. Mass Spectroscopy (MS)
Measures mass-to-charge ratio (m/z) to identify amount and type of chemicals in a sample. Used for identification or partial structural elucidation of unknown compounds.
12.2.2. Chromatography
Chromatography is the most versatile separation technique—separation by distribution between a stationary phase and a mobile phase. Used for separation, isolation, purification, and identification of components in a mixture.
12.2.2.1. Classification of Chromatography
- Adsorption Chromatography: Solid support (activated alumina, powdered cellulose, silicic acid) packed in a tube
- Columnar chromatography
- Gas solid chromatography
- Thin-layer chromatography (TLC)
- Partition Chromatography: One liquid phase held on stationary phase surface, other liquid (mobile phase) passed through
- Gas-liquid chromatography
- Liquid-liquid chromatography
- Paper chromatography (PC)
12.2.2.2. Paper Chromatography (PC)
Analysis of unknown compound by flow of solvent on specially designed filter paper. Separation occurs due to differences in partition coefficients.
Retardation Factor (Rf):
Rf = Distance moved by solute from original line / Distance moved by solvent from original line12.2.2.3. Thin Layer Chromatography (TLC)
TLC is a simple yet significant chromatographic technique for separation of compounds. Widely used in phytochemical evaluation due to:
- Rapid analysis of herbal extracts with minimum sample clean-up
- Provides qualitative and semi-quantitative information
- Allows quantification of chemical constituents
Data recorded using HPTLC scanner—includes chromatogram, Rf values, colour of separated bands, absorption spectra.
12.2.2.4. High Performance Liquid Chromatography (HPLC)
HPLC is a high resolution chromatographic technique widely used in pharmaceutical industries for:
- Process development
- Identification and detection of adulterants in herbal products
- Identification of pesticide content and mycotoxins
- Separating structurally similar components in plant extracts (digitalis, cinchona, liquorice, ergot)
- Determining stability of pharmaceuticals (e.g., atropine stability studies)
Dpharmguru’s exam insights:
Chromatographic techniques are frequently tested. Remember: Paper chromatography gives Rf values. TLC is simple, rapid, and widely used for herbal drug evaluation. HPLC is high resolution and used for identification and detection of adulterants. The Rf value is the ratio of distance moved by solute to distance moved by solvent. These are classic exam questions!
COMPARISON: SPECTROSCOPIC TECHNIQUES
| Technique | Wavelength/Region | Information Provided |
|---|---|---|
| UV-Visible Spectroscopy | 190-900 nm | Electronic transitions, identity confirmation, detector in HPLC |
| IR Spectroscopy | 4000-400 cm⁻¹ | Functional groups, finger printing analysis |
| ¹H NMR | Radio frequency | Number of hydrogen atoms, molecular structure |
| ¹³C NMR | Radio frequency | Carbon skeleton, stereochemistry, configuration |
| Mass Spectrometry | m/z ratio | Mass-to-charge ratio, structural elucidation |
COMPARISON: CHROMATOGRAPHIC TECHNIQUES
| Technique | Principle | Key Parameter | Application |
|---|---|---|---|
| Paper Chromatography | Partition | Rf value | Analysis of unknown compounds |
| Thin Layer Chromatography | Adsorption | Rf value, colour bands | Rapid analysis, semi-quantitative information |
| HPLC | Adsorption/Partition | Retention time | High resolution, process development, adulterant detection |
FREQUENTLY ASKED QUESTIONS (FAQs)
1. What is phytochemistry?
Phytochemistry involves the study of chemicals (mainly secondary metabolites) that plants produce to protect themselves. It includes structural compositions, biosynthetic pathways, functions, mechanisms of action, and medicinal applications of secondary metabolites.
2. What are the four stages of phytochemical investigation?
The four stages are: Stage-I (quality control of raw material), Stage-II (extraction, purification, characterisation), Stage-III (biosynthetic pathway investigations), and Stage-IV (quantitative and qualitative evaluations).
3. What is the difference between maceration and percolation?
Maceration is the simplest method—drug is soaked in solvent for a period. Percolation involves passing solvent slowly through a column of powdered/granulated drug under gravity.
4. What is the principle of Soxhlet extraction?
Soxhlet extraction is continuous hot extraction using a Soxhlet apparatus. The solvent is repeatedly percolated through the drug, syphoned back, and re-percolated, using limited solvent until complete extraction.
5. What is the Rf value in chromatography?
The Rf value (Retardation Factor) is the ratio of the distance moved by the solute from the original line to the distance moved by the solvent from the original line.
6. What is the difference between TLC and HPLC?
TLC (Thin Layer Chromatography) is simple, rapid, and used for qualitative and semi-quantitative analysis. HPLC (High Performance Liquid Chromatography) is high resolution, more precise, and used for quantitative analysis, process development, and adulterant detection.
SUMMARY
Phytochemical investigation is the foundation of modern pharmacognosy. This guide covered:
- Stages: Quality control, extraction/purification, biosynthetic pathway investigation, quantitative/qualitative evaluation
- Extraction Methods: Maceration, Digestion, Percolation, Soxhlet (continuous hot extraction), Supercritical fluid, Counter current, Microwave-assisted, Ultrasonic-assisted
- Spectroscopy: UV-Visible (190-900nm), IR (functional groups), ¹H NMR and ¹³C NMR (structure elucidation), Mass Spectrometry (m/z ratio)
- Chromatography: Paper (Rf value), TLC (rapid analysis, semi-quantitative), HPLC (high resolution, process development, adulterant detection)
As I always tell my students: “Phytochemical investigation is the bridge between the plant and the pharmacy—it transforms nature’s chemistry into life-saving medicines.”
REFERENCES AND FURTHER READING
- Pharmacy Council of India (PCI). (2022). Pharmacognosy Syllabus. New Delhi: PCI.
- Kokate, C. K., Purohit, A. P., & Gokhale, S. B. (2020). Pharmacognosy (50th ed.). Nirali Prakashan.
- Trease, G. E., & Evans, W. C. (2019). Trease and Evans’ Pharmacognosy (16th ed.). Elsevier.
- Skoog, D. A., West, D. M., Holler, F. J., & Crouch, S. R. (2021). Fundamentals of Analytical Chemistry (10th ed.). Cengage Learning.
- Indian Pharmacopoeia Commission. (2018). Indian Pharmacopoeia (8th ed.). Ghaziabad: IPC.
Disclaimer: This article is for educational purposes only and does not constitute medical advice. Always consult qualified healthcare professionals for medical concerns.
written by:
Dr. N. Sujith Kumar
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