WHO guidelines
»Haemolytic activity
»Swelling index
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Content
WHO guidelines for the standardization of crude drugs and extracts
» Haemolytic activity
» Swelling index
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Objectives
• At the end of this lecture, student will be able to
Discuss the principle and procedure involved in the determination
of
» Haemolytic activity
» Swelling index
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Determination of Haemolytic Activity
❖ Caryophyllaceae, Araliaceae, Sapindaceae, Primulaceae, and
Dioscoreaceae – contain Saponins
❖ Saponins – ability to cause haemolysis
❖ Comparison with that of a reference material, saponin R, which has a
haemolytic activity of 1000 units per g
❖ Suspension of erythrocytes + equal volumes of a serial dilution of the
herbal material extract
❖ Lowest concentration to effect complete haemolysis is determined
❖ Similar test is carried out simultaneously with Saponin R
Determination of Haemolytic Activity
Procedure
Erythrocyte suspension
❖ Fill a glass-stoppered flask to one tenth of its volume with sodium
citrate (36.5 g/l) TS, swirling to ensure sufficient volume of blood
freshly from a healthy ox, shake
❖ Citrated blood- can be stored for about 8 days at 2−4 °C
❖ 1 ml of citrated blood in a 50-ml volumetric flask with phosphate buffer
pH 7.4 TS – diluted blood suspension (2% solution)
Determination of Haemolytic Activity
Procedure
Reference solution
10 mg of Saponin R add phosphate buffer pH 7.4 TS to make 100 ml
Preliminary test
Serial dilution – herbal material extract with phosphate buffer pH 7.4 TS
and blood suspension (2%) using four test-tubes
Determination of Haemolytic Activity
Procedure
✓ Gently invert – mix
✓ Shake again after a 30-minute interval
✓ Allow to stand for six hours at room temperature
❖ Examine the tubes
❖ Record the dilution at which total haemolysis has occurred
➢ Total heamolysis – clear, red solution without
any deposit of erythrocytes
Determination of Haemolytic Activity
If total haemolysis
➢ only in tube 4: original herbal material extract directly for the main test
➢ In tubes 3 and 4: two-fold dilution of the original herbal material
extract
➢ In tubes 2, 3 and 4: five-fold dilution of the original herbal material
extract
➢ In all four tubes: ten-fold dilution and carry out the preliminary test
again
➢ Not observed in any of the tubes: Repeat the preliminary test using a
more concentrated herbal material extract
Determination of Haemolytic Activity
Main test
❖ Prepare a serial dilution – undiluted or diluted herbal extracts
❖ Blood suspension (2%) using 13 test-tubes
❖ Observe the results after 24 hours
Determination of Haemolytic Activity
Calculate
✓ Amount of herbal material in g or ml that produces total haemolysis
✓ Quantity of saponin R in g that produces total haemolysis
Calculation of haemolytic activity of the herbal material
1000 = the defined haemolytic activity of saponin R in relation to ox blood
a = quantity of saponin R that produces total haemolysis (g)
b = quantity of herbal material that produces total haemolysis (g)
Determination of Swelling Index
✓ Swelling properties
Especially gums, appreciable amount of mucilage, pectin or
hemicellulose
✓ Swelling index
Volume in ml taken up by the swelling of 1 g of herbal material
under specified conditions
Determination of Swelling Index
Procedure
✓ Atleast three simultaneously determinations
Accurately weighed sample
25-ml glass-stoppered measuring cylinder
(Internal diameter around 16 mm, length of graduated portion about 125 mm
marked in 0.2- ml divisions from 0 to 25 ml)
Add 25 ml of water, Shake thoroughly every 10 minutes for 1 hour
Allow to stand for 3 hours at room temperature
Measure the volume in ml occupied by the material, including any sticky mucilage
Calculate the mean value
Summary
✓ Saponins – ability to cause haemolysis, Lowest concentration to
effect complete haemolysis is determined, Similar test is carried out
simultaneously with Saponin R
✓ Swelling index – Volume in ml taken up by the swelling of 1 g of
herbal material under specified conditions
Thank You
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