U.S. patent application number 10/284158 was filed with the patent office on 2003-03-20 for method of increasing the presence of glutathione in cells.
This patent application is currently assigned to N. V. Nutricia. Invention is credited to Hageman, Robert Johan Joseph, Norren, Klaske van, Smit, Hobbe Friso.
Application Number | 20030054048 10/284158 |
Document ID | / |
Family ID | 24564219 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054048 |
Kind Code |
A1 |
Smit, Hobbe Friso ; et
al. |
March 20, 2003 |
Method of increasing the presence of glutathione in cells
Abstract
A composition for increasing the presence of glutathione in
cells and for treating or preventing impaired liver function. The
composition includes a precursor of gluthathione, and enhancer of
glutathione biosynthesis, and a third component which is a lignan
or the third component is a combination of an enhancer of
gluthathione regeneration and an enhancer of glutathione-mediated
conjugation. The precursor of gluthathione is cysteine or is a
functional equivalent of cysteine which yields cysteine when
administered to a mammal undergoing treatment.
Inventors: |
Smit, Hobbe Friso; (Utrecht,
NL) ; Norren, Klaske van; (Renkum, NL) ;
Hageman, Robert Johan Joseph; (Waddinxveen, NL) |
Correspondence
Address: |
BACON & THOMAS, PLLC
625 SLATERS LANE
FOURTH FLOOR
ALEXANDRIA
VA
22314
|
Assignee: |
N. V. Nutricia
Eerste Stationsstraat 186
Zoetermeer HM
NL
NL-2712
|
Family ID: |
24564219 |
Appl. No.: |
10/284158 |
Filed: |
October 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10284158 |
Oct 31, 2002 |
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09639469 |
Aug 16, 2000 |
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6495170 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 31/198 20130101; A61K 36/9066 20130101; A23L 33/175
20160801; A61K 36/28 20130101; A61K 36/82 20130101; A61K 36/28
20130101; A61K 31/375 20130101; A61K 31/198 20130101; A61K 36/82
20130101; A61K 36/79 20130101; A61K 36/9066 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A23L 33/105 20160801; Y10S 514/893 20130101; A61K
36/79 20130101; A61K 31/355 20130101; A23L 33/155 20160801; A23L
33/15 20160801; A61K 36/80 20130101; A61K 36/80 20130101; A61K
31/375 20130101; A61K 31/355 20130101; A23L 33/16 20160801 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 035/78 |
Claims
1. Method of increasing the presence of glutathione in cells
including treating or preventing impaired liver function by
administering to a mammal in the need thereof a composition which
contains at least one of each of the following components a) a
precursor of glutathione which is selected from the group
consisting of cysteine and functional equivalents containing said
amino acid in an amount of at least 100 mg cysteine per day,
calculated as free amino acid; b) an enhancer of the glutathione
biosynthesis; and c) a third component which is either selected
from lignans or which is a combination c1) of an enhancer of
glutathione regeneration and c2) of an enhancer of the
glutathione-mediated conjugation.
2. Method of increasing the presence of glutathione in cells
according to claim 1 by administering a composition wherein the
enhancer of the glutathione biosynthesis b) contains apocynin or
analogues; the enhancer of glutathione regeneration c1) contains an
active ingredient which is selected from the group of silymarin and
lignans; and the enhancer of the glutathione-mediated conjugation
c2) contains an active ingredient which is selected from the group
consisting of iridoid glycosides and lignans.
3. Method of increasing the presence of glutathione in cells
according to claim 1 or 2 by administering a composition wherein
the enhancer of the glutathione biosynthesis b) is a preparation
obtained from Picrorhiza species; the enhancer of glutathione
regeneration c1) is selected from the group consisting of a
preparation of Silybum marianum and a preparation of Schisandra
species; and the enhancer of the glutathione-mediated conjugation
c2) is selected from the group consisting of a preparation of
Picrorhiza species and a preparation of Schisandra species.
4. Method of increasing the presence of glutathione in cells
according to claim 1 by administering a composition wherein the
precursor of glutathione a) comprises cysteine in an amount of 100
to 2000 mg per day; the enhancer of the glutathione biosynthesis b)
is selected from the group consisting of an extract of Picrorhiza
kurrooa and an extract of Picrorhiza schrophulariiflora, whereby
said extract contains 0.01 to 100 mg per day apocynin or analogues;
the enhancer of glutathione regeneration c1) is selected from the
group consisting of an extract of Silybum marianum containing 10 to
1000 mg per day silymarin and an extract of Schisandra chinensis or
Schisandra phenanthera containing 0.1-100 mg per day schisandrins;
and the enhancer of the glutathione-mediated conjugation c2) is
selected from the group consisting of an extract, which contains 1
to 100 mg per day picrosides and has been obtained from Picrorhiza
kurrooa or from Picrorhiza schrophulariiflora or from both
Picrorhiza spezies, and of an extract, which contains 0.1 to 100 mg
per day schisandrins and has been obtained from Schisandra
chinensis or from Schisandra phenanthera or from both Schisandra
spezies.
5. Method of increasing the presence of glutathione in cells
according to claim 1 or 4 wherein a composition is administered
which contains in addition a further active ingredient d) which is
selected from the group consisting of methionine and functional
analogues containing said amino acid, folic acid, vitamin B12,
vitamin B6, selenium yeast, a zinc salt, a copper salt, and a
manganese salt.
6. Method of increasing the presence of glutathione in cells
according to claim 5 wherein the active ingredient d) is selected
from the group consisting of 100 to 1000 mg per day methionine,
calculated as free amino acid, 10 to 200 mcg per day selenium
yeast, 0.1 to 30 mg per day zinc oxide, 0.1 to 8 mg per day cupric
oxide and 0.1 to 20 mg per day manganese gluconate.
7. Dietetic or pharmaceutical composition for increasing the
presence of glutathione in cells including the prevention or
treatment of impaired liver function, characterised in that the
composition contains in combination at least one of each of the
following components: a) a precursor of glutathione which is
selected from the group consisting of cysteine and functional
equivalents containing said amino acid; b) an enhancer of the
glutathione biosynthesis; and c) a third component which is either
selected from lignans or which is a combination c1) of an enhancer
of glutathione regeneration and c2) of an enhancer of the
glutathione-mediated conjugation; and that cysteine as precursor of
glutathione a) accounts for at least 1.5 g, calculated as free
amino acid, per 100 g of solid matter contents of the
composition.
8. Dietetic or pharmaceutical composition according to claim 7,
characterised in that the enhancer of the glutathione biosynthesis
b) contains apocynin or analogues; the enhancer of glutathione
regeneration c1) contains an active ingredient which is selected
from the group of silymarin and lignans; and the enhancer of the
glutathione-mediated conjugation c2) contains an active ingredient
which is selected from the group consisting of iridoid glycosides
and lignans.
9. Dietetic or pharmaceutical composition according to claim 7 or 8
characterised in that the enhancer of the glutathione biosynthesis
b) is a preparation obtained from Picrorhiza species; the enhancer
of glutathione regeneration c1) is selected from the group
consisting of a preparation of Silybum marianum and a preparation
of Schisandra species; and the enhancer of the glutathione-mediated
conjugation c2) is selected from the group consisting of a
preparation of Picrorhiza species and a preparation of Schisandra
species.
10. Dietetic or pharmaceutical composition according to claim 7
characterised in that the enhancer of the glutathione biosynthesis
b) is selected from the group consisting of an extract of
Picrorhiza kurrooa and an extract of Picrorhiza schrophulariiflora,
whereby said extract contains at least 5 mg apocynin or analogues
per 100 g of solid matter contents of the composition; the enhancer
of glutathione regeneration c1) is selected from the group
consisting of an extract of Silybum marianum containing at least
150 mg silymarin per 100 g of solid matter contents of the
composition and an extract of Schisandra chinensis or Schisandra
phenanthera containing at least 1.5 mg schisandrins per 100 g of
solid matter contents of the compisition; and the enhancer of the
glutathione-mediated conjugation c2) is selected from the group
consisting of an extract, which contains at least 15 mg picrosides
per 100 g of solid matter contents of the composition and has been
obtained from Picrorhiza kurrooa or from Picrorhiza
schrophulariiflora or from both Picrorhiza spezies, and of an
extract, which contains at least 1.5 mg schisandrins per 100 g of
solid matter contents of the composition and has been obtained from
Schisandra chinensis or from Schisandra phenanthere or from both
Schisandra spezies.
11. Dietetic or pharmaceutical composition according to claim 7 or
10 characterised in that the composition contains in addition a
further active ingredient d) which is selected from the group
consisting of methionine and functional analogues containing said
amino acid, selenium yeast, a zinc salt, a copper salt, folic acid,
vitamin B12, vitamin B6 and a manganese salt.
12. Dietetic or pharmaceutical composition according to claim 11
characterised in that the ingredient d) is selected from the group
consisting of 1.5 g to 15 g methionine, calculated as free amino
acid, per 100 g of solid matter contents of the composition, 150 to
3000 mcg selenium yeast per 100 g of solid matter contents of the
composition; 1.5 to 450 mg zinc oxide per 100 g of solid matter
contents of the composition; 1.5 to 120 mg cupric oxide per 100 g
of solid matter contents of the composition; and 1.5 to 300 mg
manganese gluconate per 100 g of solid matter contents of the
composition.
13. Dietetic or pharmaceutical composition according to claim 7 or
10 characterised in that that cysteine as precursor of glutathione
a) accounts for at least 3.0 g, calculated as free amino acid, per
100 g of solid matter contents of the composition.
14. Dietetic or pharmaceutical composition for increasing the
presence of glutathione in cells including the prevention or
treatment of impaired liver function, characterised in that the
composition contains in combination at least one of each of the
following components: a) a precursor of glutathione which is
selected from the group consisting of cysteine and functional
equivalents containing said amino acid in an amount of at least 0.1
g cysteine per day, calculated as free amino acid; b) an enhancer
of the glutathione biosynthesis; and c) a third component which is
either selected from lignans or which is a combination c1) of an
enhancer of glutathione regeneration and c2) of an enhancer of the
glutathione-mediated conjugation.
15. Dietetic or pharmaceutical composition according to claim 14,
characterised in that the enhancer of the glutathione biosynthesis
b) contains apocynin or analogues; the enhancer of glutathione
regeneration c1) contains an active ingredient which is selected
from the group of silymarin and lignans; and the enhancer of the
glutathione-mediated conjugation c2) contains an active ingredient
which is selected from the group consisting of iridoid glycosides
and lignans.
16. Dietetic or pharmaceutical composition according to claim 14 or
15 characterised in that the enhancer of the glutathione
biosynthesis b) is a preparation obtained from Picrorhiza species;
the enhancer of glutathione regeneration c1) is selected from the
group consisting of a preparation of Silybum marianum and a
preparation of Schisandra species; and the enhancer of the
glutathione-mediated conjugation c2) is selected from the group
consisting of a preparation of Picrorhiza species and a preparation
of Schisandra species.
17. Dietetic or pharmaceutical composition according to claim 14
characterised in that the enhancer of the glutathione biosynthesis
b) is selected from the group consisting of an extract of
Picrorhiza kurrooa and an extract of Picrorhiza schrophulariiflora,
whereby said extract contains 0.01 to 100 mg apocynin or analogues
per day; the enhancer of glutathione regeneration c1) is selected
from the group consisting of an extract of Silybum marianum
containing 10 to 1000 mg silymarin per day and an extract of
Schisandra chinensis or Schisandra phenanthera containing 0.1-100
mg schisandrins per day; and the enhancer of the
glutathione-mediated conjugation c2) is selected from the group
consisting of an extract, which contains 1 to 100 mg picrosides per
day and has been obtained from Picrorhiza kurrooa or from
Picrorhiza schrophulariiflora or from both Picrorhiza spezies, and
of an extract, which contains 0.1 to 100 mg schisandrins per day
and has been obtained from Schisandra chinensis or from Schisandra
phenanthera or from both Schisandra spezies.
18. Dietetic or pharmaceutical composition according to claim 14 or
7 characterised in that the composition contains in addition a
further active ingredient d) which is selected from the group
consisting of methionine and functional analogues containing said
amino acid, folic acid, vitamin B12, vitamin B6, selenium yeast, a
zinc salt, a copper salt, and a manganese salt.
19. Dietetic or pharmaceutical composition according to claim 18,
characterised in that the ingredient d) is selected from the group
consisting of 100 to 1000 mg per day methionine, calculated as free
amino acid, 10 to 200 mcg per day selenium yeast, 0.1 to 30 mg per
day zinc oxide, 0.1 to 8 mg per day cupric oxide and 0.1 to 20 mg
per day manganese gluconate.
20. Dietetic or pharmaceutical composition for increasing the
presence of glutathione in cells including the prevention or
treatment of impaired liver function, characterised in that it
represents a unit dosage form; that it contains in combination at
least one of each of the following components: a) a precursor of
glutathione which is selected from the group consisting of cysteine
and functional equivalents containing said amino acid: b) an
enhancer of the glutathione biosynthesis; and c) a third component
which is either selected from lignans or which is a combination c1)
of an enhancer of glutathione regeneration and c2) of an enhancer
of the glutathione-mediated conjugation; and that cysteine as
precursor of glutathione a) is present in an amount of at least 30
mg, calculated as free amino acid, per unit dosage form.
21. Dietetic or pharmaceutical composition according to claim 20
characterised in that the enhancer of the glutathione biosynthesis
b) contains apocynin or analogues; the enhancer of glutathione
regeneration c1) contains an active ingredient which is selected
from the group of silymarin and lignans; and the enhancer of the
glutathione-mediated conjugation c2) contains an active ingredient
which is selected from the group consisting of iridoid glycosides
and lignans.
22. Dietetic or pharmaceutical composition according to claim 20 or
21, characterised in that the enhancer of the glutathione
biosynthesis b) is a preparation obtained from Picrorhiza species;
the enhancer of glutathione regeneration c1) is selected from the
group consisting of a preparation of Silybum marianum and a
preparation of Schisandra species; and the enhancer of the
glutathione-mediated conjugation c2) is selected from the group
consisting of a preparation of Picrorhiza species and a preparation
of Schisandra species.
23. Dietetic or pharmaceutical composition according to claim 20
characterised in that the precursor of glutathione a) comprises
cysteine in an amount of 30 to 333 mg; the enhancer of the
glutathione biosynthesis b) is selected from the group consisting
of an extract of Picrorhiza kurrooa and an extract of Picrorhiza
schrophulariiflora, whereby said extract contains 0.0033 to 33 mg
apocynin or analogues; the enhancer of glutathione regeneration c1)
is selected from the group consisting of an extract of Silybum
marianum containing 3,3 to 333 mg silymarin and an extract of
Schisandra chinensis or Schisandra phenanthera containing 0.033 to
33 mg schisandrins; and the enhancer of the glutathione-mediated
conjugation c2) is selected from the group consisting of an
extract, which contains 0,33 to 33 mg picrosides and has been
obtained from Picrorhiza kurrooa or from Picrorhiza
schrophulariiflora or from both Picrorhiza spezies, and of an
extract, which contains 0.033 to 33 mg schisandrins and has been
obtained from Schisandra chinensis or from Schisandra phenanthera
or from both Schisandra spezies.
24. Dietetic or pharmaceutical composition according to claim 20 or
23 characterised in that the composition contains in addition a
further active ingredient d) which is selected from the group
consisting of methionine and functional analogues containing said
amino acid, selenium yeast, a zinc salt, a copper salt, folic acid,
vitamin B12, vitamin B6 and a manganese salt.
25. Dietetic or pharmaceutical composition according to claim 24
characterised in that the ingredient d) is selected from the group
consisting of 33 to 333 mg methionine, calculated as free amino
acid, 3,3 to 66 mcg selenium yeast, 0.033 to 10 mg of zinc oxide,
0.033 to 2,4 mg cupric oxide and 0.033 to 6,6 mg manganese
gluconate.
26. Dietetic or pharmaceutical composition for increasing the
presence of glutathione in cells including the prevention or
treatment of impaired liver function, characterised in that the
composition contains in combination at least one of each of the
following components: a) a precursor of glutathione which is
selected from the group consisting of cysteine and functional
equivalents containing said amino acid; b) an enhancer of the
glutathione biosynthesis; and c) a third component which is either
selected from lignans or which is a combination c1) of an enhancer
of glutathione regeneration and c2) of an enhancer of the
glutathione-mediated conjugation; and that cysteine as precursor of
glutathione a) accounts for at least 25 g, calculated as free amino
acid, per 100 g of solid matter contents of the sum of the active
components a) to c2).
27. Dietetic or pharmaceutical composition according to claim 26,
characterised in that the enhancer of the glutathione biosynthesis
b) contains apocynin or analogues; the enhancer of glutathione
regeneration c1) contains an active ingredient which is selected
from the group of silymarin and lignans; and the enhancer of the
giutathione-mediated conjugation c2) contains an active ingredient
which is selected from the group consisting of iridoid glycosides
and lignans.
28. Dietetic or pharmaceutical composition according to claim 26 or
27, characterised in that the enhancer of the glutathione
biosynthesis b) is a preparation obtained from Picrorhiza species;
the enhancer of glutathione regeneration c1) is selected from the
group consisting of a preparation of Silybum marianum and a
preparation of Schisandra species; and the enhancer of the
glutathione-mediated conjugation c2) is selected from the group
consisting of a preparation of Picrorhiza species and a preparation
of Schisandra species.
29. Dietetic or pharmaceutical composition according to claim 26
characterised in that the enhancer of the glutathione biosynthesis
b) is selected from the group consisting of an extract of
Picrorhiza kurrooa and an extract of Picrorhiza schrophulariiflora,
whereby said extract contains 70 mg to 40 g apocynin or analogues
per 100 g of solid matter contents of the sum of the components a)
to c2); the enhancer of glutathione regeneration c1) is selected
from the group consisting of an extract of Silybum marianum
containing at least 150 mg silymarin per 100 g of solid matter
contents of the sum of the active components a) to c2) and an
extract of Schisandra chinensis or Schisandra phenanthers
containing at least 1.5 mg schisandrins per 100 g of solid matter
contents of the sum of the active components a) to c2); and the
enhancer of the glutathione-mediated conjugation c2) is selected
from the group consisting of an extract, which contains at least 15
mg picrosides per 100 g of solid matter contents of the sum of the
active components a) to c2) and has been obtained from Picrorhiza
kurrooa or from Picrorhiza schrophulariiflora or from both
Picrorhiza spezies, and of an extract, which contains at least 1.5
mg schisandrins per 100 g of solid matter contents of the sum of
the active components a) to c2) and has been obtained from
Schisandra chinensis or from Schisandra phenanthera or from both
Schisandra spezies.
30. Dietetic or pharmaceutical composition according to claim 26 or
29 characterised in that the composition contains in addition a
further active ingredient d) which is selected from the group
consisting of methionine and functional analogues containing said
amino acid, selenium yeast, a zinc salt, a copper salt, folic acid,
vitamin B12, vitamin B6 and a manganese salt.
31. Dietetic or pharmaceutical composition according to claim 30
characterised in that the ingredient d) is selected from the group
consisting of 1.5 g to 15 g methionine, calculated as free amino
acid, per 100 g of solid matter contents of the sum of the active
components a) to c2); 150 to 3000 mcg selenium yeast per 100 g of
solid matter contents of the sum of the components a) to c2); 1.5
to 450 mg zinc oxide per 100 g of solid matter contents of the sum
of the components a) to c2); 1.5 to 120 mg cupric oxide per 100 g
of solid matter contents of the sum of the components a) to c2);
and 1.5 to 300 mg manganese gluconate per 100 g of solid matter
contents of the sum of the components a) to c2).
32. Dietetic or pharmaceutical composition according to claim 7 or
10 characterised in that that cysteine as precursor of glutathione
a) accounts for at least 30 g, calculated as free amino acid, per
100 g of solid matter contents composed of the active components a)
to c2).
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method of increasing the
presence of glutathione in cells including treating or preventing
of impaired liver function by administering to a mammal in the need
thereof a dietetic or pharmaceutical composition and to a
corresponding dietetic or pharmaceutical composition.
BACKGROUND OF THE INVENTION
[0002] The liver is the major organ involved in metabolism of
protein, carbohydrates, and fats, but is also the major organ for
detoxification. Potential toxic compounds are converted into
inactive metabolites by phase I-metabolising enzymes and excreted.
Alternatively, metabolites are further conjugated by phase
II-metabolising enzymes and excreted after all.
[0003] Many drugs and toxins can be detoxified by conjugation with
glutathione. When the levels of these drugs or toxins, however,
exceed the liver concentration of reduced glutathione; such
components become acute hepatotoxic. Striking is that in several
kinds of liver disorders, glutathione levels are decreased, for
example: in hepatitis infection, where the grade of activity of the
liver disease is correlated with reduction of GSH in acute liver
toxification, e.g. acetaminophen intoxication, and in alcoholics
with liver failure
[0004] Both hepatitis C and the exposure to liver toxins can lead
to hepatocarcinoma, possibly via the same mechanism of incompetence
of the liver to respond to toxins, either endo-toxins or
exo-toxins.
[0005] Reduced glutathione (GSH) is the substrate of glutathione
peroxidase (GPX) and as such contributes to the antioxidant defence
mechanism as well.
[0006] Glutathione reductase enzymes reduce GSSG back to GSH,
predominantly in the presence of NADPH, which is provided by e.g.
the oxidative pentose phosphate pathway. The capacity of the
glutathione system to cope with H.sub.2O.sub.2 in liver depends on
the activity of GPX and glutathione reductase, the rate of NADPH
supply, and the GSH content. Except for its effects on hydrogen
peroxide, GSH can also react with e.g. OH.degree., HOCI,
peroxynitrite, RO.degree., and RO.sub.2.degree. in vitro. Upon
reacting with free radicals, thiyl radicals (GS.degree.) are
produced, which generate superoxide. Moreover, superoxide can also
inactivate GPX in the absence of GSH. Hence, superoxide dismutase
(SOD) or antioxidants can co-operate with GSH to remove free
radicals in vivo.
[0007] Many xenobiotics supplied to living organisms are
metabolised by conjugation with GSH. This process is catalysed by
glutathione S-transferase enzymes (GST).
[0008] GSH is synthesised in two steps, catalysed by two different
enzymes. During the first step, .UPSILON.-glutamylcysteine
synthetase (GCS) catalyses the formation of
L-.UPSILON.-glutamyl-L-cysteine from glutamate and cysteine. The
second step incorporates glycine under influence of glutathione
synthetase, yielding GSH.
[0009] One example of a drug that can deplete the concentration of
reduced glutathione in the liver is the commonly used sedative
acetaminophen (paracetamol). Acetaminophen has caused severe
hepatic necrosis when ingested in large amounts, e.g. in suicide
attempts or accidentally by children. Acetaminophen hepatotoxicity
is mediated by a toxic reactive metabolite formed from the parent
compound by the cytochrome P450 mixed-function oxidase system of
the hepatocyte. The metabolite is then detoxified by conjugation
with glutathione. If excessive amounts are formed, the glutathione
reserves of the liver are depleted, and the quinonimine reacts with
constituents of the liver cells, leading to necrosis. The hepatic
injury may be potentiated by the use of alcohol or other drugs and
also starvation and cachexia, conditions in which liver glutathione
are lower, may potentiate the effect.
[0010] Milk thistle, or Silybum marianum (L.) Gaertn. (Compositae)
is in Western countries well-known for its hepatoprotective
effects. Its key components are flavanolignans, collectively known
as silymarin. A composition for the protection, treatment and
repair of liver tissue containing Milk thistle is for instance
described in WO 99/43336.
[0011] Intraperitoneal administration of silymarin (200 mg/kg) in
rats increased the total glutathione content and improved the
reduced glutathione/oxidised glutathione ratio in the liver,
intestine, and stomach, while levels of kidney, lung, and spleen
were not affected (Valenzuela et al/., Selectivity of silymarin on
the increase of gutathione content in different tissues of the rat,
Planta Medica 55, 420-2 (1989)). The main component of silymarin,
silybin, exerted inhibitory effects on superoxide and hydrogen
peroxide production in human neutrophils and increased the activity
of both superoxide dismutase and glutathione peroxidase in human
erythrocytes Silymarin protects against acetaminophen-induced GSH
depletion and cytotoxicity in hepatoblastoma Hep G2 cells in vitro.
Silymarin and silybin protect in vivo against hepatic glutathione
depletion induced by ethyl alcohol and paracetamol in rats. It was
shown that silybin reduces the GSH depletion induced by
acetaminophen, but does not affect GSH depletion by buthionine
sulfoximide in isolated rat hepatocytes. This suggests that it
enhances GSH oxidation or conjugation, without affecting
glutamylcysteine synthetase or GSH synthesis (Garrido et al.,
Pharmacology and Toxicology 69, 9-12 (1991)).
[0012] Furthermore, silybin was found to be a potent, non-toxic,
inhibitor of glutathione-S-transferase (Bartholomeus et al.,
Xenobiotica 24, 17-24, (1994)). This observation leads to the
conclusion that, although silymarin might be effective as a
hepatoprotective agent, secondary detoxification by conjugation to
GSH, as is catalysed by GSH-transferase, is inhibited.
[0013] Silymarin treatment (Legalon, 420 mg, 6 months) in patients
with chronic alcoholic liver disease in a double blind placebo
controlled trial increased the serum level of free --SH groups and
the activity of GSH peroxidase. Other clinical trials confirm the
hepatoprotective effect of standardised Silybum extracts.
[0014] RO 102689 (Intr. Medicamente Biofarm) discloses the
extraction of silymarin from armurariu fruit. Said extract shall be
useful in the treatment of active chronic hepatitis, cirrhosis of
the liver and for the protection of liver cells during
administration of hepatoxic agents.
[0015] Picrorhiza kurrooa Royle (Scrophulariaceae) has been used in
{overscore (A)}yurveda mainly for the treatment of liver disorders.
Iridoid glucosides have been regarded as its active constituents;
however, other compounds like acetophenones might also play a
role.
[0016] Several iridoid glucosides have been isolated from P.
kurrooa, most of them conjugates of catalpol with either benzoyl-
or cinnamoyl-derived side chains.
[0017] Picroliv is a standardised fraction of Picrorhiza kurrooa
containing the iridoid glycosides picroside I and kutkoside in a
ratio of 1:1.5 to 1:2 (50-70%) and a mixture of cucurbitacin
glycosides (4-5%) (US-A 5145955). Picroside II has also been
isolated from Picrorhiza kurrooa and 50 mg/kg p.o. in mice treated
with CCl4 showed a hepatoprotective activity (DE-A 2203884).
[0018] Picroliv has been tested in a number of liver damage models
in vitro and in vivo. These studies have demonstrated its
antihepatotoxic, hepatoregenerative, choleretic, and hypolipidemic
activities (reviewed by Dhawan, Medicinal Chemistry Research 5,
595-605, (1995)). Hepatoregenerative effects were associated with
an increased recovery of the liver anti-oxidant system after
partial hepatectomy or carbon tetrachloride-induced liver damage of
Picroliv-treated rats. Furthermore, Picroliv significantly reversed
paracetamol-induced biochemical changes in several liver cell
markers after oral administration to rats (6 and 12 mg/kg) for 7
days. Injection of carbon tetrachloride in rats induced a drastic
impairment of the hepatic mixed-function oxidase system, as
indicated by several drug-metabolising enzymes such as
glutathione-S-transferase and reduced glutathione. Administration
of Picroliv (6 mg/kg) for 7 days significantly prevented liver
damage (Rastogi et al., Drug Development Research 41, 44-47,
(1997)). Picroliv, given to rats during the last 15 days (12
mg/kg/day p.o.) of a 45 days exposure to alcohol, prevented the
decrease of superoxide dismutase, catalase,
glutathione-S-transferase, and the level of reduced glutathione
(Rastogi et al., 1196). Picroliv (6 mg/kg/day p.o.) recovered the
depletion of reduced glutathione level and the inhibition of
glutathione-S-transferase, glutathione reductase, and glutathione
peroxidase activities in the livers of mice infected with
Plasmodium berghei. (Furthermore, Picroliv recovered the decreased
levels of cysteine, sulphydryl groups and glutathione synthesis by
suppressing the enhanced activation of .UPSILON.-glutamyl
transpeptidase in mice infected with Plasmodium berghei.
Pretreatment with the ethanol extract of Picrorhiza kurrooa (50
mg/kg/day, p.o.) prevented the decrease of glutathione peroxidase
and glutathione transferase activity, and the decrease of reduced
glutathione in rats with galactosamine-induced liver damage
[0019] Apocynin, an acetophenone isolated from P. kurrooa, enhances
intracellular GSH synthesis, mediated by the increased expression
of .UPSILON.-GCS mRNA and enzyme activity through activation of
transcription factor AP-1 (Lapperre et al., Febs Letters 443,
235-9, (1999)).
[0020] Schisandra chinensis (Turcz.) Baill (Schisandraceae) is a
winding herb found in the Far East. Its fruits have been used since
long in traditional medicine in this part of the world. The main
active ingredients are lignans, e.g. schisandrins (gomisins). These
compounds were shown to be potent hepatoprotective agents (Hancke
et al., Fitoterapia 70, 451-471, (1999)). They are able to enhance
the GSH/GSSG ratio and the activity of GSH reductase, increasing
the GSH status of liver cell mitochondria. Furthermore, they are
able to enhance detoxification by enhancing cytochrome
P450-mediated metabolism of hepatotoxins, followed by the induction
of GSH-S-transferase-mediated conjugation.
[0021] Pretreatment with a lignan-enriched extract of Schisandra
chinensis (1.6 g/kg p.o.) enhanced the hepatic glutathione status
and protected against physical exercise-induced muscle damage in
rats (Ko et al., Phytotherapy Research 10, 450-452, (1996)).
Treatment of rats with extract from Schisandra chinensis (1.6
g/kg/day p.o.) increased the hepatic GSH level and activities of
GSH reductase, G6PDH, and decreased the susceptibility of hepatic
tissue homogenates to peroxide-induced GSH depletion; pretreating
rats with these extracts (0.2-3.2 g/kg p.o.) dose-dependently
protected against CCl4-induced GSH-depletion and oxidative
hepatocellular damage. These data were confirmed by other
experiments, showing that Schisandra chinensis extract (1.6
g/kg/day p.o.) increases hepatic GSH levels, GSH reductase and
GSH-transferase activities and prevents liver damage in rats with
aflatoxin- and cadmium-induced liver damage. Pretreatment of
schisanhenol and schizandrin B (200 mg/kg) to mice with
ethanol-induced liver peroxidation increased dismutase and catalase
activity, while GSH-peroxidase activity was unaffected. Treatment
of schisandrin B (3 mmol/kg/day p.o.) increased the mitochondrial
GSH level and reciprocally decreased GSSG level, elevating the
GSH/GSSG ratio, and increased mitochondrial GSH reductase activity.
These effects were more pronounced in CCl4-intoxicated mice,
providing protection against liver damage. BCNU, a specific
inhibitor of GSH reductase, however, did not affect the protective
activity, although it inhibited GSH reductase activity, suggesting
that the enhancement of GSH reductase activity by Schisandrin B is
not a primary factor of hepatoprotection.
[0022] The direct antioxidants are radical scavengers by their
chemical composition or interfere with the formation of oxygen
radicals. The antioxidants most commonly known for this function
are vitamin C, vitamin E and flavonoids. Catechins from green tea
and curcumin have similar effects and enhance the antioxidant
activity.
[0023] Selenium is a component of glutathione peroxidase in
erythrocytes and other tissues. Glutathione peroxidase reduces
hydrogen peroxide to water. Selenium is thought to be well-absorbed
both as inorganic selenium salts and organic selenium compounds. A
200 .mu.g/day supplement for 2 months in selenium-deficient
patients with alcoholic cirrhosis significantly improved plasma
selenium levels and glutathione peroxidase but had no effect on
peroxidative parameters.
[0024] Zinc has a role in protection against oxygen radical damage,
being an essential component of cytoplasmic superoxide dismutase.
Zinc ions also bind to sulphydryl groups in proteins, protecting
them against oxidation. Most (80-85%) plasma zinc is bound to
albumin, 15% to alpha2-macroglobulin and <2% to RBP. Plasma zinc
levels are depressed by infections and other stressors, probably
due to systemic redistribution mediated by metallothionein, and
perhaps because plasma albumin levels are reduced in stress.
[0025] Copper is an essential part of many enzymes which are
involved in protection against free radical damage, e.g. cytochrome
oxidase, lysyl oxidase, Zn/Cu superoxide dismutase. About 30% of
dietary copper is absorbed, and there is some evidence that this
increases in deficiency. Dietary deficiency is extremely rare. Over
80% of plasma copper is bound to ceruloplasmin (a positive acute
phase protein that increases in inflammatory conditions). Free
copper ions (unlikely in normal conditions but may occur in the
case of copper excess) are a major source of oxygen radicals.
[0026] Manganese is part of a number of metalloenzymes, including
mitochondrial pyruvate carboxylase, superoxide dismutase and acetyl
CoA carboxylase.
SUMMARY OF THE INVENTION
[0027] It is a primary object of the present invention to provide a
method of increasing the presence of glutathione in cells including
the prevention or treatment of impaired liver function by
administering a certain combination of components to a mammal.
[0028] It is a further object of the present invention to provide
improved compositions for increasing the presence of glutathione in
cells including the prevention or treatment of impaired liver
function.
[0029] It was surprisingly found that a combination of components
is required for providing an optimal protection against liver
damage. It was found that, for this purpose, a combination is
required with components that enhance the glutathione status of the
cells. In order to achieve this, components are administered which
are precursors for the endogenous glutathione production. Together
therewith components are administered which enhance the endogenous
glutathione biosynthesis and which enhance the regeneration of
oxidised glutathione, and/or enhance the glutathione-mediated
conjugation.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides a method of increasing the
presence of glutathione in cells including the prevention or
treatment of impaired liver function and novel dietetic or
pharmaceutical compositions for said purpose comprising in
combination at least a) one precursor of glutathione which is
selected from the group consisting of cysteine and functional
equivalents containing said amino acid, b) one enhancer of the
glutathione biosynthesis, and c) a third component which is either
selected from lignans or which is a combination c1) of an enhancer
of glutathione regeneration and c2) of an enhancer of the
glutathione-mediated conjugation.
[0031] It was found that the ability to detoxify is crucial for
liver health in general. If the capacity of detoxification is
reduced, the liver itself becomes toxified by all the endo- and
exo-toxins it would normally neutralise.
[0032] As already pointed out above many drugs and toxins can be
detoxified by conjugation with glutathione. If the detoxifying
capacity via GSH of the body (liver) is exceeded other
detoxification pathways will be used to a greater extent. The
overall clearance will however be slower whereby body parts can be
damaged which are exposed too heavily. Said parts can be for
instance the liver, the respiratory tract, the gastrointestinal
tract, and the skin. This could lead to respiratory diseases like
COPD, emphyseme, CARA, and cancer, and liver diseases like fatty
liver, cirrhosis, hepatitis and liver cancer.
[0033] In addition many people are exposed to toxic compounds that
require an adequate glutathione metabolism. This applies for
instance to people who live in polluted areas (radiation, numerous
halogenated, nitrated chemicals), who work with dangerous chemicals
(organophosphorus insecticides or halogenated solvents) and who
smoke cigarettes. Also during chronic inflammation of tissue, which
may occur in the case of arthritis or inflammatory bowel disease,
the glutathione levels in the inflamed tissues are often depleted.
Also in this case a further stimulation of the endogenous
glutathione production will be advantageous.
[0034] The method and the compositions of the present invention can
treat and/or prevent the above mentioned diseases. According to the
invention the glutathione presence or the glutathione status,
respectively, in the cells is increased or improved, respectively.
This applies in particular for the treatment and/or prevention of
impaired liver function.
[0035] The present invention is applicable for mammals including
human beings and animals, for instance birds. Several diseases here
described can occur during intensive animal breeding due to the
consumption of an unbalanced feed diet. Liver problems occur due to
the excessive amount of fats of inferior quality or the consumption
of waste products of the human food industry which for some reason
have become contaminated or comprise too high levels of
anti-nutritional factors. This happens predominantly in pig and
poultry breeding.
[0036] According to the present invention a glutathione precursor
a) is administered in a certain minimum amount. Said precursor a)
is either cysteine or one or more functional analogue(s) containing
said amino acid cystelne or a combination of both. Said functional
analogues are for instance salts and common esters of cysteine,
especially of the L-isomer, N-alkylated derivatives of cysteine,
such as N-acetyl cysteine (NAC), cysteine-rich proteins and
peptides, such as some proteins from whey or egg, and
cystathionine, both in reduced or oxidised form Glutathione itself
in reduced or oxidised form can also increase glutathione tissue
levels. When methionine or S-adenosylmethionine (SAM) are used as
cystein analogues the risk for elevated cystein levels is high in
animals including men suffering from impaired liver function. In
case said compounds are used as cystein analogues at least folic
acid, vitamin B6 and/or zinc should be present in addition.
Preferably all three compounds are present in amounts that exceed
the recommended daily amounts.
[0037] The Glutathione precursor used according to the invention
can be a single species or a mixture of different components or
species, respectively. The minimum amount of the precursor a)
administered per day is at least 100 mg cysteine, calculated as
free amino acid. The minimum amount is preferably at least 200 mg
per day. The maximum amount of cysteine should not exceed 2000 mg.
The absolute amounts of the equivalents can be easily calculated by
using the molar weight of the above mentioned amino acid.
[0038] The precursor of glutathione a) is administered together
with an enhancer of the glutathione biosynthesis b) and together
with a third component c) described below. In a simple embodiment
of the invention the above mentioned components a) b) and c) are
administered or contained in the compositon of the invention.
[0039] The third component c) administered is either one or more
lignans or a combination of an enhancer of glutathione regeneration
c1) and of an enhancer of the glutathione mediated conjugation c2).
In this context it should be noted that lignans can act as an
enhancer of glutathione regeneration c1) as well as an enhancer of
the glutathione mediated conjugation c2). Consequently, according
to a first embodiment of the present invention, the third component
c) can consist of one lignan alone or a mixture of different
lignans. According to a second embodiment of the invention the
third component c) can be a combination of one or more lignans
either with one enhancer or more enhancers of the glutathione
biosynthesis c1) which is or are different from a lignan, or with
one enhancer or more enhancers of the gutathione-mediated
conjugation c2) which is or are different from a lignan. According
to a third embodiment of the invention the third component c) can
be a combination of one enhancer or more enhancers of the
glutathione biosynthesis c1) which is or are different from a
lignan with one enhancer ore more enhancers of the
gutathione-mediated conjugation c2) which is or are also different
from a lignan. According to a fourth embodiment of the invention
the third component c) can be a combination of i) one enhancer or
more enhancers of the glutathione biosynthesis c1) which is or are
different from a lignan, ii) one enhancer or more enhancers of the
gutathione-mediated conjugation c2) which is or are also different
from and of iii) one lignan or more lignans.
[0040] According to a preferred embodiment the enhancer of the
glutathione biosynthesis b) contains apocynin or analogues.
[0041] Apocynin can be isolated from plants such as Iris species
(Iridaceae) or from the rhizome of Apocynam cannabinum or other
Apocynaceae.
[0042] More preferably the enhancer of the glutathione biosynthesis
b) is a preparation obtained from Picrorhiza species; even more
preferably the enhancer of the glutathione biosynthesis b) is
selected from the group consisting of an extract of Picrorhiza
kurrooa and an extract of Picrorhiza schrophulariiflora, whereby
said extract contains most preferably 0.01 to 100 mg per day
apocynin or analogues.
[0043] The enhancer of glutathione regeneration c1) is preferably
selected from the group consisting of preparations rich in
silymarin, such as extracts from Silybum marianum or armurariu
fruit, and preparations rich in schisandrins, such as extracts from
Schisandra species; and the enhancer of glutathione-mediated
detoxification process c2) is preferably selected from the group
consisting of preparations rich in iridoid glycosides, especially
extracts from Picrorhiza species, and preparations rich in
Schisandrins such as extracts from Schisandra species.
[0044] The preparation of Picrorhiza species is preferably an
extract from Picrorhiza kurrooa or Picrorhiza scrophulariiflora
(also known as Neopicrorhiza scrophulariiflora), which contains
preferably 1-100 mg of total iridoid glycosides, in particular 1 to
100 mg picrosides, more preferably 1 to 50 mg picroside II, for
example about 6 mg picroside II, and 0.01-100 mg apocynin.
[0045] According to the invention, analogues of apocynin may be
used instead of or in addition to apocynin. Such analogues are in
particular those in which the 4-hydroxy group is etherified,
especially with a sugar moiety. The analogue in which the sugar
moiety is .beta.-D-glucose is commonly known as androsin.
[0046] The preparation of Silybum marianum is preferably an extract
containing 10-1000 mg silymarin, more preferably 175 mg of an
extract containing 140 mg of silymarin.
[0047] The preparation of Schisandra species is preferably an
extract from Schisandra chinensis or Schisandra sphenanthera
containing 0.1-100 mg of lignans, in particular 0.1 to 100 mg
schisandrins, more preferably 11 to 70 mg schisandrins, for example
about 5 mg schisandrins.
[0048] The different components a), b), c), c1), and, c2) are known
and described above. Several traditionally used herbs, e.g. Milk
thistle, Picrorhiza and Schisandra can be used, which are known to
exhibit liver protective activities, and induce or enhance the
endogenous glutathione production. This endogenous antioxidant
plays a central role in the metabolisation and elimination of
toxins. According to the present invention the GSH status in the
liver is increased, and in addition, the enzyme
.UPSILON.-glutamylcysteine synthetase, responsible for GSH
synthesis, is enhanced as well.
[0049] Therefore, the present invention includes precursors and
cofactors important for glutathione synthesis, compounds that
enhance glutathione regeneration, and compounds that induce the
glutathione-mediated detoxification process.
[0050] Preferably, direct antioxidants like vitamin C, E, and
flavonoids, and enzymatic antioxidants like selenium, zinc, copper,
and manganese enhancing the antioxidant activity of the liver can
be present in addition according to the present invention. This is
preferred since several toxins are capable of exhibiting oxidative
damage due to the formation of oxygen radicals.
[0051] Enzymatic antioxidants are in particular components that
improve the activity of glutathione peroxidase or superoxide
dismutase. Selenium can be used as selenium salt and preferably as
selenium yeast that provides per daily dose 10-200 mcg selenium.
Zinc can be present as an inorganic or organic zinc salt, such as
ZnO providing preferably 0.1-30 mg zinc per daily dose. Copper can
be present as a copper salt, providing preferably 0.1-8 mg copper
per daily dose. Manganese can be present as a manganese salt,
providing preferably 0.1-20 mg manganese per daily dose.
[0052] Furthermore, the composition can contain at least one
compound that further enhances the glutathione status of the liver,
as a cofactor in the biosynthesis of precursors of glutathione.
Such a compound is selected from the group consisting of
riboflavin, preferably 0.1-200 mg riboflavin, vitamin B66,
preferably 0.1-200 mg vitamin B6, vitamin B12, preferably 0.1-3000
mcg vitamin B12. and folic acid or folate, preferably 10-1000 mcg
folic acid. Preferred sources of vitamin B6 and vitamin B12 are
pyridoxine and cyanocobalamine, respectively.
[0053] In addition. the composition can contain at least one
component that enhances the antioxidant function by directly
scavenging reactive oxygen species or by interfering with their
formation. Such a component is selected from the group consisting
of vitamin C, preferably 10-1000 mg vitamin C, vitamin E,
preferably 1-800 mg .alpha.-TE, flavonoids, preferably an extract
of Matricaria recutita containing 1-100 mg apigenin, catechins,
preferably an extract of Camellia sinensis containing 1-500 mg
catechins, and curcuminoids, preferably an extract of Curcuma longa
containing 1-1000 mg curcumin.
[0054] As already pointed out above the present invention applies
also to animals and in particular to pig and poultry breeding. In
this case the components used according to the present invention
can be present in the extracts of plants which are preferably used
in animal feed. Said components can also be included in a premix
for blending with regular animal feed. The premix is preferably
based on whey proteins as source of the glutathione precursor
a).
[0055] The compositions of the invention can have a form that is
normally used for the administration of a food or of a food
supplement. They can be present for instance as powders, liquids.
emulsions, bars, tablets, and capsules which can be packaged in
cartons, cans, sachets, bottles, bags (tea bags) and can be
prepared according to methods known in the art.
[0056] A composition according to the invention in the form of a
tablet contains for instance 300 mg glutathione, 20 mg apocynin and
5 mg schisandrins.
[0057] The invention is further described by the following, non
limiting examples.
Example 1
[0058] The following composition was mixed and prepared in a
pill.
1 Amount per serving (serving size 3 tablets) amount unit Silybum
marianum (milk thistle) fruit extract 175 mg [providing silymarin
(80%) 140 mg] Camellia sinensis (green tea) leaf extract 28.5 mg
[providing catechins 20 mg] Curcuma longa (turmeric) root extract
10.5 mg [providing curcumin 10 mg] Picrorhiza kurrooa rhizome
extract 120 mg [providing picrosides 6 mg] Schisandra chinensis
fruit extract 55.6 mg [providing schisandrins 5 mg] Matricaria
recutita (camomile) flower extract 625 mg [providing apigenin 15
mg] vitamin C (ascorbic acid) 54.2 mg (providing ascorbic acid 50
mg] vitamin E acetate 6.8 mg succinate 10.9 mg [providing
tocopherols 15.5 [U] Selenium yeast 14.7 mg (prvoviding selenium 28
mcg Zinc oxide 3.8 mg [providing zinc 3 mg Cupric oxide 0.58 mg
[providing copper 0.45 mg] Manganese gluconate dihydrate 7.2 mg
[providing manganese 0.8 mcg] vitamin B12 1.3 mg [providing
cyanocobolamine 1.6 mcg] folic acid 0.27 mg [providing folate 200
mcg] N-acetylcysteine 202 mg [providing N-acetylcysteine 200 mg]
Other ingredients: Bulking agents: microcrystalline cellulose,
calcium carbonate, croscarmellose sodium; Anti-caking agents:
vegetable source stearic acid; vegetable stearate-glyceryl
monostearate, silicon dioxide; Glazing agents: shellac,
hydroxypropyl methyl cellulose, acetic acid esters of mono &
diglycerides of fatty acids; Natural source colour: titanium
dioxide; Thickeners: carnauba wax, xanthan gum; Emulsifiers:
polysorbate 80.
[0059] Three tablets were administered daily, preferably combined
with an additional multivitamin, to treat deprived liver
function.
Example 2
[0060]
2 Amount per daily dose amount unit Silybum marianum (milk thistle)
fruit extract 175 mg Curcuma longa (turmeric) root extract 10.5 mg
Picrorhiza kurrooa rhizome extract 360 mg Schisandra chinensis
fruit extract 55.6 mg Matricaria recutita (camomile) flower extract
625 mg N-acetylcysteine 600 mg Other ingredients, known in the art
of making a capsule
Example 3
[0061]
3 Amount per daily dose amount unit Silybum marianum (milk thistle)
175 mg Picrorhiza kurrooa 360 mg N-acetylcysteine 600 mg Other
ingredients known in the art of making a tablet
Example 4
[0062]
4 Amount per daily dose amount unit Silybum marianum (milk thistle)
175 mg Picrorhiza kurrooa 120 mg Schisandra chinensis 55.6 mg
Selenium yeast (about 0.2% selenium) 15 mg Zinc oxide 3.8 mg Copper
oxide 600 mcg Manganese gluconate 7.2 mg N-acetylcysteine 200 mg
Other ingredients known in the art of making a tablet
Example 5
[0063]
5 Amount per daily dose Amount unit Silybum marianum (milk thistle)
175 mg Picrorhiza kurrooa 120 mg vitamin B2 (riboflavin) 1.25 mg
vitamin B6 (pyridoxine) 1.3 mg vitamin B12 premix (1% 0.18 mg
cyanocobalamin) folic acid (monoglutamate) 200 mcg N-acetylcysteine
1000 mg Other ingredients known in the art of making a tablet
Example 6
[0064]
6 Amount per daily dose amount unit Silybum marianum (milk thistle)
175 mg Camellia sinensis (green tea) 28.5 mg Curcuma longa
(turmeric) 10.5 mg Picrorhiza kurrooa 120 mg Schisandra chinensis
55.6 mg Matricaria recutita (camomile) 625 mg vitamin C (ascorbic
acid) 50 mg Alfa-tocopherol (vitamin E) 10.4 IU N-acetylcysteine
200 mg Other ingredients known in the art of making a tablet
* * * * *