Evaluation of herbal products
1- methods of identifications
To evaluate a crude drug:
Can be established by actual collection of the drug from plants → +ve identified.
How to determine the origin of the samples:
1- "Drug gardens' established by institutions engaged in pharmacognostical research.
2- Comparing unknown sample with a- a published description drug or b- with authentic drug samples.
It is refers to the intrinsic value of the drug, i.e., the amount of medicinal principle or active constituents present.
How obtain high grade of quality?
I- Collection of the drug from the correct natural source at the proper time and in proper manner.
e.g1., Opium (morphine, codeine, thebaine, papaverine…..etc).
Origin: Papaver somniferum (induce sleep).
Time of collection: a- must collect from green unripe capsule (fruit). Why?
b- 2-3 weeks after flowering. Why? Because at this time obtain a maximum morphine content (active constituent). But before this time, alkaloids content mainly thebaine which is inactive.
Proper manner: capsules must be incision slowly in the morning, not deep and not allow aqueous juice to contaminate latex.
e.g 2., clove:
Origin: Myristica fragrance (nice aroma).
Proper time: flower buds from clove tree. Because contain high % of A.C.
Proper manner: absence of stalk. In official book say no more than 5% of stalk and mother clove to obtain high grade of quality.
II- Preparation of the collected drug by proper cleaning, drying and garbling.
e.g., 1- Under ground organs roots, rhizomes, corms and tubers.
III- Proper preservation of the cleaned, dried, pure drugs against contamination with dirt, moisture, fungi, filth and insects.
The evaluation of a drug involves a number of methods:
Definition: The entire description of official crude drug monographs.
= Organs of sense = Macroscopic appearance of drug.
1- Shape and size (for entire drugs not powdered).
Floral parts: stigmas, corollas, anther, ovary, receptacle.
Leaves and leaflets:
Length, width, apex, margin, base, venation, the texture of the leaf and the hairs in upper and lower surface. The feel of the surface described as soft, hairy smooth.
i- The barks occur in three shapes:
1- Flat or curved pieces.
2- Single quill.
3- Double quills.
ii- Barks have two surfaces, an outer and inner.
iii- The inner surface is usually lighter in color than the outer surface.
2- Odor and taste.
1- distinct 2- indistinct
[Depend on the amount of volatile constituents present in drug].
General terms used in describing odor are:
General terms used in describing taste are:
1- Acid (sour)
2- Saccharine (sweet): indicates sugar or sugar like substances e.g., liquorice.
3- Saline (salty)
5- Bitter: indicates presence of substances such as bitter principle, glycoside, alkaloids.
6- Tasteless (all substances insoluble in the salive).
7- Distinctive sensations to the tongue:
a- mucilaginous and oily (soft feeling) e.g., linseed.
b- astringent (contraction of the tissues of the mouth) indicates presence of tannin.
c- pungent (warm biting sensation) e.g., ginger.
d- acrid (irritant sensation) e.g., Aconite, coca.
e- nauseous (those tending to excite vomiting), Ipeca.
3- Color and external markings.
May help in revealing the nature of the herb.
1- White: e.g., starch, flours, gums, talk)
2- Pale yellow (yellowish white) e.g., ginger, squill, white pepper.
3- Deep yellow: e.g., peeled liquorice, calumba, hydrastis.
4- Light pale brown e.g., nux vomica, fennel, coriander, anise.
5- Dark brown: e.g., cloves.
6- Dark reddish brown: cinchona, nutmeg.
7- Red: Kamala.
8- Pale green e.g., lobelia.
9- Greenish brown: most of the leaf herbs.
4- Fracture and internal color.
Dealing with microscopic appearance of the herb in sectional view and in powdered form.
Microscopical evaluation is useful in the study of:
1- Histologic elements of herbs.
2- Detection of adulterant.
3- Quantitative microanalysis of admixed or adulterated powders.
Histology refers to the character and arrangement of these tissues as they are present in the herb. Histologic studies are made from very thin transverse (radial) or longitudinal (tangential) sections (entire organ) properly mounted in suitable stains, reagents or mounting media.
Powdered herbs posses very few macroscopic features of identification outside of color, odor and taste. Microscopically, the cells are broken, except those with lignified walls, but the cell contents are scattered in the powder and become very evident in the mounted specimen e.g., starch, calcium oxalate crystals, aleurone, phloem, glandular and nonglandular hair, pollen grain.
In some cases, the drug may have the same diagnostic element, they are known as Closely Related Species. So, microscope is not the method of choice.
It is used to identify closely related species.
1-Microscopical linear measurement
Used only in Root Rhizomes Bark
E.g1., Different between Cinnamon القرفه السيلانيه as quill and Cassiaالقرفه الصينيه as flat [Both have same diagnostic element].
So differentiate between both by:
Microscopical linear measurement
1- Diameter of starch granules
< 8 μm
2- Width of phloem fiber
Not less than 85%
Rio Ipecacuanha Cartagena Ipecacuanha
15 μm 17-20 μm
Why we need to differentiate between Rio and Cartagena Ipeca?
Because Rio Ipeca contains more emetine alkaloid.
Contain clusters of CaOx
American podophyllum Indian podophyllum
60-100 μm 30-40 μm
If closely related species are leaves: We need to
2- Ratio value: (Microscopical quantitative value)
1- Palisade ratio.
2- Stomata index.
3- Vein islet number.
4- Vein islet terminate number
1- Palisade ratio:
Def: numbers of palisade cell under one epidermal cell using four continuous epidermal cells for the count.
To do the ratio value is determined by “camera lucida” can transfer the microscopic field to microscopic stage by using mirror.
Take leave, T.S., count and draw 4 epidermal cells, then count and draw palisade cells under 4 epidermal cells.
2- Stomatal index (%):
Def: it is the percentage of the number of stomata to the total number of epidermal cells including the stomata, each stomata being counted as one cell.
Stomatal index= S/E+S x 100
(S) Number of stomata per unit area
(E) Number of epidermal cells in the same unit area.
Cassia angustifolia (both surface) 17.1-18.7-20
Cassia acutifolia (both surface) 11.4- 12.2-13
The figure obtained is constant for any species and can be used for the differentiation between the closely related species.
Determination of stomatal numbers:
It is an average number of stomata per mm2 of epidermis.
3- Vein islet number:
Def of vein islet: The small areas of green tissue outlined by the veinlets are temrmed vein islet.
Def of vein islet number: is the number of vein islet per mm2.
Cassia angustifolia 25-30
Cassia acutifolia 19-23
4- Veinislet terminate number:
Def: it is the number of veinlet termination per mm2 of leaf surface.
A veinlet termination is the ultimate free termination of a veinlet or branch of a vienlet. It can be used to distinguish between leaves of closely related species.
Atropa belladonna 6.3-10.3
Digitalis purpurea 2.5-4.2
Hyoscymus niger 12.4-19.0
This value has been shown to be constant for any species and unaffected by the age of the plant or the size of the leaves.
How to determined the mm2 ?
By using Eye-peice micrometer and stage micrometer.
It is the study of active constituents by the application of chemical and physical methods to small quantities (a few milligrams) of the drug in powdered form or to histologic sections of the drug. It offers a means by which constituents of many drugs may be isolated and purified.
It includes steps:
I- Isolation of A.C.:
A- By chemical solvents:
B- By micro-sublimation
II- Identification of constituents:
1- By crystallography
2- By melting point determination
3- By confirmative test
1- Chemical test.
2- Physical test
It is a separation of the constituents from a small quantity of the drug and depends on the solubility of the constituents in a solvent.
During microextraction procedures, various factors must be considered, such as:
1- State of division of the drug (whole, broken, powdered)
2- Type of solvent used with increase polarity [Pet.ether, chloroform, ethylacetate, butanol, ethanol, methanol, water].
3- Temperature: [increase temperature lead to increase solubility between solvent and extract].
4- Nature of impurities e.g., if impurities soluble in certain solvent do not used this solvent.
5- Nature of substances e.g., volatile oil, fixed oil, sterol, triterpenes, anthracene, coumarin, flavonoids, alkaloids, etc.
· If soluble in polar solvent means it is a polar compounds.
· If soluble in non-polar solvent means it is a non-polar compounds.
· All substances soluble in 90-95% alcohol.
To secure small quantities of the extracted substances in a clear solution generally requires microfiltration methods to obtain the extracted constituent in a pure form necessitates crystallization and re-crystallization.
If substances is soluble in methanol and insoluble in ethylacetate. How can be crystallization?
1- It is refer to a method of obtaining a constituent of a drug by heating the drug to vaporize its chief constituent to a gaseous state and then condensing the vapor back into a solid form.
2- This method is employed only when the drug or its constituents are not decomposed by heat.
3- When the constituent condenses on a cool place, the resulting crystals develop in a pure form.
4- Caffeine is sublimed from powdered Kola or from powdered coffee.
II- Identification of constituents:
1- By crystallography: It is a science dealing with:
i- Classification of crystals
iv- Properties of crystals e.g., crystal is:
· Its type of extinction
· Optic sign
· Sign of elongation
· Refractory index
2- By melting point determination:
It is very important as a means of identifying pure substances.
3- By confirmative test:
1- Chemical test.
2- Physical test
e.g., menthol is isolated from peppermint oil. It occurs as colorless, hexagonal crystals, usually needle like. The melting point of l-menthol from natural sources is between 41 and 43 ˚C. when l-menthol is triturated with an equal weight of camphor, chloral hydrate or phenol, the mixture liquefies, thus confirming the identity.
The use of the petrographic microscope is very important in the determination of the optic constants of crystalline substances.
It is a rapid method for identification of very small amounts of chemical compounds.
A series of different animals is necessary for the complete evaluation of one drug or preparation such as Corticotropin injection requires adult cats for the vasopressin activity and rats for the assays.
Before a new product is introduced on the market, it must be proven both safe and effective in preliminary pharmacologic tests on animals and in quantitative clinical tests on humans.
1- The active constituents should be extracted and purified before applying the chemical test.
Preliminary chemical test for active constituents:
1- Test for glycosides and carbohydrate:
* The test reaction is based on positive reaction to Molisch’s and Fehling’s reagents (after hydrolysis) which are not specific:
* Powdered (the powdered extract with 2% alcohol, evaporate, dissolve in water) + Few drops of α-naphthol + conc H2SO4 → violet ring at the junction.
* powdered + aniline citrate → brown color with reducing sugar (after hydrolysis).
2- Test for cardiac glycosides:
The powdered extract with 70% alcohol, evaporate, dissolve in water and purified by lead acetate then tested for cardenolides by Baljet’s reagent, Raymond’s Kedde’s reagent or legal’s test.
Test for deoxy sugar by keller killiani’s test is important for detection of glycosides containing this type of sugar.
3- Test for anthraquinone glycosides:
The powdered drug is boiled either by dilute acid, by alcohol or by acid and oxidixing agent according to anthracene glycoside then the aglycones are extracted by organic solvent followed by addition of alkali give red color.
4- Test for flavonoids:
The powdered is refluxed with alcohol, evaporate, the residue is extracted with boiling water and filtrate while hot. A portion of filtrate
Is tested with different reagents, Ammonium hydroxide, ferric chloride.
5- Test for saponine:
a- The powdered drug is extracted with 25% ethanol and filtrated. The filtrate is evaporated to dryness. The residue is dissolved in normal saline solution and added to a suspension of R.B.Cs in normal saline, if gaemolysis occurs this indicates the presence of saponins in the drug.
b- Famous forth test with water.
6- Test for tannins:
The powdered drug is extracted with 50% ethanol and filtrated. The filtrate is tested for tannins
Tannins + FeCl3→ blue color (hydrolysable tannins)
Green color (condensed tannins)
7- Test for alkaloids:
The alkaloid should be separated from other non alkaloidal material and impurity. How?
a- The alkaline nature of alkaloids have an ability to form salts with acids.
b- The relative solubility of free alkaloids and their salts in organic solvents and water.
c- Most common reagent is used for testing alkaloids by precipitation or microcrystal are: Kraut’s, Marme’s, Wagner’s, Gold chloride, platonic chloride and ammonium reineckate.
The application of physical constants is applied to the active principles of drugs such as alkaloids, volatile oils, fixed oil… etc.
-We should determine solubility of A.C. in different solvents.
- Usually expressed in the following form: 1g is soluble in ….ml of water, … ml of alcohol, etc.
e.g., 1g of atropine sulfate dissolves in 0.5ml of water, in 5 ml of alcohol and 2.5 ml of glycerin.
2- Specific gravity:
- Particularly of the fats and volatile oils.
- Weight of a given volume of liquid compared with the weight of equal volume of water at specific temperature in air.
The specific gravity of anise oil is not less than 0.978 and not more than 0.988.
Specific gravity of water =1
Most of V.O. is lighter than water, except V.O. of clove, cinnamone (heavier than water).
S.G. is not absolutely constant………….why?
Because other factors affected it:
1- maturity of drug from which v.o. is prepared.
2- Age of v.o.
3- Method of preparation and
E.g., Jalp is resin is used as purgative is heavier than water.
3- Optical rotation:
Of certain alkaloids in solutions and of v.os.
Described as dextro (+) rotary (rotate plane of polarized light to right, it is clock wise rotation). Or levo (-) rotary. rotate plane of polarized light to left, it is anti-clock wise rotation).
[α]TD = ± value of α/degree of rotation
D: sodium line ≈ 580 mm (light than make rotation).
[α]TD = ± α x 100/LXC
L: Path length of tube in cm
C: concentration in gm .
It is measured by polarimeter.
Atropine ± zero
Iso pelocarpine -
The angular rotation of peppermint oil is not less than -18◦ and not more than -32◦ in a 100 mm tube.
Automatic Digital Polarimeter, range ±45°. Polarimeter cell 100 mm, 200 mm and a sodium lamp.
Hand-Held Polarimeter, wave length 589nm, measurement range -35°… +35° optical rotation.
4- Refractive index:
- Particularly of the fixed and volatile oils (crystals, liquids).
It is a ratio of = sign angle of incidence/sign angle of refraction of monochromatic beam.
RI measure by refractometer.
- The RI of peppermint oil is not less than 1.4590 and not more than 1.4650 at 20 ◦C.
5- Congealing point:
- Particularly of fixed and volatile oils.
- The solidification range of fatty acids in olive oil is between 17 and 26◦C.
6- Melting point:
It is of extreme importance, there is a constant range of m.p. using specific solvent. Cocaine m.p. 96-98 ◦C.
UV light is f importance in several cases, notably in detecting
1- adulteration: a- of rhubarb, where rhapontic rhubarb can easily be distinguished from genuine Chinese or Indian rhubarb by its marked fluorescence. Chinese and Indian rhubarb fluorescence brown, while rhubarb rhaponticum is bright blue. b- powdered ergot exhibits a strong reddish fluirescence and can be detected in wheat flour.
2- UV light is used for determining fluorescence and a play of colors in connection with certain extracts such as chlorophyll and the extracts of certain drugs (catechu, senna).
Many alkaloids show distinctive colors under this light, aconitine (light blue), berberine (yellow), emetine (orange).
An alkaloids such as quinine show fluorescence in acid solution even in day light, exhibits a much stronger fluorescence under the quartz lamb.