Natural Products and Their Derivatives against Human Herpesvirus Infection

Herpesviruses establish long-term latent infection for the life of the host and are known to cause numerous diseases. The prevalence of viral infection is significantly increased and causes a worldwide challenge in terms of health issues due to drug resistance. Prolonged treatment with conventional antiviral drugs is more likely to develop drug-resistant strains due to mutations of thymidine nucleoside kinase or DNA polymerase. Hence, the development of alternative treatments is clearly required. Natural products and their derivatives have played a significant role in treating herpesvirus infection rather than nucleoside analogs in drug-resistant strains with minimal undesirable effects and different mechanisms of action. Numerous plants, animals, fungi, and bacteria-derived compounds have been proved to be efficient and safe for treating human herpesvirus infection. This review covers the natural antiherpetic agents with the chemical structural class of alkaloids, flavonoids, terpenoids, polyphenols, anthraquinones, anthracyclines, and miscellaneous compounds, and their antiviral mechanisms have been summarized. This review would be helpful to get a better grasp of anti-herpesvirus activity of natural products and their derivatives, and to evaluate the feasibility of natural compounds as an alternative therapy against herpesvirus infections in humans.


Introduction
Herpesviruses (Family: Herpesviridae) are enveloped viruses containing doublestranded DNA that establish lifelong infections [1]. Their life cycle can be characterized as having two phases: lytic (active viral reproduction) and latency establishment (persistence of the viral genome with no particle production and the possibility of reactivation back to lytic replication) [2]. Herpesviruses derive their name from the Greek word herpes (to creep) to describe the spreading lesions along the skin manifested during herpes simplex virus or varicella zoster virus infection [3]. Referring to the quiescent nature of herpesvirus infections, latency is the hallmark of their life cycle [4]. There are over 25 viruses in the herpes family and at least eight types that infect humans, and the review focuses only on the herpesviruses that affect humans.
Herpesviruses are classified into three subfamilies: alpha, beta, and gamma-herpesviruses. Each of them is characterized differently regarding their tropism, replication cycle length, and associated diseases [4]. Alpha-herpesvirus consists of herpes simplex virus 1 (HSV-1), HSV-2, and varicella zoster virus (VZV). These viruses have a relatively fast and short replicative cycle in epithelial cells, fibroblasts, and neurons, and latency is established in sensory neurons [5]. HSV-1 and HSV-2 are among the most common human diseases, with a prevalence of 60% to 95% of the population [6]. HSV-1 is commonly associated with oral and perioral infections, and HSV-2 is known to cause genital Cephalotaxus alkaloids have been investigated as plant secondary metabolites for over 60 years and possess diverse bioactivities including antileukemia, anticancer, antimicrobial, antiviral, immunomodulatory, and other biological effects [148]. Cephalotaxine, a benzazepine pentacyclic alkaloid, is a biologically inactive parent structure of C3-ester compounds including harringtonine (1) and homoharringtonine (2) [47]. Both active compounds isolated from Cephalotaxus harringtonii significantly inhibited the replication of recombinant VZV-pOka luciferase with 50% effective concentrations (EC50) of 9.574 and 4.654 ng/mL and 50% cytotoxic concentrations (CC50) in HFF cells of 45.82 and 74.71 ng/mL, respectively. Both compounds at a concentration of 10 ng/mL reduced VZV-pOka luciferase activity more effectively by 76.1% and 91.3% and induced a significant reduction in the clinical isolate VZV-YC01 DNA by 57.8% and 97.1% compared to 1.13 μg/mL acyclovir, respectively. Moreover, 2 actively inhibited HSV-1 DNA and pseudorabies virus (PRV) at 500 and 1000 nM, respectively, in Vero cells. Then, 2 and acyclovir demonstrated 50% inhibitory concentrations (IC50) of 139 and 789 nM against HSV-1, respectively, and 2 strongly reduced PRV glycoprotein C (gC) levels at a concentration of 500 nM [48].
Molecules 2021, 26, 6290 5 of 36 compounds inhibited the enzyme by forming hydrogen bonds and interacting hydrophobically with significant residues of the active site.

Terpenoids with Potential Anti-Herpetic Activities
Natural products derived from different medicinal plants were obtained and tested with low toxicity for antiherpetic activities. The chemical structures of seven terpenoids possessing the antiviral activity are shown in Figure 3. The root of a Kenyan medicinal plant named Maytenus heterophylla containing pristimerin (19) was reported to have the first time for anti-HCMV activity [84]. The triterpenoid quinone methoide compound using cold diethyl ether methanol as a solvent was found to be effective against HCMV in immunocompromised hosts. The IC50, CC50, and SI were 0.53, 14.8, and 27.9 μg/mL, respectively [84]. The results demonstrated that 19 Pristimerin (19) Glycyrrhizic acid (20) Triptolide (   Cis/trans-martynoside (26) and cis/trans-isomartynoside (27) were first isolated from Martynra Louisiana, and the structures were identified using low-pressure liquid chromatography, NMR spectra, and electron impact mass spectrometry [171]. They were later isolated from Lindernia crustacea and displayed a significant inhibitory effect on the EBV Rta lytic cycle at 20 μg/mL via the immunoblot analysis. Using ethyl acetate to extract pure compounds showed the most potent anti-EBV activity [79].
Resveratrol (28), trans-3,5,4-trihydroxystilbene was found in several plants, including grape vines, berries, pines, pomegranates, peanuts, legumes, and soybeans with various mechanisms of action against different human and animal viruses [105][106][107][108][109][110][111][112][113][114][115][116][117][118]172]. Compound 28 and its derivatives demonstrated anti-HSV activity both in vivo and in vitro [173], it dose-and time-dependently inhibited both HSV-1 and HSV-2 replications via inhibition of ICP4 (IE gene) expression, and the inhibition of viral replication was reversible in cell culture [105]. Cream containing 12.5% and 25% 28 inhibited HSV-induced skin lesion formation as effectively as acyclovir treatment [106], and another study reported that cream containing 19% 28 inhibited HSV-induced vaginal lesion formation throughout a 9-day assay period and increased mortality rates in SKH1 mice [107]. The compound also inhibited NF-kB activation in HSV infected cells at a concentration of 219 μM, and the inhibition of viral replication was reversible and dose-dependent, which also impaired the activity of IE, E, and L genes as well as the viral DNA synthesis [108]. Oxyresveratrol, a derivative of 28, exerted inhibitory effects against HSV-1 7401H and acyclovir-resistant strains by inhibiting the E and L phases of both HSV-1 and 2 [109]. A synergistic effect was observed when oxyresveratrol was combined with acyclovir to inhibit late protein synthesis. The IC50 values of oxyresveratrol were 19.8, 23.3, 23.5, 24.8, 25.5, and 21.7 μg/mL against three clinical isolates, TK-deficient and PAA-resistant HSV-1, respectively. In mice, topical treatment with oxyresveratrol delayed HSV cutaneous infection and decreased mortality rates better than oral administration. Compound 28 inhibited VZV major transcriptional regulatory protein (IE62) synthesis with EC50 values of 4 and 19 μM for acyclovir and 28 treatments, respectively [111]. It also inhibited 12-O-tetradeca-  formation. The results were determined by decreasing viral DNA copies of HCMV clinical strains (CH19 and BI6) in human foreskin fibroblast (HFF) cells.

Anthraquinones and Anthracyclines with Potential Antiherpetic Activities
Natural products derived from different medicinal plants were obtained and tested with low toxicity for antiherpetic activities. The chemical structures of two anthraquinones and three anthracyclines possessing antiviral activity are shown in Figure 5. Anthraquinones are a class of aromatic compounds demonstrating a broad spectrum of bioactivities. Emodin (32) and aloe-emodin (33) are active ingredients in different Chinese herbs including Aloe vera, Cassia occidentalis, Rheum palmatum, and Polygonam multiflorumto and possesses broad pharmacological effects, such as antibacterial, antiviral, antiinflammatory, anticancer, neuroprotective, hepatoprotective, and anticardiovascular effects [134][135][136][137][138][139]176,177]. At a concentration of 50 μg/mL, 32 inhibited HSV-1 and 2 in Hep-2 cells with antiviral indices of 2.07 and 3.53, respectively, and the compound with equivalent antiviral efficacy to acyclovir also increased the survival rate and time of HSV-in-  Figure 5. Anthraquinones and anthracyclines with potential antiherpetic activities. compounds demonstrated efficient lytic induction of KSHV compared with sodium butyrate.

Miscellaneous Compounds with Potential Antiherpetic Activities
Natural products derived from different medicinal plants have been obtained and tested with low toxicity for antiherpetic activities. The chemical structures of twelve miscellaneous compounds possessing antiviral activity are shown in Figure 6. Allicin (37) from fresh garlic extract displayed activity against a wide range of viruses, including enveloped and non-enveloped viruses [93,179]. The antiviral activity of compound 37 and its derivatives was pre-clinical and clinically investigated in a previous Artemisinin-egonol (38) Artemisinin-homoegonol ( Figure 6. Miscellaneous with potential antiherpetic activities. [58] 4 Biliverdin (4) A chicken bile pigment HHV-6 (HST) MT-4 -Anti-HHV-6 activity in vitro during viral adsorption.

Induced cell death and ROS
generation.

Blocking Viral Adsorption and Entry
During viral attachment, herpesviral entry is mediated by t h e interaction of multiple glycoproteins and binding receptors on the cell surface to facilitate capsid penetration. Glycoprotein C (gC) plays a vital role in terms of viral attachment in HSV by interacting with heparan sulfate carbohydrates on the host cell surface [ 90] . Glycoprotein D (gD) binds to Nectin-1, herpesvirus entry mediator (HVEM), and a modified form of heparan sulfate receptors, which triggers membrane fusion together with heterodimers (gH-gL) and gB [1]. Viral glycoproteins could be vulnerable to mutations causing resistance to viral entry by altering interactions with their receptors [91]. Natural compounds have been proven to have better inhibitory effects than conventional drugs without showing major cytotoxic effects in blocking viral adsorption and entry from drug-resistant strains in vitro. Compounds, including berberine (3), biliverdin (4), baicalin (9), and epigallocatechin-3-gallate (29), are described in Section 5. The possible mechanism of action of these compounds showed in Figure 7. Further investigation is required to clearly understand the mechanisms of action.

Inhibition of Viral Replication
The Viral enzyme DNA-polymerase is the major target of many drug-resistant strains in herpesviruses. Numerous antiviral compounds from natural sources can be effectively used against viral DNA replication without resistance in vitro. Cytarabine (5), tricin (8)

Alkaloids with Potential Anti-Herpetic Activities
Natural products derived from different medicinal plants were obtained and tested with low toxicity for antiherpetic activities. The chemical structures of six alkaloids possessing the antiviral activity are shown in Figure 1.
The isoquinoline alkaloid berberine (3), isolated from plants including Berberis vulgaris, Coptis chinensis, Hydrastis canadensis, and Rhizoma coptidis, and its derivatives demonstrated antiviral effects against alpha-, beta-, and gamma-herpesviruses [49][50][51][52][53][54][55][56][57][58]. Then, 3 inhibited HSV-1 and 2 replications with EC 50 values of 6.77 and 5.04 µM respectively, inhibited HSV immediate-early (IE) and early (E) gene expression, suppressed HSV-2-induced c-Jun N-terminal kinase (JNK) and NF-κB activation, and downregulated HSV-2-induced interleukin-8 (IL-8) and TNF-expression [49]. The combination index (CI) of 3 and acyclovir was 0.814, demonstrating a synergistic effect. Another study found that 3 inhibited HSV-1 and HSV-2 adsorption with 93.2 and 93.9% inhibition, respectively, and late gene products, gB and gE via Western blot analysis [50]. The IC 50 values were 8.2 × 10 −2 and 9.0 × 10 −2 mg/mL against HSV-1 and 2, respectively, along with a CC 50 of 13.2 mg/mL and a selectivity index (SI) of 147-161. No inhibitory effects were observed on viral penetration even at 10 times the concentration of IC 50 . The mechanisms targeting EBV and associated malignancies in cell culture and animal models have been reviewed [149]. Additionally, 3 demonstrated inhibitory effects of latent and lytic replication on EBV-positive NPC cells [53]. The IC 50 was 101.3 and 56.7 µM for HONE1 cells and 124.5 and 43.1 µM for HK1-EBV cells when treated at 24 and 48 h, respectively, and at the concentration of 50 µM, 3 induced cell cycle arrest and apoptosis and reduced EBNA1 expression and the STAT3 signaling pathway in EBV-positive cells. Tumor formation was inhibited in NOD/SCID mice. Studies on EBV efficacy of 3 were investigated on tumor growth inhibition via the MAPK/ERK signaling pathway in nude mice and on reduction of cell viability via the p53-related signaling pathway targeting XAF1 and GADD45α expressions via the mitochondria-dependent pathway [55,56]. As a broad-spectrum inhibitor, 3 has antiviral properties against several HCMV strains, and 3 dose-dependently inhibited the replication of the HCMV AD-169 strain with EC 50 , CC 50 , and SI values of 2.65 µM, 390 µM, and 147, respectively, and fully inhibited the replication of the HCMV drug-resistant strain with mutations in the UL54 gene conferring cross-resistance to ganciclovir, cidofovir, foscarnet, and acyclovir [57]. Then, 3 exhibited a clear reduction early (UL44) and late (UL99) viral protein levels and efficiently inhibited viral IE-2 protein transactivating functions. Therefore, the progression of the HCMV replication cycle was impaired.
Biliverdin (4), a bile pigment from chickens, has been reported for its anti-HHV-6 activity in vitro [59], and 10 µg/mL of 4 inhibited HHV-6 HST strain replication in MT-4infected cells only during viral adsorption, but treatment with 40 µg/mL of 4 after 3 h of viral infection demonstrated no inhibitory effect. The inhibitory effects of 4 on HSV-1 or HCMV were not observed.
Cytarabine (5) derived from Cryptotheca crypta was clinically approved by the FDA in 1969 and has been used for treating meningeal leukemias, lymphomas, and recurrent embryonal brain tumors in clinical trials [150]. In our scope of review, 5 has been used as a potent inhibitor of KSHV-induced PEL [60], and it inhibited the active KSHV replication and virion production of various PEL cells by inducing cell cycle arrest and apoptosis, inhibited host DNA and RNA syntheses, induced cellular toxicity, and induced the rapid degradation of KSHV major latent protein LANA, which is essential for replication and persistence of KSHV episomes. Additionally Frigocyclinone (6) isolated from Streptomyces griseus strain NTK 97 played a significant role in antibacterial and antitumor activities as well as a potent inhibitor of KSHV [61]. Molecular docking was performed to predict whether the compound possessed potential drug-like properties. Computational investigation of 6 exhibited the best inhibitory constant effect of 607.94 nM against KSHV LANA1 with an intermolecular energy and electrostatic energy of −8.89 and +0.04 Kcal/mol, respectively. The amino acid residues LYS1030, PRO1033, PHE1037, LYS1070, TRP1122, HIS 1126, and LEU1128 formed stable hydrogen bond interactions on the surface of the LANA1-frigocyclinone complex. Hence, 6 can be a good lead compound against molecular dynamics simulations, and further experiments are needed to achieve the best activity.

Flavonoids with Potential Anti-Herpetic Activities
Natural products derived from different medicinal plants have been obtained and tested with low toxicity for antiherpetic activities. The chemical structures of twelve flavonoids possessing antiviral activity are shown in Figure 2.
Several Kaempferia species, including K. parviflora, K. pulchra, and K. galanga, have been traditionally used for the treatment of cancer, obesity, HIV, inflammation, and other pharmaceutical activities [151]. The black rhizome extract of Kaempferia parviflora was found to contain 5-hydroxy-3,7-dimethoxyflavone (7) [62]. HCMV protease is responsible for capsid assembly, which is an essential process of viral production [152]. The IC 50 of the Thai health-promoting herb against HCMV protease was 250 µM, and more than 70% of the enzymes was inhibited by 7 at a high concentration of 200 µg/mL. The methanolic extract inhibited HCMV protease more effectively than the aqueous extract.
Baicalin (9), isolated from a classic traditional Chinese herbal formula, called Huanglian-Jie-Du-Tang (HLJDT) exhibited anti-ischemic stroke, neuroprotective, and antioxidative effects [154]. The anti-HSV-1 and 2 activities of 9 were found in Plantago major with a CC 50 of 41.1 µg/mL and EC 50 of more than 50 and 61.5 µg/mL, respectively [68]. Another study reported only anti-HSV-1 activity in a cytopathic effect inhibition assay with CC 50 , EC 50 , and SI values of 1000 µM, 5 µM, and 200, respectively [69]. Furthermore, 9 extracted from the root of the traditional Chinese herb Scutellaria baicalensis inhibited HHV-6 GS strain proliferation and adsorption with a maximum concentration of 2.8 mg/mL without any toxicity to the human T-cell strain HSB2 in vitro [70].
Apigenin (11), 4 5 7-trihydroxyflavone, was first isolated from Caesalpinia decapetala and reported to have antitumor activities [157]. The compound was also found in Ocimum basilicum and displayed anti-herpetic activity [73]. The extract containing 11 demonstrated anti-HSV-1 and 2 with EC 50 values of 6.7 and 9.7 mg/L, SI values of 9.0 and 6.2, and CC 50 values of 59.9 mg/L, respectively. Compound 11 exhibited anti-HSV-1 and 2 effects by decreasing viral progeny production with EC 50 values of 7.04 and 0.05 µg/mL in clinical strains when using chloroform as the solvent [74]. It was also inhibited with an EC 50 of 2.33 µg/mL in the acyclovir-resistant strain. The cold lyophilized aqueous extract from the leaves of Punica granatum containing 11 exhibited superior anti-VZV activity compared with the ethanolic extract in inhibiting the VZV induced cytopathic effect at minimum inhibitory concentrations (MICs) of 15.625 and 31.25 µg/mL for the clinical isolate chickenpox and human epithelial diploid (HEp-2)-infecting zoster cells, respectively [75]. The results demonstrated that the extract containing 11 had the highest binding affinity against the VZV protease, which is involved in the capsid assembly and DNA packaging of the virus with an energy of −318.299 kcal moL −1 , forming hydrogen bonds with Gly146, Leu212, Arg148, and Arg147 with a bond length between 1.9 and 3.4 Å, which suggested the potential antiviral activity of the phytochemical compound due to interaction with the VZV protease. After EBV reactivation, the IE genes Zta and Rta are expressed, and then E, L, and virion release subsequently occur. At concentrations of 20 and 50 µM, 11 completely inhibited EBV lytic proteins including Zta, Rta, EAD, and DNase, and dose-dependently blocked EBV virion production in HA and EBV-positive NPC cell lines [76]. It also inhibited EBV reactivation by suppressing EBV IE promoter Zp and Rp activities, which were induced by Zta and Rta, and demonstrated inhibitory effects against the HCMV Towne strain in colorimetric and titer reduction assays with IC 50 values of 22 and 6.4 µM, respectively [77]. Finally, 11 dose-independently induced apoptosis and autophagy, activated p53, reduced ROS production, and reduced STAT3 phosphorylation in KSHV PEL cells [78].
Apigenin-7-O-[β-D-apiofuranosyl (1→6)-β-D-glucopyranoside] (12) was first isolated from the aerial parts of Crotalaria podocarpa using methanol extract [158], and luteolin-7-O-β-D-glucopyranoside (13) was isolated as an antioxidant from the butanol fraction of the methanol-water-acetic acid extract of Marrubium vulgare [159]. Later, both bioactive constituents displayed anti-herpetic activity and were found in the plant, Lindernia crustacea in the Scrophulariaceae family [79]. Both compounds exhibited a significant inhibitory effect on the EBV lytic protein Rta, a transcription factor during the IE stage that is required for the development of the EBV lytic cycle, at 20 µg/mL in the immunoblot analysis. The anti-EBV effect was evaluated in an EBV-containing Burkitt's lymphoma cell line (P3HR1), and the extraction of pure compounds using ethyl acetate as a solvent exhibited the most potent anti-EBV activity.
The root extracts of Scutellaria baicalensis are one of the most popular traditional Chinese medicines containing wogonin (14), 5,7-dihydroxy-8-methoxyflavone as the major bioactive compound, and possess numerous pharmacological bioactivities, including anticancer, antiinflammation, antibacterial, and antiviral activities [160]. HSV glycoprotein D (gD) is the structural component of the viral envelope and L gene product, and the reduced levels of HSV gD suggest that 14 demonstrates anti-HSV effects [80]. The compound blocked both HSV-1 and HSV-2 viral replication by suppressing IE gene expression and interfering with E and L gene expression in vitro. NF-κB is a signaling pathway, that is required for preventing host cell apoptosis during the early stage of HSV infection, and it is an important pathway for certain pathogens to invade and infect, as well as the JNK/p38 MAPK signaling pathway. When these pathways are activated, viral mRNA transcription and DNA replication are expressed. The results showed that HSV significantly and dosedependently inhibited HSV-induced NF-κB and JNK/p38 MAPK activation, which is an important pathway for viral replication. Hence, the compound reduced viral mRNA transcription, viral protein synthesis, and infectious virion particle titers. Moreover, 10 µg/mL 14 reduced VZV ORF4 gene expression, inhibited VZV ORF63 (IE) and ORF14 (L) genes at 48 and 72 hpi, induced type 1 interferon (IFN-1) signaling, inhibited inflammation in VZV-infected cells, and inhibited adenylate-activated protein kinase (AMPK) activity [81]. Then, 14 dose-dependently demonstrated antiproliferative effects, induced apoptosis at the G1 phase, and inhibited NF-κB activity in Raji cells. The compound also attenuated the average tumor size and weight in BALB⁄c nude mice [82]. The mice received intraperitoneal injection of 14 with a minimal toxic concentration of 8 mg/kg every 2 days for 2 weeks. The average sizes of the control group and 14-treated group were 663.4 and 199 mm 3 , respectively, and the average tumor weight of the control group was 0.426 g compared to 14-treated groups at 0.162 g. Hence, these results demonstrated significant inhibitory effects of tumor growth.
Morus alba L. known as white mulberry, was originally used in traditional Chinese medicine for treating bacterial infection, inflammation, and viral and other various diseases [83]. After purification of the root bark of the mulberry tree, the flavonoids kuwanon C (15), T (16), E (17), and U (18) [83]. Compound 17 was strongly bound to the active site of the HSV-2 protease with a binding affinity of −7.1 kcal/mol. These results suggested that the compounds inhibited the enzyme by forming hydrogen bonds and interacting hydrophobically with significant residues of the active site.

Terpenoids with Potential Anti-Herpetic Activities
Natural products derived from different medicinal plants were obtained and tested with low toxicity for antiherpetic activities. The chemical structures of seven terpenoids possessing the antiviral activity are shown in Figure 3.
The root of a Kenyan medicinal plant named Maytenus heterophylla containing pristimerin (19) was reported to have the first time for anti-HCMV activity [84]. The triterpenoid quinone methoide compound using cold diethyl ether methanol as a solvent was found to be effective against HCMV in immunocompromised hosts. The IC 50 , CC 50 , and SI were 0.53, 14.8, and 27.9 µg/mL, respectively [84]. The results demonstrated that 19 suppressed viral replications without virucidal effects on MRC-5 cells, inhibited ganciclovir (GCV)-sensitive clinical isolate 91-7S and GCV-resistant clinical isolate 93-1R strains in a dose-dependent manner, and significantly reduced IE1 and IE2 protein synthesis in MRC-5-infecting cells at concentrations of 1.3 and 6.5 µg/mL.
The root of the traditional Chinese herb, Glycyrrhiza glabra, containing a triterpenoid compound called glycyrrhizin (20) (also known as glycyrrhizic acid or glycyrrhizinic acid), and its derivatives, exerts inhibitory effects on hepatitis C virus (HCV), HSV-1, influenza virus, and severe acute respiratory syndrome associated coronaviruses (SARS-COV) [161]. Several studies have reported that glycyrrhetinic acid, the product of body metabolism from 20, and other derivatives, demonstrated anti-HSV in vitro [85][86][87][88]162]. Then, 20 dose-dependently inhibited VZV and EBV replication with IC 50 values of 0.71 and 0.04 mM for viral inhibition and with IC 50 values of 21.3 and 4.8 mM for cell growth mainly at the early stage but not during adsorption [89]. The therapeutic index (TI) was 30 and 120 against VZV and EBV replication, respectively. Moreover, seven derivative compounds from 20 dose-dependently inhibited EBV infection, and three of the seven compounds containing the amino acid residues in the carbohydrate part of 20 exerted antiviral activity [90]. SUMOylation processes have been proposed as therapeutic targets of antiviral activity [91]. Firstly, 20 inhibited SUMOylation processes in LMP1-expressing cells without affecting ubiquitination processes, induced apoptosis in multiple cell lines, blocked proliferation, and reduced viral production without inducing viral reactivation. Moreover, 20 decreased KSHV latency transcription, disrupted the RNA polymerase 2 (RNAPII) complex, altered the enrichment of the RNAPII-pausing complex, and reduced the interaction between cohesion subunits, SMC3, RAD21, and SPT5 together with RNAPII. The compound abrogated RNAPII pausing at the intragenic CTCF-cohesin binding sites, resulting in a reduction in proper mRNA production and defects in sister chromatid cohesion [92]. Then, 20 demonstrated inhibitory effects against both lytic and latent KSHV in KSHV latently infected primary effusion lymphoma (PEL)-derived BC-3 cells in vitro [93]. The maximum concentration of 960 µM of the compound did not show major effects on cell viability, and the concentration of 480 µM exhibited the most potent effect on the reduction of virion DNA. It also downregulated the expression of latency-associated nuclear antigen (LANA) and upregulated viral cyclin (vCyclin) expression [94].
Triptolide (21), a diterpenoid triepoxide isolated from Tripterygium wilfordii, inhibited the proliferation of EBV-positive B lymphoma cells by downregulating latent membrane protein 1 (LMP1) transcriptional expression in BALB/c male mice [95]. The function of LMP1 has been reported to promote the proliferation of B lymphocytes, increase viral genome production, and induce viral release during lytic infection [163,164]. LMP1 promoter activity during EBV latency III infection was reduced by treatment with 21 in malignant human epithelial cervical cancer cells (HeLa) in vitro. The compound strongly reduced the growth of EBV-positive B lymphoma cells (B95-8 and P3HR-1) in BALB/c male mice as viral LMP1 mRNA expression was decreased. Moreover, 21 effectively inhibited proliferation, induced mitochondrial apoptosis, and decreased EBNA1 expression with low toxicity in EBV-positive and EBV-negative NPC cells, with IC 50 values of 55.43, 76.56, 1.12, 11.04, and 10.66 for C666-1, HONE1/Akata, HK1/Akata, CNE1/Akata, and CNE1 cells, respectively [96]. The 0.4 mg/kg compound also inhibited tumor growth, as suggested by its weight and volume, decreased EBNA1 expression, and increased the expression of associated apoptotic proteins. In addition, 200 nM 21 decreased the hTERT mRNA, SP1 and c-Myc expression in B95-8 and P3HR-1 cells [97]. The activity of hTERT promoter and cell proliferation were inhibited by 21, and it decreased the latency-associated nuclear antigen 1 (LANA1), which is essential for segregation, replication, and maintenance of the viral genome, and suppressed STAT3 activity and secretion of pivotal cytokines (IL-6), which plays a role in promoting the survival of KSHV-associated primary effusion lymphoma (PEL) cells [98]. Treatment with 21 dose-dependently inhibited the formation of malignant ascites and lymphomatous effusions caused by BC-3, BCBL-1, JSC-1, and BJAB cells in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.
Phytol (22), an acyclic diterpene alcohol, was one of the most significant constituents found in the essential oil of Yimucao from the herb Leonurus japonicus Houtt. [165]. This Chinese herb has been widely used to treat gynecological problems and possesses antibacterial activity against various gram-negative bacteria. Compound 22 has anti-inflammatory properties by downregulating the p38MAPK and NFκB signaling pathways [166]. Although this might link to our scope of review, no studies concerning herpetic activity has been conducted to confirm these pathways. Additionally, 22 isolated from Lindernia crustacea demonstrated an inhibitory effect of EBV lytic protein Rta at 20 µg/mL in P3HR1 cells [79].
Cypellocarpin C (24), the monoterpenoid ester extracted from the dried leaves of Eucalyptus cypellocarpa, which is commonly known as gray gum in Australia was first reported to have a structure according to spectroscopic methods to determine its antitumorpromoting effect [168]. Further study revealed that 24 was abundant in the leaves of the genus Eucalyptus [169]. Eucalyptus globulus was selected and it was found that 24 exhibited more than twice stronger anti-HSV-2 activity with an EC 50 and SI of 0.73 µg/mL and >287.7 compared to acyclovir with an EC 50 and SI of 1.75 µg/mL and >120 without showing cytotoxic effects as both have CC 50 > 210 µg/mL [104]. Furthermore, tereticornate A (25), the triterpene ester extracted from the dried leaves of Eucalyptus tereticornis was first isolated and identified for its structure [170]. The anti-HSV-1 activity of 25 compared to acyclovir showed a promising result with IC 50 values of 0.96 and 1.92 µg/mL, SI > 218.8 and >109.4, and CC 50 > 210 µg/mL, respectively, in Vero cells. It also inhibited NF-κB activity more strongly than prednisone, which is a routinely used anti-inflammatory drug. Both compounds require further research regarding their antiherpetic activity.

Polyphenols with Potential Anti-Herpetic Activities
Natural products derived from different medicinal plants were obtained and tested with low toxicity for antiherpetic activities. The chemical structures of six polyphenols possessing the anti-viral activity are shown in Figure 4.
Cis/trans-martynoside (26) and cis/trans-isomartynoside (27) were first isolated from Martynra Louisiana, and the structures were identified using low-pressure liquid chromatography, NMR spectra, and electron impact mass spectrometry [171]. They were later isolated from Lindernia crustacea and displayed a significant inhibitory effect on the EBV Rta lytic cycle at 20 µg/mL via the immunoblot analysis. Using ethyl acetate to extract pure compounds showed the most potent anti-EBV activity [79].
Resveratrol (28), trans-3,5,4-trihydroxystilbene was found in several plants, including grape vines, berries, pines, pomegranates, peanuts, legumes, and soybeans with various mechanisms of action against different human and animal viruses [105][106][107][108][109][110][111][112][113][114][115][116][117][118]172]. Compound 28 and its derivatives demonstrated anti-HSV activity both in vivo and in vitro [173], it dose-and time-dependently inhibited both HSV-1 and HSV-2 replications via inhibition of ICP4 (IE gene) expression, and the inhibition of viral replication was reversible in cell culture [105]. Cream containing 12.5% and 25% 28 inhibited HSV-induced skin lesion formation as effectively as acyclovir treatment [106], and another study reported that cream containing 19% 28 inhibited HSV-induced vaginal lesion formation throughout a 9-day assay period and increased mortality rates in SKH1 mice [107]. The compound also inhibited NF-kB activation in HSV infected cells at a concentration of 219 µM, and the inhibition of viral replication was reversible and dose-dependent, which also impaired the activity of IE, E, and L genes as well as the viral DNA synthesis [108]. Oxyresveratrol, a derivative of 28, exerted inhibitory effects against HSV-1 7401H and acyclovir-resistant strains by inhibiting the E and L phases of both HSV-1 and 2 [109]. A synergistic effect was observed when oxyresveratrol was combined with acyclovir to inhibit late protein synthesis. The IC 50 values of oxyresveratrol were 19.8, 23.3, 23.5, 24.8, 25.5, and 21.7 µg/mL against three clinical isolates, TK-deficient and PAA-resistant HSV-1, respectively. In mice, topical treatment with oxyresveratrol delayed HSV cutaneous infection and decreased mortality rates better than oral administration. Compound 28 inhibited VZV major transcriptional regulatory protein (IE62) synthesis with EC 50 values of 4 and 19 µM for acyclovir and 28 treatments, respectively [111]. It also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr early antigen activation [112], the dose-dependently reduced the EBV lytic cycle and transcription of lytic proteins, including Rta, Zta, and EA-D expression, reduced virion production in P3HR-1 cells with an EC 50 of~24 µM [113], and inhibited the EBV-induced activation of NF-kB and AP-1 [114]. Moreover, 28 induced apoptosis, inhibited the STAT3 pathway, decreased the viral LMP1 product affecting the downregulation of survivin and Mcl-1, and decreased miR-155, miR-34a, and BHRF1 expression in EBV-infected B cells [115]. Compound 28 exhibited anti-HCMV activity by blocking IE, E, and L viral proteins with an IC 50 of 1.7 µM, CC 50 > 400 µM, and SI ≥ 50. Compound 28 also lowered ERK1/2 activity and Egr1 expression in KSHV-infected cells, resulting in the suppression of viral reactivation from latency [117], and it induced apoptosis and autophagy, increased ROS generation without causing viral reactivation, disrupted latent viral infection, inhibited lytic gene expression, and decreased progeny virus production in PEL cells [118].
Epigallocatechin-3-gallate (29), a major component of the dry leaves of Camellia sinensis, was previously suggested to have potential antiviral activity against a broad range of viruses [122,174]. In our related scope, 29 exhibited greater antiviral activity than other green tea catechins by reducing HSV-1 and HSV-2 titers by 3,000-and 10,000-fold, respectively [119]. The compound directly affected the virion, which could bind to gB, gD, or another enveloped glycoprotein. Compound 29 is stable in the pH range found in the vagina and inactivated HSV-1 and 2 at pH 8.0 by 3 log 10 to 4 log 10 . Theasinensin A, P2, and TF-3, digallate dimers of 29, had antiviral activity at pH 5.7 and 8.0 and inactivated Class I, II, and III fusion proteins of enveloped viruses [120]. p-EGCG, modified from 29 inhibited infectious HSV-1 production at a concentration of 50 µM via viral adsorption in Vero cells [121]. Compound 29 also inhibited virion surface proteins and competed with heparan sulfate binding of HSV-1 [122]. MST-312 containing moieties related to 29, dose-dependently reduced plaque formation of HSV-1 virions [123]. However, 29 exhibited virucidal activity toward HSV-1 at a concentration 80 times lower than that of MST-312, and 29 also inhibited infected cell protein 0 (ICP0), which promoted transcription of viral genes. Both compounds possessed virucidal activity against HSV virions at temperatures between 25 and 37 • C. Furthermore, 29 and acyclovir at concentrations of 25 and 50 µg/mL, respectively, increased the viability of HSV-1-induced cell death, and synergistically inhibited ICP5, TK, and gD [124]. Compound 29 inhibited the expression of EBV lytic proteins, including Rta, Zta, and EA-D, and inhibited the transcription of IE genes [125], and it dose-dependently inhibited the transcriptional activity of the BZLF1 and BMRF1 genes and suppressed the MEK/ERK1/2 and PI3-K/Akt signaling pathways involved in EBV spontaneous lytic infection in CNE1-LMP1 and B95.8 cells [126]. Compound 29 inhibited EBV spontaneous lytic infection involved in downregulation of LMP1 with an IC 50 of 20 µM [127], and it inhibited KSHV replication by inducing cell cycle arrest in the G2-M phase and apoptosis, p53 activation, autophagy, and MAPKs via ROS generation [128]. It also reduced the production of viral progeny without causing reactivation in PEL cells. Compound 29 displayed activity against lytic KSHV infection without major effects on cell viability at 100 µM while at a concentration of 5 µM, the control group with 100% relative KSHV virion DNA was reduced to 30%.
Ginkgolic acid (31), a 2-hydroxy-6-alkyl benzoic acid, is an alkylphenol constituent of the leaves and fruits of Ginkgo Biloba, and it is most experimentally used to date C15:1. An extract containing < 5 ppm 31 demonstrated anti-HSV-1 and -2 activities before viral adsorption to the cell surface, disrupted the viral structure, and inhibited HSV-1 virion entry without cytotoxicity [131]. At a concentration of 50 µM, 31 blocked downstream HSV-1 protein synthesis, such as the production of HSV-1 immediate early (ICP27), early (ICP8), and late (US11) proteins [133]. It significantly reduced mortality, infection score, and durations of HSV-1 cutaneous infection in a zosteriform model in BALB/cJ mice in vivo and inhibited an acyclovir-resistant strain of HSV-1 in Vero cells [132]. Moreover, 31 inhibited HCMV DNA synthesis in a dose-dependent manner and prevented plaque formation. The results were determined by decreasing viral DNA copies of HCMV clinical strains (CH19 and BI6) in human foreskin fibroblast (HFF) cells.

Anthraquinones and Anthracyclines with Potential Antiherpetic Activities
Natural products derived from different medicinal plants were obtained and tested with low toxicity for antiherpetic activities. The chemical structures of two anthraquinones and three anthracyclines possessing antiviral activity are shown in Figure 5.
Anthracyclines are a class of drugs produced by different wild type Streptomyces bacterium strains, used in cancer chemotherapy [178]. Three FDA-approved drugs, daunorubicin (34), doxorubicin (35), and epirubicin (36), are topoisomerase II inhibitors that are capable of intercalating DNA and inducing cytotoxic effects in cancer cells [140]. At concentration of 10 µM, all three compounds induced effective luciferase expression under control of the PAN or RTA promoters of KSHV infecting BCBL1 and Vero cells. All three compounds demonstrated efficient lytic induction of KSHV compared with sodium butyrate.

Miscellaneous Compounds with Potential Antiherpetic Activities
Natural products derived from different medicinal plants have been obtained and tested with low toxicity for antiherpetic activities. The chemical structures of twelve miscellaneous compounds possessing antiviral activity are shown in Figure 6.
Allicin (37) from fresh garlic extract displayed activity against a wide range of viruses, including enveloped and non-enveloped viruses [93,179]. The antiviral activity of compound 37 and its derivatives was pre-clinical and clinically investigated in a previous review [180]. In our review, we found that compound 37 exhibited antiviral activity against both latent and lytic KSHV, but the former was more effective than the latter. Compound 37 at a concentration of 15 µM significantly reduced the virion DNA in KSHV. The compound demonstrated maximum tolerable drug concentrations without major effects on cell viability at a very low concentration of 6 µM.
Byzantionoside B (40) was first isolated from Stachys byzantina as the C-9 epimer of blumenol C glucoside [189], and the absolute configurations have been determined [190]. The compound was later found in Lindernia crustacea and demonstrated significant inhibitory effect on the EBV lytic cycle at 20 µg/mL in the immunoblot analysis. Compound 40 was extracted using ethyl acetate to exhibit the most potent anti-EBV activity [79].

Mechanisms of Action
Natural products can target different stages of herpesviral life cycles, which are summarized and shown in Figure 7.

Blocking Viral Adsorption and Entry
During viral attachment, herpesviral entry is mediated by the interaction of multiple glycoproteins and binding receptors on the cell surface to facilitate capsid penetration. Glycoprotein C (gC) plays a vital role in terms of viral attachment in HSV by interacting with heparan sulfate carbohydrates on the host cell surface [90]. Glycoprotein D (gD) binds to Nectin-1, herpesvirus entry mediator (HVEM), and a modified form of heparan sulfate receptors, which triggers membrane fusion together with heterodimers (gH-gL) and gB [1]. Viral glycoproteins could be vulnerable to mutations causing resistance to viral entry by altering interactions with their receptors [91]. Natural compounds have been proven to have better inhibitory effects than conventional drugs without showing major cytotoxic effects in blocking viral adsorption and entry from drug-resistant strains in vitro. Compounds, including berberine (3), biliverdin (4), baicalin (9), and epigallocatechin-3-gallate (29), are described in Section 5. The possible mechanism of action of these compounds showed in Figure 7. Further investigation is required to clearly understand the mechanisms of action.

Inhibition of Viral Replication
The Viral enzyme DNA-polymerase is the major target of many drug-resistant strains in herpesviruses. Numerous antiviral compounds from natural sources can be effectively used against viral DNA replication without resistance in vitro. Cytarabine (5), tricin (8) Figure 7. Further investigation is required to clearly understand the mechanisms of action.

Inhibition of NF-κB Activity
An inactive form of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) dimers is localized in the cytoplasm by binding to inhibitors of κB (IκB), and after degradation of this inhibitory protein causes translocation (during early stage of infection) of the dimers into the nucleus, NF-κB regulates the expression of numerous genes involved in apoptotic, inflammatory, and immune responses [101]. NF-κB also induces the expression of various proinflammatory genes, including cytokines and chemokines [102]. The activation of NF-κB synthesizes numerous antiapoptotic factors, which are required for preventing host cell apoptosis [103]. Based on the evidence, the compound inhibiting NF-κB activation or translocation may interfere with viral replication via programmed cell death, including berberine (3), wogonin (14), and resveratrol (28), which are described in Section 5. The possible mechanisms of action of these compounds are shown in Figure 7. Further investigation is required to clearly understand the mechanisms of action.

Compounds Affecting Viral Replication by Other Mechanisms
Natural compounds affect antiviral activity via different pathways. Berberine (3) inhibited tumor growth via the MAPK/ERK signaling pathway in nude mice and via the p53related signaling pathway and its downstream molecules targeting XAF1 and GADD45α expression via a mitochondria-dependent pathway [55,56]. Glycyrrhizin (20) inhibited small ubiquitin-like modifier (SUMO)-ylating processes as a novel therapeutic target of antiviral activity [91]. During the latency of EBV infection, LMP1 increased protein SUMOylation, aiding tumorigenesis. Then, 20 blocked cell proliferation, increased cell death without inducing viral reactivation, and impeded viral production in infected cells. Carvacrol (23) exerted anti-HSV-2 activity via inhibition of the RIP3-mediated programmed cell necrosis pathway and exhibited decreased ubiquitination levels in the ubiquitin-proteosome system, which was caused by HSV-2 infection [102]. Toll-like receptor 3 (TLR3) plays a significant role in immune responses to herpesviruses. After viral stimulation, TLR3 subsequently transmitted signals to TAK1 via TRAF6, TRADD, p38, and NF-κB, which produced the expression of inflammatory factors [136]. Emodin (32) suppressed the TLR3 pathway and its downstream molecules, trif, TRADD, traf3, TRAF6, Nemo, p38, and IRF3, as well as f IL-6, TNF-α, and IFN-β expression in HSV-infected brain tissues of mice [136]. Further investigation is required to clearly understand the mechanisms of action.

Efficacy of Natural Compounds In Vivo
Numerous natural compounds and extracts with antiherpetic activity have been analyzed in vitro. These results can be further investigated to evaluate their efficacy in living organisms via in vivo studies. However, only berberine (3), wogonin (14), triptolide (21), resveratrol (28), ginkgolic acid (31), and emodin (32) have been tested in both cell culture experiments and animal studies. The results are summarized in Table 1 and discussed in Section 5. Further investigation is required to clearly understand their mechanisms of action.

Summary and Additional Comment
Nucleoside analog drugs, including acyclovir, ganciclovir, valaciclovir, and other related prodrugs, are used as the current standard therapy for herpesvirus infection. However, after frequent and long-term treatment, drug resistance and undesirable effects occur that limit the available treatment options especially in immunocompromised patients. Hence, the search for newly developed, safe, and effective antiviral drugs is clearly needed. Most medicinal plants are promising sources of new antiviral compounds. In this review, natural compounds isolated from plants, animals, fungi, and bacteria are summarized with different antiviral mechanisms affecting the herpesviral surface, viral adsorption, entry, replication, capsid assembly, and release. The compounds are classified into alkaloids, flavonoids, terpenoids, polyphenols, anthraquinones, anthracyclines, and miscellaneous compounds. Compounds, including the alkaloid berberine (3), the flavonoid deguelin (10), the terpenoid pristimerin (19), the phenolic ginkgolic acid (31), and the polysaccharide Cucumis melo sulfated pectin (SPCm), exhibited better antiherpetic activity than conventional drugs in vitro [57,72,84,132,192]. These candidates were further investigated for alternative treatment of drug-resistant viruses. For example, 3, 10, and 19 can effectively inhibit ganciclovir-resistant strain in HCMV [57,72,84]: 3 fully inhibited the HCMV replication in ganciclovir-resistant strain due to a mutation in a DNA polymerase catalytic subunit (UL54 gene); 10 can also inhibit the drug-resistant HCMV strain at nanomolar concentration; and 19 can suppress both the ganciclovir-resistant (93-1R) and sensitive strains (91-7S) in HCMV replication. Both 31 and SPCm inhibited an acyclovir-resistant HSV-1 strain (17+ and AR-29, respectively), but the latter also inhibited the acyclovir-sensitive HSV-1 strain (KOS).
A few compounds, including berberine (3), wogonin (14), triptolide (21), resveratrol (28), ginkgolic acid (31), and emodin (32), have been evaluated for their efficacy against viral infection in vivo. For example, 3 inhibited tumor formation in NOD/SCID mice via the MAPK/ERK signaling pathway and reduced cell viability through the p53-related signaling pathway by targeting XAF1 and GADD45α expression via a mitochondria-dependent pathway [55,56]. Treatment with 14 attenuated the average tumor size and weight in BALB⁄c nude mice [82]. Then, 21 can efficiently reduce the growth of B95-8-induced B lymphoma and downregulate LANA1 expression, and viral replication in PEL cells in mice [95,96,98]. Topical application of 28 inhibited HSV-induced skin lesions and increased survivability in mice [106,107]. The mortality rate was decreased by treatment with 31, and the overall infection score and average time to death of HSV-1 cutaneous infection in female BALB/cJ mice was reduced comparing to the DMSO control group in both HEC gel and PEG [132]. Finally, 32 suppressed HSV-infected brain, heart, liver, and ganglion tissues, increased survival rate, and inhibited EBV-induced tumor growth in mice [135,136,138]. These results show that these compounds can be potential candidates for future clinical trials, and further investigations are required to compare these compounds with the standard therapy of herpesviral infection in clinical trials and in vivo studies.

Conclusions
Human herpesvirus is known to establish lifelong latent infection. Until now, no prophylactic herpesvirus vaccine has been found to be effective and completely eradicated the viral infection. In this review, we have summarized the findings of previous literature regarding natural compounds derived from medicinal plants, animals, and bacteria with promising antiviral activity against herpesvirus infections in both cell culture and animal models. Most natural compounds are briefly demonstrated as safe and effective treatments to inhibit different types of herpesviruses, in which the IC 50 , EC 50 , CC 50 , and SI are shown in Table 1. These natural compounds can target different stages of herpesviral life cycles, which are shown in Figure 7 and discussed in Section 6. For instance, tricin (8) dose-dependently inhibited the expression of immediate early 2 mRNA, 50 µM of ginkgolic acid (31) blocked downstream HSV-1 protein synthesis by inhibiting viral entry, which is involved in the production of HSV-1 immediate early (ICP27), early (ICP8), and late (US11) proteins, and treatment with 250 nM deguelin (10) significantly reduced the expression of the viral early (pUL44) and late (IE2-60) proteins. Overall, most natural compounds in the review have been studied in vitro with various mechanisms of action based on different screening methods, which are comparatively better than nucleoside analogs. Only a few studies have investigated them in animal models. The evaluations for promising antiherpetic compounds in animal trials and further clinical trials as alternative therapies against herpesvirus infections in humans would be still needed in the future.