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Selection and collectionof herbal drugs
Prof. Dr. Talal Aburjai
Plant Selection • Selection of plant material for study in drug-
discovery programs are performed by different approaches:
- Random selection- Ethnopharmacology- Chemotaxonomy- Geographical- Computer-based selection methods- Literature information selection technique (LIST
correlates biological activity, botanical facts, and chemotaxonomic information)
• ecological strategy
• ethno-medicinal strategy
• environmental strategy
• taxonomical strategy
• random strategy
Collection Strategies
• Plant selection should normally involve a literature survey of the floristic diversity of the area of interest and should include the plant medicinal uses in the region where collection will take place.
• Points to be kept in mind:• Specimens should be healthy (changing
metabolites).• Variations in the collection site altitude, plant age,
climate and soil type can influence the concentration and even kind of compounds.
• Different organs produce and/or accumulate different profiles of secondary metabolites.
• After collection the plant material should be identified in a local national herbarium or authenticated by a taxonomist.
• A voucher specimens should be kept in an appropriate protected place for further references.
Collection Card
• (i) Collection number: A serial number specific to collector.
• (ii) The name of the plant: • (iii) Locality: This should be as detailed as possible.• (iv) Description:• (v) Habitat: • (vi) Date.• (vii) Names of collector's.• (viii) Notes: Space should be left to note the name of the
person who makes the final determination (identification), the date on which it is made and the place were the specimen is sent or stored.
Most of the current ethnopharmacological studies are conventional: “listing of the plant species, their scientific and common names, their traditional uses, and methods of use”.
These studies usually lack certain specific information regarding:
1. The extent of agreement (consensus) among the informants regarding the use of certain plants for treatment of a particular ailment or ailment category................................................... .......... ICF
2. How much (how frequent) the community uses a particular plant ................................................... UV
Motives
The ICF is employed to deduce the homogeneity of the information about the use of plants to treat a particular category of ailments. It is an indicative value of how much the informants are consistent and the extent they agree about the use of certain plant species for treatment of a given ailment or ailments category. This means that as the ICF value of a given ailments category approximates 1.0 as the number of species (taxa) mentioned to be used to treat this category or (ailment) approximates 1.0 (i.e. one plant). The relative importance of each plant species known locally to be used as herbal remedy was, however, reported as Use Value (UV). This value is even helpful in determining the plants with the highest use (most frequently indicated) in the treatment of an ailment (or ailment category) with a given ICF value.
DefenitionsThus,
Use of Informant Consensus Factor (ICF) and Plant Use Value (UV) models, developed by Heinrich et al.1 and Gazzaneo et al.2, respectively, to evaluate the current ethnopharmacological knowledge and status in Jordanian communities.
Understanding the impact of these factors on the proper selection of plants for:
1. Therapeutic use2. Drug discovery
Objectives
(1) M. Heinrich, A. Ankli, B. Frei, C. Weimann, and O. Sticher. Social Science & Medicine 47(11), 1859-1871 (1998). (2) L. Gazzaneo, R. de Lucena, and U. de Albuquerque. Journal of Ethnobiology and Ethnomedicine 1(9), 1-8 (2005)
Methodology In each region, the survey collected information regarding the use of medicinal plants in the treatment of various ailments and ailment categories.
These data were utilized to calculate the ICF and UV values by applying their corresponding formulas.
Informants Consensus Factor (ICF) is calculated as follows:* FIC = (nur - nt)/(nur – 1) Where,nur: number of use reports per each category andnt: number of taxa used.
The use-value (UV) is calculated as follows:* UV = ΣU/n Where,UV: use value of a species,U: number of uses per species, andn: number of informants.
Results The ethnopharmacological information were so far collected from two Jordanian regions (Ajloun1 and Mujib2).
The ICF values calculated for different ailment categories such as liver problems, diabetes, and digestive problems were included in appropriate tables (e.g. Table I, Mujib).
The UV values of the surveyed medicinal plants were also calculated and added to the plant list (e.g. Table II, Mujib)
1 T. Aburjai, M. Hudaib, R. Tayyem, M. Yousef, M. Qishawi. Journal of Ethnopharmacology, 110(2), 294-304 (2007)2 M. Hudaib, M. Muhammad, R. Tayyem, M. Yousef, M. Aburjai, T. Aburjai. Journal of Ethnopharmacology, submitted (2007)
Conclusion
The ICF and UV values, calculated in both regions, allowed for more guided selection of the species used for treatment of different ailments, particularly those with the highest ICF values. The plant(s) selected can be, then, used as a medicine (traditional use) or as a subject for:
- further laboratory evaluation by in vitro and in vivo tests to validate their traditional uses. - further analysis and investigation for
development and discovery of new drugs.
Table I. Informant Consensus Factor (Fic) categorized by medicinal use for corporal ailment.
SN Ailment Category Species% All
SpeciesUse
Citation% All Use Citations
ICF
1 Cancer 6 10.53 8 2.61 0.29
2 Skin and scalp problems 9 15.79 14 4.58 0.38
3 Liver problems 5 8.77 11 3.59 0.60
4 Kidney problems 5 8.77 12 3.92 0.64
5 Skeletal system problems and pain 11 19.30 31 10.13 0.67
6 Vascular System problems 4 7.02 11 3.59 0.70
7 Female sterility and delivery problems 6 10.53 19 6.21 0.72
8 Respiratory problems 10 17.54 37 12.09 0.75
9 Diabetes 11 19.30 51 16.67 0.80
10 Digestive problems 18 31.58 112 36.60 0.85
Table II. Plants and herbs used for treatment of various human ailments in Mujib Reserve Study Area.
# Scientific Name (voucher
specimen)
Synonyms Arabic Family Name Status Claimed Usage
Part Use
Recommended uses UV Methods of Use
1 Achillea santolina L.
(AS-MJ)
A.damascena
A.sulpherea
Kaisoom
Asteraceae C Aerial Parts
Carminative, Depurative, Stomachaches, antispasmodic and diabetes
0.23
Infusion and Decoction are prepared in water and taken orally 3 times daily
2 Alhagi maurorum
Medik.(AM-MJ)
A.mannifera Akol Fabaceae C Roots Kidney Stones
0.03
Decoction of 100g of the root is prepared in 1L of water and taken 2-3 times daily
3Allium sativum
L. (AL-MJ)
Thom Alliaceae BulbsEdible, around the neck to treat Jaundice
0.06
Bulbs are eaten fresh (3-6) cloves or as juice mixed with milk taken orally.For treatment of jaundice a neck lace of cloves is made to be hang around the neck until improvement occurs
4 Anchusa aegyptiaca Maly ex Nyman (AA-
MJ)
El-dobba
Boraginaceae C Arial Parts
Wounds, skin infections and acne
0.06
Pads form, Juice from macerated leaves is applied externally
5 Anchusa strigosa
[Soland.](AnS-MJ)
Himhim Boraginaceae C Arial Parts
Wounds, Female sterility Anthelmentic, headache
0.09
Pads form for treatment of wounds. Decoction taken internally for treatment of headacheVapors for sterility,
6 Artemisia judaica L.
(AJ-MJ)
Beithran Asteraceae C Flowering tops
antispasmodic, antidiabetic, calming 0.34
Infusion
7 Artemisia sieberi Bess.
(ArS-MJ)
A.herba-alba
Sheih Asteraceae C Foliage Antidiabetic, Antispasmodic, pectoral, antiarthritis
0.54Infusion of 30 g in 1L of water
8 Arum dioscoridis
Sibth. & Sm. (AD-MJ)
Rqeita Araceae C, D Leaves Cancer, post-delivery helps the mother not to be infected
0.37
Decoction or cooked with eggs (edible)
Sample preparation
Drying• In most cases, plant material is dried in the
atmosphere prior to working.• Drying at RT or in Oven (30oC).• Keep the sample away from direct sun light (cmp.
Artifacts).• Well ventilated places and homogeneous
distribution of the material should be ensured.• Certain plants needed to be extracted fresh
before enzymatic reactions or pH-induced degradation, otherwise denaturing the enzymes by soaking the samples in ethanol soon after collection.
Comminution and classification
• Objective is to obtain certain fractions in high yield and as free of dust as possible.
• Large differences in particle size of the drug results in long extraction times.
• Pneumatic removal of sand, combined with magnetic removal of metals is followed by a preliminary sieving.
• Shredding,, grinding and sieving are the suitable processes done before extraction.
Extraction
Separation
Isolation
Identification
Extraction
Crude Extract
Extract Fractions
Pure Compounds
Biomass
Active Lead Compound
Determination of Biological Activity
Source Material = Biomass
• Plants
• Microorganisms– bacteria, fungus
• Marine organisms
• Animal products
Solvent Extraction
• Extraction: transfer of a solute from one phase to another.
• Can use any combination of phases (solid, liquid, gas, supercritical fluid)
• Different types of extractions: Solid-Liq Ext. (solid drug is extracted with liq medium), Liq-Liq (solvent extractions use two immiscible liquids).
• Extraction processes for drugs can be divided into two major groups:
• a- Lead to established equilibrium.• b- Exhaustive extraction.• Factors affecting extraction of herbal drugs:• Quantity of natural drug, • degree of grinding• Moisture content• Nature and solvents volume• Temperature• pH of the extracting solvent• Lipophilicity of the solvent mixture
Solvent Extraction
• Like dissolves like so ideally, the extracting solvent should be similar to the solute.
• Organic solvents less dense than water – diethyl ether, toluene, hexane
• Organic solvents more dense than water– chloroform, CCl4, dichloromethane
• The solvents chosen should be:• Dissolve the 2nd metabolites, be easy to remove,
inert, nontoxic, and not easily flammable, of good purity.
• Extraction processes:• A- With organic solvents: * Percolation * Maceration * Soxhlet extraction• B- With water: * Infusion * Decoction * Steam distillation
• Maceration: solvent extraction with several daily shaking or stirring at RT.
• Digestion: maceration at higher T. (40-50).• Percolation: exhaustive extraction by fresh
solvent (hot or cold solvents).• Infusion: milled plant material is soaked in hot or
cold water for a period of time with or without intermittent shaking.
• Decoction: the sample is boiled for about 15 min.• Steam distillation: (volatile oils)• Miscellaneous methods; (enfleurage, expression,
microwave, pervaporation (binding membranes), sublimation).
shake
add second immiscible
solvent
Solvent Extraction
• Solute partitions between the two phases
Solvent Extraction
[S]1
[S]2
Phase 1
Phase 2
Solvent Extraction
• Equilibrium constant for this partitioning is K (partition coefficient)
K=[S]2
[S]1
B + H2O BH+ + OH-Kb
HA H+ + A-Ka
Generally, neutral species are more soluble in an organic solvent and charged species are more soluble in aqueous solution
pH effects
• with organic acids/bases:
organic
HA H+ + A-Kaaqueous
HA H+ + A-
very little here, ions have poor solubility
Solvent Extraction (pH effects)
• Partitioning of organic acids between two phases:
D
pH
[H+]=Ka
pH=pKaK
[H+]>>Ka
mainlyHA
[H+]<<Ka
mainlyA-
• pH effect on D for organic acids
K1
4 8
K2
D
pH
Acid 2 stays in organic phase, acid 1 is extracted into aqueous phase
D
pH
K
[H+]=Ka
pH=pKa
[H+]>>Ka
mainlyBH+
[H+]<<Ka
mainlyB
organic bases
D
pH
Kacid base
Initial Aq. phase
Aq. PhaseOrg. acid
Aq. PhaseOrg. base
Ether PhaseOrg. acid, Org. neutral
Ether PhaseOrg. neutral
pH=1, extract with ether
extract with pH=12 Aq. Sol’n
Separate organic acid, base and neutral analytes
Isolation of Alkaloids
• Process remained unchanged >1,000 years
Plant Material
Acid solution
EtOAc: neutral/weakly basic alkaloids
1) Methanol2) Concentrate3) Partition EtOAc/2% acid
Petroleum ether extracts non-polar fats and waxes
Residue: polar material
Wash with petroleum ether
Basic aqueous solution of quaternary alkaloids
1) Ammonia2) Partition with EtOAcEtOAc: basic alkaloids
:Classical extraction method is: SOXHLET EXTRACTION
(named after developer). Apparatus
Sample in porous thimble. Exhaustive reflux for up to 1 - 2 days. Solution of analyte(s) in volatile solvent (e.g. CH2Cl2, CHCl3 etc.) Evaporate to dryness or suitable concentration, for separation/analysis.
Supercritical Fluid Extraction
• A pure supercritical fluid (SCF) is any compound at a temperature and pressure above the critical values (above critical point). The critical pressure is the vapor pressure of the gas at the critical temperature. In the supercritical environment only one phase exists. The fluid, as it is termed, is neither a gas nor a liquid and is best described as intermediate to the two extremes. This phase retains solvent power approximating liquids as well as the transport properties common to gases.
FluidCritical Temperature
(K)Critical Pressure
(bar)
Carbon dioxide 304.1 73.8
Ethane 305.4 48.8
Ethylene 282.4 50.4
Propane 369.8 42.5
Propylene 364.9 46.0
Trifluoromethane (Fluoroform)
299.3 48.6
Chlorotrifluoromethane 302.0 38.7
Trichlorofluoromethane 471.2 44.1
Ammonia 405.5 113.5
Water 647.3 221.2
Cyclohexane 553.5 40.7
n-Pentane 469.7 33.7
Toluene 591.8
Supercritical Fluids
Pressure/temperature phase diagram of carbon dioxide:
SF phase: properties intermediate between liquid and gas.
Density: 0.1 - 0.8 of typical liquid values
Diffusivity: 10 - 100 times higherthan for typical liquid.
Viscosity: 10 - 100 times lowerthan for typical liquid.
Solvating power: comparable to many
conventional solvents, e.g. CO2 ~
non-polar organic solvents.
Small changes in T and/or P (especially close to critical point) lead to largechanges in physical properties.
Dependence of CO2 density on pressure
at different temperatures:
Unlike liquids, SF’s can havetheir properties (esp. solvatingpower) tuned by quite smallchanges in T and/or P.
This is very useful in extraction
Useful properties of supercritical fluids
Solvating powers like liquids
Mobility approaching that of gases
Easy to modify (“tune”) these properties
Advantages • Dissolving power of the SCF is controlled by pressure
and/or temperature • SCF is easily recoverable from the extract due to its
volatility • Non-toxic solvents leave no harmful residue • High boiling components are extracted at relatively low
temperatures • Separations not possible by more traditional processes can
sometimes be effected • Thermally labile compounds can be extracted with
minimal damage as low temperatures can be employed by the extraction
Disadvantages • Elevated pressure required • Compression of solvent requires elaborate recycling
measures to reduce energy costs • High capital investment for equipment
Supercritical Fluid ExtractionSupercritical Fluid Extraction
Clean” technology
Liquid or supercritical CO2 provides an efficient and selective
extraction solvent with variable properties according to parameters
selected
Product degradation is minimised and solvent residues are eliminated
Product development - 5 extraction plants from
2 x 1litre to 5 x 1200 litre
Used to extract essential oils, fixed oils, pharmaceuticals and pigments
1. CO2 cylinder
2. Pump3. Pressure gauge4. Oven5. Ballast volume6. Extraction cell7. Monitor (UV)8. Back pressure regulator
Schematic Diagram of an SFE System
OBJECTIVEOBJECTIVE
• Efficient and selective extraction of a target molecule from a complex botanical matrix, with the minimum post extraction purification
Why use COWhy use CO22 as a process solvent as a process solvent
Chemically pure, stable, non-polar solventChemically pure, stable, non-polar solvent
Colourless, odourless and tastelessColourless, odourless and tasteless
Easily removed, no residue issuesEasily removed, no residue issues
Safe - not toxic or flammableSafe - not toxic or flammable
Environmentally friendlyEnvironmentally friendly
Widely availableWidely available
Further processing of residue possibleFurther processing of residue possible
Liquid COLiquid CO22 extraction extraction
Very solubleVery soluble Sparingly solubleSparingly soluble Insoluble Insoluble
Non-polar andNon-polar and Organic compounds <500MW Sugars, proteinsOrganic compounds <500MW Sugars, proteins
slightly polar molecules or with higher polarity Tannins, waxes,slightly polar molecules or with higher polarity Tannins, waxes,
<250 MW<250 MW Chlorophyll, Chlorophyll, amino amino
acids andacids and
ExamplesExamples most pesticides most pesticides
Terpenes,thiolsTerpenes,thiolsOleic acid, decanol, lipids <C18Oleic acid, decanol, lipids <C18
esters, short chainesters, short chain
organic acids, alcohols organic acids, alcohols
and ketonesand ketones
Supercritical COSupercritical CO22 extraction extraction
Very solubleVery soluble Sparingly soluble Sparingly soluble InsolubleInsoluble
Non-polar andNon-polar and Chlorophyll, waxes Sugars, proteinsChlorophyll, waxes Sugars, proteins
moderately polar molecules and carotenoidsmoderately polar molecules and carotenoids Tannins, amino Tannins, amino acids acids
<500 MW<500 MW and most pesticides and most pesticides
ExamplesExamples
Triterpenoids, alkaloidsTriterpenoids, alkaloids
lipids <C22lipids <C22
Factors influencing extraction Factors influencing extraction efficiencyefficiency
TemperatureTemperature
PressurePressure
Solubility of productSolubility of product
Structure and particle size of raw material Structure and particle size of raw material
Mass of COMass of CO22 per mass of raw material per mass of raw material
Supercritical COSupercritical CO22 extraction circuit extraction circuit
CO2 tank
Extract
CO2
Extraction columns
Extract
Pump
Heat exchanger
Reducing valve
Heatexchangers
SeparatorsChiller
Industrial Extraction with COIndustrial Extraction with CO22
Decaffeination of Tea and Coffee - SCO2
Extraction of Hops - LCO2 and SCO2
Defatting of cocoa powder - SCO2
Extraction of oil seeds - SCO2
Extraction of spices and aromatic plants - LCO2 and SCO2
Problem with CO2 - not a good solventfor more polar compounds.
Get round the problem by adding smallamounts (1 - 10%) of polar modifiers to theCO2, e.g. H2O, CH3OH, CH3CN etc.
These do not detract from the advantages ofSFE, and allow many more substances to beextracted.
Some examples of SFE Applications
Analytes Matrix S.F Extraction time (mins)
Pesticides Meat CO2 30 - 60
Terpenes etc. Lemon peel CO2 20 - 30
PAH’s, PCB’s Soil, sediment, CO2 diesel particulates CO2/MeOH 1 - 60
ethane etc. Polymer additives Polyethylene CO2 120
This is just a very small selection of published reports of SFE.Note : PAH = polycyclic aromatic hydrocarbon;
PCB = polychlorinated biphenyl(both classes include many extremely toxic environmental pollutants)
Why should we use SFE?
Conventional solvent extraction Supercritical Fluid Extraction
¶ Slow- several hours minimum¶ Non-selective¶ Hard to vary solvating power¶ Environmentally damaging solvents¶ Difficult to purify solvent¶ Produces dilute solution¶ Solvent disposal
BUT - very low capital outlay
¶ Quick - minutes to few hours maximum¶ Selective¶ Easy to vary solvating power¶ Environmentally friendly solvents¶ Easy to purify solvent¶ Produces extract in usable form¶ Solvent disposal trivial
BUT - high capital outlay
Some comparisons between SFE and conventional extraction
Conventional SFE
Sample size 1 g 20 mg
Liquid solvent required 450 ml 3 ml
Bench space
(for sample prep.) 5 m 1 m
Extraction time 48 h 1 h
Extract concentration time 3 h 0 -10 min
Shortest possible total
extraction time 2 days 2 h
Cost per extraction £6 30p
(based on data in S.B.Hawthorne et al., J. Chromatogr. Sci., 1989, 27, 347)
Case Studies in SFE
• Extraction of feverfew -
tuning solvating power
SFE of feverfew The plant feverfew, Tanacetum parthenium, is a well known herbal remedy effective in treatment of migraine etc.
O
O
O
But - very complex mixture - how to extract selectively?
Important to be able to identify active ingredient(s).
These are thought to be sesquiterpene lactones, such asparthenolide.
Carry out SFE at lower pressure (takes out most soluble species)than at higher pressure (less soluble species). Separates the very complex mixture into two simpler ones - helping subsequentanalysis. Such a separation impossible by conventionalextraction.