Scholarly article on topic 'Silymarin and its constituents in cardiac preconditioning'

Silymarin and its constituents in cardiac preconditioning Academic research paper on "Clinical medicine"

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{Ischemia / Preconditioning / Silymarin / Silybin / Quercetin / "Signalling pathways"}

Abstract of research paper on Clinical medicine, author of scientific article — A. Zholobenko, M. Modriansky

Abstract Silymarin, a standardised extract of Silybum marianum (milk thistle), comprises mainly of silybin, with dehydrosilybin (DHSB), quercetin, taxifolin, silychristin and a number of other compounds which are known to possess a range of salutary effects. Indeed, there is evidence for their role in reducing tumour growth, preventing liver toxicity, and protecting a number of organs against ischemic damage. The hepatoprotective effects of silymarin, especially in preventing Amanita and alcohol intoxication induced damage to the liver, are a well established fact. Likewise, there is weighty evidence that silymarin possesses antimicrobial and anticancer activities. Additionally, it has emerged that in animal models, silymarin can protect the heart, brain, liver and kidneys against ischemia reperfusion injury, probably by preconditioning. The mechanisms of preconditioning are, in general, well studied, especially in the heart. On the other hand, the mechanism by which silymarin protects the heart from ischemia remains largely unexplored. This review, therefore, focuses on evaluating existing studies on silymarin induced cardioprotection in the context of the established mechanisms of preconditioning.

Academic research paper on topic "Silymarin and its constituents in cardiac preconditioning"

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FITOTE-02952; No of Pages 11

Fitoterapia xxx (2014) xxx-xxx

ELSEVIER

Contents lists available at ScienceDirect

Fitoterapia

journal homepage: www.elsevier.com/locate/fitote

Review

Silymarin and its constituents in cardiac preconditioning

A. Zholobenko, M. Modriansky *

Department of Medical Chemistry and Biochemistry, School of Medicine and Dentistry, Palacky University, Hnlvotinskä 3, Olomouc 77515, Czech Republic

ARTICLE INFO

ABSTRACT

Article history:

Received 4 March 2014

Accepted in revised form 21 May 2014

Available online xxxx

Keywords:

Ischemia

Preconditioning

Silymarin

Silybin

Quercetin

Signalling pathways

Silymarin, a standardised extract of Silybum marianum (milk thistle), comprises mainly of 13

silybin, with dehydrosilybin (DHSB), quercetin, taxifolin, silychristin and a number of other 14

compounds which are known to possess a range of salutary effects. Indeed, there is evidence for 15

their role in reducing tumour growth, preventing liver toxicity, and protecting a number of 16

organs against ischemic damage. The hepatoprotective effects of silymarin, especially in 17

preventing Amanita and alcohol intoxication induced damage to the liver, are a well established 18

fact. Likewise, there is weighty evidence that silymarin possesses antimicrobial and anticancer 19

activities. Additionally, it has emerged that in animal models, silymarin can protect the heart, 20 q4

brain, liver and kidneys against ischemia reperfusion injury, probably by preconditioning. The 21

mechanisms of preconditioning are, in general, well studied, especially in the heart. On the 22

other hand, the mechanism by which silymarin protects the heart from ischemia remains 23

largely unexplored. This review, therefore, focuses on evaluating existing studies on silymarin 24

induced cardioprotection in the context of the established mechanisms of preconditioning. 25

© 2014 Published by Elsevier B.V.

Contents

1. Introduction..............................................................................................................0

2. Preconditioning and silymarin..............................................................................................0

3. Actions of silymarin that may be related to preconditioning..................................................................0

4. Future directions..........................................................................................................0

5. Conclusion................................................................................................................0

Conflicts of interest ............................................................................................................0

Acknowledgements ............................................................................................................0

References......................................................................................................................0

Abbreviations: AC, adenylyl cyclase; ALDH, aldehyde dehydrogenase; ANT, adenine nucleotide transporter; AR, aderenergic receptor; ARE, aryl hydrocarbon receptor; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; COX, cyclo-oxygenase; CsA, cyclosporine A; DAG, diacylglycerol; DHSB, dehydrosilybin; EGF, endothelial growth factor; EGFR, EGF receptor; FGF, fibroblast growth factor; GSK, glycogen synthase kinase; HIF, hypoxia induced factor; HUVEC, human umbilical vein endothelial cell; IP3K, inositol phosphate 3 kinase; IPC, ischemic preconditioning; IR, ischemia reperfusion; MMP, matrix metaloprotease; mPTP, mitochondrial permeability transition pore; mTOR, mitochondrial target of rapamycin; PDE, phosphodiestrase; PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; ROS, reactive oxygen species; SIRT, silent information regulator two ortholog; VDAC, voltage dependent anion channel; VEGF, vascular endothelial growth factor.

* Corresponding author. Tel.: +420 585632219; fax: +420 585632302. E-mail address: martin.modriansky@upol.cz (M. Modriansky).

http://dx.doi.Org/10.1016/j.fltote.2014.05.016 0367-326X/© 2014 Published by Elsevier B.V.

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1. Introduction

Silymarin, a well known, multicomponent extract from the seeds of the milk thistle (Sylibum marianum), has been used for the treatment of various ailments, mainly those of the liver, for over two thousand years [1]. Interest in this venerable remedy has not been lost with the advent of the systematic scientific approach and modern biochemical methods, and there are now over four hundred clinical trials using silymarin or its components for liver related diseases alone [2]. In this day and age, silymarin is available as an extract from several major suppliers, each with its own standard composition, which varies dramatically between suppliers and appears to depend on variety and growing condition of the crop [3-5]. Typically, silymarin contains around 50% silybin, 20% silychristin, 10% silydianin, 5% isosilybin and between 10 and 30% of a typically unidentified organic polymer fraction formed from the above compounds. Additionally, a minor fraction of other flavanols including 2,3-dehydrosilybin (DHSB), quercetin, taxifolin, kaempferol and others is present [5,6]. Some of the constituents, including silybin, are present as a mixture of stereoisomers with contrasting biological activities [7,8]. It is understandable therefore, that small changes in the chemical composition of the extract can have a strong influence on its biological activity. On the other hand, this is largely irrelevant when working with the purified, individual components of silymarin. It should be noted that as a consequence of consisting of a number of bioactive compounds, silymarin does not have a single molecular target. Indeed, many of its components, as will become apparent from the discussion below, target more than one enzyme or process. Whilst this can be viewed as a pharmacologist's nightmare, the same pharmacologist may find that it can also become a treasure trove of interesting medicinal compounds and precursors. The milk thistle would serve well for this purpose, owing partially due to its wide range and ease of cultivation.

It is understandable, therefore, that more and more attention is being devoted to the possible protective effects of silymarin on organs besides the liver. As such studies examining protection by silymarin against ischemic damage to kidney, liver, brain and heart have emerged. This is most likely tied to the discovery, and more recently improved understanding, of pre- and post-conditioning. Applicable to tissue that has been subject to ischemia, these closely related biological phenomena prevent a large part of the damage that occurs upon its reperfusion. Whilst preconditioning must be applied during the early window, at least 24 h prior to ischemia, or the late window around 30 min prior to ischemia, post-conditioning can be applied immediately upon reperfusion. Given the unpredictable nature of infarcts, post-conditioning is undoubtedly more valuable as a treatment. Preconditioning, on the other hand, could be availed of when ischemia can be anticipated, for example during surgery or transport of organs [9,10]. The most common, and most clinically relevant, examples of this kind of injury are the heart and brain, where ischemic events manifest themselves as heart attacks and strokes respectively. Arguably, due to the increased window for treatment, pre- and post-conditioning of the heart makes a better example. Both pre- and post-conditioning can be induced either by a series ofbrief ischemia-

reperfusion cycles, in which case they are known as ischemic 108

pre- or post-conditioning (IPC), or by pharmacological agents, 109

in which case they are known as pharmacological pre- or post- 110

conditioning. The former was discovered in 1986 [11] using an 111

open chest dog model, whilst the later arguably in 1984 [12]. 112

Whilst IPC is the better known of the two, pharmacological 113

preconditioning is probably more applicable in practice, as well 114

as serving as a useful tool for the study of the mechanisms 115

involved in IPC. 116

2. Preconditioning and silymarin

Following occlusion of the blood supply, ischemic tissue 118

will eventually die by necrosis (curiously the 1986 study had 119

already established a limit for the length of ischemia which 120

preconditioning can protect against [11]). It follows that 121

reperfusion became the main form of intervention for 122

myocardial infarction. This led to the discovery of ischemia 123

reperfusion (IR) injury of the heart, which occurs, as the name 124

suggests, when following a prolonged period of ischemia, 125

blood supply is restored to the ischemic tissue, paradoxically 126

causing a rise in cell death. This is proposed to occur because 127

the kick-starting of respiration, in cells where most of the ion 128

gradients have all but collapsed, sets up the perfect conditions 129

for the opening of the mitochondrial permeability transition 130

pore (mPTP) and the subsequent induction of apoptosis. In 131

accordance with this model, ischemic cells rapidly become 132

hypoxic and switch to glycolysis for their source of adenosine 133

triphosphate (ATP) hence becoming acidified. At the same time, 134

levels of reactive oxygen species (ROS) increase and levels of 135

ATP drop along with the activity of the Na+/K+ ATPase. Due to 136

the increased proton concentration, i.e. intracellular acidifica- 137

tion and reduced activity of the Na+/K+ ATPase, the Na+/H+ 138

exchanger causes an influx of Na+. This reverses ion-flux 139

through the Na+/Ca2+ antiporter, increasing the intracellular 140

concentration of Ca2+. Under normal conditions this increase 141

in ROS and Ca2+ would be sufficient to open the mPTP and 142

induce apoptosis, however, as low pH inhibits mPTP opening, 143

apoptosis does not occur in ischemic cells. Instead the damage 144

occurs upon reperfusion, when the mitochondrial pH begins to 145

normalise with the restoration of the mitochondrial H+ 146

gradient and all the conditions for the opening of the mPTP 147

have been met [13,14]. 148

Pre- and post-conditioning, must therefore function either 149

by reducing calcium concentrations in the cells [15-17], 150

limiting over-production or accumulation ofROS or increasing 151

the mPTP threshold [13,18]. In fact it has been shown that 152

pharmacological opening of mPTP with atractyloside prevents 153

preconditioning [19-21], whilst preventing this opening with 154

cyclosporin A (CsA) induces preconditioning in rabbit hearts 155

[20]. The latter prevents the binding of Cyclophilin D [22], 156

whilst the former is a direct inhibitor of the adenine nucleotide 157

transporter (ANT) [23]. Rasola et al. [18] suggest that glycogen 158

synthase kinase 3(3 (GSK3() and protein kinase Cs (PKCs) may 159

be responsible for the phosphorylation and hence modulation 160

of mPTP components. It appears that when phosphorylated 161

and hence inhibited by PKCs, GSK3( shifts from the voltage 162

dependent anion channels (VDAC) to ANT binding [24]. 163

This coincides with reduced VDAC phosphorylation and 164

may be central to pre- and post-conditioning [25] as there is 165

evidence that GSK3( inhibition is a central and crucial step in 166

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167 preconditioning [26-28]. Whilst there are studies that chal-

168 lenge this notion in favour of the model where GSK3(3 is a

169 marker of preconditioning [19-21,29-31], the evidence for the

170 central role of PKCs in preconditioning is solid [27,29,32-41].

171 It is therefore possible that other targets of PKCs, such as

172 respiratory chain components or aldehyde dehydrogenase 2

173 (ALDH2), may be responsible for raising mPTP threshold q5 [42-44]. PKCs itself can be activated by an increase in ROS

175 [45], DAG [36], or by phosphorylation by Erk or protein kinase

176 G (PKG) [15,38,46-50]. In turn, pharmacological precondition-

177 ing can be achieved through a number of receptors which are

178 known to activate Erk, PKG and phospholipase C (PLC). Thus, as

179 summarised in Fig. 1, it has been established that stimulation

180 through adrenergic, ouabain, acetylcholine, opioid, bradykinin,

181 oestrogen and adenosine receptors triggers preconditioning

[38-40,50-63]. Taking into account the number of receptors 182 and downstream components involved in pre- and post- 183 conditioning, along with the number of their possible inter- 184 actions of the main components of silymarin, the scope of 185 any attempt to pin down the mechanism responsible for 186 silymarin's cardioprotective activity becomes apparent. 187 Silymarin, and silybin in particular, is known to protect a 188 number of organs, including brain, liver, kidney and the 189 gastrointestinal tract. Silymarin's hepatoprotective proper- 190 ties are especially well researched, with over two hundred 191 clinical trials [2,64-66]. (See Fig. 2.) q6

In addition, several studies have investigated the potential 193 of silymarin in protecting gastric mucosa [67], liver [68], 194 kidney [69,70] and brain against IR injury [71,72]. Curiously, 195 unlike Wang et al. [73], Hou et al. [71] found that whilst 196

oh o Quercetin

O ,,CH2OH

Dehydrosilybin (A)

o^ _,CH2OH

Dehydrosilybin (B)

Silychristin

Siiydianin

Isosilybin B

q2 Fig. 1. A schematic diagram of several of the more important components of silymarin; taxifolin, silybin, isosilybin, quercetin, dehydrosilybin, silychristin and silydianin.

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Taxifolin

Fig. 2. Diagram outlining the pathways of preconditioning, as evidenced from various models of preconditioning, and the possible interaction points for the components of silymarin that are summarised in this paper. Black arrows indicate interactions between components of the pathways, with blue curly arrows indicating signal transduction by second messengers. Green arrows and red stubbed arrows indicate potential interaction by silymarin's components. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

197 silymarin pretreatment afforded rat brains a certain level of

198 protection against IR injury, silybin did not. This leads to the

199 fascinating possibility that components of silymarin besides

200 silybin, are primarily responsible for the cardioprotective ef-

201 fects of silymarin. As we point out in the Introduction section

202 (vide supra) it is unlikely that silymarin as a multicomponent

203 extract has a single molecular target. This is exemplified by

204 studies whose findings highlight quercetin's ability to protect

205 various tissue types against IR Injury [74-79]. To add to this, a

206 number of studies examined taxifolin's cardioprotective ef-

207 fects in diabetic cardiomyopathy [80] and its ability to pre-

208 condition against cerebral ischemia [81]. As quercetin and

209 taxifolin are relatively minor components of silymarin, the

210 importance of their contribution to preconditioning by silybin

211 remains unclear. Furthermore, unlike resveratrol, whose

212 cardioprotective effects are known to be mediated via the

213 activation of SIRT1 and inhibition of cyclo-oxygenase (COX)

214 [64-66], it is not quite so clear-cut as to which of silymarin's

215 components could be responsible for the protective effects of

216 the extract. If anything, the situation in the field is somewhat

217 similar to that of cardioprotection by garlic, where it is

218 thought that the effect is attained by a mix of anti-oxidant

219 activity, COX inhibition and potentiation of H2S signalling

220 [67-72], but definitive proof is lacking, probably because of

221 the complexity of the biochemical cocktail.

222 There are additional studies evidencing silymarin's action

223 as a cardioprotective agent. The first was a simple IR study in

224 rat hearts, published in 1992 that found silybin to cause a

225 modest reduction in infarct size [73]. This study went largely

226 unnoticed, probably due to language barriers, and the next

227 study on the cardioprotective properties of silybin was not

228 published until fifteen years later. In this study Rao and

Viswanath [74] used in vivo rat infarct models to test the 229 effect of week-long feeding of various doses of silymarin on IR 230 injury, using both biochemical markers and infarct size mea- 231 surements. Silymarin feeding was found to cause a dose de- 232 pendent decrease in infarct size, lipid peroxide level and 233 glutamate oxaloacetate transaminase levels whilst increasing 234 glutathione transferase and catalase levels [74]. Silymarin was 235 also found to normalise levels of biomarkers of Adriamycin 236 cardiotoxicity, which are elevated by this highly toxic chemo- 237 therapeutic agent [75]. Since whole silymarin was used in 238 both studies, it is unclear which of its components was re- 239 sponsible for the cardioprotective effect [74,75]. As the dam- 240 age associated with the use of Adriamycin is thought to be 241 caused by free radicals, the authors of the study suggested 242 that the antioxidant properties of silymarin are likely re- 243 sponsible for its cardioprotective effect in this instance, 244 although reduction of mPTP sensitivity to free radicals via 245 the various preconditioning pathways remains an equally 246 valid, if untested, possibility [75]. In an investigation of pre- 247 conditioning by quercetin in an open chest rat model, Jin et al. 248 [76] found a reduction in the levels of markers of inflamma- 249 tion and improved functional recovery in quercetin treated 250 rats, but no reduction in infarct size when rats were treated 251 with 1 mg/kg quercetin prior to ischemia. The study did not 252 rule out a reduction in infarct size at higher concentrations 253 of quercetin. In fact, a simulated ischemia study in cardio- 254 myocytes later found that long term quercetin treatment 255 protected the cells against simulated IR injury [77]. It is 256 noteworthy that inhibition of PKCs was found to prevent 257 cardioprotection in this study, as this suggests that quercetin 258 mediated cardioprotection is true preconditioning, rather 259 than simple reduction in the ROS levels due to its antioxidant q7

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activity. Likewise, Wang et al. [78] found an increase in Akt and mTOR phosphorylation in rat models of cerebral ischemia treated with silybin. As Akt phosphorylation should eventually lead to PKCs phosphorylation this re-enforces the hypothesis that the components of silymarin act via the canonical preconditioning pathways. It is also possible that the protective effect is due to reduction of inflammatory damage to the tissue, as a reduction in NF-kB levels was seen in models of stroke [78,79]. Conversely, a number of the studies mentioned above found increased levels of antioxi-dant levels and decreased levels of oxidative stress upon treatment with silymarin, silybin or quercetin [72,80,81]. At the same time studies of quercetin preconditioning have pointed to reduced inflammation [76], inhibition of MMP [82] and reduced oxidative stress [83], but as none of these studies compared quercetin to well established methods of preconditioning, it is unclear whether these are effects specifically caused by quercetin or are a general trait of preconditioning. There have been no studies to date testing preconditioning or cardioprotection by purified isosilybin, silychristin or silydianin. However, since silychristin and silydianin have been found to offer at least partial protection to cardiomyocytes against an-thracycline toxicity and are generally reported to be antioxi-dants, it is not unlikely that they may also offer protection against IR injury [84-87].

3. Actions of silymarin that may be related to preconditioning

Apart from the finding that PKCs activity is required for quercetin preconditioning, no study has examined the role of other preconditioning pathways in cardioprotection by this compound [77]. On the other hand, a wealth of information about silymarin's components, has been gleaned from biochemical and molecular studies. These studies have shown several promising directions for unravelling silymarin's mechanisms in preconditioning.

There is a strong argument for the involvement of silymarin in the final steps of the preconditioning pathway. Its constituent flavonolignans and flavonols, with the exception of silydianin and silychristin under certain conditions, have been found to act as antioxidants and radical scavengers, which, as reduction in free radical concentrations increases mPTP threshold, may go some way towards explaining their protective effects [87-89]. This is not, however, the whole story, as the concentrations at which these compounds begin to act as antioxidants were quite high and show a dependence on the system used to investigate them [85,87,90]. In the study by Gabrielova et al. [85], DHSB (dehydrosilybin) was found to inhibit free radical formation between 10 and 100 |jM in cell based and cell free systems, whilst effectively preventing free radical formation at sub-micromolar concentrations in isolated mitochondria. The authors suggest that as well as acting as a free radical scavenger, DHSB acts as an uncoupler, hence preventing mitochondrial free radical generation [85]. Similarly, silydianin and silychristin were found to be mildly pro-oxidative in models of copper induced LDL oxidation [87], but were found to act as antioxidants in a doxorubicin-iron based models of oxidative damage [90], as well as being capable scavengers of phenylglyoxyl ketyl radicals [91]. Curiously, silybin also proved to be a scavenger

in this model. Dorta et al. [86] investigated the antioxidant 320 properties of quercetin and taxifolin. Aside from making 321 certain structural deductions, they found quercetin to be both 322 the stronger anti-oxidant and a respiratory chain uncoupler 323 [86]. It should be noted, that working on isolated mitochon- 324 dria, similar to Gabrielova et al. [85], this group found that q8 DHSB has antioxidant activities at submicromolar concentra- 326 tions — indirect evidence that the substances interact directly 327 with the biological system. Silydianin and silychristin, on the 328 other hand, may act as pro-oxidants under certain conditions, 329 but this is unlikely to have a significant effect on the overall 330 properties of silymarin in all systems [87]. The dependence of 331 the components' activity on concentration and model system 332 is further highlighted by additional examples. Certain studies 333 have found that at 20 ^M and 50 ^M, quercetin can preserve 334 cell viability following treatment with H2O2 [92,93]. Whilst 335 another study found that at 50 |jM quercetin can increase 336 mPTP opening [94]. The possibility that high concentrations of 337 quercetin increase mPTP opening is supported by ANT in- 338 hibition by the compound [94]. There is also some evidence 339 that DHSB interacts with ANT and may be transported by the 340 ion carrier, although it is not clear whether the direct effect of 341 this interaction would be to raise or lower the threshold of 342 mPTP [85]. As such, there is evidence that the components of 343 silymarin may exert a concentration dependent effect on mPTP 344 opening, which may either assist or inhibit preconditioning. 345 Upstream of the mitochondria silymarin's components has 346 been shown to interact with a number of cellular pathways 347 and the receptors. Aside from direct evidence that silybin B 348 (but not the A stereoisomer), taxifolin and quercetin activate 349 ERs [8], which, in and of itself, should be enough to cause 350 preconditioning [62,63,95-98], there are also hints that these 351 compounds may modulate other receptors. Adenosine and 352 Ouabain receptors, for example, both bind adenosine, mark- 353 ing them as potential candidates for interaction with DHSB 354 and quercetin, as the former was found to bind the nucleotide 355 binding domain of proteins [99] and the latter appears to be 356 an inhibitor of ATPases and ANT [100]. There was also a study 357 by Angelone et al. [101] that suggests that quercetin's ino- 358 tropic and lusitropic effects are directly dependent on adren- 359 ergic receptors. This is further supported by a study by Zhou et 360 al. [102], which found silybin to reverse isoproterenol induced 361 damage in a model of cardiac hypertrophy. As adrenergic 362 stimulants such as isoproterenol, which in the long term 363 induce deleterious effects such as hypertrophy, cause ische- 364 mic preconditioning in the short term, it is possible that an 365 agent that prevents AR stimulant induced hypertrophy would 366 also antagonise the mechanisms leading to cardiac precondi- 367 tioning [102]. On the other hand, the modified coupling to G 368 proteins by long term isoproterenol exposure may make the 369 model inapplicable for preconditioning. There is also the 370 possibility that silybin also inhibits ARp3, which would lead to 371 preconditioning. It should be noted that the study by Zhou et 372 al. [102] did not elucidate whether silybin directly interacts 373 with ARps or whether more downstream components of the 374 AR signalling pathway were affected. This raises the possibil- 375 ity that this pathway could be targeted by silymarin via the 376 adenosine binding motifs of AC or PKA. In addition, at around 377 10 |jM, quercetin was found to inhibit phosphodiesterase 4 378 (PDE4) [103], which is one of the PDE isoforms responsible for 379 negative feedback in PKA/cAMP signalling. Curiously inhibition 380

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381 of PDE by silymarin components is not a recent discovery

382 [104,105]. The inhibition of PDE4 and resulting increase in

383 cAMP levels could also explain the result seen by Angelone et

384 al. [101 ]. This may also shed light on the increased cAMP levels

385 and vasodilation observed in HUVECs (human umbilical vein

386 endothelial cells) and aortic rings respectively [106]. A com-

387 prehensive study by Ai et al. [107] on the effects of silybin in

388 cardiac hypertrophy found that the compound inhibited the

389 phenomenon by blocking EGFR phosphorylation, as well as

390 that of the components of the downstream Erk and Akt path-

391 ways. GSK3(3 phosphorylation was decreased when mice were q9 subjected to aortic banding and increased in sham operated

393 mice. Whilst this effect was recapitulated by the Ang II inhibitor

394 SU1428, the deactivation of Akt and Erk pathways is the op-

395 posite effect to that observed in preconditioning. Although the

396 experimental model used here was not one of IR injury and

397 therefore might not accurately reflect silybin's effects in IR

398 injury, the study did suggest that silybin is not the component

399 of silymarin responsible for preconditioning. Incidentally, this

400 was not the only study to show reduced Akt phosphorylation

401 and cell growth upon treatment with silybin. In a study even

402 further removed from our ideal model of IR injury, Singh et al.

403 [108] showed reduced proliferation of HUVEC cells and

404 reduced phosphorylation of Akt at Ser 473 and Thr 308 fol-

405 lowing 48 h treatment with 10-50 mg/ml silybin. Similarly,

406 Deep et al. [109,110] found that in culture models of prostate

407 cancer isosilybin A decreases Akt phosphorylation, whilst

408 isosilybin B increases it. Curiously in this case upregulation of

409 Akt phosphorylation resulted in a reduction in androgen

410 expression, whilst downregulation increased stimulated apo-

411 ptosis. Likewise, both topical pretreatment with, and feeding of

412 silybin to, hairless mice prior to UV (ultraviolet) carcinogenesis

413 slowed tumour growth, reducing levels of phosphorylated Akt,

414 Erk and Jnk [111]. In another in vitro tumour model, silybin was

415 found to inhibit the activity of hypoxia induced factor 1 (HIF-1)

416 and the p70S6K/mT0R pathway, whilst paradoxically activat-

417 ing Akt [112]. Curiously, the same study found that hypoxia

418 induced vascular endothelial growth factor (VEGF) release was

419 inhibited by silybin. This echoes the investigation of Deep et al.

420 [113], where the growth of prostate cancer xenografts was

421 slowed by silybin and isosilybin with a decrease in angiogenic

422 markers such as VEGF. Whilst these cancer models are far

423 removed from our ideal models of IR injury, they nevertheless

424 demonstrate that the mechanism of action of silybin differs

425 somewhat from what one would expect of a pharmacological

426 preconditioning agent. Another component of silymarin,

427 quercetin was found to preserve cell viability following treat-

428 ment with H202, although studies reached opposite conclu-

429 sions as to the activation of Erk1/2 during these effects [92,93].

430 This could be a result of the differences in concentrations of

431 quercetin applied and cell lines used; Ishikawa and Kitamura

432 [92] used 20 |jM quercetin, whilst Youl et al. [93] showed

433 increased Erk phosphorylation at 50 |jM and above. Addition-

434 ally quercetin reduced expression of Erb2 and 3 (EGFR family)

435 in a HT-29 in vitro model of colon cancer. This was accom-

436 panied by a reduction in the activation of the IP3K/Akt path-

437 way. It is unclear, whether findings in this model would

438 translate to a reduction in EGFR expression in cardiomyocytes

439 [114]. Furthermore, in vitro taxifolin was found to alter gene

440 expression from the antioxidant response element (ARE),

441 including the down-regulation of EGF and fibroblast growth

factor (FGF), although the exact consequences of this for IR 442

preconditioning are difficult to interpret [3]. Thus, it is difficult 443

to reconcile the models showing down-regulation of Akt sig- 444

nalling with cardioprotective effects of silybin seen by Chen et 445

al. [73] and the neuroprotective effects seen by Hou et al. [79] 446

and Wang et al. [96] (especially as the last study specifically 447

observed up-regulation of Akt signalling). In fact, given the 448

weight of evidence showing silybin to be an antiproliferative 449 agent which reduces Akt activation, we can only surmise that 450

highly nuanced differences in pathway coupling, specific to the 451

different models, are responsible for the effects observed. It is 452

also possible that prior to ischemia, silybin does in fact down- 453

regulate Akt signalling in the models used by Hou et al. and 454

Wang et al. [78,79], with a rebound in Akt activation upon 455

occlusion. Another, somewhat unrelated, possibility men- 456

tioned above is that inhibition of the cyclooxygenase path- 457

way by silybin and taxifolin would reduce the damage caused 458

by inflammation [115,116]. In fact silybin and silydianin have 459

been found to reduce production of hydroxyl radicals by poly- 460 morphonuclear neutrophils (PMNs) [117]. A further study by 461

Zielinska-Przyjemska et al. [118] suggests that, at least in the 462

case of silydianin, this is due to increased apoptosis of PMNs 463

due to the induction of caspase 3. This fits in with the sugges- 464

tion that silymarin prevents IR injury, at least in part, by 465

reducing inflammation [78,79]. 466 469

Compound Effect Model(s) References 471

Silybin Cardioprotection Aortic banding [75] 473

mouse model 474

Preconditioning Open chest Sprague- [73] 477

Dawley rats, 478

Open chest Wistar [74] 481

rat model 482

Long-Evans rats [79] 483

ischemic stroke 486

BLAB/C mouse [119] 499

ischemic tourniquet- 490

gastrocnemius 491

Sprague Dawley rats [78] 494

ischemic stroke 493

Antioxidant In vitro [88] 408

assays (silybin 499

dihemisuccinate) 300

Copper induced [87] 303

oxidation model 304

(silybin, silydianin, 303

silychristin) 306

PDE inhibitor In vitro assays, [104] 300

partially purified 311

beef heart PDEs 312

HIF inhibitor HeLa and HEP-3 cell [111] 317

model 316

GLUT inhibitor 3T3-L1 and CHO cell [120] 329

models 320

ER stimulation T47D.Luc cell culture [8] 326

luciferase reporter 324

model 323

Hypoxia induced HeLa and HEP-3 cell [112] 321

VEGF release model 329

Anti-inflammatory Mouse tail-flick and [121] 333

writhing model 334

Anti-proliferative UV irradiated SKH [111] 340

activity hairless mice 338

Akt modulation HeLa and HEP3B [112] 3423

culture model 343

(stimulation) 344

HUVEC (inhibition) [108] 334

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(continued)

Compound Effect Model(s) References

UV irradiated SKH [111]

hairless mouse

(inhibition)

Wistar rat IR model [74]

(inhibition)

Antiviral (HCV) Cell culture and [122]

ex vivo models.

Dehydrosilybin Mitochondrial Rat myocyte, [85]

Uncoupling mitochondria and

in vitro models

Antioxidant Ex vivo and [123]

activity microsomal assays

Review [124]

GLUT inhibition 3T3-L1 and CHO [120]

cell models

Topoisomerase EPI and FIB cell [125]

inhibition nuclear extracts

In vitro [126]

topoisomerase

MDR inhibition T5-HeLa membrane [100]

vesicle preparations

Review [127]

Sensitive and [128]

resistant cancer

cell lines

MRP1-BHK1 cell [57]

lines and membrane

vesicle models

Plasmodium [129]

falciparum strains

Taxifolin Anti-inflammatory Rat paw oedema [99]

EGF/FGF HCT 116 cell model [3]

downregulation

ER stimulation T47D.Luc cell culture [8]

luciferase reporter

Quercetin Cardioprotection Neonatal rat [77]

myocytes, simulated

ischemia model

Open chest Sprague- [76]

Dawley rat model

Simulated ischemia, [130]

embryonic rat

ventricular cells

Preconditioning Ischemic kidney [83]

Sprague Dawley

rat model

Ischemic stroke [82]

C57BL6 mouse

Vasodilation HUVECs & aortic [106]

MPTP opening Rat kidney cortex [93]

mitochondria

IP3K inhibition X-ray crystallography [131]

ERK modulation Aortic banding mouse [111]

model. (Inhibition)

UV irradiated SKH [107]

hairless mouse

(Inhibition)

SM43 rat mesangial [94]

cell culture model

(Inhibition)

INS-1 cell culture [93]

and rat Langerhans

islet preparation

(Activation)

(continued) 692

Compound Effect Model(s) References 695

EGFR HT-29 colon cancer [114] 68759

downregulation culture model 6868

ER stimulation T47D.Luc cell culture [8] 6941

luciferase reporter 692

model 693

ANT inhibition Rat kidney cortex [101] q11

mitochondria 697

MDR inhibition T5-HeLa membrane [100] 70 IB

vesicle preparations 702

Review [127] 7065

Ca2+ channel NG108-15 cell model [133] 7000

modulation 709

PL-PK inhibition Mouse Brain Ca PL-PK [134] 713

Silychristin Pro-oxidant Ex vivo LDL oxidation [87] 719

assay. 718

Antioxidant DPTT/DPTA ex vivo [91] 723

model. 722

Rat mitochondria/ [90] 7285

microsome models, 726

DPPH ex vivo assays. 727

DPTT/ORAC/HORAC/ [135] 7330

TEAC/TAC ex vivo 731

model. 732

Huh7.5.1 cell model. [136] 7356

Chemoprotection Rat cardiomyocyte [84] 73408

model. 739

Silydianin Pro-oxidant Ex vivo LDL oxidation [87] 74352

assay. 744

Antioxidant DPTT/ORAC/HORAC/ [135] 75470

TEAC/TAC ex vivo 748

model. 749

Rat mitochondria/ [90] 7552

microsome models, 753

DPPH ex vivo assays. 754

DPTT/DPTA ex vivo [91] 7597

model. 758

Huh7.5.1 cell model. [136] 7621

Anti-inflammatory Isolated human PMNs. [118] 76548

Chemiluminescence 766

and O2^-formation. 767

Isolated human PMNs. [117] 7701

Huh7.5.1 cell model. [136] 7734

ppary 3 T3-L1 cell model. [137] 7768

downregulation 777

Chemoprotection Rat cardiomyocyte [84] 7802

model. 781

Isosilybin ppary activation HEK-293 luciferase [138] 7855

reporter assays 786

Antioxidant ORAC/HORAC/TEAC/ [135] 789

TAC ex vivo model. 790

Huh7.5.1 cell model. [136] 7934

Anti-inflammatory Human peripheral [136] 7969

blood mononuclear 797

cell model. 798

Antiviral (HCV) Huh7.5.1 cell model. [136] 8021

Cell culture and ex [122] 8046

vivo models. 805

Increased Akt Isosilybin B — [110] 818

Phosphorylation prostate cancer 800

cell culture models. 811

Decreased Akt Isosilybin A — prostate [109] 8199

phosphorylation cancer cell culture 815

models. 88122801

4. Future directions

The hypothesis that silymarin protects tissue against ische- 823 mia, as highlighted in this text, is supported by a considerable 824 collection of evidence. However, neither the role that each 825

ARTICLE IN PRESS

A. Zholobenko, M. Modriansky / Fitoterapia xxx (2014) xxx-xxx

826 component of silymarin plays in the process, nor the timing is

827 entirely clear. To compound this, prevention of IR injury by

828 purified isosilybin, silychristin and silydianin has not yet been

829 tested.

830 The component responsible could be elucidated by large

831 scale cell culture based simulated ischemia studies, or better

832 small animal studies, designed to compare each of the com-

833 ponents side by side, along with the whole extract. At the

834 same time, the mechanisms by which silymarin's components

835 cause cardioprotection should be cross-examined by observ-

836 ing the effects of inhibitors of the classic pathways of pre-

837 conditioning on silymarin induced cardioprotection. Whilst

838 cell culture models may be suitable for this type of study, they

839 possess several important disadvantages, including differ-

840 ences in metabolism and perfusion and the impossibility of

841 examining the effect of alterations in the immune system by

842 the formulation in question on IR injury. In addition, trans-

843 genic mouse models, such as those byJuhaszova et al. [27] or

844 Gomez et al. [28] would be very useful in elucidating whether

845 the effects of silymarin's components are due to true pre-

846 conditioning or antioxidant/radical scavenging effects. As

847 silymarin's components have been shown to affect markers

848 of inflammation, the use of a model that does not account for

849 the immune system when studying cardioprotection by

850 silymarin may leave the investigators with an incomplete

851 set of conclusions.

852 As there are numerous studies showing that silymarin is

853 generally safe when studying diseases of the liver [2,139],

854 clinical studies of silymarin or its components in cardiopro-

855 tection are not beyond the realms of possibility. It is ques-

856 tionable, however, whether a clinical trial that is capable of

857 advancing the field can be designed with the current under-

858 standing of the compounds in question.

859 5. Conclusion

860 Overall, the evidence seems to indicate that the constit-

861 uents of silymarin reduce the activity of both the Erk/MEK

862 and IP3K/Akt pro-survival pathways, whose activation is

863 central to ACh, bradykinin, and ouabain preconditioning. At

864 the same time, it makes sense that stimulation of oestrogen

865 receptors, inhibition of MMPs, PDEs, and mitochondrial ROS

866 generation by silymarin's components should facilitate pre-

867 conditioning. Furthermore the antiinflammatory properties of

868 certain components may also have a role to play in protecting

869 tissue from IR.

870 As we have highlighted in this review, deciphering the

871 mechanisms of action of silymarin in preconditioning is a

872 fairly involved affair. Summary of available literature shows

873 that silymarin and its components do influence signalling

874 pathways, which are involved in preconditioning. Whilst the

875 major component of silymarin, silybin, is the usual suspect

876 when it comes to these salutatory properties, other, minor

877 components of the extract have also been shown to possess

878 an important cardioprotective activity. With this in mind, it is

879 our belief that the individual components of silymarin con-

880 stitute a family of compounds worth investigating in relation

881 to ischemia reperfusion. Mechanisms of their action, if prop-

882 erly understood, promise a relatively inexpensive way of

883 broadening the spectrum of pharmacological agents available

884 for treatment of ischemia reperfusion injury.

Conflicts of interest 885

The authors have no conflicts of interest to declare. 886

Acknowledgements

This work was supported by grants P301/11/0662 and 888 CZ.1.07/2.3.00/30.0041. We thank Professor Simanek for the q12 advice and comments regarding the manuscript. 890

References

[1] Kren V, Walterova D. Silybin and silymarin — new effects and 892 applications. Biomed Pap Jul 1 2005;149(1):29-41. 893

[2] Saller R, Brignoli R, Melzer J, Meier R. An updated systematic review 894 with meta-analysis for the clinical evidence of silymarin. Forsch 895 Komplementärmed Res Complement Med 2008;15(1):9-20. 896

[3] Lee SB, Cha KH, Selenge D, Solongo A, Nho CW. The chemopreventive 897 effect of taxifolin is exerted through ARE-dependent gene regulation. 898 Biol Pharm Bull 2007;30(6):1074-9. 899

[4] Martin RJ, Lauren DR, Smith WA, Jensen DJ, Deo B, Douglas JA. Factors 900 influencing silymarin content and composition in variegated thistle 901 (Silybum marianum). N ZJ Crop Hortic Sci 2006;34(3):239-45. 902

[5] Abenavoli L, Capasso R, Milic N, Capasso F. Milk thistle in liver 903 diseases: past, present, future. Phytother Res 2010;24(10):1423-32. 904

[6] Morazzoni P, Bombardelli E. Silybum marianum (Carduus marianus). 905 Fitoterapia 1995;66(1):3-42. 906

[7] Jancovä P, Siller M, Anzenbacherovä E, Kren V, Anzenbacher P, 907 Simänek V. Evidence for differences in regioselective and stereo- 908 selective glucuronidation of silybin diastereomers from milk thistle 909 (Silybum marianum) by human UDP-glucuronosyltransferases. 910 Xenobiotica Sep 2011;41(9):743 -51. 911

[8] Pliskovä M, VondräcekJ, Kren V, Gazäk R, Sedmera P, Walterovä D, et al. 912 Effects of silymarin flavonolignans and synthetic silybin derivatives on 913 estrogen and aryl hydrocarbon receptor activation. Toxicology Nov 5 914 2005;215(1-2):80-9. 915

[9] Yellon DM, Downey JM. Preconditioning the myocardium: from cellular 916 physiology to clinical cardiology. Physiol Rev Oct 1 2003;83(4):1113-51. 917

10] Kloner rA. Clinical application of remote ischemic preconditioning. 918 Circulation Feb 17 2009;119(6):776-8. 919

11] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a 920 delay of lethal cell injury in ischemic myocardium. Circulation Nov 1 921 1986;74(5):1124-36. 922

12] Reimer KA, Jennings RB. Verapamil in two reperfusion models of 923 myocardial infarction. Temporary protection of severely ischemic 924 myocardium without limitation of ultimate infarct size. Lab Invest Dec 925 1984;51(6):655-66. 926

13] Halestrap AP. Mitochondria and reperfusion injury of the heart—a 927 holey death but not beyond salvation. J Bioenerg Biomembr Apr 1 928 2009;41(2):113-21. 929

14] Javadov S, Karmazyn M, Escobales N. Mitochondrial permeability 930 transition pore opening as a promising therapeutic target in cardiac 931 diseases. J Pharmacol Exp Ther Sep 1 2009;330(3):670-8. 932

15] Miyawaki H, AshrafM. Isoproterenol mimics calcium preconditioning- 933 induced protection against ischemia. Am J Physiol Feb 1997;272(2 Pt 934 2):H927-36. 935

16] Li S-Z, Wu F, Wang B, Wei G-Z, Jin Z-X, Zang Y-M, et al. Role of reverse 936 mode Na+/Ca2+ exchanger in the cardioprotection of metabolic 937 inhibition preconditioning in rat ventricular myocytes. Eur J Pharmacol 938 Apr 30 2007;561(1-3):14-22. 939

17] Bouwman RA, Salic K, Padding FG, Eringa EC, van Beek-Harmsen BJ, 940 Matsuda T, et al. Cardioprotection via activation of protein kinase C-8 941 depends on modulation of the reverse mode of the Na+/Ca2+ 942 exchanger. Circulation Jul 4 2006;114(1 Suppl.):I-226-32. 943

18] Rasola A, Sciacovelli M, Pantic B, Bernardi P. Signal transduction to the 944 permeability transition pore. FEBS Lett May 17 2010;584(10):1989-96. 945

19] Gross ER, Hsu AK, Gross GJ. Delayed cardioprotection afforded by the 946 glycogen synthase kinase 3 inhibitor SB-216763 occurs via a KATP- 947 and MPTP-dependent mechanism at reperfusion. Am J Physiol Heart 948 Circ Physiol Mar 1 2008;294(3):H1497-500. 949

[20] Pagel PS, Krolikowski JG, Neff DA, Weihrauch D, Bienengraeber M, 950

Kersten JR, et al. Inhibition of glycogen synthase kinase enhances 951

isoflurane-induced protection against myocardial infarction during 952

early reperfusion in vivo. Anesth Analg May 1 2006;102(5):1348-54. 953

ARTICLE IN PRESS

A. Zholobenko, M. Modriansky / Fitoterapia xxx (2014) xxx-xxx 9

954 [21] Park S-S, Zhao H, Mueller RA, Xu Z. Bradykinin prevents reperfusion [43] Lang X-E, Wang X, Zhang K-R Lv J-Y, Jin J-H, Li Q-S. Isoflurane 1034

955 injury by targeting mitochondrial permeability transition pore through preconditioning confers cardioprotection by activation of ALDH2. In: 1035

956 glycogen synthase kinase 3ß. J Mol Cell Cardiol May 2006;40(5):708-16. Singh SR, editor. PLoS One Feb 28 2013;8(2):e52469. 1036

957 [22] WoodfieldK, Ruck A, Brdiczka D, Halestrap AP. Direct demonstration of [44] Gong D, Zhang H, Hu S. Mitochondrial aldehyde dehydrogenase 2 1037

958 a specific interaction between cyclophilin-D and the adenine nucleo- activation and cardioprotection. J Mol Cell Cardiol Feb 2013;55:58-63. 1038

959 tide translocase confirms their role in the mitochondrial permeability [45] Tian J, Liu J, Garlid KD, Shapiro JI, Xie Z. Involvement of mitogen- 1039

960 transition. Biochem J Dec 1 1998;336(Pt 2):287-90. activated protein kinases and reactive oxygen species in the inotropic 1040

961 [23] Scherer B, Grebe K, Riccio P, Klingenberg M. The new atractyloside action of ouabain on cardiac myocytes. A potential role for mitochon- 1041

962 type compound as a high affinity ligand to the adenine nucleotide drial KATP channels. Mol Cell Biochem Jan 1 2003;242(1-2):181-7. 1042

963 carrier. FEBS Lett Apr 1 1973;31(1):15-9. [46] Morisco C, Zebrowski D, Condorelli G, Tsichlis P, Vatner SF, Sadoshima J. 1043

964 [24] Nishihara M, Miura T, Miki T, Tanno M, Yano T, Naitoh K, et al. The Akt-glycogen synthase kinase 3ß pathway regulates transcription of 1044

965 Modulation of the mitochondrial permeability transition pore com- atrial natriuretic factor induced by ß-adrenergic receptor stimulation in 1045

966 plex in GSK-3ß-mediated myocardial protection. J Mol Cell Cardiol cardiac myocytes. J Biol Chem May 12 2000;275(19):14466-75. 1046

967 Nov 2007;43(5):564-70. [47] Robinet A, Hoizey G, Millart H. PI 3-kinase, protein kinase C, and 1047

968 [25] Martel C, Huynh LH, Garnier A, Ventura-Clapier R Brenner C. Inhibition protein kinase A are involved in the trigger phase of ß1-adrenergic 1048

969 of the mitochondrial permeability transition for cytoprotection: direct preconditioning. Cardiovasc Res Jun 1 2005;66(3):530-42. 1049

970 versus indirect mechanisms. Biochem Res Int May 22 2012 [[Internet], [48] Peart J, Headrick JP. Adenosine-mediated early preconditioning in 1050

971 [cited 2013 Jul 8]. Available from: http://www.hindawi.com/journals/ mouse: protective signaling and concentration dependent effects. 1051 q13 bcri/2012/213403/abs/]. Cardiovasc Res Jun 1 2003;58(3):589-601. 1052

973 [26] Tong H, Imahashi K, Steenbergen C, Murphy E. Phosphorylation of [49] Krieg T, Philipp S, Cui L, Dostmann WR Downey JM, Cohen MV. 1053

974 glycogen synthase kinase-3ß during preconditioning through a Peptide blockers of PKG inhibit ROS generation by acetylcholine and 1054

975 phosphatidylinositol-3-kinase-dependent pathway is cardioprotective. bradykinin in cardiomyocytes but fail to block protection in the whole 1055

976 Circ Res Mar 8 2002;90(4):377-9. heart. Am J Physiol Heart Circ Physiol Apr 2005;288(4):H1976-81. 1056

977 [27] Juhaszova M, Zorov DB, Kim S-H, Pepe S, Fu Q, Fishbein KW, et al. [50] Oldenburg O, Qin Q, Krieg T, Yang X-M, Philipp S, Critz SD, et al. 1057

978 Glycogen synthase kinase-3ß mediates convergence of protection Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, 1058

979 signaling to inhibit the mitochondrial permeability transition pore. J and mitoKATP channel opening and leads to cardioprotection. Am J 1059

980 Clin Invest Jun 1 2004;113(11):1535-49. Physiol Heart Circ Physiol Jan 1 2004;286(1):H468-76. 1060

981 [28] Gomez L, Paillard M, Thibault H, Derumeaux G, Ovize M. Inhibition of [51] Schultz JE, Rose E, Yao Z, Gross GJ. Evidence for involvement of opioid 1061

982 GSK3ß by postconditioning is required to prevent opening of the receptors in ischemic preconditioning in rat hearts. Am J Physiol Heart 1062

983 mitochondrial permeability transition pore during reperfusion. Circu- Circ Physiol May 1 1995;268(5):H2157-61. 1063

984 lation May 27 2008;117(21):2761-8. [52] Salie R Moolman JA, Lochner A. The role of ß-adrenergic receptors in 1064

985 [29] Gross ER, Hsu AK, Gross GJ. Opioid-induced cardioprotection occurs the cardioprotective effects ofbeta-preconditioning (ßPC). Cardiovasc 1065

986 via glycogen synthase kinase ß inhibition during reperfusion in intact Drugs TherFeb 1 2011;25(1):31-46. 1066

987 rat hearts. Circ Res Apr 16 2004;94(7):960-6. [53] Takano H, Bolli R Black RG, Kodani E, Tang X-L, Yang Z, et al. A1 or 1067

988 [30] Nishino Y, Webb IG, Davidson SM, Ahmed AI, Clark JE, Jacquet S, et al. A3 adenosine receptors induce late preconditioning against infarction 1068

989 Glycogen synthase kinase-3 inactivation is not required for ischemic in conscious rabbits by different mechanisms. Circ Res Mar 16 1069

990 preconditioning or postconditioning in the mouse. Circ Res Aug 1 2001;88(5):520-8. 1070

991 2008;103(3):307-14. [54] Yang X-M, Krieg T, Cui L, Downey JM, Cohen MV.NECA and bradykinin 1071

992 [31] Rahman S, Li J, Bopassa JC, Umar S, Iorga A, Partownavid P, et al. at reperfusion reduce infarction in rabbit hearts by signaling through 1072

993 Phosphorylation of GSK-3? Mediates intralipid-induced cardio- PI3K, ERK, and NO. J Mol Cell Cardiol Mar 2004;36(3):411-21. 1073

994 protection against ischemia/reperfusion injury. Anesthesiology Aug [55] Frances C, Nazeyrollas P, Prevost A, Moreau F, Pisani J, Davani S, et al. 1074

995 2011;115(2):242-53. Role of beta 1-and beta 2-adrenoceptor subtypes in preconditioning 1075

996 [32] Garlid KD, Costa ADT, Quinlan CL, Pierre SV, Dos Santos P. against myocardial dysfunction after ischemia and reperfusion. J 1076

997 Cardioprotective signaling to mitochondria. J Mol Cell Cardiol Jun Cardiovasc Pharmacol Mar 2003;41(3):396-405. 1077

998 2009;46(6):858-66. [56] Hoffmann C, Leitz MR, Oberdorf-Maass S, Lohse MJ, Klotz K-N. 1078

999 [33] Dorn GW, Souroujon MC, Liron T, Chen C-H, Gray MO, Zhou HZ, et al. Comparative pharmacology of human ß-adrenergic receptor subtypes- 1079

1000 Sustained in vivo cardiac protection by a rationally designed peptide characterization of stably transfected receptors in CHO cells. Naunyn 1080

1001 that causes e protein kinase C translocation. Proc Natl Acad Sci Oct 26 Schmiedebergs Arch Pharmacol Feb 1 2004;369(2):151-9. 1081

1002 1999;96(22):12798-803. [57] Yao Z, Gross GJ. Role of nitric oxide, muscarinic receptors, and the 1082

1003 [34] Costa ADT, Jakob R Costa CL, Andrukhiv K, West IC, Garlid KD. The ATP-sensitive K + channel in mediating the effects of acetylcholine to 1083

1004 mechanism by which the mitochondrial ATP-sensitive K + channel mimic preconditioning in dogs. Circ Res Dec 1993;73(6):1193-201. 1084

1005 opening and H2O2 inhibit the mitochondrial permeability transition. J [58] Qian YZ, Levasseur JE, Yoshida K, Kukreja RC. KATP channels in rat 1085

1006 Biol Chem Jul 28 2006;281(30):20801-8. heart: blockade of ischemic and acetylcholine-mediated precondi- 1086

1007 [35] Budas GR, Churchill EN, Disatnik M-H, Sun L, Mochly-Rosen D. tioning by glibenclamide. Am J Physiol Heart Circ Physiol Jul 1 1087

1008 Mitochondrial import of PKC is mediated by HSP90: a role in cardio- 1996;271(1):H23-8. 1088

1009 protection from ischaemia and reperfusion injury. Cardiovasc Res Jun [59] Vegh A, Papp JG, Szekeres L, Parratt JR. Prevention by an inhibitor of 1089

1010 16 2010;88(1):83-92. the l-arginine-nitric oxide pathway of the antiarrhythmic effects of 1090

1011 [36] Lee JE, Bokoch G, Liang BT. A novel cardioprotective role of RhoA: new bradykinin in anaesthetized dogs. Br J Pharmacol 1993;110(1):18-9. 1091

1012 signaling mechanism for adenosine. FASEBJ Sep 1 2001;15(11):1886-94. [60] Goto M, Liu Y, Yang X-M, Ardell JL, Cohen MV, Downey JM. Role of 1092

1013 [37] Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the bradykinin in protection of ischemic preconditioning in rabbit hearts. 1093

1014 phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem Circ Res Sep 1 1995;77(3):611-21. 1094

1015 Aug 7 1998;273(32):19929-32. [61] Schultz JEJ, Hsu AK, Gross GJ. Morphine mimics the cardioprotective 1095

1016 [38] Morgan EE, Li Z, Stebal C, Belliard A, Tennyson G, Salari B, et al. effect of ischemic preconditioning via a glibenclamide-sensitive 1096

1017 Preconditioning by subinotropic doses of ouabain in the Langendorff mechanism in the rat heart. Circ Res Jun 1 1996;78(6):1100-4. 1097

1018 perfused rabbit heart. J Cardiovasc Pharmacol Mar 2010;55(3):234-9. [62] Nikolic I, Liu D, Bell JA, Collins J, Steenbergen C, Murphy E. Treatment 1098

1019 [39] Pierre SV, Yang C, Yuan Z, Seminerio J, Mouas C, Garlid KD, et al. with an estrogen receptor-beta-selective agonist is cardioprotective. J 1099

1020 Ouabain triggers preconditioning through activation of the Na+, Mol Cell Cardiol Apr 2007;42(4):769-80. 1100

1021 K + -ATPase signaling cascade in rat hearts. Cardiovasc Res Feb 1 [63] Weil BR, Manukyan MC, Herrmann JL, Wang Y, Abarbanell AM, 1101

1022 2007;73(3):488-96. Poynter JA, et al. Signaling via GPR30 protects the myocardium from 1102

1023 [40] Bugge E, Ytrehus K. Inhibition of sodium-hydrogen exchange reduces ischemia/reperfusion injury. Surgery Aug 2010;148(2):436-43. 1103

1024 infarct size in the isolated rat heart — a protective additive to [64] Das M, Das DK. Resveratrol and cardiovascular health. Mol Aspects 1104

1025 ischaemic preconditioning. Cardiovasc Res Feb 1 1995;29(2):269-74. Med Dec 2010;31(6):503-12. 1105

1026 [41] Lancaster TS, Jefferson SJ, Korzick DH. Local delivery of a PKCe- [65] Della-Morte D, Dave KR, DeFazio RA, Bao YC, Raval AP, Perez-Pinzon 1106

1027 activating peptide limits ischemia reperfusion injury in the aged MA. Resveratrol pretreatment protects rat brain from cerebral 1107

1028 female rat heart. Am J Physiol Regul Integr Comp Physiol Nov 1 ischemic damage via a SIRT1-UCP2 pathway. Neuroscience Mar 31 1108

1029 2011;301(5):R1242-9. 2009;159(3):993-1002. 1109

1030 [42] Chen C-H, Budas GR, Churchill EN, Disatnik M-H, Hurley TD, Mochly- [66] Simäo F, Matte A, Pagnussat AS, Netto CA, Salbego CG. Resveratrol 1110

1031 Rosen D. An activator of mutant and wildtype aldehyde dehydroge- preconditioning modulates inflammatory response in the rat hippo- 1111

1032 nase reduces ischemic damage to the heart. Science Sep 12 campus following global cerebral ischemia. Neurochem Int Oct 1112

1033 2008;321(5895):1493-5. 2012;61(5):659-65. 1113

ARTICLE IN PRESS

A. Zholobenko, M. Modriansky / Fitoterapia xxx (2014) xxx-xxx

Sener G, Sakarcan A, Ye en B£ Role of garlic in the prevention of [92

ischemia-reperfusion injury. Mol Nutr Food Res 2007;51(11):1345-52.

Aguilera P, Chánez-Cárdenas ME, Ortiz-Plata A, León-Aparicio D, Barrera D,

Espinoza-Rojo M, et al. Aged garlic extract delays the appearance of infarct [93

area in a cerebral ischemia model, an effect likely conditioned by the

cellular antioxidant systems. Phytomedicine Mar 20l0;17(3-4):241-7.

Colín-González AL, Ortiz-Plata A, Villeda-Hernández J, Barrera D,

Molina-Jijón E, Pedraza-Chaverrí J, et al. Aged Garlic extract attenuates [94

cerebral damage and cyclooxygenase-2 induction after ischemia and

reperfusion in rats. Plant Foods Hum Nutr Nov 1 2011;66(4):348-54.

Chuah SC, Moore PK, Zhu YZ. S-allylcysteine mediates cardiopro- [95

tection in an acute myocardial infarction rat model via a hydrogen

sulfide-mediated pathway. Am J Physiol Heart Circ Physiol Nov 1

2007;293(5):H2693-701. [96

Shaik IH, George JM, Thekkumkara TJ, Mehvar R Protective effects of

diallyl sulfide, a garlic constituent, on the warm hepatic ischemia-

reperfusion injury in a rat model. Pharm Res Oct 1 2008;25(10):2231-42. [97

Atif F, Yousuf S, Agrawal SK. S-Allyl L-cysteine diminishes cerebral

ischemia-induced mitochondrial dysfunctions in hippocampus. Brain

Res Apr 10 2009;1265:128-37. [98

Chen H, Su D-F, Zhang T-H, Ding H-J, Yang Y-C, Cheng S-C. Effects of

silybin on acute myocardial infarction and reperfusion injury in

anesthetized rats. Acta Pharmacol Sin Jan 1992;13(1):69-71.

Rao PR, Viswanath RK. Cardioprotective activity ofsilymarin in ischemia- [99

reperfusion-induced myocardial infarction in albino rats. Exp Clin

Cardiol 2007;12(4):179.

El-Shitany NA, El-Haggar S, El-desoky K. Silymarin prevents adriamycin- [100

induced cardiotoxicity and nephrotoxicity in rats. Food Chem Toxicol Jul 2008;46(7):2422-8.

Jin H-B, Yang Y-B, Song Y-L, Zhang Y, Li Y-R. Protective roles of quercetin in acute myocardial ischemia and reperfusion injury in rats. [101

Mol Biol Rep Dec 1 2012;39(12):11005-9.

Tang L, Peng Y, Xu T, Yi X, Liu Y, Luo Y, et al. The effects of quercetin protect cardiomyocytes from A/R injury is related to its capability to increasing expression and activity of PKCe protein. Mol Cell Biochem [102

Oct 1 2013;382(1-2):145-52.

Wang C, Wang Z, Zhang X, Zhang X, Dong L, Xing Y, et al. Protection by silibinin against experimental ischemic stroke: Up-regulated pAkt, [103

pmTOR, HIF-1a and Bcl-2, down-regulated Bax, Nf-kB expression. Neurosci Lett Oct 31 2012;529(1):45-50.

Hou Y-C, Liou K-T, Chern C-M, Wang Y-H, Liao J-F, Chang S, et al. Preventive effect of silymarin in cerebral ischemia-reperfusion- [104

induced brain injury in rats possibly through impairing NF-kB and STAT-1 activation. Phytomedicine Oct 2010;17(12):963-73. [105

Oliveira CPM, Lopasso F, Laurindo FR, Leitao RM, Laudanna A. Protection against liver ischemia-reperfusion injury in rats by silymarin or verapamil. Transplant Proc Sep 2001 ;33 (6):3010-4. [106

Turgut F, Bayrak O, Catal F, Bayrak R, Atmaca AF, Koc A, et al. Antioxidant and protective effects of silymarin on ischemia and reperfusion injury in the kidney tissues of rats. Int Urol Nephrol Jun 1 2008;40(2):453-60. [107

Cho J-Y, Kim I-S, Jang Y-H, Kim A-R, Lee S-R. Protective effect of quercetin, a natural flavonoid against neuronal damage after transient global cerebral ischemia. Neurosci Lett Sep 1 2006;404(3):330-5. [108

Inal M, Altini$ik M, Bilgin MD. The effect of quercetin on renal ischemia and reperfusion injury in the rat. Cell Biochem Funct 2002;20(4):291-6. Chlopcíková S, Psotová J, Miketová P, Simánek V. Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes. Part I. Silymarin and its flavonolignans. Phytother Res [109

2004;18(2):107-10.

Gabrielová E, Jabúrek M, Gazák R, Vostálová J, Jezek J, Kren V, et al. Dehydrosilybin attenuates the production of ROS in rat cardiomyocyte [110

mitochondria with an uncoupler-like mechanism. J Bioenerg Biomembr Dec 14 2010;42(6):499-509.

Dorta DJ, Pigoso AA, Mingatto FE, Rodrigues T, Pestana CR, Uyemura [111

SA, et al. Antioxidant activity of flavonoids in isolated mitochondria. Phytother Res 2008;22(9):1213-8.

Skottová N, Krecman V, Simánek V. Activities of silymarin and its flavonolignans upon low density lipoprotein oxidizability in vitro. [112

Phytother Res 1999;13(6):535-7.

Mira L, Silva M, Manso CF. Scavenging of reactive oxygen species by silibinin dihemisuccinate. Biochem Pharmacol Aug 17 1994;48(4):753-9. Sorata Y, Takahama U, Kimura M. Protective effect of quercetin and rutin [113

on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin. Biochim Biophys Acta Jun 29 1984;799(3):313-7. Psotová J, Sarka Chlopciková, Grambal F, Simánek V, Ulrichová J. Influence of silymarin and its flavonolignans on doxorubicin-iron [114

induced lipid peroxidation in rat heart microsomes and mitochondria in comparison with quercetin. Phytother Res Mar 2002;16(S1):63-7. Serseñ F, Vencel T, Annus J. Silymarin and its components scavenge [115

phenylglyoxylic ketyl radicals. Fitoterapia Dec 2006;77(7-8):525-9.

Ishikawa Y, Kitamura M. Anti-apoptotic effect of quercetin: intervention in the JNK- and ERK-mediated apoptotic pathways. Kidney Int Sep 2000;58(3):1078-87.

Youl E, Bardy G, Magous R, Cros G, Sejalon F, Virsolvy A, et al. Quercetin potentiates insulin secretion and protects INS-1 pancreatic ß-cells against oxidative damage via the ERK1/2 pathway. Br J Pharmacol Oct 2010;161(4):799-814.

Ortega R, García N. The flavonoid quercetin induces changes in mitochondrial permeability by inhibiting adenine nucleotide translocase. J Bioenerg Biomembr Feb 1 2009;41 (1):41 -7.

Gabel SA, Walker VR, London RE, Steenbergen C, Korach KS, Murphy E. Estrogen receptor beta mediates gender differences in ischemia/ reperfusion injury. J Mol Cell Cardiol Feb 2005;38(2):289-97. Wang M, Crisostomo PR, Markel T, Wang Y, Lillemoe KD, Meldrum DR. Estrogen receptor beta mediates acute myocardial protection following ischemia. Surgery Aug 2008;144(2):233-8. Deschamps AM, Murphy E. Activation of a novel estrogen receptor, GPER, is cardioprotective in male and female rats. Am J Physiol Heart Circ Physiol Nov 1 2009;297(5):H1806-13.

Bopassa JC, Eghbali M, Toro L, Stefani E. A novel estrogen receptor GPER inhibits mitochondria permeability transition pore opening and protects the heart against ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol Jan 1 2010;298(1):H16-23. Trompier D, Baubichon-Cortay H, Chang X-B, Maitrejean M, Barron D, Riordon JR, et al. Multiple flavonoid-binding sites within multidrug resistance protein MRP1. Cell Mol Life Sci Oct 2003;60(10):2164-77. Leslie EM, Mao Q, Oleschuk CJ, Deeley RG, Cole SPC. Modulation of multidrug resistance protein 1 (MRP1/ABCC1) transport and ATPase activities by interaction with dietary flavonoids. Mol Pharmacol May 1 2001;59(5):1171-80.

Angelone T, Pasqua T, Di Majo D, Quintieri AM, Filice E, Amodio N, et al. Distinct signalling mechanisms are involved in the dissimilar myocar-dial and coronary effects elicited by quercetin and myricetin, two red wine flavonols. Nutr Metab Cardiovasc Dis May 2011;21(5):362-71. Zhou B, Wu L-J, Tashiro S, Onodera S, Uchiumi F, Ikejima T. Silibinin protects rat cardiac myocyte from isoproterenol-induced DNA damage independent on regulation of cell cycle. Biol Pharm Bull 2006;29(9):1900-5. Ko W-C, Shih C-M, Lai Y-H, Chen J-H, Huang H-L. Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships. Biochem Pharmacol Nov 15 2004;68(10):2087-94.

Beretz A, Anton R, Stoclet JC. Flavonoid compounds are potent inhibitors of cyclic AMP phosphodiesterase. Experientia Aug 1 1978;34(8):1054-5. Koch HP, Bachner J, Löffler E. Silymarin: potent inhibitor of cyclic AMP phosphodiesterase. Methods Find Exp Clin Pharmacol Aug 1985;7(8):409-13.

Li P-G, Sun L, Han X, Ling S, Gan W, Xu J-W. Quercetin induces rapid eNOS phosphorylation and vasodilation by an Akt-independent and PKA-dependent mechanism. Pharmacology 2012;89(3-4):220-8. Ai W, Zhang Y, Tang Q-Z, Yan L, Bian Z-Y, Liu C, et al. Silibinin attenuates cardiac hypertrophy and fibrosis through blocking EGFR-dependent signaling. J Cell Biochem Jun 1 2010;110(5):1111-22. Singh RP, Dhanalakshmi S, Agarwal C, Agarwal R. Silibinin strongly inhibits growth and survival of human endothelial cells via cell cycle arrest and downregulation of survivin, Akt and NF-kB: implications for angioprevention and antiangiogenic therapy. Oncogene Nov 22 2004;24(7):1188-202.

Deep G, Gangar SC, Oberlies NH, Kroll DJ, Agarwal R. Isosilybin A induces apoptosis in human prostate cancer cells via targeting Akt, NF-kB, and androgen receptor signaling. Mol Carcinog Oct 2010;49(10):902-12. Deep G, Oberlies NH, Kroll DJ, Agarwal R. Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway. Oncogene 2008;27(28):3986-98. Mallikarjuna G, Dhanalakshmi S, Singh RP, Agarwal C, Agarwal R. Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling. Cancer Res Sep 1 2004;64(17):6349-56.

García-Maceira P, Mateo J. Silibinin inhibits hypoxia-inducible factor-1a and mTOR/p70S6K/4E-BP1 signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy. Oncogene Nov 3 2008;28(3):313-24.

Deep G, Gangar SC, Rajamanickam S, Raina K, Gu M, Agarwal C. Angiopreventive efficacy of pure flavonolignans from milk thistle extract against prostate cancer: targeting VEGF-VEGFR signaling. PLoS One Apr 2012;7(4):e34630.

Kim W, Bang M, Kim E, Kang N, Jung K, Cho H, et al. Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells. J Nutr Biochem Mar 2005;16(3):155-62. Kang JS, Jeon YJ, Park S-K, Yang K-H, Kim HM. Protection against lipopolysaccharide-induced sepsis and inhibition of interleukin-1 ß

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A. Zholobenko, M. Modriansky / Fitoterapia xxx (2014) xxx-xxx 11

1274 and prostaglandin E2 synthesis by silymarin. Biochem Pharmacol Jan [129] de Monbrison F, Maitrejean M, Latour C, Bugnazet F, Peyron F, Barron D, 1316

1275 1 2004;67(1):175-81. et al. In vitro antimalarial activity of flavonoid derivatives dehydrosilybin 1317

1276 [116] Au AY, Hasenwinkel JM, Frondoza CG. Silybin inhibits interleukin-1ß- and 8-(1;1)-DMA-kaempferide. Acta TropJan 2006;97(1):102-7. 1318

1277 induced production of pro-inflammatory mediators in canine hepa- [130] Akhlaghi M, Bandy B. Preconditioning and acute effects of flavonoids 1319

1278 tocyte cultures. J Vet Pharmacol Ther 2011;34(2):120-9. in protecting cardiomyocytes from oxidative cell death. Oxid Med Cell 1320

1279 [117] Ignatowicz E, Szaefer H, Zielinska M, Korczowska I, Fenrych W. Silybin Longev Jul 5 2012 [[Internet], [cited 2014 Jan 30]. Available from: 1321

1280 and silydianin diminish the oxidative metabolism of human poly- http://www.hindawi.com/journals/omcl/2012/782321/abs/]. q14

1281 morphonuclear neutrophils. Acta Biochim Pol 1997;44(1):127-9. [131] Walker EH, Pacold ME, PerisicO, Stephens L, Hawkins PT, Wymann MP, 1323

1282 [118] Zielinska-Przyjemska M, Wiktorowicz K. Anin vitro study of the et al. Structural determinants of phosphoinositide 3-kinase inhibition 1324

1283 protective effect of the flavonoid silydianin against reactive oxygen by wortmannin, LY294002, quercetin, myricetin, and staurosporine. 1325

1284 species. Phytother Res Feb 2006;20(2):115-9. Mol Cell 2000;6(4):909-19. 1326

1285 [119] Ergün Y, Kuruta$ EB, Atalay F, Alici T. Effects of silibinin and ethanol [132] MatterWF, Brown RF,VlahosCJ. The inhibition of phosphatidylinositol 1327

1286 on skeletal muscle ischemia-reperfusion injury. Acta Cir Bras Mar 3-kinase by quercetin and analogs. Biochem Biophys Res Commun Jul 1328

1287 2013;28(3):179-84. 31 1992;186(2):624-31. 1329

1288 [120] Zhan T, Digel M, Küch E-M, Stremmel W, Füllekrug J. Silybin and [133] Wu S-N, Chiang H-T, Shen A-Y, Lo Y-K. Differential effects of quercetin, 1330

1289 dehydrosilybin decrease glucose uptake by inhibiting GLUT proteins. J a natural polyphenolic flavonoid, on L-Type calcium current in pituitary 1331

1290 Cell Biochem 2011;112(3):849-59. tumor (GH3) cells and neuronal NG108-15 cells. J Cell Physiol 1332

1291 [121] Malekinejad H, Taheri-Broujerdi M, Moradi M, Tabatabaie S-H. 2003;195(2):298-308. 1333

1292 Silymarin potentiates the antinociceptive effect of morphine in mice. [134] Gschwendt M, Horn F, Kittstein W, Marks F. Inhibition of the calcium- 1334

1293 Phytother Res 2011;25(2):250-5. and phospholipid-dependent protein kinase activity from mouse 1335

1294 [122] Ahmed-Belkacem A, Ahnou N, Barbotte L, Wychowski C, Pallier C, brain cytosol by quercetin. Biochem Biophys Res Commun Dec 16 1336

1295 Brillet R, et al. Silibinin and related compounds are direct inhibitors of 1983;117(2):444-7. 1337

1296 hepatitis C virus RNA-dependent RNA polymerase. Gastroenterology [135] Anthony K, Saleh M. Free radical scavenging and antioxidant activities 1338

1297 Mar 2010;138(3):1112-22. of silymarin components. Antioxidants Dec 10 2013;2(4):398-407. 1339

1298 [123] Trouillas P, Marsal P, Svobodova A, Vostalova J, Gazak R, HrbacJ, etal. [136] Polyak SJ, Morishima C, Lohmann V, Pal S, Lee DYW, Liu Y, et al. 1340

1299 Mechanism of the antioxidant action of silybin and 2,3-dehydrosilybin Identification of hepatoprotective flavonolignans from silymarin. Proc 1341

1300 flavonolignans: a joint experimental and theoretical study. J Phys Natl Acad Sci Mar 30 2010;107(13):5995-9. 1342

1301 ChemAFeb 1 2008;112(5):1054-63. [137] Park H-S, Shim S-M, Kim G-H. Silydianin in chloroform soluble 1343

1302 [124] Gazak R, Walterova D, Kren V. Silybin and silymarin — new and fraction of Cirsium japonicum leaf inhibited adipocyte differentiation 1344

1303 emerging applications in medicine. CurrMedChem2007;14(3):315-38. by regulating adipogenic transcription factors and enzymes. J Korean 1345

1304 [125] Thongphasuk P, Stremmel W, Chamulitrat W. Potent direct or TNF- Soc Appl Biol Chem Dec 2013;56(6):709-13. 1346

1305 alpha-promoted anticancer effects of2,3-dehydrosilybin: comparison [138] Pferschy-Wenzig E-M, Atanasov AG, Malainer C, Noha SM, Kunert O, 1347

1306 study with silybin. Chemotherapy 2008;54(1):23-30. Schuster D, et al. Identification of isosilybin a from milk thistle seeds 1348

1307 [126] Webb MR, Ebeler SE. Comparative analysis of topoisomerase IB as an agonist of peroxisome proliferator-activated receptor gamma. J 1349

1308 inhibition and DNA intercalation by flavonoids and similar compounds: Nat Prod Apr 25 2014;77(4):842-7. 1350

1309 structural determinates of activity. Biochem J Dec 15 2004;384(3):527. [139] Jacobs BP, Dennehy C, Ramirez G, Sapp J, Lawrence VA. Milk thistle for 1351

1310 [127] Chen C, Zhou J, Ji C. Quercetin: a potential drug to reverse multidrug the treatment of liver disease: a systematic review and meta-analysis. 1352

1311 resistance. Life Sci Sep 11 2010;87(11-12):333-8. Am J Med Oct 15 2002;113(6):506-15. 1353

1312 [128] Dzubäk P, Hajdüch M, Gazäk R, Svobodova A, Psotovä J, Walterovä D, 1 Kren V, Walterova D. Silybin and silymarin — new effects and 1354

1313 et al. New derivatives of silybin and 2,3-dehydrosilybin and their applications. Biomed Pap Oct 15 2002;149(1):29-41. 1355

1314 cytotoxic and P-glycoprotein modulatory activity. Bioorg Med Chem 1356

1315 Jun 1 2006;14(11):3793-810. 1357