screening for bioactivity

Question 1

How do you extract secondary metabolites

Answer 1

Solvent extraction - course powder soaked in solvent

Question 2

Describe solubility of solvents

Answer 2

Hexane - 0.1 polarity
Propanol - 3.9 polarity
Chloroform - 4.1 polarity
Ethanol/methanol - 5.1 polarity
Water - 10.1 polarity

Question 3

How do you increase extraction efficiency

Answer 3

Small particle size - increases penetration and diffusion of metabolites
Increasing temp, agitation speed and/or duration can increase yields - cause solvent evaporation and potentially destroy sensitive compounds
Increasing volume - increase yield but also time needed for concentration
Energy source or enzymes can increase extraction efficacy at extra cost and risk

Question 4

Why extract secondary metabolites

Answer 4

potential for novel structures and functionalities not conceivable by humans (can’t make them)
Bioactive - regulating environment to increase host survival

Question 5

Describe toxicity testing

Answer 5

All compounds and extracts toxic - find out at what concentrations
Cancer treatments - can use at toxic levels
Cytotoxicity (screened in cultured cells) - immortalised cancer cell lines, primary human cells

Question 6

Describe cell viability assays

Answer 6

Suspension or adherent cells seeded into multi-well plates - incubated with dilution series - cell health determined by measuring metabolic activity, DNA and/or protein content or loss of membrane integrity

Question 7

What does a cell viability assay determine

Answer 7

Relationship between viability and concentration

Question 8

Describe crystal violet stains

Answer 8

Acidic phosphoric acid in DNA (fixed and adherent cells)

Question 9

Describe tetrozolium (MTT) dye

Answer 9

Reduced by mitochondrial enzymes in live cells

Question 10

Describe Tetrazolium (WST) dye

Answer 10

Cell lysis causes release of lactate dehydrogenase (LDH) which reduces (WST)

Question 11

Describe fluorescent calcein

Answer 11

Calcein AM is taken up by live cells and converted to fluorescent calcein after hydrolysis by intracellular enzymes

Question 12

Describe the mechanisms of toxicity

Answer 12

Hydrophobic compounds interact with cell membranes altering permeability or receptor levels
Basic compounds interact with proteins blocking their signalling or enzyme action

Question 13

Describe antimicrobial activity

Answer 13

kill or stop the growth of microorganisms
Should be more toxic for microorganism than us

Question 14

Describe antibacterial/antifungal assays

Answer 14

Determined by observation of growth or through measuring metabolic activity (tetrazolium dye)
Minimum inhibitory concentration (MIC) determined

Question 15

Describe antiviral assays

Answer 15

Adherent cells seeded into culture wells or dishes
Virus dilution added to cells
Infected cells incubated with extracted compounds
Virus plaques revealed through neutral red stain

Question 16

Describe the mechanisms of antimicrobial activity

Answer 16

Hydrophobic compounds interact with bacterial and fungal membranes and can prevent entry of viruses into cells
Phenolic compounds interact with membrane transporters and channels affecting cell integrity

Question 17

Describe antioxidant activity

Answer 17

Inhibit oxidation (produces free radicles)
Neutralise and stabilise free radicles during tissue damage and inflammation
Screened with redox reaction using coloured substrates that contain stable free radicals

Question 18

What are the coloured substrates used in antioxidant activity

Answer 18

DPPH (purple)
Folin-Ciocalteu reagent (orange/yellow)

Question 19

Describe antioxidant assays

Answer 19

Free radicals activated by removal of electrons
Substrate incubated with dilutions of extracted compounds
Reduction of substrate determined by measuring change in colour using spectrophotometry (determines rate and extent of reduction)

Question 20

Mechanisms of antioxidant activity

Answer 20

Phenolic compounds donate electrons (release H+ ions) to stabilise the free radicals
More alcohol groups the more potent compound is

Question 21

Describe high throughput screening

Answer 21

Automated and miniaturised clinical analyser technologies
Specialised detectors capable of reading multi-well microtiter plates
Increased the pace and lowered the cost - enabling screening of natural and synthetic compound libraries
Shift from measures of cell growth or function to binding of molecular targets (GPCR or enzymes)