Screening compounds of Chinese medicinal herbs anti-Marek's disease virus

Abstract

Context: Marek’s disease (MD) seriously threatens the world poultry industry and has resulted in great economic losses. Chinese medicinal herbs are a rich source for lead compounds and drug candidates for antiviral treatments.

Objective: To investigate the anti-MDV activity and mechanism of 20 compounds extracted from Chinese medicinal herbs.

Materials and methods: Antiviral assay, time of addition experiments, and virucidal assay were performed on chicken embryo fibroblast cells. The 50% cytotoxic concentration and 50% effective concentration were determined and, accordingly, selectivity index and inhibition ratio were calculated.

Results: Antiviral assay showed dipotassium glycyrrhizinate (DG) and sodium tanshinone IIA sulfonate (STS) exhibited significantly inhibitory activity against MDV in a dose-dependent manner. EC₅₀ of DG and STS were 893.5 ± 36.99 µg/mL and 54.82 ± 2.99 µg/mL, and selective index (SI) were >3.36 and >9.12, respectively. Time of addition experiment and virucidal assay demonstrated DG inhibited viral replication in the full replication cycle and inactivated MDV particles in non-time-dependent manner, but STS interfered with the early stage of MDV replication and inactivated MDV particles in a time-dependent manner. Moreover, both DG and STS promoted apoptosis of cells infected by MDV.

Discussion and conclusion: DG and STS have great potential for developing new anti-MDV drugs for clinic application.

• Antiviral
• apoptosis
• dipotassium glycyrrhizinate
• MDV
• sodium tanshinone IIA sulfonate

Introduction

Marek’s disease (MD), caused by Marek’s disease virus (MDV), is a lymphoproliferative disease characterized with formation of T-cell lymphomas in various visceral organs and tissues in birds (Cui et al., 2004). MD seriously threatens the world poultry industry, leading to huge economic losses. Vaccination is the best choice at present for controlling and preventing MD, but the application of MD vaccines has resulted in the emergence of virulent strains (Gimeno, 2008). There is an urgent need to develop new vaccines and/or anti-MDV drugs that control the new emerging virulent strains.

Chinese medicinal herbs are a rich source for lead compounds and drug candidates for antitumor, anti-infection, antiviral treatments, etc. (Tang et al., 2009). It has been proved that some of Chinese medicinal herbs and their main ingredients possess antiviral activities and have been used as antiviral agents. For example, “Tamiflu” (containing oseltamivir phosphate) is an effective medicine for H₁N₁ influenza (Newman & Cragg, 2012). Its lead compound was obtained from Illicium verum Hook.f., a Chinese medicinal herb.

In this study, the anti-MDV activity of 20 compounds and mechanism of active ones were investigated in order to find the promising drug candidate against MD. These tested compounds were extracted from Chinese medicinal herbs which were frequently used in many Chinese medicinal formulae possessed anti-virus activities. Moreover, these compounds have been proven to possess multi-pharmacological effects with definitely chemical structures.

Materials and methods

Reagents

Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% or 2% fetal calf serum (FCS, Hyclone, Sigma-Aldrich, St. Louis, MO), 100 IU/mL penicillin G and 100 µg/mL streptomycin was used for cell growth or maintenance medium (MM). About 0.25% trypsin (Amresco, LLC, Solon, OH) was prepared in PBS (pH 7.2–7.4). 3-(4,5-Dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Amresco, LLC, Solon, OH) (5 mg/mL) was prepared in PBS (pH 7.2–7.4). These solutions were sterilized through a 0.22 µm millipore membrane filter and aliquots were made for future use. Dimethyl sulphoxide (DMSO) is the product of Beijing Solarbio Science & Technology Co., Ltd, Beijing, China. DMEM and MM were stored at 4 °C; MTT and trypsin wrapped with opaque paper were stored at −20 °C. DMSO was stored at the room temperature. Annexin V-FITC/PI apoptosis detection kit was purchased from Nanjing KeyGen Biotech. Co. Ltd, Nanjing, China. The compounds extracted from Chinese medicinal herbs used in the experiment and Acyclovir were purchased from National Institute for Food and Drug Control (Beijing, China) (Table 1).

Table 1. Descriptions of the compounds used in the experiment.

Chemical name	Chemical family	Chinese medicinal herbs used for extraction of compounds	Solvent
Aesculetin	Coumarin	Cortex Fraxini	1% DMSO
Aesculin	Coumarin	Cortex Fraxini	MM
Arecoine hydrochloride	Alkaloids	Semen Arecae	MM
Baicalein	Flavonoids	Scutellariae Radix	1% DMSO
Baicalin	Flavonoids	Scutellariae Radix	MM
Berberine hydrochloride	Alkaloids	Rhizoma Coptidis,	3% alcohol
Chlorogenic acid	Phenolic acids	Flos Lonicerae Japonicae	MM
Cinnamic acid	Phenylpropanoid	Cortex Cinnamomi	3% alcohol
Dehydroandrographolide	Diterpenoids	Andrographis Herb	1% DMSO
Dipotassium glycyrrhizinate	Triterpenoid saponins	Radix et Rhizoma Glycyrrhizae	MM
Emodin	Anthraquinones	Radix et Rhizoma Rhei	MM
Geniposide	Monoterpenoids	Gardeniae Fructus	MM
Glycurrhetinic acid	Triterpenoids	Radix et Rhizoma Glycyrrhizae	3% alcohol
Ligustrazine hydrochloride	Alkaloid	Chuanxiong Rhizome	MM
Liquiritin	Flavonoid	Radix et Rhizoma Glycyrrhizae	1% DMSO
Matrine	Alkaloid	Radix Sophorae Flavescentis	MM
Polydatin	Glycosides	Rhizoma polygoni cuspidati	3% alcohol
Salvianic acid A sodium	Phenolic acid	Salvia miltiorrhiza Radix et Rhizoma	MM
Scutellarin	Flavonoid	Scutellariae Radix	MM
Sodium tanshinone IIA sulfonate	Diterpenoids	Salvia miltiorrhiza Radix et Rhizoma	1% DMSO

Cell and virus

The culture of Chicken embryo fibroblast (CEF) was performed according to the method as described by Zhao et al. (2011). Briefly, CEF was prepared with 10-day-old SPF chicken embryo (SYXK (Jin) 2010-0007, Shanxi Longker Biopharmaceutical Co., Ltd, Shanxi, China). The cells were diluted into 1.0 × 10⁶/mL with 8% DMEM, seeded onto 96-well plates and incubated at 37 °C in a 5% CO₂ atmosphere.

CVI988, an attenuated serotype 1 strain of MDV, was obtained from Beijing Lingyu Biological Ltd. (Beijing, China; Cryo-Marek Rispens, batch: XLYB003) and propagated in CEF cells. CEF cells infected with MDV were harvested when the cytopathic effect (CPE) reached 80–90% compared with CEF cell control. The virus titer was determined by plaque forming unit (pfu) assay (Hsuan et al., 2009), and the CEF was infected with 100 pfu/well.

Cytotoxicity

The cytotoxicity of 20 compounds on chicken embryo fibroblast cells (CEF) was assessed via changes in cellular morphology and was measured by MTT [3-(4, 5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Mosmann, 1983). A total of eight dilutions for each of the compounds and Acyclovir were made by serial two-fold dilution with DMEM containing 2% FCS and the proper solvent (Table 1). CEF cells were seeded into 96-well plates at a density of 1 × 10⁵ cells/well (1 × 10⁶/mL) and incubated at 37 °C in a 5% CO₂ atmosphere until the cells were at least 90% confluent. Then, the medium was removed and diluted compounds or Acyclovir were added to the wells, further incubated for 72 h. Afterwards, the medium was discarded and 20 μL MTT was added to each well. The plates were then further incubated at 37 °C for 4 h. Subsequently, the supernatant was removed and 100 μL of DMSO was added into each well to solubilize the dark blue formazan crystals. The optical density (OD) at 490 nm was measured by an automatic plate reader (Bio Tek®, ELx808, Gene Co., Ltd., Hong Kong, China) with absorbance at 630 nm as a reference (Pang et al., 2010). The 50% cytotoxic concentration (CC₅₀) was determined as the concentration of the compounds that reduced the cell viability to 50% of cell control (Li et al., 2005). The maximum safe concentration (MSC) was calculated as the concentration required retaining cell viability by 90% (Chen et al., 2010).

Antiviral activity

The anti-MDV activity of the 20 compounds was determined by standard plaque reduction assays in CEF cells with minor modifications (Li et al., 2005). In brief, the confluent monolayer of CEF cells in 24-well plates was prepared and the serial two-fold dilutions of each compound and 100 pfu/well of MDV were added, respectively. Cells control, MDV control, and Acyclovir positive control were included. The plate was further incubated at 37 °C for 96 h in a 5% CO₂ atmosphere, pfu of all groups were counted. The inhibition ratio (%I) was expressed as where pfu_virus represents the plaque numbers of MDV control. pfu_test corresponds to the plaque numbers of groups treated with compounds. The 50% effective concentration (EC₅₀) was defined as the concentration of the compound reducing the plaque numbers by 50%. The selectivity index (SI) was the ratio of CC₅₀ to EC₅₀ (Myskiw et al., 2010).

Time-of-addition

The MSC of each compound and 100 pfu virus per well were used in this assay. Procedures were followed the description of Alvarez et al. (2011) with some modifications. Briefly, CEF cells in 24-well plates were pre-incubated with MDV for 2, 4, 6, 8, 10, 12, 14, and 16 h, respectively, and then the medium was removed and co-incubated with each compound. After 96 h incubation at 37 °C in a 5% CO₂ humidified atmosphere, the plaque numbers were counted and viral inhibition ratio (%I) was assessed as described in antiviral activity section.

Virucidal

Each compound with the MSC and 100 pfu virus were mixed and incubated at 37 °C for 30, 60, and 120 min, respectively. At indicated time points, the virus/compound mixture was transferred onto the 100% confluent CEF monolayer in a 24-plate and incubated at 37 °C for 96 h in a 5% CO₂ humidified atmosphere. Then the plaque numbers were counted and viral inhibition ratio (%I) was assessed as described in the antiviral activity section.

Cell apoptosis

Cell apoptosis was analyzed using a fluorescence microscope and flow cytometry as previously described with some modifications (Sui et al., 2010). Briefly, the MSC of each compound and 100 pfu virus per well were added to the confluent monolayer of CEF cells in 12-well plates. Cell control and MDV control were set up simultaneously. After 48 h incubation at 37 °C in a 5% CO₂ humidified atmosphere, cells were separately treated according to the manufacture’s instructions (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China). One part of the cell sample was analyzed with a fluorescence microscope, and the other was used for cell apoptosis through a FAC-Sort flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Statistical analysis

The statistical analysis was performed using SPSS17.0 software (SPSS Inc., Chicago, IL). Data were from three separate experiments and expressed as mean ± SD. Student’s t-test and one-way ANOVA were used. A values of p < 0.05 was considered statistically significant. The CC₅₀ was calculated by regression analysis of the dose–response curves for MTT assay. EC₅₀ was determined using the GraphPad Prism version 5 software (San Diego, CA).

Results

Cytotoxicity

The MSC and CC₅₀ of the tested compounds were listed in Table 2. CC₅₀ values of the 20 compounds against CEF ranged from 5.6 ± 0.8 µg/mL to >3000 µg/mL. According to the values of the MSC, aesculin, dipotassium glycyrrhizinate (DG), geniposide, liquiritin, ligustrazine hydrochloride, and polydatin had no cytotoxic effect (defined as CC₅₀ >1000 µg/mL) on CEF in this assay. When the concentration of compound was gradually increased in the assay, cells underwent morphological changes such as detachment of monolayer, pyknosis, condensation, lysis, granulation, vacuolization in the cytoplasm, and darkening of cell boundaries.

Table 2. Summary of cytotoxicity and antiviral assay.

Monomers	CC50^a (µg/mL)	MSC^b (µg/mL)	%I ^c	EC50^a (µg/mL)	SI^d
Aesculetin	76.4 ± 13.4	31.2	<10	–
Aesculin	>1000	≥1000	<10	–
Arecoine hydrochloride	80.8 ± 4.8	15.6	<10	–
Baicalein	>50	25	<50	–
Baicalin	>500	62.5	<50	–
Berberine hydrochloride	71.4 ± 1.4	31.2	<10	–
Chlorogenic acid	455.1 ± 88.4	125	<10	–
Cinnamic acid	>500	250	<50	–
Dehydroandrographolide	34.9 ± 2.7	15.6	<10	–
Dipotassium glycyrrhizinate	>3000	≥3000	>90	893.5 ± 36.99	>3.36
Emodin	5.6 ± 0.8	1.95	<10	–
Geniposide	>1000	≥1000	<10	–
Glycurrhetinic acid	109.2 ± 11.2	15.6	<10	–
Ligustrazine hydrochloride	>2000	≥2000	<20	–
Liquiritin	>500	≥500	<50	–
Matrine	590.6 ± 117.8	62.5	<50	–
Polydatin	>1000	≥1000	<50	–
Salvianic acid A sodium	62.4 ± 4.6	31.2	<50	–
Scutellarin	547.5 ± 60.5	62.5	<10	–
Sodium tanshinone IIA sulfonate	>500	250	>90	54.82 ± 2.99	>9.12
Acyclovir	>1000	250	>90	1.861 ± 0.00098	>537.3

^aCC₅₀ and EC₅₀ represent as mean ± SD of three independent experiments.

^bMaximum safety concentration.

^c%I= (pfu_virus−pfu_test)/pfu_virus.

^dSelectivity index. “–”: due to no inhibition to virus replication.

Antiviral

The results demonstrated that DG and sodium tanshinone IIA sulfonate (STS) exhibited significantly inhibitory activity against MDV in a dose-dependent manner (Figure 1). The highest inhibition ratio (%I) of DG and STS reached 94.43% and 95.39%, similar with acyclovir (98.88%). The SI of STS was larger than that of DG but the SI of both was lower than acyclovir (Table 2). DG and STS possessed significant anti-MDV activity but have narrow safety margins. The inhibition ratio (%I) of the rest of compounds was lower than 50%, and no SI was available to calculate.

Figure 1. Effects of DG and STS on CEF cells infected by MDV. (A) CEF control, (B) CEF-infected MDV, plague formed in the CEF monolayer; (C–E) the curves indicated that DG, STS, and acyciovir had significant inhibitory effects on MDV in dose-dependent fashion.

Time-of-addition

We found that DG could consistently inhibit MDV replication, regardless of the time when DG was added to the virus infected CEF (p > 0.05), and the highest inhibition ratio (%I) nearly reached 100%. The highest inhibition ratio (%I) of STS was 70.41% at 2 h, and the inhibition ratio of STS less than 50% from 10 h to 16 h. The results demonstrated that DG showed a consistent inhibitory effect during a single replication cycle from 2 h to 16 h and STS only inhibited the early stage of the MDV replication cycle (Figure 2).

Figure 2. The antiviral activities of DG and STS in the viral replication cycle. The compounds were added from 2 h to 16 h post-infection. Inhibition ratio (%I) was determined after 96 h incubation. Data were presented as the mean ± SD from three separately repeated experiments.

Virucidal

When DG and STS were, respectively, mixed with MDV and interacted for 30, 60, and 120 min at 37 °C, the inhibition ratio (%I) of the two compounds at each time point was larger than 80%. The highest inhibition ratios of DG and STS were 86.92 and 97.25%, respectively. The inhibition ratio (%I) of STS increased steadily in a time-dependent manner, but that of DG was not time-dependent (Figure 3).

Figure 3. Direct virucidal activity of STS and DG on MDV. Compounds mixed with MDV and interacted 30, 60, and 120 min, respectively, then tested inhibition ratio of the two compounds at each time point.

Both DG and STS accelerated the apoptosis of cells infected by MDV

Compared with virus control, the number of apoptotic cells infected by MDV and treated with DG and STS was significantly increased, as indicated in Figure 4. Flow cytometry assay was utilized to confirm the apoptosis, as shown in Figure 5. MDV-infected cells did not display either early or late apoptosis. The early apoptosis rate of cell control and virus control was 10.43% ± 1.38% and 7.33 ± 1.25%, respectively, and the late apoptosis rate was 2.43% ± 1.54 and 3.93% ± 0.60, respectively. In contrast, the early apoptosis rate of DG and STS treated groups was much higher than virus control (p<0.05). However, the late apoptosis rate has no difference to virus control and cell control (p>0.05) (Figure 5). So both DG and STS could accelerate the early apoptosis of MDV infected cells.

Figure 4. Effect of DG and STS on cell apoptosis revealed by fluorescence staining. The red fluorescence and green fluorescence presented the late apoptosis and the early apoptosis, respectively. (A) Early apoptotic cells, only green fluorescence was found around cell membrane; (B) late apoptotic and necrotic cells, green fluorescence was found around cell membrane and red fluorescence in nucleus; (C) cell control; (D) virus control; (E) cells treated with DG and MDV; (F) cells treated with STS and MDV.

Figure 5. Cell apoptosis was analyzed by flow cytometry. The cells were dual stained with Annexin V-FITC and PI. (A) Cell control; (B) virus control; (C) and (D) cells treated with DG or STS and MDV. Data were presented as dual-parameter FL1of Annexin V-FITC versus FL2 of PI. The upper left quadrant (B1) shows non-normal necrotic cells, PI positive. The upper right quadrant (B2) represents late apoptotic cells, necrotic cells, positive for Annexin V-FITC binding, and PI uptake. The lower left quadrant (B3) contains the viable cells, Annexin V-FITC, and PI negative. The lower right quadrant (B4) indicates early apoptotic cells, Annexin V-FITC positive, and PI negative. The corresponding apoptosis rate was shown in bar chart.

Discussion

Up to date, there are still no effective methods to completely control the prevalence of MD, which is one of the major threats to the poultry industry. Chinese medicinal herbs have been used to prevent and cure diseases for a long time. It has been proven that some Chinese medicinal herbs and their main ingredients possess antiviral effects (Chen et al., 2010; Fan et al., 2011). In recent years, there has been a renaissance of interest in Chinese medicinal herbs for the treatment of the infectious diseases (Xu, 2006). One of the important reasons is that Chinese medicinal herbs are a natural combinatorial chemical library for the development of new lead chemicals and drugs.

In this experiment, the results showed that the cytotoxicity of the different compounds on CEF cells varied remarkably. The range of CC₅₀ was from 5.6 ± 0.8 µg/mL to >3000 µg/mL, and the MSC was from 1.95 µg/mL to ≥3000 µg/mL. DG had the highest CC₅₀ and MSC.

Among the 20 compounds, only DG and STS exhibited significant inhibitory effects on MDV. A dose dependency of antiviral activities was determined by serial dilutions of the two compounds. SI of DG and STS were larger than 3, which demonstrated that these two compounds possessed much wider safety margins and could be used as potential anti-MDV drug candidates (Chávez et al., 2006).

DG is obtained by chemical modification on glycyrrhizic acid, which is the most important bioactive compound of licorice root (Glycyrrhiza uralensis Fish). It has been proven that glycyrrhizic acid has an effective inhibitory activity against varicella zoster virus (VZV), SARS, Epstein–Barr Virus (EBV), herpes simplex type 1 (HSV-1), HIV-1 and influenza A virus (IAV), etc. (Hoever et al., 2005; Lin, 2003; Wolkerstorfer et al., 2009). Diammonium glycyrrhizin possessed a strong inhibitory effect on pseudorabies herpesvirus (PRV) (Sui et al., 2010). STS, the water-soluble derivative of tanshinone IIA, is one of the major effective components of Danshen. Danshen, traditional Chinese medicine derived from the dry root of Salviae miltiorrhizae, has been widely used in China, Japan, the United States of America, and European countries for the treatment of cardiovascular and cerebrovascular diseases (Chan et al., 2011). Extensive research has shown that STS has a broad range of pharmaceutical effects such as antitumor (Chen et al., 2008), antioxidation, apoptosis-inducing activity (Tian et al., 2010) and protective effects on cardiomyocyte against adriamycin-induced damage (Chan et al., 2011). Our previous study demonstrated that STS had strong antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV), and its anti-PRRSV activity could be due to inhibiting the virus replication or/and inactivating the virus directly (Sun et al., 2012).

In this research, time-of-addition assay, virucidal assay, and the effect on apoptosis were performed in order to explore the anti-MDV mechanism of DG and STS. In time-of-addition assay, these two compounds could inhibit the viral replication. DG displayed strong anti-MDV effect in the full cycle of MDV replication, but STS only showed anti-MDV effect in the early stage of MDV replication cycle. Lin (2003) reported that glycyrrhizic acid interfered with an early step of EBV replication cycle, and had no effect on viral adsorption, nor did it inactivate EBV particles.

Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited by hyaluronic acid, regardless of the time of addition (Cermelli et al., 2011). Our previous study also demonstrated that STS could inhibit PRRSV replication during a single replication cycle and may directly inactivate PRRSV within 30 min (Sun et al., 2012). In virucidal assay, when DG and STS were respectively mixed with MDV and interacted 30, 60, and 120 min at 37 °C, inhibition ratio (%I) of the two compounds at each time point was larger than 80%. The highest inhibition ratios of DG and STS were 86.92 and 97.25%, respectively. The inhibition ratio (%I) of STS increased steadily in a time-dependent manner, but that of DG was not time dependent (Figure 3). Chen et al. (2008) reported that STS could bind to DNA by intercalation due to its planar aromatic ring. Therefore, it could be reasonably speculated that STS inactivated or inhibited MDV replication by interfering the viral DNA synthesis.

Apoptosis is a physiological energy-dependent process through which cell populations regulate normal growth and morphogenesis (Kivinena et al., 2005). Sometimes, apoptosis triggered by virus infection serves as a host defense mechanism to limit viral replication (Danthi et al., 2008). MDV is an oncogenic herpesvirus, which possesses anti-apoptotic activity (Schumacher et al., 2008). In our study, compared to virus control, the number of apoptotic cells infected by MDV and treated with DG and STS was significantly increased, as indicated in Figure 4. Flow cytometry assay was utilized to confirm the apoptosis, as shown in Figure 5. MDV-infected cells did not display either early or late apoptosis. The early apoptosis rate of cell control and virus control was 10.43 ± 1.38 and 7.33 ± 1.25%, respectively, and the late apoptosis rate was 2.43 ± 1.54 and 3.93 ± 0.60%, respectively. In contrast, the early apoptosis rate of DG and STS treated groups was much higher than virus control (p<0.05). However, the late apoptosis rate has no difference with virus control and cell control (p>0.05) (Figure 5). So both DG and STS could accelerate the early apoptosis of MDV-infected cells. It was proved that Tanshinone IIA could induce the apoptosis of different cancer cells including leukemia, hepatocarcinoma, breast cancer, colon cancer, and glioma (Tian et al., 2010). And Tanshinone IIA could induce apoptosis of human hepatocellular carcinoma cells, which were associated with up-regulation of fas, p53, bax, and down-regulation of bcl-2 and c-myc (Yuan et al., 2004). Haq et al. (2010) reported that the oncogene Meq of MDV might play a role in inhibition of apoptosis through up-regulate limit apoptotic genes (SKI and BCL2), and down-regulate pro-apoptotic genes (FAS and EIF4G2). It could be speculated that DG and STS accelerated the apoptosis of cells infected by MDV through regulating the apoptotic genes or/and inhibiting the expression of oncogene Meq.

Conclusions

In conclusion, DG and STS can effectively inhibit MDV infection in vitro. Therefore, they have potential to be further developed into new anti-MDV drugs for use in clinic application. Antiviral mechanisms of DG and STS will be further explored in the future.

Declaration of interest

This project was funded by the key scientific and technological grant from Shanxi Province (Grant nos. 2010311047 and 20120311022-1). These experiments were conducted with compliance with the current laws of PR China. The authors have no conflict of interest in the research.