Current Understanding on Antihepatocarcinoma Effects of Xiao Chai Hu Tang and Its Constituents

Xiao Chai Hu Tang (XCHT), a compound formula originally recorded in an ancient Chinese medical book Shanghanlun, has been used to treat chronic liver diseases for a long period of time in China. Although extensive studies have been demonstrated the efficacy of this formula to treat chronic hepatitis, hepatic fibrosis, and hepatocarcinoma, how it works against these diseases still awaits full understanding. Here, we firstly present an overview arranging from the entire formula to mechanism studies of single herb in XCHT and their active components, from a new perspective of “separation study,” and we tried our best to both detailedly and systematically organize the antihepatocarcinoma effects of it, hoping that the review will facilitate the strive on elucidating how XCHT elicits its antihepatocarcinoma role.


Introduction
The American Cancer Society's estimation for primary liver and bile duct cancers in the United States for 2013 is about 30,640 new cases and 21,670 deaths from these cancers. Liver cancer is even more common in sub-Saharan Africa and Southeast Asia and currently is the most common type of cancers in many countries in these regions [1]. Among various types of liver cancer, hepatocellular carcinoma (HCC) is the most common one, and the majority of them are associated with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections [2,3]. Chronic viral hepatitis, chemicalinduced liver damage, could cause liver fibrosis and cirrhosis, finally leading to liver cancer.
Compound herbal formulas have been used to treat cancers, and many of them have shown the promise to improve the life of cancer patients [4][5][6]. These compound formulas are usually made of several Chinese medicinal herbs and suppress tumor progression by multiple mechanisms [7]. One of them is called Xiao Chai Hu Tang (XCHT, Sho-saiko-to, in Japanese) that was originally recorded in ancient Chinese medical book Shanghanlun. It consists of seven medicinal herbs (Bupleurum falcatum, Scutellaria baicalensis, Panax ginseng, Zizyphus jujube, Pinellia ternate, Zingiber officinale, and Glycyrrhiza glabra) and is currently used to treat chronic liver diseases especially chronic hepatitis [8][9][10][11]. Data from recent clinical trials convincingly show that XCHT can prevent the development of HCC in patients with cirrhosis, particularly those without HBs antigen [9]. Experimental studies further indicate that XCHT may achieve its effect by reducing hepatocyte necrosis and enhancing liver function. Moreover, XCHT has also been shown to exhibit various anticarcinogenic properties such as induction of apoptosis and suppression of invasion [12,13].  The difference of the mean value of AST and ALT between the XCHT group and placebo group was significant; a tendency towards a decrease of HBeAg and an increase of anti-HBe antibodies was also observed in patients with chronic active type B hepatitis Oka et al. [9]; 1995 260 cirrhotic subjects Randomized, controlled The cumulative incidence curve for 5 years of the trial group (XCHT combined with conventional drugs) was lower while the survival curve for 5 years of the trial group was higher compared with control group (conventional drugs). The difference was significant for patients without HBs antigen Deng et al. [11]; 2011 24 chronic hepatitis C subjects A single arm phase II study Improvement of AST (16 subjects) and ALT (18 subjects) was observed; 9 subjects showed improvement in histology activity index scores Bo and Du [14]; The liver function was improved and serum liver fibrosis indexes were decreased; the difference was significant between XCHT treatment group and controlled group (hepatic protective drug and antifibrosis drug treatment) Chinese herbal medicines are usually used to counteract tumor progression by a formula of multiple herbs rather than a single one. Unfortunately, studies have been mainly focused on defining the mechanism of a single herb or its ingredients. As each herb in XCHT can potentially exert its effect in a distinct mechanism, a formula of seven herbs is expected to reach its full effect by targeting multipathways and multitargets. So we firstly introduce a new perspective of "separation study, " that is, from the entire formula to single herb and their active components, both detailedly and systematically organize the antihepatocarcinoma effects of XCHT. We hope that this review will help XCHT to receive its well-deserved global recognition and to be better appreciated for its clinical use to treat liver cancers.

Xiao Chai Hu Tang (XCHT)
2.1. Clinical Trials of XCHT. XCHT has long been used in clinical trials for the study and treatment of liver diseases. Some XCHT clinical trials (including the clinical trials mentioned above and some other trials [14][15][16][17] conducted in China) are summarized in Table 1.

Experimental Studies of XCHT.
The potential tumorsuppressing effect of XCHT was reported in 1994, in which XCHT was found capable of inhibiting the proliferation of KIM-1, a human hepatocellular carcinoma cell line and KMC-1, a cholangiocarcinoma cell line [18]. Later on, XCHT was shown to diminish not only the growth of various cancer cell lines but also in vivo tumor outgrowth in xenograft model [19][20][21].
In addition to its tumor-suppressing role, Chang et al. studied the effect of XCHT on HBV replication in HepG2 2.2.15 cell model [22]. Their study showed that XCHT reduced HBV production and HBeAg expression without altering the level of HBsAg. Although XCHT can also block Coxsackie B type 1 virus infection in CCFS-1 cells through the induction of Type I interferon expression [23], the mechanism responsible for XCHT-mediated suppression of HBV production awaits being defined.
Hepatic fibrosis and liver cirrhosis result from wound healing of ongoing hepatocellular damage caused by chronic liver injuries [13]. Most of HCCs ensue in a cirrhotic liver [24]. Kusunose et al. created an animal model that reflected various stage-liver injuries and used this model to determine under what condition XCHT extract could improve hepatic inflammation and fibrosis [25]. Their study found that the ability of XCHT extract was limited to a certain degree which was expressed by levels of respective parameters (AST, ALT, TGF-, hydroxyproline, and the ratio of liver fibrosis area). Chen et al. later elucidated the mechanism pertinent to XCHT's antifibrosis capability by assessing its effect on the expression of those growth factors and cytokines important for the activation of hepatic stellate cells (HSCs) [26]. They showed that XCHT downregulated the levels of stellate cell activation-essential TGF 1, platelet derived growth factor (PDGF), and IL-1 while stimulated the production of stellate cell activation-inhibitory TNF .

Individual Herbs and Active Components in XCHT
In TCM, XCHT is a classical formula to treat the typhoid lesser yang syndrome. The compound prescription has its formulating principle. Among the seven herbs included in XCHT, Bupleurum falcatum is the "monarch" and Scutellaria baicalensis is the "minister, " which are the principal herbs in this formula. Panax ginseng, Zizyphus jujube, Pinellia ternate, and Zingiber officinale are the "assistant" while Glycyrrhiza glabra is the "guide" [27]. Bupleurum has the effect of upraising and dispersing the pathogen and soothing the meridian Qi. Scutellaria has the effect of clearing and down-sending heat with bitter-cold, as well as eliminating the heat. Panax ginseng and Zizyphus jujube work compatibly to tonify Qi and fortify the spleen, thus strengthening the body and eliminate pathogens; Pinellia ternate and Zingiber officinale work together to regulate the stomach Qi and prevent vomit; and the "guide" Glycyrrhiza glabra is used to harmonize the other herbs. Nowadays, XCHT is used to treat common cold, chronic hepatitis, hepatic cirrhosis, bile reflux gastritis, cholecystitis, acute pancreatitis, and so forth, which belong to lesser yang syndrome.
(Umbelliferae), especially B. chinense from mainland China and B. falcatum from Japan [28,29], is also called Bupleuri Radix ((BR) Chaihu, in Chinese and Saiko in Japanese) and is one of the principal herbs in XCHT. Early study examined the hepatoprotective effect of several BR extracts with dimethylnitrosamine-(DMN-) induced hepatic fibrosis rat model and these extracts appeared to prevent fibrosis by improving liver function and modulating the levels of relevant cytokines [29]. Recent studies also suggest BR extracts as potent antioxidant agents because they are able to decrease Lthyroxine-induced hypothyroidism and to enhance the liver antioxidant defense systems [30]. Some saikosaponins, which are the active ingredients of BR, have been found to suppress hepatic fibrosis [31,32], hepatocarcinoma [33][34][35][36], and HBV infection [37] and improve chemotherapy [38]. The different mechanisms through which saikosaponins suppressed hepatocarcinoma were summarized in Figure 1. Saikosaponin a (SSa) was shown to effectively inhibit CCl 4 -induced liver inflammation and fibrosis in SD rats by simultaneously blocking the production of hepatic proinflammatory cytokines/growth factors (TGF 1 and hydroxyproline) and increasing the expression of antiinflammatory cytokine IL-10 [31]. Saikosaponin d (SSd) was found to suppress hepatic fibrosis through the downregulation of TNF-, IL-6, and NF-B activities [32]. SSa may inhibit HepG2 growth by increasing the levels of p-15INK4a and p-16INK4b (cyclin-dependent kinase inhibitors) in a protein kinase C (PKC) [33] and/or extracellular signalregulated kinase (ERK) signaling pathway-dependent manner [34]. SSd markedly reduced the liver nodule, tumor cell invasion while increased cellular atypia in xenograft model [35]. It appeared that SSd exerted its action by diminishing the expression of highly expressed cyclooxygenase 2 (COX-2) and CCAAT/enhancer-binding protein (C/EBP ) in tumor cells and macrophages of liver tumors [35]. In a study reported by Zhu et al. [36], SSd was shown capable of reversing the malignant phenotype of HepG2 cells. SSd-treated HepG2 cells grew and migrated at slower rate, had decreased volume ratios of nucleus to plasma and small round cell shape. At molecular level, SSd decreased the level of alphafetoprotein (AFP) and enhanced the expression of cell cycle inhibitor p27. Moreover, Chiang et al. showed that HBVcontaining human hepatoma cells (2.2.15 cells) treated with saikosaponin c (SSc) secreted significantly less HBeAg into culture medium and had reduced HBV DNA replication [37]. Although not directly using liver cancer cells, SSa and SSd were also shown to sensitize cervical (HeLa and Siha), ovarian (SKOV3), and lung cancer cells (A549) to cisplatin-induced cell death by inducing the production of reactive oxygen species (ROS) and activation of caspases [38]. another principal herb in XCHT. Accumulating evidences indicate that wogonin, baicalein, and baicalin are the principal active components in SR [39]. SR has been widely used to treat hyperlipemia, atherosclerosis, and hypertension. Recent studies with various model systems suggest that SR also possesses a potent cytostatic [40][41][42], anti-inflammatory [43] and antiviral capabilities [44,45].
Antitumorigenesis ability of SR was reported by Gao et al. in human lung cancer cells (SK-MES-1, SK-LU-1, and A549) [42]. Their study showed that the absolute ethanol extracts of Scutellaria baicalensis, baicalin, baicalein, and wogonin all displayed a concentration-and time-dependent cytotoxicity to lung cancer cells while were only weakly cytotoxic to the normal human lung fibroblasts. Jung et al. later discovered that Scutellaria baicalensis is an anti-inflammatory agent because it decreases histamine release and inhibits the passive cutaneous anaphylaxis reaction in SD rats [43]. Antiviral effect of Scutellaria baicalensis was shown by Tang et al., in which it was shown to significantly inhibit the replication of HCV RNA in HCV-infected nude mice [44]. Later study revealed that the aqueous extract of Scutellaria baicalensis was also able to suppress the replication of lamivudine-resistant HBV mutant in human hepatoma cells by suppressing HBV core promoter activity [45].
Besides the studies performed with Scutellaria baicalensis, active components of it have also been extensively investigated ( Figure 2). TNF-related apoptosis-inducing ligand (TRAIL) has been recognized as a promising anticancer agent because it kills tumor cells without damaging normal tissues [46,47]. However, resistance to TRAIL is frequently seen in various tumor types. Ding et al. found that wogonin and structurally related natural flavones apigenin and chrysin overcame TRAIL resistance by downregulating the level of c-FLIP (a key inhibitor of death receptor signaling) and up-regulating TRAIL receptor 2 (TRAIL-R2) expression in human T-cell leukemia virus type 1-(HTLV-1-) associated adult T leukemia/lymphoma (ATL) cells [48]. They further showed that these flavones could enhance TRAIL-mediated apoptosis in a wide variety of cancer cell types including hepatocellular carcinomas (HepG2), breast (MDA-MB-231), colon (HT-29), and pancreatic cancer cells (Capan-1) as well as melanoma cells (SK-MEL-37) [48], implicating the use   -1), leading to significant apoptosis in a variety of human cancer cells [49]. However, wogonin-induced apoptosis of human hepatocarcinoma cells was found to be accompanied with Bax increase and Bc1-2 decrease [50,51]. Anti-HBV effect of wogonin was also found in vitro and in vivo [52], with the HBV antigen and HBV DNA level reduction.
Baicalein, a flavonoid extracted from SR, has been shown to possess potent antitumorigenesis capability toward liver cancer cells. For example, baicalein is highly cytotoxic to HCC cell lines and exerts its cytotoxicity by reducing mitochondrial transmembrane potential and subsequent cytochrome c release and caspase-3/9 activation. Disruption of MEK-ERK signaling pathway is at least partially responsible for baicalein-induced cytotoxicity [53,54]. When used in vivo, baicalein can significantly inhibit tumor growth of HCC xenografts [53]. In another study, baicalein was reported to block cell migration and invasion of human hepatoma cells through multiple mechanisms including the suppression of MMP-2, MMP-9, and uPA expressions, blockage of NF-B  activation, and decreasing the phosphorylation levels of PKC and p38 MAPK activities [55]. In a recent study, Sun et al. showed that baicalein dose dependently decreased AST, ALT, hyaluronic acid, laminin, and procollagen type III (PCIII) in serum as well as hydroxyproline and MMPs in liver in CCl 4 -induced liver fibrosis model [56]. Moreover, baicalein also alleviated inflammation, destruction of liver architecture, collagen accumulation and expression of PDGF receptor, thus preventing the activation of stellate cells and liver fibrosis [56]. Baicalin is also an important active component included in SR. Zhang et al. [57] found that baicalin induced apoptosis with downregulation of glycosylated immunoglobulin superfamily transmembrane protein CD147 expression in SMMC-7721 cells, and interestingly, this effect was accompanied with cell autophagy. This study firstly suggested that baicalin induced autophagy cell death in SMMC-7721 cells and revealed a new mechanism for the anticancer effects of baicalin. Qiao et al. [58] studied the antihepatic fibrosis effect of baicalin and found that transplantation with baicalintreated mesenchymal stem cells in combination with baicalin administration had the best therapeutic effect for hepatic fibrosis. This may further introduce a new therapeutic regimen for some liver diseases. Baicalin combined with oxymatrine showed better effect against HBV replication than oxymatrine in vitro, which was proved by Cheng et al. [59]. In aspects of the potential protective effect on liver injury, baicalin was also researched in many experiments [60][61][62].
Activation of peroxisome proliferator-activated receptor (PPAR ) signaling pathway, and Toll-like receptor 4-(TLR4-) mediated inflammatory responses were involved in the protective effect.

Panax ginseng.
Ginseng products are regularly consumed worldwide for the purpose of increasing vitality [63]. Recently, many studies have shown the chemopreventive or adjuvant effect of it [64]. A study involving two cases of control (905 pairs and 1987 pairs, resp.) and a cohort (4675 subjects) demonstrated the benefit of ginseng use for cancer prevention as ginseng use was found to be nonorgan-specific cancer preventive, and its effect depends on the frequency of ginseng intake [65].
In addition to ginseng's preventive effect toward cancer, evidences from experimental studies also suggest its direct role to suppress liver tumorigenesis. Wu et al. showed that ginseng lowered the rate of hepatoma development and prolonged life span on diethylnitrosamine (DEN) rat liver cancer model [66]. Kwon  mice [68]. The effect of ginseng appears to be mediated by a simultaneous inhibition of JNK, ERK, and p38 activities and upregulated expression of antioxidant enzyme expression in the liver. Components of ginseng have also been investigated for their inhibitory effect on liver tumorigenesis (Figure 3). Lee et al. showed that 20(S)-ginsenoside Rg3, a steroidal saponin was able to sensitize HCCs, but not normal hepatocytes to TRAIL-induced cell death [69]. Importantly, Rg3 was found to be well tolerated in animals and significantly enhance the therapeutic efficacy of TRAIL in xenograft models [69]. And other studies [70,71] suggested that intrinsic apoptotic pathway may be involved in the inhibitory effect of Rg3 on hepatocellular carcinoma cell lines. To elucidate the mechanism associated with ginseng extract-induced cell death, Park et al. showed that primary ginsenosides Rg3 and Rh2 are mainly responsible for ginseng's effect and they act by directly activating mitochondrial-dependent apoptotic pathway and inducing the production of intracellular ROS [72].
Components of ginseng also exhibit their tumorsuppressing capability by blocking cell migration and invasion. Yoon et al. found that ginsenoside Rh1 inhibited HepG2 cell migration and invasion by abrogating MAPK-dependent MMP-1 expression [73]. Similar effect was also observed with ginsenoside Rd in HepG2 cells [74]. As excess production of extracellular matrix by activated hepatic stellate cells (HSCs) is the major cause of liver fibrosis, Lo et al. determined the potential protective effect of ginseng components toward liver fibrosis. They revealed that ginsenoside Rb1 exerted an antifibrotic effect under H 2 O 2 oxidative stress by inhibiting HSC activation/proliferation [75]. Another ginsenoside, Rg1, has also been shown to prevent thioacetamide-induced hepatic fibrosis in rats by intercepting NF-B-mediated PDGF receptor expression [76].

Other Herbs in XCHT.
Besides the three herbs that have been described above, the remaining herbs in XCHT are Zizyphus jujube, Pinellia ternate, Zingiber officinale, and Glycyrrhiza glabra and act as adjuvant herbs in this compound formula. Zizyphus jujube in XCHT is prescribed by Traditional Chinese Medicine doctors to calm mind based on its ability to invigorate the spleen and nourish the blood. Recent experimental evidences showed that it was able to attenuate chemical-induced liver injury in rats [77,78]. Pinellia ternate is another herb in XCHT while the mechanism study about its antihepatocarcinoma effect is rare. Although it appears to boost the efficacy of XCHT, how it does this awaits being further explored. Zingiber officinale, a species used for over thousand years, appears to display anticancer, antiinflammatory, and chemopreventive effects in both in vitro and in vivo models [79,80]. 6-shogaol and 6-gingerol are the two active compounds of ginger, and their effects of apoptosis induction [81], hepatocarcinoma invasion inhibition [82] and anti-hepatoxicity [83] were also studied. Licorice is the dried root of Glycyrrhiza uralensis Fisch, and both the extract [84,85] and its active component glycyrrhizin [86,87] were explored for their hepatoprotective capability. Though these adjuvant herbs are also essential in XCHT from the view point of TCM, modern mechanism studies about their antihepatocarcinoma effects are relatively less than Bupleurum falcatum, Scutellaria baicalensis, and Panax ginseng. So we did not summarize the antihepatocarcinoma-related effects of these adjuvant herbs as detailedly as the former three herbs here.

Summary and Prospect
Besides the summary above, experimental studies of the active components in the herbs on antihepatocarcinomarelated effects are further summarized in Tables 2, 3, 4,   8 Evidence-Based Complementary and Alternative Medicine    figures cannot present all the targets summarized in our paper, they still could illustrate the mechanism from a more systematical aspect. On the basis of Figures 1 to 5, we depicted Figure 6 to better display the antihepatocarcinoma effects at three different levels of formula, herbs, and components. From them, it is apparent that components of XCHT possess a broad spectrum of activities ranging from antitumor, antiinflammation to fibrosis-protective effects. Some of the components directly target on tumor growth, metastasis, and invasion, while others act on inflammation and fibrosis related pathways or antiviral process to prevent further virus-facilitated tumorigenesis. Particularly, many of these components share analogous factors and pathways. Besides tumor growth, metastasis and invasion, angiogenesis is also an essential pathological component of cancer. Antiangiogenic therapy is considered to limit tumor progression [88]. Research about the anti-angiogenic effect on hepatocarcinoma of XCHT has not been seen, while some reseachers have studied the effect of herbs and active compounds in it on the angiogenic action [89][90][91]. Interestingly, different compounds in ginseng (ginsenoside Rg3 and Rg1) possessed contrary angiogenic action (antiangiogenic and angiogenic effects) [89,90]. What is more, as we summarized in our review, components of XCHT may affect various biochemical pathways, many of which are related to angiogenesis [92]. So it is also worthy to deeply study XCHT's effect on the angiogenic action for tumor treatment.
Our overview is based on a perspective of "separation study, " that is, arranging from the entire formula to each herb and their active components. This method may comprehensively utilize and deeply excavate the existing researches. It is difficult to directly elucidate a complex formula, while it will be easier when we separately study the constituents in it. Currently, to unequivocally interpret the antihepatocarcinoma effect of XCHT and the active ingredients contained in it is still difficult. The underlying reasons are severalfold. The first is that the knowledge on how each component works is still not sufficient and their respective targets are still needed to be identified. The second is how these components work in concert to achieve therapeutic effect is not understood. As the philosophy of Traditional Chinese Medicine, compound herbal formulas are used to treat disease by regulating human body globally, targeting multiple pathways and targets. This characteristic may be better coincident with cancer which may be induced by multiple factors. Tumor growth, metastasis, and invasion are the final features that we notice, but the underlying mechanism may be related with many factors. The method of "separation study" should be combined with bioinformatics, meaning that we may use bioinformatics to integrate these separated studies. So more innovative researches and novel strategies will have to be employed to fully understand the mechanisms responsible for XCHT's therapeutic effects.