The American Journal of Chinese Medicine, Vol. 40, No. 1, 151–162 2012 World Scientiﬁc Publishing Company
Institute for Advanced Research in Asian Science and Medicine
Lien Chai Chiang§ and Chun Ching Lin* †
†School of Pharmacy, College of Pharmacy
§Department of Microbiology, College of Medicine
Abstract: Human respiratory syncytial virus (HRSV) causes serious pediatric infection of thelower respiratory tract without effective therapeutic modality. Sheng-Ma-Ge-Gen-Tang(SMGGT; Shoma-kakkon-to) has been proven to be effective at inhibiting HRSV-inducedplaque formation, and Cimicifuga foetida is the major constituent of SMGGT. We tested thehypothesis that C. foetida effectively inhibited the cytopathic effects of HRSV by a plaquereduction assay in both human upper (HEp2) and lower (A549) respiratory tract cell lines. Itsability to stimulate anti-viral cytokines was evaluated by an enzyme-linked immunosor-bent assay (ELISA). C. foetida dose-dependently inhibited HRSV-induced plaque formationð p < 0:0001Þ before and after viral inoculation, especially in A549 cells ð p < 0:0001Þ.
C. foetida dose-dependently inhibited viral attachment ð p < 0:0001Þ and could increaseheparins effect on viral attachment. In addition, C. foetida time-dependently and dose-dependently ð p < 0:0001Þ inhibited HRSV internalization. C. foetida could stimulateepithelial cells to secrete IFN-β to counteract viral infection. However, C. foetida did notstimulate TNF- secretion. Therefore, C. foetida could be useful in managing HRSVinfection. This is the ﬁrst evidence to support that C. foetida possesses antiviral activity.
Keywords: Cimicifuga foetida; Plaque Reduction; Anti-Viral Activity; Respiratory SyncytialVirus.
Correspondence to: Dr. Chun-Ching Lin, Graduate Institute of Natural Products and School of Pharmacy, College
of Pharmacy, Kaohsiung Medical University, 100 Shin-Chuan 1st Road, Kaohsiung 80708, Taiwan. Tel: (þ886)7-312-1101 (ext. 2122), Fax: (þ886) 7-313–5215, E-mail: firstname.lastname@example.org
Human respiratory syncytial virus (HRSV) is a major cause of lower respiratory tractinfections in infants, young children, and adults HRSV is themost major viral pathogen of the respiratory tract in infants younger than one year old; , Infection and re-infection withHRSV are most frequent during the ﬁrst few years of life (, Therefore, of all children that are infected by 24 months, half experienced two infections, Effective therapeutic modalities are highly needed. However,only supportive care is given to manage HRSV-induced severe lower respiratory tractinfection Ribavirin is a guanosine analogue that is aninhibitor of inosine monophosphate (IMP) dehydrogenase. It interferes with early events inviral transcription and inhibits ribonucleoprotein synthesis , ). Althoughit was effective in experimentally infected animals, ribavirin has shown little effecton treating HRSV ; Pali-vizumab (Synagis) is effective at preventing HRSV infection , However, it is very expensive and is not effective at the therapy of an established infection). Therefore, effective chemotherapeutic agents are still urgentlyneeded.
Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) has been used to manage
pediatric viral infection. It has been proven to be effective at inhibiting HRSV-inducedplaque formation in vitro ). Cimicifuga foetida L. is a major constituentof SMGGT. C. foetida has been used as a medical plant for anti-pyretic and detoxiﬁcativepurposes in ancient China for thousands of years. C. foetida has anti-bacterial, anti-inﬂammatory, and anti-neoplastic activities (). Several constituents ofC. foetida have been proven to have anti-cancer ; , collagenolytic (, ), and anti-complement activities However, its anti-viral activity has not been examined. We hypothesized that C. foetida, amajor constituent of SMGGT, might have activities against HRSV. We used both humanupper (HEp2) and low (A549) respiratory tract cell lines to prove that C. foetida waseffective on cytopathic effects induced by HRSV.
Preparation of Hot Water Extracts of Cimicifuga foetida L.
Water extract of air-dried C. foetida L. Rhizoma was prepared as reported previously(, The authenticity of C. foetida was conﬁrmed by Professor M.H. Yenat the Graduate Institute of Natural Products of Kaohsiung Medical University. Brieﬂy,100 g of C. foetida was shade-dried and decocted for 1 h with 1 L of boiling reverse-osmotic water three times. The decoctions were mixed, ﬁltered, concentrated and lyo-philized. The w/w yield of C. foetida was 10.7%. The extract of C. foetida was thendissolved in minimum essential medium (MEM, Gibco BRL, Grand Island, NY, USA)
and supplemented with 2 or 10% fetal calf serum (FCS) into the ﬁnal concentrations
ð10, 30, 100, 300 g/ml for bioactivity assay and up to 3000 g/ml for cytotoxicity test)before experiments.
Human larynx epidermoid carcinoma cells [HEp-2; ATCC (the American Type CultureCollection) CCL 23] and human lung carcinoma cells (A549 cells; ATCC CCL-185) wereused to culture human respiratory syncytial virus (RSV Long strain: ATCC VR-26).
Reagents and medium for cell culture were purchased from Gibco BRL. Cells were pro-pagated at 37C under 5% CO2 in minimum essential medium (MEM) supplemented with10% fetal calf serum (FCS), 100 U/ml penicillin G sodium, 100 g/ml streptomycin sulfateand 0.25 g/ml amphotericin B. Virus was propagated on 90% conﬂuent cell monolayer inMEM with 2% FCS and antibiotics as described above. Viral titer was determined byplaque assays and expressed as plaque forming units per ml (pfu/ml). Virus was stored atÀ70C until use.
Cytotoxicity of C. foetida on proliferating cells was assayed by XTT-based methodBrieﬂy, cells ð1 Â 104 cells/well) were seeded into 96-well cultureplates (Falcon; BD Biosciences, USA) and incubated overnight at 37C under 5% CO2.
Then, the medium was removed and different concentrations ð30, 100, 300, 1000, 3000 g/ml) of C. foetida were applied in triplicate. After three days of incubation, the cytotoxicityof C. foetida was determined by XTT (sodium 30-[1-(phenylamino-carbonyl)-3,4-tetra-zolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid) kits (Roche Diagnostics GmbH,Mannheim, Germany) according to the manufacturer’s instructions. The 50% cytotoxicconcentration (CC50Þ of C. foetida was calculated by regression analysis of the dose-response curve generated from the data.
Antiviral Effectiveness Assay by Plaque Reduction Assay
Antiviral activity of C. foetida was examined by a plaque reduction assay modiﬁed fromprocedures previously described (, Brieﬂy, cells
ð1 Â 105/well) were plated in 12-well culture plates for 24 h and were inoculated with amixture of 200 pfu/well HRSV and various concentrations of C. foetida for 1 h. Ribavirin(Sigma, St. Louis, USA) was used as a positive control. After supplement of overlaymedium (MEM plus 2% FCS in 1% methylcellulose), they were cultured at 37C under5% CO2 for three days. The monolayers were ﬁxed with 10% formalin, stained with 1%crystal violet, and the plaques were counted. The minimal concentration required to inhibit50% cytopathic effect (IC50Þ of C. foetida was calculated by regression analysis of thedose-response curve generated from the data.
Antiviral activity of C. foetida was examined before and after viral inoculation by plaquereduction assay modiﬁed from procedures mentioned above (, Brieﬂy, cells were seeded and incubated for 24 h aspreviously described. C. foetida of various concentrations was supplemented at À2 h (2 hbefore viral inoculation), À1 h (1 h before viral inoculation), or 1 h or 2 h (1 h or 2 h afterviral inoculation). Supernatant was removed before supplement of overlay medium. Theywere incubated for a further 72 h as mentioned above. After ﬁxation, crystal violet wassupplemented and the plaques were counted.
The effect of C. foetida on viral attachment was evaluated by a plaque reduction assaymodiﬁed from procedures previously described ; ,). Heparin (Sigma, St. Louis, USA) was used as a positive control. Brieﬂy, cells wereseeded and incubated for 48 h. The cells were pre-chilled at 4C for 1 h and the mediumwas removed. The cells were infected with a mixture of 200 pfu/well HRSV and variousconcentrations of C. foetida. After incubation at 4C for another 3 h, the free virus wasremoved. The cell monolayer was washed with ice-cold phosphate-buffered saline (PBS)thrice, covered with overlay medium, incubated for further 72 h at 37C under 5% CO2,and examined by plaque assay as described earlier.
The effect of C. foetida on viral internalization was also evaluated by a plaque reductionassay described earlier , ). Brieﬂy, the cell monolayer was grown in12-well culture plates and pre-chilled at 4C for 1 h. Cells were infected with 200 pfu/wellHRSV and incubated at 4C for 3 h to allow virus binding without internalization.
The virus-containing medium was replaced with fresh medium containing variousconcentrations of C. foetida and cultured at 37C. In 20 min intervals, acidic PBS (pH 3)was supplemented for one minute to deactivate un-internalized virus followed byalkaline PBS (pH 11) for neutralization. Then, PBS was replaced by fresh overlay medium.
After incubation at 37C for further 72 h, the cell monolayer was examined by theplaque assay.
Interferon-ß (IFN-ß) and Tumor Necrosis Factor- (TNF-) Assay
After the experiment of antiviral effectiveness assay mentioned above, the culture mediumwas collected and assayed by the IFN-β ELISA kit (PBL Biomedical Laboratories, Pis-cataway, USA) and TNF- ELISA kit (R&D Systems, Minneapolis, USA) according to themanufacturer’s instruction. The A450 nm was determined with ELISA reader (Multiskan EX,Labsystems).
Results were expressed as mean Æ standard deviation (S.D.). Percentage of the control(infection rate; %) was calculated from the plaque counts of C. foetida groups divided bythat of viral control group. Data were analyzed with ANOVA by JMP 7.0.1 software (SASInstitute, Cary, NC, USA). Tukey honestly signiﬁcant difference (HSD) test was used tocompare all pairs of groups in the ANOVA test. p < 0:05 was considered statisticallysigniﬁcant.
C. foetida did not show any cytotoxicity against both HEp-2 and A549 cells at concen-trations up to 3000 g/ml (Fig. Instead, C. foetida might slightly increase theproliferation of HEp-2 cells. The estimated CC50 was more than 3000 g/ml. The higherCC50 proved its safety.
C. foetida and ribavirin were dose-dependently (Fig. p < 0:0001) effective againstHRSV in both HEp2 cells and A549 cells. C. foetida was more effective in A549 cells(Fig. p < 0:0001). However, the effect of ribavirin was similar in both HEp-2 cells andA549 cells (Fig. ). The IC50 of C. foetida was 67.3 g/ml in HEp-2 cells and 31.0 g/mlin A549 cells.
Figure 1. C. foetida did not show any cytotoxicity up to 3000 g/ml. Data were presented as mean Æ S.D. of 3independent experiments.
Figure 2. C. foetida were effective against HRSV in antiviral effectiveness assay. Both C. foetida (A) and
ribavirin (B) were dose-dependently ðp < 0:0001Þ effective against HRSV determined by the plaque reductionassay. C. foetida was more effective in A549 cells ðp < 0:0001Þ. However, ribavirin did not show the difference.
Data were presented as mean Æ S.D. of nine tests. *p < 0:05;**p < 0:001;***p < 0:0001 compared to the viralcontrol.
C. foetida was effective ðp < 0:0001Þ both before and after viral inoculation in both HEp-2cells and A549 cells in a dose-dependent manner. C. foetida had a better effect when givenafter viral inoculation in HEp-2 cells (Fig. In HEp-2 cells, the IC50 was 261.0 g/ml(2 h before viral inoculation), 232.8 g/ml (1 h after viral inoculation), and 151.3 g/ml(2 h after viral inoculation). Its effect on A549 cells was similar (Fig. ); however, withno time-dependent effect. When it was supplemented 2 h after viral inoculation,C. foetidacould show a better anti-HRSV activity at concentrations higher than 100 g/ml in A549cells. Its IC50 was 205.4 g/ml (2 h before viral inoculation), 268.5 g/ml (1 h before viralinoculation), 212.4 g/ml (1 h after viral inoculation), and 150.0 g/ml (2 h after viralinoculation) in A549 cells.
Since C. foetida could effectively inhibit HRSV-induced plaque formation when givenbefore HRSV infection, C. foetida was hypothesized to be effective on viral attachmentand/or internalization. The results of attachment assay conﬁrmed this assumption. C.
foetida dose-dependently inhibited viral attachment in both HEp-2 cells and A549 cells(Fig. p < 0:0001), with a better effect on A549 cells ðp < 0:0001Þ. The IC50 was82.1 g/ml in HEp-2 cells and 70.6 g/ml in A549 cells. Heparin could dose-dependentlyprevent RSV attachment (Fig. p 0:0001). C. foetida could further improve the effectof heparin (Fig. ; p < 0:0001) in both cells. The estimated IC50s of C. foetida with
Figure 3. C. foetida was effective in both before and after viral inoculation in time course assay. C. foetida seemed
to be better given after viral inoculation in HEp-2 cells (A). However, this effect was not clear in A549 cells (B).
Nevertheless, C. foetida was dose-dependently ðp < 0:0001Þ effective against HRSV in both HEp-2 (A) and A549(B) cells. Data were presented as mean Æ S.D. of nine tests. *p < 0:05;**p < 0:001;***p < 0:0001 compared tothe viral control.
0.01 g/ml heparin were 19.5 g/ml and 13.1 g/ml in HEp-2 and A549 cells, respectively.
It is interesting to note that C. foetida initially had a higher IC50 on viral attachment inHEp-2 cells. However, when it was combined with heparin, C. foetida had a lower IC50 inA549 cells. Moreover, 0.001 g/ml heparin unexpectedly showed a synergistic effect with
Figure 4. C. foetida inhibited viral attachment. (a) SMGGT was dose-dependently effective against viral
attachment in both HEp-2 cells and A549 cells ðp < 0:0001Þ, with a better effect on A549 cells. (b) C. foetidacould further increase the effect of heparin at concentrations higher than 30 g/ml. Data were presented asmean Æ S.D. of nine tests. *p < 0:05; **p < 0:001; ***p < 0:0001 compared to the viral control group.
Figure 5. C. foetida inhibited viral internalization. C. foetida was time-dependently and dose-dependentlyðp < 0:0001Þ inhibited plaque formation caused by HRSV in both HEp-2 (A) and A549 cells (B). The effect wassimilar in both cell lines. Data were presented as mean Æ S.D. of nine tests. *p < 0:05;**p < 0:001; ***p < 0:0001compared to the viral control.
30 g/ml C. foetida. The effects of different concentrations of heparin with 30 g/mlC. foetida were similar (Fig. ).
C. foetida was time-dependently and dose-dependently (Fig. p < 0:0001) effectiveon HRSV internalization in both HEp-2 and A549 cells. The effects were quite similar inboth cells. C. foetida had a IC50 of 118.7 g/ml (20 min incubation), 37.4 g/ml (40 min),25 g/ml (60 min) in A549 cells, and 180.9 g/ml (20 min incubation), 94.9 g/ml (40min), 29.6 g/ml (60 min) in HEp-2 cells.
Interferon-ß (IFN-ß) and Tumor Necrosis Factor- (TNF-) Assay
The basal IFN-β and TNF- secretion in both A549 and HEp-2 cells was similar (Fig. After HRSV infection, IFN-β and TNF- secretion could be stimulated (Fig. p < 0:05).
C. foetida could stimulate IFN-β secretion in both HEp-2 and A549 cells with or withoutHRSV infection (Figs. and ; p < 0:0001). This effect was more prominent on A549cells ðp < 0:0001Þ. In contrast, C. foetida did not stimulate TNF- secretion in both HEp-2and A549 cells with or without HRSV infection (Figs. and ).
HRSV can infect upper respiratory mucosa and replicate initially in the nasopharynx). HRSV can spread rapidly to the lower respiratory tract possibly
Figure 6. The effect of C. foetida on the secretions of interferon (IFN) and tumor necrosis factor (TNF) in HEp-2
cells (A) and (C) and A549 cells (B) and (D). RSV infection might increase IFN-β/TNF- secretion. C. foetidadose-dependently ðp < 0:0001Þ stimulated both cell lines (A) and (B) to secrete IFN-β with or without HRSVinfection. In contrast, C. foetida did not induce TNF- secretion with or without HRSV infection in both cells(C) and (D). Data were presented as mean Æ S.D. of 12 tests. *p < 0:05; **p < 0:001; ***p < 0:0001 compared tothe control group. #p < 0:05 compared to the cell control.
by aspiration of secretions (). HRSV primarily causes morbidityand mortality by the pathology of the lower respiratory tract (, ).
Therefore, management of HRSV infection needs an effective strategy to inhibit viralinfection of both upper and lower respiratory tracts. This experiment showed that C. foetidawas effective at inhibiting RSV-induced plaque formation in both human upper (HEp-2)and lower (A549) respiratory tract cells. Therefore, C. foetida could inhibit viral replicationin the nasopharynx. Furthermore, 300 g/ml C. foetida could inhibit HRSV-induced plaque
formation to less than 10% of the control in lower respiratory tract (A549) cells. Therefore,higher concentrations of C. foetida could largely inhibit HRSV-induced morbidity andmortality. When given after viral inoculation, C. foetida had a similar effect to that ofgiving after HRSV inoculation in the time course assay. Its preventive activity was furthersupported by an attachment assay and an internalization assay. Therefore, C. foetida waseffective at preventing and managing HRSV infection. Heparin is highly effective at pre-venting HRSV attachment C. foetida could further increase theeffect of heparin. Furthermore, C. foetida had a synergistic effect with 0.001 g/ml heparin.
Therefore, C. foetida could have mechanisms difference from those of heparin. Other thanpreventing viral attachment and internalization, C. foetida could stimulate IFN-β to preventHRSV infection. HRSV infection will induce cellular production of IFN-β and TNF-, Both IFN and TNF contribute to innate immunity against viralinfection , , ). C. foetida couldstimulate both HEp-2 and A549 cells to secrete IFN-β with or without HRSV infection.
Therefore, along with direct cytoprotection, C. foetida could be useful for preventing andmanaging viral infection by stimulating IFN-β. Although it is a potent antiviral cytokine,TNF- can activate p38MAPK to induce apoptosis of bronchial epithelia (,). C. foetida could not induce TNF- secretion in both HEp-2 and A549 cells. Therefore,C. foetida could be active against HRSV infection without inducing apoptosis of respiratorymucosa. Most of the therapeutic reagents under development aimed at inhibiting viralentrance , However, C. foetida was also effective afterviral inoculation, especially on HEp-2 cells, a human larynx epidermoid carcinoma cells.
HRSV replicates initially in the upper respiratory mucosa (, Therefore, C. foetida could be a better candidate to manage RSV infection.
In this study, C. foetida had low IC50s in antiviral effectiveness assay in which both
HRSV and C. foetida were concomitantly supplemented. Therefore, the IC50s should showa logical trend between antiviral effectiveness assay and time course assay. However, theIC50 was 31.0 g/ml in A549 cells when C. foetida was concomitantly supplemented withHRSV. When C. foetida was supplemented 2 h before viral inoculation in A549 cells, theIC50s were 205.4 g/ml. The IC50 did not change much from 268.5 g/ml (1 h before viralinoculation), 212.4 g/ml (1 h after viral inoculation), to 150.0 g/ml (2 h after viralinoculation). The IC50 in antiviral effectiveness assay was much lower than that of timecourse assay. It lacked a logical trend between the time course assay and the antiviraleffectiveness assay. This might raise a question about the validity of the experiment.
However, our results clearly showed C. foetida was also effective when supplemented afterviral inoculation. During the time course assay, C. foetida was removed before the sup-plement of overlay medium. Nevertheless, C. foetida remained there in the antiviraleffectiveness assay to exert its antiviral effect. Therefore, the results of the antiviraleffectiveness assay were the summation of all effects in the time course assay, so it wasreasonable to have a lower IC50 in the antiviral effectiveness assay.
C. foetida could prevent RSV infection by inhibiting viral attachment, internalization,
and by stimulating IFN-β secretion. Furthermore, C. foetida could be effective at inhibitingplaque formation after HRSV inoculation. C. foetida was quite different from therapeutic
reagents under development that aimed at inhibiting viral entrance only (,). Therefore, C. foetida is worthy to be further evaluated for itsactivity and mechanisms against HRSV.
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