Evaluation /Quality Control / assessment of crude drug

 according to WHO guidelines 

(Part-IV)

 (Chemical evaluation)

(S.Y. B. Pharm Unit -IV ) Unit -I


(T.Y. B. Pharm Sem -VI ) -Unit - IV


(Final Yr. B. Pharm) Section -II

As per PCI Syllabus 

METHODS OF DRUG EVALUATION
The evaluation of a drug is done by following methods.  

The evaluation of a drug is done by following methods.
1. Organoleptic evaluation
2. Morphological evaluation
3. Microscopic evaluation
4. Physical evaluation
5. Chemical evaluation
6. Analytical evaluation
7. Biological evaluation

Chemical evaluation:

Fixed oils & fats can be determined by acid value, iodine value, saponification value, acetyl value etc,
Volatile oils by ester value, acetyl value,

Balsams by ester value, acetyl value, saponification value.
In case of drugs containing organic acid, non-aqueous titration method is used for the analysis. Gravimetric analysis Where we are going to determine the weight of substance. The substance is converted into highly colored complex or chelate, which is analyzed by colorimetric method.

But easier & faster way of evaluating the presence of group of constituents in the extract of the drug is by Phytochemical screening in which simple chemical tests are used for the identification of them

Preliminary chemical test:

Extract obtained using petroleum ether, chloroform, ethanol and water was prepared using the respective solvent. These extracts along with positive and negative controls were tested for the presence of active phytochemicals viz:

tannins, alkaloids, phytosterols, triterpenoids, falvonoids, cardiac glycosides, anthroquinone glycosides, saponins, carbohydrates, proteins, amino acids and fixed oils and  fats following standard methods. 

A) Tannins

Ferric chloride Test:
Added a few drops of 5% ferric chloride solution to 2 m1 of the test solution. Formation of blue color indicated the presence of hydrolysable tannins.

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.

B) Alkaloids

50 mg of extract was diss. in 5 ml of dis. water. Further 2M HCL was added until  acid reaction occurred & filtered. The filtrate was tested for the presence of alkaloids as detailed below:

•Dragendorff’s Test:

•To 2 ml of the filtrate was added 1 ml of Dragendorff’s reagent. Formation of orange or reddish brown precipitate indicated the test as positive.

•Mayer’s Test:

•To 1 ml of test solution or filtrate was added a drop or two of the Mayer’s reagent. White or a creamy precipitate confirmed the test as positive. 

•Hager’s Test:

To 1 ml of test solution or filtrate, a drop or two of Hager’s reagent formation of yellow precipitate indicated the test as positive.

•Wagner’ s 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.  

C) Phytosterols

i. 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 conc. H2SO4 was added. A brown ring formation at the junction and the turning of the upper layer to dark green color confirmed the test for the presence of phytosterols. 

D) Triterpenoids

i.  Salkowski 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 indicated the test as positive for Triterpenords. 

E) Flavonoids

Shinoda test: 
A few magnesium turnings and 5 drops of conc.HCL  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 confirmed the test.

Alkaline reagent test:
Addition of 5 drops of 5% NaOH to 1 ml of the test solution results in increased intensity of the yellow color which become colorless on addition of a few drops of 2 M HCL which indicated the presence of Flavonoids.

Lead acetate test: 

A few drops of 10% lead acetate added to lml of the test solution resulted in the formation of yellow precipitate confirmed the presence of Flavonoids.

F) 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.

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. 

G) Cardiac glycosides

Keller -Killiani test: 

Added 0.4 ml of glacial acetic acid and a few drops of 5% ferric chloride solution to a little of dry extract. Further 0.5 ml of concentrated sulfuric acid was added .The formation of blue color in acetic acid layer confirmed the test. 

H) Test for carbohydrates

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 confirmed the test.
Barfoed’s test: 2ml of reagent was added to 2 ml of the test solution, mixed & kept in a boiling water bath for 1 min. Red precipitate formed indicates the presence of monosaccharide. 

Seliwanoff’s test: To 3 ml of Seliwanoff’s 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.
 
Fehling’s test: Dissolved 2 mg dry extract in 1 ml of distilled water and added lml of Fehling’s(A+B) solution, shooked and heated on a water bath for 10 minutes. The brick red precipitate formed confirmed the test. 

I) Anthraquinone glycosides

i. Hydroxyanthraquinone Test: To 1 ml of the extract, added a few drops of 10% potassium hydroxide solution. The Formation of red color confirmed the test.
J) Test for proteins

i. Biuret test: To 2 m1 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. 

K) Test for amino acids :

i. 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 added 1% sodium nitrite solution. Appearance of red color confirmed the test.

L) Fat and Fixed Oils:
To 5 drops of the sample were added 1ml of 1% CuSO4 solution and a few  drops of 10% NaOH. The formation of a clear blue solution confirmed the test.


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