Fighting Liver Fibrosis with Naturally Occurring Antioxidants

Abstract

Liver fibrosis is a wound-healing response characterized by the accumulation of extracellular matrix following various liver injuries, which results in the deformation of the normal liver architecture and the development of liver cirrhosis and even hepatocellular carcinoma. Numerous in vitro and in vivo studies indicated that oxidative stress mediates the initiation and progression of liver fibrosis. Overaccumulation of reactive oxygen species disrupts macromolecules, induces necrosis and apoptosis of hepatocytes, stimulates the production of pro-fibrogenic mediators, and directly activates hepatic stellate cells, thereby resulting in liver damage and initiating liver fibrosis. Ameliorating oxidative stress is a potential therapeutic strategy for the treatment of liver fibrosis. Natural antioxidants have attracted increasing attention in treating liver fibrosis due to their safety and efficacy. In this review, the pathogenesis of liver fibrosis and the role of oxidative stress in liver fibrosis were discussed. Naturally occurring antioxidants that can treat and prevent liver fibrosis were summarized. Advances in clinical trials were also presented. The main purpose of this review is to provide a comprehensive and up-to-date knowledge from the biological importance of oxidative stress in liver fibrosis to representative antioxidants for treating liver fibrosis. Naturally occurring antioxidants show a potential for further investigations as lead compounds in fighting liver fibrosis.

Key words

liver fibrosis - antioxidants - oxidative stress - reactive oxygen species - hepatic stellate cells - natural products

Abbreviations

ALP: alkaline phosphatase

ALT: alanine aminotransferase

AMPK: AMP-activated protein kinase

APAP: acetaminophen

ARE: antioxidation response element

AST: aspartate aminotransferase

BDL: bile duct-ligated

DEN: diethylnitrosamine

DMN: dimethylnitrosamine

DPPH: 2,2-diphenyl-1-picrylhydrazyl

ECM: extracellular matrix

EGCG: epigallocatechin gallate

ET-1: endothelin-1

FFA: free fatty acid

γ-GCS: γ-glutamylcysteine synthetase

GPx: glutathione peroxidase

GR: glutathione reductase

GSH: glutathione

GSK-3β : glycogen synthase kinase 3β

GST: glutathione transferase

HBV: hepatitis B virus

HCC: hepatocellular carcinoma

HCV: hepatitis C virus

HFD: high-fat diet

HO: heme oxygenase

HSC: hepatic stellate cell

ICR: Institute of Cancer Research

iNOS: inducible NO synthase

KEAP-1: kelch-like ECH-associated protein-1

LPS: lipopolysaccharides

MAO: monoamine oxidase

MAPK: mitogen-activated protein kinase

MDA: malondialdehyde

MMP: matrix metalloproteinase

NADPH: nicotinamide adenine dinucleotide phosphate

NAFLD: nonalcoholic fatty liver disease

NAS: NAFLD activity score

NASH: nonalcoholic steatohepatitis

NDMA: N’-nitrosodimethylamine

NF-κB: nuclear factor κ-light-chain-enhancer of activated B

NO: nitric oxide

NQO1: NAD(P)H quinine oxidoreductase 1

NRF2: the nuclear factor erythroid 2-related factor 2

3-NT: 3-nitrotyrosine

OPN: osteopontin

OS: oxidative stress

PAI-1: plasminogen activator inhibitor-1

PCNA: proliferating cell nuclear antigen

PDGF: platelet-derived growth factor

ROS: reactive oxygen species

α-SMA: α-smooth muscle actin

SOD: superoxide dismutase

SP-1: the specificity protein-1

TAA: thioacetamide

TBARS: thiobarbituric acid reactive substances

TGF-β1: transforming growth factor-β1

TIMP: tissue inhibitor of metalloproteinase

VLDL-TG: very-low-density lipoprotein-triglycerides
 

Introduction

Liver diseases, including chronic hepatitis, steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), have become continuous and increasing threats for public health [1], [2], [3], [4], [5]. Liver fibrosis is a wound-healing process during liver injury caused by viral infection, inflammatory response, high lipid diet, drugs, environmental pollutants, excessive alcohol intake, and autoimmune response, which is characterized by the deposition of collagen and accumulation of extracellular matrix (ECM) [6], [7]. Liver fibrosis results in the deformation of the normal liver architecture, HCC, and ultimately liver failure that is accompanied by remarkable morbidity and mortality. Fibrotic diseases are a major cause of mortality in industrialized nations [8], [9]; therefore, anti-fibrotic therapies are urgently needed.

In this review, scientific studies from 1980 to 2018 were searched using Google Scholar, PubMed, Web of Science, and Scopus, with “liver fibrosis”, “natural products”, and “antioxidant” as keywords. Only English publications were selected. Herbal extracts without clear components and those with inadequate or insufficient data in terms of examination assays with controls are excluded as described in [Fig. 1]. Review articles are also excluded. Thirty-five naturally occurring antioxidants with anti-liver fibrotic property from 57 studies were summarized in this review.

Fig. 1 Criteria used to select the articles for the data presented in this review.

The Pathogenesis of Liver Fibrosis

Many factors, such as hepatic viral infection, excessive alcohol consumption and drug intake, and high-fat diet (HFD) feeding, can cause temporary (acute) or self-limited lesion in the liver, characterized by the induction of a focal inflammatory response, enzymatic evidence of liver damage, and hepatic necrosis and apoptosis [6]. Acute or self-limited lesion in the liver provokes ECM deposition and transiently changes the liver architecture for the sake of healing. These lesions are potentially reversible responses to the injury at the early stage of liver fibrosis. However, these situations could aggravate and develop into irreversible liver cirrhosis and ultimately into HCC with sustained liver injury and overaccumulation of ECM [7]. In normal liver, ECMs maintain a state of dynamic equilibrium [10]. To maintain the balance of production and degradation of ECM, matrix metalloproteinases (MMPs) are activated to remove the over-deposited ECM and protect the liver against irreversible harm. The TIMPs (tissue inhibitor of metalloproteinases) hinder the clearance of ECM, which worsens the situation ([Fig. 2]) [11], [12]. Hepatic stellate cells (HSCs), contributing to approximately 90% of ECM production in myofibroblasts, play a pivotal role during ECM generation and fibrotic scar formation ([Fig. 2]) [13]. In normal conditions, HSCs store vitamin A, control the production of ECM, and regulate local vascular contractility, which are important functions in liver development, metabolism, immune response, and angiogenesis. Upon stimulation, quiescent HSCs are induced into activated HSCs, which are proliferating, fibrogenic, and contractile [13]. Cells derived from the bone marrow derived cells, including circulating fibrocytes and portal fibroblasts, are also transdifferentiate into fibrogenic myofibroblasts during liver injury ([Fig. 2]) [13].

Fig. 2 The general pathogenesis of liver fibrosis.

Regulation of HSCs activation and ECM deposition is a potential strategy for the treatment of liver fibrosis [7], [14]. A number of studies have proved the therapeutic agents suppressing HSC activation and ECM accumulation are promising for the treatment of liver fibrosis [15], [16], [17].

Oxidative Stress in Liver Fibrosis

Oxidative stress (OS) is a disturbance in the balance between the production of free radicals and antioxidant defenses and is involved in the pathogenesis of various liver diseases [18]. In liver, many factors, including chronic and excessive alcohol consumption [19], HFD [20], hepatic viral infection [21], and autoimmune response [22], contribute to the onset of OS. OS disrupts the structure and function of biologically relevant macromolecules such as nucleic acids, proteins, lipids, and carbohydrates. In hepatocytes, reactive oxygen species (ROS) are mainly generated by the electron transport chain in the mitochondria, in the endoplasmic reticulum during protein folding and detoxification by cytochrome P450 systems, in the lysosomes during the removal of damaged cellular components, and in the peroxisomes during metabolic or detoxification activities [23], [24]. The prototypic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the plasma membrane and phagosomes also contributes to the production of ROS in Kupffer cells [25]. When the intracellular antioxidant defense system is overwhelmed, excessive ROS induce liver dysfunction and injury to stimulate inflammatory responses and the infiltration of neutrophils [26]. Kupffer cells, endothelial cells, and infiltrating inflammatory cells then secrete transforming growth factor-β1 (TGF-β1) following the liver injury. The cross-talk between hepatocytes and HSCs is bidirectional. On the one hand, the injured hepatocytes, activated Kupffer cells, and infiltrating neutrophils generate excessive ROS and secrete inflammatory cytokines and TGF-β1 to activate HSCs, which results in overproduction of ECM and the specific inhibitors of MMP, such as TIMP-1 and TIMP-2 [27], [28]. On the other hand, the activated HSCs produce ROS to destroy hepatocytes and activate Kupffer cells. In addition, TGF-β1 induces its own expression in activated HSCs, thereby creating an autocrine loop [29].

Challenging with free radicals from cellular and xenobiotic metabolism and immune process, liver cells develop their enzymatic and non-enzymatic antioxidative defense systems ([Fig. 3]). Superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), heme oxygenase (HO), and catalase are commonly involved in enzymatic antioxidant defense [30]. Some signaling pathways, including NF-κB (nuclear factor κ-light-chain-enhancer of activated B) and NRF2 (the nuclear factor erythroid 2-related factor 2)/HO-1, are involved in fighting against oxidative damage. The NF-κB subunits are oxidized by ROS, which impair the DNA binding and transcriptional activity of NF-κB. Its inhibitor IκBα is degraded with the help of IKK kinase complexes [31]. Hence, the activation of IKK kinase activity is a potent strategy to enhance the NF-κB activity and strengthen its antioxidant capacity [32]. As a transcription factor, NRF2 dissociates from its inhibitor kelch-like ECH-associated protein-1 and binds to antioxidation response element (ARE) in nucleus to activate the transcription of its target genes, including NAD(P)H quinine oxidoreductase 1, HO-1, and γ-glutamylcysteine synthetase under OS [33].

Fig. 3 The non-enzymatic and enzymatic antioxidative defense systems.

Apart from the enzymatic antioxidant defense, some endogenous antioxidants, such as ascorbic acid (vitamin C), carotenoids, α-tocopherol (vitamin E), and glutathione (GSH), participate in non-enzymatic antioxidant defense ([Fig. 3]) [30]. Hence, antioxidants strengthening the cellular antioxidative capacity are promising strategies for treatment of liver fibrosis [18], [34].

Antioxidants for the Treatment of Liver Fibrosis

Considering that OS has been implicated in fibrogenic stimulation for decades and overaccumulation of ROS is a crucial part of fibrotic pathway, the application of antioxidants for the treatment of liver fibrosis has been well documented [35], [36]. Recent findings contributed a concept of redox-fibrosis, in which the cellular oxidant and antioxidant systems could serve as potential therapeutic targets [37], [38]. Several anti-fibrotic therapies aimed to modulate OS and the generation of ROS have been put forward, and some of them have already been involved in clinical trials [39]. The safety and efficacy of natural antioxidants have attracted increasing attention. Hence, naturally occurring antioxidants for the treatment of liver fibrosis and the potential mechanisms were summarized ([Table 1]).

Table 1 Naturally occurring antioxidants for the treatment of liver fibrosis.

Compound	Source	Model	Dosage	Mechanisms	Clinical trials	References
SRE (wogonin as a major component)	Scutellariae radix and Rhei rhizome, 1: 2	DMN-induced liver fibrosis in rats	1.25 and 6.25 mg/kg, oral administration daily for 3 wk	↑SOD, GPx, and catalase; ↓MDA; ↓oxidation of protein and DNA; ↓ROS; ↑NRF2-ARE signaling; ↓NF-κB pathway	no report	[44]
Morin	Maclura pomifera, Maclura tinctoria and Psidium guajava	LX-2 cells; DEN-induced liver fibrosis in rats	50 µM; 50 mg/kg, orally by gavage thrice per week for 6 wk	↓proliferation; ↓Wnt signaling; ↓GSK-3β, β-catenin, and cyclin D1	no report	[45]
		Chronic CCl4-induced liver fibrosis in rats	30 mg/kg, oral administration daily for 8 wk	↓MDA and NO; ↑GSH; ↓hepatic hydroxyproline content		[46]
Galangin	Alpinia officinarum (lesser galangal) and Helichrysum aureonitens	CCl4-induced liver fibrosis in rats	40 and 80 mg/kg, orally by gavage daily for 12 wk	↓MDA and hydroxyproline concentration; ↑SOD and GPx; ↓α-SMA and TGF-β1	no report	[47]
Isorhamnetin	Yellow and red onions	Murine HSCs and LX-2 cells; CCl4-induced liver fibrosis in rats	50 and 100 µM; 10 and 30 mg/kg, oral administration 5 d per week for 4 wk	↓α-SMA, PAI-1, and collagen; ↓ROS; ↓4-hydroxynonenal and nitrotyrosine-positive cells and GSH depletion; ↓TGF-β/Smad signaling	no report	[48]
Myricitrin	Myrica rubra (Chinese bayberry)	CCl4-induced liver fibrosis in mice	30 and 100 mg/kg, orally by gavage daily for 2 d	↓lipid peroxidation; ↑GSH level and CYP2E1 expression; ↓TGF-β1 and α-SMA; ↑proliferating cell nuclear antigen expression	no report	[49]
Naringin	Grapes, oranges and tomatoes	Nickel- and cadmium-induced toxicity in rats	orally administrated 80 mg/kg for 20 d or 50 mg/kg for 4 wk, respectively	↓lipid peroxidation; ↑antioxidant defense	no report	[53], [54]
Naringenin		Ethanol-induced liver fibrosis in rat	50 mg/kg, oral administration daily for 30 d	↓expression of collagen; ↓HSCs activation	no report	[55], [56]
		Fructose-treated rat	50 mg/kg, oral administration daily for 45 d	↓lipid peroxidation and protein oxidation; ↑SOD, catalase, GPx, GST, and GR; ↑GSH, vitamins C and E		[57]
		Lead acetate-treated rats	50 mg/kg, orally by gavage daily for 4 wk	↑GSH, catalase, and GPx		[58]
Pinocembrin	Penthorum chinense	HSC-T6 and LX-2 cells	10, 20, and 40 µM	↓ROS; ↓TGF-β1; ↓SMAD nuclear translocation	no report	[62]
Silymarin	The seeds of Silybum marianum (milk thistle)	CCl4-induced liver fibrosis in rats	100 mg/kg, oral administration daily for 10 d, or 200 mg/kg, oral administration three times daily for 8 wk	↓MDA; ↑GSH; ↑Mn-SOD, Cu/Zn-SOD, and GPx activities; ↓connective tissue growth factor	NCT02006498, NCT00680407, NCT00389376	[64], [70]
		Sodium nitrite-induced liver fibrosis in rats	10 and 25 mg/kg, oral administration daily for 12 wk	↓MDA; ↑hepatic GR and GPx; ↑mitochondrial cytochrome C oxidase function		[71]
EGCG	Green tea	HFD-fed female Sprague-Dawley rats	50 mg/kg, intraperitoneal injection, 3 times per week for 8 wk	↓expressions of key pathological oxidative and pro-inflammatory markers; ↓TGF/SMAD, PI3K/Akt/FoxO1, and NF-κB pathways	no report	[75]
Glycyrrhetinic acid	The roots of Glycyrrhiza glabra (licorice)	CCl4-induced chronic liver fibrosis in mice	25, 50, and 100 mg/kg, subcutaneous injection daily for 30 d	↓MAO and MDA; ↑NRF2, Cu/Zn-SOD, GPx, and catalase	no report	[78]
Glycyrrhizin		CCl4-induced liver fibrosis in rats	12.5 and 25 mg/kg, intraperitoneal injection daily for 8 wk	↓SP-1 expression	NCT03349008	[80]
		Fructose-induced metabolic syndrome in rats	50 mg/kg, single intraperitoneal injection	↓NF-κB signaling; ↓phosphorylation of MAPKs		[81]
Astragaloside IV	Radix Astragali	HSCs from male Sprague-Dawley rats	3 – 100 µM	↓ROS and lipid peroxidation; ↑GSH; ↑NRF2 gene expression	no report	[82]
Ursolic acid	Apples, basil, cranberries, peppermint, rosemary, oregano and prunes	CCl4-induced liver fibrosis in ICR mice	25 and 50 mg/kg, oral injection daily for 6 wk	↓ROS; ↑SOD, catalase, and GPx; ↑NRF2 expression	no report	[84]
Andrographolide	Andrographis paniculata	APAP-induced liver fibrosis in mice	20 and 40 mg/kg, oral administration daily for 4 wk	↓hepatic collagen deposition and HSCs activation; ↓OS; ↑NRF2 nuclear translocation and the expression of antioxidant genes	no report	[86]
Geniposide	Fruits of Gardenia jasminoides Ellis	HFD-induced NASH in rats	50 and 100 mg/kg, oral administration daily for 6 wk	↓FFA content; ↑SOD and GPx levels; ↓MDA level	no report	[90]
		Ethanol- tripterygium glycosides- and CCl4-induced liver injury in mice models	20, 40, and 80 mg/kg, intragastrical administration daily for 7 d; 400 mg/kg, oral administration daily for 6 d	↑SOD, GSH, and catalase		[91], [92], [93]
β-carotene	Dunaliella salina	CCl4-induced hepatotoxicity male ICR mice	71, 355, and 710 mg/kg, oral treatment daily for 8 wk	↑SOD, catalase, GPx, and GSH	no report	[94]
Matrine	Dried roots of Sophora flavescens Ait	HSC-T6 cells, CCl4-induced liver fibrosis in rats	1 – 2 mM; 50 and 100 mg/kg, intragastrical administration daily for 12 wk	↓collagen synthesis; ↓serum hyaluranic acid levels and hepatic hydroxyproline contents	no report	[100]
		High-fructose-diet-induced NAFLD in rats	40, 80, and 160 mg/kg, oral administration daily for 4 wk	↓MDA level; ↑GSH content; ↑NRF2 activity; ↓NF-κB activation		[101]
Berberine	European barberry, goldenseal, goldthread, Oregon grape, phellodendron, and tree turmeric	CFSC-2G HSC and CCl4-induced liver fibrosis in mice	50 mg/kg, oral administration daily for 6 wk	↓proliferation of HSC; ↓expression of α-SMA; ↑SOD; ↓MDA content; ↑AMPK; ↓TGF-β1	no report	[102]
Armepavine	Nelumbo nucifera	TNF-α- or LPS-stimulated HSC-T6 cells and BDL-induced liver fibrosis in rats	1 – 10 µM; 3 and 10 mg/kg, oral administration twice daily for 3 wk	↓α-SMA AP-1 and collagen; ↓TGF-β1, TIMP-1 and iNOS gene expression; ↑metallothionein genes	no report	[105]
Caffeine	Coffee beans	TAA-induced liver fibrosis in rats and HSC-T6 cells	50 mg/kg, oral administration daily for 8 wk	↓expression of TGF-β and pro-fibrogenic proteins; ↓HSCs activation; ↓expression of α-SMA	no report	[106]
Nicotinic acid	Plants and animals	TAA-induced liver fibrosis in rats	50 mg/kg, oral administration daily for 8 wk	↑GPx levels; ↓TGF-β levels; ↓HSC activation and ECM deposition; ↓activities of MMP-2 and -9	no report	[107]
Betaine	Sugar beet	Ethanol plus CCl4-treated liver fibrosis in rats	2% w/w in food for 14 wk	↓α-SMA and TGF-β protein expressions; ↓MMP-2, TIMP-1, and TIMP-2 mRNA expressions	no report	[108]
Curcumin	Rhizomes of Curcuma longa (turmeric)	LPS-treated rats	30 or 60 mg/kg, oral administration daily for 7 d	↓TBARS level; ↑GSH and SOD levels	NCT02908152	[112]
		CCl4-induced liver fibrosis in rats and HSCs	200 and 400 mg/kg, oral administration daily for 8 wk	↓OS; ↓HSC activation		[113]
Resveratrol	Polygonum cuspidatum Grapes and berries	DMN-induced liver fibrosis in rats	10 mg/kg, oral gavage daily for 7 d	↓MDA level; ↑SOD and GPx levels	NCT02030977, NCT01446276, NCT01464801, NCT02216552	[119]
		LPS-induced OS in rats	20 mg/kg, intraperitoneal injection daily for 7 d	↓lipoperoxidation; ↓SOD, catalase, and GPx activities		[120]
		NDMA-induced liver fibrosis in rats	10 mg/kg, intraperitoneal injection three times per week for 3 wk	↓α-SMA and MDA; ↑liver glycogen, SOD, and ATPases		[121]
Polydatin	Polygonum cuspidatum	Alcohol-induced hepatic injury C57BL/6 mice	50 or 100 mg/kg, oral administration daily for 8 d	↓ALT and AST; ↓OS; ↑mitochondrial content and MMPs levels	no report	[122]
Rosmarinic acid	Plants from the families of Blechnaceae and Lamiaceae	CCl4-induced liver fibrosis in rats	10, 25, and 50 mg/kg, oral gavage daily for 3 d	↓3-NT and TBARS formation; ↑SOD activity; ↓TGF-β1 and α-SMA expression; ↑NRF2 and HO-1	no report	[123]
Chlorogenic acid	Coffee beans and many plants	PDGF-treated HSC-T6 cells and CCl4-treated liver fibrosis in rats	30 and 60 mg/kg, intragastrical administration daily for 8 wk	↓ROS; ↓p38 and ERK1/2 phosphorylation; ↓proliferation and pro-fibrotic genes expression; ↓hydroxyproline content and expression of α-SMA, collagen I, collagen III, and TIMP-1; ↑nuclear NRF2; ↓MDA level; ↑GSH, SOD, and catalase levels	no report	[124], [125], [126]
Salidroside	Rhodiola rosea	High-fat- and high-cholesterol-fed NASH rat	150 and 300 mg/kg, oral administration daily for wk	↓lipid accumulation; ↑antioxidant enzyme levels; ↓CYP2E1 and Nox2 mRNA	no report	[128]
Cinnamaldehyde	In the bark of cinnamon trees and other Species of the genus Cinnamomum	CCl4-injured rats	10 mg/kg, oral administration daily for 6 consecutive d	↓ALT, AST, and lactate dehydrogenase activities; ↓OS and inflammation mediated through TLR2 pathway	no report	[129]
Apocynin	Apocynum cannabinum	CCl4-induced liver fibrosis in female Long Evans rats	100 mg/kg, oral administration daily for 2 wk	↓AST, ALT, and ALP activities; ↓OS markers; ↑catalase and SOD activity	no report	[130]
Cichoric acid	A variety of plant species	Methionine- and choline-deficient diet-fed mice	10 and 30 mg/kg, oral administration daily for 4 wk	↑antioxidant enzymes; ↓inflammation; ↓fibrosis, apoptosis, and lipogenesis-related gene expression; ↑AMPK	no report	[131]
Rhein	Rheum palmatum	APAP-induced hepatotoxicity in rats	10, 20, and 40 mg/kg, oral administration daily for 2 d	↓ROS, NO, and MDA; ↑GSH content	no report	[134]
Sauchinone	Saururus chinensis	LX-2 cells, CCl4-induced liver fibrosis in mice	10 and 20 mg/kg, oral administration four times per week for 4 wk	↓phosphorylation of Smad2/3; ↓the transcript levels of PAI-1 and MMP-2; ↓α-SMA; ↓4-hydroxynonenal and nitrotyrosine	no report	[135]
Osthole	Fruits of Cnidium monnieri Cusson	TAA-induced liver fibrosis in rats and activated HSCs (HSC-T6 and LX-2)	10 mg/kg, oral administration twice per day for 4 wk; 3 and 10 µg/mL	↓hepatic collagen and α-SMA; ↓expression of fibrosis-related genes; ↓production of fibrosis-related cytokines and chemokines; ↓TGF-β1-induced migration and invasion; ↓TGF-β1- or ET-1-induced HSCs contractility	no report	[137]

Flavonoids

Flavonoids are a group of polyphenolic compounds with a C6-C3-C6 core structure, and most flavonoids possess antioxidant property. Some flavonoids have been reported with protective effect against liver fibrosis ([Fig. 4] and [Table 1]).

Fig. 4 Flavonoids for the treatment of liver fibrosis.

Wogonin is a major flavonoid from the dried roots of Scutellaria baicalensis Georgi (Lamiaceae) [40]. Wogonin possesses the hepatic protective abilities including anti-virus, anti-inflammation, apoptosis induction of HCC cells, and free radical scavenging [41], [42], [43]. The ethanol extract from a herbal combinatorial formula (SRE, 1.25 and 6.25 mg/kg body weight, oral administration daily for 3 wk), containing wogonin as a major ingredient, showed a protective effect against dimethylnitrosamine (DMN)-induced liver fibrosis in rats [44]. Furthermore, the anti-fibrotic effect of SRE is mediated by elevating the levels of SOD, GPx, and catalase in DMN-exposed liver to prevent oxidative damage, decreasing the malondialdehyde (MDA) level to protect liver from DMN-induced lipid peroxidation, and ameliorating the oxidation of protein and DNA to restore liver injury and improve organ functions. SRE attenuates DMN-mediated liver injury by removing the excess accumulated ROS and inducing NRF2-ARE signaling to stimulate the expression of antioxidative enzymes [44].

Morin is a flavonoid isolated from Maclura pomifera (Raf.) C. K. Schneid. (Osage orange, Moraceae), Maclura tinctoria (L.) Steud. (old fustic, Moraceae), and the leaves of Psidium guajava L. (common guava, Myrtaceae). Morin inhibits the proliferation of human HSCs LX-2 cells, suppresses Wnt signaling, and induces G1 cell cycle arrest at the concentration of 50 µM. Morin (50 mg/kg body weight, orally by gavage thrice per week for 6 wk) ameliorates diethylnitrosamine (DEN)-induced liver fibrosis in rats by downregulating the expression levels of glycogen synthase kinase 3β (GSK-3β), β-catenin, and cyclin D1 [45]. The treatment of morin (30 mg/kg body weight, oral administration daily for 8 wk) attenuates the liver index and serum biomarkers of liver function that were enhanced by chronic CCl₄ intoxication, with silymarin (100 mg/kg body weight) as a positive control. Furthermore, morin inhibits the elevated levels of MDA and nitric oxide (NO) and restores GSH to its normal level in hepatocytes. The increased hepatic hydroxyproline content is markedly decreased by the administration of morin [46]. Hence, morin could be employed as a promising preventive natural supplement for liver fibrosis.

Galangin is a flavonol, present in Alpinia officinarum Hance (lesser galangal, Zingiberaceae) and Helichrysum aureonitens Sch. Bip. (Compositae). In liver fibrotic rats induced by the subcutaneous injection of CCl₄, galangin (40 and 80 mg/kg body weight, orally by gavage daily for 12 wk) reverses the CCl₄-induced increase of hyaluronic acid, laminin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) and decrease of total protein and albumin in serum; colchicin (0.2 mg/kg body weight) was used as a positive control [47]. Galangin markedly reduces hepatic MDA and hydroxyproline concentration and increases the activities of liver SOD and GPx compared with CCl₄-treated rats. In addition, galangin significantly downregulates the expression levels of α-smooth muscle actin (α-SMA) and TGF-β1 [47]. Hence, galangin might inhibit the CCl₄-induced liver fibrosis in rats, probably by removing oxygen free radicals, decreasing lipid peroxidation, and inhibiting HSCs activation and proliferation.

Isorhamnetin commonly exists in pungent yellow and red onions (Amaryllidaceae). Isorhamnetin (50 and 100 µM) inhibits the TGF-β1-induced expression of α-SMA, plasminogen activator inhibitor-1 (PAI-1), and collagen in primary murine HSCs and LX-2 cells [48]. Isorhamnetin increases the nuclear translocation of NRF2 and blocks the TGF-β1-induced ROS production in HSCs. Furthermore, isorhamnetin (10 and 30 mg/kg body weight, oral administration 5 d/wk for 4 wk) prevents the CCl₄-induced increase in serum ALT and AST levels and causes histopathological changes characterized by the decrease in collagen accumulation [48]. Isorhamnetin attenuates the CCl₄-induced increase in the number of 4-hydroxynonenal and nitrotyrosine-positive cells and prevented GSH depletion. Isorhamnetin can inhibit the TGF-β/Smad signaling pathway and relieve OS, thus inhibiting HSC activation and preventing liver fibrosis.

Myricitrin (myricetin-3-O-α-rhamnoside) is a flavonoid from Myrica rubra Sieb. et Zucc. (Chinese bayberry, Myricaceae), possessing antioxidant and anti-inflammatory activities. Myricitrin (30 and 100 mg/kg body weight, orally by gavage daily for 2 d) ameliorates the CCl₄-induced increase in serum AST and ALT levels and the histopathological changes in the liver with silymarin (100 mg/kg body weight) as a positive control. Liver OS is reduced by myricitrin, as evidenced by the decrease in lipid peroxidation with a concomitant increase in GSH level and CYP2E1 expression. TGF-β1 and α-SMA expression is markedly ameliorated by myricitrin, indicating the inhibition of pro-fibrotic response. Myricitrin also improves the regeneration of hepatic tissue after CCl₄-intoxication, as evidenced by increased proliferating cell nuclear antigen expression [49], indicating its significant anti-fibrotic activity.

Naringenin and its glycoside naringin are naturally occurring citrus flavanones predominantly found in grapes (Vitaceae), oranges (Rutaceae), and tomatoes (Solanaceae), possessing have a wide range of pharmacological properties, including anti-dyslipidemia, anti-obesity, and anti-fibrosis [50], [51], [52]. Naringin attenuates the nickel- or cadmium-induced liver toxicity in rats when orally administrated with 80 mg/kg body weight for 20 d or 50 mg/kg body weight for 4 wk, respectively, by significantly reducing lipid peroxidation and restoring the levels of antioxidant defense [53], [54]. Oral treatment of 50 mg/kg naringenin for 30 d decreases the expression of collagen in ethanol-induced liver fibrotic rats, which is associated with reduced OS and HSC activation [55], [56]. In fructose-administered rats, naringenin (50 mg/kg body weight/day for 45 d) reduces the levels of oxidative markers by inhibiting liver cell leakage, lipid peroxidation, and protein oxidation, and enhances the antioxidant potential by elevating enzymatic antioxidant activities including SOD, catalase, GPx, GST, and GR in liver. Moreover, naringenin supplementation increases the levels of non-enzymatic antioxidants such as GSH and vitamins C and E in rats [57]. Histopathological evidence indicates that naringenin (50 mg/kg body weight, orally by gavage daily for 4 wk) reduces the liver damage in lead acetate-administered rats by improving GSH, catalase, and GPx levels. This finding demonstrates that naringenin serves as an antioxidant and chelating agent to compete against lead acetate-induced OS and damage in liver [58]. Hence, naringenin and naringin are potential antioxidants for treatment of liver fibrosis.

Pinocembrin is a flavonoid isolated from Penthorum chinense Pursh (Penthoraceae), which has been widely used for liver protection for thousands of years [59], [60]. The water extract of P. chinense (5.15 and 10.30 g/kg body weight/day for 4 wk) protects the liver against chronic ethanol-induced injury in mice by attenuating ROS generation and MDA level, restoring GSH depletion, and increasing SOD and GPx activities with silymarin (86 mg/kg body weight) as a positive control [61]. In a recent study, pinocembrin from P. chinense suppresses the activation of both human HSC LX-2 and rat HSC-T6 cells at the concentrations of 10, 20, and 40 µM, mediating through ROS production alleviation, TGF-β1 inhibition, and prevention of Smad nuclear translocation, which suggest its potential for protection against liver fibrosis [62]. Further in vivo studies are needed to verify the anti-fibrotic effect of P. chinense and pinocembrin.

Silymarin, a mixture of flavonolignans consisting of silibinin, isosilibinin, silicristin, and silidianin, is isolated from the seeds of Silybum marianum (L.) Gaertn. (milk thistle, Compositae) [63], [64]. Silymarin has been used for centuries to treat liver, spleen, and gallbladder disorders [64], [65]. As the most well-studied natural product in the treatment of liver disease, silymarin has been recognized as “liver tonics” [66]. Silymarin is used to combat various liver conditions in both clinical settings and experimental models [63], [64], [67], [68]. As an antioxidant, silymarin mainly reduces free radical production and lipid peroxidation [69]. Silymarin (100 mg/kg body weight, oral administration daily for 10 d) significantly decreases the MDA level and increases the GSH level in CCl₄-induced liver damage, indicating that this substance protects the liver from damage with notable redox functions [64]. Silymarin (200 mg/kg body weight, oral administration 3 times daily for 8 wk) also improves liver fibrosis in CCl₄-treated rats by decreasing the connective tissue growth factor [70]. Silymarin increases the activities of MnSOD, Cu/ZnSOD, and GPx in liver [70]. Silymarin (10 and 25 mg/kg body weight, oral administration daily for 12 wk) also prevents sodium nitrite-induced liver fibrosis in rats by reducing hepatic MDA levels, restoring the activity of hepatic GR and GPx, and inhibiting the deactivation of mitochondrial cytochrome C oxidase function [71].

Several studies have revealed the protective effects of epigallocatechin gallate (EGCG) or green tea polyphenols against liver fibrosis on various animal models [72], [73], [74]. Intraperitoneal injection with 50 mg/kg of EGCG thrice per week for 8 wk improves the hepatic histology (decreased number of fatty score, necrosis, and inflammatory foci), reduces liver injury, and attenuates hepatic changes, including fibrosis, by downregulating the expression levels of key pathological oxidative and pro-inflammatory markers in HFD-fed female Sprague-Dawley rats [75]. EGCG treatment also counteracts the activity of TGF/Smad and NF-κB pathways [75]. For the in vitro and in vivo models of thioacetamide (TAA)-induced hepatic fibrosis, EGCG inhibits the osteopontin (OPN)-dependent injury and fibrosis, primarily by upregulating miR-221 to accelerate OPN degradation [76]. Thus, green tea polyphenols and EGCG are useful supplements in the prevention of nonalcoholic fatty liver disease (NAFLD).

Terpenoids

Terpenoids are a large and diverse class of natural products derived from isoprene units. Some terpenoids have been reported with anti-fibrotic effects ([Fig. 5] and [Table 1]).

Fig. 5 Terpenoids for the treatment of liver fibrosis.

Glycyrrhetinic acid and glycyrrhizin are the major bioactive constituents isolated from the roots of Glycyrrhiza glabra L. (Leguminosae) [77]. In CCl₄-treated mice, glycyrrhetinic acid (25, 50, and 100 mg/kg body weight, subcutaneous injection daily for 30 d) reverses the CCl₄-induced increase in serum monoamine oxidase and MDA and decrease in nuclear NRF2 expression and its target genes, including Cu/Zn-SOD, catalase, and GPx, with silymarin (100 mg/kg body weight) as a positive control. In addition, glycyrrhetinic acid exhibits the antioxidant effects in vitro on FeCl₂-ascorbate-induced lipid peroxidation in mouse liver homogenates and on 2,2-diphenyl-1-picrylhydrazyl-scavenging activity [78]. These results suggest that glycyrrhetinic acid may be an effective hepatoprotective agent and a viable candidate for treating liver fibrosis. Glycyrrhizin has various pharmacological effects and has been used to treat chronic hepatitis, especially hepatitis C virus (HCV) infection and associated diseases [79]. Glycyrrhizin (12.5 and 25 mg/kg body weight, intraperitoneal injection daily for 8 wk) attenuates CCl₄-induced liver fibrosis in rat by downregulating the expression of the specificity protein-1, the critical factor for the initiation of OS, in both transcriptional and translational levels [80]. In a fructose-induced metabolic syndrome rat model, a single intraperitoneal injection of 50 mg/kg body weight of glycyrrhizin prevents several complications of metabolic syndrome, including lipid peroxidation, protein carboxylation, and mitochondrial ROS generation, which resulted in attenuation of OS in liver by inhibiting the NF-κB inflammatory pathway and preventing the phosphorylation of MAPKs (mitogen-activated protein kinases) signaling pathway [81]. Glycyrrhetinic acid, glycyrrhizin, and the extract of G. glabra have shown protective effects against liver fibrosis in various in vitro and in vivo studies. Clinical trials are needed to further verify the anti-fibrotic effects.

Astragaloside IV, the active component of Radix Astragali (Leguminosae), possesses antioxidant property and anti-fibrotic potential for renal fibrosis, and 3 – 100 µM astragaloside IV attenuates OS in activated HSCs from male Sprague-Dawley rats by scavenging ROS, reducing lipid peroxidation, elevating the cellular level of GSH, and stimulating NRF2 gene expression. The depletion of cellular GSH by buthionine sulfoximine or abrogation of p38 MAPK with SB-203580 eliminated the inhibitory effects of astragaloside IV on genes relevant to HSC activation [82]. These studies provide novel insights into the mechanisms of astragaloside IV as an anti-fibrogenic candidate in the prevention and treatment of liver fibrosis.

Ursolic acid, a natural pentacyclic triterpenoid, has been found in various plants including apples (Rosaceae), basil (Lamiaceae), cranberries (Ericaceae), peppermint (Lamiaceae), rosemary (Lamiaceae), oregano (Lamiaceae), and prunes (Rosaceae) and possesses many biological activities, including antioxidation and anti-inflammation [83]. Ursolic acid (25 and 50 mg/kg body weight, oral injection daily for 6 wk) prevents CCl₄-induced hepatotoxicity and fibrosis in ICR (Institute of Cancer Research) mice with colchicine (1 mg/kg body weight) as a positive control. The CCl₄-induced profound elevations of OS, as well as inflammation and apoptosis in liver, are suppressed by ursolic acid through modulating the NRF2/ARE signaling pathway [84]. These results suggest that ursolic acid has hepatoprotective actions.

Andrographolide is a labdane-type diterpenoid isolated from Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae), which has a broad range of therapeutic applications including anti-inflammatory and anti-platelet aggregation activities and potential antineoplastic properties [85]. Andrographolide (20 and 40 mg/kg body weight, oral administration daily for 4 wk) decreases hepatic collagen deposition and HSCs activation in APAP (acetaminophen)-induced mice [86]. Andrographolide alleviates liver OS and reduces ROS formation in HSCs. Andrographolide enhances the nuclear translocation of NRF2 and increases the expression of its downstream genes both in vitro and in vivo. Andrographolide might be clinically applied for the treatment of liver fibrosis.

Geniposide is an iridoid glycoside from the fruits of Gardenia jasminoides Ellis (Rubiaceae), which is useful against hyperlipidemia and fatty liver diseases [87], [88]. Geniposide at 5 and 20 µM effectively prevents TGF-β1-induced fibrotic responses in AML12 cells [89]. In HFD-induced nonalcoholic steatohepatitis (NASH) rats, the free fatty acid content is reduced by geniposide (50 and 100 mg/kg body weight, oral administration daily for 6 wk), suggesting its potential to prevent HFD-induced liver injury [90]. Geniposide markedly increases endogenous antioxidants and SOD and GPx levels but decreases the MDA level to protect liver cells from oxidative damage [90]. The antioxidant property of geniposide is related to its ability to reduce free radical formation and enhance free radical scavenging. The hepatoprotective activities of geniposide (20, 40, and 80 mg/kg body weight, intragastrical administration daily for 7 d) are also identified in mice models treated with alcohol, tripterygium glycosides, or CCl₄ with bifendate (150 mg/kg body weight) as a positive control [91], [92]. The ALT, AST, and alkaline phosphatase (ALP) levels are significantly decreased by geniposide in the above models [91], [92]. Moreover, geniposide (400 mg/kg body weight, oral administration daily for 6 d) remarkably elevates the GSH level and increases the SOD and catalase activities in CCl₄-induced liver damage mice with biphenyldicarboxylate pills (100 mg/kg body weight) as positive controls [93]. Thus, geniposide could be a potential candidate for the treatment of liver fibrosis.

Dunaliella salina (Dunal) Teodoresco (Dunaliellaceae) is a unicellular biflagellate green alga from the Chlorophyceae class, which is rich in β-carotene. In CCl₄-induced hepatotoxicity male ICR mice, the oral treatment of D. salina extract (71, 355, and 710 mg/kg body weight) daily for 8 wk reverses the decreases in SOD, catalase, GPx, and GSH content and the increase in MDA content in liver. Liver histopathology shows that β-carotene reduces the incidence of liver lesions induced by CCl₄ with silymarin (200 mg/kg) as a positive control [94]. The results suggest that β-carotene exhibits potential protective effects against CCl₄-induced liver damage in mice.

Alkaloids

Alkaloids with protective effects against liver fibrosis were listed in [Fig. 6] and [Table 1]. Matrine is a primary active alkaloid isolated from the dried roots of Sophora flavescens Ait (Leguminosae), a commonly used traditional herb to cure hemafecia, dysentery, jaundice, and anuresis [88], [95]. Matrine has a variety of pharmacological properties, including anti-inflammatory, immunity regulatory, antiviral, and anti-fibrotic effects [96], [97], [98], [99]. Matrine (1 – 2 mM) markedly reduces serum- or TGF-β1-driven collagen synthesis in HSC-T6 cells; matrine (50 and 100 mg/kg body weight, intragastrical administration daily for 12 wk) significantly decreases serum hyaluronic acid levels and hepatic hydroxyproline contents to attenuate CCl₄-induced liver fibrosis [100]. In a high-fructose-diet-induced NAFLD rat model, matrine (40, 80, and 160 mg/kg body weight, oral administration daily for 4 wk) retards the histopathological progression by restoring the increased MDA level, depleting GSH content, facilitating NRF2 translocation to the nuclei, and inhibiting hepatic NF-κB activation [101].

Fig. 6 Alkaloids for the treatment of liver fibrosis.

Berberine is an alkaloid found in several plants including European barberry (Berberidaceae), goldenseal (Ranunculaceae), goldthread (Ranunculaceae), Oregon grape (Berberidaceae), phellodendron (Rutaceae), and tree turmeric (Berberidaceae). Oral administration of 50 mg/kg berberine daily for 6 wk ameliorates CCl₄-induced liver fibrosis in mice by decreasing the enzyme release of ALT, AST, and ALP in the serum and elevating SOD and reducing MDA content in the liver tissue. Moreover, berberine treatment activates AMP-activated protein kinase (AMPK), decreases the expression levels of TGF-β1 and α-SMA, and inhibits the proliferation of CFSC-2G HSCs [102]. These results may benefit the development of berberine in the prevention of chronic liver disease.

Armepavine is an active compound from Nelumbo nucifera Gaertn. (Nelumbonaceae), exerting anti-inflammatory effects on human peripheral blood mononuclear cells [103] and immunosuppressive effects on T lymphocytes and lupus nephritic mice [104]. In HSC-T6 cells, armepavine (1 – 10 µM) attenuates TNF-α- and LPS-stimulated α-SMA protein expression and AP-1 activation. Armepavine (3 and 10 mg/kg body weight, oral administration twice daily for 3 wk) suppresses the TNF-α-induced collagen deposition. In bile-duct-ligated-treated rats, armepavine treatment reduces the plasma AST and ALT levels, hepatic α-SMA expression and collagen contents, and fibrosis scores. Moreover, armepavine attenuates the mRNA expression levels of TGF-β1, TIMP-1, and inducible NO synthase but upregulates metallothionein gene expression [105]. Hence, armepavine has anti-fibrotic effects.

The support on the beneficial effects of caffeine on the liver is increasing. Caffeine (50 mg/kg body weight, oral administration daily for 8 wk) protects against TAA-induced liver cirrhosis in rats by restoring the redox equilibrium and inhibiting the expression levels of TGF-β and pro-fibrogenic proteins. Caffeine also inhibits HSCs activation and suppresses the expression of α-SMA [106]. Caffeine may attenuate liver fibrotic processes.

Chronic TAA administration induces liver fibrosis, which is prevented by nicotinic acid. The oral administration of 50 mg/kg body weight nicotinic acid daily for 8 wk prevents the elevation of liver enzymes and restored the GPx levels. Additionally, nicotinic acid decreases the TGF-β levels and attenuates the oxidative processes, reducing the HSC activation and ECM deposition. Nicotinic acid decreases MMP-2 and − 9 activities [107]. Hence, nicotinic acid can be an anti-fibrotic agent against liver injury.

Betaine is an amino acid firstly discovered in sugar beet (Beta vulgaris L., Amaranthaceae), together with other beet cultivars. In ethanol with CCl₄-treated liver fibrosis rats, betaine treatment (2% w/w in food for 14 wk) diminishes the triglyceride level, the α-SMA and TGF-β protein levels, and MMP-2, TIMP-1, and TIMP-2 mRNA levels. Hence, the anti-fibrotic effect of betaine may be related to its suppressive effects on oxidant and inflammatory processes together with the HSC activation in alcoholic liver fibrosis [108].

Other compounds

In addition to the above-mentioned compounds, several other compounds have been reported to protect against liver fibrosis ([Fig. 7] and [Table 1]).

Fig. 7 Other natural compounds for the treatment of liver fibrosis.

Curcumin is a polyphenolic compound isolated from the rhizomes of Curcuma longa L. (turmeric, Zingiberaceae) [109]. Curcumin can treat a wide variety of inflammatory diseases including cancer, diabetes, and fibrosis [109], [110]. Curcumin has excellent antioxidant properties [111]. In LPS-challenged rats, the oral administration of 30 or 60 mg/kg curcumin daily for 7 d decreases the thiobarbituric acid reactive substances (TBARS) level and elevates GSH and SOD levels [112]. Curcumin (200 and 400 mg/kg body weight, oral administration daily for 8 wk) also protects the liver from CCl₄-induced injury by attenuating OS in vivo and inhibits HSC activation in vitro [113].

Resveratrol is a polyphenolic compound isolated from Polygonum cuspidatum Sieb. et Zucc (Polygonaceae). As a natural phytoalexin, resveratrol is also present in various plant species including grapes and berries and acts against environmental stress and fungal infection [114]. Resveratrol possesses anti-aging, anti-carcinogenic, anti-inflammatory, and antioxidant properties [115], [116], [117], [118]. In the rat model, the oral gavage of 10 mg/kg resveratrol daily for 7 d protects DMN-induced liver fibrosis by decreasing the MDA and increasing the SOD and GPx levels in liver [119]. Resveratrol (20 mg/kg body weight, intraperitoneal injection daily for 7 d) has a protective effect against LPS-induced OS in rat liver by reversing the LPS-induced lipoperoxidation and offsetting the depletion of SOD, catalase, and GPx activities [120]. In N’-nitrosodimethylamine-induced liver fibrosis rats, resveratrol supplement (10 mg/kg body weight, intraperitoneal injection 3 times per week for 3 wk) refurbishes the liver architecture by significantly restoring the levels of biomarkers of oxidative damage (MDA, SOD, protein carbonyls, and membrane-bound ATPases) and inhibits the α-SMA expression and HSC activation to obstruct liver fibrosis [121].

Polydatin is a resveratrol glucoside isolated from P. cuspidatum. The oral administration of 50 or 100 mg/kg polydatin daily for 8 d alleviates the alcohol-induced hepatic injury by reducing the liver injury markers, ALT and AST, attenuating OS, and restoring the antioxidant balance in the hepatic tissue. Polydatin pre-treatment prevents alcohol-induced mitochondrial damage and refurbishes the MMP levels in the liver [122]. Hence, polydatin may have a potential benefit in preventing alcohol-induced acute hepatic injury.

Rosmarinic acid is a natural phenolic acid found in a variety of plants, especially the families of Blechnaceae and Lamiaceae. Rosmarinic acid (10, 25, and 50 mg/kg body weight, oral gavage daily for 3 d) decreases 3-nitrotyrosine and TBARS formation but increases SOD activity to ameliorate OS in the liver tissue from CCl₄-treated rats. Additionally, rosmarinic acid downregulates the expression levels of TGF-β1 and α-SMA, suggesting the suppression of pro-fibrotic response. The hepatoprotective activity of rosmarinic acid occurs with enhanced NRF2 and HO-1 expression [123]. Thus, rosmarinic acid possesses anti-fibrotic activity against acute liver toxicity.

Chlorogenic acid is a phenolic compound and exerts anti-inflammatory and antioxidant activities. Chlorogenic acid (30 and 60 mg/kg body weight, intragastrical administration daily for 8 wk) prevents CCl₄-induced liver fibrosis by attenuating the inflammation and OS in rats [124], [125], [126]. Liver fibrosis is induced in CCl₄-injected rats, characterized by increased hydroxyproline content and the expression of α-SMA, collagen I, collagen III, and TIMP-1, which are alleviated markedly by chlorogenic acid [124]. Furthermore, chlorogenic acid increases the expression of nuclear NRF2 and its related antioxidant genes and suppresses the expression of NLRP3 inflammasome [127]. Chlorogenic acid decreases the MDA level and increases GSH, SOD, and catalase levels in liver tissues. In HSC-T6 cells, platelet-derived growth factor induces ROS production, p38 and ERK1/2 phosphorylation, proliferation and pro-fibrotic genes expression, which were reversed by chlorogenic acid treatment (12.5, 25, and 50 µg/mL) [126]. Hence, chlorogenic acid protects against liver fibrosis by suppressing OS.

Salidroside is a glucoside of tyrosol found from Rhodiola rosea L. (Crassulaceae). In high-fat and high-cholesterol-fed NASH rat model, salidroside treatment (150 and 300 mg/kg body weight, oral administration daily for weeks) effectively reduces lipid accumulation and inhibits liver injury in a dose-dependent manner [128]. Salidroside treatment restores the antioxidant enzyme levels and inhibits the expression of CYP2E1 and Nox2 mRNA in the liver, which prevents the initial step of free radical generation from NASH.

Cinnamaldehyde is a polyphenol possessing anti-inflammatory and antioxidant properties. In CCl₄-induced liver injury rats, daily oral administration of 10 mg/kg cinnamaldehyde for 6 consecutive days significantly reverses the CCl₄-induced elevation in serum ALT, AST, and lactate dehydrogenase activities [129]. Furthermore, cinnamaldehyde significantly reduces CCl₄-induced OS and inflammation mediating through toll-like receptor-4 (TLR-4) signaling pathway, as well as the expression of downstream transcription factors such as NF-κB. The protective effect of cinnamaldehyde is comparable to that of the positive control silymarin (100 mg/kg body weight, oral administration). Hence, cinnamaldehyde is a promising candidate for hepatoprotective therapy.

Apocynin, also known as acetovanillone, is a natural organic compound structurally related to vanillin. Female Long Evans rats were administered with CCl₄ orally (1 mL/kg) twice a week for 2 wk and were treated with apocynin (100 mg/kg, orally) daily for 2 wk. Apocynin significantly reduces serum AST, ALT, and ALP activities and inhibits OS markers (MDA and NO levels) in CCl₄ treated rats [130]. Apocynin treatment also restores the catalase and SOD activity in CCl₄ treated rats. Hence, apocynin protects liver damage induced by CCl₄ by inhibiting lipid peroxidation and stimulating the cellular antioxidant system.

Cichoric acid is a hydroxycinnamic acid, an organic compound of the phenylpropanoid class, which occurs in a variety of plant species. The oral administration of chicoric acid (10 and 30 mg/kg) daily for 4 wk reduces the OS by upregulating antioxidant enzymes and decreases the inflammation by inhibiting pro-inflammatory cytokines and NF-κB activation in mice fed with a methionine- and choline-deficient diet [131]. In addition, chicoric acid reduces the fibrosis, apoptosis, and lipogenesis-related gene expression and increases the AMPK activation. Chicoric acid may be effective in the treatment of NAFLD and NASH.

Rhein is a lipophilic anthraquinone derivative found in Rheum palmatum L. (Polygonaceae), which has been used as traditional herbal medicine in China for thousands of years [132], [133]. Rhein has various pharmacological effects including anti-inflammatory, antioxidant, and antimicrobial activities [132], [133]. The potential protective effects of rhein were investigated on APAP-induced hepatotoxicity in rats [134]. The results show a reduction in ROS production, NO level, and MDA level and the restoration of GSH content by oral rhein administration (10, 20, and 40 mg/kg body weigh daily for 2 d) in APAP-induced liver injured rat [134]. The anti-fibrotic effect of rhein might be connected to its property in ameliorating OS.

Sauchinone is a lignan found in Saururus chinensis (Lour.) Baill. (Saururaceae). Sauchinone (10 and 30 µM) blocks the TGF-β1-induced phosphorylation of Smad2/3 as well as the transcript levels of PAI-1 and MMP-2 in LX-2 cells. Sauchinone (10 and 20 mg/kg body weight, oral administration 4 times per week for 4 wk) significantly inhibits liver fibrosis in CCl4-injured mice, as indicated by the decrease in the regions of hepatic degeneration, inflammatory cell infiltration and the intensity of α-SMA staining in mice. Furthermore, sauchinone inhibits OS, as assessed by the staining of 4-hydroxynonenal and nitrotyrosine [135]. Sauchinone attenuates liver fibrosis and HSCs activation, which might be mediated by suppressing OS.

Osthole is an active component present in many medicinal plants especially in the fruits of Cnidium monnieri L. Cusson (Apiaceae), which has been clinically applied due to its various pharmacological properties, such as antioxidation and anti-inflammation [136]. In TAA-treated Sprague-Dawley rats, the oral administration of 10 mg/kg osthole twice per day for 4 wk significantly reduced liver injury by diminishing the plasma AST and ALT levels, improving the histological architecture, decreasing the collagen and α-SMA accumulation, and improving hepatic fibrosis scores. In HSCs (HSC-T6 and LX-2), osthole (3 and 10 µg/mL) reduces the expression of fibrosis-related genes, suppresses the production of fibrosis-related cytokines and chemokines, attenuates the TGF-β1-induced migration and invasion in HSCs, and alleviates the TGF-β1- or endothelin-1-induced HSCs contractility [137].

Clinical Trials

With the growing demands for a safe, effective, and economic treatment of liver fibrosis, an increasing number of researchers focused their studies in the research and development of agents against liver fibrosis in recent years. By searching the database of clinical registration in the United States (https://ClinicalTrials.gov/) by using “liver fibrosis” as the keyword, several natural compounds have been involved in clinical trials, including silymarin, glycyrrhizin, curcumin, and resveratrol. The single- and multiple-dose pharmacokinetics of silymarin were examined in patients with NAFLD or HCV to determine whether the disposition of silymarin and therefore its potential efficacy vary among liver disease populations. The efficacy of silymarin may be more readily observed in NAFLD patients because of their higher flavonolignan plasma concentrations and more extensive enterohepatic cycling compared with those in HCV patients [138]. A randomized, double-blind, placebo-controlled trial (NCT02006498) was performed on consecutive adults with biopsy-proven NASH and a NAFLD activity score (NAS) of 4 or more to verify the efficacy of silymarin in treating NASH. Patients were randomly assigned to groups given silymarin (700 mg) or placebo 3 times daily. After 48 wk, a significantly higher proportion of patients in the silymarin group had reductions in fibrosis based on histology and liver stiffness measurements. The silymarin group also had significant reductions in mean AST-to-platelet ratio index, fibrosis-4 score, and NAFLD fibrosis score [139]. Additionally, silymarin has been involved in a phase II clinical trial for noncirrhotic patients with nonalcoholic steatohepatitis (NCT00680407). Taken together, silymarin has been validated as a potential anti-fibrotic agent, but a larger trial is needed to confirm the clinical application of silymarin to reduce liver fibrosis.

A clinical trial was carried out to determine the addition of glycyrrhizin to entecavir in the treatment of chronic HBV (hepatitis B virus) and advanced fibrosis or cirrhosis (NCT03349008), which has not reported its results.

A clinical trial was performed to evaluate the effects of curcumin supplement on the metabolic factors and hepatic fibrosis in NAFLD patients with type 2 diabetes (NCT02908152); however, the results were not reported.

In a randomized, double-blinded, controlled clinical trial (NCT02030977), 50 NAFLD patients were supplemented with either a 500-mg resveratrol capsule or a placebo capsule for 12 wk. Both groups were advised to follow an energy-balanced diet and received physical activity recommendations. In both groups, anthropometric measurements (weight, body mass index, and waist circumference), liver enzymes, and steatosis grade were improved (p < 005). Resveratrol supplementation was associated with a significant reduction in liver enzyme ALT, inflammatory cytokines, NF-κB activity, serum cytokeratin-18, and hepatic steatosis grade, as compared with placebo supplementation. For the treatment of NAFLD, 12 wk of supplementation of 500 mg resveratrol, along with lifestyle modification, is superior to lifestyle modification alone [140]. In another clinical trial (NCT01446276), a long-term (6 mo) and high-dose (500 mg 3 times daily) resveratrol treatment did not improve either basal or insulin-mediated VLDL-TG (very-low-density lipoprotein-triglycerides) secretion, oxidation, or clearance rates, nor did it affect palmitate or glucose turnover in nondiabetic, upper-body obese men with NAFLD. Likewise, no changes in body composition or liver fat content occurred following resveratrol compared with placebo treatment [141]. Therefore, more studies are needed to confirm the clinical application of resveratrol in treatment of liver fibrosis.

A Chinese herbal formula Fuzheng Huayu (NCT00854087) has completed the phase II clinical trial. In China, 2 products derived from traditional Chinese herbal medicine Fufang Biejia Ruangan Tablet (NCT01965418) and Fuzheng Huayu Tablet combined with Huangqi Decoction Granule (NCT00540397) have been involved in clinical trials. These agents target the treatment of liver fibrosis, NASH, HCV/HBV infections, NAFLD, or cholestasis. Some natural compounds and herbal medicine formula have been authenticated as anti-fibrotic agents in clinical trials, but further studies should be performed with a large trial.

Conclusion and Perspective

The authors retraced the panorama of liver fibrosis and the role of OS in the development of liver fibrosis. OS, a critical factor that mediates inflammatory response and triggers HSC activation, is a potential therapy target for treating fibrotic liver diseases. Naturally occurring antioxidants have been verified as potential therapeutic agents to balance the intracellular redox state. Plenty of evidence, both basic and clinical, provides a bright perspective on the antioxidative strategy for the treatment and prevention of liver fibrosis.

However, the clinical application of antioxidants in treatment of liver fibrosis is still far away.

1. Redox balance plays a key role in many physiological and pathological processes, and the liver is a central organ for metabolism. Thus, the determination of dosage, period, and route of administration of antioxidants in treating liver fibrosis involves a challenging translational research.

2. Most of the compounds possess a variety of pharmacological activities, indicating that the mechanisms for the treatment of liver fibrosis by antioxidants might partly be attributed to the antioxidative ability. Other mechanisms should also be considered. Natural compounds are supplied in high dosage in cell and animal experiments, and most compounds did not show a dose-effect manner for treating liver fibrosis, resulting in low possibility for clinical application.

3. Most of the reviewed antioxidants have limited oral bioavailability due to their hydrophobicity, quick degradation in the gastrointestinal tract, poor permeation through the intestinal membrane, extensive metabolism in the gut, and transport mediated by efflux pumps [142]. It might yield failed results on clinical trials. Several strategies could be employed to improve the poor oral bioavailability [142]. Micro and nanonization can improve the aqueous solubility and intestinal absorption. The encapsulation of lipophilic compounds into cyclodextrins can enhance the aqueous solubility and stability. Chitosan-based delivery systems can afford gastric stability, enhanced penetration through the intestinal membrane, and protection against intestinal metabolism. The co-administration of metabolic enzyme and P-glycoprotein inhibitors may enhance oral bioavailability. Despite the significant anti-fibrotic effect of naturally occurring antioxidants in animal models, these limitations render them ineffective in humans. More translational studies are needed to evaluate the effective and safe dose, the duration of treatment, and formulation strategy to realize the clinical application of antioxidants in treating liver fibrosis.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the Research Fund of University of Macau (MYRG2017-00109-ICMS and MYRG2018-00037-ICMS) and Science and Technology Development Fund, Macao S. A.R (FDCT 102/2017/A).

• References

• 1 Thrift AP, El-Serag HB, Kanwal F. Global epidemiology and burden of HCV infection and HCV-related disease. Nat Rev Gastroenterol Hepatol 2017; 14: 122-132
• 2 Rinella ME. Nonalcoholic fatty liver disease: a systematic review. J Am Med Assoc 2015; 313: 2263-2273
• 3 Scaglione S, Kliethermes S, Cao G, Shoham D, Durazo R, Luke A, Volk ML. The epidemiology of cirrhosis in the United States: a population-based study. J Clin Gastroenterol 2015; 49: 690-696
• 4 Caballeria L, Pera G, Arteaga I, Rodriguez L, Aluma A, Morillas RM, de la Ossa N, Diaz A, Exposito C, Miranda D, Sanchez C, Prats RMA, Urquizu M, Salgado A, Alemany M, Martinez A, Majeed I, Fabrellas N, Graupera I, Planas R, Ojanguren I, Serra M, Toran P, Caballeria J, Gines P. High prevalence of liver fibrosis among European adults with unknown liver disease: a population-based study. Clin Gastroenterol Hepatol 2018; 16: 1138-1145
• 5 Singal AG, El-Serag HB. Hepatocellular carcinoma from epidemiology to prevention: translating knowledge into practice. Clin Gastroenterol Hepatol 2015; 13: 2140-2151
• 6 Friedman SL. Liver fibrosis-from bench to bedside. J Hepatol 2003; 38 (Suppl. 01) S38-S53
• 7 Hernandez-Gea V, Friedman SL. Pathogenesis of Liver Fibrosis. In: Abbas AK, Galli SJ, Howley PM. eds. Annual Review of Pathology: Mechanisms of Disease, vol. 6. Palo Alto: Annual Reviews; 2011: 425-456
• 8 Lim YS, Kim WR. The global impact of hepatic fibrosis and end-stage liver disease. Clin Liver Dis 2008; 12: 733-746
• 9 Ley B, Collard HR. Epidemiology of idiopathic pulmonary fibrosis. Clin Epidemiol 2013; 5: 483-492
• 10 Yang C, Zeisberg M, Mosterman B, Sudhakar A, Yerramalla U, Holthaus K, Xu L, Eng F, Afdhal N, Kalluri R. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology 2003; 124: 147-159
• 11 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003; 92: 827-839
• 12 Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007; 8: 221-233
• 13 Friedman SL. Evolving challenges in hepatic fibrosis. Nat Rev Gastroenterol Hepatol 2010; 7: 425-436
• 14 Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, Yee H, Zender L, Lowe SW. Senescence of activated stellate cells limits liver fibrosis. Cell 2008; 134: 657-667
• 15 Jiang Y, Wang C, Li YY, Wang XC, An JD, Wang YJ, Wang XJ. Mistletoe alkaloid fractions alleviates carbon tetrachloride-induced liver fibrosis through inhibition of hepatic stellate cell activation via TGF-beta/Smad interference. J Ethnopharmacol 2014; 158: 230-238
• 16 Liu M, Xu Y, Han X, Yin L, Xu L, Qi Y, Zhao Y, Liu K, Peng J. Dioscin alleviates alcoholic liver fibrosis by attenuating hepatic stellate cell activation via the TLR4/MyD88/NF-kappaB signaling pathway. Sci Rep 2015; 5: 18038
• 17 Zhou L, Dong X, Wang L, Shan L, Li T, Xu W, Ding Y, Lai M, Lin X, Dai M, Bai X, Jia C, Zheng H. Casticin attenuates liver fibrosis and hepatic stellate cell activation by blocking TGF-beta/Smad signaling pathway. Oncotarget 2017; 8: 56267-56280
• 18 Li S, Tan HY, Wang N, Zhang ZJ, Lao L, Wong CW, Feng Y. The role of oxidative stress and antioxidants in liver diseases. Int J Mol Sci 2015; 16: 26087-26124
• 19 Louvet A, Mathurin P. Alcoholic liver disease: mechanisms of injury and targeted treatment. Nat Rev Gastroenterol Hepatol 2015; 12: 231-242
• 20 Kakimoto PA, Kowaltowski AJ. Effects of high fat diets on rodent liver bioenergetics and oxidative imbalance. Redox Biol 2016; 8: 216-225
• 21 Ivanov AV, Valuev-Elliston VT, Tyurina DA, Ivanova ON, Kochetkov SN, Bartosch B, Isaguliants MG. Oxidative stress, a trigger of hepatitis C and B virus-induced liver carcinogenesis. Oncotarget 2017; 8: 3895-3932
• 22 Sutti S, Jindal A, Locatelli I, Vacchiano M, Gigliotti L, Bozzola C, Albano E. Adaptive immune responses triggered by oxidative stress contribute to hepatic inflammation in NASH. Hepatology 2014; 59: 886-897
• 23 Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, Kaufman RJ. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc Natl Acad Sci U S A 2008; 105: 18525-18530
• 24 Murphy MP. How mitochondria produce reactive oxygen species. Biochem J 2009; 417: 1-13
• 25 Ushio-Fukai M. Localizing NADPH oxidase-derived ROS. Sci STKE 2006; 349: re8
• 26 Mathews S, Feng D, Maricic I, Ju C, Kumar V, Gao B. Invariant natural killer T cells contribute to chronic-plus-binge ethanol-mediated liver injury by promoting hepatic neutrophil infiltration. Cell Mol Immunol 2016; 13: 206-216
• 27 Poli G. Pathogenesis of liver fibrosis: role of oxidative stress. Mole Aspects Med 2000; 21: 49-98
• 28 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115: 209-218
• 29 Itagaki T, Shimizu I, Cheng X, Yuan Y, Oshio A, Tamaki K, Fukuno H, Honda H, Okamura Y, Ito S. Opposing effects of oestradiol and progesterone on intracellular pathways and activation processes in the oxidative stress induced activation of cultured rat hepatic stellate cells. Gut 2005; 54: 1782-1789
• 30 Sánchez-Valle V, Chavez-Tapia NC, Uribe M, Méndez-Sánchez N. Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem 2012; 19: 4850-4860
• 31 Nishi T, Shimizu N, Hiramoto M, Sato I, Yamaguchi Y, Hasegawa M, Aizawa S, Tanaka H, Kataoka K, Watanabe H. Spatial redox regulation of a critical cysteine residue of NF-κB in vivo . J Biol Chem 2002; 277: 44548-44556
• 32 Korn SH, Wouters EF, Vos N, Janssen-Heininger YM. Cytokine-induced activation of nuclear factor-κB is inhibited by hydrogen peroxide through oxidative inactivation of IκB kinase. J Biol Chem 2001; 276: 35693-35700
• 33 Kaspar JW, Niture SK, Jaiswal AK. Nrf2: INrf2 (Keap1) signaling in oxidative stress. Free Radic Biol Med 2009; 47: 1304-1309
• 34 Salomone F, Godos J, Zelber-Sagi S. Natural antioxidants for non-alcoholic fatty liver disease: molecular targets and clinical perspectives. Liver Int 2016; 36: 5-20
• 35 Parola M, Robino G. Oxidative stress-related molecules and liver fibrosis. J Hepatol 2001; 35: 297-306
• 36 Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int 2011; 31: 1432-1448
• 37 Richter K, Konzack A, Pihlajaniemi T, Heljasvaara R, Kietzmann T. Redox-fibrosis: impact of TGFbeta1 on ROS generators, mediators and functional consequences. Redox Biol 2015; 6: 344-352
• 38 Richter K, Kietzmann T. Reactive oxygen species and fibrosis: further evidence of a significant liaison. Cell Tissue Res 2016; 365: 591-605
• 39 Nanthakumar CB, Hatley RJ, Lemma S, Gauldie J, Marshall RP, Macdonald SJ. Dissecting fibrosis: therapeutic insights from the small-molecule toolbox. Nat Rev Drug Discov 2015; 14: 693-720
• 40 Dong LL, Fu YJ, Zu YG, Luo M, Wang W, Li CY, Mu PS. An enhanced preparation and purification of the major antioxidants baicalein and wogonin from Scutellariae radix. Food Chem 2012; 133: 430-436
• 41 Lee H, Kim YO, Kim H, Kim SY, Noh HS, Kang SS, Cho GJ, Choi WS, Suk K. Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia. FASEB J 2003; 17: 1943-1944
• 42 Khan NM, Haseeb A, Ansari MY, Devarapalli P, Haynie S, Haqqi TM. Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human osteoarthritis chondrocytes. Free Radic Biol Med 2017; 106: 288-301
• 43 Li-Weber M. New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents wogonin, baicalein and baicalin. Cancer Treat Rev 2009; 35: 57-68
• 44 Pan TL, Wang PW, Huang CH, Leu YL, Wu TH, Wu YR, You JS. Herbal formula, Scutellariae radix and Rhei rhizoma attenuate dimethylnitrosamine-induced liver fibrosis in a rat model. Sci Rep 2015; 5: 11734
• 45 MadanKumar P, NaveenKumar P, Manikandan S, Devaraj H, NiranjaliDevaraj S. Morin ameliorates chemically induced liver fibrosis in vivo and inhibits stellate cell proliferation in vitro by suppressing Wnt/beta-catenin signaling. Toxicol Appl Pharmacol 2014; 277: 210-220
• 46 Heeba GH, Mahmoud ME. Therapeutic potential of morin against liver fibrosis in rats: modulation of oxidative stress, cytokine production and nuclear factor kappa B. Environ Toxicol Pharmacol 2014; 37: 662-671
• 47 Wang X, Gong G, Yang W, Li Y, Jiang M, Li L. Antifibrotic activity of galangin, a novel function evaluated in animal liver fibrosis model. Environ Toxicol Pharmacol 2013; 36: 288-295
• 48 Yang JH, Kim SC, Kim KM, Jang CH, Cho SS, Kim SJ, Ku SK, Cho IJ, Ki SH. Isorhamnetin attenuates liver fibrosis by inhibiting TGF-beta/Smad signaling and relieving oxidative stress. Eur J Pharmacol 2016; 783: 92-102
• 49 Domitrovic R, Rashed K, Cvijanovic O, Vladimir-Knezevic S, Skoda M, Visnic A. Myricitrin exhibits antioxidant, anti-inflammatory and antifibrotic activity in carbon tetrachloride-intoxicated mice. Chem Biol Interact 2015; 230: 21-29
• 50 Erlund I. Review of the flavonoids quercetin, hesperetin naringenin. dietary sources, bioactivities, and epidemiology. Nutr Res 2004; 24: 851-874
• 51 Wilcox LJ, Borradaile NM, Huff MW. Antiatherogenic properties of naringenin, a citrus flavonoid. Cardiovasc Drug Rev 1999; 17: 160-178
• 52 Mir IA, Tiku AB. Chemopreventive and therapeutic potential of “naringenin”, a flavanone present in citrus fruits. Nutr Cancer 2015; 67: 27-42
• 53 Pari L, Amudha K. Hepatoprotective role of naringin on nickel-induced toxicity in male Wistar rats. Eur J Pharmacol 2011; 650: 364-370
• 54 Renugadevi J, Prabu SM. Cadmium-induced hepatotoxicity in rats and the protective effect of naringenin. Exp Toxicol Pathol 2010; 62: 171-181
• 55 Jayachitra J, Nalini N. Effect of naringenin (citrus flavanone) on lipid profile in ethanol-induced toxicity in rats. J Food Biochem 2012; 36: 502-511
• 56 Jayaraman J, Namasivayam N. Naringenin modulates circulatory lipid peroxidation, anti-oxidant status and hepatic alcohol metabolizing enzymes in rats with ethanol induced liver injury. Fundam Clin Pharmacol 2011; 25: 682-689
• 57 Kannappan S, Palanisamy N, Anuradha CV. Suppression of hepatic oxidative events and regulation of eNOS expression in the liver by naringenin in fructose-administered rats. Eur J Pharmacol 2010; 645: 177-184
• 58 Ozkaya A, Sahin Z, Dag U, Ozkaraca M. Effects of naringenin on oxidative stress and histopathological changes in the liver of lead acetate administered rats. J Biochem Mol Toxicol 2016; 30: 243-248
• 59 Wang A, Lin L, Wang Y. Traditional Chinese herbal medicine Penthorum chinense Pursh: a phytochemical and pharmacological review. Am J Chin Med 2015; 43: 601-620
• 60 Wang A, Wang S, Jiang Y, Chen M, Wang Y, Lin L. Bio-assay guided identification of hepatoprotective polyphenols from Penthorum chinense Pursh on t-BHP induced oxidative stress injured L02 cells. Food Funct 2016; 7: 2074-2083
• 61 Cao YW, Jiang Y, Zhang DY, Wang M, Chen WS, Su H, Wang YT, Wan JB. Protective effects of Penthorum chinense Pursh against chronic ethanol-induced liver injury in mice. J Ethnopharmacol 2015; 161: 92-98
• 62 Zhou F, Wang A, Li D, Wang Y, Lin L. Pinocembrin from Penthorum chinense Pursh suppresses hepatic stellate cells activation through a unified SIRT3-TGF-beta-Smad signaling pathway. Toxicol Appl Pharmacol 2018; 341: 38-50
• 63 Gazak R, Walterova D, Kren V. Silybin and silymarin-new and emerging applications in medicine. Curr Med Chem 2007; 14: 315-338
• 64 Shaker E, Mahmoud H, Mnaa S. Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food Chem Toxicol 2010; 48: 803-806
• 65 Crocenzi FA, Roma MG. Silymarin as a new hepatoprotective agent in experimental cholestasis: new possibilities for an ancient medication. Curr Med Chem 2006; 13: 1055-1074
• 66 Hackett ES, Twedt DC, Gustafson DL. Milk thistle and its derivative compounds: a review of opportunities for treatment of liver disease. J Vet Intern Med 2013; 27: 10-16
• 67 Rastogi R, Srivastava AK, Rastogi AK. Long term effect of aflatoxin B(1) on lipid peroxidation in rat liver and kidney: effect of picroliv and silymarin. Phytother Res 2001; 15: 307-310
• 68 Saller R, Meier R, Brignoli R. The use of silymarin in the treatment of liver diseases. Drugs 2001; 61: 2035-2063
• 69 Abenavoli L, Milic N, Capasso F. Anti-oxidant therapy in non-alcoholic fatty liver disease: the role of silymarin. Endocrine 2012; 42: 754-755
• 70 Tzeng JI, Chen MF, Chung HH, Cheng JT. Silymarin decreases connective tissue growth factor to improve liver fibrosis in rats treated with carbon tetrachloride. Phytother Res 2013; 27: 1023-1028
• 71 Sherif IO, Al-Gayyar MM. Antioxidant, anti-inflammatory and hepatoprotective effects of silymarin on hepatic dysfunction induced by sodium nitrite. Eur Cytokine Netw 2013; 24: 114-121
• 72 Zhen MC, Wang Q, Huang XH, Cao LQ, Chen XL, Sun K, Liu YJ, Li W, Zhang LJ. Green tea polyphenol epigallocatechin-3-gallate inhibits oxidative damage and preventive effects on carbon tetrachloride-induced hepatic fibrosis. J Nutr Biochem 2007; 18: 795-805
• 73 Kim HK, Yang TH, Cho HY. Antifibrotic effects of green tea on in vitro and in vivo models of liver fibrosis. World J Gastroenterol 2009; 15: 5200-5205
• 74 Tipoe GL, Leung TM, Liong EC, Lau TY, Fung ML, Nanji AA. Epigallocatechin-3-gallate (EGCG) reduces liver inflammation, oxidative stress and fibrosis in carbon tetrachloride (CCl4)-induced liver injury in mice. Toxicology 2010; 273: 45-52
• 75 Xiao J, Ho CT, Liong EC, Nanji AA, Leung TM, Lau TY, Fung ML, Tipoe GL. Epigallocatechin gallate attenuates fibrosis, oxidative stress, and inflammation in non-alcoholic fatty liver disease rat model through TGF/SMAD, PI3 K/Akt/FoxO1, and NF-kappa B pathways. Eur J Nutr 2014; 53: 187-199
• 76 Arffa ML, Zapf MA, Kothari AN, Chang V, Gupta GN, Ding X, Al-Gayyar MM, Syn W, Elsherbiny NM, Kuo PC, Mi Z. Epigallocatechin-3-gallate upregulates miR-221 to inhibit osteopontin-dependent hepatic fibrosis. PLoS One 2016; 11: e0167435
• 77 Graebin CS, Verli H, Guimaraes JA. Glycyrrhizin and glycyrrhetic acid: scaffolds to promising new pharmacologically active compounds. J Brazil Chem Soc 2010; 21: 1595-1615
• 78 Chen S, Zou L, Li L, Wu T. The protective effect of glycyrrhetinic acid on carbon tetrachloride-induced chronic liver fibrosis in mice via upregulation of Nrf2. PLoS One 2013; 8: e53662
• 79 Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361: 2045-2046
• 80 Qu Y, Zong L, Xu M, Dong Y, Lu L. Effects of 18α-glycyrrhizin on TGF-β1/Smad signaling pathway in rats with carbon tetrachloride-induced liver fibrosis. Int J Clin Exp Pathol 2015; 8: 1292-1301
• 81 Sil R, Ray D, Chakraborti AS. Glycyrrhizin ameliorates metabolic syndrome-induced liver damage in experimental rat model. Mol Cell Biochem 2015; 409: 177-189
• 82 Li X, Wang X, Han C, Wang X, Xing G, Zhou L, Li G, Niu Y. Astragaloside IV suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway. Free Radic Biol Med 2013; 60: 168-176
• 83 Wu HY, Chang CI, Lin BW, Yu FL, Lin PY, Hsu JL, Yen CH, Liao MH, Shih WL. Suppression of hepatitis B virus x protein-mediated tumorigenic effects by ursolic acid. J Agric Food Chem 2011; 59: 1713-1722
• 84 Ma JQ, Ding J, Zhang L, Liu CM. Protective effects of ursolic acid in an experimental model of liver fibrosis through Nrf2/ARE pathway. Clin Res Hepatol Gastroenterol 2015; 39: 188-197
• 85 Dai Y, Chen SR, Chai L, Zhao J, Wang Y, Wang Y. Overview of pharmacological activities of Andrographis paniculata and its major compound andrographolide. Crit Rev Food Sci Nutr 2018; DOI: 10.1080/10408398.2018.1501657.
• 86 Yan H, Huang Z, Bai Q, Sheng Y, Hao Z, Wang Z, Ji L. Natural product andrographolide alleviated APAP-induced liver fibrosis by activating Nrf2 antioxidant pathway. Toxicology 2018; 396 – 397: 1-12
• 87 Shan M, Yu S, Yan H, Guo S, Xiao W, Wang Z, Zhang L, Ding A, Wu Q, Li SFY. A review on the phytochemistry, pharmacology, pharmacokinetics and toxicology of geniposide, a natural product. Molecules 2017; 22: E1689
• 88 Zhang WL, Zhu L, Jiang JG. Active ingredients from natural botanicals in the treatment of obesity. Obes Rev 2014; 15: 957-967
• 89 Park JH, Yoon J, Lee KY, Park B. Effects of geniposide on hepatocytes undergoing epithelial-mesenchymal transition in hepatic fibrosis by targeting TGFbeta/Smad and ERK-MAPK signaling pathways. Biochimie 2015; 113: 26-34
• 90 Ma T, Huang C, Zong G, Zha D, Meng X, Li J, Tang W. Hepatoprotective effects of geniposide in a rat model of nonalcoholic steatohepatitis. J Pharm Pharmacol 2011; 63: 587-593
• 91 Wang J, Miao M, Qu L, Cui Y, Zhang Y. Protective effects of geniposide against tripterygium glycosides (TG)-induced liver injury and its mechanisms. J Toxicol Sci 2016; 41: 165-173
• 92 Wang J, Zhang Y, Liu R, Li X, Cui Y, Qu L. Geniposide protects against acute alcohol-induced liver injury in mice via up-regulating the expression of the main antioxidant enzymes. Can J Physiol Pharmacol 2015; 93: 261-267
• 93 Chen P, Chen Y, Wang Y, Cai S, Deng L, Liu J, Zhang H. Comparative evaluation of hepatoprotective activities of geniposide, crocins and crocetin by CCl₄-induced liver injury in mice. Biomol Ther 2016; 24: 156-162
• 94 Hsu YW, Tsai CF, Chang WH, Ho YC, Chen WK, Lu FJ. Protective effects of Dunaliella salina – a carotenoids-rich alga, against carbon tetrachloride-induced hepatotoxicity in mice. Food Chem Toxicol 2008; 46: 3311-3317
• 95 Qiu S, Sun H, Zhang AH, Xu HY, Yan GL, Han Y, Wang XJ. Natural alkaloids: basic aspects, biological roles, and future perspectives. Chin J Nat Med 2014; 12: 401-406
• 96 Sun N, Sun P, Lv H, Sun Y, Guo J, Wang Z, Luo T, Wang S, Li H. Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2. Sci Rep 2016; 6: 24401
• 97 Zhang YB, Zhan LQ, Li GQ, Wang F, Wang Y, Li YL, Ye WC, Wang GC. Dimeric matrine-type alkaloids from the roots of Sophora flavescens and their anti-hepatitis B virus activities. J Org Chem 2016; 81: 6273-6280
• 98 Feng Y, Ying HY, Qu Y, Cai XB, Xu MY, Lu LG. Novel matrine derivative MD-1 attenuates hepatic fibrosis by inhibiting EGFR activation of hepatic stellate cells. Protein Cell 2016; 7: 662-672
• 99 Zhang Y, Cui L, Guan GC, Wang JK, Qiu C, Yang TL, Guo Y, Liu ZW. Matrine suppresses cardiac fibrosis by inhibiting the TGF-β/Smad pathway in experimental diabetic cardiomyopathy. Mol Med Rep 2018; 17: 1775-1781
• 100 Zhang JP, Zhang M, Zhou JP, Liu FT, Zhou B, Xie WF, Guo C. Antifibrotic effects of matrine on in vitro and in vivo models of liver fibrosis in rats. Acta Pharmacol Sin 2001; 22: 183-186
• 101 Zhang HF, Shi LJ, Song GY, Cai ZG, Wang C, An RJ. Protective effects of matrine against progression of high-fructose diet-induced steatohepatitis by enhancing antioxidant and anti-inflammatory defences involving Nrf2 translocation. Food Chem Toxicol 2013; 55: 70-77
• 102 Li J, Pan Y, Kan M, Xiao X, Wang Y, Guan F, Zhang X, Chen L. Hepatoprotective effects of berberine on liver fibrosis via activation of AMP-activated protein kinase. Life Sci 2014; 98: 24-30
• 103 Liu CP, Kuo YC, Shen CC, Wu MH, Liao JF, Lin YL, Chen CF, Tsai WJ. (S)-armepavine inhibits human peripheral blood mononuclear cell activation by regulating Itk and PLCgamma activation in a PI-3K-dependent manner. J Leukoc Biol 2007; 81: 1276-1286
• 104 Liu CP, Tsai WJ, Shen CC, Lin YL, Liao JF, Chen CF, Kuo YC. Inhibition of (S)-armepavine from Nelumbo nucifera on autoimmune disease of MRL/MpJ-lpr/lpr mice. Eur J Pharmacol 2006; 531: 270-279
• 105 Weng TC, Shen CC, Chiu YT, Lin YL, Kuo CD, Huang YT. Inhibitory effects of armepavine against hepatic fibrosis in rats. J Biomed Sci 2009; 16: 78
• 106 Arauz J, Zarco N, Segovia J, Shibayama M, Tsutsumi V, Muriel P. Caffeine prevents experimental liver fibrosis by blocking the expression of TGF-beta. Eur J Gastroenterol Hepatol 2014; 26: 164-173
• 107 Arauz J, Rivera-Espinoza Y, Shibayama M, Favari L, Flores-Beltran RE, Muriel P. Nicotinic acid prevents experimental liver fibrosis by attenuating the prooxidant process. Int Immunopharmacol 2015; 28: 244-251
• 108 Bingul I, Basaran-Kucukgergin C, Aydin AF, Coban J, Dogan-Ekici I, Dogru-Abbasoglu S, Uysal M. Betaine treatment decreased oxidative stress, inflammation, and stellate cell activation in rats with alcoholic liver fibrosis. Environ Toxicol Pharmacol 2016; 45: 170-178
• 109 Kunnumakkara AB, Bordoloi D, Padmavathi G, Monisha J, Roy NK, Prasad S, Aggarwal BB. Curcumin, the golden nutraceutical: multitargeting for multiple chronic diseases. Br J Pharmacol 2017; 174: 1325-1348
• 110 Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol 2009; 41: 40-59
• 111 Masuda T, Maekawa T, Hidaka K, Bando H, Takeda Y, Yamaguchi H. Chemical studies on antioxidant mechanism of curcumin: analysis of oxidative coupling products from curcumin and linoleate. J Agric Food Chem 2001; 49: 2539-2547
• 112 Kaur G, Tirkey N, Bharrhan S, Chanana V, Rishi P, Chopra K. Inhibition of oxidative stress and cytokine activity by curcumin in amelioration of endotoxin-induced experimental hepatoxicity in rodents. Clin Exp Immunol 2006; 145: 313-321
• 113 Lin X, Zhang S, Huang R, Wei L, Tan S, Liang S, Tian Y, Wu X, Lu Z, Huang Q. Helenalin attenuates alcohol-induced hepatic fibrosis by enhancing ethanol metabolism, inhibiting oxidative stress and suppressing HSC activation. Fitoterapia 2014; 95: 203-213
• 114 Athar M, Back JH, Tang X, Kim KH, Kopelovich L, Bickers DR, Kim AL. Resveratrol: a review of preclinical studies for human cancer prevention. Toxicol Appl Pharmacol 2007; 224: 274-283
• 115 Athar M, Back JH, Kopelovich L, Bickers DR, Kim AL. Multiple molecular targets of resveratrol: anti-carcinogenic mechanisms. Arch Biochem Biophys 2009; 486: 95-102
• 116 Brasnyo P, Molnar GA, Mohas M, Marko L, Laczy B, Cseh J, Mikolas E, Szijarto IA, Merei A, Halmai R, Meszaros LG, Sumegi B, Wittmann I. Resveratrol improves insulin sensitivity, reduces oxidative stress and activates the Akt pathway in type 2 diabetic patients. Br J Nutr 2011; 106: 383-389
• 117 Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer Lett 2008; 269: 243-261
• 118 Park SJ, Ahmad F, Philp A, Baar K, Williams T, Luo H, Ke H, Rehmann H, Taussig R, Brown AL, Kim MK, Beaven MA, Burgin AB, Manganiello V, Chung JH. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell 2012; 148: 421-433
• 119 Hong SW, Jung KH, Zheng HM, Lee HS, Suh JK, Park IS, Lee DH, Hong SS. The protective effect of resveratrol on dimethylnitrosamine-induced liver fibrosis in rats. Arch Pharm Res 2010; 33: 601-609
• 120 Sebai H, Sani M, Yacoubi MT, Aouani E, Ghanem-Boughanmi N, Ben-Attia M. Resveratrol, a red wine polyphenol, attenuates lipopolysaccharide-induced oxidative stress in rat liver. Ecotoxicol Environ Saf 2010; 73: 1078-1083
• 121 Ahmad A, Ahmad R. Resveratrol mitigate structural changes and hepatic stellate cell activation in N′-nitrosodimethylamine-induced liver fibrosis via restraining oxidative damage. Chem Biol Interact 2014; 221: 1-12
• 122 Koneru M, Sahu BD, Gudem S, Kuncha M, Ravuri HG, Kumar JM, Kilari EK, Sistla R. Polydatin alleviates alcohol-induced acute liver injury in mice: relevance of matrix metalloproteinases (MMPs) and hepatic antioxidants. Phytomedicine 2017; 27: 23-32
• 123 Domitrovic R, Skoda M, Vasiljev Marchesi V, Cvijanovic O, Pernjak Pugel E, Stefan MB. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem Toxicol 2013; 51: 370-378
• 124 Shi H, Dong L, Bai Y, Zhao J, Zhang Y, Zhang L. Chlorogenic acid against carbon tetrachloride-induced liver fibrosis in rats. Eur J Pharmacol 2009; 623: 119-124
• 125 Shi H, Dong L, Jiang J, Zhao J, Zhao G, Dang X, Lu X, Jia M. Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4 signaling pathway. Toxicology 2013; 303: 107-114
• 126 Shi H, Shi A, Dong L, Lu X, Wang Y, Zhao J, Dai F, Guo X. Chlorogenic acid protects against liver fibrosis in vivo and in vitro through inhibition of oxidative stress. Clin Nutr 2016; 35: 1366-1373
• 127 Shi A, Shi H, Wang Y, Liu X, Cheng Y, Li H, Zhao H, Wang S, Dong L. Activation of Nrf2 pathway and inhibition of NLRP3 inflammasome activation contribute to the protective effect of chlorogenic acid on acute liver injury. Int Immunopharmacol 2018; 54: 125-130
• 128 Yang ZR, Wang HF, Zuo TC, Guan LL, Dai N. Salidroside alleviates oxidative stress in the liver with non- alcoholic steatohepatitis in rats. BMC Pharmacol Toxicol 2016; 17: 16
• 129 Zakaria S, Mahmoud AAA, Hasan RA, Mahmoud MF, El Fayoumi HM. Cinnamaldehyde mitigates carbon tetrachloride-induced acute liver injury in rats through inhibition of toll-like receptor 4 signaling pathway. Int J Pharmacol 2016; 12: 851-862
• 130 Rahman MM, Muse AY, Khan D, Ahmed IH, Subhan N, Reza HM, Alam MA, Nahar L, Sarker SD. Apocynin prevented inflammation and oxidative stress in carbon tetra chloride induced hepatic dysfunction in rats. Biomed Pharmacother 2017; 92: 421-428
• 131 Kim M, Yoo G, Randy A, Kim HS, Nho CW. Chicoric acid attenuate a nonalcoholic steatohepatitis by inhibiting key regulators of lipid metabolism, fibrosis, oxidation, and inflammation in mice with methionine and choline deficiency. Mol Nutr Food Res 2017; 61: 1600632
• 132 Sun H, Luo G, Chen D, Xiang Z. A comprehensive and system review for the pharmacological mechanism of action of rhein, an active anthraquinone ingredient. Front Pharmacol 2016; 7: 247
• 133 Wu C, Cao H, Zhou H, Sun L, Xue J, Li J, Bian Y, Sun R, Dong S, Liu P, Sun M. Research progress on the antitumor effects of rhein: literature review. Anticancer Agents Med Chem 2017; 17: 1624-1632
• 134 Zhao YL, Zhou GD, Yang HB, Wang JB, Shan LM, Li RS, Xiao XH. Rhein protects against acetaminophen-induced hepatic and renal toxicity. Food Chem Toxicol 2011; 49: 1705-1710
• 135 Lee JH, Jang EJ, Seo HL, Ku SK, Lee JR, Shin SS, Park SD, Kim SC, Kim YW. Sauchinone attenuates liver fibrosis and hepatic stellate cell activation through TGF-beta/Smad signaling pathway. Chem Biol Interact 2014; 224: 58-67
• 136 Basnet P, Yasuda I, Kumagai N, Tohda C, Nojima H, Kuraishi Y, Komatsu K. Inhibition of itch-scratch response by fruits of Cnidium monnieri in mice. Biol Pharm Bull 2001; 24: 1012-1015
• 137 Liu YW, Chiu YT, Fu SL, Huang YT. Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation. J Biomed Sci 2015; 22: 63
• 138 Schrieber SJ, Hawke RL, Wen ZM, Smith PC, Reddy KR, Wahed AS, Belle SH, Afdhal NH, Navarro VJ, Meyers CM, Doo E, Fried MW. Differences in the disposition of silymarin between patients with nonalcoholic fatty liver disease and chronic hepatitis C. Drug Metab Dispos 2011; 39: 2182-2190
• 139 Wah Kheong C, Nik Mustapha NR, Mahadeva S. A randomized trial of silymarin for the treatment of nonalcoholic steatohepatitis. Clin Gastroenterol Hepatol 2017; 15: 1940-1949
• 140 Faghihzadeh F, Adibi P, Rafiei R, Hekmatdoost A. Resveratrol supplementation improves inflammatory biomarkers in patients with nonalcoholic fatty liver disease. Nutr Res 2014; 34: 837-843
• 141 Poulsen MK, Sorensen BN, Stodkilde-Jorgensen H, Pedersen SB, GronBaek H, Nielsen S. No effect of long-term high-dose resveratrol on VLDL-TG kinetics, insulin sensitivity, and liver fat content in obese men with nonalcoholic fatty liver disease. Diabetes Obes Metab 2018; 20: 2504-2509
• 142 Ting YW, Jiang Y, Ho CT, Huang QR. Common delivery systems for enhancing in vivo bioavailability and biological efficacy of nutraceuticals. J Funct Foods 2014; 7: 112-128