Diosgenin, a steroidal sapogenin obtained from Dioscorea species, is a valuable pharmaceutical precursor in steroid synthesis. Isolation of diosgenin requires careful selection of Dioscorea tubers, drying and powdering, defatting, optimized solvent extraction, saponin enrichment, and hydrolysis (acidic or enzymatic) followed by extraction and purification using chromatography and recrystallization. Key keywords include diosgenin, Dioscorea, saponins, extraction, hydrolysis, chromatography, solvent polarity, phytochemistry, and analytical techniques (TLC, HPLC, NMR). Understanding yields, quality control and scale-up challenges links plant chemistry to drug development for B. Pharm students. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. Which initial pretreatment step is most important to improve solvent penetration and extraction efficiency from Dioscorea tubers?
- Freezing whole tubers
- Drying and powdering the tubers
- Boiling the tubers in water
- Fermenting fresh tubers
Correct Answer: Drying and powdering the tubers
Q2. What is the primary purpose of a defatting step (e.g., with petroleum ether) before extracting saponins from plant powder?
- To hydrolyze glycosides to aglycones
- To remove nonpolar lipids that interfere with later extraction
- To precipitate saponins selectively
- To convert diosgenin into a more soluble form
Correct Answer: To remove nonpolar lipids that interfere with later extraction
Q3. Which solvent system is commonly used for initial extraction of steroidal saponins from Dioscorea?
- Hexane only
- Absolute acetone
- Aqueous ethanol or methanol
- Pure water at room temperature
Correct Answer: Aqueous ethanol or methanol
Q4. Why are saponins typically enriched by partitioning into n-butanol after aqueous extraction?
- Butanol selectively extracts nonpolar waxes
- Butanol concentrates glycosides due to moderate polarity and partitioning behavior
- Butanol hydrolyzes the glycosidic bonds
- Butanol converts diosgenin into a salt
Correct Answer: Butanol concentrates glycosides due to moderate polarity and partitioning behavior
Q5. Which reaction condition is most commonly used to cleave sugar moieties from saponins to release diosgenin?
- Neutral aqueous extraction
- Strong base (NaOH) reflux
- Acid hydrolysis using dilute HCl or H2SO4
- Cold enzymatic digestion with lipases
Correct Answer: Acid hydrolysis using dilute HCl or H2SO4
Q6. During acid hydrolysis of steroidal saponins, which organic solvent is typically used to extract liberated diosgenin?
- Chloroform or dichloromethane
- Water
- n-Butanol
- Petroleum ether only
Correct Answer: Chloroform or dichloromethane
Q7. Which analytical technique is most suitable for monitoring purity and separation of diosgenin during isolation?
- Thin-layer chromatography (TLC) followed by visualization
- Gravimetric analysis only
- Simple frothing test for saponins
- Colorimetric starch test
Correct Answer: Thin-layer chromatography (TLC) followed by visualization
Q8. A commonly used TLC solvent system for steroidal aglycones like diosgenin is:
- n-Hexane : ethyl acetate (9:1)
- Chloroform : methanol (9:1) or chloroform : acetone mixtures
- Water : methanol (1:1)
- Acetone : acetic acid (1:1)
Correct Answer: Chloroform : methanol (9:1) or chloroform : acetone mixtures
Q9. Which color reagent test is commonly used to indicate the presence of steroidal sapogenins like diosgenin?
- Liebermann-Burchard test producing blue-green colors
- Biuret test for proteins
- Fehling’s test for reducing sugars
- Kjeldahl test for nitrogen
Correct Answer: Liebermann-Burchard test producing blue-green colors
Q10. What is the structural classification of diosgenin?
- Alkaloid
- Terpenoid steroidal sapogenin
- Flavonoid glycoside
- Simple phenolic acid
Correct Answer: Terpenoid steroidal sapogenin
Q11. Which instrumental method provides definitive structural confirmation of diosgenin after isolation?
- Melting point determination only
- Nuclear magnetic resonance (NMR) and mass spectrometry (MS)
- Paper chromatography
- Boiling point measurement
Correct Answer: Nuclear magnetic resonance (NMR) and mass spectrometry (MS)
Q12. In scale-up of diosgenin isolation, which factor most critically impacts yield and reproducibility?
- Source of glassware
- Selection and consistency of plant material and standardized extraction parameters
- Order of adding indicators in TLC
- Type of pipettes used
Correct Answer: Selection and consistency of plant material and standardized extraction parameters
Q13. Enzymatic hydrolysis of saponins to produce diosgenin uses which type of enzyme to remove sugar residues?
- Lipases
- Glycosidases (e.g., β-glucosidase)
- Proteases
- Cellulases
Correct Answer: Glycosidases (e.g., β-glucosidase)
Q14. What is a common impurity that co-extracts with diosgenin and must be removed during purification?
- Simple sugars only
- Chlorophylls and other nonpolar plant pigments
- Inorganic salts
- Heavy metals exclusively
Correct Answer: Chlorophylls and other nonpolar plant pigments
Q15. Which chromatographic technique is best for preparative purification of isolated diosgenin at gram scale?
- Analytical paper chromatography
- Open column chromatography on silica gel followed by recrystallization
- Thin-layer chromatography only
- Gas chromatography without derivatization
Correct Answer: Open column chromatography on silica gel followed by recrystallization
Q16. During acidic hydrolysis, prolonged exposure to strong acid can cause which undesirable change in diosgenin?
- Increase in water solubility
- Acid-catalyzed degradation and structural rearrangement
- Conversion to a protein conjugate
- Spontaneous polymerization into polysaccharides
Correct Answer: Acid-catalyzed degradation and structural rearrangement
Q17. Which solvent is commonly used to recrystallize diosgenin to improve purity?
- Water
- Ethanol or methanol with controlled cooling
- n-Hexane only
- Conc. HCl
Correct Answer: Ethanol or methanol with controlled cooling
Q18. Which safety consideration is essential when using chloroform or dichloromethane during extraction of diosgenin?
- No ventilation required for short times
- Use in a fume hood with appropriate PPE due to toxicity and volatility
- They are non-toxic water-safe solvents
- They can be heated in open air without risk
Correct Answer: Use in a fume hood with appropriate PPE due to toxicity and volatility
Q19. The major advantage of HPLC analysis for diosgenin over TLC is:
- Lower cost of equipment
- Quantitative, higher resolution and reproducible retention times
- HPLC does not require solvents
- HPLC can be performed on a paper strip
Correct Answer: Quantitative, higher resolution and reproducible retention times
Q20. In the context of diosgenin isolation, what does the term “sapogenin” refer to?
- The sugar moiety attached to a steroid
- The aglycone (non-sugar) part of a saponin, e.g., diosgenin
- A synthetic steroid precursor unrelated to plants
- An alkaloid class from Dioscorea
Correct Answer: The aglycone (non-sugar) part of a saponin, e.g., diosgenin
Q21. Which parameter is NOT typically critical when optimizing solvent extraction for diosgenin?
- Solvent polarity
- Extraction temperature and time
- Particle size of powdered plant material
- Brand of glass bottles used to store solvent
Correct Answer: Brand of glass bottles used to store solvent
Q22. A typical Rf value for diosgenin on silica TLC in an appropriate solvent system helps to:
- Determine molecular weight directly
- Track diosgenin during purification and compare standards
- Convert it into a glycoside
- Measure the melting point
Correct Answer: Track diosgenin during purification and compare standards
Q23. What is the role of acid neutralization and washing after hydrolysis when isolating diosgenin?
- To enhance acid strength for further hydrolysis
- To remove residual acid and salts that may contaminate organic extracts
- To precipitate diosgenin as a sulfate salt
- To oxidize diosgenin to an inactive form
Correct Answer: To remove residual acid and salts that may contaminate organic extracts
Q24. Which physical property of diosgenin assists in its identification during purification?
- Typical steroidal melting point and crystalline appearance
- Strong fruity odor
- High solubility in cold water
- Bright green fluorescence under UV
Correct Answer: Typical steroidal melting point and crystalline appearance
Q25. When quantifying diosgenin content in extracts, which approach provides accurate percentage yield determination?
- Estimating by eye during recrystallization
- Using HPLC with an authentic diosgenin standard for calibration
- Counting number of TLC spots
- Measuring color change in the Liebermann-Burchard test only
Correct Answer: Using HPLC with an authentic diosgenin standard for calibration
Q26. Which environmental or ethical consideration is important when sourcing Dioscorea for diosgenin isolation?
- Overharvesting wild populations without sustainable practices
- Using nonrenewable solvents only
- Preferentially selecting endangered species to increase yield
- Ignoring local regulations on plant collection
Correct Answer: Overharvesting wild populations without sustainable practices
Q27. Chemical derivatization of diosgenin for GC analysis is necessary because:
- Diosgenin is too volatile for GC
- Diosgenin lacks sufficient volatility and may need silylation for GC-MS
- Diosgenin is ionic and cannot enter the GC column
- Derivatization converts it to a sugar
Correct Answer: Diosgenin lacks sufficient volatility and may need silylation for GC-MS
Q28. Which of the following best improves selectivity in column chromatography for diosgenin purification?
- Using a highly polar stationary phase like silica with gradient elution from nonpolar to moderately polar solvents
- Running the column with pure water only
- Using paper as stationary phase in a column
- Skipping chromatography and relying only on filtration
Correct Answer: Using a highly polar stationary phase like silica with gradient elution from nonpolar to moderately polar solvents
Q29. During method validation for an assay of diosgenin, which parameter is essential to establish?
- Color of the reagent bottle cap
- Linearity, accuracy, precision, limit of detection and quantitation
- Number of TLC plates used historically
- Whether the investigator likes the smell of solvent
Correct Answer: Linearity, accuracy, precision, limit of detection and quantitation
Q30. In an experimental protocol, an unexpectedly low diosgenin yield might result from which common issue?
- Inadequate hydrolysis conditions leading to incomplete cleavage of glycosides
- Using an excess of authentic standard
- Too vigorous TLC visualization
- Overuse of plastic gloves during extraction
Correct Answer: Inadequate hydrolysis conditions leading to incomplete cleavage of glycosides
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