Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Hepatoprotective properties of extensively studied

medicinal plant active constituents: Possible
common mechanisms

Hassan Farghali, Nikolina Kutinov Canov & Samir Zakhari

To cite this article: Hassan Farghali, Nikolina Kutinov Canov & Samir Zakhari
(2015) Hepatoprotective properties of extensively studied medicinal plant active
constituents: Possible common mechanisms, Pharmaceutical Biology, 53:6, 781-791, DOI:
10.3109/13880209.2014.950387

To link to this article: http://dx.doi.org/10.3109/13880209.2014.950387

Published online: 09 Dec 2014.

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 http://informahealthcare.com/phb
ISSN 1388-0209 print/ISSN 1744-5116 online
Editor-in-Chief: John M. Pezzuto
Pharm Biol, 2015; 53(6): 781791
! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.950387

REVIEW ARTICLE

Hepatoprotective properties of extensively studied medicinal plant

active constituents: Possible common mechanisms
Hassan Farghali1, Nikolina Kutinova Canova1, and Samir Zakhari2
1
First Faculty of Medicine, Institute of Pharmacology, Charles University in Prague, Czech Republic and 2Science DISCUS, Washington, DC, USA

Abstract Keywords
Context: We focused on certain plant active constituents considered to be the most promising/ Clinical trials, evidence-based
studied for liver disease and that were critically investigated from the basic science point of hepatoprotectants, hepatotoxicity models,
view and, to some extent, the clinical one. Due to insufficient pharmacological data, most of the herbal medications, liver disease, oxidative
herbal formulations containing these molecules cannot be recommended for the treatment of stress
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liver disease.
Objective: To present the most promising compounds tested experimentally and/or clinically History
and describe in brief popular models in experimental testing of potential hepatoprotective
compounds. Received 4 April 2014
Methods: A literature search using Web of Science (WOS), PubMed, and Google search was Revised 26 June 2014
performed. Accepted 25 July 2014
Results: Focusing on a few herbal hepatoprotective active constituents is useful to health Published online 9 December 2014
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.

Introduction (Rayfield, 2013). Moreover, liver diseases such as fibrosis

are a major worldwide health problem, with high prevalence
Globally, there are numerous causes for liver disease,
in developing countries where hundreds of millions are
including viral hepatitis and HIV, obesity with the consequent
afflicted (Sanchez-Valle et al., 2012). In fact, with the global
non-alcoholic fatty liver disease (NAFLD), excessive chronic
obesity epidemic, NAFLD, and the ensuing non-alcoholic
alcohol consumption, immune and cholestatic disorders,
steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular
inherited metabolic disorders, numerous medications, hemo-
carcinoma have become a worldwide health concern of all
chromatosis, schistosomiasis, and fungi infections, among
ages and ethnicities (Alonso et al., 2010; Nobili et al., 2011;
others. Liver disease is a considerable health burden across
Torres et al., 2012).
Europe (Blachier et al., 2013) and globally. In England, 2% of
In addition to regulating energy homeostasis, the liver
total deaths between 2001 and 2009 were due to liver disease
performs essential functions that sustain life, including food
(National End of Life Care Intelligence Network, 2013). The
digestion (absorption of fat and fat-soluble vitamins through
report states that, in the same period, alcoholic liver disease
bile production), metabolism, and detoxification of xeno-
accounted for well over a third (37%) of liver disease deaths.
biotics that enters the body through the digestive system or
In the United States, over 25 million Americans are afflicted
lungs, or absorbed through the skin. The liver also creates
with liver disease and more than 27 000 die from cirrhosis, the
new molecules (proteins, cytokines, etc.) that are essential for
seventh leading cause of death. Additionally, 43 000 die of
physiological and immunological functions. The liver also
liver disease, of which about 50% are alcohol related
acts as a blood filter, by converting ammonia into urea, and
by removing and excreting substances from the blood that
Correspondence: Hassan Farghali, Professor of Pharmacology, First otherwise would be toxic. In addition, the liver makes proteins
Faculty of Medicine, Institute of Pharmacology, Charles University in
Prague, Albertov 4, 128 00 Prague 2, Czech Republic. Tel/Fax: +420 that regulate blood clotting and influence the immune
224968106. E-mail: hfarg@lf1.cuni.cz system, among others. The liver has the extraordinary ability
 782 H. Farghali et al.
to regenerate its own damaged tissue. This allows transplant-
ing part of ones 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
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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 hepatopro-
tection 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;
Pharm Biol, 2015; 53(6): 781791
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 poten-
tial 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 anti-
oxidant and cytoprotective properties, but their use as
hepatoprotectants has not been extensively investigated
clinically. However, curcumin and quercetin exhibited sig-
nificant 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 proper-
ties (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 prepar-
ations 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
 DOI: 10.3109/13880209.2014.950387
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 hepa-
toprotective 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
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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 hepatopro-
tective 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 proper-
ties 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
Hepatoprotective properties of plant active constituents 783
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 anti-
hepatotoxic 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 experi-
mental 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).
Next we describe in brief the most popular models in
experimental testing of potential hepatoprotective com-
pounds. 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 inju-
ries due to various chemicals (e.g., hepatotoxins, hypoxia or
anoxia, anoxia/reoxygenation models in perfused immobi-
lized 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.
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
organisms aging and with numerous chronic diseases, such
as diabetes, atherosclerosis, macular degeneration, cataract,
and Alzheimers 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
 784 H. Farghali et al. Pharm Biol, 2015; 53(6): 781791

Table 1. Most frequently studied hepatoprotective phytochemicals.

Compound Chemical class
Silymarin Mixture of flavonolignans
(Silybin) consisting of among others
of silibinin (7080%), isosi-
libinin, silicristin, and
silidianin
Curcumin Polyphenol: (1E,6E)-1,7-bis(4- Rhizomes turmeric
hydroxy-3-methoxyphenyl)-
1,6-heptadiene-3,5-dione
Quercetin Flavonol: 2-(3,4-dihydroxy-
phenyl)-3,5,7-trihydroxy-
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Major food resources Major biological activity
Seeds of milk thistle Antioxidant, anti-inflammatory,
Silybum marianum anti-fibrotic, protein synthesis
increasing/regenerative, mem-
brane stabilizing, and toxin
blocking activities; it reduces
tumor cell proliferation, angio-
genesis as well as insulin
resistance
Antioxidant, anti-inflammatory,
(Curcuma longa), antifibrotic, anticancer, anti-
yellow spice agreggatory, and potent cyto-
chrome P450 inhibitory
activities
Fruits (apples, cranber- Antioxidant, anti-inflammatory,
ries), vegetables and anticancer activities
References
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)
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)
Chirumbolo (2010), Egert et al.
(2009), Lekic et al. (2013),

4H-chromen-4-one (broccoli, onion), tea and Zhao et al. (2011)

Resveratrol Polyphenol: trans-3,5,4-
trihydroxystilbene
Glycyrrhizin Triterpenic glycoside: Root of Indian licorice
(3b,18a)-30-hydroxy-11,30-
dioxoolean-12-en-3-yl 2-O-
b-D-glucopyranuronosyl-
b-D-glucopyranosid-uronic
acid
Colchicine Tricyclic polymethoxy-deriv- Plants of the genus
ate: N-[(7S)-1,2,3,10-tetra-
methoxy-9-oxo-5,6,7,9-tetra-
hydrobenzo[a]heptalen-7-
yl]acetamide
leaves (Camellia
sinensis), grains
(buckwheat)
Berries, grapes, wine, Antioxidant, anti-inflammatory,
peanuts antiaging, antithrombotic, anti-
fibrogenic, and regenerative
properties; it reduces tumor cell
proliferation, angiogenesis as
well as insulin resistance
Antioxidant, anti-inflammatory,
(Glycyrrhiza glabra) anticancer, antiviral, and immu-
nomodulatory activities
Anti-inflammatory, antifibrotic,
Colchicum (autumn and immunomodulatory
crocus, Colchicum activities
autumnale)
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)
Jayaprakasam et al. (2009),
Ni et al. (2011), Orlent et al.
(2006), Wan et al. (2009), Yasui
et al. (2011), Zhang et al.
(2013a)
Arrieta et al. (2006), Leung et al.
(2011), Muntoni et al. (2010),
and Nikolaidis et al. (2006)

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 inflamma-
tory response. ROS also stimulates the production of the pro-
fibrogenic mediator TGF-b 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 thera-
peutic 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
 DOI: 10.3109/13880209.2014.950387
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Figure 1. Common in vivo models of liver damage.
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 num-
bers 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, quer-
cetin, 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 (7080%) 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.,
100150 mg daily in divided doses) (Ferenci et al., 1989).
Silymarins 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
Hepatoprotective properties of plant active constituents 785
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-kB) (Salamone et al., 2012b) pathways.
In a clinical trial, silibinin given for 12 months in combin-
ation 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 IkBa (NF-kB inhibitor) which
resulted in reduced activity of NF-kB (Li et al., 2014). Several
thousand articles deal with the biology and pharmacology of
resveratrol, including many reports dealing with the molecu-
lar mechanisms of resveratrols 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)
 786 H. Farghali et al. Pharm Biol, 2015; 53(6): 781791
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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-50 -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-kB, nuclear factor kappa-B; NRS, nitrogen reactive species; ONOO,
peroxynitrite anion; p53, protein p53; PPAR-a/g, peroxisome proliferator-activated receptor-a/g; ROS, reactive oxygen species; SIRT1, silent
information regulator T1; SOD, superoxide dismutase; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a, VCAM-1, vascular cell
adhesion molecule-1.

and AMPK (adenosine-50 -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.
Resveratrol, a SIRT1 activator, increased AMPK phos-
phorylation 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-kB 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-kB-mediated vascular cell
adhesion molecule (VCAM-1) induction was reported
(Carluccio et al., 2007). Recently, NF-kB was suggested as
a target for drug therapy in liver diseases where resveratrol
was among several agents that inhibits the NF-kB
transcription factor. The fact that NF-kB has been associated
with the induction of pro-inflammatory gene-expression
makes research on agents which inhibit NF-kB 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 morpho-
logical 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
 DOI: 10.3109/13880209.2014.950387
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 chal-
lenges for translational research (Wang et al., 2012; Zakhari &
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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
Hepatoprotective properties of plant active constituents 787
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 silybinvitamin Ephospholipid complex treat-
ment 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, g-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-g, TNF-a, 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 b-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 b-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 resveratrols safety and bioavailability, reaching a
consensus that it is generally well tolerated, but poor
bioavailability. The relatively recent GlaxoSmithKlines
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).
 788 H. Farghali et al.
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 hepato-
protective 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-inflam-
matory 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
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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 recom-
mended for the treatment of liver diseases. Hence, this review
article is intended to focus briefly on few herbal hepatopro-
tective 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 pre-
clinical 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.
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