Natural Products: Review for Their Effects of Anti-HBV

Hepatitis B is a global infectious disease, seriously endangering human health. Currently, there are mainly interferons and nucleoside analogues treatment of hepatitis B in the clinic, which have certain therapeutic effects on hepatitis B, but their side effects and drug resistance are increasingly prominent. Therefore, it is urgently needed to discover and develop new anti-HBV drugs, especially natural products, which have novel, high efficiency, and low toxicity anti-HBV compounds with novel antiviral mechanisms. In this manuscript, the natural products (polysaccharides and 165 compounds) with the activity of antihepatitis B virus are discussed according to their chemical classes, including 14 phenylpropanoids, 8 flavonoids,12 xanthones, 13 anthroquinones, 47 terpenoids, 6 alkaloids, 15 enediynes, 11 aromatics, 18 phenylalanine dipeptides compounds, and 13 others. In addition, the anti-HBV mechanism and targets of natural product were also discussed. The aim of this review is to report new discoveries about anti-HBV natural products and to provide reference for researchers.


Introduction
Viral hepatitis B, referred to as hepatitis B, is a disease caused by the infection of hepatitis B virus (HBV, Figure 1). The infection of HBV can cause liver failure, acute or chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). About 2 billion people worldwide are infected with HBV, of which 400 million are long-term carriers [1,2]. According to research reports by the World Health Organization (WHO), about 600,000 people die of HBV infection or liver diseases related to HBV infection every year [3,4]. China has the largest population of HBV-infected people worldwide and is confronting this large disease burden with efficient antiviral drugs.
At present, there are mainly two kinds of drugs used in the clinic, namely, interferons (INFs) with antiviral and immunoregulatory functions and nucleoside analogues that can inhibit the reverse transcription of HBV [5,6]. In recent years, although these drugs have a certain therapeutic effect on HBV infection in the clinic, there are serious side effects and drug resistance [7,8]. Thus, there are more and more researchers focus on natural product [9][10][11]. Some researches report a variety of natural medicines with novel structure and anti-HBV activity, including some candidate drugs with good anti-HBV effects. However, these reports were mainly involved in isolation and identification of compounds with anti-HBV activity; the mechanisms and targets of compounds were less. The mechanism of clinical medicines (nucleoside analogues and interferon) on anti-HBV is basically clear, but the emergence of drug-resistant HBV mutants weakens the clinical effects. Thus, the development of safe and effective anti-HBV drugs with novel mechanism is the top priority in the current research [7,12]. In this manuscript, in order to help researchers understand HBV and develop the anti-HBV drugs, all kinds of natural products (Table 1) with anti-HBV effects and the infection process of HBV ( Figure 1) [13][14][15][16][17] were summarized. The types of natural products with anti-HBV activity include phenylpropanoids, flavonoids, alkaloids, terpenes, glycosides, and others (such as lactones and organic acids).

The Natural Products of Anti-HBV
2.1. Phenylpropanoids. Phenylpropanins have a wide range of biological activities, including antitumor, antivirus, liver protection, and antioxidation. For example, a variety of lignans in fruits of Schisandra chinensis have liver protective effects and can reduce serum alanine aminotransferase level. Schisandrae esteril A and its analogues have been used in the treatment of chronic hepatitis in China [18].
6-Hydroxyl-7-methoxyl-coumarin (1), isolated from the Streblus asper Lour core material, had significant anti-HBV effect on HepG 2.2.15 cells [19]. And the mechanism of compound 1 on anti-HBV effect may be related to its inhibition on secretion of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg), and the IC 50 were 29.60 μM (selective index, SI = 6:76) and 46.41 μM (SI = 4:31), respectively. Esculetin (2) from Microsorium fortunei (Moore) Ching. could not only inhibit the expression of the HBV antigens and HBV-DNA but also inhibit the expression of hepatitis B virus X(HBx) protein in a dose-dependent manner [20]. Chen et al. [21] isolated a series of phenylpropanins from the core material, bark and root of S. asper, all of which had significant anti-HBV activity. Among them, Magnatriol B (3) showed moderate anti-HBV activity by inhibiting the secretion of HBsAg and HBeAg with low cytotoxicity. Honokiol (4) showed significant anti-HBV activity and strong inhibition on HBsAg and HBeAg with IC 50 of 3.14 μM (SI = 21:47) and 4.74 μM (SI = 14:22), respectively. The inhibition effect of honokiol on HBsAg and HBeAg was stronger than that of positive control, lamivudine. Isomagnolol (5) and isocarpine (6) from the bark and roots of S. asper showed significant anti-HBV activity by HepG 2.2.15 cell assay and significantly inhibited HBsAg secretion with IC 50 of 10.34 μM and 3.67 μM, respectively. For inhibiting the secretion of HBeAg, IC 50 was 8.83 μM and 14.67 μM, respectively, without cytotoxicity. Honokiol (7) and (7 ′ R, 8 ′ S, 7 ′ R, 8 ′ S)-erythron-Strebluslignanol G (8), isolated from methanol extract of roots of S. asper, have strong anti-HBV activity by inhibiting the secretion of HBsAg and HBeAg. In addition, compounds 7and 8 could significantly inhibit the replication of HBV-DNA, with IC 50 of 9.02 and 8.67 μM, respectively [22,23]. Coumarin lignan (9) isolated from the stem of Kadsura heteroclita could inhibit the production of HBsAg and HBeAg with concentration of 25 μg/mL. The inhibition of compound 9 (57% and 48%) was even better than that of positive control, lamivudine (10% and 46%) [24]. Niranthin (10), isolated from Phyllanthus niruri, could inhibit the secretion of HBsAg and HBeAg in dose-dependent, with IC 50 values of 16.5 μM and 25.1 μM. The inhibition rates of 10 on HBsAg and HBeAg were 90.4% and 83.1% with 55.5 μM while the inhibition rates of lamivudine were 55.6% and 44.5% with 43.6 μM. The anti-HBV effect of compound 10 was better than that of lamivudine. The inhibition rates of compound 10 on DHBV-DNA, HBsAg, and HBeAg were higher than that of lamivudine, and the recovery rate was smaller after drug withdrawal, indicating that compound 10 had a good prospect in the development of new anti-HBV drugs  Figure 1: HBV life cycle and therapeutic targets. HBV life cycle: adsorption, penetration, biosynthesis, assembly, and secretion; therapeutic targets: entry inhibitors (NTCP and HSPG as the receptor-virus binding), cccDNA inhibitors (inhibiting the information of cccDNA), Epidrugs (inhibiting the viral RNA synthesis), endoplasmic reticulum inhibitors (inhibiting the viral capsid assembly), and glucosidese inhibitors (inhibiting the secretion of HBV proteins).    Matijin-Su HBV-DNA D. repens [68] 134

Flavonoids.
Flavonoids have a wide range of biological activities, including anti-inflammatory, anticancer, and anti-bacterial. It has a prominent role in protecting liver; for example, silymarin shows significant effect on protecting liver and has successfully developed into a protect liver medicine [28].
Recently, flavonoids have been reported with good anti-HBV effect. Luteolin (15), isolated from Swertia macrosperma C. B. Clark, could significantly inhibit the secretion of HBsAg and HBeAg with IC 50 values of 0.02 mM on HepG 2.2.15 cells in vitro [29]. Isovitexin (16) isolated from S. yunnanensis had good anti-HBV effect, which could not only inhibit the secretion of HBsAg and HBeAg, with IC 50 values of 0.04 mM, <0.03 mM, and 0.23 mM, but also significantly inhibit the replication of HBV-DNA, with the IC 50 values of 0.09 mM, <0.01 mM, and 0.05 mM [30]. Huang et al. [31] isolated LPRP-Et-97543 (17) from Liriopemuscari (Decne.) L.H.Bailey, which had significant anti-HBV activity and could significantly reduce the activity of Core, S, and Compound 9 c HBV-DNA Synthesis [75] 148 Cyclic (glycine-L-proline) HBsAg, HBeAg, and HBV-DNA [76] 149 Cyclic (4-hydroxy proline-phenylalanine) HBsAg, HBeAg, and HBV-DNA [76] 150 Cyclic (L-2-hydroxy proline-phenylalanine) HBsAg, HBeAg, and HBV-DNA [76] 151 N-acetyl phenylalanine HBsAg and HBeAg P. crinitum [77] 152 7 BioMed Research International preS promoters. Moreover, the mechanism may be that it inhibited the replication of viral DNA by regulating viral proteins. In recent years, molecular docking technology was used to screen the active ingredients against HBV, a 3D structure of HBV polymerase (Pol/RT) was modeled and docked with the active compounds, and quercetin (18) was proved that could enhance its anti-HBV activity up to 10% [32]. In addition, some researchers found that glabaarachalcone (19) and isopongachromene (20), isolated from P. pinnata, could bound with HBV-DNA polymerase protein target [33]. Isooriention (21), isolated from S. mussotii, displayed significant anti-HBV activities against the secretions of HBsAg and HBeAg with IC 50 value of 0.79 and 1.12 mM, as well as HBV-DNA replication with IC 50 value of 0.02 mM [34]. Epimedium Hyde II (22), a potential Chinese herbal active ingredient against HBV, could inhibit the replication of HBV-DNA and the expression of HBsAg and HBeAg in the serum of HBV-replicated C57BL/6 mice [35]. The chemical structures of compounds 15~22 showed in   .04 mM for HBeAg, respectively. It was deduced that hydroxy groups in the xanthone structure were essential for maintaining the inhibitory effects on the secretion of HBsAg and HBeAg. Glycosidation of hydroxy groups led to activity decreasing against HBsAg and HBeAg by comparing the activity of compounds 24, 26, and 27. It was concluded that two or more hydroxy groups were essential for inhibiting HBV-DNA replication, and methylation of hydroxy groups decreased or abolished anti-HBV activity. In addition, the position of the hydroxy groups of the isolated xanthones did not significantly affect the inhibition on HBV-DNA replication. The preliminary structure-activity relationships were deduced as (1) the anti-HBV activity of xanthones depends on the structure and substitution pattern of the hydroxy groups; (2) the hydroxy groups play very important roles in the anti-HBV activity; (3) the anti-HBV activity will be decreased after methylation orglycosidation [36]. Methyl6,8-dihydroxy-3-methyl-9-oxo-9H-xanthene-1-carboxylate (31), isolated from mangrove-derived aciduric fungus Penicillium sp., inhibited HBsAg secretion more effectively than that of the positive control, 3TC, in a dosedependent manner [37].  (43)    These results showed that extremophiles are a valuable resource of bioactive compounds, and that pH regulation is an effective strategy to induce metabolite production in aciduric fungi [37]. Peng et al. [40] found that 1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone (44), Rubiadin (45), and Anthraquinone bile acid conjugates (46) have significant anti-HBV effects on HepG2.2.15 cells. The IC 50 values of them were 12.41, 8.03, 17.05, and 8.13 g/mL, respectively. When the drug concentrations were 8 g/mL, the inhibitory rates of HBeAg were 61.42%, 43.79%, and 69.30%, respectively. The inhibitory rates of HBsAg secreted by cells were 6.15%, 23.34%, and 43.38%, respectively. Particularly, compound 45 could not only significantly decrease HBeAg and HBsAg secretion level and inhibit HBV-DNA replication but also inhibit the proliferation of the cells and HBx protein expression in a dose-dependent manner, which might become a novel anti-HBV drug candidate. Mohammad K et al. [41] reported that anti-HBV potential of AV-derived anthroquinones, possibly via HBV-DNA polymerase inhibition for the first time. Although aloin B (47) exhibited novel antiviral effect, aloe-emodin (48) appeared as the most promising anti-HBV natural drug with CYP3A4 activating property towards its enhanced therapeutic efficacy. Lan et al. [42] found that hypericin (49) could significantly reduce the expression of HBV-DNA and the expression level of HBsAg and HBeAg, which was similar to lamivu-dine, 3TC. The chemical structures of compounds 36~49 are shown in Figure 5.

Terpenoids.
Terpenes are a kind of compounds with isoprene as the basic structural unit. Terpenes have extensive biological activities, mainly including anti-inflammatory and antiviral effects [43].
Li et al. [19] isolated ursolic acid (50) from S. asper core material. Compound 50 had strong anti-HBV activity by inhibiting the production of HBsAg and HBeAg, with IC 50 of 89.91 and 97.61 μM. A triterpenoid, named MH (51), was isolated from the Vicia tenuifolia Roth, which had significant inhibitory effect on the secretion of HBsAg and HBeAg in a dose-dependent manner [44]. Sweriyunnangenin A (52), 3-epitaraxerol (53), oleanolic acid (54), and erythrocentaurin (55), isolated from S. yunnanensis, could inhibit the secretion of HBsAg with IC 50 values of 0.28, 0.70, and 1.26 mM, respectively. They also had good inhibitory effects on the secretion of HBeAg, with the IC 50 values of 0.29, 1.41, and 0.94 mM, respectively. Especially, compound 55 could effectively inhibit the secretion of HBsAg and HBeAg, as well as the replication of HBV-DNA, due to its aldehyde group [26]. Zhou et al. [45] isolated a series of heptane terpenoids from the roots and rhizomes of Aster tataricus L. f. Among them, astataricusones B andepishionol (56)(57) (65)) of the Paeonia sinjiangensis K. Y. Pan had anti-HBV activity and could inhibit the secretion of HBsAg and HBeAg as well as the replication of HBV-DNA. Among them, compound 59 had the highest anti-HBV activity, which was even better than that of positive drug, 3TC. Perovskatone A and demethylsalvicanol (66-67), isolated from Perovskia atriplicifolia, had anti-HBV activity. It was for the first report on the anti-HBV effect of P. atriplicifolia [48].  Geng et al. [54] found that the anti-HBV activity of erythrocentaurin (ET) derivatives was significantly improved. In particular, ET derivatives 1e and 1f ( [57]. The chemical structures of compounds 50~97 showed in Figure 6. 2.6. Alkaloids. Alkaloids, a kind of natural nitrogen heterocyclic, have complex ring structure, most of which have physiological activity [58]. Jiang et al. [59] found that the ethanol extract of Piper longum L. fruit had good anti-HBV effect, and erythro-1-[1-oxo-9(3,4-methylenedioxyphenyl)-8,9-dihydroxy-2E-nonenyl]-piperidine (98)

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BioMed Research International (PHAP) (131) from A. morrisonensis, which could significantly inhibit the replication of HBV. The mechanism may be that PHAP was involved in regulating the expression of surface protein genes and blocks the release of virus particles by interfering with the signaling pathway of endoplasmic reticulum. Zhao et al. [67] found that PHAP and derivatives have good anti-HBV activity, and structural modification on p-HAP and its glycoside led to a series of derivatives; among them, p-HAP derivative 2f (132) had the strongest effect on inhibiting the replication HBV-DNA (IC 50 = 5:8 μM, SI = 160:3). The primary structure-activity relationships suggested that the conjugated derivatives of p-HAP glycoside and substituted cinnamic acids obviously enhanced the activity against HBV-DNA replication. The chemical structures of compounds 121~132 are shown in Figure 9.    . Zhang et al. [82] isolated cichoric acid (158) from the leaves of Chicory intybus L and found that it had significant anti-HBV activity. Rosmarinic acid (159) inhibits HBV replication in HBVinfected cells by specifically targeting ε-Pol binding. In addi-tion, they analyzed an additional 25 rosmarinic acid derivatives and found that the "two phenolic hydroxyl groups at both ends" and the "caffeic acid-like structure" of rosmarinic acid are critical for the inhibition of ε-Pol binding [83]. It is well known that phenolic acids have better antiviral activity. The studies showed that 3-caffeoylquinicacid (160) [  In addition, carboxyl group is closely associated to the antiviral activity [85]. The chemical structures of compounds 152~165 are shown in Figure 11.

Polysaccharides.
Natural polysaccharide is mainly referred to widely exists in the nature of cellulose and its derivatives, chitin, and other natural polymer materials.
Polysaccharides have a wide range of biological activities, such as enhanced immunity, antiviral, and antiinflammatory [86,87]. In recent years, clinical researches of natural polysaccharides on anti-HBV have increased gradually; they have been proved to have significant anti-HBV effect [88]. Lentinan polysaccharide has a prominent effect on antiviral and immune regulation and is also used as an auxiliary drug for cancer and HBV [89,90]. Zhao et al. [91] obtain two polysaccharide fractions (LEP-1 and LEP-2) from Lentinus edodes (Berk.) sing. They found that LEPs possess potent anti-HBV activity in vitro. In addition, the polysaccharides from Hedyotis caudatifolia Merr.et Metcalf (50, 100, and 200 mg/L) significantly inhibited the secretion and expression of HBV-DNA on HepG 2.2.15 cells and effectively inhibited the secretion of HBsAg and HBeAg. Its mechanism may be related to the activation of JAK/STAT signaling pathway and the promotion of antiviral protein expression [92]. Zhan et al. [93] found that snail polysaccharides have a certain inhibitory effect on the replication of HBV-DNA (P < 0:01), which indicated that the maximum inhibition rate of HBsAg and HBeAg in HepG 2.2.15 cells is 42.8% and 52.1%, respectively, slightly below the positive control group (P < 0:05), and the inhibition effect of snail polysaccharide on HBeAg was better than that of HBsAg. The results of real-time fluorescence quantitative PCR test showed that snail polysaccharide had a certain inhibitory effect on the replication of HBV-DNA (P < 0:011). The anti-HBV effect of polyporus polysaccharide may be related to the regulation of the body's immune function, breaking the body's immune tolerance or low state [94]. Angelica sinensis polysaccharide [95] could promote DC mature of HBV transgenic mice, raise its coordinated stimulus molecules on the surface, enhance its promoting lymphocyte proliferation and secretion, strengthen its antigen oral ability, induce cellular immune response, reduce serum concentrations of HBsAg, and play a role in antiviral immunity. Liu et al. [96] extracted Chinese whelk polysaccharide by water extraction and transfected human hepatocellular carcinoma cells with HBV-DNA cloning as an experimental model. The results showed that PCC significantly inhibited HBV-DNA in HepG 2.2.15 cells at 0.1 mg·mL -1 and 1 mg·mL -1 . Xia et al. [97] investigated the effect of polysaccharides of Sipunculus nudus Linnaeus on anti-HBV; the results showed that polysaccharide with different dose groups were different degree of inhibition of HBV-DNA replication (P < 0:05), and the effects of high, middle dose group were similar to acyclovir.

Conclusion and Perspectives
At present, a variety of natural products with novel structure and high anti-HBV activity were isolated from natural resources. Among them, we found that terpenoids with antihepatitis B activity are the most ( Figure 6 and Table 1), and the activity is more significant.
However, the research content were disorderly and mainly focus on the simple isolation and identification of anti-HBV activity ingredients; the in-depth studies of anti-HBV mechanisms and targets are relatively rare. Moreover, most of the studies are limited to cell level, lack of animal model experiments, and no in-depth research of ingredients with significant antihepatitis B activity. Therefore, there are three suggestions for product research and development: 3.1. Search for New Natural Product Resources. The research on natural products against hepatitis B mainly focuses on the field of traditional Chinese medicine on land. The research on traditional Chinese medicine against hepatitis b has been very matured. However, it is still difficult to develop active natural products against HBV. In addition, there are few researches on marine natural products, microbial fermentation products, plant polysaccharides, and other aspects. In recent years, studies have found that marine natural products have good biological activity due to their special growth environment. Huang et al. [76] screened significant anti-HBV active ingredients from small marine molecules. Microbial fermentation products are a novel source of natural products. In recent years, many novel compounds are derived from microbial fermentation products. It is an interesting way to study the anti-HBV activity of microbial fermentation products. Plant polysaccharides have a wide range of biological activities, and studies [88][89][90][91][92][93][94][95] have shown that the chemical components of polysaccharides have a good anti-HBV activity. It is of great significance to search for anti-HBV active ingredients from novel natural products.

Novel
Method for Screening. The traditional screening of anti-HBV activity involves the separation and identification of chemical components in traditional Chinese medicinal materials and then the screening of their activity, which often takes time and effort and is difficult to obtain accurate screening results. In recent years, researchers used computer-aided drug design (molecular simulation docking) to screen out suitable compounds from the database and then carried out screening in vitro. This method has strong purpose and high accuracy. A series of derivatives with good anti-HBV activity were obtained by modifying the structure of known compounds with anti-HBV activity, and the derivatives with the best activity were screened out through activity test. This method also provides a new idea for discovering anti-HBV compounds with better activity [71][72][73][74][75].
3.3. Synergy Effect. Single-chemical components of natural products are no longer effective against HBV, and drug resistance will appear. For example, artemisinin is combined with other components to fight malaria. In anti-HBV studies, treatment methods of combination drugs are also widely used [98].

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflict of interest.