Phenolic Bitter Principles

2.1 Phenolic Bitter Principles

The crystalline acidic bitter principles having a phenolic function are found in naturally occurring plant sources, such as: humulon, lupulon.

2.1.1 Humulon

Synonyms Humulone; α-Lupulic acid, α-Bitter acid.

Biological Source It is obtained as an antibiotic constituent from the strobiles of Humulus lupulus L. belonging to the natural order Moraceae (Hops).

Chemical Structure

(R)-3, 5, 6-Trihydroxy-4, 6-bis (3-methyl-2-butenyl)-2-(3-methyl-1-oxobutyl)-2, 4-cyclohexadien-1-one; (C₂₁H₃₀O₅)

Isolation The various steps involved in the isolation of humulon from hops strobiles are:

1. Hops strobiles are extracted with ethanol for several hours and the alcoholic extract is filtered.

2. The filtrate is heated with animal charcoal and then cooled. In this manner, the charcoal adsorbs the bitter principles.

3. The charcoal is filtered and the adsorbed bitter principle is extracted with ethanol.

4. The alcoholic extract is evaporated over an water-electric bath and humulon is subsequently extracted from the resulting resinous residue by the help of boiling water repeatedly.

5. The combined aqueous fraction is cooled to 20°C and extracted with solvent ether successively.

6. The ethereal layer is filtered and evaporated in a Thin-Film Rotary Evaporator to obtain the bitter principle humulon as a residue.

Characteristic Features

1. The crystals obtained from ether have mp 65-66.5°C

2. It has a distinct bitter taste especially in alcoholic solution.

3. Humulon is observed to be more stable to air than lupulon.

4. It is a monobasic acid.

5. It has a specific optical rotation [α]²⁰_D – 212° (1.0 g in 15.5 g 96% v/v ethanol).

6. It has uv_max (ethanol): 237, 282 nm (ε 13, 760; 8330).

7. It is found to be soluble in usual organic solvent.

8. It is slightly soluble in boiling water from which it normally separates out as a milky precipitate on cooling.

9. It readily forms a sodium salt which is rapidly soluble in water.

10. Bacteriostatic Potency: Humulon suffers no loss of bacteriostatic potency against

Staphylococcus aureus upon autoclaving 40 ppm in phosphate buffer at pH 6.5 or 8.5. However, the addition of ascorbic acid in low concentrations extends the duration of bacteriostatic action.

Identification Tests These are as follows:

1. An ethanolic solution of humulon gives a reddish-violet colouration with a few-drops of FeCl₃ solution (0.5% w/v).

2. A few mg of humulon when dissolved in 0.5-1 ml NaOH solution (0.1 N) it produces a yellow colour.

3. Humulon reduces Tollen’s Reagent (i.e., ammoniaco silver nitrate solution) in cold and gives a silver mirror.

4. Humulon on being heated with a alcoholic solution of NaOH (0.5 N) undergoes complete decomposition to yield: humulinic acid (an unsaturated acid), acetic acid, isobutyric aldehyde and an unsaturated liquid volatile acid.

Uses

1. Humulon exerts bacteriostatic action.

2. It contributes to the bitterness of hops extract used in making beer.

2.2.2 Lupulon

Synonyms β-Bitter Acid; β-Lupulic Acid.

Biological Sources The biological sources of lupulon are the same as for humulon given under sections 2.1.1.

Chemical Structure

3, 5-Dihydroxy-2, 6, 6-tris (3-methyl-2-butenyl)-4-(3-methy-1-oxobutyl)-2, 4-cyclohexadien-1-one; (C₂₆H₃₈O₄).

Isolation Lupulon may be isolated from the commercial hops Humulus lupulus L. (Moraceae) by the method suggested by Lewis et al.*

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* Lewis et al., J. Clin. Invest., 28, 916 (1949).

Characteristic Features

1. It is obtained as prisms from 90% (v/v) methanol having mp 92-94°C.

2. It possesses a distinct bitter taste especially in alcoholic solutions.

3. It behaves as a monobasic acid.

4. It is perfectly stable in vacuo even upto a temperature of 60°C.

5. It exhibits a slight acid reaction.

6. It is found to be optically inactive.

7. It is freely soluble in ethanol, methanol, hexane, petroleum ether, isooctane; and slightly soluble in either neutral or acidic aqueous solutions.

8. It readily forms a sodium salt which is rapidly soluble in water.

9. The addition of 0.1% solution of ascorbic acid affords a marked and pronounced protective action upon the bacteriostatic activity of lupulon steamed or autoclaved at a concentration of 4 ppm in phosphate buffer at pH 6.5 and 8.5.

Identification Tests

1. Lupulon turns yellow and amorphous in nature within a few days with the development of a characteristic odour.

2. Lupulon on being subjected to oxidation with potassium permanganate solution gives rise to the formation of valerianic acid.

Uses

1. Lupulon contributes exclusively to the bitterness of hops extract and is employed in the manufacture of beer.

2. It possesses the properties of an aromatic bitter and are said to have a sedative activity.

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Source:Pharmacognosy And Pharmacobiotechnology By Ashutosh Kar