Chinese herbal medicines as a source of molecules with anti-enterovirus 71 activity

Enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease (HFMD), which sometimes leads to severe neurological disease and death in the Asia–Pacific region. In Chinese medicine, HFMD is caused mainly by an accumulation of damp-heat and toxicity in the body. No effective drugs are currently available for the treatment and prevention of EV71 infection. This review summarizes the potential Chinese herbal extracts and isolated compounds with antiviral activity against EV71 and their clinical applications, especially those categorized as heat-clearing and detoxifying.


Background
Enterovirus 71 (EV71) is a non-enveloped, positive-sense, single-stranded RNA virus that is 7.4 kb in length and belongs to the family Picornaviridae [1]. Infection with EV71 commonly causes mild hand, foot, and mouth disease (HFMD), which sometimes leads to serious neurological complications such as aseptic meningitis, brain stem encephalitis, pulmonary edema, and poliomyelitislike paralysis, and eventually causes death especially in infants and children [2]. The potential fatal implications pose a great threat to infants and children under 5 years of age [3]. Since the first isolation of EV71 in the United States in 1969, there have been several outbreaks of EV71 in Bulgaria, Malaysia, Taiwan, and China that caused considerable levels of infection and mortality [4][5][6][7]. However, neither a precautionary vaccine nor a specific antiviral drug is available for the treatment of EV71 infection [6].
Interferons (IFNs) are a group of antiviral proteins (mainly glycoproteins) that regulate host cytokines and chemokines [8]. Infection with EV71 attenuates the IFN response, and reduces the antiviral effect of IFNs [9]. Although ribavirin, a nucleoside analog, has been clinically used as a broad-spectrum antiviral drug, treatment of EV71 infection by ribavirin has been unsatisfactory with considerable side effects in infants and children [10,11]. Pleconaril, an antipicornavirus capsid-binding agent, shows moderate efficacy in anti-EV71 treatment, but cannot reduce the cytopathic effect (CPE) induced by some EV71 strains [12,13]. Rupintrivir, an inhibitor of human rhinovirus (HRV) 3C protease, specifically binds to the 3C protease of EV71, inhibits the replication of EV71 in vitro, and strongly contains the spread of EV71 infection in vivo [14]. However, this drug has not yet been used for clinical treatment of EV71 infection.
In addition to synthetic compound design, Chinese herbal medicines (CHMs) contain a wide range of phytochemicals and comprise a potential source of anti-EV71 active agents [15]. According to Chinese medicine (CM) theory, diseases occur and develop through the effects of pathogenic factors on the human body that lead to an imbalance of qi, xue, yin, and yang, or organs and meridians in the body [16]. Herbs have different impacts on the human body to regulate qi, xue, yin, and yang, and balance the whole body. HFMD is caused mainly by an accumulation of damp-heat and toxicity in the body, and therefore its treatment may involve the usage of heatclearing and detoxifying medicines. In this review, studies on CHM extracts and compounds with anti-EV71 activity are summarized, based on CHMs categorized into heat-clearing and detoxifying medicines. Future perspectives and challenges in anti-EV71 drug development involving herbal medicines are also discussed.

Open Access
Chinese Medicine *Correspondence: xuhongxi88@gmail.com 1 School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Full list of author information is available at the end of the article leaves exerted antiviral effects against EV71 and CVA16 with EC 50 values of 35.88 and 42.91 µg/mL, respectively [36]. The extract inhibited virus-induced apoptosis, inactivated viral 2A protease, and reduced the expression of IL-6 and RANTES. The extract also reduced the virus yield in the intestine of EV71-infected suckling mice. The ethyl acetate (EA) fraction of the extract showed greater antiviral activity than the n-butanol or aqueous fractions, exhibiting EC 50 values of 4.21 µg/mL against EV71 and 9.08 µg/mL against CVA16 [37]. Eupafolin, a major component of the EA fraction, showed EC 50 values of 1.39 µM against EV71 and 5.24 µM against CVA16. Eupafolin attenuated the virus-induced upregulation of IL-6 and RANTES by inhibiting the virus-induced ERK1/2, AP-1, and STAT3 signals.
Saururus chinensis (Lour.) Baill (San Bai Cao) exhibited a variety of bioactivities for the treatment of edema, jaundice, cancer, and inflammatory diseases [38]. The water extract of S. chinensis showed significant antiviral activity against EV71 with an EC 50 of 8.9 µg/mL by inhibiting the activation of the MEK1/ERK signaling pathway, and rutin was identified as the major component responsible for this activity [36].
The bark, leaves, and flowers of Paulownia tomentosa (Pao Tong) have been applied to the treatment of infections and inflammatory diseases in CM [39]. The methanol extract of P. tomentosa flowers demonstrated anti-EV71 activity with a dose-dependent reduction of the CPE and an EC 50 of 65 µg/mL. Further bioactivity-guided isolation led to the discovery of the pure anti-EV71 compound apigenin, which inhibited viral replication [40].
The EA and butanol extract of Phyllanthus urinaria (Zhen Zhu Cao) was reported to possess antiviral activity against EV71 and CVA16 based on CPE reduction assays, with corilagin identified as the major active component [41].

Other medicines
Salvia miltiorrhiza (Dan Shen) has been widely used in CM to improve blood circulation, relieve blood stasis, and treat coronary heart disease [42]. Wu et al. [43] obtained seven extracts of S. miltiorrhiza, and two fractions derived from water extracts showed anti-EV71 activity in CPE inhibition assays with EC 50 values of 0.742 mg/mL for fraction SA1 and 0.585 mg/mL for SA2. Both fractions exhibited antiviral activity against three strains of EV71 in Vero, RD, and MRC-5 cell lines. The extracts also inhibited EV71-induced plaque formation and apoptosis.
Pueraria lobata (Ge Gen) is commonly used for the treatment of cold, fever, and dysentery [44]. P. lobata exhibited antidiabetic, anti-inflammatory, antioxidant, and antiviral activities against respiratory syncytial virus (RSV) [45][46][47]. The water extract of P. lobata, which is the main component of Ge Gen Tang, inhibited the CPE induced by EV71 when given before, simultaneously with, or after infection, with an EC 50 of 0.028 µg/mL and an SI of >107,000 in a human foreskin fibroblast cell line [48]. The extract inhibited viral attachment and penetration of the host cell and decreased EV71-induced IFN production.
Glycyrrhiza uralensis (Gan Cao) is a tonic herb with a wide range of bioactivities, such as antiulcer, anti-inflammatory, spasmolytic, antioxidative, antiviral, anticancer, and hepatoprotective effects [49]. The water extract of G. uralensis inhibited the EV71-induced CPE in a human foreskin fibroblast cell line with an EC 50 of 0.056 µg/ mL, and treatment after viral infection provided a better protection rate than treatment before infection [50]. The protective mechanism might have involved the prevention of viral attachment and penetration, but did not involve activation of the IFN pathway.
Ampelopsis brevipedunculata Trautv (Shan Pu Tao) is used for the treatment of liver disease and inflammation, and as a food ingredient [51]. The herb also exhibited antioxidative, hepatoprotective, and antiviral activities toward HBV [52][53][54]. The extracts of A. brevipedunculata Trautv showed the most potent anti-EV71 activity among 58 Taiwanese folk medicinal plants examined [51]. The EC 50 of the acetone extract was 26.11 µg/mL, and the inhibitory effects may be related to effects on viral infection, activity, and replication. The extract may also significantly upregulate IL-6 and IL-1β levels and downregulate IL-8 levels.
The extract inhibited viral attachment and penetration, though it did not significantly change the IFN level.
GuiQi polysaccharides (GQP) are derived from the water extract and alcohol precipitation of mixtures of Angelica sinensis (Dang Gui) and Astragalus membranaceus (Huang Qi) roots in a ratio of 1:5, and this formula is used for the tonification of qi and xue in the body. GQP significantly reduced EV71 induced-CPE with EC 50 below 31.2 μg/mL and blocked EV71 adsorption rather than inhibiting EV71 replication [62].

Pure compounds derived from CHMs with anti-EV71 activity
Extracts of CHMs show a variety of bioactivities and contain diverse mixtures of essential bioactive compounds. Pure compounds with anti-EV71 activity are summarized in Table 2.

Flavones
Apigenin is widely distributed in a variety of plants, such as Ocimum basilicum (Luo Le), parsley, artichoke, basil, and celery. Apigenin showed anti-EV71 activity at approximately 25 µM, and inhibited viral protein expression, reactive oxygen species (ROS) generation, and cytokine upregulation [63,64]. Apigenin also interfered with viral internal ribosome entry site (IRES) activity and JNK activation [65]. The study also suggested that apigenin inhibited the association of EV71 RNA with RNAediting-related hnRNP proteins [65].
Chrysosplenetin and penduletin are two flavonols isolated from the leaves of Laggera pterodonta (Chou Ling Dan), which is used for clearing heat and detoxification [66]. These flavonols exhibited potent anti-EV71 activity with low EC 50 values of 0.20 µM for chrysosplenetin and 0.17 µM for penduletin, and had SI values of >100 [67]. The flavonols showed strong antiviral potency by targeting the viral post-attachment stage. Flavonoids with 3-methoxy, 5-hydroxy, and 4′-hydroxyl groups showed antipicornavirus activity by targeting the phosphatidylinositol 4-kinase IIIβ (PI4KB) pathway, and being within the same group, chrysosplenetin and penduletin might inhibit PI4KB, which would contribute to their anti-EV71 activity [68]. The PI4KB/oxysterolbinding protein (OSBP) pathway was the major target of the anti-picornavirus activity of enviroxime-like compounds and flavonoids with 3-methoxy, 5-hydroxy, and 4′-hydroxyl groups [69][70][71][72]. Inhibition of the PI4 KB/ OSBP pathway might contribute to the anti-EV71 activity of many uncharacterized compounds [69,73,74]. However, as reported, consecutive administration of two different structural PI4 KB inhibitors in SJL mice exhibited certain toxicity, which limited their further application [75].       Kaempferol also significantly reduced the viral yield by its regulatory effects on IRES function and EV71 replication through changes to the IRES-associated trans-acting factors FUBP1, FUBP3, HNRPD, HNRH1, and HNRPF.
Chrysin (CR) is a flavone extracted from the seeds of Oroxylum indicum (L.) Vent. (Mu Hu Die) and other plants, and exhibited antitumor and antidiabetic bioactivities [77,78]. CR was indicated to show possible binding to EV71 protease 3C in Autodock 4.0 simulations [79]. CR exhibited strong anti-EV71 activity with an EC 50 of 10 µM in CPE inhibition assays, while its phosphate ester (CPI) showed a more potent effect with a lower EC 50 of 6 µM.
Luteolin can be found in many plants, such as Lonicera japonica (Jin Yin Hua) and Perilla frutescens (L.) Britt (Bai Su). This flavonoid exhibited various pharmacological activities, including inhibition of EV71 and CVA16 with EC 50 values of approximately 10 µM [80]. Xu et al. [80] reported that luteolin targeted the post-attachment stage of EV71 and CVA16 infection by inhibiting viral RNA replication, while Lv et al. [64] reported that it might act on viral polyprotein expression after viral entry of EV71, and prevent EV71-induced cell apoptosis, intracellular ROS generation, and cytokine upregulation.
Formononetin can be extracted from many herbs and plants, such as leguminous plants [81]. It exhibited various bioactivities including anti-inflammatory, antioxidative, and anticancer effects [82]. In a large-scale screening, formononetin demonstrated significant anti-EV71 activity [82]. Specifically, it inhibited the EV71-induced CPE with an EC 50 of 3.98 µM, reduced virus RNA replication and protein expression in a dose-dependent manner, and exerted antiviral activity by application before and after EV71 infection. The mechanism of the formononetin activity involved the suppression of ERK, p38 MAPK, and JNK activation as well as the suppression of EV71induced COX-2/PGE 2 expression.

Terpenes
Raoulic acid is the main component of Raoulia australis, a perennial shrub plant from New Zealand. Raoulic acid reduced the EV71-induced CPE [83], and possessed broad-spectrum antiviral activity against six HRVs with EC 50 values of less than 0.1 µg/mL [84].
Ursolic acid from O. basilicum was reported to possess antitumor activity [85]. It showed strong anti-EV71 activity with an EC 50 of 1.1 µM and SI of >200, and might inhibit viral infection and replication processes [63].
Glycyrrhizic acid is a major bioactive compound found in G. uralensis (Gan Cao), which is used for the treatment of sore throat, cough, peptic ulcers, and other ailments in CM [49]. The compound exhibited anti-inflammatory, antidiabetic, antioxidative, anticancer, anti-microbial, and antiviral properties [86]. Glycyrrhizic acid suppressed the EV71-induced CPE and plaque formation at 3 and 5 mM, respectively, and might target post-viral entry processes [87].
Geniposide is a primary component of Fructus gardeniae (Zhi Zi), a fruit that can be used for its laxity and anti-inflammatory effects [88]. Geniposide protected more than 80 % of cells against EV71 infection at a concentration of 3 mg/mL, and reduced the EV71-induced CPE by approximately 80 % at a concentration of 2 mg/ mL [89]. Geniposide might block the translation of viral proteins.
Hederasaponin B can be isolated from Hedera helix (Chang Chun Teng). The isolate inhibited the CPE induced by the C3 and C4a types of EV71 with EC 50 values of <0.16 µg/mL and reduced viral capsid protein expression [92].
Ginsenosides are major active components of Panax ginseng Meyer (Ren Shen). Ginsenosides possesses antiaging, antidiabetic, anticancer, and antiviral activities [93,94]. In a cell-based screening of seven ginsenosides, a CPE reduction assay was applied and quantified with the sulforhodamine B method [95]. Among the substances examined in the screening, only ginsenoside Rg2 showed moderate dose-dependent anti-EV71 effects.

Polyphenols
Epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG) are two tea catechins that significantly reduced EV71-induced plaque formation, while EGCG also reduced the viral RNA levels of EV71 [96]. EGCG and GCG at concentrations of 25 µM increased the cell survival rate by approximately fourfold compared with the rate in mock-infected Vero cells. The anti-oxidative activity of EGCG might contribute to the anti-EV71 activity.
Chebulagic acid, a hydrolysable tannin, is isolated from the fruits of Terminalia chebula (He Zi), and used for its spasmolytic, anti-diarrheal, anti-bacterial, anti-hyperglycemic, and broad-spectrum antiviral activities [100,101]. Chebulagic acid showed anti-EV71 activity in vitro with an EC 50 of 13.1 µM, and reduced the mortality and relieved the symptoms of EV71-infected mice by inhibiting viral replication in vivo [102].
Punicalagin was examined for its antiviral effects, and reduced both the CPE and viral RNA levels in vitro with an EC 50 of 15 µg/mL. Furthermore, punicalagin reduced the mortality and relieved the clinical symptoms, such as hind limb paralysis, of mice in vivo [103].

Steroids
Components of Anemarrhena asphodeloides (Zhi Mu) exhibited significant pharmacological effects on the nervous system and blood, and displayed antitumor, antioxidative, antimicrobial, antiviral, anti-inflammatory, antiosteoporotic, skin-protective, and anti-aging effects [104]. By applying an isolation method called folding fan mode counter-current chromatography and CPE reduction assays, six anti-EV71 saponins were identified in A. asphodeloides, among which timosaponin B-II displayed the best medicinal potential with an EC 50 of 4.3 µM and the highest SI of 92.9 [105]. Further improvements of the isolation method were achieved using two-phase solvent systems in sample pre-treatment, which increased the production yield of the active compound [106].
Resveratrol is contained in grapes, mulberries, peanuts, Polygonum cuspidatum (Hu Zhang), and several other sources. Resveratrol exhibited antioxidant and antiinflammatory activities and improved glucose and lipid metabolism [114]. Resveratrol also acted on cardiovascular parameters and modified some pathways involved in carcinogenesis [115]. Resveratrol increased the survival rate of EV71-infected Vero cells with an EC 50 of 20.2 mM and SI of 15.2 [116]. Resveratrol inhibited the virus titer and protein expression by blocking the IKK/NF-κB signaling pathway [117]. However, polydatin is the most abundant form of resveratrol that exists naturally, and this compound did not show significant anti-EV71 activity.
Allophycocyanin is a fluorescent protein derived from the blue-green alga Spirulina platensis, and exhibited antioxidant and anticancer activities [118,119]. The protein exerted anti-EV71 activity in CPE and plaque reduction assays with an EC 50 of 0.045 µM, delayed viral RNA synthesis, and inhibited EV71-induced apoptosis [120]. Allophycocyanin also showed antiviral activity against CVA16.
Aloe-emodin is a free anthraquinone isolated from Rheum palmatum (Da Huang) that is used for purgation, clearing heat, and detoxification, and exhibited antibacterial, hepatoprotective, antitumor and antiangiogenic effects [121][122][123]. Aloe-emodin showed antiviral activity against EV71 in HL-CZ and TE-671 cells with EC 50 values of 0.5-1.9 µM [124]. Aloe-emodin induced the expression of IFNs, and might be involved in the activation of the type I and II IFN signaling pathways against viral replication.
Oblongifolin J, oblongifolin M, and euxanthone are isolated from the leaves of Garcinia oblongifolia Champ. ex Benth (Ling Nan Shan Zhu Zi), which have anti-inflammatory and analgesic activities and can be used to treat myogenic convergence, allergies, rash, itching, ulcers, hepatitis, laryngitis, and hemoptysis. The isolates inhibited the CPE in EV71-infected Vero cells with EC 50 values of 31.1, 16.1, and 12.2 µM, respectively [128].
Gramine, a natural indole alkaloid, can be isolated from various raw plants and coal tar, and exhibited broad pharmaceutical activities, such as relaxation of bronchial smooth muscle, vasorelaxation, blood pressure elevation, relief of bronchitis nephritis, and relief of bronchial asthma [129]. Gramine did not exhibit anti-EV71 activity [129]. However, Wei et al. [130] performed a series of chemical modifications on gramine, and showed that 18 of 21 derivatives displayed some degree of anti-EV71 effect. Among the derivatives, 4 s had a relatively low EC 50 of 9.1 µM and the highest SI of 14.3. In their study, 4 s inhibited the virus-induced cell apoptosis, viral RNA replication, and viral protein expression, and may therefore target the early stage of the EV71 lifecycle.
Magnesium lithospermate B (MLB) and rosmarinic acid (RA) are two compounds found in S. miltiorrhiza. The results from pGS-EV71 IRES-based bicistronic reporter assays suggested that MLB and RA inhibited EV71 IRES activity [132], and further inhibited the EV71induced CPE with EC 50 values of 0.09 and 0.50 mM, plaque formation, and viral protein expression. These compounds exerted their antiviral effects during the viral absorption stage.
Matrine is isolated from Sophora flavescens (Ku Shen), and used for its heat-clearing and detoxifying properties. Matrine exhibited anticancer, antidiabetic, hepatoprotective, and cardioprotective effects [133][134][135][136]. It also showed significant inhibitory effects on EV71 in vitro and in vivo [137]. Matrine reduced the viral RNA levels in RD cells, and protected mice from a lethal dose of EV71 virus while relieving the clinical symptoms of infection.
As one of the most abundant alkaloids in the Amaryllidaceae family, lycorine exhibited anticancer and anti-inflammatory properties and conferred antiviral effects against human immunodeficiency virus (HIV), hepatitis C virus, and HSV-1 [138][139][140][141][142][143][144]. In RD cells, lycorine exhibited a dose-dependent reduction of the EV71-induced CPE with an EC 50 of 0.48 µg/mL. Treatment of cells with 1.0 µg/mL lycorine significantly inhibited the viral RNA level. The antiviral mechanism might be related to interference with viral polyprotein translation. Lycorine inhibited EV71 replication in muscle tissues of mice, resulting in reduced mortality, dosedependent increases in clinical scores, and reduced pathological changes including virions in tissues, moderate inflammation, and necrotizing myositis in muscle [145].

Application of CM in treatment of HFMD
In the past three decades, there have been several outbreaks of HFMD in Taiwan, Singapore, Australia, Japan, and China, leading to millions of infections and thousands of deaths [2,[146][147][148][149]. EV71 is mainly responsible for the severe symptoms caused by HFMD. No specific antiviral agent is available, making clinical management of HFMD largely supportive in nature [150]. The Ministry of Health of China issued "Guidelines for the diagnosis and treatment of hand, foot, and mouth disease" in 2010, and recommended a series of CHMs for the treatment of HFMD [151].
According to CM syndrome differentiation, HFMD has been classified into different groups with corresponding CM treatment recommendations (Table 3).
For general cases, decoctions with effects of clearing heat, dampness, and toxicity were recommended, including Forsythia suspense (Lian Qiao), L. japonica (Jin Yin Hua), Scutellaria baicalensis (Huang Qin), Artemisia apiacea (Qing Hao), and Fructus arctii (Niu Bang Zi), as well as tonic medicines such as G. uralensis (Gan Cao) and red peony root (Paeoniae rubrathe) (Chi Shao), and diuretic medicines such as barley (Semen coicis) (Yi Mi). For severe cases involving symptoms such as convulsions, CM preparations that soothe the nerves were recommended, such as Uncaria tomentosa (Gou Teng), Gastrodia elata (Tian Ma), silkworm larvae (Bai Jiang Can), and concha ostreae (Ostrea gigas thunberg) (Sheng Mu Li). In urgent cases, potent tonic medicines such as P. ginseng (Ren Shen) and Aconiti carmichaeli (Fu Zi) were recommended for use with caution. For oropharyngeal ulcers, several CM powders were recommended to alleviate the symptoms, and for HFMD-induced combined flaccid paralysis associated with the recovery period, acupuncture and massage were recommended for inclusion in the treatment.
Although the Chinese Government recommended these CMs in the clinical treatment of HFMD, insufficient evidence is available to support their extensive application.
Xi Yan Ping injection is composed mainly of andrographolide sulfonate, which is used clinically to treat bronchitis, amygdalitis, and bacillary dysentery. Andrographolide is a diterpene lactone with a variety of bioactivities, including anti-inflammatory, anticancer, and immunoregulatory effects, and is isolated from Andrographis paniculata (Burm) Nees. (Chuan Xin Lian), a heat-clearing and detoxifying medicine. Andrographolide prevented infectious diseases by inhibiting the multiplication of infectious agents, including Pseudomonas aeruginosa, Escherichia coli, Candida albicans, influenza virus, RSV, and adenovirus [152]. In 2012, a review article on the clinical application and research progress of CMs in the treatment of HFMD was published [153], in which the research progress on application of CM to the treatment of HFMD was systematically analyzed. In their study, 76 trials were included, and 18 trials met the criteria for inclusion in a meta-analysis. Compared with conventional therapy, Xi Yan Ping injection significantly reduced the time required for fever clearance and the skin eruption-eliminating time, with no obvious side effects [154]. A further study provided evidence that andrographolide sulfonate decreased ROS production in vitro by inhibiting lipopolysaccharide-stimulated neutrophil activation, while at 5 days post-medication with andrographolide sulfonate, the plasma myeloperoxidase, S100A8/A9, histone, and IL-6 levels were markedly lower in the combination therapy group than in the conventional therapy group [155].
Yan Hu Ning injection is composed of potassium sodium dehydroandrographolide succinate, with defervescent, antibacterial, antiviral, and sedative effects. This injection promoted adrenal function in the treatment of upper respiratory tract infection, viral pneumonia, and child epidemic parotitis [156][157][158]. Yan Hu Ning injection reduced the time required for fever clearance and rash subsidence [153]. Re Du Ning injection, which is included in three heatclearing and detoxifying medicines, Qing Hao, Jin Yin  Hua, and Zhi Zi, has been studied for its antiviral and anti-inflammatory effects [159,160]. Re Du Ning injection appeared to significantly reduce the time required for fever clearance and rash subsidence compared with conventional therapy [161].
Pu Di Lan is prepared as oral tablets or a liquid, and mainly consists of Taraxacum mongolicum (Pu Gong Ying), S. baicalensis (Huang Qin), Corydalis bungeana Turcz. (Ku Di Ding), and Baphicacanthis cusiae Rhizoma et Radix (Ban Lan Gen). Pu Di Lan treatment possessed superior efficacy compared with conventional drug therapy in the time required for fever clearance, rash subsidence, and oral ulcer treatment [153].

Kappa carrageenan
Kappa carrageenan is water-soluble, sulfated galactan existing in a variety of seaweeds and is widely applied as a food additive [162]. It showed anti-EV71 activity with little associated toxicity [163]. The anti-EV71 activity might be attributable to the direct binding of carrageenan with virus particles.

Perspectives
In recent years, attention has been increasingly drawn to the screening of natural products and especially CM for anti-EV71 active components. The anti-EV71 mechanisms of action of these components have been summarized in Fig. 1. Common strategies for identifying anti-EV71 components are shown in Fig. 2. One strategy is bioactivity-guided isolation, which has been represented in the isolation of Garcinia oblongifolia, Hedera helix and Anemarrhena asphodeloides. Another strategy is systematic identification of the chemical constituents from a natural source and subsequent testing of each purified compound for antiviral activity, with more time and cost consumption. Additionally, in the case where the active compound is known and a standard substance is available, a fingerprint profile can be used for identification of them in active extracts. Furthermore, chemical modification of known active natural compounds may lead to better structural optimization to yield higher efficiency and lower toxicity, thus promoting anti-EV71 drug development [130].
Development of computer simulations allows the indepth study of EV71 infection and pathogenic mechanisms; with the expansion of compound libraries, the anti-EV71 compound targeting of viral proteins becomes increasingly predictable, thus enabling direct synthesis of predicted bioactive molecules [79]. An alternative for cell-based screening involves a two-step platform that Fig. 1 Mechanisms of anti-EV71 components. Mechanisms of anti-EV71 activities associated with the shown CM-derived extracts and molecules were classified into six categories uses two types of reporter viruses, specifically a pseudovirus with luciferase-encoding RNA replicons encapsulated by viral capsid proteins and a full-length reporter virus expressing enhanced green fluorescent protein. The two reporters can be used to screen for possible hits and then to conduct a cell-based assay to confirm the activity [80].
Current research on anti-EV71 drug development is mostly carried out in vitro with a few in vivo studies. The current evidence for the clinical application of CM in HFMD treatment is still insufficient to determine the efficacy, due to the numerous factors, such as the quality control of CM [164,165], and sufficient sample sizes, improved randomization, and better group organization in clinical studies.

Conclusion
This review summarized the group of anti-EV71 molecules that have been isolated from CHM and have been applied clinically for this purpose.