key: cord-0745701-t2d2egyx authors: Cho, Won-Kyung; Kim, Hyunil; Choi, Yu Jeong; Yim, Nam-Hui; Yang, Hye Jin; Ma, Jin Yeul title: Epimedium koreanum Nakai Water Extract Exhibits Antiviral Activity against Porcine Epidermic Diarrhea Virus In Vitro and In Vivo date: 2012-11-29 journal: Evid Based Complement Alternat Med DOI: 10.1155/2012/985151 sha: 5e3b24f55fdc84cfc263d4784c6ad6349d212866 doc_id: 745701 cord_uid: t2d2egyx Porcine epidemic diarrhea virus (PEDV) causes diarrhea of pigs age-independently and death of young piglets, resulting in economic loss of porcine industry. We have screened 333 natural oriental herbal medicines to search for new antiviral candidates against PEDV. We found that two herbal extracts, KIOM 198 and KIOM 124, contain significant anti-PED viral effect. KIOM 198 and KIOM 124 were identified as Epimedium koreanum Nakai and Lonicera japonica Thunberg, respectively. The further plaque and CPE inhibition assay in vitro showed that KIOM 198 has much stronger antiviral activity than KIOM 124. Additionally, KIOM 198 exhibited a similar extent of antiviral effect against other subtypes of Corona virus such as sm98 and TGE viruses. Cytotoxicity results showed that KIOM 198 is nontoxic on the cells and suggest that it can be delivered safely for therapy. Furthermore, when we orally administered KIOM 198 to piglets and then infected them with PEDV, the piglets did not show any disease symptoms like diarrhea and biopsy results showed clean intestine, whereas control pigs without KIOM 198 treatment exhibited PED-related severe symptoms. These results imply that KIOM 198 contains strong antiviral activity and has a potential to be developed as an antiviral phytomedicine to treat PEDV-related diseases in pigs. Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, dehydration, vomiting, and high mortality in the piglets [1, 2] . PEDV is known for the family of Coronaviridae containing enveloped, singlestranded RNA [3] [4] [5] . Most of newborn piglets infected by PEDV would die and pigs of all ages are also affected and exhibit severe symptom like massive diarrhea and dehydration, resulting in serious damage in the swine industry [6] [7] [8] . Until now, there are no effective treatments or vaccines developed to prevent economic loss by PEDV. Traditionally, natural oriental herbal medicines have been used for relieving and curing many kinds of symptoms arisen from viral infection including cold, flu, and other virus-related diseases [9] [10] [11] [12] [13] [14] . A lot of studies demonstrated that herbal extracts from oriental medicinal plants exert significant antiviral effects on infectious viruses including Influenza virus [15, 16] , Coronavirus [17] , Human immunodeficiency virus (HIV) [18] , Hepatitis B [19] or C [20] virus (HBV or HCV), Herpes simplex virus (HSV) [21] , Poliovirus [22] , Human adenovirus type 1 [23] , and Dengue virus type 2 [24] . Nevertheless, herbal extract with antiviral effect on PED virus has not been reported. Several compounds including Mizoribine, Deoxynojirimycin, and Ribavirin are available commercially as antiviral drugs. A lot of reports demonstrated that they have the inhibitory effect on various viruses, including cytomegalovirus (CMV) [25] , HIV [26] , HCV [27] , Respiratory Syncytial Virus (RSV) [28] , HSV [29] , Influenza B virus [30] , and ovine viral diarrhea virus (BVDV) [31] . Especially, both Mizoribine and Ribavirin were known to contain 2 Evidence-Based Complementary and Alternative Medicine the inhibitory activity on severe acute respiratory syndrome (SARS-) associated coronavirus [32] . The antiviral effect of Ribavirin on PEDV was weakly observed in vitro [33] . There is no information on the effect of Mizoribine and Deoxynojirimycin on PEDV. Traditionally, Epimedium Koreanum Nakai has been used to treat aphrodisiac, hypotensives, and neurasthenia. Epimedium Koreanum Nakai contains a lot of flavonoids including Icariin, Icariside II, Epimedin, Epimedosides, Hyperoside, Qercetin, and Chlorogenic acid. Recent report showed icariin in Epimedium Koreanum Nakaistimulated angiogenesis [34] . Other researcher reported that flavonoids and icariin of Epimedium Koreanum Nakai improved the development of osteroblast [35] . Also, Icariside II was found to induce apoptosis in human prostate cancer cells [36] . But, the antiviral effect of Epimedium Koreanum Nakai was not reported until now. In the present study, we first demonstrate the water extract of Epimedium Koreanum Nakai inhibits PED viral production in vitro and in vivo and has a potential to be develop as a phytomedicine for treatment of diseases arisen from PED viral infection in pigs. Vero cells (African green monkey kidney cell line; ATCC CCR-81) and ST-cells (pig testis cell line; ATCC CRL-1746) were purchased from KCLB, Korean Cell Line Bank (Seoul, Republic of Korea) and maintained in alpha-minimum essential medium (Hyclone, Logan, UT) with 5% fetal bovine serum and 100 U/mL of Penicillin and Streptomycin at 37 • C with 5% CO 2 . Two strains of PED, KPEDV-9 and sm98 and other subtype TGE viruses were obtained from National Veterinary Research and Quarantine Service in Korea. Plaque assay was performed as below. The supernatants harvested were serially diluted up to 10 6 and added to Vero cells seeded in 6 well (1 × 10 6 cell/well). After 1 h incubation, media were removed from infected cells and over-layered with 0.5% agar-containing alpha-MEM media, and incubated for 4-5 days at 37 • C. Plaques were fixed with 7% formalin, stained with crystal violet, and counted. Virus-containing supernatants were collected from the cells infected with virus (0.01 or 0.1 MOI) with or without KIOM 198 or KIOM 124 at 0, 24 h, 40 h, and 48 h postinfection. Total RNA was isolated using viral Gene-spin Viral DNA/RNA Extraction Kit (European Biotech Network, Belgium) and used for cDNA synthesis using iScript Reverse transcriptase (iNtRON Biotech, Daejeon, Korea) according to manufacturer's instruction. Real-time PCR using Bio-Rad iQ5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was performed by subjecting the reaction mixtures to initial denaturation at 94 • C for 3 min, followed by 40 cycles of 94 • C for 20 sec, 65 • C for 20 sec, and 72 • C for 30 sec. The primer sequences specific for Nucleocapsid gene of PED virus are used for PCR and as follows: 5 -CGCAAAGACTGAACCCACTAATTT-3 for forward, 5 -TTGCCTCTGTTGTTACTTGGAGAT-3 for reverse [37] . To evaluate toxicity of natural herbal extract on Vero cells, lactate dehydrogenase (LDH) assay was performed according to manufacturer's recommendation (Roche, Mannheim, Germany). The level of LDH released into the media is used as a marker of dead cells. Cells seeded at 96 wells (3 × 10 4 cells/well) were incubated with diluted herbal extracts for 48 h. After incubating with 5 μL of lysis solution for 15 min, 50 μL of reaction mixture was added and more incubated for 5-10 min. Reaction was terminated with addition of 50 μL stop solution and absorbance at 492 nm was measured using spectrophotometer. For checking cytotoxicity of KIOM 198 on mouse liver primary cells, MTT assay was used. MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma (St. Louis, MO, USA) and dissolved in a phosphatebuffered saline (PBS) at a concentration of 5 mg/mL. KIOM 198 extract at different concentrations was added to the cells and incubated for 48 h. And then MTT solutions were added to each well and the cells were incubated for another 4 h. The formazan melted in dimethyl sulfoxide (DMSO) was determined with absorbance at 570 nm. Herbal Extract. The procedures used in this study were Table 1 : Screening of herbal extract containing antiviral activity against PED virus. 333 herbal extracts were evaluated for the inhibitory effect on PED viral production and 27 herbal extracts having anti-PED viral activity were selected. Antiviral activity was examined by plaque assay and determined as a PFU (plaque forming unit). KIOM 124 and KIOM 198 exhibited strong inhibitory effect on PED viral propagation. (Figure 1(a) , middle panel) but cells containing KIOM 124 did not show CPE effect until 72 h of postinfection (Figure 1(a) , right panel). Figure 1(b) shows KIOM 198 also 30 32 43 44 48 49 51 55 56 57 58 60 71 77 78 124 130 143 198 208 223 232 274 297 324 325 326 Herbal extract number (Figure 3(a), top panel) . On the contrary, when we added KIOM 198 to the cells, CPE by viral propagation was completely blocked (Figure 3(a) , bottom panel). We also tested whether KIOM 198 could protect the cells from CPE by TGE, other subtype of coronavirus. As presented in Figure 3 (b), at 24 h postinfection, KIOM 198 inhibited a little CPE by TGE viral propagation. Be Developed for a Safe Antiviral Drug. The antiviral activity test showed that KIOM 198 has a remarkable ability to inhibit coronavirus propagation. Next, we investigated the toxicity of KIOM 198 using lactate dehydrogenase (LDH) assay in Vero cells and MTT assay in mouse primary liver cells. Vero cells were lysed, mixed with 20 fold-diluted herbal extract, and the activity was measured using substrate. Figure 4 represents the cytotoxicity result of 27 antiviral herbal extracts in Vero cells. The extent of cytotoxicity of KIOM 198 is close to nothing, compared to other herbal extracts. And, when we checked the cytotoxicity of KIOM 198 on mouse liver primary cells, in the presence of 1.5 mg/mL, cell viability was more than 90% (Figure 4(b) ). These data suggest KIOM 198 could be a safe and nontoxic drug when it is used for antiviral therapy. 198, the intestine of piglet was free of disease symptom ( Figure 5(b) , right panel) compared to intestine of control pigs, which was thinned and filled with diarrheal material ( Figure 5(b) , left panel). Furthermore, we compared viral replications in the piglets, at 24 h, 48 h postinfection, and on necropsy. As shown in Figure 5 (c), no virus was detected in the feces of piglet dieted with KIOM 198 at 24 h postinfection. From 48 h of postinfection, viral number in the KIOM 198-dieted piglets was increased, but it was still 10-fold lower than control group. Taken together these results, KIOM 198 has strong inhibitory effect on PED viral growth in the pigs. The main component profile of KIOM 198 was analyzed using HPLC. As shown in Figure 6 , among marker compounds of Epimedium Koreanum Nakai, quercetin and icariin were representatively identified at 270 nm based on comparison to the standard compounds. Several unidentified peaks were detected. In this study, we have represented the antiviral activity of KIOM 198, water extract of Epimedium Koreanum Nakai, on PED virus. PEDV causes severe damage to pig-industry. It is inevitable to develop a novel, strong viral inhibitor to prevent economical loss from PED viral infection. Although some inhibitors were screened and tested for antiviral effect on PED virus, new effective antiviral remedy without toxicity should be developed. KIOM 198 exhibited antiviral activity on not only PED virus but other corona virus like sm98 and TGE virus. We examined whether antiviral agents available commercially, such as Deoxynojirimycin, Mizoribine, and Ribavirin, could inhibit PED viral growth. Each compound at concentration of 100 μg/mL was treated to the Vero cells infected with PED virus and compared their antiviral activity with KIOM 198. These agents except for KIOM 198 did not significantly inhibit PED viral growth in this study (data not shown). These results mean KIOM 198 contains specific, remarkable antiviral activity on PED virus, which is not inhibited by other well-known antiviral drug. Antiviral activity of KIOM 198 was confirmed in vivo disease model. Piglets infected with PED virus expressed disease symptoms like diarrhea, but piglet oral-administered with KIOM 198 had normal feces and no virus was detected in the early time and 10fold lower viruses were found in the feces later. This result suggests the antiviral effect of KIOM 198 on the virus is very effective in the early stage. Importantly, the cytotoxicity of antiviral reagents should be considered prior to use. Traditional oriental herbal medicines have been used for human being for a long time and any severe side-effect after dose did not reported. KIOM 198 showed the least toxicity among 27 antiviral candidates extracted from herbal medicines and also did not have any significant toxicity on normal primary cells. We analyzed KIOM 198 using HPLC and detected several peaks including two known compounds, icariin and quercetin. Recent reports demonstrated that Quercetin 7rhamnoside reduces PED viral replication [33, 38] . They also showed quercetin, apigenin, luteolin, and catechin contained moderate anti-PEDV activity, and ribavirin, coumarin, and tannic acid exhibited weak efficacy on PEDV. Based on these reports, further studies are needed to examine whether active compounds including Quercetin 7-rhamnoside are present in KIOM 198 or which new compounds in KIOM 198 are responsible for anti-PED viral effect. Finally, we have demonstrated that KIOM 198, water extract of Epimedium Koreanum Nakai, exerts a potent antiviral activity on PED virus in vitro and in vivo animal model. Despite the fact that the underlying mechanism of KIOM 198 action in details should be addressed, we assume KIOM 198 exerts strong antiviral effect through modulating immune response such as macrophage and lymphocyte stimulation. 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Won-Kyung Cho and Hyunil Kim contributed equally in this work. All authors have no conflict of interests.