R. tanguticum were collected from some high mountainous areas in Qinghai provinces, China. The plant was authenticated by Prof. Keli Chen, Department of Identification and Assessment of TCM, Hubei University of Chinese Medicine (Hubei, China). A voucher specimen (RQ/20140315) was deposited in School of Basic Medical Sciences, Wuhan University. R. tanguticum nanoparticles were produced from Shenzhen Lvwa Biological Technology Co., Ltd. The dried R. tanguticum roots were grounded to produce coarse powders using medicinal herb grinder (CW700, Shandong, China). Then the coarse powders were passed through a 100 mesh sieve, mixed with a quantity of pure water and subjected to a gel grinding (JMS-240, Hebei, China) with wet grinding to the required particle size less than 50 µm. Then these mixed particles were smashed to cell breaking as nanoparticles using the high pressure homogenizer (APV-2000, Germany). Finally, the R. tanguticum nanoparticles were obtained by drying process before usage.

The content of main components of R. tanguticum nanoparticles was performed by high-performance liquid chromatography (HPLC). Chromatography analysis was performed on a Waters e2695 Alliance HPLC system equipped with an Agilent Zorbax SB-C18 column (150 mm × 4.6 mm, 3.5 µm). The mobile phase composed of A) methanol and B) 0.1% (v/v) phosphoric acid water solution was delivered at a rate of 1 ml/min. The reference marker compounds of aloe-emodin, rhein, emodin, chrysophanol, and physcion for high-performance liquid chromatography (HPLC) assay were purchased from the Shanghai Standard Technology Co., Ltd. (Shanghai, China) and the purity of all these compounds were higher than 98.0%.

R. tanguticum nanoparticles were characterized by a transmission electron microscope (TEM, HT7700, Hitachi). A drop of R. tanguticum nanoparticles suspension was placed on a carbon coated-copper grid and evaporated to form a monolayer at room temperature and then mounted on a specimen stub in the transmission electron microscope. The elemental analysis was carried out with an accelerating voltage of 80 kV.

The particle size distribution of R. tanguticum nanoparticles was measured via dynamic light scattering (DLS) technique using a Nano ZS90 Zetasizer (Malvern Instruments, Malvern, UK) at 633 nm. The zeta potential (ZP) of R. tanguticum nanoparticles was measured with the same instrument. The samples were suspended in distilled water and were analyzed in triplicate at a temperature of 25°C.

HEp-2 (human laryngeal carcinoma) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin (Gibco) and 100 µg/ml streptomycin (Gibco) at 37°C in 5% CO₂. The virus strains used were HSV-1 F gifted by Professor Rapp. F from Hershey Medical Center, USA in 1990 and stored in our laboratory ever since. The virus was propagated in HEp-2 cells and stored at −80°C for further studies. R. tanguticum nanoparticles were dissolved in (DMSO) (Sigma-Aldrich, St Louis, MO) followed by dilution in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) to prepare a stock concentration of 1 mg/ml solution.

The 50% cytotoxic concentration (CC₅₀) of R. tanguticum nanoparticles on HEp-2 cells was performed by MTT assay (Shi et al., 2007). HEp-2 cells in 96-well plates were incubated with various concentrations of R. tanguticum nanoparticles (0, 31.25, 62.50, 125, 250, 500, and 1,000 µg/ml) at 37°C in an atmosphere of 5% CO₂. After incubation, an MTT assay (Sigma-Aldrich) was performed according to the manufacturer’s protocol. The optical density at 570 nm was then determined by a conventional microplate reader (Multiskan^TM GO; Thermo Fisher Scientific, Waltham, MA). The CC₅₀ was calculated using a regression analysis.

The antiviral activity was performed using the plaque reduction assay (PRA) as previously described (Prichard et al., 1990). Briefly, HEp-2 cell monolayers seeded on 24-well plates were infected with HSV-1 (100 PFU/well). After 2 h incubation, the cells were washed twice with pre-warmed PBS and overlaid with 1% methylcellulose containing different concentrations of R. tanguticum nanoparticles as indicated. The plaques developed after 72 h of incubation were fixed with 4% paraformaldehyde for 10 min and were stained with 1% crystal violet solution. The plaques were counted and the percentages of inhibition were calculated. The effective concentration of test compound that reduced plaques number by 50% (EC₅₀) was calculated.

Time of addition assay was performed as described previously (Bertol et al., 2011). Briefly, 350 µg/ml R. tanguticum nanoparticles were added to HEp-2 cell monolayers at indicated time periods after HSV-1 infection (MOI = 0.01). After 72 h incubation at 37°C, total HEp-2 cells were collected and frozen and thawed for three times. Virus titer was determined by plaque assay.

Equal volumes of diluted virus (2.0 × 10⁵ PFU/ml) were mixed with each drug dilution and were incubated at 37°C for 1 h. The mixture was then 1,000 times diluted with fresh DMEM containing 2% fetal bovine serum (FBS) to yield a sub-therapeutic concentration of the test compound and to generate 100 PFU/ml HSV-1. The virus inocula were then added to HEp-2 cell monolayers. After adsorption for 2 h, the viral inoculum were discarded, the infected cells were washed once with PBS, and then overlaid with overlay media. Plates were incubated at 37°C for 72 h and were subjected to the plaque assay, as described previously (Lin et al., 2011).

The attachment and penetration assays were performed as described previously (Gong et al., 2002) with minor modifications. For the attachment assay, HEp-2 cell monolayers were pre-chilled at 4°C for 1 h. The medium was replaced with different concentrations of cold R. tanguticum nanoparticles and was incubated for 2 h at 4°C. Then, 100 PFU of virus was added to the cells. After infection for 2 h, the unbound virus was removed. The cells were washed twice with cold PBS and were covered with methylcellulose to allow plaque formation. For the penetration assay, HEp-2 cell monolayers were cooled to 4°C for 1 h and then incubated with 100 PFU of HSV-1 at 4°C for 2 h to allow viral adsorption. The unbound viruses were removed and HEp-2 cells were incubated with different concentrations of R. tanguticum nanoparticles at 37°C for 2 h to facilitate viral penetration. At the end of the incubation, the cells were washed with PBS, acidic glycine (pH 2.2) for 1 min, and DMEM (no serum), respectively. Then the cells were overlaid with methylcellulose for virus plaque formation.

HEp-2 cells were infected with HSV-1 at an MOI of 0.01 and were treated with R. tanguticum nanoparticles (350 µg/ml) at 6, 12, 18, and 24 h intervals, and total RNA was extracted with TRIzol reagent (Ambion^®, Life Technologies Pty Ltd., Carlsbad, CA) according to the manufacturer’s instructions. The cDNA was synthesized in a reverse-transcriptase reaction by using Thermo cDNA Synthesis Kit (Thermo Scientific, Rockford, IL). The immediate early gene α4 (encoding ICP4), early gene U _L 29 (encoding ICP8) and late gene U _S 6 (encoding glycoprotein gD) were determined by using a Bio-Rad CFX96 instrument according to a two-step protocol (95°C for 2 min, 45 cycles of 5 s at 95°C, 10 s at 60°C, and 15 s at 72°C). The transcription levels of total RNA in each sample were standardized against the GAPDH gene. The sequences of Primers used were as follows: ICP4 (5’-CGACACGGATCCACGACCC-3’ and 5’-GATCCCCCTCCCGCGCTTCGTCCG-3’);ICP8 (5’-CGACAGTAACGCCAGAAG-3’ and 5’-GGAGACAAAGCCCAAGAC-3’); gD(5’-GCCCCGCTGGAACTACTATG-3’ and 5’-TTATCTTCACGAGCCGCAGG-3’);GAPDH(5’-GGTGGTCTCCTCTGACTTCAACA-3’ and 5’-GTTGCTGTAGCCAAATTCGTTGT-3’). Relative quantification was carried out based on the 2^−ΔΔCT threshold cycle method.

The HSV-1(MOI = 0.01) infected HEp-2 cells were in the presence or absence of R. tanguticum nanoparticles (350 µg/ml). At 6, 12, 18, and 24 h.p.i., the cells were fixed with pre-chilled 4% paraformaldehyde for 30 min and were permeabilized in PBS containing 0.25% Triton X-100 for 10 min at room temperature and then rinsed thrice in PBS, following by blocked in 1% BSA for 30 min. Cells were further washed with PBS, then incubated for 2 h at 37°C with anti-ICP4 antibody (Abcam, ab6514, 1:1000) and anti-ICP8 (Abcam, ab20194, 1:500) antibody, respectively. The cells were then rinsed three times for 5 min in PBS, followed by incubation with the 488-conjugated goat anti-mouse IgG (H+L) secondary antibody (diluted 1:200) for 60 min at 37°C and counterstained with DAPI. Images were observed using a fluorescence microscope (TE2000; Nikon, Tokyo, Japan) (Roberts and Baines, 2011).

HEp-2 cells were infected with HSV-1 at an MOI of 0.01 and were incubated with or without R. tanguticum nanoparticles (350 µg/ml) at intervals of 6, 12, 18, and 24 h post-infection. Cells were harvested in RIPA Lysis Buffer (Biosharp, Hefei, China) and the soluble fraction was then clarified by centrifugation at 12,000 × g for 5 min at 4°C. Equal amounts of protein (40 µg/sample) were then isolated by 8% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a pre-equilibrated PVDF membrane (Thermo Scientific). Membranes were blocked with 5% BSA for 2 h, rinsed and followed by incubation with anti-ICP4 antibody (Abcam, ab6514, 1:1000), anti-ICP8 antibody (Abcam, ab20194, 1:500) and anti-GAPDH antibody (Tianjin Sungene Biotech KM9002T, Beijing, China, 1:5,000) in 5% BSA at 4°C overnight, respectively. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature and visualized by using an ECL Western Blot Detection Kit (Millipore Corp., Bedford, MA).

Male Kunming mice (weight: 15–17 g) were purchased from the Animal Research Center of Wuhan University (Certificate No. SCXK 2015-0018, Hubei, China). The animal study protocols were approved by the Institutional Animal Care and the Ethics Committee of Wuhan University School of Medicine. Mice were anesthetized intraperitoneally by ketamine (100 mg/kg) and injected intracerebrally with 20 μl 10-fold serial dilutions of HSV-1(HSV-1 F strain). The 50% lethal dose (LD₅₀) of virus was calculated by Reed-Muench method (Ling et al., 2012). Clinically, according to human dosage of rhubarb recommended in Chinese Pharmacopoeia (Commission, 2015), the equal doses of 3–15 g should be suggested to 0.46–2.28g/kg of mice. We set R. tanguticum nanoparticles groups with five doses of 2,500 mg/kg/day, 1,250 mg/kg/day, 625 mg/kg/day, 312.5 mg/kg/day, 156.3 mg/kg/day to determine the drug toxicity in mice. No death and obvious weight loss were seen when R. tanguticum nanoparticles were at dosages below 2,500 mg/kg/day. The daily animal doses of 312.5 and 625 mg/kg/day of R. tanguticum nanoparticles used in the study corresponded to doses of 2.06 and 4.12g of rhubarb translated from the clinical dosage for an adult human (60 kg) (Reagan-Shaw et al., 2008).

To investigate the protective activity of R. tanguticum nanoparticles against HSV-1 in vivo, the mice were randomly assigned to 5 groups and inoculated intracerebrally with 20 μl of DMEM (Gibco, Grand Island, NY) containing either no virus (vehicle only) or 5LD₅₀ of HSV-1(HSV-1 F strain). After 5 h, the inoculated mice received the following treatment: R. tanguticum nanoparticles were dissolved in Saline (0.9%) before administration to mice with the indicated doses at 625 mg/kg/day or 312.5 mg/kg/day, respectively; the positive controls received 100 mg/kg/day acyclovir. Saline (0.9%) was used in normal and viral controls. The drugs were administered via oral gavage once daily or acyclovir twice daily at 12 h intervals for five consecutive days (Quenelle et al., 2010).

For survival curve experiments, 10 mice per group were observed for mortality and weighed daily for 14 days. The protection was estimated by body weight evaluation, the survival time and the reduction of mortality. For viral titer analysis, four mice per group were sacrificed on day 5 post infection. Brains were then harvested and subsequently homogenized to 10% (w/v) suspensions in test medium. The homogenates were frozen and thawed twice to release the virus and centrifuged and the supernatant was used to test the virus titers in the HEp-2 cells by plaque assay. For histological examination, on day 5 post infection, brains of mice were removed and fixed (n = 4/group) with 4% paraformaldehyde, and then embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H&E). For gene expression and protein levels assays, brains of four mice per group were collected on day 5 post infection and total RNA was extracted from brain tissues with TRIzol reagent following the manufacturer’s instructions. The expression of two viral genes (ICP4 and ICP8) was determined using RT-qPCR. Furthermore, the ICP4 proteins of the treated and untreated mice with virus infection were analyzed using Western blot as previously described with minor modifications (Menasria et al., 2013).

Data were presented as the mean ± standard deviation (mean ± SD) and analyzed with the GraphPad Prism software v.5 (GraphPad Software Inc., La Jolla, CA). Statistic differences between groups were determined by one-way analysis of variance (one-way ANOVA) with Bonferroni’s multiple comparison tests. The probability of the mouse survival was estimated using Log-rank test. A value of p < 0.05 was considered significant.

To determine the main chemical profile of the R. tanguticum nanoparticles, the five bioactive constituents of R. tanguticum nanoparticles, the aloe-emodin, rhein, emodin, chrysophanol, and physcion were quantified using HPLC in comparison with standard reference compounds ( Figure 1 ). The concentrations of aloe-emodin, rhein, emodin, chrysophanol, and physcion in R. tanguticum nanoparticles were 0.226, 0.443, 0.503, 0.853 and 0.252%, respectively. The result revealed that the content of the five anthraquinone compounds in R. tanguticum nanoparticles was in accord with the quality standard of Chinese Pharmacopoeia (Commission, 2015).

The morphology and size of R. tanguticum nanoparticles were characterized by using TEM and DLS techniques. Transmission electron microscopy (TEM) images showed the R. tanguticum nanoparticles distributed in spherical shape, with sizes ranging from 50 to 219 nm ( Figure 2A ). The mean particle size of R. tanguticum nanoparticles calculated by TEM was around 123.2 nm ( Figure 2B ). The average hydrodynamic size of R. tanguticum nanoparticles determined by DLS was found to be 1794.7 ± 21.7 nm ( Figure 2C ). Polydispersity index (PDI) and zeta potential were 0.13 ± 0.05 and −19.93 ± 0.38 mV, respectively ( Figure 2D ). The small Polydispersity index (PDI) value, which is less than 0.3, indicates the R. tanguticum nanoparticles are uniform in particle size with a narrow particle size range (Yuan et al., 2008). The zeta potential values were a little low which may induce agglomeration in nano system (Gan et al., 2005). While the particle size measured by DLS showed a much larger diameter than the TEM observation, probably because of the R. tanguticum nanoparticles tend to aggregate in aqueous state.

In order to evaluate the antiviral efficacy of R. tanguticum nanoparticles on HSV-1 infectivity, we first determined their cytotoxicity by MTT assays. HEp-2 cells were treated with serial dilutions of R. tanguticum nanoparticles at 37°C for 72 h. As shown in Figure 3A , the 50% cytotoxic concentration (CC₅₀) of R. tanguticum nanoparticles on HEp-2 cells was 415.3 µg/ml. Thus, a safe and low-toxicity concentration 350 µg/ml was selected to determine the anti-HSV-1 efficacy of R. tanguticum nanoparticles using a plaque reduction assay (PRA). The anti-HSV-1 efficacy of R. tanguticum nanoparticles was further conducted using a plaque reduction assay (PRA). The results showed that R. tanguticum nanoparticles could suppress HSV-1 viral plaque formation in a dose-dependent manner ( Figure 3B ) and with the increase of drug concentrations, a noted increased antiviral activity was observed. The EC₅₀ of R. tanguticum nanoparticles determined by PRA was 194.1 µg/ml. To investigate the activity of R. tanguticum nanoparticles on viral replication cycle, time-of-addition assay was performed ( Figure 3C ). R. tanguticum nanoparticles (350 µg/ml) were added to HEp-2 cell monolayers at indicated times post-infection (h.p.i.) and the cells were harvested at 72 h.p.i. The inhibitory effect was significant when R. tanguticum nanoparticles were added at 0–15 h.p.i. Moreover, the results of the inactivation, attachment and penetration assays suggested that R. tanguticum nanoparticles also show effect on directly inactivate HSV-1 particles, prevent viral adsorption and block HSV-1 entry into cells. As shown in Figure 4 , compared with those in the virus control, the 200–350 µg/ml concentrations of R. tanguticum nanoparticles inhibited both the inactivation, attachment and penetration processes of HSV-1 and the viral activities were dramatically reduced. These findings suggested that the antiviral activity of R. tanguticum nanoparticles was due to the interference with the whole phase of viral replication.

To investigate the effect of R. tanguticum nanoparticles on HSV-1 genes expression, we examined the mRNA levels of HSV-1 immediate-early (IE), early (E) and late (L) genes by RT-qPCR. As shown in Figure 5B , the HSV-1 infected HEp-2 cells were incubated with or without R. tanguticum nanoparticles (350 µg/ml) at different time point (6, 12, 18, and 24 h) and the results showed that the mRNA levels of the IE gene ICP4, E gene ICP8 and L gene gD were significantly reduced in a time-dependent manner when treated with R. tanguticum nanoparticles. ICP4 is a transcription activator that is an essential factor for early and late promoter, while ICP8, the viral major DNA-binding protein and an E protein, is necessary for viral DNA replication. Therefore, the effect of R. tanguticum nanoparticles on their production was further determined by Western blot analysis and IFA. As shown in Figure 5A , the western blot results showed that ICP4 and ICP8 fails to express in virus-infected R. tanguticum nanoparticles treated cells which were significantly lower than in the virus-infected group. Besides, the results of IFA indicated that although the expression levels of ICP4 treated with R. tanguticum nanoparticles at 350 µg/ml were similar to the virus control at 6 and 12 h.p.i., ICP4 production was strongly suppressed by R. tanguticum nanoparticles at 18 and 24 h.p.i. ( Figure 6A ). At the same time, the production of ICP8 in the virus control group was significantly more abundant than that in the R. tanguticum nanoparticles-treated cells at 18 and 24 h ( Figure 6B ). These results are consistent with those found and indicated that R. tanguticum nanoparticles could strongly affect HSV-1 replication through interfering with viral IE, E, and L gene expressions.

The experimental mice were monitored for 14 successive days. Clinical signs of changes in behavior of murine HSV-1 infection, such as lack of appetite hyperactivity and weight loss, a hunched posture, and inactivity were observed. The survival time, lethality and viral titers in the brains were recorded to determine the antiviral efficiency of R. tanguticum nanoparticles. As shown in Figure 7A , the survival rates of the R. tanguticum nanoparticles-treated groups at dosages of 312.5 mg/kg/day and 625 mg/kg/day were 30.0% and 60.0%, which are higher than the viral control group (20.0%) on the 14th day post-infection (d.p.i.). However, there was no significant difference of weight loss between all drug-treated groups and the virus control group (p > 0.05) (Supplementary Figure S1). Compared with the virus control group, R. tanguticum nanoparticles treated mice died later and the progress time course of clinical signs was slowed. Additionally, the protective rate of acyclovir at 100 mg/kg/day was shown to be 70%. The changes of the virus titers at the 5th d.p.i. were also detected to confirm the protective efficacy of R. tanguticum nanoparticles against HSV-1infection in vivo. Oral gavage by R. tanguticum nanoparticles in 625 mg/kg/day significantly reduced the virus titers compared to the virus control group (p < 0.01) ( Figure 7B ). Moreover, the treatment with R. tanguticum nanoparticles (625 mg/kg/day) showed no significant difference with the acyclovir group. Taken together, the results suggest that R. tanguticum nanoparticles could protect mice from HSV-1infection and prolong the lifespan.

We examined the pathological changes in mice brains harvested at the 5th d.p.i. In virus group, HE staining showed tissue hyperemia, inflammatory cell aggregation, degeneration of neural cells and virus-induced diffuse necrosis [ Figure 7C (b)]. Following R. tanguticum nanoparticles (625 mg/kg/day) or acyclovir intervention, the damage of brain lesion was relieved and only mild tissue hyperemia could be found [ Figure 7C (c and e)]. Furthermore, the level of neuronal degeneration and aggregation of inflammatory cell in brain tissue were significantly lower than that in virus group. However, the histological changes with the treatment with R. tanguticum nanoparticles at 312.5 mg/kg/day are similar to those in the virus control group [Figure 7C (d)]. These results indicated that post-treatment with R. tanguticum nanoparticles (625 mg/kg/day) can effectively alleviate the severity of the brain damage in the virus-infected mice.

We then assessed the expression of the ICP4 and ICP8 at mRNA levels in vivo. The result showed that when treated with R. tanguticum nanoparticles in 625 mg/kg/day, the mRNA expression levels of both ICP4 and ICP8 could be down-regulated in brain tissues of mice compared to virus control (p < 0.01) ( Figure 8B ). The groups treated with 312.5 mg/kg/day R. tanguticum nanoparticles could reduce the amount of ICP4 mRNA levels (p < 0.01), while it showed no effect on the expression levels of ICP8 with respect to the expression in virus control. The brain tissues were also analyzed to determine the expression levels of the ICP4 protein by western blot. As shown in Figure 8A , treatment with R. tanguticum nanoparticles (625 mg/kg/day) significantly decreased the expression of the ICP4 protein levels compared with non-treated infected groups. These results suggested that R. tanguticum nanoparticles could alter the transcription levels and suppress the expression of the virus protein in vivo.