Hepatoprotective properties of extensively studied medicinal plant active constituents: Possible common mechanisms

Abstract

Context: We focused on certain plant active constituents considered to be the most promising/studied for liver disease and that were critically investigated from the basic science point of view and, to some extent, the clinical one. Due to insufficient pharmacological data, most of the herbal formulations containing these molecules cannot be recommended for the treatment of liver disease.

Objective: To present the most promising compounds tested experimentally and/or clinically and describe in brief popular models in experimental testing of potential hepatoprotective compounds.

Methods: A literature search using Web of Science (WOS), PubMed, and Google search was performed.

Results: Focusing on a few herbal hepatoprotective active constituents is useful to health professionals working in the field of therapeutics to develop evidence-based hepatoprotective agents by conducting research on pure chemical structures or on molecular modifications using computational chemistry. This review demonstrates that multi-pathways in the liver pathobiology can be interrupted at one or more levels by natural hepatoprotective studied, such as interference with the oxidative stress at multiple levels to reduce reactive oxygen/nitrogen species, resulting in ameliorating hepatotoxicity.

Conclusion: Hepatoprotective constituents of herbal medications are poorly absorbed after oral administration; methods that can improve their bioavailability are being developed. It is recommended that controlled prospective double-blind multicenter studies on isolated active plant constituents, or on related newly designed molecules after structural modifications, should be performed. This effort will lead to expanding the existing, limited drugs for the vast majority of liver diseases.

• Clinical trials
• evidence-based hepatoprotectants
• hepatotoxicity models
• herbal medications
• liver disease
• oxidative stress

Introduction

Globally, there are numerous causes for liver disease, including viral hepatitis and HIV, obesity with the consequent non-alcoholic fatty liver disease (NAFLD), excessive chronic alcohol consumption, immune and cholestatic disorders, inherited metabolic disorders, numerous medications, hemochromatosis, schistosomiasis, and fungi infections, among others. Liver disease is a considerable health burden across Europe (Blachier et al., 2013) and globally. In England, 2% of total deaths between 2001 and 2009 were due to liver disease (National End of Life Care Intelligence Network, 2013). The report states that, in the same period, alcoholic liver disease accounted for well over a third (37%) of liver disease deaths. In the United States, over 25 million Americans are afflicted with liver disease and more than 27 000 die from cirrhosis, the seventh leading cause of death. Additionally, 43 000 die of liver disease, of which about 50% are alcohol related (Rayfield, 2013). Moreover, liver diseases such as fibrosis are a major worldwide health problem, with high prevalence in developing countries where hundreds of millions are afflicted (Sanchez-Valle et al., 2012). In fact, with the global obesity epidemic, NAFLD, and the ensuing non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma have become a worldwide health concern of all ages and ethnicities (Alonso et al., 2010; Nobili et al., 2011; Torres et al., 2012).

In addition to regulating energy homeostasis, the liver performs essential functions that sustain life, including food digestion (absorption of fat and fat-soluble vitamins through bile production), metabolism, and detoxification of xenobiotics that enters the body through the digestive system or lungs, or absorbed through the skin. The liver also creates new molecules (proteins, cytokines, etc.) that are essential for physiological and immunological functions. The liver also acts as a blood filter, by converting ammonia into urea, and by removing and excreting substances from the blood that otherwise would be toxic. In addition, the liver makes proteins that regulate blood clotting and influence the immune system, among others. The liver has the extraordinary ability to regenerate its own damaged tissue. This allows transplanting part of one’s liver to a blood-related relative who is a matching type, and both people will have healthy livers that will grow to its original size. Of course, liver regeneration works in healthy livers. Thus, it is imperative to highlight few aspects of liver diseases.

An outline of liver diseases

Numerous diseases and environmental factors can affect liver function resulting in gallstones, liver steatosis, fibrosis, cirrhosis, and even cancer. By clearing xenobiotics through biotransformation (in most instances by detoxification or sometimes by production of active or even more toxic metabolites), the liver itself can be injured by these xenobiotics. Due to its high blood perfusion rate and high metabolic capacity, the liver is continuously exposed to high levels of xenobiotics and to their metabolites. Fortunately, the liver has a high capacity of regeneration and ability to repair any underlying damage. Generally speaking, hepatotoxicity occurs when liver regeneration capabilities are exhausted and cell damage ensues. Hepatic injury due to xenobiotic exposure involves inflammation, oxidative stress, and lipid peroxidation reactions that can result in mitochondrial damage and cell death. The resulting pathological alterations of the endogenous substances such as proteins, nucleic acids, and lipids can harm the proper functioning of the liver (Hong et al., 2009).

Scientific rationale for the traditional use of some plants in the management of liver diseases

Generally, the medicinal use of plants in their natural and unprocessed form began when it was first observed that certain food plants altered particular body functions. Historically, plants were sources of medications for millennia. Purified active constituents such as alkaloids, glycosides, and flavonoids were isolated and their pharmacologic activities were assessed, and they constituted the base for modern drug development. The Indians, Egyptians, Chinese, and Sumerians are just a few civilizations that have provided evidence suggesting that plants can be effectively used as medications to treat or prevent disease. Ayurveda, a 5000-year-old system of natural healing that has its origins in the Vedic culture of India described the use of food for therapeutic purpose.

Therefore, the potential use of these plants for hepatoprotection makes them an attractive target for future studies, and for the identification of their active constituents. This effort may probably lead to widening the horizon of existing therapies for liver diseases. Future studies will be necessary to expand the existing, limited therapeutic drugs for the vast majority of liver diseases. Reports of medicinal plants for the treatment of liver disease are numerous; however, due to space limitations, we will focus on only a few (Ansari et al., 2011; Asgarpanah & Kazemivash, 2012; Asuku et al., 2012; Avila et al., 2011; Ghosh et al., 2011; Gutierrez et al., 2013; Haddad et al., 2011; Ho et al., 2012; Lee et al., 2012; Mihailovic et al., 2013; Nagalekshmi et al., 2011; Nithianantham et al., 2011; Paul et al., 2013; Shivananjappa et al., 2013; Singab et al., 2010; Mukherjee et al., 2009; Rana et al., 2011; Subramanian et al., 2012; Torres-Gonalez et al., 2011; Upadhyay et al., 2013; Xavier et al., 2012; Zhang et al., 2012).

It is understandable that the use of substances isolated from natural sources for the phytotherapy of various diseases, including liver diseases, is desirable because they are relatively inexpensive and are widely available. Silymarin and resveratrol are two of many examples of natural substances that have shown a strong hepatoprotective potential due to their antioxidant, anti-inflammatory, and liver regenerative capabilities (Farghali et al., 2000a, 2009; Glauert et al., 2010; Haddad et al., 2011; Pradhan & Girish, 2006).

Substances such as curcumin and quercetin exhibit antioxidant and cytoprotective properties, but their use as hepatoprotectants has not been extensively investigated clinically. However, curcumin and quercetin exhibited significant hepetoprotection in experimental setups as reported previously (Cerny et al., 2011; Chirumbolo, 2010; Lekic et al., 2013; Zhao et al., 2011; Zhou et al., 2011). In addition, medicinal plants play a key role in human health care. The World Health Organization (WHO) estimates that 80% of the populations of some Asian and African countries presently use herbal medications for some aspect of primary health care. Studies in the United States and Europe have shown that the use of herbal therapy is less common in clinical settings, but has become increasingly more so in recent years as scientific evidence about their effectiveness has become more widely available. Scientific studies available on medicinal plants indicate that promising phytochemicals can be developed for many diseases (Gupta, 1994).

In a report by Adewusi and Afolayan (2010), who reviewed natural products with hepatoprotective activity, more than one hundred plants with 58 compounds classified into appropriate chemical group were addressed. Therefore, these plants are attractive targets for future studies, and the identification of their active constituents will probably lead to new therapies for liver disease. The efficacy of herbal medications is influenced by factors such as variations in the soil and climate, period and place of collection, age and part of plants used, which influence the hepatoprotective properties (Valan et al., 2010).

It is important to realize that despite the great advances in modern medicine, there is no effective drug available that stimulates liver function, offer liver protection, or help to regenerate hepatic cells (Chattopadhyay, 2003). In fact, we need to seek novel potential hepatoprotective substances and to search for alternative drugs for the treatment of liver diseases to replace currently used medications of doubtful efficacy and safety. Notwithstanding their potential health effects, there could be potential harm from herbal preparations either per se (Khan et al., 2008) or due to their interaction with conventional medicines. For instance, Bilgi et al. (2010) reported on a case of imatinib-induced exaggerated hepatotoxicity after concurrent ginseng ingestion in a patient with chronic myelogenous leukemia. Our interest in hepatoprotective agents from various origins – whether synthetic, natural, or drugs used for various therapeutic indications but possess potential liver protection properties – stems from more than two decades of our research, as reflected by our publications. In those articles, we looked for possible underlying common mechanisms of cytoprotection from molecular levels to whole animal studies of various structurally unrelated molecules. We found that several compounds experimentally showed some hepato-ameliorative properties and enumerated possible explanations (Canova et al., 2007; Cerny et al., 2009; Farghali & Masek, 1998; Farghali et al., 1991a,b, 1994, 1996a,b, 1997, 2000b, 2002, 2003; Gasbarrini et al., 1992a,b, 1993; Kmonickova et al., 2001; Lekic et al., 2011; Sakr et al., 1991a,b).

Aim of the article

This review highlights the literature about purified or semi-purified chemicals of herbal origins (i.e., chemically defined molecules) with reported experimental and/or clinical hepatoprotective activity. The findings are likely to help further to investigate hepatoprotective agents at both the experimental and the clinical levels. In addition to our personal experience, we have performed a literature search using mainly Web of Science (WOS), PubMed, and Google search. An extensive number of articles which address many plant constituents with reported experimental (in vitro or in vivo) effects are summarized in various reviews (Alvari et al., 2012; Dhiman et al., 2012; Ding et al., 2012; Girish & Pradhan, 2012; Muriel & Rivera-Espinoza, 2008; Zhang et al., 2013a). In this article, we only consider literature related to the most widely studied active plant constituents of chemically defined molecules with potential hepatoprotective activity and the models most commonly used for their validation. We believe that these findings will certainly increase the likelihood of using hepatoprotective agents of well-defined molecules with less adverse effects and will help to design new molecules by using computational and synthetic chemistry. Naturally, this requires examination of the molecular aspects of mode of actions of these compounds. This may seem more intricate since both the impairment of the liver is multifactorial (viral or protozoal infections, chronic use of excessive alcohol, drugs, xenobiotics, etc.) and the chemoprotective agents are different in their chemical structures. However, there is an urgent need to find out a range of efficient drugs that may be classified as hepatoprotective agents. In the literature, the use of terms such as nutraceuticals, functional foods, herbal extracts, bioactive dietary constituents, phytochemicals, and similar terms is becoming widespread.

Potential beneficial plant constituents for liver diseases

Natural products from plants used traditionally as hepatoprotective have been reviewed by several authors (Adewusi & Afolayan, 2010; Alvari et al., 2012; Dhiman et al., 2012; Ding et al., 2012; Girish & Pradhan, 2012; Muriel & Rivera-Espinoza, 2008; Valan et al., 2010; Zhang et al., 2013a), who described the phytoconstituents with hepatoprotective properties and classified them under phenyl compounds, coumarins, essential oils, monoterpenoids, diterpenoids, triterpenoids, steroids, alkaloids, and others.

In the present article, we focused on certain plant active constituents considered to be most promising/studied for liver diseases, and were critically investigated from the basic science point of view and, to some extent, the clinical one. We also recognize that experimental pharmacological studies on medicinal plant constituents are being done, but clinical studies lack behind and need completion. Hence this review deals with constituents that are being tested experimentally with or without clinical studies. They are arranged according to the wide use and the relevant experimental and/or clinical trials in Table 1. For instance, silymarin obtained from the seeds of Silybum marianum (L.) Gaertner (Asteraceae) is the most thoroughly investigated compound with potent antihepatotoxic activity. Silymarin is a mixture of isomeric flavolignans – silybin, silydianin, and silychristen. It is important to study the hepatic effects of these pure substances using in vitro and in vivo models, followed by genomic and proteomic studies, and finally complemented with clinical evaluation for some of them. Here we give a simple table for the most promising compounds that were tested at experimental levels and some at clinical levels. In addition, recent advances in several natural products from plant origin for the potential treatment of several liver diseases were described as wogonin, naringenin, geniposide, rhein, and the list is increasing (Alvari et al., 2012; Zhang, 2013a).

Table 1. Most frequently studied hepatoprotective phytochemicals.

Compound	Chemical class	Major food resources	Major biological activity	References
Silymarin (Silybin)	Mixture of flavonolignans consisting of among others of silibinin (70–80%), isosilibinin, silicristin, and silidianin	Seeds of milk thistle Silybum marianum	Antioxidant, anti-inflammatory, anti-fibrotic, protein synthesis increasing/regenerative, membrane stabilizing, and toxin blocking activities; it reduces tumor cell proliferation, angiogenesis as well as insulin resistance	Abenavoli et al. (2010), Ding et al. (2012), El-Kamary et al. (2009), Falasca et al. (2008), Farghali et al. (2000a), Ferenci et al. (1989), Gharagozloo et al. (2009), Glauert et al. (2010), Haddad et al. (2011), Loguercio et al. (2012), Mereish et al. (1991), Pradhan and Girish (2006), Salamone et al. (2012a, b), Singh et al. (2012), Thabrew and Huges (1996), Wagner et al. (1968, 1974), and Zhang et al. (2013a,b)
Curcumin	Polyphenol: (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione	Rhizomes turmeric (Curcuma longa), yellow spice	Antioxidant, anti-inflammatory, antifibrotic, anticancer, antiagreggatory, and potent cytochrome P450 inhibitory activities	Cerny et al. (2011), Ding et al. (2012), Gupta et al. (2013), Maiti et al. (2007), Kim et al. (2013), Singh et al. (2012), Zhang et al. (2013a), Zhou et al. (2011)
Quercetin	Flavonol: 2-(3,4-dihydroxy-phenyl)-3,5,7-trihydroxy-4H-chromen-4-one	Fruits (apples, cranberries), vegetables (broccoli, onion), tea leaves (Camellia sinensis), grains (buckwheat)	Antioxidant, anti-inflammatory, and anticancer activities	Chirumbolo (2010), Egert et al. (2009), Lekic et al. (2013), and Zhao et al. (2011)
Resveratrol	Polyphenol: trans-3,5,4’-trihydroxystilbene	Berries, grapes, wine, peanuts	Antioxidant, anti-inflammatory, antiaging, antithrombotic, antifibrogenic, and regenerative properties; it reduces tumor cell proliferation, angiogenesis as well as insulin resistance	Aftab et al. (2010), Athar et al. (2009), Bishayee et al. (2010), Cerny et al. (2009), Farghali et al. (2009), Glauert et al. (2010), Li et al. (2014), Patel et al. (2011), Smoliga and Rundell (2011), Smoliga et al. (2012), Suchankova et al. (2009), Timmers et al. (2012), Wong et al. (2009), and Zhang et al. (2013a)
Glycyrrhizin	Triterpenic glycoside: (3β,18α)-30-hydroxy-11,30-dioxoolean-12-en-3-yl 2-O-β-d-glucopyranuronosyl-β-d-glucopyranosid-uronic acid	Root of Indian licorice (Glycyrrhiza glabra)	Antioxidant, anti-inflammatory, anticancer, antiviral, and immunomodulatory activities	Jayaprakasam et al. (2009), Ni et al. (2011), Orlent et al. (2006), Wan et al. (2009), Yasui et al. (2011), Zhang et al. (2013a)
Colchicine	Tricyclic polymethoxy-derivate: N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acetamide	Plants of the genus Colchicum (autumn crocus, Colchicum autumnale)	Anti-inflammatory, antifibrotic, and immunomodulatory activities	Arrieta et al. (2006), Leung et al. (2011), Muntoni et al. (2010), and Nikolaidis et al. (2006)

Next we describe in brief the most popular models in experimental testing of potential hepatoprotective compounds. Drug constituents used for the treatment of viral hepatitis that exhibit very favorable results are extensively reviewed elsewhere (Ahmed et al., 2008; Chen & Lai, 2013; Thabrew & Hughes, 1996).

In vitro and in vivo experimental hepatotoxic models in liver research

Successful development of therapy for the liver depends on the suitability of in vitro and in vivo test model systems for hepatic injury. Several models are available to screen the antihepatotoxic activity of any substance. Since there are limitations of the outcomes in each model, it is important to combine different methods for confirmation of the findings (Muriel, 2008).

There are several in vitro models (e.g., hepatocyte cultures, perfused hepatocytes) that examine pathophysiological injuries due to various chemicals (e.g., hepatotoxins, hypoxia or anoxia, anoxia/reoxygenation models in perfused immobilized hepatocytes) (Cerny et al., 2009; Farghali et al., 1991a, 2000b; Gasbarrini et al., 1992b).

A number of in vivo models are depicted in Figure 1. Most of the studies investigating the hepatoprotective potential are of experimental nature. Some clinical studies were performed using certain constituents (see later section); however, the majority of the studies are performed on herbal extracts or powdered dry plants for a specific liver disease.

Figure 1. Common in vivo models of liver damage.

Are there possible common mechanisms of action in hepatoprotection among various plant constituents?

The answer is probably yes. In general, oxidative cell injury is well known to be associated both with the process of organism’s aging and with numerous chronic diseases, such as diabetes, atherosclerosis, macular degeneration, cataract, and Alzheimer’s disease, among others. In addition, oxidative stress which is the cardinal mechanism that can be induced by toxins and environmental factors leads to the accumulation of reactive oxygen/nitrogen species (ROS/RNS), further causing imbalance in pro-oxidant/antioxidant steady state, known as oxidative stress. Protein cross-linking, lipid peroxidation, mitochondrial dysfunction, and induction of cell death pathways are among the identified mechanisms of cellular damage due to oxidative stress (Jaeschke, 2011; Jaeschke et al., 2012; Yeum et al., 2010). In this review, we discuss the role of ROS in liver fibrosis, a liver disease with significant morbidity and mortality that affects 100 million people worldwide. The role of oxidative stress in liver fibrosis, and pathways involved in hepatic fibrosis, are well covered in a recent review article by Sanchez-Valle et al. (2012). ROS play an important role in initiating hepatic fibrogenesis. Fibrosis, manifested in several chronic liver diseases, is characterized by excessive collagen deposition in the extracellular matrix in response to activation of hepatic stellate cells. Clinical and experimental data suggest that oxidative stress mediates the progression of fibrosis and that oxidative stress-related molecules may act as mediators of molecular and cellular events implicated in liver fibrosis. Oxidative stress causes lipid peroxidation resulting in the formation of highly immunogenic molecules, damages proteins and DNA, induces necro-apoptosis of hepatocytes, and amplifies the inflammatory response. ROS also stimulates the production of the pro-fibrogenic mediator TGF-β from Kupffer cells and the circulating inflammatory cells, as well as directly activates hepatic stellate cells, resulting in the initiation of fibrosis. Advances in understanding the mechanisms involved in fibrosis have identified new molecular targets with therapeutic potential for more targeted intervention of this disease. In this regard, it is established that food rich in antioxidants, such as vegetables and fruits, could reduce risk of several chronic diseases. Therefore, it is reasonable to assume that physiological doses of antioxidants that might be present in an appropriate fruit and vegetable diet can establish an effective antioxidant network in vivo with consequent cytoprotective effects on liver.

Various basic science literature contains enormous numbers of molecules of plant origin that have the promise to ameliorate diseases such as cancer to slowing the aging process. However, most of these compounds were not sufficiently evaluated in humans. A wide variety of plant constituents, including silymarin, resveratrol, curcumin, quercetin, and glycyrrhizin, have been reported to have multiple biological activities, mainly due to their antioxidant and anti-inflammatory properties (Alvari et al., 2012; Dhiman et al., 2012; Ding et al., 2012; Girish & Pradhan, 2012; Muriel & Rivera-Espinoza, 2008; Zhang et al., 2013a). The present article focuses on only few well-studied naturally occurring hepatoprotective (cytoprotective) compounds.

The seeds of milk thistle have been used for over 2000 years for the treatment of liver diseases. Silybin, also known as silibinin, is the major constituent (70–80%) as well as the most active biological component of milk thistle. Laboratory studies revealed there is no real difference in activity between silymarin and silibinin in some experimental models. Pharmacological and clinical studies showed that silymarin is relatively safe at the dosage range studied clinically (e.g., 100–150 mg daily in divided doses) (Ferenci et al., 1989). Silymarin’s protective effect against oxidative stress was partially attributed to a reduction in the intracellular calcium in a model of perfused immobilized hepatocytes, resulting in better functioning hepatocytes (Farghali et al., 2000b). The possible mechanism(s) that contribute to the hepatoprotective effect of silymarin and the role played by intracellular calcium was investigated using tert-butyl hydroperoxide and d-galactosamine toxicity in the isolated immobilized and perfused hepatocytes model. Silibinin was also found to be hepato-protective against steatosis and insulin resistance both in vivo and in vitro, partly through regulating the IRS-1/PI3K/Akt pathway which is involved in the pathogenesis of NAFLD (Zhang et al., 2013b). Silibinin was also reported to improve hepatic oxidative stress and inflammation through the c-Jun NH2-terminal kinase (Salamone et al., 2012a) and nuclear factor kappa B (NF-κB) (Salamone et al., 2012b) pathways. In a clinical trial, silibinin given for 12 months in combination with vitamin E and phosphatidylcholine improved hepatic enzymes and insulin resistance in NAFLD patients (Loguercio et al., 2012). The anti-inflammatory, immune-modulating activity, antifibrotic, and antioxidant effects of silymarin that contribute to its hepatoprotective effect are summarized in a review by Abenavoli et al. (2010).

Resveratrol, a polyphenolic compound with antioxidant properties, has been studied for the treatment of numerous pathologies, including NAFLD. Treatment with resveratrol ameliorated NAFLD features, including insulin resistance, histological changes, glucose tolerance, inflammation, and oxidative stress, and restored IκBα (NF-κB inhibitor) which resulted in reduced activity of NF-κB (Li et al., 2014). Several thousand articles deal with the biology and pharmacology of resveratrol, including many reports dealing with the molecular mechanisms of resveratrol’s cytoprotection (Suchankova et al., 2009; Wong et al., 2009). The vast interest in resveratrol, which inspired an enormous number of studies, has led to the identification of multiple molecular targets of resveratrol (Aftab et al., 2010; Athar et al., 2009; Bishayee et al., 2010; Smoliga & Rundell 2011; Smoliga et al., 2012). Resveratrol also acts as anti-inflammatory (Bereswill et al., 2010), alters drug metabolizing enzymes (Chow et al., 2010), inhibits cyclooxygenases (Wendeburg et al., 2009), and, importantly, has specific effects on proteins and/or signaling cascades such as SIRT1 (silent information regulator T1) and AMPK (adenosine-5′-monophosphate-activated protein kinase) (Fullerton & Steinberg, 2010; Xiong et al., 2011) that are summarized by Smoliga et al. (2012). The most studied molecular mechanisms which might represent part of common pathway responsible for a hepatoprotective action that controls a number of regulatory factors associated with metabolism, inflammation, and other pathophysiological pathways, e.g., SIRT1 and AMPK are depicted in Figure 2.

Figure 2. The most important multiple pathways in the liver pathobiology that can be interrupted at one or more levels by hepatoprotective plant constituents (not shown) to interfere with oxidative stress. ACC, acetyl-CoA carboxylase; AMPK, adenosine-5′-monophosphate-activated protein kinase; ATP, adenosine triphosphate; Bax, proapoptotic protein of Bcl-2 family; BSEP, bile salt export pump; CAT, catalase; CO, carbon monoxide; COX-2, inducible cyclooxygenase; CYP, cytochrome P450; eCa2+, extracellular calcium; ECM, extracellular matrix; eNOS (NOS-1), endothelial nitric oxide synthase; ER, endoplasmic reticulum; GPx, glutathione peroxidase; GSH, reduced glutathione; GST, glutathione-S-transferase; HO-1, inducible heme oxygenase; IL, interleukin; iCa2+, intracellular calcium; iNOS (NOS-2), inducible nitric oxide synthase; MDR3, multidrug resistance protein 3; MRP2, multidrug resistance-related protein 2; NO, nitric oxide; NF-κB, nuclear factor kappa-B; NRS, nitrogen reactive species; ONOO−, peroxynitrite anion; p53, protein p53; PPAR-α/γ, peroxisome proliferator-activated receptor-α/γ; ROS, reactive oxygen species; SIRT1, silent information regulator T1; SOD, superoxide dismutase; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α, VCAM-1, vascular cell adhesion molecule-1.

Resveratrol, a SIRT1 activator, increased AMPK phosphorylation and reduced oxidative damage biomarkers during aging in F 2 hybrid mice (Suchankova et al., 2009). In addition, recent studies indicate intricate relationships among resveratrol, nuclear factors, autacoids, and cytoprotection in various cells, tissues, or organs. For instance, in one study, resveratrol suppressed lipopolysaccharide (LPS)-induced nuclear translocation and activation of NF-κB thus inhibiting pro-inflammatory responses in C6 microglia (Kim et al., 2007). A similar finding about the protective effect of resveratrol as an inhibitor of NF-κB-mediated vascular cell adhesion molecule (VCAM-1) induction was reported (Carluccio et al., 2007). Recently, NF-κB was suggested as a target for drug therapy in liver diseases where resveratrol was among several agents that inhibits the NF-κB transcription factor. The fact that NF-κB has been associated with the induction of pro-inflammatory gene-expression makes research on agents which inhibit NF-κB an interesting topic.

Studies on resveratrol pretreatment effects on the synergy between d-galactosamine (d-GalN) and LPS-induced liver failure in rats, and its effects on chemical prooxidants in immobilized perfused hepatocytes was investigated in our laboratory (Cerny et al., 2009; Farghali et al., 2009). Parameters studied included liver function; plasma nitrite as a measure of nitric oxide (NO); non-enzymatic and enzymatic antioxidants in plasma and liver homogenate; and morphological examinations were performed using light and electron microscopy. Observations related to pharmacological increases of inducible nitric oxide synthase (NOS-2)/NO and inducible hemeoxygenase (HO-1)/carbon monoxide (CO) in fulminant hepatic failure and modulation by resveratrol were followed up by real-time reverse transcription PCR (RT-PCR) in liver tissue. In the last study, we found that reduction in NO production, down-regulation of NOS-2 expression, modification of oxidative stress parameters, and modulation of HO-1 are among the mechanisms responsible for the cytoprotective effect of resveratrol in the LPS/d-GalN liver toxicity, and in tert-butylhyroperoxide-induced hepatocyte toxicity models. Resveratrol pretreatment led to the overall improvement in hepatotoxic markers and morphology after the hepatic insult. Several studies have also highlighted the hepatoprotective properties of resveratrol (Bishayee et al., 2010).

SIRT1 plays a cardinal role in the mode of action of resveratrol through AMPK activity as shown in Figure 2. Moghe et al. (2011) presented a relationship between histone modification and alcohol-induced liver disease with a focus on epigenetic changes, including histone modifications that regulate gene expression during pathogenesis. As reported, chronic alcohol consumption also sensitizes non-immune cells such as hepatocytes, in addition to immune cells, to inflammatory signals and impairs their ability to respond to protective signals. Based on these advances, a number of inflammatory targets have been identified with the potential for therapeutic intervention in alcoholic liver disease by using such compounds, presenting new opportunities, and challenges for translational research (Wang et al., 2012; Zakhari & Li, 2007).

Oxidative stress and liver inflammation are also linked to diet-induced obesity, thus emphasizing the assumption that hepatoprotectors should have common pathways at certain level(s) (Peng et al., 2012). At this juncture, it is important to emphasize the importance of SIRT1 allosteric modulators and cytoprotection including hepatoprotection.

To summarize, Figure 2 demonstrates that multipathways in the liver pathobiology that can be interrupted at one or more levels, practically, by all natural hepatoprotective studied leading to interference with the oxidative stress.

Importance of and emphasis on controlled clinical trials on active plant constituents

Most clinical trials were conducted on herbal powders or extracts, and a relatively low number of studies used pure active constituents. In spite of the several thousand reports that deal with favorable effects on various liver injury models, clinical trials are lagging behind and relatively small numbers were done on active constituents (Akachi et al., 2010; Kim et al., 2013; Panahi et al., 2012; Sarwar et al., 2011; Singh & Sharma, 2011; Wong et al., 2013).

The safety and efficacy of silymarin on signs and symptoms, and biomarkers were investigated in randomized controlled clinical trials on patients with acute hepatitis. The standard dose of silymarin (140 mg) produced earlier improvement in subjective and clinical markers of biliary excretion. With a modest sample size, and multiple etiologies for acute clinical hepatitis, the results suggest that standard recommended doses of silymarin are safe and may be potentially effective in improving symptoms of acute clinical hepatitis (El-Kamary et al., 2009).

A randomized clinical trial was published on silybin combined with phosphatidylcholine and vitamin E in patients with non-alcoholic fatty liver disease. A preparation with the name of Realsil (RA) which comprises the silybin phytosome complex (silybin plus phosphatidylcholine) co-formulated with vitamin E was tested. Treatment for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight (Loguercio et al., 2012).

Although the present review is not dealing with antiviral agents for liver infection as mentioned before, we cite the example of silybin–vitamin E–phospholipid complex treatment in patients with HCV (Falasca et al., 2008). The treated group of HCV patients showed an improvement in hepatic indices and viral load, and had a significant and persistent reduction of ALT and AST serum levels. In this group, cytokines showed a statistically significant modulation of IL-2 and IL-6. After the treatment, the hepatic steatosis group showed a significant decrease in ALT, AST, γ-GT, alkaline phosphatase, total cholesterol, fasting glucose, insulinemia, HOMA (the homeostatic model assessment) value, and C-reactive protein. There was a significant reduction of IFN-γ, TNF-α, and IL-6. The data suggest that the complex exerts hepatoprotective, anti-inflammatory, and antifibrotic effects. The authors suggested that this treatment may, therefore, be useful in clinical practice in patients with chronic hepatitis C who cannot undergo conventional antiviral therapy.

In a double-blind clinical trial, the combined therapy of silymarin and desferrioxamine in patients with β-thalasemia (iron-loaded patients) was beneficial (Gharagozloo et al., 2009). Because of the poor absorption of silymarin, critical reassessment of its bioavailability (using an array of methods that can improve its bioavailability, including complexion with β-cyclodextrins, solid dispersion method, formation of microparticles and nanoparticles, self-microemulsifying drug delivery systems, micelles, liposomes, and phytosomes) were published (Javed et al., 2011). Even a sensitive LC-MS/MS assay for the simultaneous analysis of the major active components of silymarin in human plasma has been developed to ensure regularity of plasma level of this agent (Brinda et al., 2012).

Resveratrol also received widespread attention as either a potential therapeutic or a preventative agent for several diseases, which resulted in thousands of reports that described the effects of resveratrol administration on physiological responses in animals and humans. The limited numbers of human clinical trials available have largely described various aspects of resveratrol’s safety and bioavailability, reaching a consensus that it is generally well tolerated, but poor bioavailability. The relatively recent GlaxoSmithKline’s phase IIa study of SRT501 (a resveratrol pharmaceutical formulation in relatively high dose of resveratrol – 5 g of SRT501) in advanced multiple myeloma has been concluded. It was explained by the manufacturers that SRT501 offers minimal efficacy while having a potential to indirectly exacerbate a renal complication common in the patient population with multiple myeloma. Therefore, researchers are hoping to find a way to concentrate the effect into a safe dose within an effective therapeutic range (Smoliga et al., 2012). Given the worldwide increase in age-related metabolic diseases, the beneficial effects of resveratrol on metabolism and healthy aging in humans are currently a topic of intense investigation. In fact, the occurrence of mild-to-moderate side effects is likely to limit the doses employed in future trials to significantly less than 5 g/d and additional information is needed to increase the chances of success in future clinical trials (Patel et al., 2011; Timmers et al., 2012).

Other molecules such as curcumin, quercetin, glycerhyzin, or colchicine were clinically investigated in liver diseases. According to recent report, more than 65 human clinical trials of curcumin, which included more than 1000 patients, have been completed, and as many as 35 clinical trials are underway (Gupta et al., 2013). Curcumin was suggested to counteract the adverse hepatotoxic side effects of anti-tuberculosis drug combinations, a possible valuable hepatoprotective application (Singh et al., 2012). In fact, clinical trials are underway using these plant constituents for various health applications including liver diseases (Del Prete et al., 2012; Egert et al., 2009). Clinical trials on licorice or glycyrrhizin are increasing specifically in viral hepatitis adjuvant therapy and autoimmune hepatitis (Orlent et al., 2006; Yasui et al., 2011).

Colchicine is a well-established drug for its anti-inflammatory actions and is used as a medication for several diseases. It is effective clinically with ursodiol in a subset of patients with primary biliary cirrhosis and was found to be an effective and safe antifibrotic drug for long-term treatment of chronic liver disease in which fibrosis progresses towards cirrhosis (Leung et al., 2011; Muntoni et al., 2010).

Conclusion

This article examines the current state-of-knowledge of the effects of pure natural plant constituents and possible derivatives and their potential applications to human liver diseases. It utilizes this information to develop further guidance for encouraging human clinical trials. From various literature and our own studies, it seems that all these active herbal constituents act at some point to reduce ROS/RNS, leading to the hepato-ameliorative effect. Due to insufficient scientific-based pharmacological data, most of the herbal formulations containing these molecules cannot be recommended for the treatment of liver diseases. Hence, this review article is intended to focus briefly on few herbal hepatoprotective active constituents that may be useful to health professionals, scientists working in the field of pharmacology and therapeutics to develop evidence-based hepatoprotective agents. This can be accomplished by research on pure chemical structures derived from plants or after structural modifications using computational chemistry to prepare new lead compounds.

The future is to carry out controlled prospective double-blind multicenter studies with the isolated active constituents or related newly synthesized drugs with proven beneficial preclinical in vitro and in vivo effects where basic hepatobiology should also be encouraged.

Declaration of interest

This work was fully supported by the Charles University institutional program PRVOUK-P25/LF1/2.