METHODOLOGY OF PHARMACOGNOSY
BASED ON METHODOLOGY OF BASIC SCIENTIFIC DISCIPLINES
1. Corrected botanic determination (each expedition should „hire“ systematic botanist)
2. Macroscopic analysis of drug
3. Microscopic analysis of drug
4. Chemical control of drug
- proof of content compounds
(physical methods – fluorescence, microsublimation, chromatography)
(biological methods – haemolytic activity, agglutination effect)
(chemical methods – colour and precipitation reaction after previously carried out extraction of
tested compounds, histochemistry)
- determination of compounds content (quantification)
methods derived from characteristic of quantified compound
5. Study of mechanisms of biosynthesis of effective compounds
METHODOLOGY OF PHARMACOGNOSY
6. Preliminary assays of biological activity (selection of material for further studies)
(effect antibacterial, antimycotic, antiviral, cytotoxic, antihypertensive, antiphlogistic,
spasmolytic ….)
(Selection based on relations of species and genera, based on folk medicine, random
selection - intuition)
7. Separation and isolation of content compounds from experimental
material
(distillation, crystallization, liquid-liquid extraction, preparative chromatography)
8. Detection of isolátes and proof of purity
physical: melting point, index lomu, optical rotation, CD, GC, HPLC)
chemical: creation of color products with specific reagents, degradation
biologic: haemolysis, agglutination of erythrocytes, determination of bitterness
9. Characterization and identification of isolates
STUDIES OF BIOLOGIC ACTIVITIES
CHARAKTERISATION AND IDENTIFICATION
Chemical structure
• Mass spectrometry
• Electron spectrometry
• Infrared spectrometry
• Raman spectrometry
• 1H NMR, 13C NMR
• ORD
• CD
• RTG
• Degradation
• Derivatives
• Synthesis
Parameters
Necessary amounts of compound
μg mg g
% to be sure _ RTG TOTAL SYNTHESIS
IR
90 _ DEGRADATION
PREPARATION OF DERIVATIVES
_
70 _ HRMS
ORD
_ C,H,N,S
13C NMR CD
50 _ 1H NMR
_ Raman
30 _
MS UV
_ HPLC
GC
10 _
PC, TLC
_
ISOLATION OF NATURAL COMPOUNDS
1. EXTRACTION: (series of solvents with different polarity – eluotropic series)
solid compound with liquid
• periodical (maceration, digestion)
• repeated
• continual (percolation, Soxhlet)
liquid with liquid (perforation, shaking)
2. DISTILLATION
• at normal pressure (with capillary, with boiling stones)
• vacuum (RVO)
• molecular
• with rectification column (rectification)
3. SUBLIMATION (purines, quinones)
4. CRYSTALLISATION
5. PRECIPITATION
• change of solvent polarity, change of pH (purification of alkaloids)
• salting
ISOLATION OF NATURAL COMPOUNDS
6. FILTRATION, ULTRACENTRIFUGATION, DIALYSIS
7. CHROMATOGRAPHIC METHODS (Cvět, Reichstein, Mistrujakov, Stahl, Hostetmann)
• Classification according principles: adsorption, partitioning, liquid, gas, ion exchange, gel filtration,
affinity
• Classification according to the process: column, paper, thin layer, high performance, droplet counter-
current
• Classification according to the polarity: at polar stationary phases – normal, at non-polar stationary
phases - reversed
8. ELECTROPHORETIC METHODS
• without bearer
• paper (mobility is caused by charge, molecule size…)
• in gels (starch, agarose, polyacrylamid)
• isotachoforesis (separates anions or cations only)
• isoelectric focusation (pH gradient)
9. COMBINATION OF SEPARATION METHODS
10. SEPARATION BASED ON DIFFERENT PRINCIPLES
• counter-current shaking (Creig)
• centrifugation (separation according to the density – high-molecular compounds, in gradient of sacharose)
• flotation (enrichment of surface active compounds in interface of gas-liquid system
ISOLATION OF NATURAL COMPOUNDS
11. CHEMICAL METHODS
• derivatisation (acetylation, etherification)
• Preparation of molecular compounds - adducts (for example sterols + digitonin)
• clathrates
12. FERMENTATION METHODS
(purification of plant extracts from sugars with help of yeast)