STP of Fungal Diastase and Papain capsules

Description : Blue/white coloured ‘2’ size capsule with cream coloured powder.

Weight of 20 Capsules : 7.3 gm. Limits +/- 7.5%

Average weight of Capsule : 0.365 gm. Limits +/- 7.5%

Average net content of Capsule : 0.300 gm. Limits +/- 7.5%

Disintegration : NMT 30 Minutes.

Each Capsule Contains Lable Claim Result Limits
Fungal Diastase (1:800) I.P. 100 mg NLT 90 %
(Fungal Diastase derived from aspergillus oryzae. Disests not less than 80 gms of cooked starch.)
Papain I.P. 60 mg NLT 90 %

Fungal Diastase

Reagents:
1. Freshly preparation normal saline solution (0.9%), PH 5.0
2. Soluble starch having about 15% moisture content – BDH grade starch is most sutable.
3. Buffer solution, PH 5.0 : Weigh accurately 2.05 gm of Sodium Acetate, dissolve in sufficient

quantity of water and make the volume to 250 ml. When 5.9 ml of 0.1 M Acetic Acid is mixed

with 14 ml of 0.1 M Sodium Acetate, solution will attain PH 5.0
4. Iodine Solution : 1.276 gm of Iodine and dissolve in 15 ml of water containing 4 gms.

Of Potassium Iodide and make the volume to 100 ml. Standardise this solution to get a solution of exactly 0.1 M.
5. Iodine Solution, 0.02 M : Dilute 20 ml of the Stock solution (0.1 M) with 80 ml of water

to obtain 0.02 M Iodine solution. Prapare 0.02 M Iodine solution just before use, and use 1 drop of this solution, per tube.
6. 10 M Hydrochloride, to arrest the enzyme reaction.
7. Substrate : Weigh 200 mg of soluble starch accurately ( on Dry Weight basis) and transfer

into 200 ml measuring flask with little quantity of water. Boil the suspension till gets into solution,

constant shaking the flask while boiling. Cool under tap water, add 5.9 ml of 0.1 M Acetic Acid , 14 ml of 0.1 M Sodium

Acetate and finally make the volume to 200 ml with normal saline, PH 5.0. Dispense this solution in 5 ml

aliquot to each to each tube. Thus each tube contains 5 mg of Soluble starch.

Dilution of Sample : All dilutions are to be made with normal saline solution, PH 5.0.
The Sample is diluted so that the final solution containing 5 mcg/ml ( 1:2000).

This Solution is used as test enzyme solution in greaded amounts of 0.1 ml to 1.0 ml per tube.

Procedure:
Despence substrate (Starch Solution) and saline solution as shown in the following table, and pre- Incubate the tubes for 5 min in a water- bath at 370 C (+/-0.50 C). Then, add greaded amount of diluted enzyme solution, shake to mix well and incubate for 1 hour at 370 C. At the end of incubation period, Stop the enzyme action by adding 5 drops of 10 M Hydrochloric Acid. Shake the tube well after adding Hydrochloric Acid. Add to each tube 1 drop of 0.002 M Iodine and shake well. Note the change of colour in the tubes. With Increasing concentration of enzyme, there will be fall in the intensity of the colour from light purple, red to colourless. The tube showing no colour on the addition of iodine is to be taken as the end point.

It is assumed that amount of enzyme present in the tube showing no colour on the addition of iodine solution hasfully digested the quantity of starch present,

Papain:

Reagents:
1.Cystein Hydrochloride : Dissolve 0.5 gms. Of Cystein Hydrochloride accurately weighed in 10 ml of water, adjust

to PH 7.0 with solution of Sodium Hydroxide. It should freshly prepared.
2. Casein Solution : Dissolve 4.0 gms. Of purified Casein accurately weighed by shaking with 90 ml of water.

Adjust to PH 7.0 with 1 M Sodium Hydroxide and dilute to 100 ml with water.

Assay preparation : Take 100 mg of sample add 10 ml of Cystein Hydrochloride and dilute to 100 ml of water to get 1 mg/ml .

Procedure:
To 10 ml of water in each of two flasks, add 15 ml accurately measured solution of casein and maintain at 600 C by heating

on water-bath. To the first flask, add 25 ml of accurately measured assay preparation and to the second flask,

add 25 ml accurately measured same assay solution previously boiled for 3 minutes and cooled.

Maintain the solution at 600 C for 30 minutes, cool rapidly to room temparature and add to each

flask 10 ml of solution of formaldehyde previously neutralised to Phenolpthalene. Titrate both solutions

with 0.1 M Sodium Hydroxide to PH 8. The difference between the two titrations is not less than 4.5 ml.

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