Bitter Principles

Lecture-3

By

Dr. Ahmed Metwaly

Objectives:Non–Terpenoid Bitters

• Phenolic bitters

I. Humulone(α-Lupulinic acid)

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

II. Lupulone (β- Lupulinic acid)

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

• Chromone bitters

I. Khellin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Mechanism of action

7. Chemical test

II. Visnagin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

• Coumarin bitters

I. Xanthotoxin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Mechanism of action

7. Chemical test

II. Imperatorin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

Phenolic bitters

Humulone (α-Lupulinic acid) and Lupulone (β- Lupulinic acid)

Structure

These are crystalline phloroglucinol derivatives, the α-acids e.g. humulone

and the β-acids e.g. lupulone

Humulone; (6S)-3,5,6-Trihydroxy-2-(3-methylbutanoyl)-4,6-bis(3-methylbut-2-en-1-

yl)cyclohexa-2,4-dien-1-one

Lupulone; 3,5-dihydroxy-2-(3-methylbutanoyl)-4,6,6-tris(3-methylbut-2-

enyl)cyclohexa-2,4-dien-1-one

Phloroglucinol Humulone Lupulone

O

ROHHO

O

HC

CCH3

CH3CH2

Humulone R = OH

Lupulone R =

Biological Source;

Humulone and lupulone are the

chief active constituents

obtained from the dried flowers

(strobiles) of Humulus lupulus

(hops) family Cannabinaceae

Moraceae.

The bitterness of hops is mainly

due to acidic bitter principles

humulone, lupulone and about

10 % of resins

Hops bear glandular hairs

which are known as lupulin,

which contain most of the bitter

principles that account for the

use of the product in brewing

and in medicine.

Humulus lupulus

Isolation of humulone

Hops strobiles are macerated with alcohol and filtered.

The filtrate is heated with animal charcoal to adsorb the bitter

principle, the suspension cooled and filtered.

The charcoal is boiled with alcohol to extract the bitter principles.

The alcohol extract is evaporated and the residue treated with boiling

water that extracts humulone.

The aqueous solution is cooled and humulone extracted by shaking

with ether.

The ether extract is evaporated to yield humulone.

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 uvmax (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. However, the addition of ascorbic

acid in low concentrations extends the duration of bacteriostatic action.

Test for identification;

An alcoholic solution of humulone gives a reddish-violet color with alcoholic

ferric chloride (phenolic).

Uses;

Humulone and lupulone contribute to the bitterness of hops.

They are used as aromatic bitters and exert a mild sedative action.

Hops are used in the manufacture of beer for taste improvement

(flavoring agent).

Humulone was found to exert a bacteriostatic activity

Chromone bitters

O O

OCH3

R

O

CH2R1

R R1

Khellin OCH3 H

Visnagin H H

Khellol glucoside H O-Glucose

Khellin and Visnagin

Khellin ; 4,9-Dimethoxy-7-methylfuro[3,2-g]chromen-5-one; (Khellin is a

Furanochromone compound.

Visnagin; 4-methoxy-7-methylfuro[3,2-g]chromen-5-one;

Khellol glucoside; 7-[(β-D-glucopyrano)-methyl 4-methoxy-7-

methylfuro[3,2-g]chromen-5-one

Biological source;

Fruits of Ammi visnaga

family Umbelliferae.

The fruit contains about 1

% of khellin beside two

other crystalline

compounds, visnagin

(about 0.1 %) and khellol

glycoside (about 0.3 %).

Ammi visnaga

Powdered Ammi visnaga fruits

Extract with ether.

Ether extract

- Concentrate - Cool (refrigerator)

Green

Oily

Cream colored

Fatty

Green

Crystalline

Discard by filtration (suction)

Discard by dissolvingin Petroleum ether

Repeated crystallization from metahnol

Mother liquor Crystals

Khellin- Evaporate to dryness- Benzene

Visnagin &

traces of Khellin

Benzene solution

- Petroleum ether - Cool- Filter

Crystals

Khellin

Filtrate

Visnagin

Dry & recrystallize

from methanol

Upper layer Lower layer Middle layer

Isolation;

Properties;

Khellin:

1.Khellin occurs as bitter needle crystals, m.p. 153ºC.

2.It is freely soluble in CHCl3 and alcohol, less soluble in ether, sparingly

soluble in petroleum ether and cold H2O, but more soluble in hot water

and hot CH3OH.

Visnagin:

1.Visnagin occurs as colorless thread-like needles.

2.It is freely soluble in CHCl3, sparingly soluble in alcohol, insoluble in cold

water and petroleum ether

Khellol glucoside;

1. It is obtained as crystals from ethanol having mp 179°C.

2. It has uvmax (ethanol): 250, 325 nm.

3. It is found to be soluble in acetic acid, hot ethanol slightly soluble in hot

methanol; and almost

insoluble in acetone, ethyl acetate, ether, chloroform, cold alkali.

Tests for identification;

Khellin:

1.Mix few crystals of khellin with solid KOH or NaOH a rose red color is

produced (not given with K2CO3 or Na2CO3 or bicarbonate).

2.Mix few crystals of khellin in a porcelain dish with a drop of H2SO4 or of

phosphoric acid, a reddish-orange color is produced, that turn to bright yellow

on addition of 2-3 drops of H2O.

3.Add about 1-ml nitric acid (1:1) to few mg of khellin in a porcelain dish, then

add 5 ml NaOH solution, violet color is produced.

4.To few crystals of khellin, add an equal quantity of ninhydrin followed by 2 –

3 drops of concentrated H2SO4, an emerald green color is produced on

stirring.

Visnagin:

Triturate few crystals of visnagin with solid KOH or NaOH, a faint rose red

color is produced (paler than that produced with khellin)

Identification Test Khellol glucoside may be identified by making its

following tetraacetate derivative due to the presence of four OH moieties

Khellol Glucoside Tetracetate It is obtained as flakes from ethanol having

mp 153°C. It is freely soluble in acetone, ethanol, ethyl acetate; and almost

insoluble in petroleum ether.

Uses ;

• Khellin can be used for Vitiligo

Vitiligo is a disease which causes loss of

pigmentation in portions of skin.

Mechanism of Khellin action is;

When khellin is applied topically in

combination with UVA light, it is able to

stimulate melanocytes (cells that produce

melanin) in hair follicles for successful

treatment of vitiligo

Khellin can be used for Kidney Stones and Renal colic

Khellin has also been used to treat renal

colic, which is due mostly to stone

formation. It were seen to relieve renal

colic by relaxing the ureter and acting as

a diuretic.

As khellin act as vasodilator, It is used in treatment of coronary

insufficiency, angina pectoris and in bronchial asthma.

Vinagin and kellol glucosides also have a vasodilator effects.

Side effects;

Unwanted side effects include dizziness, reversible cholestatic jaundice,

pseudoallergic reaction, and elevated levels of liver enzymes

Coumarin bitters

Xanthotoxin, Imperatorin and Bergapten

OO O

R

R1

R R1

Psolaren H HXanthotoxin H OCH3

Imperatorin H OCH2- CH=C(CH3)2Bergapten OCH3 H

Xanthotoxin 9-methoxy-7H-furo[3,2-g]chromen-7-one

9-(3-Methylbut-2-enoxy)-7-furo[3,2-g]chromenone

Biological source;

Xanthotoxin (Ammoidin or Methoxsalen) is obtained from the fruits of Ammi

majus (Umbelliferae).

Imperatorin (Ammidin) occurs in the fruits of Ammi majus (Umbelliferae).

Bergapten was first isolated from the oil of bergamot from Citrus bergamia

(Rutaceae) and fruits of Ammi majus and some other plants belonging to

family Umbelliferae .

Ammi majus

Isolation of Xanthotoxin and Imperatorin;

Xanthotoxin and Imperatorin are isolated from the petroleum ether

extract of the fruits of Ammi majus according to the following scheme:

Powdered Ammi majus fruits

- Extract with petroleum ether - Concentrate - Decantation while hot

Crystalline mass

Dark green , semi-solid

Crude xanthotoxin

- Dissolve in boiling alcohol- Cool

Pale green crystals

Mother liquor- Concentrate- Cool

Crude imperatorin

Xanthotoxin

- Dissolve in ether- Concentrate and cool

Imperatorin

Properties;

Xanthotoxin occurs as a crystalline solid substance with bitter taste. It is

insoluble in cold water, more soluble in boiling H2O, petroleum ether,

ether, benzene, CHCl3, ethyl acetate and glacial acetic acid.

Imperatorin occurs as prisms or needle crystals with the same

characters as xanthotoxin.

Test for Identification;

Test Xanthotoxin Imperatorin

Triturated few crystals in a

porcelain dish with

concentrated H2SO4.

Test with Marquis reagent.

Reduction of ammoniacal

silver nitrate and

Fehling's solution.

Test with Wagner's reagent.

Boil with dilute HNO3.

Orange yellow

color that turns to

light green.

Orange color that

turns to grass

green.

No reduction

Precipitate

Yellow color turns

to crimson on

treatment with

alkali.

Deep orange color

that turns to brown.

Orange color that

turns to brown.

Reduction

No precipitate

Yellow color turns to

purple on treatment

with alkali.

Xanthotoxin used to treat psoriasis

psoriasis

Uses

Xanthotoxin used to treat psoriasis, eczema and vitiligo in conjunction

with exposing the skin to UVA light from lamps or sunlight.

The dosage comes in 10 mg tablets, which are taken in the amount of 30 mg

75 minutes before a PUVA (psoralen + UVA) light treatment.

Mechanism of action;

The exact mechanism of action of methoxsalen with the epidermal

melanocytes and keratinocytes is not known.

Psoralens given orally are preferentially taken up by epidermal cells. The

best known biochemical reaction of methoxsalen is with DNA. Methoxsalen,

upon photoactivation, conjugates and forms covalent bonds with DNA which

leads to the formation of both monofunctional (addition to a single strand of

DNA) and bifunctional adducts (crosslinking of psoralen to both strands of

DNA). Reactions with proteins have also been described.

The exact Topical mechanics are unknown but it has been suggested that

melanocytes in the hair follicles are stimulated to move up the follicle and to

repopulate the epidermis. Methoxsalen modifies the way skin cells receive

the UVA radiation, allegedly clearing up the disease.

Contraindications; Patients with high blood pressure or a history

of liver problems are at risk for inflammation and irreparable damage to

both liver and skin. The eyes must be protected from UVA radiation.

Side effects include nausea, headaches, dizziness, and in rare

cases insomnia.

Summary:Non–Terpenoid Bitters

• Phenolic bitters

I. Humulone(α-Lupulinic acid)

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

II. Lupulone (β- Lupulinic acid)

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

• Chromone bitters

I. Khellin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Mechanism of action

7. Chemical test

II. Visnagin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test

• Coumarin bitters

I. Xanthotoxin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Mechanism of action

7. Chemical test

II. Imperatorin

1. Biological Sources

2. Chemical structure

3. Characteristic features

4. Isolation

5. Uses

6. Chemical test