Bitter principles Lec.2 (2017)

Bitter Principles
Lecture-2
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. Uses5. Uses
6. Chemical test
II. Lupulone (β- Lupulinic acid)
4. Isolation
5. Uses
6. Chemical test

• Chromone bitters
I. Khellin
4. Isolation
5. Uses
6. Mechanism of action
7. Chemical test7. Chemical test
II. Visnagin
4. Isolation
5. Uses
6. Chemical test

• Coumarin bitters
I. Xanthotoxin
4. Isolation
5. Uses
6. Mechanism of action
7. Chemical test
II. Imperatorin
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
R
OHHO
O
H
C
C
CH3
CH3
C
H2
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 resins10 % 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. 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.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

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 dissolving
in Petroleum ether
Repeated
crystallization from
metahnol
Mother liquor Crystals
Upper layer Lower layerMiddle layer
Isolation;
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

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.1.Visnagin occurs as colorless thread-like needles.
2.It is freely soluble in CHCl3 and ether, 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, then3.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, ethyl acetate; and almost insoluble
in petroleum ether.

Uses ;
• Khellin can be used for Vitiligo

Bitter principles Lec.2 (2017)

Vitiligo is a disease which causes loss of
pigmentation in different 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
R1R1
R R1
Psolaren H H
Xanthotoxin H OCH3
Imperatorin H OCH2- CH=C(CH3)2
Bergapten 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, hot 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
Orange yellow
color that turns to
light green.
Orange color that
turns to grass
green.
No reduction
Deep orange color
that turns to brown.
Orange color that
turns to brown.
ReductionReduction of ammoniacal
silver nitrate and
Fehling's solution.
Test with Wagner's reagent.
Boil with dilute HNO3.
No reduction
Precipitate
Yellow color turns
to crimson on
treatment with
alkali.
Reduction
No precipitate
Yellow color turns to
purple on treatment
with alkali.

Psoriasis is a chronic autoimmune condition that causes hyper proliferation
of outer skin layers

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.

The exact Topical mechanics in treatment of vitiligo 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.
Psoralens given orally are preferentially taken up by epidermal cells.
The best known biochemical reaction of methoxsalen is with DNA.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.

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.
Skin burning and increase in risks of squamous cell carcinoma (9 folds
more)
A prospective study of 1380 patients over 5 years revealed an approximately
nine-fold increase in risks of squamous cell carcinoma among PUVA treated
patients

Summary:
Non–Terpenoid Bitters
• Phenolic bitters
I. Humulone(α-Lupulinic acid)
4. Isolation
5. Uses5. Uses
6. Chemical test
II. Lupulone (β- Lupulinic acid)
4. Isolation
5. Uses
6. Chemical test

Bitter principles Lec.2 (2017)

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Bitter principles Lec.2 (2017)