key: cord-1035179-vvjdzf29 authors: Li, Ting; Peng, Tao title: Traditional Chinese herbal medicine as a source of molecules with antiviral activity date: 2012-11-12 journal: Antiviral Res DOI: 10.1016/j.antiviral.2012.10.006 sha: 19e9f6a8e0880d10527cc9d1fd75aa64793ee0c6 doc_id: 1035179 cord_uid: vvjdzf29 Traditional Chinese herbal medicine (TCHM) is widely used in the prevention and treatment of viral infectious diseases. However, the operative mechanisms of TCHM remain largely obscure, mainly because of its complicated nature and the fragmented nature of research. In recent years, systematic methodologies have been developed to discover the active compounds in TCHM and to elucidate its underlying mechanisms. In this review, we summarize recent progress in TCHM-based antiviral research in China and other Asian countries. In particular, this review focuses on progress in targeting key steps in the viral replication cycle and key cellular components of the host defense system. Recent developments in centralized and standardized TCHM screening and databases are also summarized. Traditional Chinese herbal medicine (TCHM) is the most important component of the traditional Chinese medicine system, which has long been used for its multiple combinations of compounds in the form of processed natural products. Similar to conventional medicine, TCHMs are prescription or over-the-counter drugs. Today, TCHMs account for 10% of the prescription drugs in China. Because of the long history of medical usage, from the drug discovery point of view, screening for active lead compounds from TCHMs extracts is considered more efficient compare to random screening from a standard combinatorial chemical library. More functional compounds (''hits'') are likely to be discovered from TCHM extracts in biological screening assays, and the chemical properties of these compounds are often more ''drug-like'' (e.g. with better pharmacokinetics and bioavailability). TCHM-derived active compounds are thus often better lead compounds for further chemical improvements. These characteristics of TCHMs offer major opportunities for finding novel chemical structures active against a variety of therapeutic targets. However, even with these unique advantages, modernization and globalization of TCHM have been slow. Some of the most difficult issues have been understanding the operative mechanisms of TCHMs and identify their active components. This review summarizes recent progress and advantages of TCHM-based antiviral research in China. In particular, this paper follows the steps of the generalized virus life cycle and reports progress in assay development and in knowledge of the antiviral mechanisms of TCHMs or TCHM-derived compounds. TCHMs are widely used for the prevention and treatment of viral infectious diseases in China and many other Asian countries. However, the international community remains uncertain about the efficacy of TCHMs, because of the lack of supporting clinical evidence collected under international standards (randomized, placebo-controlled, double-blind and multicentered clinical studies). Governments have put forward support aimed at international regulatory approval of TCHMs. Leading the pack is the compound T89 (also known as Dantonic Ò , a THCM product by Tasly Pharmaceuticals, China), which may become the first traditional Chinese medicine to receive Food and Drug Administration (FDA) approval in the United States. T89 is a TCHM used in China for the management of ischaemic heart disease. It is currently under a global phase III trial (ClinicalTrials.gov identifier: NCT01659580). A growing number of TCHMs with antiviral activity is also garnering evidence of experimental and/or clinical efficacy. Table 1 shows a partial list of antiviral TCHMs approved by the China Food and Drug Administration (SFDA). TCHMs for respiratory viral infections represent the majority of drugs in the market. The viral replication cycle includes attachment and entry into the host cell ( Fig. 1, 1-3) , transcription of viral mRNA, viral genome replication ( Fig. 1 and 4-6) , protein synthesis and the assembly and budding of progeny virus particles (Fig. 1, 7 and 8 ). These steps provide targets for inhibitors of entry, replication (e.g., protease inhibitors, viral polymerase inhibitors, and integrase inhibitors, among others), assembly and budding. Such inhibitors are classified as direct antiviral agents. Previous studies have provided evidence of the direct antiviral activity of many medicinal herbs used in TCHMs (Sun, 2007; Wang et al., 2007 Wang et al., , 2008 Zhao and Han, 2009) . By definition, a virus depends on the cellular machinery to complete its replication cycle (e.g., cellular peptidase, transcription factors, and elongation factors). Following co-evolution with the host, many viruses have established sophisticated mechanisms to interact with the host immune system for immune evasion. These mechanisms provide cellular targets for antiviral drug intervention. Among the classes of antiviral agents, immunomodulators are the most abundant in TCHM. Based on TCM theory, a remedy contains multiple active components (mainly herbs) with multiple targets. Some of these components work directly on the therapeutic targets, whereas others counteract drug toxicity or enhance the bioavailability of the medicine. Thus, a TCHM remedy is often composed of a hierarchy of different components, the so-called ''monarch,'' ''minister,'' ''assistant,'' and ''guide components'' (Yu et al., 2006) . Considering the complicated nature of TCHM, experiments in laboratory animals have been considered the ''gold standard'' for pharmacological screening. The process is very important for medical evaluation, because it reflects the efficacy, side effects, and toxicity of medicines as a whole. In general, TCHM whole extracts are often tested first for their ability to protect animals against viral challenges (Fig. 2) . However, such in vivo methods are costly and have low throughput. For TCHM testing, optimized cell-based assays are often carried out directly for the initial evaluation of whole extracts that show clinical evidence of antiviral activity. This practice is based on the assumption that compounds with direct antiviral activity are present in whole TCHM extracts. These compounds are measured by their ability to protect cells against virus-induced cytotoxicity (Fig. 2) . Activity-guided fractionation (AGF) is often performed for subsequent identificaton of active fractions and further isolation of pure compounds (Koehn and Carter, 2005) (Fig. 2) . The basic principle of AGF is that a TCHM fraction is further separated only when its antiviral activity is confirmed. In recent years, with improved understanding of viral replication mechanisms at the cellular and molecular level, highly specific assays with Xia-sang-ju granule, Guang-yao-xing-qun-xiasang-ju Flu, RSV Influenza Huang et al. (2007) and Zhan and Dong (2006) high-throughput capabilities have been developed (Fig. 3) . These assays enhance the chances of success of AGF and provide data for understanding the mechanisms of action of the identified compounds. In addition to classical bioscreening, computer-aided molecular design and docking-based virtual screening technologies are also being applied to the antiviral screening of TCHM. Progress in this area depends heavily on the availability of structural databases and bioinformatics. In the past, databases were scattered among individual laboratories, and included an insufficient number of compounds and limited associated information. However, several larger databases have recently been constructed. The TCM Data-base@Taiwan (http://tcm.cmu.edu.tw), built by a team led by Prof. Calvin Yu-Chian Chen from China Medical University in Taiwan contains the chemical structures of over 20,000 compounds (Chen, 2011) . Using this database, the team has identified quinic acid, Protection? Virus challenge in animal models In vitro evaluation genipin, syringic acid, cucurbitine, fagarine, methyl isoferulate and their derivatives as potent anti-influenza compounds, through blocking of the viral M2 ion channel (Lin et al., 2011) . Using the same approach, they also identified xynopine-2, rosmaricine-14 and rosmaricine-15 as strong antagonists of the binding of hemagglutinin subtype H1 to sialic acid (Chang et al., 2011b) . Entry into host cells is the first step of the viral life cycle, and its machinery has been proven an excellent target for antiviral therapeutics. Advanced assays have been developed to identify compounds that inhibit this critical step of the viral life cycle (Peng, 2010) . For many viruses, cell-surface attachment is accomplished through interaction with cell surface glycans. Polysaccharides have been observed to saturate the cell surface of viral attachment proteins and inhibit viral entry, as confirmed by antiviral TCM studies (Table 2) . Polysaccharides and their derivatives are the most frequently found viral entry inhibitors. Mechanism studies show that these sugars target the viral attachment and/or internalization steps mediated by specific interactions with viral particles or cell-surface molecules, resulting in viral serotype-or host cell type-dependent activity (Baba et al., 1988; Marchetti et al., 1995) . The composition of the sugar units and the diversity of the linkage chemistry are also factors that determine the functional properties and the target specificity of these compounds. Thus, while polysaccharides are considered to be broad-spectrum virus entry inhibitors, their derivatives display significant levels of virus-specific activity (Zhou and Meng, 1997) . Because polysaccharides are also ligands for immunoregulatory cell-surface receptors such as the toll-like receptors, they might also function as immunomodulators (Takeda et al., 2003) . After attachment, viral surface proteins interact with cell-surface receptors, triggering conformational changes which initiated the entry process. Inhibition of formation of the entry machinery Tannin Inhibits the binding to histo-blood group antigens (HBGAs) Zhang et al. (2012) or of required conformational changes can prevent viral entry. As indicated in Table 2 , aside from polysaccharides, tannins are the most identified entry inhibitors. Multiple mechanisms have been proposed for this activity, including the ability of tannins to interact with and precipitate proteins. Tannins have been shown to inhibit fusion completion in HIV infection (Liu et al., 2002) . Although polysaccharides and tannins are not typical drug-like molecules, they display broad antiviral activity. Their development as topically applied medicines such as microbicides is actively pursued. Replication represents the core of the viral life cycle, and involves most viral protein functions. Inhibitors of viral proteases, polymerases, integrases (helicases), and reverse transcriptases of HIV, HCV, and herpesviruses have been clinically successful, and most current antiviral agents target this stage. Considering these unique scenarios, development of TCHMs with antiviral activity is focused principally on this stage of infection (Table 3) . Compared with anti-entry TCHMs, compounds targeting replication are more chemically diverse and more virus-specific. Furthermore, considering that cellular machinery is required for viral replication, the mechanisms of many antiviral TCHMs involve cellular factors. The assembly and release of infectious virions is the final step in the viral life cycle. In this stage, vial structural proteins (often as pre-structural proteins such as P1 of enterovirus 71) mature until they are assembled into viral capsids. During this step, viral genomes are packaged into capsids for intracellular transport, enveloped (for enveloped viruses), then released. Despite the absolute requirement for sustained viral infection, no antiviral agents that target this stage have been developed. This limitation is partially Glycyrrhizin Inhibits viral replication Chen et al. (2004) due to limited knowledge of the packaging and assembly mechanisms of most viruses, resulting in a limited number of specific assays available. Studies of some TCHMs have revealed that their mechanisms of action involve viral packaging and assembly (summarized in Table 4 ), but the number remains limited, and the level of understanding is still preliminary. As host cell invaders, viruses must escape the immune response to survive. Host innate and adaptive responses against viral infection and replication oppose viral strategies (escaping and blocking) against the host immune response. An excessive reaction of the host immune response may also lead to tissue damage and multi-organ injury (Ferrero-Miliani et al., 2007; La Gruta et al., 2007) , which in turn may cause related diseases. TCHMs that enhance host antiviral immune responses or block viral immune escape mechanisms therefore display antiviral activity through immunoregulatory mechanisms. Considering that many TCHMs have immunoregulatory activities (Table 5 ), many such remedies also display antiviral activities. This class of TCHMs includes multi-target compounds. For example, polysaccharides are potent interferon inducers and good viral entry inhibitors. Another example is glycyrrhizin, which has activity against entry, replication (Chen et al., 2004) , and immunomodulation (Shinada et al., 1986) . The major goal of current research is to meet international standards for the modernization of TCHMs. To achieve this goal, a TCHM must satisfy all requirements set by international standards, including evidence-supported efficacy (particularly through randomized, double-blind, placebo-controlled, multicenter clinical trials), safety assessment, and quality control. A centralized and standardized research system, aimed at achieving a better understanding of medicinal chemistry and the mechanism of action of TCHMs, is fundamental to achieving this goal. Realizing these needs, the Twelfth Five-Year (2011-2016) Plan for the National Economic and Social Development of the People's Republic of China laid out a national strategy for TCM development. Compared with former Plans, it reflects the equal importance of TCM and Western medicine at the national level. The project for ''Supporting the Development of TCM'' stipulates that ''the protection, research, and rational utilization of Chinese materia medica resources, and establishment of quality evaluation and standardization system'' has the highest priority in terms of government support (http://www.news.cn, 2011). This initiative shows a determination to solve the bottleneck of underdeveloped Chinese materia medica. Thus, based on the Plan, it is expected that TCM-based medical systems will be greatly enhanced through increased funding for basic research and improved education. This government support will undoubtedly result in advanced phytochemistry, assay development, and bioinformatics, which will in turn provide platform technologies and tools for the modernization and commercialization of TCM. Supported by central and local governments, drug screening centers have been established in China in recent years (Table 6 ). These centers are operated by scientists with extensive experience in global pharmaceutical industries, and are equipped with state-of-the-art equipment, including robots capable of highthroughput screening. Large pharmaceutical companies such as Novartis have also set up research centers in China. Compounds originating from TCHMs are among their foci for drug discovery. Information fragmentation poses a significant challenge to TCM research. Benefiting from strong financial support, large TCM-focused databases are now becoming available (Table 7) . Comprehensively integrated databases are foreseen to greatly enhance TCHM-based drug discovery. 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