Bromelain

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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CAS RN: 9001-00-7.

A glycoprotein that is highly active thiol proteinase. It is found in the leaves and stems of the pineapple plant. Yellowish-white to tan powder.

ACTIVITY DETERMINATION

pH 4.5 water: Adjust with 0.1 N hydrochloric acid to a pH of 4.5.

Gelatin substrate: Dissolve 25 g of gelatin in 375 mL of hot water. Bring to a boil. Cool to 45°. Adjust the pH to 4.5 with 0.1 N hydrochloric acid. Dilute with pH 4.5 water to 500 mL. Keep it at 45°. This substrate should be prepared fresh daily.

Buffer solution: Add 150 g of sodium chloride to 700 mL of pH 4.5 water, stir to dissolve, then add 5.7 mL of acetic acid. Adjust with 50% sodium hydroxide to a pH of 4.5, if necessary. Dilute to 1 L.

3% Hydrogen peroxide solution: Transfer 2.5 mL of hydrogen peroxide to a 25-mL volumetric flask. Dilute with pH 4.5 water to volume.

pH 9.0 formaldehyde solution: Adjust a 100-mL formaldehyde solution to a pH of 9.0 with 0.1 N sodium hydroxide VS.

Bromelain preparation: Weigh 100 mg of bromelain with a theoretical activity of 2400 GDU/g. If the sample activity differs by more than 10% from 2400 GDU/g, determine the sample weight:

mg of sample = (2400 × 100)/theoretical activity

Transfer the sample to a 50-mL volumetric flask. Add 8.3 mL of Buffer solution. Let stand for 30 min at room temperature. Dilute with pH 4.5 water to volume. Add a small stir bar and stir for 10-15 min, resulting in a suspension as bromelain will not dissolve. _{(USP 1-Dec-2023)}

Procedure: Transfer 25 mL of Gelatin substrate to each of two 100-mL beakers containing stir bars and place them in a water bath at 45° for 5 min. One beaker is for the Test solution and the other for the Blank solution.

Test solution: Add 1.0 mL of Bromelain preparation into the beaker, start timing, and swirl. After exactly 20 min of incubation at 45°, add 0.1 mL of 3% Hydrogen peroxide solution, and swirl. Incubate for an additional 5 min. Remove the beaker from the water bath and, with constant stirring, insert the pH electrode. Record the pH after 10 s (initial pH). Adjust with 0.1 N sodium hydroxide VS to a pH of 6.0. [NOTE -When adjusting the pH to 6.0 be cautious at pH 5.8; the pH increases slowly but minute additions of sodium hydroxide at this point will significantly increase the pH.] Add 10 mL of pH 9.0 formaldehyde solution with constant stirring. Titrate to a pH of 9.0 with 0.1 N sodium hydroxide VS. Record the volume of titrant used. This is the test titer, T.

Blank solution: Run concurrently with the Test solution by starting the Blank solution determination 12 min after the Test solution is started. Add 0.1 mL of 3% Hydrogen peroxide solution to the Blank solution beaker, and swirl. After exactly 20 min of incubation at 45°, add 1.0 mL of Bromelain preparation, and swirl. Incubate for an additional 15 min. Remove the beaker from the water bath and, with constant stirring, insert the pH electrode. Record the pH after 10 s (initial pH). Adjust with 0.1 N sodium hydroxide to a pH of 6.0. See the Note under Test solution. Add 10 mL of pH 9.0 formaldehyde solution with constant stirring. Titrate to a pH of 9.0 with 0.1 N sodium hydroxide VS. Record the volume of titrant used. This is the blank titer, B.

Calculation: 1 Gelatin Digestion Unit (GDU) is the amount of enzyme that, after 20 min of digestion at 45°, will liberate 1 mg of amino nitrogen from a standard gelatin solution at a pH of 4.5.

GDU/g = (T − B) × 14 × N × (50/W)

T = mL of 0.1 N sodium hydroxide used with the Test solution

B = mL of 0.1 N sodium hydroxide used with the Blank solution

N = actual normality of 0.1 N sodium hydroxide VS from standardization

W = weight of bromelain taken (g)