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F. Fraschini, G. Demartini, Department of Pharmacology, University of Milan, Milan, Italy; D. Esposti, Institute of Human Physiology II, University of Milan, Milan, Italy Clin Drug Invest 22(1):51-65, 2002. © 2002 Adis International Limited
Abstract and Introduction
Abstract
The flavonoid silymarin and one of its structural components,
silibinin, are substances with documented hepatoprotective properties.
Their mechanisms of action are still poorly understood. However, the data
in the literature indicate that silymarin and silibinin act in four different
ways: (i) as antioxidants, scavengers and regulators of the intracellular
content of glutathione; (ii) as cell membrane stabilisers and permeability
regulators that prevent hepatotoxic agents from entering hepatocytes; (iii)
as promoters of ribosomal RNAsynthesis, stimulating liver regeneration;
and (iv) as inhibitors of the transformation of stellate hepatocytes into
myofibroblasts, the process responsible for the deposition of collagen
fibres leading to cirrhosis. The key mechanism that ensures hepatoprotection
appears to be free radical scavenging. Anti-inflammatory and anticarcinogenic
properties have also been documented.
Silymarin is able to neutralise the hepatotoxicity of
several agents, including Amanita phalloides, ethanol, paracetamol (acetaminophen)
and carbon tetrachloride in animal models. The protection against A. phalloides
is inversely proportional to the time that has elapsed since administration
of the toxin. Silymarin protects against its toxic principle -amanitin
by preventing its uptake through hepatocyte membranes and inhibiting the
effects of tumour necrosis factor- , which exacerbates lipid peroxidation.
Clinical trials have shown that silymarin exerts hepatoprotective
effects in acute viral hepatitis, poisoning by A. phalloides, toxic hepatitis
produced by psychotropic agents and alcohol-related liver disease, including
cirrhosis, at daily doses ranging from 280 to 800mg, equivalent to 400
to 1140mg of standardised extract. Hepatoprotection has been documented
by improvement in liver function tests; moreover, treatment with silymarin
was associated with an increase in survival in a placebo-controlled clinical
trial in alcoholic liver disease.
Pharmacokinetic studies have shown that silymarin is
absorbed by the oral route and that it distributes into the alimentary
tract (liver, stomach, intestine, pancreas). It is mainly excreted as metabolites
in the bile, and is subject to enterohepatic circulation. Toxicity is very
low, the oral 50% lethal dose being 10 000 mg/kg in rats and the maximum
tolerated dose being 300 mg/kg in dogs. Moreover, silymarin is devoid of
embryotoxic potential.
In conclusion, silymarin is a well tolerated and effective
antidote for use in hepatotoxicity produced by a number of toxins, including
A. phalloides, ethanol and psychotropic drugs. Numerous experimental studies
suggest that it acts as a free radical scavenger, with other liver-specific
properties that make it a unique hepatoprotective agent.
Introduction
Flavonoids belong to the family of the benzo gamma-pyrones.
More than 4000 different flavonoids are currently known; they are ubiquitous
not only in the plant kingdom, where they are particularly abundant in
the photosynthetic cells of higher plants, but also in the animal kingdom.
For centuries they have been attributed numerous therapeutic properties
and many have been used as popular therapeutic remedies. Compounds such
as quercetin, taxifolin and silymarin have been used as active ingredients,
both alone and as components of complex chemical preparations.
Silymarin is a flavonolignan that has been introduced
fairly recently as a hepatoprotective agent. It is the most well known
compound of the flavonoids, thanks to its well defined therapeutic properties.
It is extracted from the seeds and fruit of Silybum marianum (Compositae)
and in reality is a mixture of three structural components: silibinin,
silydianine and silychristine. The structure of the constituents of silymarin
was clarified in the 1960s (figure 1).[1,2]
The main chemical difference between silymarin and other
flavonoids is that its isomers are substituted by a coniferyl alcohol group.
Of the three isomers that constitute silymarin, silibinin is the most active.[3,4]
From a medical point of view, silymarin and silibinin have been found to
provide cytoprotection and, above all, hepatoprotection.[2,5]
Silymarin is used for the treatment of numerous liver
disorders characterised by degenerative necrosis and functional impairment.[3]
Furthermore, it is able to antagonise the toxin of Amanita phalloides[6,7]
and provides hepatoprotection against poisoning by phalloidin,[8] galactosamine,[9]
thioacetamide,[10] halothane[11] and carbon tetrachloride.[12] The compound
also protects hepatocytes from injury caused by ischaemia, radiation, iron
overload and viral hepatitis.[13]
Figure 1. (click image to zoom) The three structural
components of silymarin: silibinin, silydianine, and silychristine.
Silymarin is included in the pharmacopoeia of many countries
under the trademark LegalonTM or HepatronTM and is often used as supportive
therapy in food poisoning due to fungi and in chronic liver disorders,
such as steatosis[14] and alcohol-related liver disease.[15]
1. Pharmacodynamics
1.1 Antioxidant Properties
Flavonoids usually possess good antioxidant activity.
The water-soluble dehydrosuccinate sodium salt of silibinin
is a powerful inhibitor of the oxidation of linoleic acid-water emulsion
catalysed by Fe2+ salts.[15] It also inhibits in a concentration-dependent
way the microsomal peroxidation produced by NADPH-Fe2+ -ADP, a well known
experimental system for the formation of hydroxy radicals.[16] In studies
performed in rat hepatic microsomes, it has been demonstrated that lipid
peroxidation produced by Fe(III)/ascorbate is inhibited by silibinin dihemisuccinate;
the inhibition is concentration-dependent.[17,18]
It has been shown that silymarin is as active as quercetin
and dihydroquercetin, and more active than quercitrin, in terms of antiperoxidant
activity, independent of the experimental model used to produce peroxidation.[19]
It has recently been reported that in rat hepatocytes
treated with tert-butyl hydroperoxide (TBH), silymarin reduces the loss
of lactate dehydrogenase (LDH), increases oxygen consumption, reduces the
formation of lipid peroxides, and increases the synthesis of urea in the
perfusion medium. Furthermore, silymarin is able to antagonise the increase
in Ca2+ produced by TBH, reducing ion levels down to below 300 nmol/L.
The protective effect of silymarin is mediated by the inhibition of lipid
peroxidation, and the modulation of hepatocyte Ca2+ content seems to play
a crucial role.[20]
1.2 Protective Effects in Models of Oxidative Stress
Oxidative stress is defined as structural and/or functional
injury produced in tissues by the uncontrolled formation of pro-oxidant
free radicals. Oxidative stress usually develops when the pro-oxidant action
of an inducer exceeds the anti-oxidant capacity of the cell defence system,
altering its homeostatic capacity. Numerous substances induce oxidative
stress, including carbon tetrachloride, TBH, ethanol, paracetamol (acetaminophen)
and phenylhydrazine. It has been shown in rats that silibinin protects
neonatal hepatocytes from cell damage produced by erythromycin, amitriptyline,
nortriptyline and TBH.[21]
Erythrocytes obtained from rats treated with silymarin
exhibited high resistance against the haemolysis produced by phenylhydrazine[22,23]
and the lysis induced by osmotic shock.[1] This suggests that silymarin
may act by increasing the stability of the erythrocyte membrane.
The cytoprotective activity of silymarin has also been
shown in hepatocytes of rats subjected to osmotic stress produced by hypotonic
saccharose solutions.[24]
The perfused liver is a valid experimental model for
the evaluation of the effect of substances that induce oxidative stress
and of the protection provided by scavengers. Using this experimental model,
it has been shown that phenylhydrazine produces an increase in oxygen consumption
in rat liver in vitroand in the release of thiobarbituric acid reactive
substances (TBARS) in the perfusate.[25] This stress is associated with
a reduction in the amount of reduced glutathione (GSH) in the liver; GSH
exerts important protective activity against chemically induced oxidative
stress.[26,27] Using liver from rats pretreated in vivo with silibinin
50 mg/kg intravenously, a significant reduction in the oxygen consumption
stimulated by phenylhydralazine and in the release of TBARS was observed,
without any changes in GSH levels.[22,25]
The antioxidant effect of silibinin was observed in rats
with acute intoxication caused by ethanol[1,26] or paracetamol,[28] which
are peroxidation inducers that produce marked GSHdepletion in the liver.
Treatment with silymarin or silibinin was able to protect animals from
oxidative stress produced in the liver by ethanol or paracetamol.[2,26,28]
Furthermore, it has been reported that treatment with silibinin attenuates
the increase in plasma levels of AST, ALT and gammaglutamyl transpeptidase
(GGT) observed after intoxication by paracetamol.[1]
The hepatoprotective activity of silibinin has also been
studied in rats with liver cirrhosis induced by the long-term administration
of carbon tetrachloride. Muriel & Mourelle[29] have shown that silibinin
preserves the functional and structural integrity of hepatocyte membranes
by preventing alterations of their phospholipid structure produced by carbon
tetrachloride and by restoring alkaline phosphatase and GGT activities.
Another interesting property of silibinin and silymarin
is their role as regulators of the content of GSH in various organs. In
rats treated with silibinin intravenously or silymarin intraperitoneally,
a significant increase in the amount of the GSH contained in the liver,
intestine and stomach was found, whereas there were no changes in the lungs,
spleen and kidneys (table I).[30]
1.3 Activity against Lipid Peroxidation
Lipid peroxidation is the result of an interaction between
free radicals of diverse origin and unsaturated fatty acids in lipids.
Lipid peroxidation involves a broad spectrum of alterations, and the consequent
degeneration of cell membranes may contribute towards the development of
other disorders of lipoprotein metabolism, both in the liver and in peripheral
tissues.
Silymarin appears to act as an antioxidant not only because
it acts as a scavenger of the free radicals that induce lipid peroxidation,[17,31]
but also because it influences enzyme systems associated with glutathione
and superoxide dismutase.[30]
It has been shown that all the components of silymarin
inhibit linoleic acid peroxidation catalysed by lipoxygenase[37] and that
silymarin protects rat liver mitochondria and microsomes in vitro against
the formation of lipid peroxides induced by various agents.[38]
1.4 Effects on Liver Lipids
The influence of silymarin on cellular permeability is
closely associated with qualitative and quantitative alterations of membrane
lipids (both cholesterol and phospholipids).[29,39,40] This suggests that
silymarin may also act on other lipid compartments in the liver; this may
influence lipoprotein secretion and uptake. It has been shown that silymarin
and silibinin reduce the synthesis and turnover of phospholipids in the
liver of rats. Furthermore, silibinin is able to neutralise two effects
of ethanol in rats: the inhibition of phospholipid synthesis and the reduction
in labelled glycerol incorporation into lipids of isolated hepatocytes.[14,27,32]
In addition, silibinin stimulates phosphatidylcholine synthesis and increases
the activity of cholinephosphate cytidyltransferase in rat liver both in
normal conditions and after intoxication by galactosamine.[41]
Data on the influence of silymarin on triglyceride metabolism
in the liver are scanty. It is known that in rats silibinin is able to
partly antagonise the increase in total lipids and triglycerides produced
in the liver by carbon tetrachloride[12] and, probably, to activate fatty
acid ß-oxidation.[1] It has also been suggested that silymarin may
diminish triglyceride synthesis in the liver.[14]
Letteron et al.[31] studied the mechanisms of action
of silymarin that provide protection against lipid peroxidation and the
hepatotoxicity of carbon tetrachloride in mice, and came to the conclusion
that silymarin works by reducing metabolic activation by carbon tetrachloride
and by acting as an antioxidant that prevents chain rupture.
Other authors have shown that silymarin affords hepatoprotection
against specific injury induced by microcystin (a hepatotoxin), paracetamol,
halothane and alloxan in several experimental models (table I).[11,35,36,42]
1.5 Effects on Plasma Lipids and Lipoproteins
The administration of silymarin reduces plasma levels
of cholesterol and low-density lipoprotein (LDL) cholesterol in hyperlipidaemic
rats, whereas silibinin does not reduce plasma levels of cholesterol in
normal rats; however, it does reduce phospholipid levels, especially those
transported in LDL.[14]
Data obtained in experimental models of hepatic injury
have shown that silymarin is able to normalise the increase in plasma lipids
observed after administration of carbon tetrachloride and to antagonise
the reduction in serum free fatty acids induced by thioacetamide. In the
experimental model of hepatic injury produced by thioacetamide, silymarin
did not appear to be able to normalise the reduction in triglycerides in
serum. In the experimental model of hepatic injury produced by paracetamol
in rats, it was evident that silymarin improves LDL binding to hepatocytes,
an important factor for the reduction of LDL in plasma.[14]
1.6 Stimulation of Liver Regeneration
One of the mechanisms that can explain the capacity of
silymarin to stimulate liver tissue regeneration is the increase in protein
synthesis in the injured liver. In in vivo and in vitro experiments performed
in the liver of rats from which part of the organ had been removed, silibinin
produced a significant increase in the formation of ribosomes and in DNA
synthesis, as well as an increase in protein synthesis.[43] Interestingly,
the increase in protein synthesis was induced by silibinin only in injured
livers, not in healthy controls.[44] The mechanism whereby silibinin stimulates
protein synthesis in the liver has not been defined; it may be the physiological
regulation of RNA polymerase I at specific binding sites, which thus stimulates
the formation of ribosomes.[13] In rats with experimental hepatitis caused
by galactosamine, treatment with intraperitoneal silymarin 140 mg/kg for
4 days completely abolished the inhibitory effect of galactosamine on the
biosynthesis of liver proteins and glycoproteins.[34]
These data support the results of previous experiments
in a similar model of acute hepatitis in the rat, in which silymarin protected
hepatic structures, liver glucose stores and enzyme activity in vivo from
injury produced by galactosamine.[9]
The capacity of silymarin to stimulate protein synthesis
has also been studied in neoplastic cell lines, in which no increase in
protein synthesis, ribosome formation or DNA synthesis has been found after
treatment with silymarin.[44]
1.7 Effects during Experimental Intoxication with
Amanita phalloides
The therapeutic activity of silymarin against mushroom
poisoning is worthy of particular attention. The hepatoprotective properties
of silymarin have been tested in dogs, rabbits, rats and mice. A dose of
15 mg/kg of silymarin was administered intravenously 60 minutes before
intraperitoneal administration of a lethal dose of phalloidin, and was
able to protect all animal species tested (100% survival) from the action
of the toxin.
When it is injected 10 minutes after phalloidin, silymarin
affords similar protection only at doses of 100 mg/kg. The longer the time
that has elapsed after administration of the toxin, the less effective
the drug becomes, and after 30 minutes it is no longer effective even at
high doses. Histochemical and histoenzymological studies have shown that
silymarin, administered 60 minutes before or no longer than 10 minutes
after induction of acute intoxication with phalloidin, is able to neutralise
the effects of the toxin and to modulate hepatocyte function.[6,7]
Similar results were obtained in dogs treated with sublethal
oral doses of A. phalloides, in which hepatic injury was monitored by measuring
enzymes and coagulation factors. Amongst the numerous substances tested
(prednisolone, cytochrome c, benzylpenicillin, silymarin), only benzyl-penicillin
(1000 mg/kg intravenous infusion after 5 hours) and silymarin (50 mg/kg
intravenous infusion after 5 hours and 30 mg/kg after 24 hours) were able
to prevent the increase in hepatic enzymes and the fall in coagulation
factors induced by experimental intoxication (table II).[45]
The cyclopeptides of fungi of the genus Amanita, including
amatoxins and fallotoxins, are captured by hepatocytes through the sinusoidal
system, which is also involved in the mediation of liver uptake of biliary
salts. It has been demonstrated that silibinin is able to inhibit uptake
of amanitin in isolated preparations of hepatocyte membranes, and the same
effect has been shown for taurocholate, antamanide, prednisolone and phalloidin.
The effect of silibinin appears to be competitive.[2]
Recently, the role of tumour necrosis factor- (TNF
) in hepatic injury produced by -amanitin has been investigated in
primary cultures of rat hepatocytes. At a concentration of 0.1 µmol/L,
the toxin inhibits RNA and protein synthesis within 12 hours, but cytotoxicity
appears only much later (36 hours). TNF is not indispensable for
the development of cytotoxicity, but exacerbates it and markedly increases
lipid peroxidation. The addition of silibinin at a concentration of 25µmol/L
to the culture medium prevented the effects of TNF (50µg/L)
1.8 Anti-Inflammatory and Anticarcinogenic Properties
A significant anti-inflammatory effect of silymarin has
been described in liver tissue. Studies have shown that silymarin exerts
a number of effects, including inhibition of neutrophil migration, inhibition
of Kupffer cells, marked inhibition of leukotriene synthesis and formation
of prostaglandins.[13,47-49]
The protection afforded by silymarin against carcinogenic
agents has been studied in various experimental animal models. A series
of experiments have been performed in nude mice with nonmelanoma skin cancer
produced by UVB radiation, studying its initiation, promotion and complete
carcinogenesis. In all the stages studied, silymarin applied onto the skin
at different doses appeared to reduce significantly the incidence, multiplicity
and volume of tumours per animal. Furthermore, in a short-term experiment
(using the same experimental model), the application of silymarin significantly
reduced apoptosis, skin oedema, depletion of catalase activity and induction
of cyclo-oxygenase and ornithine decarboxylase activity. This effect provides
protection against photocarcinogenesis.[50] Similar results were also obtained
in the model of skin carcinogenesis produced by chemical carcinogenic agents
in carcinogenesis-sensitive (SENCAR) mice.[51,52]
The molecular bases of the anti-inflammatory and anticarcinogenic
effects of silymarin are not yet known; they might be related to the inhibition
of the transcription factor NF- B, which regulates the expression of various
genes involved in the inflammatory process, in cytoprotection and carcinogenesis.[53-55]
It has also been hypothesised that silymarin may act by modulating the
activation of regulating substances of the cellular cycle and of mitogen-activated
protein kinase.[56]
1.9 Antifibrotic Effects
Stellate hepatocytes have a crucial role in liver fibrogenesis.
In response to fibrogenic influences (for example protracted exposure to
ethanol or carbon tetrachloride), they proliferate and transform into myofibroblasts
responsible for the deposition of collagen fibres in the liver. Recently,
the effects of silibinin on the transformation of stellate cells into myofibroblasts
have been investigated. The results have shown that silibinin, at a concentration
of 100µmol/L reduces the proliferation of stellate cells isolated
from fresh liver of rats by about 75%, reduces the conversion of such cells
into myofibroblasts, and downregulates gene expression of extracellular
matrix components indispensable for fibrosis.[57]
Furthermore, it has been demonstrated that silymarin
improves hepatic fibrosis in vivo in rats subjected to complete occlusion
of the biliary duct, a manoeuvre that causes progressive hepatic fibrosis
without inflammation. Silymarin, administered at a dosage of 50 mg/kg/day
for 6 weeks, is able to reduce fibrosis by 30 to 35% as compared with controls.
A dose of 25 mg/kg/day is not effective.[58]
Colchicine and silymarin, administered at a dose of 50
mg/kg orally for 55 days, were able to prevent completely all the alterations
induced by carbon tetrachloride in rats (peroxidation of lipids, Na+ ,K+
-and Ca2+ -ATPase), except for the hepatic content of collagen, which was
reduced only by 55% as compared with controls; moreover, alkaline phosphatase
and ALT were unchanged as compared with controls. In the group of rats
treated with silymarin, the loss of glycogen was inhibited completely.[59]
1.10 Inhibition of Cytochrome P450
Silymarin can inhibit the hepatic cytochrome P450 (CYP)
detoxification system (phase I metabolism). It has been shown recently
in mice that silibinin is able to inhibit numerous hepatic CYP enzyme activities,[60]
whereas other researchers have not detected any effect of silymarin on
the CYP system.[61-63]
This effect could explain some of the hepatoprotective
properties of silymarin, especially against the intoxication due to A.
phalloides. The Amanita toxin becomes lethal for hepatocytes only after
having been activated by the CYP system. Inhibition of toxin bioactivation
may contribute to the limitation of its toxic effects. Additionally, silymarin,
together with other antioxidant substances, could contribute towards protection
against free radicals generated by enzymes of the CYP system.
1.11 Overview of Mechanisms of Action
The hepatoprotection provided by silymarin appears to
rest on four properties:
· activity against lipid peroxidation as a result
of free radical scavenging and the ability to increase the cellular content
of GSH;
· ability to regulate membrane permeability and
to increase membrane stability in the presence of xenobiotic damage;
· capacity to regulate nuclear expression by means
of a steroid-like effect; and
· inhibition of the transformation of stellate
hepatocytes into myofibroblasts, which are responsible for the deposition
of collagen fibres leading to cirrhosis.
Silymarin and silibinin inhibit the absorption of toxins,
such as phalloidin or -amanitin, preventing them from binding to
the cell surface and inhibiting membrane transport systems. Furthermore,
silymarin and silibinin, by interacting with the lipid component of cell
membranes, can influence their chemical and physical properties. Studies
in erythrocytes, mast cells, leucocytes, macrophages and hepatocytes have
shown that silymarin renders cell membranes more resistant to lesions (figure
2).[1,2,13]
Furthermore, the well documented scavenging activity
of silymarin and silibinin can explain the protection afforded by these
substances against hepatotoxic agents. Silymarin and silibinin may exert
their action by acting as free radical scavengers and interrupting the
lipid peroxidation processes involved in the hepatic injury produced by
toxic agents. Silymarin and silibinin are probably able to antagonise the
depletion of the two main detoxifying mechanisms, GSH and superoxide dismutase
(SOD), by reducing the free radical load, increasing GSH levels and stimulating
SOD activity.
Furthermore, silibinin probably acts not only on the
cell membrane, but also on the nucleus, where it appeared to increase ribosomal
protein synthesis by stimulating RNA polymerase I and the transcription
of rRNA.[13,34,44] The stimulation of protein synthesis is an important
step in the repair of hepatic injury and is essential for restoring structural
proteins and enzymes damaged by hepatotoxins.[1,2]
Figure 2. (click image to zoom) Mechanism of action
of silymarin as proposed by Valenzuela and Garrido.[1]
2. Pharmacokinetics
Silymarin is not soluble in water and is usually administered
in capsules as a standard extract (70 to 80% silymarin).
Absorption after oral administration is rather low, with
recovery in the bile in rats ranging from 2 to 3%. Peak plasma concentrations
are achieved in 4 to 6 hours, both in animal sand in humans. Silymarin
is mainly excreted in the bile and, to a lesser degree, in the urine. Its
elimination half-life ranges from 6 to 8 hours.[64-66] However, other authors[67]
reported plasma levels of 500 mg/L (as silibinin) 90 minutes after oral
administration of 200 mg/kg of silymarin or of purified S. marianum extract
in mice.
Silibinin and other components of silymarin are rapidly
conjugated with sulfate and glucuronic acid in the liver. The conjugates
pass into the plasma and into the bile, where they are found in amounts
corresponding to 80% of the total dose administered. An egligible portion
is eliminated in the urine. These findings suggest the existence of enterohepatic
circulation: intestinal absorption, conjugation in the liver, excretion
in the bile, hydrolysis by the intestinal flora, and reuptake in the intestine.[69]
The presence of this cycle makes the study of the intestinal
absorption of the natural products very difficult. However, the use of
labelled silibinin in the rat has made it possible to show that intestinal
absorption of a dose of 20 mg/kg amounts to about 35%. Peak radioactivity
is found in the plasma 30 minutes after ingestion.[3]
In 1975, Bülles et al.[68] showed that silibinin
is excreted mainly unmodified in the urine after oral or intravenous administration
of silibinin N-methylglucamine (2 to 120 mg/kg and 20 mg/kg as silibinin,
respectively) in the rat, whereas in the bile it is excreted as metabolites,
independently of the route of administration. Silibinin is excreted in
minimal quantities in the urine during the 48 hours following oral (2 to
5%) or intravenous (8%) administration. On the contrary, biliary excretion
is fairly high during the same period (about 40 to 45% after oral administration
of up to 20 mg/kg, and about 80% after intravenous administration). The
ratio between dose and quantity excreted in the bile is linear for doses
up to 20 mg/kg. Study of the kinetics of biliary excretion after oral administration
of 20 mg/kg showed that peak excretion occurs 1 hour after administration.
Similar results have been reported by Mennicke.[69]
Tissue distribution of silibinin was studied in SENCAR
mice (6 to 7 weeks old) after oral administration of 50 mg/kg.[70] Peak
concentrations of free silibinin were recorded after 0.5 hours in the liver,
lungs, stomach and pancreas, with values of 8.8 ± 1.6, 4.3 ±
0.8, 123 ± 21, 5.8 ± 1.1µg/g of tissue, respectively.
In the skin and prostate, peak concentrations of silibinin were 1.4 ±
0.5 and 2.5 ± 0.4µg/g, respectively, and were reached 1 hour
after administration. With regard to sulfate conjugates and ß-glucuronides
of silibinin, excluding the lungs and stomach, in which peak values were
reached after 0.5 hours, all the other tissues analysed exhibited peak
tissue concentrations after 1 hour. The concentrations of free and conjugated
silibinin diminished exponentially after 0.5 or 1 hour, with an elimination
half-life of 57 to 127 minutes for the free portion and 45 to 94 minutes
for the conjugated portion. Assessment of the effects of silibinin 100
and 200 mg/kg/day orally on phase II enzymes revealed a dose-and time-dependent
increase in glutathione-S-transferase and quinone reductase activity that
was moderately or markedly significant in the liver, lungs, stomach, skin
and small intestine.[70]
3. Therapeutic Activity
The results of most clinical trials with the thistle
(Silybum marianum) extract, silymarin, are difficult to interpret because
of a variety of reasons: small sample size, variability in type and severity
of the liver disorders, heterogeneous dosages, inconsistent use of a control
group, poorly defined objectives. Furthermore, the intrinsic capacity of
the liver to improve after exposure to hepatotoxins was not always taken
into consideration.
Acute Viral Hepatitis
The results of double-blind clinical trials in patients
with acute viral hepatitis indicate that therapy with silymarin reduces
complications, reduces the duration of hospital stay and promotes recovery.
In patients with acute hepatitis randomly allocated to receive silymarin
140mg or placebo three times daily for at least 3 weeks, the proportion
of patients in whom AST normalised was much higher in the treated group
(82%) than in controls (52%). The percentage of patients in whom bilirubin
normalised was 40% in the active treatment group versus 11% in the control
group.[71]
Hepatitis Induced by Toxins or Drugs
It has been shown that silymarin reduces the hepatic
injury produced by poisoning with A. phalloides, phenothiazines and butyrophenones
in humans.[13] Generally, the mortality rate among patients poisoned with
A. phalloides and treated with various drugs, except silymarin, ranges
from 22 to 40% in adults, and is higher in children.[71]
In a retrospective study performed in patients with intoxication
caused by A. phalloides, the severity of hepatic injury was found to be
closely related to the time that had elapsed between ingestion and treatment
with silibinin: the shorter the interval, the less severe the injury.[72]
Silibinin was administered by intravenous infusion at a mean dosage of
33 mg/kg/day for on average 81.6 hours. All the 18 patients included in
the study survived, except one who had taken a high dose to commit suicide.
Other treatments were not related to reduction in liver injury.
Patients exposed long term to organic phosphates and
treated with silymarin for 1 month did not exhibit any improvement in liver
function as compared with controls, although serum levels of pseudocholinesterase
were considerably increased. This may reflect blockage of toxin anti-cholinesterase
activity by silymarin.[73]
The few studies on silymarin in toxic hepatitis in the
literature have yielded positive results. An interesting clinical trial
was performed in patients being treated with psychotropic agents (phenothiazines
or butyrophenones). Patients were subdivided into two groups: in the first
group treatment was discontinued, and in the other group treatment was
continued at the same dose. The groups were further subdivided into two
subgroups: one was given silymarin 800mg daily for 90 days, the other was
given placebo. The results showed that silymarin is able to improve liver
function, independently of the discontinuation of psychotropic therapy.[74]
Similar results were obtained by other authors.[71]
Chronic Hepatitis and Cirrhosis
In a clinical trial performed in 170 patients with a
positive biopsy for cirrhosis, followed up for 2 to 6 years and given oral
silymarin 140mg three times daily (87 patients, of whom 46 had cirrhosis
due to alcohol abuse) or placebo (83 patients, of whom 45 had cirrhosis
due to alcohol abuse) the mean survival rate after 4 years was significantly
(p = 0.036) higher in patients treated with silymarin (58 ± 9%)
as compared with those treated with placebo (39 ± 9%), whereas no
significant difference was found in biochemical markers. Analysis of subgroups
revealed that treatment was more effective in patients with alcohol-related
cirrhosis (p = 0.01) and in groups of patients with nonalcoholic cirrhosis,
whereas it was ineffective in patients with class B or C portal hypertension.[15]
Another two interesting studies are reported in the review
by Flora et al.[71] The first study was performed in 2637 patients with
chronic liver disease, treated with high doses of silymarin (560 mg/day)
for 8 weeks. Resolution of subjective symptoms was achieved in 63% of cases;
AST diminished on average by 36%, ALT by 34% and GGT by 46%. Furthermore,
the investigators reported a reduction in hepatomegaly upon palpation.
The second study was performed under double-blind conditions in patients
with persistent or aggressive chronic hepatitis, with or without cirrhosis,
monitored for 3 to 12 months and treated with silymarin. Treatment did
not produce any signs of improvement in liver function; however, histological
examination revealed an improvement in portal inflammation, parenchymal
alterations and necrosis.
4.4 Alcohol-Related Liver Disease
A randomised clinical trial was performed in patients
with moderate alcohol-related liver disease (ALT and AST <200 U/ml)
and persistent abnormalities of liver function after abstinence from alcohol
for at least 1 month. Patients were treated with silymarin 420 mg/day or
with placebo for 4 weeks. At the end of the study period, mean AST and
ALT levels diminished by 30.1% and
40.8%, respectively, in patients treated with silymarin
who had completed the study as compared with increases of 5.4% and 2.8%,
respectively, in patients treated with placebo (p >/=0.001). There was
no significant difference in bilirubin levels.[75]
Not all the clinical trials performed with silymarin
in this indication have yielded positive results.[76] The results of a
recent randomised double-blind study in 125 patients with histologically
documented alcohol-related cirrhosis did not show any significant benefit
on survival after 2 years of treatment with silymarin 450 mg/day by the
oral route.[77]
4. Dosage and Administration
In the clinical trials described, the daily oral dose
of silymarin used ranged from 280 to 800mg. This is equivalent to 400 to
1140mg of standardised extract containing 70% silymarin. The recommended
dosage for active disease is 140mg of silymarin (200mg of extract) three
times daily. If the preparation silipide (silymarin-phosphatidylcholine)
is used, 100mg three times daily is the appropriate dosage.[71] At higher
dosages (>1500 mg/day) silymarin may have a laxative effect due to an increase
in secretion and bile flow.[13] Moderate allergic reactions have also been
reported.[13,66]
5. Antioxidant and Hepatoprotective Effects of Other
Flavonoids
Flavonoids are a large group of phenolic compounds ubiquitously
distributed in the plant kingdom. More than 4000 flavonoids belonging to
different classes have been identified so far. They are important for the
normal growth, development and defence of plants. Important constituents
of the human diet, they are present in fruit and vegetables. Multiple biological
effects of flavonoids have been described, including anti-inflammatory,
antiallergic, antihaemorrhagic, anti-mutagenic, antineoplastic and hepatoprotective
activities.[78] The biological and pharmacological effects of flavonoids
in mammals are assumed to result mainly from two properties: modulation
of certain enzymes (hexokinase, aldose reductase, phospholipase C, protein
kinase C, cyclo-oxygenase, lipoxygenase, myeloperoxidase, NADPH oxidase
and xanthine oxidase) and their antioxidant activity. The various flavonoids,
however, vary greatly in their efficacy, and a single flavonoid can inhibit
one enzyme at a certain concentration while inhibiting another enzyme at
a 100-fold higher concentration.[46,47]
Some flavonoids, including quercetin and silibinin, can
protect cells and tissues against the effects exerted by reactive oxygen
species. Their antioxidant activity results from the scavenging of free
radicals and other oxidising intermediates, from the chelation of iron
or copper ions, and from inhibition of oxidases.[79] As discussed in the
preceding sections, flavonoids from Silybum marianum have been widely used
for the treatment of liver disorders. In experimental animal models they
were demonstrated to exert not only a positive effect on intact liver cells
or cells not yet irreversibly damaged, but also to stimulate their regenerative
capacity after partial hepatectomy.[41]
Antihepatotoxic activity was also demonstrated for kolaviron,
a defatted alcoholic extract of the seeds of Garcinia kola, and for Garcinia
biflavonones, in mice intoxicated with phalloidin.[80] Other flavonoids
extracted from Baccharis trimera were reported to protect mice from hepatic
damage; hispidulin appeared to be the most active compound, and quercetin,
luteolin, nepetin and apigenin were less active or inactive.[81] Quercetin,
however, was demonstrated to exert some ameliorative effects on tissue
damage induced by cigarette smoke[78] and to reduce the cytotoxic effect
of T-2 mycotoxin.[82] Gossypin and hydroxyethyl rutosides significantly
reduced the toxic effect of dermal application of sulphur mustard on hepatic
lipid peroxidation in mice. Moreover, it increased the survival rate of
the animals.[83]
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