Vol. 34 No Journal of South-Central University for Nationalities Nat. Sci. Edition Mar MTT 3 CVB 4 R373 A

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1 34 1 Vol 34 No Journal of South-Central University for Nationalities Nat Sci Edition Mar 2015 B B 4 CVB 4 MTT 3 CVB 4 PCR CVB 4 mrna IC 50 = ± 0 74 μm TI = 4 35 ± 0 60 CVB 4 mrna p < ~ 4 h CVB 4 B 4 R373 A Antiviral Effect of Emodin on Coxsakievirus B 4 in vitro Liu Zhao 1 Ma Nian 2 Zhong Yan 2 Yang Zhanqiu 2 1 Journal Editorial Department South-Central University for Nationalities Wuhan China 2 State Key Laboratory of Virology Institute of Medical Virology School of Medicine Wuhan University Wuhan China Abstract To investigate the antiviral effect of emodin on Coxsakievirus B 4 ribavirin was used as the positive control and MTT assay was used to evaluate its anti-cvb 4 effect through 3 manners including propagation inhibition direct inactivation and anti-absorption Then real-time PCR were performed to determine the effect of emodin on the mrna expression of CVB 4 and plaque forming assay were applied to detect the effect of emodin on the plaque formed rate after virus infection at different time The results indicated that emodin could not directly inactivate the viruses or block their absorption to cells but it may act as a biological synthesis inhibitor The IC 50 was detected as ± 0 74 μm and TI were detected as 4 35 ± 0 6 Emodin could significantly reduce the mrna expression of CVB 4 gene and may act its inhibitory effect during 0 ~ 4 h postinfection p < 0 05 So emodin may act its antiviral activity of inhibiting CVB 4 replication after virus penetration in a concentration- and time-dependent manner Keywords emodin Coxsakievirus B 4 antiviral effect Coxsackie Virus B 4 Picornaviridas Enterovirus g 200 μl DMSO ddh 2 O @ mail scuec edu cn 2009ZX

2 1 B ml μm 10 mg / ml 0 04 ~ 0 06 MPa TCID 50 CVB 4 HEp-2 min 4 2 h g / μm 100 mg / ml 0 08 MPa 15 min 1 2 HEp-2 2% III CCTCC CVB 4 HEp-2 CPE ~ DMEM Trizol Invitrogin MTT dntp mix PCR Markers Amresco RNA AMV Reverse Transcriptase dntps SYBR GREEN qpcr Super Mix Promega DEPC Sigma CKX41 Olympus Japan OD 570 DG3022-A Rayto PCR My cycler Bio Rad USA Real-time PCR CFX96 Bio Rad USA I II 100 μl 100 TCID 50 CVB h HEp-2 2 h 37 5% CO 2 6 h 100 TCID 50 CVB 4 2 h 2% 80% MTT OD 570 /% = OD % OD 570 OD Real-time PCR CVB 4 mrna RNA HEp μl 100 TCID 50 CVB % CO h PBS ~ μm 50 μl μm 37 5% CO 2 48 h Trizol 2 h 200 μl RNA CPE 9 24 h cdna +++~++++ RNA 20 PBS 3 MTT μl 1 μg RNA 4 % = OD 570 / μl 5 RT Buffer 1 μl dntp 1 μl AMV Reverse OD % Transcriptase 0 4 μl Radom Primer RNase Free 2 2 dh 2 O 20 μl min min 94 3 min 4 4 min 96 cdna 1 HEp μl 4 PCR 25 μl 2 5 DMSO μl cdna 9 0 μl sterile H 2 O 12 5 μl PCR Super ddh 2 O h Mix 0 5 μl Primer S 0 5 μl Primer A MTT 10 OD min s s s min 4 PCR 1%

3 58 34 Tab 1 1 CVB 4 GAPDH The primer sequences for CVB 4 and GAPDH / bp CVB 4 -A CVB 4 -S GAPDH-A GAPDH-S '-GTAGTCCTCCGGCCCCT-3' 5'-AATTGTCACCATAAGCAGCCA-3' 5'-TCATTGACCTCAACTACATGGTTT-3' 5'-GAAGATGGTGATGGGATTTC-3' DNA 42 ~ % 37 PCR μm 512 μm 37 5% CO 2 DNA CVB 4 GAPDH 3 NCBI Blast 100% PCR min 1% 10 min Real-time PCR CVB 4 GAPDH PCR cdna ~ 10 1 copies /μl 8 PCR 20 μl TCID 50 TC 50 sterile H 2 O 7 2 μl qpcr Super Mix 10 0 μl 0 4 μl Primer S 0 4 μl Primer A cdna 2 0 μl 95 3 min s s s s 65 5 s Ct CVB 4 GAPDH Ct value y log 10 RNA copies numbers / μl x / h MTT SPSS μm CVB 4 TCID μm Real-time PCR 100 TCID 50 Ct y 3 2 HEp-2 copy 10 x 2 5 OD μl 100TCID 50 CVB h 1 PBS 3 0 h h 72 h 4% 30 % = / 100% SPSS Probit IC 50 TI = TC 50 / IC 50 One-Way ANOVA LSD ~ 10-8 CVB 4 HEp-2 HEp-2 Fig 1 a ~ d μm 1 HEp-2 The toxic effect of emodin with different concentrations on HEp-2 cells MTT 2 2

4 1 B 4 59 p < 0 05 TC ± 4 40 μm TC ± μm 37μM 10 μg /ml 0 5 V /V HEp-2 DMSO < 5 V /V OD DMSO 2 Fig CPE +++~++++ a b HEp-2 The cytotoxic effect of emodin a and ribavirin b on HEp-2 cells CPE MTT ± 1 64 μm TI 4 35 I ± ± TI p > ~ 40 μm CVB 4 p < 0 05 IC ± 0 74 μm Fig 3 3 a b CVB 4 The inhibitory effect of emodin a and ribavirin b on CVB 4 with different concentrations II III 4c CVB 4 GAPDH y = 0 ~ x y = x 40 μm CPE OD p > CVB 4 GAPDH 0 05 CVB 4 I CVB 4 GAPDH 3 4 copy μm CVB 4 CVB 4 / 4 4a 4b GAPDH copies S VC CVB 4 /GAPDH copies μm p

5 CVB h 16 5% 20 5% 40 0% p < 0 01 p < h p > ~ 12 h p < 0 01 CVB 4 0 ~ 4 h Fig 6 6 ** p < 0 01 n = 3 CVB 4 The inhibitory effects of drug on CVB 4 at different time Fig 4 a CVB 4 b GAPDH 4 c CVB 4 GAPDH CVB 4 GAPDH Amplificatin curve and standard curve for CVB 4 and GAPDH SARS 13 < μm 512 μm p > 0 05 Fig 5 5 ** p < 0 01 n = 3 CVB 4 /GAPDH The copies of CVB 4 /GAPDH under different drug treatment 4 1'3' SARS-CoV S ACE CVB 4 CVB 4 / IC 50 TI ± 0 74 μm 4 35 ± 0 6 TI p > ΔΔCT CVB 4 GAPDH

6 1 B 4 61 copy cancer cell proliferation in vitro J JPEN μm p < μM 512μM 8 Alves D S Pérez-Fons L Estepa A et al Membranerelated effects underlying the biological activity of the p > 0 05 anthraquinones emodin and barbaloin J Biochem Pharmacol ~ 4 h 9 B 4 J CVB 4 RNA ATP RNA 10 J CVB Hsiang C Y Ho T Y Emodin is a novel alkaline CVB 4 p < 0 01 nuclease inhibitor that sup-presses herpes simplex virus type 1 yields in cell cultures J Br J Pharmacol 0 ~ 4 h CVB 4 12 J Schwarz S Wang K Yu W et al Emodin inhibits current through SARS-associated coronavirus 3a protein J Antivir Res Crowell R L Landau B J A short history and introductory 14 Lu Y Jeong Y T Li X et al Emodin isolated from background on the coxsackieviruses of group B J Curr Top Microbiol Immunol Polygoni Cuspidati Radix inhibits TNF-α and IL-6 release by blockading NF-κB and MAP kinase pathways 2 Ramsingh A I CVB-induced pancreatitis and alterations in gene expression J Curr Top Microbiol Immunol Huber S Ramsingh A I Coxsackievirus-induced pancreatitis J Viral Immunol Chapman N M Kim K S Persistent coxsackievirus infection enterovirus persistence in chronic myocarditis and dilated cardiomyopathy J Curr Top Microbiol Immunol Feigelstock D A Mihalik K B Feinstone S M Selection of hepatitis C virus resistant to ribavirin J Virol J Nomura H Tanimoto H Kajiwara E et al Factors contributing to ribavirin-induced anemia J J Gastroen Hepatol Cai J Razzak A Hering J et al Feasibility evaluation of emodin rhubarb extract as an inhibitor of pancreatic in mast cells stimulated with PMA plus A23187 J Biomol Ther Yim H Lee Y H Lee C H et al Emodin an anthraquinone derivative isolated from the rhizomes of Rheum palmatum selectively inhibits the activity of casein kinase II as a competitive inhibitor J Planta Med J Ho T Y Wu S L Chen J C et al Emodin blocks the SARS coronavirus spike protein and angiotensinconverting enzyme 2 interaction J Antivir Res PCR Grb10 J