complementary and alternative therapies in the

[Downloaded free from http://www.hepatitisbannual.org on Thursday, November 1, 2018, IP: 47.9.175.245]

COMPLEMENTARY AND ALTERNATIVE THERAPIES IN THE TREATMENT OF CHRONIC HEPATITIS B Jia-Ming Chang, Kai-Ling Huang1 Division of Research and Development, Development Center for Biotechnology, Xizhi City, Taipei County, Taiwan 221, R.O.C. 1 Department of Life Sciences and Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C. Correspondence: Dr. Jia-Ming Chang, Division of Research and Development, Development Center for Biotechnology, Xizhi City, Taipei County, Taiwan 221 R.O.C. E-mail: [email protected]

ABSTRACT Hepatitis B virus (HBV) infects approximately more than 350 million people worldwide, especially in Asia, Africa, southern Europe and Latin America. Except for interferon-α, most anti-HBV drugs are derived from the anti-herpes and antiHIV drugs. Because of the high cost of hepatitis B medications, herbs-also called ‘complementary and alternative therapies’ in modern Western science-are widely used for treatment of chronic hepatitis B in developing countries. Herbals confer their activities not only by inhibiting HBV secretion but also by building up immunity against viruses. After studying the anti-HBV mechanism of herbs, scientists were encouraged to find that novel anti-HBV drugs target viral secretion, whereas nucleoside analogues target viral polymerase. The complementary and alternative anti-HBV therapies published in scientific peer-reviewed journals are reviewed and discussed in this article. Keywords: Chinese medicinal herbs, complementary and alternative medicine, hepatitis B, herbal medicine

72


INTRODUCTION Hepatitis B virus (HBV) infection is known to produce chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) and there is a high rate of HBV infection (more than 350 million) worldwide, especially in Asia, Africa, southern Europe and Latin America.1 Although the natural history of HBV carriers is complicated and remains unclear, vaccination is still the most important strategy to protect healthy persons from becoming chronic HBV carriers.2 On the other hand, the introduction of antiviral agents to people with chronic hepatitis B could attenuate or stop the Þbrotic progression, thereby preventing the development of HCC and fulminant hepatitis. Before the Þrst anti-HBV drug, 3TC, was approved by the US Food and Drug Administration (FDA) in 1998, there was no available small-molecule drug for treating chronic hepatitis B disease. Interferon was the only choice for treating this infection. Although interferon can reduce the activity of HBV, the side effects of interferon remain a concern to physicians with regard to its clinical use in treating chronic hepatitis B patients. Unfortunately, most anti-HBV drugs are derived from herpes and human immunodeÞciency virus (HIV) drug development and only a few efÞcacious anti-HBV drugs are available for clinical use. The large repertoire of herbal compounds has shown potential in developing new ways to combat previously considered ‘incurable’ disease, provided that these compounds (or often, mixtures of compounds) can satisfy current government regulations. At present, alternative or traditional medical resources are used by more than 80% of the population in developing countries and by an increasing number of people in other parts of the globe.3 For modern Western science, the use of herbs is termed ’complementary and alternative therapies’. These kinds of therapies for chronic hepatitis also are being extensively explored and the results appear promising.4 The complementary and alternative anti-HBV therapies published in scientiÞc peer-reviewed journals are reviewed and discussed in this article. OVERVIEW OF HBV LIFE CYCLE HBV is a DNA virus that consists of a double-shelled structure (a nucleocapsid covered with surface/envelope proteins) enclosing a circular, partial double-strand DNA genome, also called ‘Dane particle’. The HBV genome is approximately 3.2 kb and encodes 73


the viral envelope (preS/S), core (preC/C), polymerase and X proteins. In the HBV replication cycle, the viral pregenomic RNA (pgRNA) and polymerase, together with cellular factors including chaperones and phosphorylated core proteins, are encapsidated by viral core proteins to form immature nucleocapsids.5-7 Within the nucleocapsid, a complete HBV negative-strand DNA is Þrst synthesized via reverse transcription of an RNA intermediate and then converted into partial double-strand DNA by viral polymerase to form mature nucleocapsids. These nucleocapsids are packaged by transmembrane envelope proteins in the post-endoplasmic reticulum/pre-Golgi membrane, where the assembled virions are secreted via the constitutive secretion pathway.8 Alternatively, the mature nucleocapsids can return to the nucleus to amplify the intracellular genome copy number.9 Excess middle- and smallsurface proteins could self-assemble into subviral quasi-spherical or Þlamentous particles without envelopment of nucleocapsids that are secreted efÞciently in relation to the virions.10 THE STRATEGY OF CHRONIC HEPATITIS TREATMENT The current hepatitis B treatments are categorized into two strategies to Þght HBV viral infection: (1) Induction of certain cellular immune responses including activation of speciÞc cytotoxic T lymphocytes that target infected cells and/or inhibition of virus replication in the infected cells by speciÞc cytokines such as interferons. In addition, vaccines made of viral recombinant protein or viral DNA plasmid can induce speciÞc immune responses involving both cellular and humoral immunity against HBV or HBVinfected cells via neutralization of anti-envelope antibodies or by destruction of infected liver cells. (2) Inhibition of viral replication by targeting viral molecules or by affecting cellular regulatory factors that are associated with HBV replication and the processes for viral nucleocapsid formation, envelopment and secretion. On the basis of the cellular and molecular mechanism of HBV replication, a number of potential targets for anti-HBV drugs were developed for chronic hepatitis B treatment (summarized in Figure 1) and are described as follows. THE IMMUNOMODULATORS Alpha-interferon (IFN-α), the Þrst drug approved by FDA for the treatment of chronic hepatitis B inhibits viral replication 74


and acts as an immunomodulator. Treatment with IFN-α.achieves persistent loss of HBV in about 30% of patients with chronic hepatitis B. However, the disadvantages of interferon treatment include its limited efÞcacy, the fact that only about one third of patients with a sustained infection are eligible for trials, its low efÞcacy with respect to cost and its serious side effects.11 Recently, a pegylated interferon, PEG-IFN-α2a was approved and this seems to be superior to IFN-α. PEG-IFN-α2a has a prolonged half-life in serum and is also effective against a broader range of cases than the other interferons.2 THE VIRAL POLYMERASE INHIBITORS HBV encodes multifunctional polymerase, which is a signal for viral pgRNA encapsidation; catalyzes RNA- and DNA-dependent DNA synthesis and has an intrinsic RNase H activity that degrades pgRNA during reverse transcription.12 Therefore, it is already a promising target for antiviral agents and for the nucleoside or nucleotide analogues that target selectively the viral RNA-dependent DNA polymerase, which can suppress viral replication.2 Lamivudine (3TC) was approved by FDA in 1998 for the treatment of chronic hepatitis B. However, short-term monotherapy is not sufÞcient to clear viral infection and long-term monotherapy has been associated with the emergence of resistant mutants in the tyrosine-methionineaspartic acid-aspartic acid (YMDD) motif of the viral polymerase (during 2-3 years of 3TC administration).13,14 Recently, adefovir and entecavir were approved and used against 3TC-resistant viruses; unfortunately, resistances to these two drugs were reported in 3TCresistant patients.15-17 Research efforts are still ongoing to improve current antiviral drugs by developing novel nucleoside analogues. At present, two nucleotide analogues, emtricitabine and tenofovir, are at the phase III clinical trial stage. THE NUCLEOCAPSID ASSEMBLY INHIBITORS Two critical early steps in HBV replication, the initiation of reverse transcription, via protein priming and nucleocapsid assembly, take place in the cytoplasm of infected cells. Binding of the polymerase to ε region, a short RNA signal located at the 5’ end of pgRNA, leads to the selective incorporation of both polymerase and pgRNA together with the 90-kDa heat shock protein (Hsp90) chaperone complex into the viral core proteins.18-21 It has been 75


reported that some host chaperones are involved in the assistance of viral protein folding and assembly in the viral life cycle. At present, three host chaperones are known to be required for the early steps in HBV replication. In a dynamic process that is dependent on ATP hydrolysis, Hsp90 facilitates HBV polymerase-pgRNA interaction and is associated with HBV polymerase conformational changes for replication, whereas Hsp60 activates HBV polymerase both in vitro and ex vivo.5,22,23 The chaperone 94-kDa glucose-regulated protein (GRP94) is also involved in stabilization of HBV polymerase as an active form.24 In light of the importance of HBV nucleocapsid as a process necessary for genome replication, an experimental approach that targets the HBV core protein to interfere with the nucleocapsid formation would possibly inhibit HBV production. Recently, heteroaryldihydropyrimidines, bis-ANS and alkylated imino sugar were reported to reduce the virus production from the virus-producing cells by preventing the maturation of HBV nucleocapsids or by destabilizing the formed nucleocapsids.25-29 In addition, suppression of Hsp90 or Hsp60 has similar anti-HBV effects in reducing the viral nucleocapsid formation.5,22 Targeting nucleocapsid formation may serve as a good approach for antiHBV drug development. THE VIRAL ENVELOPMENT INHIBITORS The HBV envelope consists of three structurally related HBV proteins, large-surface, middle-surface and small-surface proteins, which are individually translated from one open reading frame of the viral genome by using different translation initiations.30 These proteins share a common carboxyl terminus. The large-surface protein contains additional pre-S1 and pre-S2 domains, whereas the middle-surface protein contains more pre-S2 domains than the smallsurface protein. All these proteins are cotranslationally integrated into the endoplasmic reticulum (ER) membrane by the topogenic signals of the small-surface protein domain and are glycosylated as they are transported through the secretory pathway.31 These glycoproteins are probably involved in virion assembly and secretion and/or infectivity.32,33 One glucosidase function is to remove terminal glucose residues from N-linked oligosaccharides attached to nascent glycoproteins, which in turn assists in protein folding via the interaction oßectin-like chaperone proteins (calnexin and calreticulin) with nascent glycoproteins.34 The chemicals for the inhibition of the 76


N-linked glycosylation pathway, such as N-butyl-deoxynojirimycin (NB-DNJ) for glucosidase I and II and N-nonyl-DNJ (NN-DMJ) for mannosidase I, can cause some proteins to misfold and be retained in the ER.32,35 NB-DNJ is also reported to suppress secretion of HBV particles and to cause intracellular retention of HBV DNA in HepG2 2.2.15 cells; site-directed mutagenesis experiments prove that virion secretion requires the glycosylation sequence in the pre-S2 domain of middle-surface proteins.35,36 This Þnding highlights the potential role in the glycosylation of the middle-surface protein with oligosaccharide, which may serve as a therapeutic target in the treatment of chronic hepatitis B. THE NOVEL TARGET IN THE SECRETION OF HBV The pre-S (pre-S1 plus pre-S2) domain of the largesurface protein is needed for the translocation process during HBV morphogenesis. 37 About half of large-surface proteins posttranslationally translocate their pre-S domains into the ER, whereas the other pre-S domains appear on the cytosolic side, which yields a dual topology maintained in the secreted viral and subviral particles.38-40 The dual topology of pre-S domains of largesurface proteins may perform a different function in the viral lifecycle. Large-surface proteins containing pre-S domains oriented at the cytosolic site (external) participate in virus-host receptor binding and those with pre-S domains oriented at the luminal site (internal) are probably involved in envelopment of the nucleocapsid.37,41,42 In addition, both large- and small-surface proteins are required for virion secretion.43 Over-expression of large-surface proteins relative to small-surface proteins can inhibit virion secretion and restrict the subviral particles in the ER lumen, suggesting that a small relative ratio of large-surface proteins to middle- and small-surface proteins is crucial for virion secretion.44-46 Recently, the GRP78 was found to play an important role in the suppression of HBV production by Boehmeria nivea. This Þnding also suggests that inhibiting the function of chaperones associated with HBV replication may alter the production of HBV virion. COMBINATION APPROACH FOR ANTIVIRAL THERAPY At present, the major target of anti-HBV drug development is to emphasize inhibition of the HBV multifunctional viral polymerase. An objective to develop other nucleotide analogue drugs, with the 77


expectation of a synergistic antiviral effect, may prevent or attenuate the emergence of resistant mutants and improve therapeutic effects. Clinically, the use of combination therapy, such as IFN-α plus 3TC, 3TC plus adefovir or 3TC plus entecavir, may yield additive or synergistic effects and/or reduce the emergence of resistance, even though the problems of serious side-effects and unsatisfactory efÞcacy are still arising. A high mutation rate of HBV results from the lack of a proofreading function in HBV polymerase, which is the main reason for drug resistance and suggests that combining drugs with different pharmacological action modes beneÞts the effectiveness of anti-HBV therapy.47 STUDY MODEL FOR HBV For the development of anti-HBV drugs, a number of in vitro and in vivo models of HBV infection are established for drug screening. In vitro cell culture models, including infection of primary hepatocytes and transient or established HBV DNA-transfected cell lines, have become widely used in the laboratory for antiviral research because of their convenient manipulation and maintenance. However, the disadvantages of in vitro cell culture models are the poor data for reproducibility of infection of hepatocytes with infected sera and non-natural viral production from chromosomally integrated viral cDNA of established cell lines. Because hepatitis viruses have a restricted host range, chimpanzee and tree shrews (Tupaia belangeri sinensis) are the few animal models for HBV infection.48,49 Chimpanzees develop acute hepatitis and evoke an immune response after HBV infection that could be used to evaluate the safety and immunogenicity of HBV vaccines, but the limited availability and the high cost of these primates severely restrict their use for such purposes.50 In surrogate animal models, including the woodchuck, the Pekin duck and the ground squirrel, which can be infected by host-speciÞc genus hepadnaviruses, are used to simulate the condition of human HBV infection to evaluate the efÞcacy of antiviral agents.51-53 In 2002, Huang and his co-workers Þrst introduced a novel murine model by using the transfection of HBV-expressing plasmid for studying human HBV replication, immunogenicity and control.54 However, the major drawback of these animal models in testing antiviral agents is that they may produce aberrant results as a consequence of virus-specific differential susceptibility to the drugs. Transgenic mouse model 78


and human-mouse chimeric liver model have been established as suitable laboratory small-animal models for investigators to study the immunopathogenesis of HBV infection.55,56 Although there is no particular cell culture model or animal model for studying all features of HBV, researchers can choose suitable models to investigate different aspects of pathology, prevention and therapy of HBV. HERBAL MEDICINES USED IN CHRONIC HEPATITIS B TREATMENT Because chronic hepatitis B is widely spread in Asia, Africa, southern Europe and Latin America, it is urgent to develop antiHBV drugs in these countries. One possibility is to Þnd useful therapeutic agents from herbs for treating chronic hepatitis B. The empirical experiences of these medicinal substances have been widely used in folk medicine and studied as modern drugs by employing science-based methodology. Chemists continue to identify active compounds from plants and attempt to discover their lead compounds. Before the etiology of chronic hepatitis B was understood, many plants and remedies were used to treat liver-related diseases, including hepatitis, cirrhosis and cancer. According to historical experiences, some of these treatments are highly relevant to alleviate “liver inßammation syndrome” no matter whether or not the hepatitis is caused by viruses. For the past decade, researchers have turned to systemically screening these medicinal plants for anti-HBV activity. The anti-HBV efÞcacy of these medicinal plants are investigated by using established in vitro and in vivo models; these results are summarized and listed in Table 1. Since the 1990s, many assay models have been developed for evaluating anti-HBV activity. The HepG2 2.2.15 cells can consistently secrete infectious viral particles that are acceptable for investigating antiHBV herbals in vitro and DHBV-transfected ducks are promising for investigating the metabolism and efÞcacy of anti-HBV drugs in vivo. In some of these herbs, the active compounds that are responsible for their anti-HBV activities have been identiÞed and some of them are still under investigation. The anti-HBV mechanism of these herbs remains mostly unclear; however they do inhibit the production of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HbeAg) or HBV DNA in the HepG2 2.2.15 cell model or suppress the serum DHBV in the DHBV-transfected duck animal 79

80

Ganoderic acid

Cultured broth with aqueous herbal extract

Ganoderma lucidum and Sophorae ßavescentis

Aqueous extract Ethanolic extract

Extract/active compound Osthole

Ganoderma lucidum

Angelica pubescens Maxim Ardisia chinensis Boehmeria nivea

Herbals

HepG2 2.2.15 cells

HepG2 2.2.15 cells

Huh7 cells transfected with cloned HBV DNA; MS-G2 cells Primary duck hepatocyte/DHBV Hep3B cells; HepG2 2.2.15 cells; HepG2 2.2.15-bearing animal model

Study model

Observation/possible mechanism Suppression of secretion of HBV via increase in glycosylation of HbsAg Inhibition of DHBV DNA secretion Inhibition of secretion of HBsAg in Hep3B cells; inhibition of secretion of HBV DNA in HepG2 2.2.15 cells; suppression of the serum HBV DNA of HepG2 2.2.15-bearing animal model; blocking of secretion of HBV virion via down-regulation of chaperones interacting with HBV surface protein Inhibition of HBV replication and both HBsAg and HBeAg production Inhibition of HBsAg and HBeAg production

Li, Yang, et al (2006)

Li, Wang, et al (2006)

Leung, Chiu, et al (2006) Huang KL (2005) Huang, Lai, et al (2006) Chang, Huang, et al (2006) Huang, Lai, et al (2007)

Huang, Chen, et al (1996)

References

Table 1: Summary of complementary and alternative medicines studied for treating chronic hepatitis B


81

Phyllanthus amarus

Mallotus apelta (Lour) Ocimum basilicum Oenanthe javanica

Genera: Rheum; Polygonum; Rhamnus; Senna Glycyrrhiza uralensis (licorice)

Herbals

Aqueous extract

Apigenin; linalool; ursolic acid Hyperoside

Glycyrrhizin; glycyrrhetic acid 3-Omonoglucuronide Aqueous extract

Extract/active compound Emodin

Huh7 cells/HBV luciferase plasmids HepA2 cells transfected with HBV DNA plasmid

HepG2 2.2.15 cells; duck/DHBV infection

HepG2 2.2.15 cells

Duck/DHBV

secretion PLC/PRF/5 cells

HepG2 2.2.15 cells

Study model

Inhibition of HBsAg and HBeAg production Inhibition of HBeAg secretion in HepG2 2.2.15 cells and DHBV DNA levels in DHBV-infected duck Down-regulation of HBV mRNA via inhibition of C/EBP transcription factors binding to HBV enhancer I Suppression of gene expression of HBsAg

Inhibition of serum DHBV

Observation/possible mechanism Inhibition of HBV DNA replication and HBsAg et al (2006) Inhibition of HBsAg expression and sialylation

Ott, Thyagarajan, et al (1997) Yeh, Hong, et al (1993)

Xu, Lu, et al (2006) Chiang, Ng, et al (2005) Wang, Yang, et al (2005)

Sato, Goto, et al (1996)

Shuangsuo, Zhengguo,

References

Table 1 continued: Summary of complementary and alternative medicines studied for treating chronic hepatitis B


82

Salvia miltiorrhiza

Pithecellobium clypearia Rheum palmatum Rhizoma rhei

Phyllanthus nanus

Phyllanthus uriraria.

Herbals

Study model

Observation/possible mechanism Blocking of immune tolerance caused by HBeAg

C57ML/6, HBeAgproducing transgenic mice Ethanolic HepG2 2.2.15 cells; Suppression of HBsAg secretion extract duck hepatocyte/DHBV; and HBsAg mRNA expression PLC/PRF/5 cells in HepG2 2.2.15 cells; suppression of viral replication of DHBV in DHBV-infected primary duck hepatocytes; induction of annexin A7 is correlated to anti-HBV activity Aqueous extract Primary duck Inhibition of DHBV DNA hepatocyte DHBV secretion Volatile oil HepG2 2.2.15 cells Inhibition of HBsAg and HBeAg production Emodin; physcion; HepG2 2.2.15 cells Inhibition of HBsAg and rhein; citreorosein HBeAg production Protocatechuic HepG2 2.2.15 cells; Down-regulation of HBsAg and aldehyde ducklings/DHBV HBeAg secretion and HBV DNA; reduction of viremia in DHBV-infected ducks

Extract/active compound Ellagic acid

Leung, Chiu, et al (2006) Zhang, Chen, et al (1998) Liu, Zhu, et al (2004) Zhou, Zhang, et al (2007)

Lam, Leung, et al (2006)

Kang, Kown, et al (2006)

References



83

Daphnoretin

Wikstroemia indica C.A. Mey

Clinical trial: (1) 46 patients with chronic hepatitis B; (2) 94 patients with chronic hepatitis B Clinical trial: 120 patients with HBV infection Hep3B cells

Kurarinone

Matrine

HepG2 2.2.15 cells

HepG2 2.2.15 cells; Duck/DHBV; HBV transgenic mice

Hep 3B cells; HepA2 cells

Study model

Aqueous extract

Extract/active compound Costunolide; dehydrocostus lactone Wogonin

Sophorae ßavescentis

Serissa serissoides (DC) Druce Sophora ßavescens Ait

Scutellaria radix

Saussurea lappa Clarks

Herbals

Serum conversion from HBeAg to HBe antibody and serum HBV DNA from positive to negative Inhibition of HBsAg secretion via PKC activation

Serum conversion in HBeAg and HBV DNA

Observation/possible mechanism Inhibition of HBsAg gene expression; inhibition of HBsAg and HBeAg secretion Suppression of secretion of the HBsAg and HBV DNA level in HepG2 2.2.15; reduction of DHBV DNA in the liver of DHBV-transfected duck; reduction of plasma HBsAg level in HBV transgenic mice Inhibition of HBsAg and HBeAg production

Chen, Chou, et al (1996)

Long, Lin, et al (2004)

Pan, Yu, et al (2005) Chen, Guo, et al (2000)

Chen, Yu, et al (1999)

Guo, Qinglong, et al (2007)

Chen, Chou, et al (1995)

References




model. It is believed that these herbs may have anti-HBV action modes that differ from that of the nucleoside inhibitors which target viral polymerase. SINGLE HERB IN THE TREATMENT OF CHRONIC HEPATITIS B The genus Phyllanthus (Euphorbiaceae) is traditionally used for the treatment of virus-associated diseases and has been extensively investigated for its anti-HBV activity.57 In China, herbs are permitted to be used clinically in treating chronic hepatitis B patients without precise chemical and manufacturing controls. Despite the poor quality of clinical trials on Phyllanthus herbs for chronic hepatitis, the authors of a review of information on the use of this genus of herbs alone or in combination with interferon in humans from 22 RCTs suggested that they may be effective against HBV.58 In laboratory research, ellagic acid was identiÞed from P. uriraria, which is not able to inhibit either HBV polymerase activity or HBV replication or block HBsAg secretion.59,60 Instead, ellagic acid blocks effectively HBeAg secretion in HepG2 2.2.15 cells and shows a low IC50 (0.07 µg/ml). Kang and his co-workers concluded that the function of ellagic acid in treatment of chronic hepatitis B is to block the immune tolerance caused by HBeAg.61 The ethanolic extract of Phyllanthus nanus has been studied for anti-HBV activity.62 P. nanus suppresses the HBsAg secretion and HBsAg mRNA expression in HepG2 2.2.15 cells and the viral replication of DHBV in DHBV-infected primary duck hepatocytes. A microarray analysis consisting of 800 human cDNA clones showed that induction of annexin A7 can be correlated with the anti-HBV activity of this herb.62 Later, Niu et al, demonstrated the utility of P. amarus in anti-HBV treatment.63 The researchers in this Þeld are interested in the anti-HBV activity of this herb and are starting to extensively investigate it. With the use of a different assay model, P. amarus was shown to suppress the gene expression of HbsAg64,65 and studies have shown that P. amarus suppresses hepatitis B virus by interrupting interactions between HBV enhancer I and cellular transcription factors.64,65 Recently, many active compounds were identiÞed from this genus, such as niranthin, nirtetralin, hinokinin, geraniin, etc and all of them show anti-HBV activities.66 Radix Sophorae flavescentis is recorded in the Chinese Divine Husbandman’s Classic of the Materia Medica and has been traditionally used in treatment of chronic liver diseases.67 Recently, 84


pharmacological studies have shown that S. flavescentis possesses anti-HBV activity. Clinical trials with S. flavescentis conÞrmed that this herb possessed antiviral activity, as this treatment achieved seroconversion of HBeAg to anti-HBe antibody and of HBV DNA from positive to negative.68 The extract of S. flavescentis is also reported to improve the therapeutic effect of IFN-α.69 The active ingredients identified from S. flavescentis, such as matrine, oxymatrine, sophoranol, anagyrine, sophocarpine and spophordine were studied for their individual anti-HBV activity.70 Among these compounds, matrine has more potent anti-HBV activity and is being extensively studied for treatment of chronic hepatitis B in clinics.71 No matter whether used alone or in combination with either IFN-α or other herbs, the outcomes are beneÞcial in the clearance of serum HBeAg. Another active compound, kurarinone, found in Sophora flavescens Ait, is also being investigated for its efÞcacy in clinical treatments of chronic hepatitis B.69 The outcomes of clinical trials reveal that kurarinone used alone is equivalent to IFN-α; the seroconverion of HBeAg and HBV DNA was observed in chronic hepatitis B patients.68,72 Glycyrrhiza uralensis is widely used in modulation or improvement of the other herbs in many Chinese prescriptions. The active compounds in G. uralensis are identiÞed as glycyrrhizin, glycyrrhetic acid 3-O-monoglucuronide and glycyrrhetic acid. All these compounds have anti-HBV activity in PLC/PRF/5 cells that inhibits the expression of HBsAg and sialylation of HBsAg.73 Because of its modest effect on the suppression of HBV, glycyrrhizin usually is used in combination with other potent anti-HBV drugs, such as 3TC.74 In a pilot clinical trial, the combination of intravenous glycyrrhizin and oral lamivudine demonstrated safety and efÞcacy of anti-HBV activity.75 Regardless of its anti-HBV activity, glycyrrhzin showed its effect on the modulation of the immune system, with enhanced interleukin-12 production in peritoneal macrophages.76 Because of this characteristic, G uralensis is used widely in many complex remedies for supporting the immune system against infectious diseases, such as hepatitis B infection.77 Boehmeria nivea has been used therapeutically in China and Taiwan for diuretic and antipyretic purposes and for liver protection.78 Recently, the anti-HBV activity of B. nivea was Þrst demonstrated by Huang et al for the Þrst time.79 B. nivea strongly 85


inhibits the HBV virion secretion in HepG2 2.2.15 cells, but this inhibitory effect was not related to its cytotoxicity or inhibition of viral DNA replication and RNA expression. Furthermore, B. nivea can also reduce the level of serum HBV DNA in a HepG2 2.2.15 tumor-bearing SCID model.80 Similar to other anti-HBV herbs, B. nivea’s primary mechanism of anti-HBV effect was not through inhibition of the viral polymerase. It was later demonstrated that B. nivea inhibits HBV virion secretion via down-regulation of a chaperone interacting with HBV surface proteins.81 In the early screening of anti-HBV herbs, many active compounds were identiÞed and proposed for hepatitis B treatment: costunolide and dehydrocostus lactone in Saussurea lappa Clarks, daphnoretin in Wikstroemia indica and osthole in Angelica pubescens.64,82 These compounds exert anti-HBV activities by inhibition of HBsAg gene expression and HBsAg secretion via protein kinase C activation or HBsAg glycosylation.83 Wogonin, an active compound, from Scutellaria radix is reported to be able to inhibit secretion of HBsAg and reduce HBV DNA in HepG2 2.2.15 cells as well as serum DHBV and HBsAg levels in DHBV-infected ducks.84 COMPLEX HERB FORMULA IN THE TREATMENT OF CHRONIC HEPATITIS B Although a single herb can be used to treat a disease, traditional physicians have been treating chronic hepatitis patients with complex herbs, a so-called decoction or remedy. Generally, different herbs with a consistent proportion are extracted with hot water and then evaporated to approximately 4-fold concentration. In traditional Chinese medical theory, each herb confers its activity to eliminate symptoms, enhances the therapeutic effect of the other herbs and supports the patients’ tolerance to side effects or balances the body’s milieu. Some decoctions or remedies have been studied for a novel ‘indication’ of not only reducing the levels of alanine transaminoferase or aspartate aminotransferase but also reducing the serum HBsAg, HBeAg and HBV DNA levels, which in turn reßects the suppression of replication of HBV. Because of the difÞculties and complexities in the control of manufacturing, evaluation of the most complex remedies include only authentication of an individual herb with very limited chemical control. In addition, the remedy is designed for human use and there is no suitable 86


assay model to evaluate the therapeutic efÞcacy. Many clinical trials have been conducted to evaluate the efÞcacy of these complex remedies for treatment of chronic hepatitis B [Table 2]. Most of these anti-HBV complex remedies, such as Hejie decoction, Bushen recipe and sho-saiko-to, are not designed to clear serum HBV but support patients in evoking an immune response against HBV.85,86 These remedies have been shown to reduce the serum HBV DNA of chronic patients in controlled and uncontrolled clinical trials. According to the results of these clinical trails, these remedies may stimulate the immune system via the expression of TCRVβ7 in T cells or may polarize Th1 cytokine secretion followed by activation of cytotoxic T lymphocytes to eliminate HBV.87,88 In addition, the Bushen recipe combined with 3TC not only enhances the therapeutic effect but also reduces mutation of the YMDD motif.86 This Þnding implies that the Bushen recipe prevents the mutation of HBV in long-term chronic hepatitis B treatment. The Chinese remedy xiao-chai-hu-tang, also called sho-saiko-to and TJ9 in Japan, has been extensively studied for its therapeutic purposes. In treatment of chronic hepatitis B, xiao-chai-hu-tang can eliminate the serum HBeAg in children infected with HBV and can induce anti-HBe antibody development in some chronic hepatitis patients. 89,90 Experimental evidence has also revealed that xiao-chai-hu-tang improved the effect of vaccination in an HBV transgenic animal model.91 Unfortunately, there are reports that xiao-chai-hu-tang may induce acute hepatitis and acute respiratory distress syndrome.92-95 Hejie decoction, a complex formula of Chinese traditional medicine, is composed of nine herbs and its composition is similar to that of Xiao-Chai-hu-Tang, which contains the additional herbs Polygonum cuspidatum, Morinda officinalis How and Hedyotis diffusa Willd instead of Zingiber officinale [Table 2]. Clinical trails reveal that Hejie decoction is effective for treating chronic hepatitis B; its effect in eliminating HBV is through activating T cells by increasing the expression of TCRVβ7.85,87 So far, no observed adverse effect has been reported while chronic hepatitis B patients were treated with Hejie decoction. Although Hejie decoction, Bushen recipe and sho-saiko-to are designed to improve the immune system against infectious diseases, several kinds of complex formulas of herbs are designed to clear HBV, such as Denshao Huaxian capsule and New LivFit. Danshao Huaxian capsule contains Þve medicinal herbs that inhibited replication of HBV DNA and reduced serum HBV DNA in 87

Herbal composition

Morinda officinalis How; Cistanche salsa; Lycium chinense Mill; Rehmannia glutinosa; Sophora flavescens Ait; Citrus sinensis L); Panax pseudoginseng Wall var notoginseng

Stephaniae tetrandrae; Salviae miltiorrhizae; Paeoniae rubra; Radix astragal; Folium Ginkgo Bupleurum chinense; Scutellaria baricalensis; Pinellia ternate; Codonopsis pilosula; Glycyrrhiza uralensis; Zizyphus jujuba; Polygonum cuspidatum; Morinda officinalis How; Hedyotis diffusa Willd

Formulation name

Bushen recipe (BSR)

Danshao Huaxian capsule Hejie decoction (HJD)

88 Clinical trial: 45 patients with chronic hepatitis B Clinical trial: 65 patients with chronic hepatitis B

Clinical trial: 35 patients

Clinical trial: 30 patients Clinical trial: 88 patients

Clinical trial/ assay model

Increase of TCRVβ7 expression in PBMCs Activation of T cells to eliminate HBV via increase of TCRVβ7 expression

Polarization of Th1 cytokine secretion cytokines; increase in cytotoxic T-lymphocyte production Enhancement of therapeutic effect of 3TC; reduction in mutation of YMDD motif Inhibition of HBV DNA replication

Outcome

Zhang, Chen, et al (2002) Zhang, Chen, et al (2004)

Cheng, Lu, et al (2006)

Gao, Chang, et al (2005) Gao YQ, Sun XH (2005) Zhou, Wang, et al (2003)

References

Table 2: Summary of complementary and alternative medicines clinically used for the treatment of chronic hepatitis B


New LivÞt (NLF)

Formulation name KYH-1

Eclipta alba; Phyllanthus niruri; Rheum emodi; Tephrosea purpurea; Cichorium intybus; Tinospora ordifolia; Treminalia chebula; Boerrhaavia diffusa; Andrographis paniculata; Picorrhiza kurroa; Fumaria officinalis

Cucumis melo L var ma-gua Cucubitaceae; Laminaria japonica Aresch; Pueraria lobata (Willd); Youngia sonchifolia Max; Lactuca indica L var laciniata; Lxeris dentate (Thunb)

Herbal composition

Clinical trial/ assay model HepG2 2.2.15 Primary woodchuck hepatocyte/ woodchuck hepatitis virus (WHV) HBV polymerase gene expression assay/ Sf9 insect cells Clinical trial: end-stage renal disease patients, with HBV infection

89

Clearance of serum HBV DNA

Inhibition of HBV replication in HepG2 2.2.15 cells Inhibition of expression of WHV RNA, pregenomic WHV RNA and WHsAg mRNA Inhibition of activity of HBV polymerase

Outcome

Katiyar CK, Arora D (2005)

Jacob, Korba, et al (2004)

References

Table 2 continued: Summary of complementary and alternative medicines clinically used for the treatment of chronic hepatitis B


VI-28

Formulation name Sho-saiko-to; Xiao-chaihu-tang; TJ9

Radix ginseng; Cervi pantotrichum; Salvia miltiorrhizae; Cordyceps sinensis; Semen allii; Fructus cnidii; Fructus evodiae; Rhizoma kaempferiae

Bupleurum chinense; Scutellaria baricalensis; Pinellia ternate; Zingiber officinale; Zizyphus jujube; Codonopsis pilosula; Glycyrrhiza uralensis

Herbal composition

Clinical trial/ assay model Clinical trial: 8 patients with chronic active hepatitis Clinical trial: 14 children with chronic hepatitis HBV transgenic mice PBMCs and spleen cells

Kakumu, Yoshioka, et al (1991) Tajiri, Kozaiwa, et al (1991) Akbar, Abe, et al (1999)

Modulation of both cellular and humoral immune responses speciÞc for HBV -associated antigens Development of antiHBe antibody Improvement in effect of vaccination Up-regulation of IFN-r and IL-2Ra Enhancement of both innate and acquired immunity

PanHammarstrum, Wen, et al (2006) Lee, Wong, et al (2006)

References

Outcome

Table 2 continued: Summary of complementary and alternative medicines clinically used for the treatment of chronic hepatitis B


90


a 35-patient clinical trial.96 New LivFit contains six medicinal herbs that demonstrate the effect of hepatic protection and eliminate circulating HBV DNA in end-stage renal disease patients with HBV infection.97,98 It is not surprising that the formulation contains more than one herb with effective anti-HBV activity. Other complex herbal formulas, such as VI-28 and KYH-1, are also being studied at the pre-clinical stage. VI-28 can up-regulate the level of IFN-γ and IL-2Ra and can enhance both innate and acquired immunity, which is supposed to eliminate HBV.99,100 KYH-1 has been shown to inhibit HBV replication in HepG2 2.2.15 cells, expression of woodchuck hepatitis virus (WHV) RNA, pregenomic WHV RNA and WHV surface antigen mRNA in WHV-infected primary woodchuck hepatocytes and also inhibits the activity of HBV polymerase in an in vitro assay.101 STRATEGY OF HERBAL MEDICINES AGAINST HBV To Þght HBV infection, one must adopt two strategies: inhibit HBV replication and evoke immunity against HBV infection. Unlike nucleoside analogue drugs that target viral polymerase, herbal medicines may confer their anti-HBV activity in any step of the HBV lifecycle. Because of the high mutation rate of viral polymerase, the use of nucleoside analogue drugs, such as 3TC, will select the resistant mutation clone. Nevertheless, there are other targets that could affect the lifecycle, including viral encapsidation, assembly, envelopment and so forth. Herbal medicines elicit an unknown mechanism to inhibit replication and secretion of HBV, which is reßected in the reduction of HBsAg, HBeAg and HBV DNA. Because of the lack of a drug-screening system for anti-HBV effect, the herbal medicines still languish in the early stage of anti-HBV drug development. Although the mechanism of anti-HBV remains unclear, the effectiveness of these herbal medicines encourages scientists to identify the novel anti-HBV drug components from herbs that differ from the nucleoside analogues. In fact, once a novel viral target of the HBV lifecycle can be established, the herbs could be screened by using this target to facilitate the development of an anti-HBV drug. Of the traditional herbal medicines, some may confer an important function in promotion of immunity against HBV. By means of increasing the antigen presentation or inducing the expression of immune-related genes, the herbs could even substitute the costly IFN-α therapy. 91


Moreover, the herbs that are used as immunomodulators may yield additive or synergistic effects in combination with vaccinations or drugs in different pharmacological action modes.69,86,90 CRUDE MIXTURE VERSUS ACTIVE COMPOUNDS Among the herbals studied against HBV infection in the past decades, some of them have known chemical constituents. Some active compounds have been identiÞed and reported for their potent anti-HBV activity. However, continued research on these potent compounds is no longer pursued, which implies that inhibition of HBV by medicinal plants in the studies is not reproducible or is not feasible for pharmaceutical purposes. In fact, medicinal plants or herbs exhibit their anti-HBV as a result of a mixture of their constitutive components. When scientists try to fractionate the extract of herbs into partial puriÞed fractions or to identify the single active compounds, such action results in loss of the synergistic effects from a set of similar compounds. In most cases, this Þnding also explains the fact that herbs in crude extract form are effective and partially puriÞed fractions are less effective. COMBINATION OF HERBAL MEDICINE AND CURRENT ANTIHBV DRUG So far, there is no generally effective therapy for chronic hepatitis B because most of the present clinical regimens for treatment, such as interferon and nucleoside analogues, have had limited success.47 Therefore, some scientists are putting in a lot of effort into searching for novel anti-HBV agents from the empirically traditional herbal medicines. Medicinal herbs are widely used in the treatment of chronic hepatitis B in developing countries and many clinical trials are being conducted to verify the anti-HBV efÞcacy and safety of these herbal medicines individually or combined with current therapeutic drugs.68,69,72,86,102 The quality of these clinical trials on herbal medicines for hepatitis B is poor in that they do not follow the randomized, placebo-controlled, double-blind model, suggesting that the results may lead to misunderstandings.103 There are obstacles in conducting a good double-blind and placebocontrolled clinical trial because of the taste, ßavor and color of herbal medicine products. However, the outcomes of these limited clinical trials have encouraged physicians and guided scientists to continue the search for active compounds or the optimal formula 92


for the treatment of chronic hepatitis B. REGULATION OF COMPLEMENTARY AND ALTERNATIVE MEDICINES Complementary and alternative medicine (CAM), also termed ‘herbal medicines’, has a long history of use worldwide; even in Western countries, ancient people used herbs to treat many diseases. In China, multiple herbs are used to treat diseases, especially to cure a ’syndrome’, whereas Western industries emphasize use of a single herb to treat the etiology of a disease. Traditionally, appropriate herbs are combined to synergize the therapeutic effects and reduce adverse effects. However, the use of multiple herbs may lead to quality-control problems in manufacturing. In developing countries, patients can visit traditional physicians and accept the medication in herb forms. Most herbs are brought home to make a decoction or have already been made into capsule, solution and tablet form by a pharmaceutical company. Unfortunately, it is difÞcult to achieve consistency in these botanical products from the initial control of botanical raw materials to standardization of active ingredients. This problem may invoke a question: Do physicians use uncertain drugs to treat patients? In the treatment of chronic hepatitis B, complex herbs have been used clinically in treating patients and have yielded good therapeutic effects in clinical trials. In fact, CAM can be used as an adjuvant or adjunctive therapy; for example, Bushen recipe synergizes the therapeutic effect of 3TC and Sophora flavescens Ait synergizes the function of IFN-α.67,69,86 Most herbs exert antiviral activity in the treatment of chronic hepatitis B in a manner different to that of nucleotide drugs [Figure 1]. In China, many clinical trials have been conducted and regulated by the State Administration of Traditional Chinese Medicine (under the Ministry of Public Health) and a number of herbal medicines are already approved and commercially available. Although these herbal medicines have effects on the clearance of HBV markers and/or the restoration of liver function in patients with chronic hepatitis B, their reliability is still doubted because of the poor methodological qualities of the trials. Moreover, the organic chemicals contained in the herbs vary substantially because of many factors including the growth condition of the plant, the method of extraction and the type of formulation, which is correlated highly with the effectiveness of botanical products. Therefore, herbal medicines should be subject to 93


Figure 1: The targets for anti-HBV agents.

more stringent regulation to meet the requirements of the Western pharmaceutical market. In 1998, the National Center for Complementary and Alternative Medicine (NCCAM), was founded in the United States. This medical institute published a book titled Complementary and Alternative Medicine in the United States, which stated that more than one third of American adults had used CAM and that visits to CAM providers each year exceeded those to primary care physicians.104 About 19% of the people surveyed in the United States used natural products. This implies that CAM has already been accepted by technology-rich countries or areas. For regulation of natural products, FDA published guidance on botanical drug products in June 2004, which formally opened the regulatory door for the approval of botanical drugs. According to a 2005 report from the FDA Center for Drug Evaluation and Research (CDER) on preclinical issues and the status of botanical drug products in the United States, 192 botanical drugs were submitted as investigational new drugs (INDs) compared with 50 botanical IND drugs in 2000, showing that the number of botanical IND drugs has gradually increased in recent years. For the global pharmaceutical market, the herbal medicine manufactures would have to meet the FDA regulations for chemistry, manufacturing and controls (CMC), toxicology and

94


pharmacology to provide the world with evidence-based, sciencebased, clinically-driven and veriÞable botanical drugs. CONCLUSIONS Because of the low response rate of 3TC therapy and the serious side-effects of IFN treatment, the development of herbal medicines is assuming greater importance. Herbal medicines not only inhibit HBV replication but also lower the mutation rate in the YMDD sequence. Most herbs inhibit replication of the HBV virus but unlike 3TC, which inhibits only the viral polymerase, multiple herbal compounds in addition affect multiple intracellular targets to elicit synergistic anti-HBV effect. It is also believed that a combination of two anti-HBV drugs with different pharmacological actions will improve the therapeutic effect in the treatment of chronic hepatitis B. In many clinical trials, the effectiveness of herbs combined with IFN has been intensively evaluated and veriÞed. The results suggest that herbs can be integrated into the current Western anti-HBV therapy to achieve a good therapeutic effect. For this purpose, a novel type of anti-HBV herb or drug should be developed as the leading drug for the treatment of chronic hepatitis B. REFERENCES 1.

Chang MH. Impact of hepatitis B vaccination on hepatitis B disease and nucleic acid testing in high-prevalence populations. J Clin Virol 2006;36:S45-50.

2.

Farrell GC, Teoh NC. Management of chronic hepatitis B virus infection: A new era of disease control. Intern Med J 2006;36:100-13.

3.

Bent S, Ko R. Commonly used herbal medicines in the United States: A review. Am J Med 2004;116:478-85.

4.

Coon JT, Ernst E. Complementary and alternative therapies in the treatment of chronic hepatitis C: A systematic review. J Hepatol 2004;40:491-500.

5.

Hu J, Flores D, Toft D, Wang X, Nguyen D. Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function. J Virol 2004;78:13122-31.

6.

Lott L, Beames B, Notvall L, Lanford RE. Interaction between

95


hepatitis B virus core protein and reverse transcriptase. J Virol 2000;74:11479-89. 7.

Kann M, Gerlich WH. Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus. J Virol 1994;68:7993-8000.

8.

Vanlandschoot P, Cao T, Leroux-Roels G. The nucleocapsid of the hepatitis B virus: A remarkable immunogenic structure. Antiviral Res 2003;60:67-74.

9.

Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 1986;47:451-60.

10.

Patzer EJ, Nakamura GR, Simonsen CC, Levinson AD, Brands R. Intracellular assembly and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum. J Virol 1986;58:884-92.

11.

Niederau C, Heintges T, Lange S, Goldmann G, Niederau CM, Mohr L, et al. Long-term follow-up of HBeAg-positive patients treated with interferon alfa for chronic hepatitis B. N Engl J Med 1996;334:1422-7.

12.

Zoulim F. Therapy of chronic hepatitis B virus infection: Inhibition of the viral polymerase and other antiviral strategies. Antiviral Res 1999;44:1-30.

13.

Fischer KP, Gutfreund KS, Tyrrell DL. Lamivudine resistance in hepatitis B: Mechanisms and clinical implications. Drug Resist Updat 2001;4:118-28.

14.

Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM, Tyrrell DL. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996;24:714-7.

15.

Angus P, Vaughan R, Xiong S, Yang H, Delaney W, Gibbs C, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 2003;125:292-7.

16.

Fung SK, Chae HB, Fontana RJ, Conjeevaram H, Marrero

96


J, Oberhelman K, et al. Virologic response and resistance to adefovir in patients with chronic hepatitis B. J Hepatol 2006;44:283-90. 17.

Tenney DJ, Levine SM, Rose RE, Walsh AW, Weinheimer SP, Discotto L, et al. Clinical emergence of entecavirresistant hepatitis B virus requires additional substitutions in virus already resistant to Lamivudine. Antimicrob Agents Chemother 2004;48:3498-507.

18.

Nassal M, Rieger A. A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous Þrst-strand DNA synthesis. J Virol 1996;70:2764-73.

19.

Pollack JR, Ganem D. Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis. J Virol 1994;68:5579-87.

20.

Bartenschlager R, Schaller H. Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. Embo J 1992;11:3413-20.

21.

Hu J, Toft DO, Seeger C. Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. Embo J 1997;16:59-68.

22.

Cho G, Park SG, Jung G. Localization of HSP90 binding sites in the human hepatitis B virus polymerase. Biochem Biophys Res Commun 2000;269:191-6.

23.

Hu J, Seeger C. Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase. Proc Natl Acad Sci USA 1996;93:1060-4.

24.

Kim SS, Shin HJ, Cho YH, Rho HM. Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli. Arch Virol 2000;145:1305-20.

25.

Weber O, Schlemmer KH, Hartmann E, Hagelschuer I, Paessens A, Graef E, et al. Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a 97


transgenic mouse model. Antiviral Res 2002;54:69-78. 26.

Stray SJ, Bourne CR, Punna S, Lewis WG, Finn MG, Zlotnick A. A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly. Proc Natl Acad Sci USA 2005;102:8138-43.

27.

Hacker HJ, Deres K, Mildenberger M, Schroder CH. Antivirals interacting with hepatitis B virus core protein and core mutations may misdirect capsid assembly in a similar fashion. Biochem Pharmacol 2003;66:2273-9.

28.

Zlotnick A, Ceres P, Singh S, Johnson JM. A small molecule inhibits and misdirects assembly of hepatitis B virus capsids. J Virol 2002;76:4848-54.

29.

Lu X, Tran T, Simsek E, Block TM. The alkylated imino sugar, n-(n-Nonyl)- deoxygalactonojirimycin, reduces the amount of hepatitis B virus nucleocapsid in tissue culture. J Virol 2003;77:11933-40.

30.

Cattaneo R, Will H, Hernandez N, Schaller H. Signals regulating hepatitis B surface antigen transcription. Nature 1983;305:336-8.

31.

Huovila AP, Eder AM, Fuller SD. Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment. J Cell Biol 1992;118:1305-20.

32.

Lu X, Mehta A, Dwek R, Butters T, Block T. Evidence that N-linked glycosylation is necessary for hepatitis B virus secretion. Virology 1995;213:660-5.

33.

Macrae DR, Bruss V, Ganem D. Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly. Virology 1991;181:359-63.

34.

Zapun A, Petrescu SM, Rudd PM, Dwek RA, Thomas DY, Bergeron JJ. Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. Cell 1997;88:29-38.

35.

Block TM, Lu X, Platt FM, Foster GR, Gerlich WH, Blumberg BS, et al. Secretion of human hepatitis B virus is inhibited

98


by the imino sugar N-butyldeoxynojirimycin. Proc Natl Acad Sci USA 1994;91:2235-9. 36.

Mehta A, Lu X, Block TM, Blumberg BS, Dwek RA. Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing:evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion. Proc Natl Acad Sci USA 1997;94:1822-7.

37.

Bruss V. Hepatitis B virus morphogenesis . World J Gastroenterol 2007;13:65-73.

38.

Bruss V, Lu X, Thomssen R, Gerlich WH. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. Embo J 1994;13:2273-9.

39.

Ostapchuk P, Hearing P, Ganem D. A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. Embo J 1994;13:1048-57.

40.

Prange R, Streeck RE. Novel transmembrane topology of the hepatitis B virus envelope proteins. Embo J 1995;14:24756.

41.

Gripon P, Cannie I, Urban S. EfÞcient inhibition of hepatitis B virus infection by acylated peptides derived from the large viral surface protein. J Virol 2005;79:1613-22.

42.

Urban S, Gripon P. Inhibition of duck hepatitis B virus infection by a myristoylated pre-S peptide of the large viral surface protein. J Virol 2002;76:1986-90.

43.

Ueda K, Tsurimoto T, Matsubara K. Three envelope proteins of hepatitis B virus: Large S, middle S and major S proteins needed for the formation of Dane particles. J Virol 1991;65:3521-9.

44.

Xu Z, Jensen G, Yen TS. Activation of hepatitis B virus S promoter by the viral large surface protein via induction of stress in the endoplasmic reticulum. J Virol 1997;71:738792.

45.

Xu Z, Bruss V, Yen TS. Formation of intracellular particles by

99


hepatitis B virus large surface protein. J Virol 1997;71:548794. 46.

Chisari FV, Filippi P, McLachlan A, Milich DR, Riggs M, Lee S, et al. Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol 1986;60:880-7.

47.

Sheldon J, Rodes B, Zoulim F, Bartholomeusz A, Soriano V. Mutations affecting the replication capacity of the hepatitis B virus. J Viral Hepat 2006;13:427-34.

48.

Guha C, Mohan S, Roy-Chowdhury N, Roy-Chowdhury J. Cell culture and animal models of viral hepatitis. Part I: hepatitis B. Lab Anim (NY) 2004;33:37-46.

49.

Walter E, Keist R, Niederost B, Pult I, Blum HE. Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology 1996;24:1-5.

50.

Prince AM, Brotman B. Perspectives on hepatitis B studies with chimpanzees. Ilar J 2001;42:85-8.

51.

Summers J, Smolec JM, Snyder R. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci USA 1978;75:4533-7.

52.

Mason WS, Seal G, Summers J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J Virol 1980;36:829-36.

53.

Marion PL, Oshiro LS, Regnery DC, Scullard GH, Robinson WS. A virus in Beechey ground squirrels that is related to hepatitis B virus of humans. Proc Natl Acad Sci USA 1980;77:2941-5.

54.

Yang PL, Althage A, Chung J, Chisari FV. Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci USA 2002;99:1382530.

55.

Larkin J, Clayton M, Sun B, Perchonock CE, Morgan JL, Siracusa LD, et al. Hepatitis B virus transgenic mouse model of chronic liver disease. Nat Med 1999;5:907-12.

100


56.

Ilan E, Burakova T, Dagan S, Nussbaum O, Lubin I, Eren R, et al. The hepatitis B virus-trimera mouse: A model for human HBV infection and evaluation of anti-HBV therapeutic agents. Hepatology 1999;29:553-62.

57.

Unander DW, Webster GL, Blumberg BS. Usage and bioassays in Phyllanthus (Euphorbiaceae), IV: Clustering of antiviral uses and other effects. J Ethnopharmacol 1995;45:1-18.

58.

Liu J, Lin H, McIntosh H. Genus Phyllanthus for chronic hepatitis B virus infection: A systematic review. J Viral Hepat 2001;8:358-66.

59.

Heo NY, Lim YS, Kang JM, Oh SI, Park CS, Jung SW, et al. Clinical features of fulminant hepatic failure in a tertiary hospital with a liver transplant center in Korea. Korean J Hepatol 2006;12:82-92.

60.

Shin SJ, Ahn SH, Kim HM, Kim JK, Kim BC, Lee JH, et al. Clinical features and prognostic factors of fulminant hepatic failure in Koreans. Korean J Hepatol 2004;10:298-307.

61.

Kang EH, Kown TY, Oh GT, Park WF, Park SI, Park SK, et al. The ßavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis B virus-e antigen. Antiviral Res 2006;72:100-6.

62.

Lam WY, Leung KT, Law PT, Lee SM, Chan HL, Fung KP, et al. Antiviral effect of Phyllanthus nanus ethanolic extract against hepatitis B virus (HBV) by expression microarray analysis. J Cell Biochem 2006;97:795-812.

63.

Niu JZ, Wang YY, Qiao M, Gowans E, Edwards P, Thyagarajan SP, et al. Effect of Phyllanthus amarus on duck hepatitis B virus replication in vivo. J Med Virol 1990;32:212-8.

64.

Chen HC, Chou CK, Lee SD, Wang JC, Yeh SF. Active compounds from Saussurea lappa Clarks that suppress hepatitis B virus surface antigen gene expression in human hepatoma cells. Antiviral Res 1995;27:99-109.

65.

Ott M, Thyagarajan SP, Gupta S. Phyllanthus amarus 101


suppresses hepatitis B virus by interrupting interactions between HBV enhancer I and cellular transcription factors. Eur J Clin Invest 1997;27:908-15. 66.

Huang RL, Huang YL, Ou JC, Chen CC, Hsu FL, Chang C. Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro. Phytother Res 2003;17:449-53.

67.

Liu J, Zhu M, Shi R, Yang M. Radix Sophorae ßavescentis for chronic hepatitis B: A systematic review of randomized trials. Am J Chin Med 2003;31:337-54.

68.

McCulloch M, Broffman M, Gao J, Colford JM Jr. Chinese herbal medicine and interferon in the treatment of chronic hepatitis B: A meta-analysis of randomized, controlled trials. Am J Public Health 2002;92:1619-28.

69.

Chen C, Guo SM, Liu B. A randomized controlled trial of kurorinone versus interferon-alpha2a treatment in patients with chronic hepatitis B. J Viral Hepat 2000;7:225-9.

70.

Ding PL, Huang H, Zhou P, Chen DF. Quinolizidine alkaloids with anti-HBV activity from Sophora tonkinensis. Planta Med 2006;72:854-6.

71.

Long Y, Lin XT, Zeng KL, Zhang L. EfÞcacy of intramuscular matrine in the treatment of chronic hepatitis B. Hepatobiliary Pancreat Dis Int 2004;3:69-72.

72.

Pan ZS, Yu QH, Yan H, Zhang Y. Clinical study on treatment of chronic hepatitis B by kurarinone combined with interferon alpha-1b. Zhongguo Zhong Xi Yi Jie He Za Zhi 2005;25:700-3.

73.

Sato H, Goto W, Yamamura J, Kurokawa M, Kageyama S, Takahara T, et al. Therapeutic basis of glycyrrhizin on chronic hepatitis B. Antiviral Res 1996;30:171-7.

74.

Sumiyama K, Kobayashi M, Miyashiro E, Koike M. Combination therapy with transfer factor and high dose stronger neo-minophagen C in chronic hepatitis B in children (HBe Ag positive). Acta Paediatr Jpn 1991;33:327-34.

102


75.

Tandon A, Tandon BN, Bhujwala RA. Treatment of subacute hepatitis with Lamivudine and intravenous Glycyrrhizin:a pilot study. Hepatol Res 2001;20:1-8.

76.

Dai JH, Iwatani Y, Ishida T, Terunuma H, Kasai H, Iwakula Y, et al. Glycyrrhizin enhances interleukin-12 production in peritoneal macrophages. Immunology 2001;103:235-43.

77.

Sitia G, Iannacone M, Muller S, Bianchi ME, Guidotti LG. Treatment with HMGB1 inhibitors diminishes CTL-induced liver disease in HBV transgenic mice. J Leukoc Biol 2007;81:100-7.

78.

Lin CC, Yen MH, Lo TS, Lin JM. Evaluation of the hepatoprotective and antioxidant activity of Boehmeria nivea var. nivea and B. nivea var. tenacissima. J Ethnopharmacol 1998;60:9-17.

79.

Shuangsuo D, Zhengguo Z, Yunru C, Xin Z, Baofeng W, Lichao Y, et al. Inhibition of the replication of hepatitis B virus in vitro by emodin. Med Sci Monit 2006;12:BR302-6.

80.

Chang JM, Huang KL, Yuan TT, Lai YK, Hung LM, Establishment of a convenient Hepatitis B virus-producing xenograft model for validation of anti-HBV drug efÞcacy, in The American Society for Cell Biology 46th annual meeting. Mol Biol Cell 2006;17:L139.

81.

Huang KL, Lin CC, Lai YK, Chang JM. Involvement of GRP78 in Inhibition of Hepatitis B Virus Secretion by Boehmeria nivea Root Extract in Human HepG2 2.2.15. Cells 2007.

82.

Chen HC, Chou CK, Kuo YH, Yeh SF. IdentiÞcation of a protein kinase C (PKC) activator, daphnoretin, that suppresses hepatitis B virus gene expression in human hepatoma cells. Biochem Pharmacol 1996;52:1025-32.

83.

Huang RL, Chen CC, Huang YL, Hsieh DJ, Hu CP, Chen CF, et al. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro. Hepatology 1996;24:508-15.

84.

Guo Q, Zhao L, You Q, Yang Y, Gu H, Song G, et al. Anti-hepatitis B virus activity of wogonin in vitro and in vivo. 103


Antiviral Res 2007;74:16-24. 85.

Zhang SJ, Chen ZX, Lao SX, Huang BJ. Effect of Hejie decoction on T cell immune state of chronic hepatitis B patients. World J Gastroenterol 2004;10:1436-9.

86.

Zhou F, Wang LT, Chen JJ. Therapeutic efÞcacy of combined application of lamivudine and bushen recipe in treating chronic hepatitis B and its inßuence on YMDD motif. Zhongguo Zhong Xi Yi Jie He Za Zhi 2003;23:417-20.

87.

Zhang SJ, Chen ZX, Huang BJ. Effect of hejie decoction on T-cell receptor V beta 7 gene expression in patients of chronic hepatitis B. Zhongguo Zhong Xi Yi Jie He Za Zhi 2002;22:499-501.

88.

Gao YQ, Sun XH, Zhang XY. Effect of Bushen recipe on T-cell subsets and their function in patients with chronic hepatitis B. Zhongguo Zhong Xi Yi Jie He Za Zhi 2005;25:408-11.

89.

Tajiri H, Kozaiwa K, Ozaki Y, Miki K, Shimuzu K, Okada S. Effect of sho-saiko-to (xiao-chai-hu-tang) on HBeAg clearance in children with chronic hepatitis B virus infection and with sustained liver disease. Am J Chin Med 1991;19:121-9.

90.

Kakumu S, Yoshioka K, Wakita T, Ishikawa T. Effects of TJ-9 Sho-saiko-to (kampo medicine) on interferon gamma and antibody production speciÞc for hepatitis B virus antigen in patients with type B chronic hepatitis. Int J Immunopharmacol 1991;13:141-6.

91.

Akbar SM, Yamamoto K, Abe M, Ninomiya T, Tanimoto K, Masumoto T, et al. Potent synergistic effect of sho-saiko-to, a herbal medicine, during vaccine therapy in a murine model of hepatitis B virus carrier. Eur J Clin Invest 1999;29:786-92.

92.

Hsu LM, Huang YS, Tsay SH, Chang FY, Lee SD. Acute hepatitis induced by Chinese hepatoprotective herb, xiaochai-hu-tang. J Chin Med Assoc 2006;69:86-8.

93.

Sakamoto O, Ichikado K, Kohrogi H, Suga M. Clinical and CT characteristics of Chinese medicine-induced acute respiratory distress syndrome. Respirology 2003;8:344-50.

104


94.

Akira M, Ishikawa H, Yamamoto S. Drug-induced pneumonitis:thin-section CT Þndings in 60 patients. Radiology 2002;224:852-60.

95.

Kaneko M, Kawakita T, Tauchi Y, Saito Y, Suzuki A, Nomoto K. Augmentation of NK activity after oral administration of a traditional Chinese medicine, xiao-chai-hu-tang (shosaiko-to). Immunopharmacol Immunotoxicol 1994;16:41-53.

96.

Cheng ML, Lu T, Yao YM, Geng XX. Danshao huaxian capsule in treatment of decompensated cirrhosis resulting from chronic hepatitis B. Hepatobiliary Pancreat Dis Int 2006;5:48-51.

97.

Gupta YK, Sharma M, Chaudhary G, Katiyar CK. Hepatoprotective effect of New LivÞt, a polyherbal formulation, is mediated through its free radical scavenging activity. Phytother Res 2004;18:362-4.

98.

Katiyar CK, Arora D, Mehrotra R, Nandi AR, Dutta A, Jain AK. Management of chronic hepatitis B with New LivÞt in end stage renal disease. Indian J Physiol Pharmacol 2005;49:83-8.

99.

Pan-Hammarstrom Q, Wen S, Hammarstrom L. Cytokine gene expression proÞles in human lymphocytes induced by a formula of traditional Chinese medicine, vigconic VI-28. J Interferon Cytokine Res 2006;26:628-36.

100. Lee SK, Wong CK, Poon PM, Ip PS, Che CT, Fung KP, et al. In vitro immunomodulatory activities of a newly concocted traditional Chinese medicine formula:VI-28. Phytother Res 2006;20:883-8. 101. Jacob JR, Korba BE, You JE, Tennant BC, Kim YH. Korean medicinal plant extracts exhibit antiviral potency against viral hepatitis. J Altern Complement Med 2004;10:1019-26. 102. Liu KZ, Xu CH, Zhang MT. Clinical and experimental studies on effects of chronic hepatitis B treated with astragali composita. Zhongguo Zhong Xi Yi Jie He Za Zhi 1996;16:394-7. 103. Liu J, Kjaergard LL, Gluud C. Misuse of randomization: A 105


review of Chinese randomized trials of herbal medicines for chronic hepatitis B. Am J Chin Med 2002;30:173-6. 104. Board on Population Health and Public Health Practice, Institute of Medicine Io, Complementary and Alternative Medicine in the United States: National Center for Complementary and Alternative Medicine; 2005. p. 34-5. Source of Support: Nil, Conflict of Interest: None declared.

106

complementary and alternative therapies in the

complementary and alternative therapies in the

Suggest Documents

Complementary and Alternative Therapies in the ...

Complementary and Alternative Therapies in the ...

Complementary and alternative cancer therapies

Complementary and alternative therapies - MS International Federation

Complementary and alternative therapies for ...

Complementary and alternative therapies - MS International Federation

Download Complementary and Alternative Therapies for Nursing ...

Complementary and Alternative Medicine Therapies ...

The use of complementary and alternative therapies in Western Saudi ...

0133346501-complementary-alternative-therapies-nursing-practice ...

[PDF] Download Complementary Alternative Therapies for Nursing ...

Importance of Complementary and Alternative Cancer Therapies in ...

Complementary and Alternative Therapies in Surgical Care - GUPEA

Practitioner-based complementary and alternative therapies for the ...

The Use of Complementary and Alternative Medicine Therapies by ...

[PDF] Complementary Alternative Therapies in Nursing - Google Sites

Pdf Downlaod Complementary Alternative Therapies in ... - Google Sites

Complementary and Alternative Cancer Therapies - Dr. Sanjoy Kumar ...

Use of Complementary and Alternative Therapies by Asian Americans ...

Complementary Therapies in Allergic Rhinitis

Complementary Therapies in Medicine - BioMedSearch

Complementary Therapies in Medicine - BioMedSearch

Complementary Therapies in Allergic Rhinitis

alternative therapies