BIO127 UNIT 5B: Secondary Metabolites (Bioassay Techniques)

Medicinal plants

Any plant that contains substances in its parts that can be used therapeutically or as precursors for drug synthesis. This can include the entire plant or specific parts (like roots, leaves, or flowers) that have medicinal properties

Bio-active

Simply a substance with biological activity, also known as “biologically active“

Plant extract

Substance or an active substance with desirable properties removed from the tissues of a plant, frequently by treating it with a solvent, to be used for a particular purpose

Phytochemicals

Bioactive compounds obtained from crude extracts or essential oils

1. Plant collection

2. Extraction

3. Isolation

4. Bioassay

5. Structural characterization

6. Lead optimization

6 step-process of botanical bioassay:

Plant collection

Proper identification and collection of plant species with potential therapeutic properties, using traditional knowledge from indigenous and local communities (ethnobotanical research and field surveys)

Extraction

Separating the medicinally active mixture of many naturally active compounds usually contained inside plant materials (tissues) using selective solvents through the standard procedure

Isolation

Techniques that separate and purify bioactive components from non- active substances (HPLC, solvent partitioning or crystallization).

Bioassay

Evaluates the pharmacological efficacy of the isolated compounds through in vitro (lab-based experiments) and in vivo (tests on living organisms). designed to assess the compounds' effectiveness, safety, and possible mechanisms of action,

in vitro

Latin for "in glass," referring to scientific experiments, tests, or processes conducted outside a living organism, typically in controlled laboratory environments like test tubes, petri dishes, or flasks

in vivo

Latin for "within the living," refers to biological experiments or procedures conducted on whole, living organisms—such as animals, plants, or humans—rather than partial, dead, or isolated cells.

Structural characterization

Techniques such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry are used that offers detailed information on the molecular structure, which is crucial for understanding how these molecules can be developed into medicines.

Lead optimization

The final, iterative phase of drug discovery where chemists modify a "lead compound's" structure to improve its effectiveness, safety, and pharmacokinetic properties

menstruum

Solvent of extraction of medicinal plants. The choice of solvent depends on the type of plant, part of plant, nature of the bioactive compounds, and the availability of solvent.

Liquid-Liquid Extraction

Miscible solvent combinations (e.g., water with dichloromethane, water with ether) are commonly employed to facilitate the separation of compounds.

dichloromethane, ether

Miscible solvent combinations: Water with ____, (or) ____.

polar solvents

These include water, methanol, and ethanol, which are typically used to extract polar compounds from plant materials

Nonpolar solvents

Examples include hexane and dichloromethane, which are effective for extracting nonpolar compounds.

Selectivity

The solvent should preferentially extract the desired active compounds while leaving inert materials behind.

Safety

Non-toxic and non-flammable solvents are preferred for safe handling.

Cost

Economical choices are essential for large-scale extraction.

Reactivity

The solvent should not chemically interact with the extract

Recovery

The solvent should be easily recoverable after extraction

Viscosity

Low viscosity facilitates better penetration into the plant material.

Boiling temperature

A lower boiling point is preferred to prevent degradation of heat-sensitive compounds during the extraction process.

1. Maceration

2. Infusion

3. Digestion

4. Decoction

5. Percolation

6. Soxhlet

6 steps of conventional method of extraction

Maceration

• Coarsely powdered plant material (leaves, stem bark, or root bark) soaked in menstruum, 3+ days, with periodic agitation (stirring or shaking)

• Micelle is separated from the solid residue (marc) through filtration or decantation.

• Micelle is concentrated by evaporating the solvent.

• Advantages: Low investment cost, modulation of selectivity by solvent choice

• Limitation: Thermal destruction of some compounds

Infusion

• Similar to maceration

• Short-term steeping in solvent

• Extracting readily soluble bioactive constituents

• Advantages: Less time, less heat consumed to make concentrated extract

• Limitation: Large amounts of solvent required

Digestion

• Heated in a water bath at 50°C heat to reduce the viscosity of the solvent and increas extraction efficiency

• Ideal for easily soluble materials

• Limitation: Large amounts of solvent are required

Decoction

• Continuous hot extraction with water solvent to speed up extraction for about 15 minutes.

• Advantages: for heat-stable, water-soluble materials

• Limitation: Large amounts of solvent are required

Hydrodistillation

• Steam/water separation

• Bioactive compounds separate automatically from the water

• Limitation: Volatile components may be lost if extraction temperature is too high

Soxhlet Extraction

• Continuous hot extraction using Soxhlet extractor

• Plant material in porous bag; solvent is heated in a flask

  • Vaporized solvent travels through condenser, returning to the extraction chamber, where it extracts the active compounds.

• Advantages: Simple, applicable at high temp, for materials partially-soluble in solvent, and insoluble impurities

• Limitation: not suitable for heat-sensitive materials

Percolation

• Utilizes a percolator

• Material moistened with the solvent and left for some time before transferred to the percolator.

• Mixture is saturated with solvent that then flows through the material due to gravity, continuously adding more solvent until a desired volume is extracted.

• Extract is then filtered and decanted

• Advantages: increased yield due to better contact time between solvent and material

• Limitation: time consuming, lots of solvents

1. Microwave-Assisted, MAE

2. Enzyme-Assisted, EAE

3. Ultrasound-Assisted, UAE

4. Supercritical Fluid, SFE

4 novel methods:

(Full, acronym)

MAE

Acronym only

• Uses microwave energy by heating the solvent and plant material

• effective for extracting flavonoids by enabling quick penetration of solvent into plant matrix

• Advantage: less time, less solvent

• Limitation: NOT for nonpolar solvents, degradation of sensitive compounds due to high temp

EAE

Acronym only

• Environmentally friendly, efficient, and mild technique

• Extract essential oils from bay leaves, lycopene from tomatoes, curcumin from turmeric

• Use of cellulases and pectinases that break down cell walls

• Limitation: Additional long operation in wet conditions

UAE

Acronym only

• High-frequency sound waves disrupt the cell walls

• Mixed in an ultrasonic extractor.

• Advantage: low cost, effective

• Limitation: High power consumption, challenging to reproduce, degradation of sensitive compounds

SFE

Acronym only

• uses substances like carbon dioxide compressed and heated beyond their critical point—to extract bioactive compounds based on solubility differences

• extract essential oils, flavonoids, phenolic compounds

• rosemary, sage, savoury, Catharanthus roseus, and radish leaves

• Advantages: non-toxic CO2, avoids organic solvents, low temp., selective extraction, prevents oxidation, automated

• Limitation: equipment cost

1. Dragendorff’s Test

2. Wagner’s Test

3. Mayer’s Test

4. Hager’s Test

4 types of phytochemical screening/assay:

Dragendorff’s Test

Introduced by an Estonian-German Professor Johann Georg Noel Dragendorff (1836-1898) in the middle of the 19th century (1866). It is a solution of potassium bismuth iodide composing of basic bismuth nitrate (Bi(NO₃)₃), tartaric acid, and potassium iodide (KI), and when contact with alkaloids produces an orange or orange red precipitate, indicating the presence of alkaloids

Wagner’s Test

Reagent is prepared by dissolving 1 gm of iodine and 3 gm of potassium iodide in 50 mL of distilled water. This test is utilized to detect alkaloids, yielding a reddish- brown precipitate by reaction.

Mayer’s Test

Reagent a solution of potassium mercuric iodide This test is utilized to detect alkaloids, yielding a whitish or cream colored precipitate.

Hager’s Test

Adding a saturated ferric solution will yield a yellow precipitate if alkaloids are present.

potassium bismuth iodide

Dragendorff's reagent (DR) composed of basic bismuth nitrate (Bi(NO₃)₃), tartaric acid, and potassium iodide (KI).

iodine, potassium iodide, distilled water.

Wagner reagent constituents (separate into commas)

potassium mercuric iodide

Mayer’s reagent

ferric solution

Hager’s reagent

1. Bontrager’s test

2. Legals test

3. Keller-Killiani test

3 tests for glycosides, help distinguish between various glycoside classes, such as cardiac glycosides, anthraquinone glycosides, and flavonoid glycosides.

1. Libermann Burchard’s test

2. Salkowski’s test

2 tests for steroids and triterpenoids

Bontrager's Test

• Used to identify anthracene glycosides/anthraquinone glycosides

• Extract dissolved in dilute HCl, followed by adding FeCl3

• Pink color or deep red coloration of aqueous layer

Legals Test

• indicating cardiac glycoside

• Mixture of sodium nitroprusside, sodium hydroxide,

• pink to blood-red precipitate

Keller-Killiani Test

• identifies digitoxose (cardiac glycoside)

• reddish-brown layer that changes to bluish-green when H2SO4 is added

Libermann Burchard’s Test

• presence of unsaturated steroids and triterpenes, particularly cholesterol

• violet to blue-colored ring at the junction of two liquids after addt’n of acetic anhydride and H2SO4

Salkowski’s Test

• used to detect the presence of triterpenes like sterols, particularly cholesterol

• golden-yellow precipitate appears upon adding H2SO4 to an extract filtered through chloroform

1. Gold Beater’s Skin Test

2. Gelatin's Test

2 Tannin tests

Gold Beater’s Skin Test

• using a piece of a membrane from ox intestine to react with a solution w/ tannin

• skin darkens or turns brown

Gelatin's Test

• solution w/ tannins mixed with a gelatin solution

• white or buff-colored precipitate forms

1. Shinoda’s test

2. Lead acetate test

3. Alkaline reagent test

3 flavonoids tests

Shinoda’s Test

• magnesium and HCl to flavonoid extract

• color change to pink or magenta

Lead Acetate Test

• lead acetate w/ sol’n of flavonoid

• yellow ppt.

Alkaline Reagent Test

• addition of NaOH w/ flavonoid extract gives a yellow color

• decolorization with HCl

1. Ferric chloride test

Phenols test

Ferric Chloride Test

• dilute FeCl3 and phenols extract

• blue, purple, green, red-brown colored complex

1. Biuret Test

2. Ninhydrin Test

2 protein tests

Ninhydrin Test

• reaction with amino group results in a deep blue color called Ruhemann's purple

• used in forensic science to develop latent fingerprints on porous surfaces like paper

Biuret Test

• used to detect the presence of peptide bonds (bonds between amino acids in proteins)

• NaOH and copper sulfate

• violet color

1. Saponin test

2. Xanthoproteic test

2 saponin tests

Saponin test

• “frothing test”, used to detect the presence of saponins in plant extracts

• formation of persistent, stable foam after shaking sample with water

Xanthoproteic Test

• presence of aromatic amino acids (like tyrosine and tryptophan) in a protein solution

• concentrated nitric acid.

• yellow color