Biological screening of herbal drugs: in vitro antioxidant, antimicrobial, anticancer assays MCQs With Answer

Biological screening of herbal drugs: in vitro antioxidant, antimicrobial, anticancer assays MCQs With Answer

Introduction: This quiz set targets M.Pharm students studying Advanced Pharmacognosy-II and focuses on practical and theoretical aspects of in vitro biological screening of herbal drugs. It covers antioxidant assays (DPPH, ABTS, FRAP, ORAC, Folin–Ciocalteu), antimicrobial methods (disk diffusion, broth microdilution, MIC/MBC, bioautography), and anticancer cell-based assays (MTT, SRB, clonogenic, Annexin V/PI, caspase, flow cytometry). Questions emphasize assay principles, standard controls, interpretation of results, critical parameters, and best laboratory practices for reliable, reproducible data. This resource helps consolidate core concepts needed for experimental design and result evaluation in herbal drug screening.

Q1. Which wavelength is typically monitored for the DPPH radical scavenging assay?

• 734 nm
• 593 nm
• 517 nm
• 450 nm

Correct Answer: 517 nm

Q2. The FRAP (Ferric Reducing Antioxidant Power) assay is based on which principal reaction?

• Scavenging of DPPH radicals with decrease in absorbance
• Reduction of Fe3+-TPTZ complex to Fe2+-TPTZ measured at 593 nm
• Generation of peroxyl radicals measured by fluorescein decay
• Colorimetric reaction with Folin–Ciocalteu reagent

Correct Answer: Reduction of Fe3+-TPTZ complex to Fe2+-TPTZ measured at 593 nm

Q3. Which standard antioxidant is commonly used to express results in the ABTS radical cation decolorization assay?

• Ascorbic acid
• Trolox
• BHT (butylated hydroxytoluene)
• Gallic acid

Correct Answer: Trolox

Q4. The Folin–Ciocalteu reagent is primarily used to quantify which component in herbal extracts?

• Total flavonoid content (expressed as quercetin equivalents)
• Total phenolic content (expressed as gallic acid equivalents)
• Protein concentration (expressed as BSA equivalents)

Correct Answer: Total phenolic content (expressed as gallic acid equivalents)

Q5. The ORAC (Oxygen Radical Absorbance Capacity) assay measures antioxidant activity by monitoring:

• Reduction of ferric to ferrous ions
• Inhibition of DPPH radical at 517 nm
• Protection of fluorescein from peroxyl-radical-induced decay (area under the curve)
• Color development with Folin reagent

Correct Answer: Protection of fluorescein from peroxyl-radical-induced decay (area under the curve)

Q6. In broth microdilution antimicrobial testing, the MIC (Minimum Inhibitory Concentration) is defined as:

• The lowest concentration that kills 99.9% of the initial inoculum
• The lowest concentration that inhibits visible growth
• The highest non-toxic concentration for mammalian cells
• The zone diameter measured in millimeters on agar plates

Correct Answer: The lowest concentration that inhibits visible growth

Q7. What does MBC (Minimum Bactericidal Concentration) represent in antimicrobial assays?

• The concentration that inhibits visible growth after 24 hours
• The concentration that prevents biofilm formation
• The lowest concentration that kills ≥99.9% of the initial bacterial inoculum
• The highest concentration showing no cytotoxicity to mammalian cells

Correct Answer: The lowest concentration that kills ≥99.9% of the initial bacterial inoculum

Q8. Results from an agar disk diffusion antimicrobial assay are reported as:

• MIC values in µg/mL
• Zone of inhibition diameters in millimeters
• Percent reduction in CFU/mL
• Optical density at 600 nm

Correct Answer: Zone of inhibition diameters in millimeters

Q9. Which technique combines chromatographic separation and microbial overlay to localize antimicrobial constituents in a complex extract?

• Broth microdilution
• Bioautography (TLC‑bioautography)
• Time-kill assay
• MIC gradient strip test (Etest)

Correct Answer: Bioautography (TLC‑bioautography)

Q10. Which culture medium is commonly used for routine antifungal susceptibility testing of yeasts and molds?

• MacConkey agar
• Mueller‑Hinton agar
• Sabouraud dextrose agar
• Blood agar

Correct Answer: Sabouraud dextrose agar

Q11. The MTT assay assesses cell viability by measuring the reduction of MTT to formazan by:

• DNA polymerases in the nucleus
• Lysosomal hydrolases
• Mitochondrial dehydrogenases in metabolically active cells
• Extracellular proteases released during necrosis

Correct Answer: Mitochondrial dehydrogenases in metabolically active cells

Q12. The SRB (sulforhodamine B) assay quantifies cell growth by binding to which cellular component?

• Cell membrane lipids
• Intracellular ATP
• Total cellular protein content
• Genomic DNA content

Correct Answer: Total cellular protein content

Q13. The IC50 value in anticancer in vitro assays is defined as:

• The concentration required to kill 100% of cells
• The concentration causing 50% inhibition of the measured biological response (e.g., cell viability)
• The maximal non-toxic concentration to normal cells
• The time point at which half the cells undergo apoptosis

Correct Answer: The concentration causing 50% inhibition of the measured biological response (e.g., cell viability)

Q14. How is Selectivity Index (SI) commonly calculated for anticancer screening of herbal extracts?

• IC50 (cancer cell) / IC50 (normal cell)
• IC50 (normal cell) / IC50 (cancer cell)
• MIC / MBC
• Zone of inhibition (mm) / IC50 (µg/mL)

Correct Answer: IC50 (normal cell) / IC50 (cancer cell)

Q15. In flow cytometric Annexin V/PI staining, a cell population that is Annexin V positive and PI negative is interpreted as:

• Viable and healthy cells
• Early apoptotic cells
• Late apoptotic or necrotic cells
• Necrotic only

Correct Answer: Early apoptotic cells

Q16. The clonogenic assay is particularly useful for assessing which property of cancer cells after treatment?

• Short-term metabolic activity after 24 hours
• Immediate membrane permeability changes
• Long-term reproductive viability and colony-forming ability
• Acute ROS generation within minutes

Correct Answer: Long-term reproductive viability and colony-forming ability

Q17. LDH release assays are commonly used to detect which cellular event following cytotoxic treatment?

• Apoptosis via caspase activation
• DNA fragmentation measured by comet assay
• Membrane damage and necrotic cell death
• Mitochondrial membrane potential hyperpolarization

Correct Answer: Membrane damage and necrotic cell death

Q18. In bactericidal time‑kill studies, bactericidal activity is conventionally defined as:

• Any decrease in CFU compared with control
• ≥1 log10 reduction in CFU/mL over 24 hours
• ≥3 log10 reduction in CFU/mL from the starting inoculum
• Complete eradication to 0 CFU/mL

Correct Answer: ≥3 log10 reduction in CFU/mL from the starting inoculum

Q19. The comet assay (single-cell gel electrophoresis) is primarily used to detect:

• Protein oxidation levels in tissue homogenates
• DNA strand breaks and alkali-labile sites at single-cell level
• Membrane lipid peroxidation measured by TBARS
• Mutagenicity in Salmonella typhimurium strains

Correct Answer: DNA strand breaks and alkali-labile sites at single-cell level

Q20. When preparing test samples for cell‑based anticancer assays, what is the commonly recommended maximum final concentration of DMSO in culture to avoid solvent-induced cytotoxicity?

• ≥5% v/v
• ≤0.5–1% v/v (commonly ≤0.5%)
• ≈2–3% v/v
• 10% v/v

Correct Answer: ≤0.5–1% v/v (commonly ≤0.5%)

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