Anti-Enterovirus 71 Agents of Natural Products

This review, with 42 references, presents the fascinating area of anti-enterovirus 71 natural products over the last three decades for the first time. It covers literature published from 2005–2015 and refers to compounds isolated from biogenic sources. In total, 58 naturally-occurring anti-EV71 compounds are recorded.


Introduction
Human enterovirus 71 (EV71) is one of the major causative agent of hand, foot and mouth disease (HFMD) in infants and children aged <10 years, and is a positive-sense, single-stranded RNA virus in the genus Enterovirus (family Picornavirus). About 7400-base genomes contain a single long open reading frame (ORF) with untranslated regions (UTR) at the 50 and 30 ends and a variable length poly-A tail at OPEN ACCESS the terminus of the 30UTR. The ORF is divided into three consecutive parts, P1, P2 and P3. The viral RNA encodes a large polyprotein which is cleaved by virus-encoded and host proteases to produce the mature proteins. EV71 is divided into four genotypes, A, B, C and D. The most common form of EV71 infection is HFMD. Initial signs and symptoms include fever, headache, sore throat and a flu-like syndrome. Within a few days, patients develop painful ulcerated lesions in the nose, mouth and throat, accompanied by a rash that typically affects the hands and feet. In addition to HFMD, EV71 infection may involve the upper respiratory tract, the gastrointestinal tract, the cardiovascular system and the central nervous system. Neurological diseases range in severity from aseptic meningitis to acute flaccid paralysis and fatal encephalitis [1].
EV71 has emerged as a clinically important neurotropic virus that can cause acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. This illness has caused mortalities in large-scale outbreaks in the Asia-Pacific region in recent years, with widespread occurrence in China [2,3], creating a need to develop new anti-EV71 agents. Accordingly, exploring anti-EV71 agents is important. Several strategies have been used to develop antiviral drugs on the basis of the molecular characteristics of the virus [4]. Currently, no direct targeting vaccines or antivirals are available to treat severe EV71 infections.
At present, the prevention of EV71 epidemics mainly depends on public surveillance, and more effort should be made to develop drugs to conquer EV71 infections. Many compounds from various pharmacological medicinal plants have been extensively researched, not only for their potential inhibitory properties against virus invasion, but also for their low toxicity in cells. Therefore, it is essential to identify novel anti-EV71 agents as candidates for further research and optimization.

Flavonoids
Flavonoids are widely distributed natural products with broad biological and pharmacological activities, including anti-EV71 activity.

Flavonols
Chrysosplenetin (1) and pendulentin (2), two flavonols ( Figure 1) isolated from the leaves of Laggera pterodonta, showed strong activity against EV71 in Vero and RD cell-based infection systems and inhibited viral RNA replication [8]. Vero cells are isolated from African green monkey and used as host cells for growing viruses.

Tannins
A hydrolyzable tannin, chebulagic acid (22), was isolated from Terminalia chebula fruits found in Asia and Africa. Treatment with chebulagic acid (22) reduced the viral cytopathic effect on rhabdomyosarcoma cells with an IC50 of 12.5 μg/mL. Chebulagic acid (22) efficiently reduced mortality and relieve clinical symptoms through the inhibition of viral replication. [19] Geraniin (23) effectively inhibited virus replication in rhabdomyosarcoma cells with an IC50 of 10 µg/mL. Moreover, geraniin (23) treatment of mice that were challenged with a lethal dose of EV71 resulted in a reduction of mortality, relieved clinical symptoms, and inhibited virus replication in muscle tissues [20]. The corilagin (24) was a major component of Phyllanthus urinaria extract. Corilagin (24) inhibited EV71 infection in vitro. Corilagin (24) reduces the cytotoxicity induced by EV71 on Vero cells with an IC50 value of 5.6 µg/mL [21]. Punicalagin (25) reduced the viral cytopathic effect on rhabdomyosarcoma cells with an IC50 of 15 µg/mL, as well as reducing mortality and relieving clinical symptoms by inhibiting viral replication [22].

Sesterterpenoids
Raoulic acid (36), purified from a whole-plant extract of a New Zealand plant, Raoulia australis, was tested for antiviral activity in Vero cells and inhibited EV71 with an EC50 of less than 0.1 µg/mL and a CC50 of more than 65 µg/mL, giving it a therapeutic index >650 [31].

Triterpenoids
A large number of triterpenoids have been shown to have multiple biological activities.

Triterpenoids Glycosides
Glycyrrhizic acid (43) is considered the principal component in Glycyrrhiza spp. with a wide spectrum of antiviral activity. 43 dose-dependently blocked viral replication of EV71. At 3 mM, 43 reduced infectious EV71 production by 2.2 logs. At 5 mM, EV71 production was reduced by 6.0 logs 4.0 logs [35]. Ginsenosides are the major components responsible for the biochemical and pharmacological actions of ginseng, and have been shown to have various biological activities. The antiviral activities of three protopanaxatriol (PT) type ginsenosides, Re (44), Rf (45) and Rg2 (46), were demonstrated against EV71. The antiviral efficacies of PT-type ginsenosides were comparable to those of ribavirin, a commonly used antiviral drug [36]. The antiviral activity of hederasaponin B (47) from Hedera helix against EV71 subgenotypes C3 and C4a was evaluated in vero cells. Hederasaponin B (47) showed potent antiviral activity against EV71 subgenotypes C3 and C4a ( Figure 5). Hederasaponin B (47) also inhibited the viral VP2 protein expression and inhibition of viral capsid protein synthesis [37].

Alkaloids
Matrine (56) could suppress the viral RNA on rhabdomyosarcoma cells, reducing the mortality and relieving clinical symptoms [40]. Lycorine (57) blocks elongation of the viral polyprotein during translation. Lycorine (57) treatment of mice challenged with a lethal dose of EV71 resulted in reductions in mortality, improved clinical scores and fewer pathological changes in the muscles, associated with inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine (57) treatment prevented paralysis [41]. Deferoxamine (58), a marine microbial natural product, compensated for the decreased levels of B cells caused by EV 71 infection. The neutralizing antibody titer was also improved after deferoxamine treatment. Deferoxamine relieved symptoms and reduced mortality and muscle damage, and has the potential for development as a B cell-immunomodulator [42].

Conclusions
Since the 1980s, EV71 epidemics have occurred in Asian countries and regions, causing a wide range of human diseases. There is no clinical approved antiviral drug currently available for the prevention and treatment of the EV71 viral infections. The few examples of anti-EV71 natural products can be grouped into five main structural classes: flavonoids, polyphenols, terpenoids, steroids and alkaloids. Of all the anti-EV71 NPs, most of them (53/58) have been derived from territorial plants while the remaining five were of marine origins sources. The small number of anti-EV71 NP being discovered over the last 30 years is possibly due to the significant reduction of natural product screening campaigns undertaken by academia and the pharmaceutical industry. One of the technical hurdles is to develop an assay amendable for HTS screening. One plausible reason for the loss in interest by companies is that they cannot patent the structures of NPs. The uniqueness of many NP core structures (or templates) makes these compounds of interest for use as starting points for semi-synthesis and total synthesis. More effort should be put into the screening of NP libraries for anti-EV71 activity.

Conflicts of Interest
The authors declare no conflict of interest.

Abbreviations
Enterovirus 71 EV71 Natural product NP Hand, foot and mouth disease HFMD Cytopathic effect CPE