Myocarditis caused by coxsackie virus B3 (CVB3) infec-tion is a common cause of acute heart failure and dilated car-diomyopathy, especially in young patients. Immunosuppres-sive agents, intravenous immunoglobulin, antiviral agents,and natural medicines have been used to treat the disease;however, the efficacy of these agents has not been well estab-lished.1) Overall, specific therapy for viral myocarditis is stilllacking.

In China, natural products have been widely used in theclinics. Radix astragali, the dried root of Astragalus mem-branaceus (FISH.) BGE., or Astragalus membranceus var.,known as “Huangqi” in Chinese, is one of the most popularherbal medicines, owing to its well-known immune-stimulat-ing and antioxidant activities, as well as activities against awide variety of viruses.2,3) It has also been shown to improvecardiac functions, possibly through its diuretic effects.4,5)

Since Chinese medicines are natural products with mixtureof chemical components, defining their active ingredientsand mechanisms of actions have been always the challenges.Aponins, isofavones, and amino acids have been reported tobe active components in Astragalus membranaceus.6) For ex-amples, astragaloside IV has been demonstrated for antiviralactivities in vitro and in vivo,7) other isoflavones in Radix as-tragali have also been shown to possess biological activitiessuch as removing oxidative radicals, immune modulation andantiviral activity.4,8,9)

Calycosin-7-O-b-D-glucopyranoside (CCGR) is the mainisoflavonoid isolated from Astragalus membranaceus var.Mongholicus (BGE.) HSIAO,10) but its biological functions arenot well studied. In order to determine whether it is one ofthe active components in Radix astragali for the treatment ofviral myocarditis, we investigated its antiviral effects againstCVB3 using both in vitro and in vivo approaches.

MATERIALS AND METHODS

Chemicals CCGR was isolated from Astragalus mem-

branaceus var. Mongholicus (BGE.) HSIAO in our labora-tory,11) and its purity was determined to be higher than 98%by HPLC. The chemical structure was shown in Fig. 1. Foruse in vitro, CCGR was dissolved in 5% dimethylsulfoxide(DMSO) with culture medium. For use in vivo studies, theformulation for oral administration of CCGR was preparedby mixing CCGR with sodiumcarboxymethyl cellulose dis-solved in distilled water (0.5%, w/w). Ribavirin, a commonantiviral drug (Guangdong, China) was used in the antiviralscreening tests with CVB3 as the control compound in vitro.According to the guideline of the State Food and Drug Ad-ministration (SFDA) in China, there is no effective medicineavailable for use as the control of screening anti-coxsackievirus B3 in vivo. Since CCGR was the active component iso-lated from Radix astragali, Radix astragali oral solution(HQ), which has been shown to enhance cardiac function ofchronic viral myocarditis and be widely accepted in clinicalpractice in China,3) was used as the positive control for invivo study.

CVB3 and Titration in Vero Cells A Nancy variant ofCVB3 was propagated in Vero cells cultured as monolayers,and stored in a �70 °C freezer. Cell cultures and viral infec-tion will be described later. The supernatant from infectedcell culture was collected, and viral titers were determined in96-well plates by the end-point dilution method. Briefly, 10-

68 Vol. 32, No. 1

In Vivo and in Vitro Antiviral Activities of Calycosin-7-O-bb-D-glucopyranoside against Coxsackie Virus B3

Haiyan ZHU,a,b Yuanyuan ZHANG,c Guan YE,a Zhixiong LI,a Pei ZHOU,*,b and Chenggang HUANG*,a

a Shanghai Institute of Materia Medica, Chinese Academy of Sciences; No. 555 Zuchongzhi Road, Zhangjiang, Pudong,Shanghai 201203, P. R. China: b Department of Biosynthetic Medicinal Chemistry, School of Pharmaceutical Sciences,Fudan University; and c Department of Microbiology and Microbial Engineering, School of Life Science, FudanUniversity; Shanghai 200032, P. R. China.Received August 14, 2008; accepted October 30, 2008; published online November 4, 2008

Calycosin-7-O-bb-D-glucopyranoside (CCGR) is the main isoflavonoid compound isolated from Astragalusmembranaceus var. Mongholicu (BGE.) HSIAO, a Chinese herb medicine traditionally used to treat viral myocardi-tis. In this study, its antiviral activities against coxsackie virus B3 (CVB3) causing myocarditis were investigated.In vitro assay showed that CCGR displayed a low cytotoxicity and effectively inhibited CVB3-mediated cyto-pathic effects on Vero cells with an IC50 value of 25 mmg/ml. In an acute myocarditis murine model, treatment with24 mg/kg CCGR for 14 d significantly improved the survival rate of mice infected with CVB3, alleviated patho-logical damages of cardiac muscles in the myocarditis mice, reduced the virus titers in the heart, decreased heartindexes and improved left ventricular function. These results showed that CCGR exerted significant antiviral ac-tivities against CVB3 both in vitro and in vivo, and identified CCGR as one of active ingredients in Astragalusmembranaceus for the treatment of viral myocarditis.

Key words Astragalus membranaceus; calycosin-7-O-b-D-glucopyranoside; coxsackie virus B3; viral myocarditis

Biol. Pharm. Bull. 32(1) 68—73 (2009)

© 2009 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: cghuang@mail.shcnc.ac.cn; pz19444@yahoo.com.cn

Fig. 1. The Chemical Formula and Structure of CCGR

fold serial dilutions (1 : 10 to 1 : 106) of the culture mediawere prepared, and 50% tissue culture infectious dose(TCID50) titer/0.2 ml was determined by the cytopathic ef-fects (CPE) visible after 72 h. Virus titers were determined tobe 10�4.95 in terms of viral CPE assay , and expressed as theTCID50.

In Vitro Experiments. Cytotoxicity Assay Vero cellswere routinely cultured in 10-cm cell culture dishes as mono-layers in Dulbecco’s modified Eagle medium (DMEM,GIBCO, U.S.A.) supplemented with 10% (v/v) fetal bovineserum (GIBCO) in a humidified incubator at 37 °C under 5%CO2. CCGR was diluted in the culture medium at differentconcentrations, and 50 m l was added onto the cell monolay-ers. The cytotoxic CC0 value was determined by the maximalconcentration of CCGR that did not reduce the amount oflive cells, and CC50 was determined by the concentration thatkilled 50% of the live cells by toxicity.

CPE Assay Vero cells were grown to confluence in a 96-well tissue culture microplate, and then infected by CVB3 atthe titer of 100�TCID50. Cells receiving no virus served asnormal cell control. Virus-induced CPE in Vero cells wereexamined under a light microscope. Virus-containing mediawere exchanged to fresh media following a 1.5 h virus ad-sorption period, and test compounds were added into culturemedia at serial dilutions. Infected cells receiving no drugsserved as the virus control, and infected cells treated by rib-avirin were included as the positive control. When 100%CPE was observed at 3—4 d post-infection,12) the drug con-centration that achieved 50% inhibition of the observed CPEwas determined as IC50, which was calculated from three in-dependent assays. The selectivity index (SI), or the ratio ofCC50 to IC50, was also calculated to show the specificity ofthe drug’s inhibitory effect.

In Vivo Experiments. Animals One hundred andforty pathogen-free inbred male BALB/c mice, 4-weeks old,weighing 14—16 g, were used in this study. They were pur-chased from Shanghai Experimental Animal Center, ChineseAcademy of Sciences, Shanghai, China (Certificate No.153 -SCXK(SH)2007-0005), and maintained at the animal facilityof Fudan University. They were housed under consistent conditions at a temperature of 23�1 °C, the humidity of40�5%, and on a 12 : 12 h light–dark cycle. They had freeaccesses to food pellets and tap water. All animals werecared for in accordance with the policies and guidelines ofEthic Committee for Animal Use in Shanghai Institute ofMateria Medica.

Inoculation of Viruses Briefly, 2�LD50 of CVB3(Nancy Strain) was diluted in phosphate-buffered saline to afinal volume of 0.2 ml, and intraperitoneally injected intomice of the myocarditis model and treatment groups(n�120). Mice in the normal control group were similarlyinjected with 0.2 ml PBS without viruses (n�20).

Treatment and Observation Mice were divided intothree groups of 40 mice. In the CCGR-treated group, theywere orally administered with daily dose of 24 mg/kg CCGRfor 14 d; in the infected control group, they received men-struum (5‰ CMC) without any drug; in the HQ group, in-fected mice received HQ (10 ml/kg) alone. All mice were ob-served daily for morbidity and mortality, and were recordedduring the 14-d experiment. The body weight (BW) andheart weight (HW) of each animal were measured to calcu-

late the HW/BW as the heart index. From each group, 18mice were evaluated for mortality; and the others were usedfor echocardiography, histology and viral titer examinationson days 7 and 14.

M-Mode Echocardiography M-mode echocardiogra-phy was performed with the small animal echocardiographyanalysis system (Vevo770, Canada) in the Institute of Bio-medical Sciences, Fudan University. Briefly, mice werelightly anesthetized by inhalation of 1—1.5% isoflurane, andthe upper sternal and subxiphoid areas were shaved and thenmoistened for better acoustic coupling. Electrode was at-tached to mouse limbs and parameters of the cardiac cyclewere recorded by M-mode echocardiography of the left ven-tricle at the tip of the mitral valve apparatus with a 30 MHztransducer. The interventricular septal (IVS) thickness, leftventricular internal diameter (LVID), posterior wall (PW)thickness, left ventricular volume (LV Vol) were measured inboth systole and diastole phases. Percentages of ejectionfraction (EF%) and percent fractional shortening (FS%) werecalculated by Vevo770 analysis software. To minimize heartrate variability during the procedure, mouse-specific anes-thetic dosages were strictly monitored and maintained.13,14)

Histology On days 7 and 14 after viral inoculation,hearts were dissected from mice of each group. Inflammatorylesions in the myocardium were recorded by a scale of 1 to 4based on the sizes of the inflammatory foci, from the small-est to the biggest. Then the hearts were fixed in 10% forma-lin solution, embedded in paraffin, sectioned serially to 5 mmthickness, and stained with hematoxylin–eosin. Sectionswere examined under a light microscope blindly and inde-pendently for signs of myocarditis. The combination ofpathological features of lymphocytic infiltration and myocar-dial necrosis was used to determine the histological scores ofthe hearts on a scale of 1 to 4 in terms of severity: 0, no infil-tration or necrosis; 1, 1—25% infiltration or necrosis per sec-tion; 2, 26—50% infiltration or necrosis per section; 3, 51—74% infiltration or necrosis per section; 4, 75—100% infil-tration or necrosis per section. Photographs were taken witha Cannon camera.

Virus Titers of the Heart On day 7 and day 14 afterviral inoculation, 5 mice from each group were dissected forinfectivity assays. Their hearts were removed, weighted, andhomogenized aseptically in 2 ml of MEM. After centrifuga-tion at 1200�g for 15 min at 4 °C, the supernatant was 10-fold serially diluted with MEM and then used to inoculateVero cell monolayers, as described previously.15) The 96-welltissue culture microplates were observed daily for cytopathiceffects for 5 d, and viral titers were determined by Reed–Munch method.16)

Statistics Data were analyzed with SPSS 11.5 softwarepackage (SPSS Inc., Chicago, IL, U.S.A.). All experimentalvalues obtained from above assays were expressed asmean�S.D., and examined by the two-sample test or one-way analysis of variance. Mean virus titers of the treated anduntreated groups were compared with Mann–Whitney non-parametrical test; survival rates were compared by nonpara-metric c2-test. A probability of less than 0.05 was consideredto be statistically significant.

January 2009 69

RESULTS

In Vitro Experiments The cytotoxicity and the antiviralactivity of CCGR against CVB3 were evaluated in Vero cells,and the results were reported in Table 1. CCGR showed a lowtoxicity to Vero cells with the CC0 value 125 mg/ml and bet-ter antiviral activity with the IC50 value 25 mg/ml. In the anti-coxsackie virus B3 assay, the selectivity index (ratio of cyto-toxicity to antiviral activity) of CCGR and ribavirin was 5.7and 6.3, respectively. This observation indicated that CCGRinhibited virus replication in Vero cells, and this activity isstronger and more specific than that of the common antiviraldrug ribavirin.

In Vivo Experiments. Survival Rate After mice wereinoculated with viruses, they became morbid on day 4,started to die on day 7. The death rate reached a peak on day10 and stabilized after day 14. During this process, a series ofmyocarditis symptoms occurred before the death of animals,such as weight loss, reduced appetite and body movement,hair loss, low body temperature, and muscle paralysis. Ourobservation showed that CCGR treatment alleviated theseverity of all these symptoms and reduced the animal mor-bidity. The survival rate of mice in the CCGR-treated groupwas 77.7%, significantly higher than that of the infectedgroup (44.4%). While, in the HQ-treated group, only 50% in-fected mice were protected from death during 14-d treatment(Fig. 2, Table 3).

M-Mode Echocardiography M-mode echocardiogra-phy analysis was performed with the small animal echocar-diography analysis system. The stable beat rates of all ani-mals in each group indicated the appropriate anaesthetic stateduring the operation (Table 2). All the infected mice exhib-ited the severe cardiac dysfunctions. The echocardiographyshowed the severely depressed movement of the ventricularwall, while strong echo on focus of cardiac muscle in the in-

fected mice (Fig. 5). The EF%, FS%, systolic IVS, systolicLVPW, systolic LV Vol were all lower significantly in in-fected mice than in uninfected mice, while diastolic LV Vol,diastolic LVID, systolic LVID were all higher significantly(Fig. 5, Table 2) compared with the uninfected mice. Treat-ments with CCGR and HQ enhanced cardiac functionsthrough enhancing contractility of cardiac muscles that re-sulted in higher percentages of ejection fractions. The pro-gression of heart failure in the infected mice was inhibited byCCGR and HQ through increased IVS, LVPW, LV Vol in thesystole phase. The LV systolic volume of CCGR-treatedmice were about 2-fold higher on day 14 than untreated-con-trol mice while EF% was higher by 63.6% and FS% waslower by 82.3% compared with those of the untreated-controlmice (Table 2).

Histology The pathological process of VMC is often ac-companied by the inflammation in cardiac muscles. In themouse acute viral myocarditis model, the inflammatory focidispersed in ventricular muscles and epicardium and weredetected more frequently in the ventricles and less in theatria. They were mostly white, appeared as stripes or oneconverged big focus in the heart.

In our study, we observed that CCGR prevented swellingof myocardial cells, enhanced eosinophil and inhibited my-ocardial necrosis and mononuclear cell infiltration caused byCVB3 infection in the mice (Fig. 3, Table 3). On both days 7and day 14, the scores of apparent pathological changes of

70 Vol. 32, No. 1

Table 1. Inhibitory Effects of CCGR on CVB3 Replication in Vero Cells

Drug CC50 (mg/ml) CC0 (mg/ml) IC50 (mg/ml) SI

Ribavirin 750 500 120 6.3CCGR 143 125 25 5.7

CC50 is the concentration of the drug showing 50% cytotoxicity. CC0 is the maximalconcentration of the drug showing 0% cytotoxic effect. IC50 is the concentration of drugrequired to inhibit 50% cytotoxic effect. SI�selective index�CC50/IC50.

Fig. 2. Change in the Survival Rate of CVB-Infected Mice in Response toTreatment with 5‰CMC, CCGR (24 mg/kg) or HQ (10 ml/kg)

After 14-d treatment, CCGR elevates significantly the survival rate of infected miceof acute VMC (p�0.05).

Table 2. Echocardiographic Evaluation for the Left Ventricular Functions in Viral-Infected Mice 14 d after CCGR or HQ Treatment

IndicesNormal control Infected control CCGR-treated (24 mg/kg) HQ-treated (10 ml/kg)

n�7 n�6 n�6 n�6

EF (%) 81.05�6.84a),# 35.96�7.90b),# 58.97�13.99a),#,b),# 53.53�11.14a),#b),#

FS (%) 49.04�7.24a),# 16.99�4.19b),# 31.02�9.55a),#,b),# 27.40�7.03a),*,b),#

IVS; dc) (mm) 0.81�0.09 0.75�0.20 0.94�0.11a),* 0.76�0.17IVS; sc) (mm) 1.30�0.13a),# 0.94�0.29b),# 1.24�0.19a),* 1.11�0.23LVPW; dc) (mm) 0.91�0.18 0.97�0.09 0.86�0.23 0.90�0.10LVPW; sc) (mm) 1.45�0.18a),# 1.1�0.10b),# 1.25�0.25 1.19�0.23b),*LV Vol; dc) (m l) 39.45�17.02a),* 61.55�5.06b),* 45.90�20.17 51.80�9.46LV Vol; sc) (m l) 7.12�2.58a),# 39.55�6.76b),# 20.33�3.18a),#,b),* 25.30�0.09a),*,b),#

LVID; dc) (mm) 3.17�0.51a),* 3.79�0.13b),* 3.29�0.62 3.51�0.27LVID; sc) (mm) 1.60�0.22a),# 3.14�0.23b),# 2.30�0.67a),#,b),# 2.56�0.43a),*,b),#

HR (beats/min) 315.6�43.56 321�16.19 329�21.13 304�25.90

Data were represented as mean�S.D. of values obtained from replicated samples in each group. a) Values were compared with those in the infected control group; b) valueswere compared with those in the normal control group. c) “d”: the diastole phase; “s”: the systole phase; ∗ p�0.05, # p�0.01.

the myocardium, cellular infiltration and necrosis in CCGR-treated group were all significantly lower than those in the in-fected-control group without drug treatment (Fig. 4, Table 3).However, there was no difference found in pathologicalscores between CCGR- and HQ-treated groups.

Myocardial Virus Titers Tissue extracts from the heartsof infected mice were transferred to cultures of Vero cells.Viral titers were then determined by viral cytopathic effects,and expressed as the tissue culture mean infectious dose(TCID50). There was no significant difference in the virus

January 2009 71

Table 3. Effects of CCGR on the Survival Rate, the Heart Index, Myocardial Lesions and Virus Titers in Infected Mice 14 d after Treatment with CCGR

Survival HW/BW Pathological scores of Virus titers in the heart

Groups (n) myocardium (day 14) TCID50/mg tissue (day 14) rate (%) (mean�S.D.)

(1—4, mean�S.D.) (mean�S.D.)

Normal control 8 100% (8/8) 5.04�0.41 0�0 0�0Infected control 18 44.4% (8/18) 5.95�0.29 2.33�1.03 4.10�0.32HQ-treated (10 ml/kg) 18 50% (9/18) 5.71�0.48 2.0�0.89 3.28�0.48*CCGR-treated (24 mg/kg) 18 77.7% (14/18)* 5.23�0.68 1.50�0.5* 3.06�0.56*

The viral infected mice were sacrificed for assays of the heart index (HW/BW), the pathological feature and the virus titer of the cardiac muscles. Data were represented as themean�S.D. from values obtained from replicated samples in each group. Statistical significance: ∗ p�0.05, as compared with the infected control.

Fig. 3. Histology of Transverse Section of Heart Muscles from Mice inDifferent Groups

A, from a normal mouse (400�); B, C, E, the viral-infected mouse on day 7 (C,400�) and day 14 (B, 40� and E, 400�); or from CCGR-treated mouse on day 7 (D,400�) and on day 14 (F, 400�). The arrows indicate foci of the myocardial necrosisand mononuclear cell infiltration.

Fig. 4. Histological Scores of Heart Muscles from Mice in DifferentGroups

Pathologic features of hearts of BALB/c mice in the normal control, the infectedcontrol, CCGR-treatment group and the HQ-treatment control were shown quantita-tively. Typically the scores of lesion of mononuclear cell infiltration and necrosis weresignificantly alleviated in CCGR group. ∗ p�0.05, compared with the infected controlgroup.

Fig. 5. M-Mode Tracings and Echocardiographic Analyses of Mice inDifferent Groups

A normal BALB/c mouse (A), a mouse from CCGR-treated group (B), and a mousefrom the infected control group (C). The infected heart showed strong echo and de-pressed movement of the ventricular wall. Left ventricular function was improved in theCCGR group (B) as demonstrated by increased ejection fraction and fractional shorten-ing.

titers in the same group between days 7 and 14 after CVB3infection. However, the cardiac viral titers were significantlylower in CCGR-treated group than those in the untreatedgroup on both days 7 and 14, but not in the HQ-treated or theinfection control groups. This indicated that HQ did not havesignificant antiviral activity (Table 3).

DISCUSSION

Coxsackieviruses belonging to enteroviruses are composedof class A with 23 serotypes and class B of 6 serotypes.17)

Coxsackievirus subtype B (CVB) is the leading viralpathogen for myocarditis.18,19) Although exact mechanism isstill not clear, coxsackievirus B-induced damages in my-ocytes are likely involved in the immune-mediated and directviral cytotoxicities.20)

Flavonoid compounds are the main active components inAstragalus that protect the cardiac functions in the process ofischemia reperfusion.21) Several flavonoids have been iso-lated and identified from traditional Chinese medicines, how-ever, their antiviral activities have not been reported. In thisstudy, CCGR, one important isoflavonoid from Astragalus,was assayed for such activities both in vitro and in vivo.

CVB3-infected mice are common experimental models foracute and chronic myocarditis. In such models, mice withviral infection showed various symptoms from death tosymptom-free living. The variation in the manifestations ofthe disease reflects the diversity of individual traits in ani-mals; therefore, sufficient numbers of animals were used ineach treatment to ensure statistically meaningful evaluationof the antiviral activities of the drug. In these murine acuteVMC models, viral replication, left Ventricular dysfunctionand pathologic remodeling resulted in high mortality. In ourstudy, pathological evolvement of cardiac muscle was corre-lated with LV dysfunction and the mortality rate. The cardiacfunction by echocardiography was useful in determiningphysiological functions of antivirus medicine as a whole.

The numbers of infectious CVB3 virus in the heart de-pended on the rate of viral infection in the cardiac musclecells. The cellular uptakes and replication of viruses result ininflammation and dysfunction of heart, therefore, inhibitionof CVB3 replication in vivo may offer an effective therapy. Inour study, CCGR was evaluated as a new candidate anti-CVB3 medicine by its effects on the intuitionistic surviralrate and virus titers in heart, as well as the pathologicalchange and holistic hemodynamics.

CCGR-treatment group showed a significant improvementin the overall survival rate and the reduction of adversepathological changes. These effects of CCGR were mediatedby blockade in virus replication in the heart and the protec-tion of cardiac muscles. Interestingly, certain attenuation ofmyocardial inflammation seems to be helpful for the im-provement of survival rate in the acute viral myocarditismodel. Together, our in vitro and in vivo results demonstratedCCGR’s potent antiviral activity against coxsackie virus B3.The datum implied that anti-CVB3 activity was the key foun-dation to protect the infected mice, resulting in improved car-diac function and survival rate. The results also suggestedthat CCGR was one of the active substances in Radix astra-gali to treat viral myocarditis. HQ has been widely used to al-leviate symptoms of chronic viral myocarditis in clinical

practice in China.3) Radix astragali itself was raw material.As a raw preparation of traditional Chinese medicine, despiteits known effects in chronic myocarditis, in this study, HQdid not show significant activity in acute myocarditis. Thismay simply indicate the different mechanism of viral actionsor pathogenesis underlying in the acute and chronic my-ocarditis and the difference between human and mouse. Inthis study, we only tested oral administration of CCGR. Dueto its known low cumulative urinary excretion and lowplasma concentration in animals,11) the antiviral activity ofCCGR observed in this study may be further improved if ad-ministration routes and approach can be optimized.

Together, results presented here demonstrated that CCGRis a promising anti-viral agent against coxsackie B3 virus,evidenced by its significant effect in the improvement of sur-vival rate and left ventricular function, and attenuation ofpathological evolvement of the infected mice. Viral infectionof the myocardium and the ensuing inflammatory reactionconstitute the major pathogenic mechanisms, which ulti-mately lead to left ventricular dysfunction. The balance be-tween the protective and deleterious immune mechanismsmay eventually determine the course of the disease. It hasbeen reported that Radix Astragali control viral infection andimprove heart function, probably by regulating the cellularimmunity.2,3) In the study, CCGR as a main component inRadix Astragali had been approved that it inhibited the viralreplication and improved the cardiac function. It was specu-lated that CCGR modulate cellular immunity and maintain-ing the balance. We believed that this study will providevaluable data for future study of its anti-inflammation andimmune-modulation functions.

Acknowledgements We sincerely thank National Nat-ural Science Foundation of China (30500655) and Scienceand Technology Commission of Shanghai Municipality(06DZ22906 and 02DZ19114) for their financial supports.

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