Current Status of Herbal Medicines in Chronic Liver Disease Therapy: The Biological Effects, Molecular Targets and Future Prospects

2.1.1. The Epidemiological and Pathological Characteristics of Chronic Hepatitis and Current Therapeutic Strategy

Chronic hepatitis is the inflammation of the liver; the common causes of chronic hepatitis include viral infection, autoimmune diseases and toxic substances such as drugs or alcohol. Viral hepatitis is the most common liver diseases which may progress over time to fibrosis and cirrhosis [9]. The well-known pathogens for chronic viral hepatitis include Hepatitis viruses B (HBV) and Hepatitis viruses C (HCV). At present, more than 30% of the world’s population is infected with the HBV and 5% is considered as chronic HBV carriers. In some developed countries, due to the widespread vaccination of HBV, Hepatitis C has become the most common cause of viral hepatitis since the 1980s [10]. Hepatitis induced by toxins, such as alcohol or drugs, has also significantly increased in recent years. For alcoholic hepatitis, recent studies have indicated that younger people, females, and binge drinkers have a higher incidence of suffering from this disease and are associated with higher mortality rates [11]. A large number of drugs such as acetaminophen, antibiotics or other chemical agents are also common causes of hepatitis. Age, genetic variability, female sex, and previous drug-induced hepatitis are the key predisposing risk factors to drug-induced hepatitis [12]. The occurrence of autoimmune hepatitis is relatively rare; it is thought to have a genetic predisposition and is more likely to occur in young women. Patients with autoimmune hepatitis are often accompanied with other autoimmune diseases [13].

The specific mechanism of chronic hepatitis depends on the underlying causes of the disease. In viral hepatitis, the immune system is activated by the hepatic virus, resulting in inflammation and liver damage. In autoimmune hepatitis, the autoimmune disorder causes the abnormal immune response against liver cells. In alcoholic hepatitis, the damage is usually found in association with fatty liver which we will in a subsequent paragraph [14,15]. In general, the common characteristics of chronic hepatitis are inflammation, necrosis and fibrosis in liver tissues. The macrophages (Kupffer cells) and other liver-resident cells such as hepatic stellate cells and sinusoidal endothelial cells promote the formation and emitting of pro-inflammatory signals of cytokines, chemokines, lipid messengers and reactive oxygen species (ROS) and activate the inflammatory response that leads to hepatic cells oncotic necrosis and apoptosis [16,17,18]. Apoptotic bodies derived from the damaged hepatic cells can further activate Kupffer cells to secrete transforming growth factor beta 1, endothelial growth factor and platelet-derived growth factor, which can promote the transformation of activated hepatic stellate cells into myofibroblasts [19].

Currently, alpha-Interferon is the most widely used drugs for chronic Hepatitis B and C, although only a few patients can respond to interferon therapy. So far, several novel anti-viral agents such as nucleoside analogue have been applied to patients. However, these drugs cannot completely eradicate the hepatitis virus, while at the same time they may induce significant adverse effects and drug resistance [20]. For example, in patients with alcoholic hepatitis, pentoxifylline and prednisolone are the first-line recommended therapy. Unfortunately, a large proportion of patients do not respond to these drugs [21]. Budesonide with azathioprine may have potential therapeutic effects for autoimmune hepatitis, but the durability of response and target population still need further study [22]. Due to the limited therapeutic effect of conventional chronic hepatitis treatment, the use of complementary and alternative medicine is expanding throughout the world. In some Asian countries, people have treated chronic hepatitis with herbs or phytochemicals since ancient times; some herbal products have shown remarkable therapeutic effects in both basic and clinical studies (Table 1).

2.1.2. Herbs and Phytochemicals in the Treatment of Chronic Hepatitis

Glycyrrhizin, an active component in Glycyrrhiza uralensis Fisch, has been used widely as a folk medicine agent for hepatitis in China and Japan. In Japan, glycyrrhizin injection has been used as an approved preparation for allergy inflammation since 1948 and for chronic hepatitis since 1979 [23]. Recently, glycyrrhizin has been found to suppress HCV particle release as well as the activity of phospholipase A2 (PLA2) [24]. As PLA2 has been proved to be related with HCV particles’ release, the researchers concluded that suppression of Hepatitis C by glycyrrhizin may be attributed to the reduced activity of PLA2. According to these studies, glycyrrhizin may be a promising agent for Hepatitis C when it is used alone or combined with interferon. According to the study by Fujisawa et al., glycyrrhizin also showed significant anti-inflammatory effects in liver and other tissues. The mechanisms may be related to suppression of the cytolytic activity of complement by activating both classical and alternative pathways. Further studies demonstrated that glycyrrhizin suppresses the lytic pathway in which the membrane attack complex is formed [25]. In vivo studies also confirmed that glycyrrhizin can inhibit liver inflammation by enhancing the secretion of interleukin-10 (IL-10) in concanavalin-A (Con A)-induced hepatitis mice model which closely imitated the pathology of human autoimmune hepatitis. Further mechanism study revealed that glycyrrhizin can modulate the function of dendritic cells in mouse with autoimmune hepatitis and further promote the production of anti-inflammatory cytokines such as IL-10 [26]. There has a clinical case report on treating chemotherapy-induced HBV hepatitis with lamivudine combined with glycyrrhizin for a non-Hodgkin lymphoma patient. The result showed that glycyrrhizin combined with nucleotides’ analogue may be effective for controlling HBV replication in cancer patients [27]. Another clinical study in Europe also confirmed the therapeutic effect of glycyrrhizin in viral hepatitis. Sixty-nine out of 72 treatment courses were completed in this study. In the placebo group, there were no obvious changes of alanine aminotransferase (ALT) level. The mean percentage of ALT decrease by the end of treatment was 47% in the six-time glycyrrhizin treatment per week group and 26% for the three-time treatment per week group (p < 0.001). For the six-time per week treatment group, 20% (three of 15) patients got normal serum ALT levels and for the three-time per week treatment group, the result was about 10% (four of 41). No obvious side effects were detected in this study. The serum ALT levels went up again when treatment stopped [28].

Silymarin is a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn (milk thistle seeds) Major ingredients in silymarin include silibinin, isosilibinin, silicristin and silidianin. Silymarin is a time-honored herbal remedy for chronic liver diseases worldwide. Silibinin is the major active compound in silymarin, which has been widely studied for its hepatoprotective effects and anti-cancer effects both in vitro and in vivo. The main problems in the clinical application of silymarin include poor solubility and bioavailability. Recently, some modified derivatives of silymarin, such as the siliphos, have been developed for liver disease treatment. This new chemically synthesized agent contains lecithin which can significantly improve the solubility and bioavailability of silymarin [29]. Bonifaz et al. explored the antiviral effects of silymarin, and their results showed that silymarin can down-regulate the copy number of HCV core mRNA and protein expression. The antiviral mechanism of silymarin is different from interferon which inhibits the JAK/STAT signaling pathway [30]. Silymarin can block HCV entry and transmission by inhibiting the activity of microsomal triglyceride transfer protein and suppressing apolipoprotein B production. However, it seems that silymarin may not decrease the virus content in vivo, which has been proved by several clinical studies [31]. Mayer et al. developed a systematic review for the therapeutic effects of silymarin in treating chronic viral Hepatitis B and C, and the results demonstrated that silymarin can down-regulate serum transaminases in chronic viral hepatitis patients, but the liver histology or serum viral content have not been affected by silymarin [32]. A 12-month randomized controlled trial comparing the effects of silymarin with a multi-vitamin placebo in chronic HCV patients also showed that silymarin at the recommended dose has no better effect in clearing HCV RNA than the vitamin placebo. One hundred and fourty-one patients were evaluated in this study. After two years, 64 patients in the silymarin treatment group (n = 68) were HCV RNA positive and in 71 patients in the placebo group (n = 73) were HCV RNA positive (p = 0.61). Sixty-five patients in the silymarin treatment group were anti-HCV positive while 72 patients in the placebo group were anti-HCV positive (p = 0.56). [33]. Another clinical study explored the immunomodulatory properties of oral silymarin in patients with Hepatitis C, and their results revealed that silymarin can inhibit inflammation in vitro and in vivo. The mechanisms may be associated with inhibition of T-cell proliferation and production by suppressing the pro-inflammatory cytokine as well as up-regulating the anti-inflammatory IL-10 [34]. A meta-analysis demonstrated that silymarin combined with other anti-viral drugs may be beneficial for the chronic Hepatitis B patients [35]. Silibinin, the major active ingredient in silymarin, also exhibited the inhibitory effects on Hepatitis C virus by blocking the clathrin-dependent trafficking. Silibinin can influence the formation of both clathrin-coated pits and vesicles in liver cells and destroy the clathrin endocytic pathway by interfering with the absorption and trafficking of transferrin [36]. A short-term pilot study on 20 patients with chronically active hepatitis was carried out to assess the liver protective activity and the antioxidant properties of a new silybinin phosphatidylcholine complex, and the results showed that after treatment with this new silybinin complex, there was a remarkable reduction of serum aspartate transaminase (AST) from 88.0 (95% CI 65.52–113.48) to 65.9 (95% CI 52.23–78.57) µ/L, (p < 0.01), a decrease of ALT from 115.9 (95% CI 94.1–147.7) to 82.5 (95% CI 64.59–100.41) µ/L (p < 0.01), and a decrease of total bilirubin from 0.76 (95% CI 0.63–0.89) to 0.53 (95% CI 0.47–0.59) mg/dL (p < 0.05) and of gamma-glutamyltranspeptidase from 51.4 (95% CI 35.69–67.11) to 41.3 (95% CI 36.01–50.19) µ/L (p < 0.02). Decrease of Alkaline phosphatase was not obviously, from 143.4 (95% CI 132.59–154.21) to 137.5 (95% CI 124.32–150.68) µ/L [37].

Phyllanthus niruri L is a subtropical plant widely distributing in China and South Asia, which has been used as traditional medicines to treat chronic hepatitis as well as intestinal infections and kidney disease [38,39,40]. The main active compounds in Phyllanthus niruri L including quercetin rhamnoside, gallic acid, geraniin and quercetin glucoside have been identified and proved with the properties of anti-bacterial, anti-viral and anti-hepatotoxic effects [41]. In recent years, several researches for evaluating the anti-viral effects and safety of Phyllanthus niruri L have been conducted both in vitro and in vivo. Lam et al., found that the ethanolic extract of Phyllanthus niruri L could suppress HBsAg secretion and down-regulate the expression of HBsAg mRNA, with a mechanism that may be related to up-regulation of annexin A7 protein [42]. A systematic review of 22 randomized trials (n = 1,947) on using Phyllanthus niruri L for treating chronic Hepatitis B showed that Phyllanthus niruri L were effective in clearance of serum HBsAg, HBeAg and HBV DNA without serious side-effects. When compared with interferon (IFN) and placebo, the therapeutic effects of combination treatment of Phyllanthus niruri L and IFN was better [43].

The rhizome of Polygonum cuspidatum Willd. ex Spreng. has been used as folk medicine for treating chronic hepatitis, jaundice, cough, hyperlipidemia and arthralgia in some Eastern Asian countries for thousands of years. This herbaceous perennial plant is widely distributed in the world and prefers to grow in humid environment such as the valley or forest [44]. Recent pharmacological studies have shown that Polygonum cuspidatum Willd. ex Spreng. has antiviral and hepatoprotective effects, and the major active ingredients may include anthraquinones, resveratrol and polydatin [45]. According to a recent meta-analysis review study, Polygonum cuspidatum Willd. ex Spreng. is one of the most frequently used herbs in the traditional Chinese medicine for chronic hepatitis treatment [46]. In vitro studies also confirmed that the water extract of Polygonum cuspidatum Willd. ex Spreng. at the concentration of 30 μg/mL could suppress the expression of Hepatitis B e antigen (HBeAg). For the ethanol extract of Polygonum cuspidatum Willd. ex Spreng., it could inhibit the production of HBV DNA at a low dose (10 μg/mL) [47].

Bupleurum chinense DC is one of the most frequently used herbs for relieving exterior syndrome in traditional Chinese medicine. When applying bupleurum decoction in combination with long-term exercise training, rats with obstructive jaundice exhibited a significant reduction in inflammatory cytokines expression, which in turn alleviated the liver hepatitis [48]. The leaf of Bupleurus has been regarded as the useless part in Chinese medicine, but a recent study has demonstrated that the saikosaponins extracted from Bupleurus leaves exhibit significant antioxidant effects and hepatoprotective activity. In vitro study proved that the saikosaponins exhibited free radical scavenging activity in diphenyl picryl hydrazinyl radical and can suppress the superoxide anion formation and the activity of superoxide anion scavenging in liver cells [49]. There are many types of saikosaponin and each type may have different biological effects in vivo. Previous study on saikosaponin C has found that the HBV-infected liver cells cultured with saikosaponin C exhibited a remarkable lower expression of HBV antigen in culture medium, and this antiviral effect was not due to the cytotoxicity of saikosaponin C [50]. Saikosaponin B2 was also an effective antiviral agent. In vitro study confirmed that saikosaponin B2 can suppress early HCV entry, such as neutralization of virus particles, inhibiting viral attachment, preventing viral fusion, and finally blocking HCV infection of primary human hepatocytes [51].

Salvia miltiorrhiza Bunge, also known as Chinese sage, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. Recent studies found the Salvia miltiorrhiza Bunge can increase blood flow into the liver to reduce the potential damage by clearing the harmful substance in the liver. Active ingredients of Salvia miltiorrhiza Bunge can also exert hepatoprotective effect from CCl₄-induced liver injury in rats. The protective effect may be due to inhibition of NF-kB and p38 signaling in liver Kupffer cells [52]. Treatment of chronic iron-overloaded mice with Salvia miltiorrhiza Bunge will improve the hepatic morphology, decreasing iron deposition as well as inhibiting the expression of type I and type III collagen, TGF-beta mRNA, and increase the expression of matrix metalloprotein-9 (MMP-9) mRNA in the liver. Moreover, Salvia miltiorrhiza Bunge treatment can decrease malondialdehyde content as well as increase glutathione (GSH) content and superoxide dismutase (SOD) activity while it reduced expression of TNF-alpha, IL-1alpha and caspase-3 [53].

Matrine and oxymatrine are both alkaloids with antiviral and anti-inflammation effects, which can be isolated from the herb of Sophora flavescens Aiton. Ma et al. found that the single use of oxymatrine (100 mug/mL), matrine (200 mug/mL), and the nucleoside analogs lamivudine (30 mug/mL) did not show any inhibition effects on the content of HBsAg, HBeAg, and HBV-DNA in culture media. However, the combination of lamivudine (30 mug/mL) with oxymatrine (100 mug/mL) or matrine (100 mug/mL) can significantly suppress the content of HBsAg, HBeAg, and HBV-DNA, the effects of combination treatment were even more remarkable than that from using lamivudine alone at 100 mug/mL [54]. A recent study has analyzed the molecular mechanisms underlying matrine’s anti-inflammatory effects in hepatic cells. The results found that matrine could increase the concentration of nitric monoxide (NO) in culture supernatant of rat intestinal microvascular endothelial cells (RIMECs) in a dose-dependent manner and up-regulate the endothelial nitric oxide synthase (eNOS) concentration, which further improves the vasomotion in liver tissue. Additionally, matrine reduced the secretion of IL-8, IL-6, and sICAM-1 induced by lipopolysaccharide (LPS) in RIMECs. These results indicated that matrine could inhibit inflammation in liver and might be beneficial for patients with hepatitis [55]. Yao et al. investigated the anti-HBV immunomodulatory mechanism of oxymatrine in vitro. Their results showed that oxymatrine could activate the peripheral lymphocytes and induce antiviral cytokine secretion. Pretreatment with oxymatrine could modulate toll-like receptor 9(TLR9) signal pathway and promote the immune function of the TLR9 ligand against virus [56].

Periplocoside A (PSA), a pregnane glycoside, is a new phytochemicals isolated from P. sepium Bge which is widely used for treating rheumatoid arthritis in traditional Chinese medicine. A recent study has examined the protective effects of PSA on T cell-mediated hepatitis induced by concanavalin A (ConA) in murine model. Pretreatment with PSA significantly ameliorated liver damage induced by ConA, the secretion of interleukin (IL)-4, interferon (IFN)-gamma and serum alanine transaminase (ALT) levels were dramatically decreased, which further inhibited the hepatocyte necrosis and protected the liver function in vivo. In vitro studies demonstrated that PSA suppressed the secretion of inflammatory cytokines such as IL-4 and (IFN)-gamma produced by Natural killer T cells upon stimulation with anti-CD3 mAb or α-galactosylceramide. In addition, no obvious toxicity has been observed both in vitro and in vivo with PSA treatment. These results suggested that the new phytochemicals PSA may have therapeutic potential for treating human autoimmune-related hepatitis [57,58].

Baicalin, which is extracted from Scutellaria baicalensis Georgi, was proved to be able to protect hepatocytes from oxidative stress by up-regulating both liver fatty acid binding protein expression and activity of intracellular SOD and GSH [59]. Animal experiments confirmed that baicalin presents anti-inflammatory, anti-oxidant, and anti-apoptotic effects, which offered its hepatoprotective effects against hepatocellular ischemia/reperfusion-induced injury [60]. Wan et al. found that 50-day treatment of the baicalin-containing diet (0.25% and 1%) also exhibited protective effects on liver injury in iron overload mouse, the mechanism of which may be associated with the iron chelation and antioxidant activities of baicalin [61]. Baicalein, another active compound isolated from Scutellaria baicalensis Georgi, may be a promising agent for liver injury. According to a recent study, it can accelerate liver cells’ regeneration by modulating IL-6 and TNF-alpha mediated signaling pathways [62]. Another study found that the molecular mechanisms involved in baicalein-inhibited apoptosis of liver cells were the protective effect on mitochondria, inhibition of the release of cytochrome c, decrease of the Bax/Bcl-2 ratio, and suppression of the phosphorylation of NF-kB, JNK and ERK [63].

Schisandra chinensis (Turcz.) Baill is a frequently used herbal medicine in China, where it has a common name Wuweizi (five-flavor berry). According to the theory of traditional Chinese medicine, Schisandra chinensis (Turcz.) Baill can supplement Qi and promote the production of body fluid. Modern pharmacological studies confirmed that Schisandra chinensis (Turcz.) Baill may regulate the body's humoral balance, promote cell-mediated immune responses and has anti-HBV properties [64]. Xue et al. isolated and investigated seven unknown lignans and a schischinone, together with several known lignans from the fruits of Schisandra chinensis (Turcz.) Baill. Their study found that two lignans from the Schisandra chinensis (Turcz.) Baill exhibited significant anti-Hepatitis B virus activity by suppressing HBV DNA replication without obvious cell toxicity on hepatic cells [65]. Schisandrin B, a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis (Turcz.) Baill, has shown therapeutic efficiency for the treatment of hepatitis. The anti-inflammatory mechanisms of Schisandrin B were investigated by Checker et al. The results showed that Schisandrin B can induce nuclear translocation of redox sensitive nuclear factor erythroid-derived factor 2-related factor (Nrf2) and increase the transcription of Heme Oxygenase-1(HO-1). Schisandrin B can also inhibit the nuclear translocation of NF-kB in activated lymphocytes and suppress the expression of downstream inflammation-related genes [66].

Astragalus membranaceus (Fisch.) Bunge has been one of the most widely used herbs in China for thousands of years. According to the traditional Chinese medicine theory, it could increase metabolism, improve the functions of lungs and the gastrointestinal tract, promote wound healing and provide fatigue relief. The main active compounds in Astragalus membranaceus (Fisch.) Bunge are flavonoids and saponins, such as calycosin, formononetin, astragaloside, and calycosin-7-Obeta-D-glucoside [67,68]. Modern researches have shown that Astragalus membranaceus (Fisch.) Bunge could modulate immunologic system, inhibit virus growth and suppress cancer proliferation [69,70]. A recent study has indicated that Astragalus membranaceus (Fisch.) Bunge has potential therapeutic effects in patients with viral hepatitis. In vivo studies showed that Astragalus membranaceus (Fisch.) Bunge could suppress duck HBV DNA replication. The liver pathological changes in duck viral Hepatitis B models with Astragalus membranaceus (Fisch.) Bunge treatment were also milder than the control group. Clinical study was conducted by the same group, in which Astragalus membranaceus (Fisch.) Bunge was used for treating 116 patients with chronic viral hepatitis while 92 patients were given regular antiviral agents. The proportion of negative seroconversions of HBeAg and HBV DNA were significantly higher in the Astragalus membranaceus (Fisch.) Bunge group than in the control group (p < 0.01 and p < 0.05 respectively). These results showed that the clearance of serum HBeAg and HBV DNA was better in Astragalus membranaceus (Fisch.) Bunge treatment group [71]. Further studies have identified that the active ingredients in Astragalus membranaceus (Fisch.) Bunge for inhibiting HBV activities may be triterpenoid saponin [72].

2.2.1. The Epidemiological and Pathological Characteristics of Fatty Liver Disease and Current Therapeutic Strategy

Fatty liver disease, or steatosis, is a reversible pathological process wherein large vacuoles of triglyceride fat accumulate in liver cells. Fatty liver disease is usually induced by excessive alcohol consumption or metabolic disorders such as obesity, insulin resistance and dyslipidemia [73]. Alcoholic fatty liver (AFL) refers to the alcohol-related steatosis while nonalcoholic fatty liver (NAFL) means the original cause of steatosis is not alcohol. In Western countries, fatty liver disease is the most common liver disease, the prevalence of which ranges from about 20%–60% [74]. In 1992, some researchers in Italy started the Dionysos Study that intended to evaluate the prevalence and incidence of chronic liver disease in two communities of Northern Italy. According to their results, 45% of the residents had fatty liver disease identified by liver ultrasonography. As fatty liver diseases cannot be distinguished at liver biopsy, normally, their distinction depends on alcohol consumption. Using a cut-point of 20 g/d for ethanol consumption, about 25% residents had NAFL while 20% had AFL [75]. In patients with NAFL, approximately 30% patients will progress to non-alcoholic steatohepatitis (NASH). In those with NASH, approximately 20% will further progress to cirrhosis, in which the histological lesions of the liver are irreversible. For patients with AFL, nearly 20%–40% of these patients will progress to alcoholic steatohepatitis (ASH). Of those with ASH, approximately 40% will develop cirrhosis [76].

Defects in fatty acid metabolism are responsible for the initial stage of fatty liver disease, which may lead to lipid overstorage in the liver. For AFL, alcoholism may damage mitochondria and other cellular structures, and further leads to cellular fatty acid metabolism disorder. For NAFL, insulin resistance and other metabolism disorder induced the lipid storage and progress to steatosis. In both AFL and NAFL, steatosis will sensitize the liver tissue to the induction of inflammation by oxidative stress and will further develop steatohepatitis [77]. Actually, only a part of patients with steatosis will progress to steatohepatitis, and the mechanisms are still unclear. The inflammation and a high degree of steatosis will induce hepatocyte necrosis, lipid peroxidation and activation of stellate cells, which play an important role in liver fibrosis [78].

The therapeutic strategy for fatty liver depends on its cause, treating the underlying cause at an early stage of steatosis may reverse the process and inhibit steatohepatitis. Thus, strict restriction of alcohol consumption and avoiding high calorie containing foods are important interventions for patients with fatty liver. For those with early stages of non-alcoholic fatty liver disease, a gradual weight loss is the only solution for preventing disease progression. Conventional medications such as ursodeoxycholic acid, insulin sensitizers, antioxidants or lipid-lowering drugs for decreasing insulin resistance, hyperlipidemia, and inducing weight loss may have certain therapeutic effects on pure steatosis without inflammation. For advanced patients with steatohepatitis, there are still no effective treatments [79,80]. According to recent studies, some herbal products seemed to have positive effects on this potentially reversible disease. Both basic and clinical studies suggested that herbal medicines may have modest benefit for fatty liver disease treatment (Table 2).

2.2.2. Herbs and Phytochemicals in the Treatment of Fatty Liver Disease

Berberine is a quaternary ammonium salt isolated from the plant of Berberis such as Coptis chinensis Franch, which is one of the 50 fundamental herbs used in traditional Chinese medicine. The alkaloids in Coptidis Rhizoma aqueous extract (CRAE) were found to be effective in reducing triglyceride (TG) accumulation in the FFA-induced hepatic steatosis in liver cells. Berberine has been confirmed as the major compartment in inhibition of TG accumulation [81]. Berberine can also enhance insulin resistance of nonalcoholic fatty liver disease by increasing the expression of insulin receptor substrate-2 (IRS-2) and regulating the insulin signaling pathway [82]. Xue et al. found that the berberine-loaded solid lipid nanoparticles (BBR-SLNs) can relieve hepatosteatosis in db/db mice by suppressing lipogenesis and promoting lipolysis in the liver. Further study of the mechanisms revealed that BBR-SLNs can inhibit the expression of lipogenic genes such as stearoyl-CoA desaturase (SCD1), fatty acid synthase (FAS), and sterol regulatory element-binding protein 1c (SREBP1c), and increase the expression of lipolytic gene such as carnitine palmitoyltransferase-1 (CPT1) [83].

Gynostemma pentaphyllum (Thunb.) Makino (GP) is a dioecious, herbaceous climbing vine distributed in Eastern Asian. It is widely used for lowering cholesterol, and preventing fatty liver and obesity in China and Japan. Wang et al. found that GP extracts can promote lipid metabolism, and decrease serum lipids level by down-regulating the production of trimethylamine N-oxide (TMAO) and up-regulating the secretion of phosphatidylcholine [84]. In a study with a NAFL cellular model, GP extract was efficient in inhibiting the accumulation of cholesterol and triglycerides as well as preventing oxidative stress in liver cells. Further study of the mechanisms revealed that GP enhances the production of nitric oxide (NO) which reduces oxidative stress damage in liver cells and affects the molecular composition of the mitochondrial phospholipid cardiolipin (CL) [85]. A randomized, single-blind, controlled clinical trial showed that six-month dietary GP treatment can significantly reduce the level of serum AST, ALP, insulin, and decrease the body mass index (BMI) and insulin resistance index. In the GP group, BMI had reduced remarkably from 28.4 at month 0 to 27.5 at month 6 (p = 0.018), total cholesterol had dropped from 228.5 (95% CI 148.06–308.94) to 206.1 (95% CI 139.35–272.85) (p = 0.004), and ALT had decreased from 80.2 (95% CI 19.53–140.87) to 63.2 (95% CI 3.99–122.51) (p = 0.004) [86]. These studies demonstrated that GP extracts can be an effective adjunct treatment for NAFL patients. Ombuine is a flavonoid isolated from GP. Recent studies confirmed that ombuine treatment remarkably decreased intracellular concentrations of triglyceride and cholesterol in liver cancer cells and reduced the expression of some lipogenic genes through activating PPARα and β/δ. Furthermore, Ombuine can also decrease cellular cholesterol concentrations by activating ATP binding cassette cholesterol transporter A1 and G1 protein expression [87].

Panax notoginseng (Burkill) F.H.Chen (PNS) is a species of the genus Panax, which grows naturally in China and Japan. Ding et al. found that PNS extracts can attenuate the ethanol induced hepatic lipid accumulation by inhibiting the production of malondialdehyde (MDA), glutathione (GSH) l and reactive oxygen species (ROS), reducing TNF-alpha and IL-6 levels, as well as enhancing the superoxide dismutase (SOD) activity in liver, and abrogating cytochrome P450 2E1 (CYP2E1) induction. In addition, PNS can protect liver cells from CYP2E1-mediated oxidative stress [88,89]. In vivo studies from the same group also found that total saponins of PNS can relieve oxidative stress and insulin resistance in NAFLD rat models. Concentrations of malondialdehyde (MDA), the hydroxy radical level (–OH), and TNF-alpha decreased after PNS treatment, while the activities of total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) were recovered, and insulin resistance improved significantly.

Penta-oligogalacturonide is isolated from the fruit of Crataegus pinnatifida Bunge, which is the most often used herb in the treatment of NAFLD according to the systematic review study by Shi et al. [90]. Recently, the anti-lipidemic and antioxidant effects of penta-oligogalacturonide were investigated both in vitro and in vivo. The results showed that penta-oligogalacturonide was effective in scavenging hydroxyl, superoxide anion and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals in liver cells. Additionally, penta-oligogalacturonide can enhance the antioxidant enzyme activities of superoxide dismutase, catalase, and glutathione peroxidase, increase the levels of glutathione and the total antioxidant capacity, but lower the production of malondialdehyde in the liver of high-fat fed mice. Furthermore, penta-oligogalacturonide significantly decreased the triglyceride levels, expression of phosphatidate phosphohydrolase (PAP) and glycerol 3-phosphate acyltransferase (GPAT) in mice livers. Moreover, the liver biopsy results confirmed that penta-oligogalacturonide treatment at a dose of 300 mg/kg can inhibit liver steatosis in mice with high-fat diet [91]. Another study from the same research group also found that penta-oligogalacturonide remarkably increased the liver fatty acid oxidation-related enzyme activities of acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, 2,4-dienoyl-CoA reductase, and carnitine palmitoyltransferase I, which may prevent hyperlipemia in the liver of high fat diet mice [92].

Dioscin, a natural steroid saponin isolated from Dioscorea opposita Thunb, has been shown to have a remarkable protective effect against fatty liver disease. Recent study has demonstrated that dioscin can reduce body weight and lipid accumulation in liver, increase oxygen consumption and energy expenditure and decrease the levels of serum ALT, AST. Further study of the mechanisms revealed that dioscin can relieve oxidative damage, inhibit inflammation, suppress cholesterol and triglyceride synthesis, decrease mitogen-activated protein kinase (MAPK) phosphorylation levels, promote fatty acid beta-oxidation, and induce autophagy to improve fatty liver conditions. These results indicated that dioscin may be a potential candidate for obesity and NAFLD prevention [93]. Dioscin also showed an excellent protective effect against alcoholic fatty liver by relieving alcohol-induced oxidative stress, inflammatory cytokine production, mitochondrial function, apoptosis and liver steatosis in vivo [94].

Gallic acid is a trihydroxybenzoic acid found in the pericarp of Punica granatum L. Previous studies have confirmed that gallic acid has potent anti-oxidative and anti-obesity activity. Chao et al. found that gallic acid can improve the glucose and lipid metastasis disorder in NAFLD mice. The hepatoprotective effect by gallic acid may be related to regulation of the metabolism of choline, amino acids, glucose, lipid and gut-microbiota in mice [95]. Histological study in the high fat diet-induced NAFLD rat showed that the lipid droplets in gallic acid treatment group were significantly smaller than the control group. When compared with the control group, cholesterol level and hepatic triacylglycerol (TAG) in gallic acid treatment groups were remarkably reduced. Furthermore, gallic acid treatment decreased oxidized glutathione (GSSG) content and increased the levels of anti-oxidative enzyme in liver. These results indicated that gallic acid treatment can inhibit NAFLD-related hepatosteatosis, dyslipidaemia and oxidative stress in rats [96].

Compound glycyrrhizin has been widely used in clinical practice for chronic liver disease. An in vivo study has proved the therapeutic effects of compound glycyrrhizin liposome on nonalcoholic steatohepatitis in rats in inhibiting the process of fibrosis [97]. Another study also confirmed that the glycyrrhizin-containing preparation can suppress the development of hepatic steatosis in a dose-dependent manner and attenuate ultrastructural alterations of mitochondria of the hepatocytes. In addition, it can down-regulate the reactive oxygen species in mice liver [98]. These results demonstrated that the glycyrrhizin-containing preparation can prevent hepatic steatosis possibly by preventing mitochondria damage from oxidative stress.

According to previous studies, the curative effects of silymarin in alcoholic and non-alcoholic steatohepatitis are widely recognized [99]. Recently, siliphos, a modified derivative of silymarin, has been proved to effectively suppress severe oxidative stress and to protect hepatic mitochondrial bioenergetics in nonalcoholic steatohepatitis [100]. Through protecting mitochondrial function, the damage of lipid membranes by oxidation is alleviated by siliphos. Clinical trials also confirmed that after siliphos treatment, ultrasonographic score for liver steatosis has decreased remarkably in patients with nonalcoholic fatty liver disease. In addition, a significant improvement in liver enzyme levels, indexes of liver fibrosis, and hyperinsulinemia have been observed in siliphos treatment group [101].

The kernels of Prunus armeniaca L are used as a common drug in traditional Chinese medicine. As a popular fruit, dietary intake of Prunus armeniaca L kernels can lower cholesterol levels in the body [102]. Recent studies have found that feeding rats with the kernels of Prunus armeniaca L had protective effects against CCl₄-induced liver steatosis due to its remarkable antioxidant and radical-scavenging capacity. The rich content of beta-carotene and vitamin may be associated with the anti-steatosis effect by the kernels [103]. In a clinical study, the extract of Prunus armeniaca L kernels also exhibit protective effects on fatty liver disease patients. The results showed that serum ALT and AST levels were remarkably reduced compared with pre-intake baseline levels from 103.5 (95% CI 4.03–202.87) IU/L to 71.8 (95% CI 5.39–138.21) IU/L (p < 0.05) and from 93.5 (95% CI 0.02–187.72) IU/L to 65.5 (95% CI 6.69–123.31) IU/L (p < 0.05). A decrease of more than 30% from the pre-study baseline ALT and AST levels were detected in 45% and 43% of the patients (n = 58), respectively. These results indicated that after treating these patients with Prunus armeniaca L extracts, the serum AST and ALT levels were significantly decreased compared with the control group [104].

Baicalin was also found to be effective in modulating lipid metabolism and suppressing systemic inflammation in high-fat feeding rat liver and the mechanism may be related to the regulation of AMPK-alpha signaling pathway [105,106]. Further study confirmed that baicalin can decrease the levels of serum total cholesterol, triglycerides, low density lipoprotein (LDL), ALT and AST, and increase the levels of serum HDL. Pathological analysis revealed a higher dose of baicalin diminished both macrovesicular and microvesicular steatoses while a low dose of baicalin only suppressed macrovesicular steatosis. The mechanism may be associated with the regulation of hepatic CaMKKbeta/AMPK/ACC pathway [107]. For alcoholic fatty liver disease, baicalin is also effective in attenuating the ischemia/reperfusion injury in alcoholic fatty liver. In ethanol-fed animals, baicalin attenuated inflammatory responses by suppressing of myeloid differentiation factor 88 and toll-like receptor 4 expressions and the nuclear translocation of NF-kB after reperfusion [108].

2.3.1. The Epidemiological and Pathological Characteristics of Cirrhosis and Current Therapeutic Strategy

Cirrhosis is a condition in which the liver does not function properly due to long-term damage; it is a terminal complication of chronic liver diseases. According to a study in Germany, the most common etiologies of liver cirrhosis in German were related to alcoholic liver disease (52%), chronic Hepatitis C (28%) or Hepatitis B (14%) infection and NASH (6%) [109]. However, the situation in developing countries is quite different; for example, in China, the distribution of etiological agents for cirrhosis was as follows: HBV 77.22%, alcohol 5.68%, HCV 2.80%, and the other etiologies accounted for about 15% [110]. Cirrhosis-related deaths increased from about 676,000 cases in 1980, or 1.54% of total global deaths, to more than 1,000,000 deaths in 2010, or 1.95% of the global deaths [111].

The pathological hallmark of cirrhosis is the development of fibrosis that replaces normal parenchyma. Fibrosis means overabound scar tissue builds up in the liver during wound healing and damage repair. The extracellular matrix (ECM) proteins such as collagens are excessively produced or deficiently degraded in this process, which may be due to the chronic inflammation or fatty liver. Fibrosis is potentially reversible if the cause is removed at this stage, but advanced fibrosis may further develop to cirrhosis which involves loss of hepatic cells and irreversible scarring in liver. Recent studies show the pivotal role of the stellate cells in the development of cirrhosis. Inflammation in the liver parenchyma can cause activation of hepatic stellate cells, which further leads to fibrosis formation and blood circulation obstruction. In addition, it secretes cytokines such as TGF-β1, which further promotes the fibrotic response and proliferation of connective tissue. Furthermore, it secretes tissue inhibitor of metalloproteinases (TIMPs) which inhibit the activity of matrix metalloproteinases (MMPs) and suppress them from resolving fibrotic material in the ECM [112]. Several mitogen-activated protein kinases regulate major fibrogenic actions of stellate cells. For example, c-Jun N-terminal kinase regulates apoptosis of hepatic cells and induces the secretion of inflammatory cytokines by stellate cell [113]. The focal adhesion kinase PI3K-Akt–signaling pathway regulates agonist-induced fibrogenic actions in stellate cell [114]. The TGF-β1–activated Smad-signaling pathway modulates experimental hepatic fibrosis in vitro and is a potential target for anti-fibrosis [115]. The PPAR pathway modulates stellate cell activation and fibrosis process. PPAR-γ ligands suppress the fibrogenic process in stellate cell in vitro and in vivo [116]. Recent studies suggest that NF-κB, Toll-like receptors and β-cathepsin may be involved in fibrosis regulation [117,118].

So far, there is no treatment that can radically reverse cirrhosis except for liver transplantation, but the supply of liver allografts is far short of the number of potential recipients [119]. Key prevention strategies for current cirrhosis management are through further prevention of liver damage by hepatitis or fatty liver disease. Unlike the irreversible process of cirrhosis, recent researches have proved that even advanced fibrosis is remediable. The appropriate anti-fibrosis therapy should target the reduction of lavish collagen deposition specifically without destroying normal ECM. Although no drugs have been approved for treating fibrosis or cirrhosis in USA, some herbal medicines have shown remarkable efficiency in preventing cirrhosis and treating fibrosis or relieving symptoms of cirrhosis (Table 3).

2.3.2. Herbs and Phytochemicals in the Treatment of Cirrhosis

The hepatoprotective effect by CRAE on CCl₄-induced chronic liver hepatotoxicity such as fibrosis has been reported by many studies [120,121]. These results showed that serum ALT and AST activities were remarkably decreased in CCl₄-induced rats when treated with CRAE. The significant increase of serum SOD activity and the histological results proved that CRAE could recover CCl₄-induced chronic oxidative stress by anti-oxidant mechanisms. In addition, another study found that the myofibroblast proliferation and the expression of TGF-b1 and a-SMA were remarkably decreased after berberine treatment [122]. These studies proved that CRAE and berberine could be promising anti-fibrosis agents in preventing and treating liver disease. The further mechanism study showed that the anti-fibrosis effects by berberine may be induced by activation of AMPK, blockade of Nox4 and Akt expression [123]. A comparative research on the hepatoprotective action of bear bile and CRAE on experimental liver fibrosis in rats demonstrated that berberine, CRAE, and bear bile all exhibited anti-fibrotic properties on liver fibrosis [124]. All of these agents could remarkably increase the SOD activity and reduce the peroxidative stress in rat liver. CRAE and berberine can protect hepatocytes from cholestatic damage through excreting bilirubin products from the liver. These results illustrated that CRAE and berberine may be promising agents to replace the valuable bear bile for liver fibrosis in clinical practice.

Puerarin is a kind of isoflavones isolated from Pueraria lobata. Recent studies have found that puerarin could promote the liver metabolic function and reduce the levels of ALT, AST and total-bilirubin, ECM contents and up-regulate the levels of albumin and total-protein in liver fibrosis rats. Further studies showed that puerarin could relieve the CCl₄-induced liver fibrosis in vivo; the mechanisms may be related with modulation of the expression of PPAR-γ protein and PI3K/Akt pathway [125]. Another study found the TNF-alpha/NF-kB pathway was also involved in puerarin mediated anti-fibrosis effects in hepatic fibrotic rats. Through down-regulating the TNF-alpha and NF-kB expression by puerarin treatment, the inflammation response in liver tissue was relieved and the metabolic function was also improved [126]. Puerarin can also induce apoptosis of hepatic stellate cells, which plays a pivotal role in liver fibrosis. Further study of the mechanisms confirmed that down-regulation of bcl-2 mRNA are involved in puerarin induced hepatic stellate cells apoptosis [127].

Saururus chinensis (Lour.) Baill has been used in Chinese medicine for treating jaundice, gonorrhea and edema as well as reversing liver fibrosis [128,129]. Saururus chinensis (Lour.) Baill extract can effectively reduce the elevated levels of liver indexes such as serum ALT, AST, hyaluronic acid (HA), and hepatic MDA contents in hepatic fibrotic rats, it can also enhance the hepatic SOD activity after CCl₄-treatment. The histopathological results proved that Saururus chinensis (Lour.) Baill extract significantly relieved the CCl₄-induced liver fibrosis. These results demonstrated the hepatoprotective effect of Saururus chinensis (Lour.) Baill extract on CCl₄-induced liver fibrosis [130].

The protective effect of glycyrrhizin on liver cirrhosis has been proved by both in vitro and in vivo studies. The mechanism of the anti-cirrhosis effect of glycyrrhizin perhaps has an inhibitory effect on NF-kB binding activity [131]. In this research, both the serum ALT detection and histological analysis results proved the anti-cirrhosis effect by glycyrrhizin. In addition, NF-kB binding activity was significantly decreased after glycyrrhizin treatment in the liver specimens of cirrhosis rats. 18alpha-glycyrrhizin, a modified derivative of glycyrrhizin, also showed obvious anti-fibrosis effects in CCl₄-induced liver damage in rats. 18alpha-glycyrrhizin treatment improved histological changes and inhibited collagen deposition by decreasing the expressions of Smad2, Smad3, SP-1 and TGF-beta1 in both mRNA and protein level in the liver [132]. A phase III clinical study also confirmed the anti-fibrosis efficacy and safety of glycyrrhizin in 379 chronic Hepatitis C patients who failed to respond to interferon-based treatment. Results showed that 12-week treatment with 5×/week glycyrrhizin injections could decrease ALT level ≥50% in 28.7% patients and in the effect of 3×/week glycyrrhizin injections group the proportion was 29.0%, which were much more significant than placebo group (7.0%, p < 0.0001). Fifty-two week treatment can relieve the necro-inflammation in liver in 44.9% patients with 5×/week and in 46.0% patients with 3×/week, respectively. These results indicated that glycyrrhizin can reduce serum ALT level after 12 weeks of treatment and suppress necro-inflammation and fibrosis after 52 weeks of treatment compared to placebo group. In addition, no obvious side-effect by glycyrrhizin was detected in the clinical trial [133].

Silybinin, which is also known as silybin, exhibits antioxidant effect and induces mitochondrial biogenesis in cirrhotic livers [134]. Silybinin can prevent the production of mitochondrial reactive oxygen species (ROS) and inhibit the cardiolipin oxidation or citrate carrier failure in the liver of cirrhosis rat model. Silybinin also exhibits significant anti-inflammatory effect in cirrhotic rat liver by decreasing the expression of lysophosphatidylcholine acyltransferase (LPCAT) while increasing platelet-activating factor level [135]. A clinical survey indicated that silymarin can reduce symptoms and quality-of-life in cirrhosis patients. Long-term use of silymarin may provide benefits to patients with chronic Hepatitis C [136]. However, the effects of silymarin on survival rate or the disease progression of liver cirrhosis remains controversial in clinical practice [137].

Bupleurum kaoi Liu, C.Y.Chao & Chuang, a plant of endemic Bupleurum species in Taiwan, exhibits anti-fibrotic and anti-inflammatory activities in liver cells. These effects are proved to be associated with the anti-oxidant activity by observing the increased glutathione expression. It can also regulate the liver cell regeneration through the increased expression of IL-10 and INF-gamma. In addition, when compared with the Bupleurum chinense DC, the anti-fibrotic and hepatoprotective effects of Bupleurum kaoi are more significant [138]. Saikosaponin A, the major active ingredients in Bupleurum chinense DC, was found to increase the expression of bone morphogenetic protein 4 (BMP-4) and inhibit the activation of hepatic stellate cells [139]. When combining IFN-alpha treatment with saikosaponin, it could significantly reduce the damage of immune hepatic injury. In this study, the combination therapy can increase peripheral blood CD4+ T cell and CD8+ T cell ratios more significantly than using IFN-alpha alone. Meanwhile, the IL-18 and TNF-alpha levels both decreased obviously in combination therapy [140]. A recent study has indicated that saikosaponin A can be used for treatment of BMP-4 induced liver damage. Their result showed that the development of liver fibrosis was remised in the saikosaponin treatment group. The saikosaponin treatment can down-regulate plasma aspartate aminotransferase and alanine aminotransferase activities. In addition, the plasma and hepatic cholesterol and triglyceride levels also decreased after saikosaponin treatment [141].

The isolated Salvianolic acid B from Salvia miltiorrhiza Bunge has anti-fibrosis effect in liver diseases. Salvianolic acid B can inhibit intracellular signal transduction of TGF-β1 in hepatic stellate cells and suppress the expression of its receptor protein, thereby antagonizing the hepatic stellate cells activation, and inhibiting the fat-storing cell proliferation as well as reducing the deposition of collagen fiber in liver. A related animal study also confirmed that the Salvia miltiorrhiza Bunge treated liver tissues exhibited little fibrous tissue deposition in the portal areas, presented normal tissue morphology, and obviously decreased collagen deposition. In addition, decreased tissue inhibitor of metalloproteinase (TIMP)1 and collagen 1(alpha) protein also supports the anti-fibrotic effect of Salvia miltiorrhiza Bunge [142]. In animal studies, Salvia miltiorrhiza Bunge extracts have appeared to inhibit the process of liver fibrosis and improve liver function by reducing the nonfunctioning fibers in the liver. For people who have chronic hepatitis or excessive consumption of alcohol, Salvia miltiorrhiza Bunge is recommended for protecting their liver function [143].

The increase of glutathione S-transferase A5 expression and decrease of P450 cytochrome 3A2 by Scutellaria baicalensis Georgi extracts was also observed in liver cells. Treatment with methanolicextracts of Scutellaria baicalensis Georgi significantly reduced the levels of liver malondialdehyde and hydroxyproline with ameliorative histological results, which indicated the anti-fibrosis effect of Scutellaria baicalensis Georgi [144]. Scutellaria baicalensis Georgi extract also suppresses the proliferation and activation of hepatic stellate cells by inducing cell cycle arrest in G2/M phase and stellate cell apoptosis via caspases and Bax pathway [145]. Baicalein may be the main active component in Scutellaria baicalensis Georgi for prevention of cirrhosis and fibrosis. In vivo studies confirmed that baicalein can inhibit hypertrophic scar formation by suppressing TGF-beta/Smad2/3 signaling pathway in mice with mechanical load-induced scars [146]. Long-term administration of baicalein may suppress stellate cell activation by decreasing the expression of PDGF-beta receptor, and thus it can restrain the development process of liver fibrosis in animal models [147].

2.4.1. The Epidemiological and Pathological Characteristics of Primary Liver Cancer and Current Therapeutic Strategy

The incidence of primary liver cancer is increasing in many industrialized countries, it is the sixth most frequent diagnosed cancer globally, and has become the second leading cause of cancer death since 2014. In 2010, primary liver cancer become the third leading cause of cancer death worldwide, with 754,000 deaths related with this disease. In 2012, about 782,000 people were diagnosed with primary liver cancer and 746,000 people eventually died from this disease. The occurrence of liver cancer may be associated with hepatitis virus infection (71%) and alcoholic abuse (20%). In 2014, liver cancer resulted in more than 750,000 deaths globally [148].The incidence of liver cancer is higher in those countries where hepatitis virus infection are common, such as East-Asia and sub-Saharan Africa [149]. The five year survival rate of primary liver cancer is only 17% in the United States in 2014 and even lower in developing countries [150]. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, which shows an obvious geographical distribution. For example, 50% of HCC are diagnosed in China [151].

The leading cause of liver cancer is cirrhosis induced by viral hepatitis or fatty liver disease. Hepatitis or fatty liver promote the development of HCC through repeatedly inducing the immune system to attack the hepatic cells, while this constant cycle of damage followed by repair can lead to mistakes during repair which in turn lead to carcinogenesis. For chronic Hepatitis B, the integration of the viral genome into the host liver cells can directly induce gene mutation and thereby develop HCC. The disorders in Wnt, TGF-β, Hedgehog and Notch signaling pathways play a pivotal role in the oncogenesis process of HCC. Some other factors such as microRNAs may also regulate the liver oncogenesis process [152,153].

So far, the treatment for liver cancer is limited, the later stage or higher-grade liver cancer patients will ordinarily have poor prognosis [154]. Surgical resection may be the best therapy for promoting long-term survival, but because most patients are diagnosed at the later stage of disease, therefore only 10% of the patients are suitable for surgical resection. Sorafenib, a receptor tyrosine kinase inhibitor which has been approved in the US and Europe, may be beneficial for patients with advanced HCC. Unfortunately, Sorafenib increases lifespan by only two months for late stage HCC patients with satisfactory liver function according to a phase III clinical trial [155]. Under these circumstances, herbal medicines are recommended as an alternative treatment for liver cancer patients, in order to reduce the side-effects from conventional therapy and prolong survival time as well as improve life quality (Table 4).

2.4.2. Herbs and Phytochemicals in the Treatment of Primary Liver Cancer

The recent pharmacological studies have demonstrated that both Coptidis rhizoma and berberine have a prominent hepatoprotective effect, especially the anti-cancer effect on hepatocellular carcinoma. A recent research has shown that the alkaloids in Coptidis rhizoma aqueous extract (CRAE) inhibited vascular endothelial growth factor (VEGF) by suppressing the activity of eukaryotic elongation factor 2 (eEF2) in liver cancer [156]. The phytochemical study showed that the major alkaloids in CRAE are berberine, jatrorrhizine, magnoflorine and palmatine. These alkaloids could suppress VEGF secretion in HepG2 and MHCC97L cells while no significant toxicity has been observed in normal liver cells. This inhibition effect was mainly observed for protein level rather than mRNA transcriptional level. The alkaloids in CRAE also inhibited the activation of eEF2 by raising the phosphorylation levels of eEF2 and thereby blocked the nascent protein synthesis. Berberine was found to be the main active ingredient in these alkaloids but the total alkaloids in CRAE is more effective in inhibiting the activity of eEF2. The in vivo study also confirmed that the tumor size of xenograft mice was significantly reduced after Coptidis rhizoma treatment. According to another study by our research group, miR-23a expression can be upregulated by berberine treatment in human HCC cells and may activate the transcription of p53-related tumor suppressive GADD45alpha and p21 genes. In vivo study also showed that when miR-23a expression was inhibited, the berberine-induced tumor suppression in mice was attenuated [