Influence of the Flavonolignan Silibinin of Milk Thistle on Hepatocytes

Chapter 18

Influence of the Flavonolignan Silibinin of Milk Thistle on Hepatocytes and Kidney Cells

Downloaded by STANFORD UNIV GREEN LIBR on September 28, 2012 | http://pubs.acs.org Publication Date: April 15, 1998 | doi: 10.1021/bk-1998-0691.ch018

J. Sonnenbichler, I. Sonnenbichler, and F. Scalera Max Planck Institut für Biochemie, D-82152 Martinsried, Germany

The pharmacologically active components of the plant Silybum marianum are theflavonolignans,silibinin and silichristin. It has been shown that these compounds can be used successfully in therapy to promote faster regeneration of diseased liver. The biochemical mechanism for this cell-regenerating power has been elucidated. It has been demonstrated that silibinin stimulates the activity of the DNA-dependent RNA-polymerase I, thus causing an increase in rRNA synthesis and an accelerated formation of intact ribosomes. The consequence of this stimulation is a general increase in the rate of synthesis of all cellular proteins. Molecular modelling revealed that silibinin may imitate a steroid hormone by binding specifically to polymerase I, thus stimulating the enzyme activity. The molecular mechanism described has been demonstrated in experiments with rat and mice liver in vivo, with hepatocyte cultures, isolated liver nuclei, and purified enzyme and receptor proteins in vitro. The increase in protein synthesis offers a good explanation for the liver -regenerating power of the plant extract. Similar results have been recently found with human and monkey kidney cells.

Extracts of the flowers and leaves of Silybum marianum (milk thistle or St. Mary's thistle), Figure 1, have been used for centuries to treat liver diseases. In the 1960s, scientists began to isolate the most important ingredients of the extracts of milk thistle, and their chemical structures were elucidated in the study-groups of Pelter and Hansel (7), as well as of Wagner et aL (2, 3). The efforts at isolation first lead to a mixture, which was named silymarin. It was with this mixture that most of the clinical studies were carried out. We know today that the constituents are the compounds shown in Figure 2, and that silibinin makes up approximately 60% of the main components. These compounds are flavonolignans and are produced in the plant under conditions of strong solar radiation. Every physician knows the abundance of clinical pictures associated with the term "liver diseases". These clinical pictures are just as varied as are the causes of the diseases—starting with viral infections and continuing on to toxic damage, for example,

©1998 American Chemical Society

In Phytomedicines of Europe; Lawson, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

263


264

Figure 1. Silybum marianum.

OH

Figure 2. Constituents of Silymarin.



265 from alcohol intoxication. It is, therefore, not surprising that one faces considerable difficulties in selecting a suitable group of patients for double-blind clinical trials. Nevertheless, the therapeutic efficacy of silymarin has been convincingly verified. Fintelmann and Albert (4) observed already in 1980 that after toxic liver damage, administration of sttyrnarin results in a significantly accelerated normalization of GOT (glutamate-oxaloacetate trarisaminase) and GPT (glutamate-pyruvate transaminase), Figure 3. A 1989, a double-blind study by Feher (5) likewise demonstrated that after admiriistration of silymarin to patients with alcohol-induced liver diseases, a distinctly more rapid normalization of the transaminases and bilirubin takes place. A multicenter study by Ferenci and colleagues (6) in Vienna found that the average survival rate of cirrhotic patients in the Child A stage could be markedly lengthened if they were given silymarin, Figure 4. Berenquer and Carrasco (7) observed an accelerated normalization in the plasma albumin values in patients with chronic inflammatory liver diseases after treatment with silymarin. One could summarize all thesefindingsunder the term "increased regenerative ability". Studies on the Mechanism of Action of Silibinin Irrespective of the noxious agents causing the damage (Le. virus or toxin), in biochemical terms, "regenerative ability" means the restoration of damaged cellular components. Above all, defective macromolecules with biochemically meaningful functions must be replaced in order to restore normal cell function. Besides nucleic acids, these include, above all, proteins as building blocks of cell walls, organelles and as enzymes which in the end deterrnine the entire primary metabolism. The synthesis of proteins is managed according to the scheme in Figure 5. In the past 20 years, we have been able to clarify in considerable detail how the flavonolignans of milk thistle influence this mechanism. Among other things, it was necessary to measure the rate of synthesis of the various macromolecules. This is done generally in the following manner: a specific building block of the macromolecule is radioactive-labelled and then employed in the experiment. For example, in the case of quantitative determination of protein biosynthesis, this would involve labelling an amino acid, or in the case of nucleic acid measurements, a nucleotide unit would be thus labelled. After isolation of the high-molecular weight products, the time-dependent incorporation of the precursor can then be measured. We carried out experiments on the livers of rats and rnice in vivo, as well as with isolated cells - particularly with hepatocyte cultures, with isolated organelles (e.g., with cell nuclei in vitro), and finally with isolated enzymes and receptor proteins. The following was found under the influence of siHbinin: 1. Protein biosynthesis, measured in units of time, proceeds approximately 25-30% faster under the influence of the flavonolignan sihbinin compared to controls (8), Figures 6 and 7. 2. After determining the specific radioactivity of hundreds of different newly synthesized cellular proteins, it could be seen that the rate of synthesis of all the cellular proteins is increased equally without any preference or de novo synthesis of certain proteins. 3. We found that preceding this stimulation of protein synthesis is a great increase in the rate of RNA synthesis (9), Figure 8. We differentiate here three species of RNAs, which are indicated in Figure 9. Although the rates of synthesis of amino acid-activating tRNAs and of the heterogenic m-RNAs are not influenced by silibinin, the synthesis of the rRNAs



266

1

3

7

10** 1 4 *

2V

28*

35

days Figure 3. Time dependent normalization of a liver-specific enzyme after toxic liver disease in men, in the presence and absense of silymarin. (Reproduced with permission from reference 4. Copyright 1980 Therapiewoche.) ALCOHOLIC CIRRHOSIS

O

2 0,3

I P-« 0,4

1

B °-

•

SILYMARIN

•

PLACEBO

AT RISK S 47 AT RISK P 4i

Figure 4. Survival curves for 170 patients with cirrhosis of the liver treated with silymarin or placebo, data analyzed according to the etiology of liver disease. Placebo vs. silymarin in alcoholic cirrhosis: Wilcox-Breslov test, p=0.011; Mantel-Cox test, p=0.012. In Phytomedicines of Europe; Lawson, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

267

DMA

Nucleus

DOOOOOOOOOOOC

Transcription


RNAA A A A A A

Translation

Cytoptasma m-RNA

growing protein chain Figure 5. Scheme for the protein biosynthesis in eucaryotes including the transcriptional and translational process.

Figure 6. Time dependent incorporation of methionine into synthesized proteins in isolated rat hepatocytes in the presence of 10 ug/ml silibinin (o) and without silibinin (•).


268

219 -

I

150 %]


100

50

n

c

s

14

Figure 7. Incorporation of C-leucine into rat liver proteins 1.5 h after application of silibinin in vivo, and 3 h before sacrifice. S=10 mg/kg silibinin, C=control.

time [h]

Figure 8. Time dependent incorporation of uridine into newly synthesized RNA in rat livers in vivo with (A)and without (A) silibinin application (a). Precursor pools (b). (Reproduced with permission from reference 10. Copyright 1984 Walter de Gruyter.)


269

is strongly stimulated (10), Table I. These rRNAs consist of a 5.8S, 18S and 28S-RNA species and are essential building blocks of the ribosomes—those "sewing machines" on which the proteins are synthesized.


Table I. Influence of silibinin on the rate of synthesis of different RNA species in rat liver in vivo (%). total RNA rRNA (5.8S) rRNA (18S) rRNA (28S) tRNA mRNA (from nuclei)

+99 +33 +74 +29 +1 -7

4. It was also shown that the synthesis of the mature—those with the associated ribosomal proteins—more complex ribosomes is distinctly increased, Figure 10. Upon reflection of these findings, it becomes clear that with the increase in "sewing machines"—that is to say, the ribosomes—also the protein synthesis of the entire cell is increased, which then accelerates the cell regeneration. 5. Finally, the cause for the stimulation was found to be a specific enzyme, namely DNAdependent RNA-polymerase I, whose activity is greatly increased by silibinin (77), Figure 11. This enzyme catalyzes the transcription of rRNA, and silibinin acts as a positive effector on this biocatalyst, but specifically with eucaryotes. With procaryotic RNApolymerases (Gram-positive and Gram-negative bacteria), we could not find such a stimulation (72). In the case of partially hepatectornized rats even the DNA-replication is stimulated (75). In enzymology one can recognize negative and positive effectors, for example in the case of pacemaker enzymes. It is astonishing though why a plant metabolite can exert such a specific effect on the molecular biology of an animal celL In order to clarify this question, we investigated around 30 structurally related compounds with respect to these properties. Only a few of the tested substances exhibited a similar effect (14), Table n. Structural comparisons and molecular modelling of the biologically active compounds revealed a similarity to steroids, which is indicated in Figure 12. In fact, it is known that besides their specific ability for genetic induction (sihbinin does not do this!), steroids can stimulate also the activity of RNA-polymerase I, Le. they can stimulate the general rate of rRNA-transcription. In cooperation with Prof. Jungblut of the Max-Planck-Institute for Endocrinology, Braunschweig, we were able to demonstrate that sihbinin binds competitively to a purified, isolated steroid receptor, albeit at a reduced affinity (15), Figure 13. Sihbinin thus imitates a regulator of the cell itself, resulting in a stimulation of the entire cellular protein synthesis. We can now summarize the mechanism of action in Figure 14.


270

TRANSCRIPTION X D O O O O O O O O O O O O C


\

DNA

DNA dependent

XRNA Polymerases 5.8,18,285 ribosomal RNA's (structural elements of ribosomes)

5-10 5 messenger RNA's (genetic message)

4-5S transfer RNA's (activation of amino acids)

Figure 9. Different RNA types that are transcribed from the DNA template.

cpm

time [ h ] 14

Figure 10. Incorporation of C-uridine into RNA of complex ribosomes. C-Orotic acid (50 pCi) was injected into Wistar-rats (80-120 g) i.p. 3 h before sacrifice. Silibinin-hemisuccinate (1.5 mg) (•) was previously injected at differenttimes.Controls (o) were treated only with solvent 14



271

Figure 11. Transcription activities of the isolated and purified DNAdependent RNA-polymerases in vitro with and without addition of silibinin, with calf thymus DNA as the template. (Reproduced with permission from reference 11. Copyright 1977 Walter de Gruyter.)

OH

Silibinin Figure 12. Comparison of silibinin to the steroid skeleton. The steroid skeleton is enclosed in the dashed box.


272

• A •

V.


o

Estradiol Silibinin Wedelo-lacton E A3

• H-Estradiol 3

50

Competitor concentration IM]

Figure 13. Binding of radioactive labelled estradiol (%) in the presence of unlabelled estradiol and unlabelled silibinin as a competitor to the isolated and purified steroid-receptor from pig uteri. Wedelo-lactone and EA3 are inactive controls.

Nucleus

(genetic information)

(amino acid activation)

(ribosome structure)

1

J. '

ribbsomes

proteins Figure 14. Summary scheme for the interaction of silibinin with the molecular biology of the cell.



273

Table n. Effects of various flavonoids on protein and RNA synthesis in liver cells (+ stimulation, - inhibition, -0 no effect, () marginal effects). Flavanones: Flavonoles: Pinocembrin Gossypetin 0 (+) Isokuranetin 0 Galangin 0 Homoeryodictyol 0 Fisetin (+) Naringenin 0 Kampferol (+) Hesperitin ++ Luteolin 0 Flavanone 0 Morin (+) Rhamnetin 0 Quercetin 0 Flavanonoles Taxifolin ++ Flavonolignans Flavones Acacetin Apigenin

0 0

Isoflavones Prunetin Irigenin Genistein

0 (+)

Catechin

++

-

Silibinin Silidianin Silichristin Isosilibinin Others: Benzalacetophenone Chalcone Cynarin Curcumin

++ 0 + (-)

0 0 0 0

This mechanism applies for the period of time in which silibinin is available to the celL The absorption of current preparations after oral adrriinistration is approximately 5060%. We were able to confirm this in rats with radioactive-labelled silibinin, Figure 15. Two maxima of silibinin concentrations occur in the liver via enterohepatic circulation. Then, after approximately 40 hours, the flavonolignan is excreted in the form of glucoside and sulfate metabolites (16). Considering the biochemical mechanism described, it would be expected that the stimulation of protein biosynthesis also occurs in non-hepatocytes, even though the flavonoid concentrations are by far highest in the hepatocytes. In fact, we have been able to ascertain in the past months that also in cell cultures of human and monkey kidney cells, silibinin causes increased protein synthesis, nucleic acid synthesis and also increased replication of dividing cells by approximately 25-30%, Table HI. Already, positive clinical findings have also been obtained in this regard. Our experiments with liver and kidney cells demonstrated that only silibinin and silichristin exhibit the stimulatory effect, but not isosilibinin or sihdianin, Table IV. We have also worked with tumor cells, particularly human hepatoma cells (Alexander cells), rat hepatoma cells (Raji cells), Burkitt lymphoma cells, and HeLa cells, Figure 16. No stimulation was found in any of these cases. An obvious explanation would be that the transcription and translation in malignant cell lines already proceed at maximal rates and cannot be accelerated any further.



274

0

5

10

15

20

25

30

time [hours]

Figure 16. Time course of protein synthesis in cultures of malignant cell lines (for example, Raji-cells from rat hepatoma) in the presence of different concentrations of silibinin-hemisuccinate.


275

Table m . Effect of various silibinin-hemisuccinate concentrations on the cell line Vero from green monkey kidneys. Four days after transfer of 7.5xl0 cells/ml in clusters with 400 pi medium 199, the cells were counted. Silibinin-hemisuccinate 4


silibininhemisuccinate (Hg/mL) 0

10

20

50

final cell counts

cell vitality

growth

(%) 92 93

(%)

2.86xltf 2.86x10 3.30x10 3.26x10

s

s

s

3.50x10 3.44x10

s

s

2.60x10 2.58x10

s

s

100

93 94

116

94 94

123

91 92

92

Table IV. Effect of 10 ug/ml silibinin-hemisuccinate, silidianin, silichristin and iso-silibinin on the kidney cell line Vero. Details described in Table HI. growth probe cell vitality final cell counts (%) (%) control 4.26X10 95 100 4.34x10 96 5

s

control +1% ethanol

4.26x10 4.28x10

silibinin hernisuccinate

4.96x10 4.98x10

silidianin

4.10x10 4.92x10

silichristin

isosilibinin

s

s

s

s

s

s

4.92x10 4.88x10

s

3.90x10 3.72x10

s

s

s

96 96

99

96 96

116

96 96

94

96 96

114

96 95

89



276 Additional mechanisms of action have been proposed for the flavonolignans of milk thistle, and some have been verified in experiments. For example, these polyphenols have the ability to intercept radicals, such as radicals formed from oxidative alcohol degradation or from other noxious agents in the liver which can cause cell damage. Data for the scavenger function of the silymarin components have been obtained in in vitro experiments primarily by the study-groups of Feher (77), Mira (18), Valenzuela and Guerra (19), and GyOrgy (20). However, the flavonoid concentrations required in this case are so high, that, in my opinion, they cannot be achieved in the organism through oral application of silymarin preparations. Furthermore, we know that in high concentrations silibinin diminishes the membrane permeability of the hepatocytes, so that many toxins can hardly then penetrate the cell (27). For example, the absorption of amanitine, the poison of the green deathhead, is slowed down so much that the excretion of the toxin takes place before the actual toxic effect can set in. One can also employ this possibility for the successful therapy against mushroom poisoning (22). The silibinin concentrations required in such a case are around 10-20 times higher than those needed for stimulation of protein synthesis and can be achieved only via intravenous adrriinistration. Conclusion If one considers all of the biochemical effects described here, then the clinical results obtained with silibinin and silymarin can be explained from the view of the molecular biologist and confirm that theflavonolignansof milk thistle—above all, sihbinin and silichristin—are indeed interesting drugs. Acknowledgement. Sihbinin (Legalon) and the flavonolignans were a gift of Dr. Madaus A G , KOln, FRG, which is gratefully mentioned. Literature cited 1. Pelter, A.; Hänsel, R. Tetrahedron 1968, 25, 2911 2. Wagner, H.; Hörhammer, R.; Münster, R. Naturwissenschaften 1965, 52, 305 3. Wagner, H.; Seligmann, O.;Hörhammer,L.; Seitz,M.;Sonnenbichler, J. TetrahedronLetters1971, 22, 1895 4. Fintelmann, V.; Albert, A. Therapiewoche 1980, 30, 5589 5. Feher, J. Orvosi Hetilap 1989, 130, 2723 6. Ferenci, P.; Fragosics, B.; Dittrich, H.; Frank, H.; Benda, L.; Lochs, H.; Meryn, S.; Base, W.; Schneider, B. J. of Hepatology 1989, 2, 105 7. Berenquer, J.; Carrasco, D. MünchnerMed.Wochenschr. 1977, 119 8. Sonnenbichler, J.; Zetl I. Plant Flavonoids in Biology and Medicine II 1986, 369 9. Sonnenbichler, J.; Mattersberger, J.; Rosen, H. Hoppe-Seyler's Z. Physiol.Chem. 1976, 357, 1171 10. Sonnenbichler, J.; Zetl, I. Hoppe-Seyler'sZ.Physiol.Chem.1984, 365, 555 11. Machicao, F.; Sonnenbichler, J. Hoppe-Seyler'sZ.Physiol.Chem.1977, 358, 141 12. Schnabel, R.; Sonnenbichler, J.; Zillig, W. FEBS Letters 1982, 50, 400 13. Sonnenbichler, J.; Goldberg,M.;Hane, L.; Madubunyi, I.; Vogl, S.; Zetl, I. Biochemical Pharmacology 1986, 35, 538



277 14. Sonnenbichler,J.;Pohl, A. Hoppe-Seyler'sZ.Physiol.Chem.1980, 361, 1757 15 Sonnenbichler, J.; Zetl, I. In Progress in Clinical and Biological Research, Cody, V.; Middleton, E.; Harborne, J.B., Eds., Alan R. Liss, Inc. 1986, Vol. 213; p. 319 16. Sonnenbichler, J.; Mattersberger, J.; Hanser, G. Hoppe-Seyler'sZ.Physiol.Chem. 1980, 361, 175 17. Feher, J. Free Rad. Res. Commun. 1987, 3, 373 18. Mira, M.L. Free Rad. Res. Commun. 1987, 4, 125 19. Valenzuela, A.; Guerra, R. Experientia 1986, 42, 139 20. György, I. Radiat. Phys.Chem.1992, 39, 81 21. Sonnenbichler, J.; Sonnenbichler, I.; Scalera, F. in press. 22. Hruby, K.; Fuhrmann, M.; Csomos, G.; Thaler, H. Wiener Klin. Wochenschr. 1983, 95, 225


Influence of the Flavonolignan Silibinin of Milk Thistle on Hepatocytes

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