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.