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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5987_Библиотеки_им_академика_М_И_Перельмана.pdf
472
Pharmaceutical Chemistry of Natural Products
R
OHC
H
O
H
H
O
R
UV
H
H
R
O
HO
H
H
R
O
HO
H
H
R
O
HO
(3)
OAc
OH
R = C H
917
Ph P CH
32
Epi-ergocalciferol Ergocalciferol
11. X-ray analysis: X-ray analysis also confi rms the above structure (2) for vitamin D
2
.
Structure
of vitamins D
3
, D
4
and D
5
The structure of all these vitamins can be derived in the same manner like vitamin D
2
. Hence, the
structure of various vitamins D is as follows:
HO
7-Dehydrocholesterol
Irradiation
HO
Vitamin D
(5,6- -cholecalciferol)
3
cis
H
Vitamins
473
HO
22,23-Dihydro ergosterol
Irradiation
HO
Vitamin D
(22,23-Dihydro-5,6- -ergocalciferol)
4
cis
HO
7-Dehydro sitosterol
Irradiation
HO
Vitamin D
5
H
A detailed biological investigation has shown that vitamin D in CLO is not identical with
ergocalciferol, and the vitamin D activity could be conferred on cholesterol or on some impurity
in cholesterol other than ergosterol. In 1935, Windaus suggested that natural vitamin D present
in CLO is derived from 7-dehydro cholesterol. The following chart shows the method of preparing
7-dehydrocholesterol [originated by Windaus (1935), improved by Buser (1947) and Fieser et al.
(1950)].
H COCO
3
CrO
3
Cholesteryl acetate
H COCO
3
O
LiAlH
4
OH
C H COCl
65
OCOC H
65
C H OCO
65
C H N(CH )
65 32
C H OCO
65
KOH
7-Dehydro cholesterol
HO
HO
Irradiation of 7-dehydro cholesterol with UV light gives a product that is more active as
ergocalciferol (D
2
). In 1936, Windaus et al. found that this product was impure and the pure active
474
Pharmaceutical Chemistry of Natural Products
constituent was isolated as 3,5-dinitro benzoate. They named this vitamin D with a cholesterol
side chain as vitamin D
3
, and it has been shown to be identical with the natural vitamin that
is isolated from other fi sh liver oils such as tunny liver oil and halibut. In 1951, the chemical
society proposed the name cholecalciferol for vitamin D
3
. Now it was understood that the irradiation
of 7-dehydro cholesterol (at low temperature) fi rst produces provitamin D
3
, and this on gentle
heating is converted into vitamin D
3
.
Irradiation of 22,23-dihydro ergosterol gives a compound with antirachitic properties which is
named as vitamin D
4
by Windaus et al. in 1937.
VITAMIN E (TOCOPHEROLS)
Vitamin E, otherwise known as tocotrienol or tocopherol (TCP), is a naturally occurring antioxidant.
It is essential for normal reproduction in many animals, and hence, tocopherol is known as
antisterility vitamin. Tocopherol is described as a vitamin in search of a disease because of lack of
any specifi c vitamin E defi ciency disease in humans.
Vitamin E is the name given to a group of tocopherol which possesses vitamin E activity. Up
to now about eight tocopherols (e.g. as α, β, γ, δ) have been identifi ed. Among these tocopherols,
α-tocopherol is the most active vitamin and β- and γ-tocopherol possess half the activity of α-isomer.
Tocopherols are the derivatives of 6-hydroxy chromane (tocol) ring with three isoprenoid side
chain. The antioxidant property of tocopherol is due to the presence of chromane ring. According
to Karrer, tocol is the parent compound of all tocopherols. For example, α-tocopherol is 5,7,8-
trimethyl tocol, β-tocopherol is 5,8-dimethyl tocol and γ-tocopherol is 7,8-dimethyl tocol.
History
In 1922, Evans and Bishop observed that for normal reproduction in rats, a fat-soluble factor
present in vegetable oil was required. It was named as vitamin E. In 1936, compounds of vitamin E
activity was isolated and named as tocopherol (Greek: tokos means child birth; pheros means to bear;
ol means alcohol) by Evans and his associates. Finally, the structure of tocopherol was elucidated
by Fernholz in 1937 and its synthesis was achieved by Karrer.
Dietary Sources
Vegetable oils (olive, soya beans, palm, corn, saffl ower, sunfl ower, etc.), nuts, whole grains and
wheat germ are the most important sources of vitamin E. Other sources are seeds and green leafy
vegetables. The vitamin E content of vegetables, fruits, dairy products, fi sh and meat is relatively
low.
Vitamins
475
RDA
In humans, symptoms of vitamin E defi ciency are rare. Hence, it is rather diffi cult to fi nd
out its requirements accurately. Intake of vitamin E is directly related to the consumption of
polyunsaturated fatty acid (PUFA), i.e. requirement increases with increased intake of PUFA.
About 10 mg (15 IU) and 8 mg (12 IU) of α-tocopherol is recommended per day for men and
women, respectively. About 1 mg of α-tocopherol is equal to 1.5 IU. For pregnant and lactating
woman, vitamin E–supplemented diet is advisable.
Deficiency Symptoms
As depletion of vitamin E tissue stores takes a very long time, no clinical defi ciency symptoms
have been noted in healthy adults. Symptoms of vitamin E defi ciency are seen in patients with
fat malabsorption syndromes or liver disease, in individuals with genetic defects affecting the
α-tocopherol transfer protein and in newborn infants, particularly premature infants.
The symptoms of vitamin E defi ciency vary from one species to another. In many animals,
the defi ciency is associated with sterility. Vitamin E defi ciency results in neurological symptoms
(neuropathy), myopathy (muscle weakness) and pigmented retinopathy. Early diagnostic signs
are leakage of muscle enzymes, increased plasma levels of lipid peroxidation products and
increased haemolysis of erythrocytes (red blood cells or RBCs). In premature infants, vitamin E
defi ciency is associated with haemolytic anaemia, intraventricular haemorrhage and retrolental
fi broplasias.
Toxicity of Vitamin E
Vitamin E is the least toxic among all fat-soluble vitamins because no toxic effect has been reported
even after ingestion of 300 mg/day for 23 years.
Isolation
There are three methods to isolate vitamin E and their explanations are as follows:
Method 1
The wheat germs are dried and pressed to get oil. It is then saponifi ed with 20% alcoholic KOH
in the absence of oxygen. The saponifi ed portion is removed by precipitating with digitonin and
the unsaponifi ed portion is removed which contains sterols and vitamin E. Now the unsaponifi ed
portion subjected to distillation yields vitamin E fraction at 200–300°C under 0.1 mm pressure.
During saponifi cation there is loss of vitamins, hence this method is not economical.
476
Pharmaceutical Chemistry of Natural Products
Method 2
In this method, the oil left after removing sterol is treated with cyanic acid gas which converts
tocopherol into their respective esters. By fractional crystallization method, both α-tocopherol and
β-tocopherol esters are separated. Saponifi cation of these esters yields the respective tocopherol.
In the similar manner γ-tocopherol is obtained from cotton seed oil. This method is also not
economical because of loss of vitamins during saponifi cation.
Method 3
This method is called adsorption method. In this method, the wheat germ oil is dissolved in light
petroleum. Then, this is adsorbed on alumina to remove sterols. Further it was treated with cyanic
acid gas which coverts tocopherol into allophenates which are separated as above and converted
into tocopherol. This method is an economical one, because there no saponifi cation is involved.
Biochemical Function
Many biochemical functions of vitamin E are due to its antioxidant property. Generally, it prevents
the nonenzymatic oxidation of various cell components such as unsaturated fatty acid by molecular
oxygen and free radical such as superoxide (O
2
–
) and hydrogen peroxide (H
2
O
2
). Selenium helps
in this function of vitamin E.
Vitamin E is lipophilic in character and is found in association with lipoproteins, fat deposits
and cellular membranes. It protects the PUFAs from peroxidation reaction, and also acts as a
scavenger and gets itself oxidized to quinone form by free radicals (R) and spares PUFA.
Biochemical functions of vitamin E, directly or indirectly to its antioxidant property, are listed
below:
1. Vitamin E is closely associated with reproductive function and prevents sterility. It preserves
and maintains germinal epithelium of gonads for proper reproductive function.
2. It protects the liver from being damaged by toxic compounds such as CCl
4
.
3. Vitamin E works in association with vitamins A, C and β-carotene to delay the onset of
cataract.
4. Vitamin E increases the synthesis of haeme by enhancing the activity of enzymes
δ-aminolevulinic acid (ALA) synthase and ALA dehydratase.
5. Vitamin E prevents the oxidation of vitamin A and carotenes.
6. Vitamin E is required for optimum absorption of amino acids from the intestine.
7. Vitamin E is involved in proper synthesis of nucleic acid.
8. It is essential for the membrane structure and integrity of the cell.
9. Vitamin E prevents the peroxidation of PUFA in various tissues and membranes and protects
RBC from haemolysis by oxidizing agents such as H
2
O
2
.
10. Vitamin E is required for cellular respiration through electron transfer chain reaction (believed
to stabilize co-enzyme Q).
11. Recent studies reveal that high intake of vitamin E (about 200–300 mg/day) protects the
development of heart disease. It is believed that vitamin E prevents the oxidation of low-
density lipoproteins (LDLs). The oxidized LDLs have been implicated to promote heart
diseases.
Chemically, α-tocopherol may undergo the following sequence of reactions leading to the
formation of α-tocopherol quinone.
Vitamins
477
HC
3
HO
CH
3
CH
3
O
..
CH
3
R
H
.
TH
HC
3
.O
CH
3
CH
3
O
..
CH
3
R
T
.
H
H
3
C
CH
2
O
CH
3
O
..
CH
3
R
HO
2
R
CH
3
OH
C
O
CH
3
CH
3
O
HC
3
-Tocopherol quinone
It is now known that selenium is an essential component of the enzyme glutathione peroxidase
that scavenges toxic hydroperoxide compounds in tissues by the following reaction:
2GSH ROOH
Glutathione Alkyl
hydroperoxide
Glutathione
peroxidase
G-S-S-G
Oxidised
glutathione
ROH + H O
2
Therefore the body has two lines of defence against toxic hydroperoxides.
1. α-Tocopherol, which prevents the part of formation of these compounds.
2. Glutathione peroxidase, which converts toxic hydroperoxides (ingested or formed endogenously)
to harmless primary and secondary alcohols.
Structural elucidation of ␣-Tocopherol
In 1937, Fernholz elucidated the structure of α-tocopherol on the basis of following synthetic
and chemical evidences.
6
1
2
3
4
5
7
8
HC
3
HO
CH
3
CH
3
O
CH
3
CH CH CH —CH—
222
CH
3
CH CH CH —CH—
222
CH
3
CH CH CH —CH—
222
CH
3
CH
3
( ) -Tocopherol-
478
Pharmaceutical Chemistry of Natural Products
1. Molecular formula: The molecular formula of α-tocopherol has been found to be C
29
H
50
O
2
from elementary analysis and molecular weight determination.
2. Nature of fi rst oxygen atom: α-Tocopherol forms monoesters and monoether, which
suggests that one of the oxygen atoms is present as hydroxyl group. Further the hydroxyl
group was found to be phenolic in nature by the colour reaction with neutral ferric chloride,
its acetate formation and through UV spectra of α-tocopherol and its acetate.
CHO
29 50 2
-Tocopherol
Mono ester derivative
Acid chloride
3. Nature of the second oxygen atom: Chemical evidences confi rmed that the second
oxygen atom is present as cyclic ether.
4. Thermal decomposition of ␣-tocopherol: When α-tocopherol is heated under different
conditions, different products are obtained.
(a) When α-tocopherol is heated at 350°C, duroquinol is obtained.
CHO
29 50 2
350°C
-Tocopherol
Duroquinol
OH
OH
HC
3
HC
3
CH
3
CH
3
(b) When α-tocopherol is heated with selenium, duroquinone is obtained.
CHO
29 50 2
Heat
-Tocopherol
Duroquinone
O
O
HC
3
HC
3
CH
3
CH
3
Se
(c) When α-tocopherol is heated with HI, Ψ-cumenol is obtained.
CHO
29 50 2
HI
-Tocopherol
-Cumenol
OH
HC
3
HC
3
CH
3
Vitamins
479
From the above reactions, it is suggested that α-tocopherol is the monoether of the duro-
quinol and not the diether of duroquinol. The possibility of α-tocopherol as diether was
eliminated by the fact that α-tocopherol yields an allophanate which confi rms the presence
of one free hydroxyl group. The UV spectrum of α-tocopherol shows the band due to the
presence of a free hydroxyl group and also because it was phenolic.
Later it was reported that α-tocopherol as monoether of duroquinol is not correct because
the UV spectrum of α-tocopherol is different from the spectra of various monoethers of
duroquinol.
5. Oxidation of ␣-tocopherol: Dimethyl maleic anhydride and a compound (A) with the
composition C
21
H
40
O
2
were yielded when α-tocopherol was oxidized with chromic acid
under mild conditions. Then it was shown that the compound (A) is an optically active
saturated lactone. The lactone has been also derived from γ-hydroxy acid. Later the acid
lactonizes rapidly, which indicates that the hydroxy acid and the lactone is a γ-lactone. The
methyl ester of the hydroxy acid cannot be hydrolysed and the hydroxy acid is resistant to
oxidation and also could not be oxidized to a keto acid. These facts reveal that the hydroxy
group in the γ-hydroxy acid is tertiary in nature.
CHO
29 50 2
CrO
3
-Tocopherol
Dimethyl maleic anhydride
O
HC
3
HC
3
O
O CHO
21 40 2
Lactone
(A)
Hence, the structure of the lactone may be written as [R
1
+ R
2
= 17 atoms] I.
CH CH COOH
22
C
OH
R
1
R
2
Lactonization
HO
2
CH CH CO
22
C
R
1
R
2
O
-Lactone (I)
Hydroxy acid-
CH OH
3
CH CH COOH
22 3
C
OH
R
1
R
2
Hydrolysis
Not easily hydrolysed
Methyl ester
480
Pharmaceutical Chemistry of Natural Products
A mixture of an acid C
16
H
32
O
2
(II) and a ketone C
18
H
36
O (III) is obtained when α-tocopherol
acetate is oxidized with chromic acid under more vigorous conditions. According to
Fernholz, the formation of the above compounds can be explained by the oxidation of the
lactone at different points in the chain if the lactone is having R
1
= C
16
H
37
and R
2
= CH
3
.
CH CH CO
22
C
CH
16 33
CH
3
O
CrO
3
[O ]
CHO
16 32 2
(II)
CH CH CO
22
C
CH
16 33
CH
3
O
CrO
3
[O ]
C H COCH
16 33 3
(III)
By using the Kuhn–Roth method, Fernholz showed that the acid contains three –C–CH
3
groups. The naturally occurring compound contains the isoprene units. This led Fernholz
to postulate the structure of the acid as follows (II).
CH CH(CH ) CH(CH ) CH(CH ) COOH
3232322
CH
3
CH
3
CH
3
Structure of acid (II)
Since the acid is the degraded product of the lactone (I), the structure of the γ-lactone (I)
may be written as follows.
HC
2
OC
O
HC
2
C
CH (CH ) CH (CH ) CH (CH ) CH
2222323
CH
3
CH
3
CH
3
CH
3
CH
3
-Lactone (I)
6. Structure of ␣-tocopherol: From the above reactions, it is clear that α-tocopherol
contains a substituted benzene ring and a long side chain. Hence, α-tocopherol may be
having either chroman structure (IV) or coumarin structure (V).
CH
3
CH
3
HC
3
HO
O
CH
3
CH CH CH
222
CH CH CH CH
222
CH CH CH CH
222
CH CH
3
CH
3
CH
3
CH
3
(IV)
Vitamins
481
CH
3
CH
3
HC
3
HO
O
CHCH CH CH
222
CH CH CH CH
222
CH CH CH CH
222
CH CH
3
CH
3
CH
3
CH
3
(V)
CH
3
The above structure (IV) is acceptable because chromas are formed from phenols and γ,
γ-disubstituted allyl bromides while coumarins are formed from phenols and unsubstituted
allyl bromide. In 1938, Karrer synthesized α-tocopherol from a trimethyl quinol (phenolic
compound) and phytyl bromide (γ,γ-disubstituted allyl bromide).
CH
3
CH
3
HC
3
HO
O
CH
3
CH CH CH
222
CH CH CH CH
222
CH CH CH CH
222
CH CH
3
CH
3
CH
3
CH
3
( ) -Tocopherol-
CH
3
CH
3
HC
3
HO
OH
CH
3
CH CH CH
222
CH CH CH CH
222
CH CH CH CH
222
CH CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
HC
3
HO
OH
HC
3
C CH CH CH
222
CH CH CH CH
222
CH
CH
3
CH
3
CH
3
CH
HC
2
Br
CH CH CH
222
CH CH
3
Trimethylquinol
ZnCl
2
Phytyl bromide
Ring closure reaction
The above reaction is not completed because phenols may undergo condensation with allyl
bromide to form coumarins.