WHO & ICH GUIDELINES (CHEMICAL EVALUATION) FOR ASSESSMENT OF HERBAL DRUGS (PART - 4) T.Y B.PHARM | SEM VI | HERBAL DRUG TECHNOLOGY | PCI SYLLABUS | UNIT IV.

WHO & ICH GUIDELINES FOR ASSESSMENT OF HERBAL DRUGS (PART - 4) 

CHEMICAL EVALUATION OF CRUDE DRUGS

CHEMICAL EVALUATION OF CRUDE DRUGS:-

     Determination of the active constituent in a drug by chemical tests is referred to as

chemical evaluation. Chemical evaluation comprises different chemical test and

chemical assays.

CHEMICAL TEST:- are carried out using various chemical reagents to identify the nature

and quantity of chemical constituents present in a crude drugs. It may be

1. Qualitative chemical test

2. Quantitative chemical test.

QUALITATITVE CHEMICAL TEST:- involved identification test for various

phytochemical constituents.  These test provide Information regarding nature of active

principles.

Method for chemical evaluation:-Extract obtained using petroleum ether, chloroform,

ethanol and water was prepared using the respective solvent. These extracts were tested for

the presence of active phytochemicals viz: carbohydrates, proteins, amino acids. fixed oils &

fats, alkaloids, glycosides, tannins, phytosterols, triterpenoids, falvonoids,,  saponins, by

following standard methods.

A] TEST FOR CARBOHYDRATES:-

1.Molisch’s test:- To 1 ml of test solution added a few drops of 1 % alpha-napthol and 2-3

ml concentrated sulfuric acid. The reddish violet or purple ring formed at the junction of two

liquids.

2.Barfoed’s test:- 2ml of reagent was added to 2 ml of the test solution, mixed & kept in

boiling water bath for 1 min. Red precipitate formed indicates the presence of

monosaccharide's.

3.Seliwanoffs test:- To 3 ml of Seliwanoffs reagent was added to 1 ml of the test sample

and heated on a water bath for one minute. The formation of rose red color confirmed

carbohydrates.

4. Fehlings test:- Dissolved 2 mg dry extract in 1 ml of distilled water and added 1ml of

Fehling’s (A+B) solution, and heated on a water bath for 10 minutes. The brick red

precipitate formed.

B] TEST FOR PROTEINS:-

1.Biuret test:- To 2 ml of the test solution added 5 drops of 1% copper sulphate solution

and 2 ml of 10% NaOH .Mix thoroughly. Formation of purple or violet color confirmed

proteins.

C] TEST FOR AMINO ACIDS:-

1.Millon’s test:- Added 5 drops of millons reagent to 1 ml of test solution and heated on a

water bath for 10 min, cooled and added 1% sodium nitrite solution. Appearance of red color

confirmed the test.

D] FATS AND FIXED OILS:-

To 5 drops of the sample was added 1 ml of 1% copper sulphate solution and a few drops of

10% sodium hydroxide. The formation of a clear blue solution confirmed the test.

E] TEST FOR ALKALOIDS:-

1.Dragendorff’s Test:- 2 ml of the filtrate+1 ml of Dragendorff’s reagent. Formation of

orange or reddish brown  precipitate.

2.Mayer’s Test:-1 ml of test solution + a drop or two of the Mayer’s reagent. white or a

creamy precipitate.

3.Hager’s Test:- 1 ml of test solution + a drop or two of Hager’s reagent formation of

yellow precipitate.

4.Wagner Test:- Two drops of Wagner’s reagent was added to 1ml of the test solution. The

formation of yellow or brown precipitate confirmed the test as positive for alkaloids.

F] TEST FOR GLYCOSIDES:-

a] Chemical Tests for Anthraquinone Glycosides:-

1.Borntrager’s test:- To 1 gm of drug add 5–10 ml of dilute HCl boil on water bath for 10

min and filter. Filtrate was extracted with CCl4/ benzene and add equal amount of ammonia

solution to filtrate and shake. Formation of pink or red colour in ammoni-cal layer due

to presence of anthraquinone moiety.

2.Modified borntrager’s test:- Same as above only difference is that add 5 ml ferric

Chloride (5% w/v) after addition of dilute Hcl.

b] Chemical Tests for Steroid and Triterpenoid Glycosides

1.Libermann burchard test:- Alcoholic extract of drug was evaporated to dryness and

extracted with CHCl3, add few drops of acetic anhydride followed by conc. H2 SO4 from

side wall of test tube to the CHCl3 extract. Formation of violet to blue coloured ring at the

junction of two liquid, indicate the presence of steroid moiety.

2. Salkowaski test:- Alcoholic extract of drug was evaporated to dryness and extracted

with CHCl3, add conc. H2 SO4 from sidewall of test tube to the CHCl3 extract. Formation of

yellow coloured ring at the junction of two liquid, which turns red after 2 min, indicate the

presence of steroid moiety.

c] Chemical Tests for Cardiac Glycosides :-

1. Keller-kiliani test:- To the alcoholic extract of drug equal volume of water and 0.5 ml of

strong lead acetate solution was added, shaked and filtered. Filtrate was extracted with

equal volume of chloroform. Chloroform extract was evaporated to dryness and residue was

dissolved in 3 ml of glacial acetic acid followed by addition of few drops of FeCl3 solution.

The resultant solution was transferred to a test tube contain-ing 2 ml of conc. H2SO4.

Reddish brown layer is formed, which turns bluish green after standing due to presence of

digitoxose.

2. Legal test:- To the alcoholic extract of drug equal volume of water and 0.5 ml of strong

lead acetate solution was added, shaked and filtered. Filtrate was extracted with equal

volume of chloroform and the chloroform extract was evaporated to dryness. The residue

was dissolved in 2 ml of pyridine and sodium nitropruside 2 ml was added followed by

addition of NaOH solution to make alkaline. Formation of pink colour in presence of

glycosides or aglycon moiety.

d] Chemical Tests for Flavonoid Glycosides :-

1. Ammonia test:- Filter paper dipped in alcoholic solution of drug was exposed to

ammonia vapor. Formation of yellow spot on filter paper.

2. Shinoda test:-To the alcoholic extract of drug magnesium turning and dil. HCl was

added, formation of red colour indicates the presence of flavonoids. To the alcoholic extract

of drug zinc turning and dil. HCl was added, formation of deep red to magenta colour

indicates the presence of dihydro flavonoids.

e] Chemical Tests for Saponin Glycosides 

1. Haemolysis test:- A drop blood on slide was mixed with few drops of aq. Saponin

solution, RBC’s becomes ruptured in presence of saponins.

2. Foam test:- To 1 gm of drug add 10–20 ml of water, shake for few minutes, formation

frothing which persists for 60–120 s in presence of saponins.

G]CHEMICAL TEST FOR TANNIN'S:-

1.Ferric chloride Test:- Added a few drops of 5% ferric chloride solution to 2 ml of the

test solution. Formation of blue color indicated the presence of hydrolysable tannins.

2.Gelatin Test:- Added five drops of 1% gelatin containing 10% sodium chloride to 1 ml of

the test solution. Formation of white precipitates confirmed the test. 

H] TEST FOR PHYTOSTEROLS:-

1.Liebermann-Burchard’s Test:- The extract (2 mg) was dissolved in 2 ml of acetic

anhydride, heated to boiling, cooled and then 1 ml of concentrated sulfuric acid was added.

A brown ring formation at the junction and the turning of the upper layer to dark green

color.

I] TEST FOR TRITERPENOIDS:-

1. Salkowaski Test:- Approximately 2 mg of dry extract was shaken with 1 ml of

chloroform and a few drops of concentrated sulfuric acid were added. A red brown color

formed at the interface.

J] TEST FOR FLAVONOIDS:-

1.Shinoda test:- A few magnesium turnings and 5 drops of concentrated hydrochloric acid

was added drop wise to 1 ml of test solution. A pink, scarlet, crimson red or occasionally

green to blue color appeared after few minutes.

2. Alkaline reagent test:- Addition of 5 drops of 5% sodium hydroxide to 1 ml of the test

solution resulted an increase in the intensity of the yellow color which became colorless on

addition of a few drops of 2 M hydrochloric acid which indicated the presence of falvonoids.

3.Lead acetate test:- A few drops of 10% lead acetate added to

1ml of the test solution resulted in the formation of yellow

precipitate confirmed the presence of falvonoids.

K] CHEMICAL TEST FOR SAPONINS:-

1.Foam Test:- 5 ml of the test solution taken in a test tube was shaken well for five

minutes. Formation of stable foam confirmed the test.

2. Olive oil test:- Added a few drops of olive oil to 2ml of the test solution and shaken well.

The formation of a soluble emulsion confirmed the test.

QUANTITAVE CHEMICAL TEST:- These test give the value or amount of active

constituent in the crude drugs. Fixed oil, volatile oil & fats can be determined by this

method.  These include…..

1. Acid value:- The no of mg of potassium hydroxide required to neutralize the free acid

present in 1 gm sample of fat or oil. Used for resin.

2. Saponification value:-The no mg of potassium hydroxide require to neutralize the

fatty acid resulting from complete hydrolysis of 1 gm of the sample of oil or fats. Used for

lipids.

3. Ester value:- The no mg of potassium hydroxide require to combined with fatty acid

which are present in glyceride form 1 gm sample of fat or oil. Used for volatile oil.

4. Iodine value:- The wt of iodine absorbed by 100 parts by wt of the sample of fat or oil.

CHEMICAL ASSAY:- These test give an approximate value of total phytochemical

constituents in a crude drugs. The conventional trimetric estimation as applicable to the

estimation of alkaloids from drugs, ester & aldehyde content of volatile oil.

gravimetric methods are used for technique of chemical assay.

Eg. The alkaloidal drugs  which are routinely analyzed for their total alkaloid content such as

Morphine in opium , atropine in belladonna, Strychnine in nux vomica, Reserpine in

rauwlfia.etc.

Cineole in eucaleptos oil, aldehyde in lemon oil, Vitamins in cod liver oil.

The extraction ,isolation and purification of drug are also a part of chemical evaluation.

Extraction methods:- maceration, percolation, Decoction etc.

Purification methods:- Sublimation, distillation, crystallization etc.