APJTM148_proof ■ 26 December 2015 ■ 1/7

Asian Pacific Journal of Tropical Medicine 2015; ▪(▪): 1–7 1

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H O S T E D BY
Contents lists available at ScienceDirect 57
58
Asian Pacific Journal of Tropical Medicine 59
60
journal homepage: http://ees.elsevier.com/apjtm 61
62
1 63
2 Review http://dx.doi.org/10.1016/j.apjtm.2015.12.002 64
3 65
4 Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and 66
5 cinanserin on murine coronavirus and dengue virus infection 67
6 68
7 Q4 Q1 K.H. Chiow1,2,3, M.C. Phoon1,2,3, Thomas Putti1,2,3, Benny K.H. Tan1,2,3, Vincent T. Chow1,2,3* 69
8 1 70
Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore,
9 Kent Ridge, Singapore 71
Q2
10 2 72
Department of Pathology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore,
11 73
Kent Ridge, Singapore
12 74
3
13 Department of Pharmacology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore,
Kent Ridge, Singapore 75
14 76
15 77
A R TI C L E I N F O ABSTRACT
16 78
17 79
Article history: Objective: To evaluate the in vitro activities of the ethyl acetate (EA) fraction of
18 80
Received 15 Oct 2015 Houttuynia cordata (H. cordata) Thunb. (Saururaceae) and three of its constituent fla-
19 81
Received in revised form 20 Nov vonoids (quercetin, quercitrin and rutin) against murine coronavirus and dengue virus
20 82
2015 (DENV).
21 Methods: The antiviral activities of various concentrations of the EA fraction of 83
Accepted 3 Dec 2015
22 Available online xxx H. cordata and flavonoids were assessed using virus neutralization tests against mouse 84
23 hepatitis virus (MHV) and DENV type 2 (DENV-2). Cinanserin hydrochloride was also 85
24 tested against MHV. The EA fraction of H. cordata was tested for acute oral toxicity in 86
25 Keywords:
C57BL/6 mice. 87
26 Houttuynia cordata
Murine coronavirus
Results: The EA fraction of H. cordata inhibited viral infectivity up to 6 d. Cinanserin 88
27 hydrochloride was able to inhibit MHV for only 2 d. The 50% inhibitory concentrations 89
Dengue virus
28 (IC50) of the EA fraction of H. cordata added before the viral adsorption stage were 90
Quercetin
29 0.98 mg/mL for MHV and 7.50 mg/mL for DENV-2 with absence of cytotoxicity. The 91
Quercetrin
30 mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute 92
Cinanserin
31 toxicity, with normal histological features of major organs. Certain flavonoids exhibited 93
32 comparatively weaker antiviral activity, notably quercetin which could inhibit both MHV 94
33 and DENV-2. This was followed by quercitrin which could inhibit DENV-2 but not
95
34 MHV, whereas rutin did not exert any inhibitory effect on either virus. When quercetin
96
35 was combined with quercitrin, enhancement of anti-DENV-2 activity and reduced
97
36 cytotoxicity were observed. However, the synergistic efficacy of the flavonoid combi-
98
37 nation was still less than that of the EA fraction.
Conclusions: The compounds in H. cordata contribute to the superior antiviral efficacy 99
38 100
of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vivo. H. cordata
39 101
has much potential for the development of antiviral agents against coronavirus and
40 102
dengue infections.
41 103
42 104
43 105
44 106
45 1. Introduction emerged in 2003 and rapidly spread throughout the world, with a 107
46 mortality rate of 10%–15%. Although the disease disappeared in 108
47 Severe acute distress syndrome (SARS) is a highly conta- mid-2003, its re-emergence cannot be excluded since SARS-like 109
48 gious respiratory illness caused by SARS coronavirus, which coronaviruses are zoonotic and exist in animal reservoirs (e.g., 110
49 bats, raccoon dogs and palm civets), thus posing a potential risk 111
50 *Corresponding author: Vincent T. Chow, Host and Pathogen Interactivity for future epidemics [1–3]. SARS coronavirus is a large, 112
51 Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, enveloped, single-strand, positive-sense RNA virus. The viral 113
National University of Singapore, 5 Science Drive 2, Kent Ridge 117545, Singapore.
52 Tel: +65 65163691
genome is about 30 kbp, containing open reading frames that 114
53 Fax: +65 67766872 encode the polymerases required for viral RNA synthesis, while 115
54 E-mail: micctk@nus.edu.sg the remaining open reading frames encode structural proteins 116
Peer review under responsibility of Hainan Medical College.
55 117
1995-7645/Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Chiow KH, et al., Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection,
Asian Pacific Journal of Tropical Medicine (2015), http://dx.doi.org/10.1016/j.apjtm.2015.12.002
 APJTM148_proof ■ 26 December 2015 ■ 2/7

2 K.H. Chiow et al./Asian Pacific Journal of Tropical Medicine 2015; ▪(▪): 1–7

1 (i.e., spike, envelope, membrane and nucleocapsid) and acces- be consumed daily [15]. Flavonoids are known to display 63
2 sory proteins. antiviral activities, e.g., glabranine and 7-O-methyl-glabranine 64
3 First identified in 2012, the emerging Middle East respiratory against dengue virus, procyanidin and pelargonidin against 65
4 syndrome (MERS) coronavirus causes severe pneumonia HSV, and catechins against influenza virus [16,17]. 66
5 with multiorgan involvement (including acute renal failure), and Using virus-specific neutralization tests, this study tested 67
6 a mortality rate of 36% (446 deaths out of 1 244 cases docu- the EA fraction of H. cordata for its antiviral efficacy against 68
7 mented by WHO as of 9 June 2015). It is considered to be MHV (for the first time) and DENV-2 (as further verification 69
8 a zoonotic virus being transmitted from camels in the Middle using a different batch of H. cordata specimen). Three flavo- 70
9 East. Currently, no specific treatment exists against MERS noid components of H. cordata, i.e., quercetin, quercitrin 71
10 coronavirus. and rutin, were also investigated for their antiviral activities 72
11 The murine coronavirus, mouse hepatitis virus (MHV), is a against both viruses. They were also selected since they are 73
12 coronavirus that causes an epidemic murine illness with high common and naturally occurring flavonoids, whose molecular 74
13 mortality. Generally, MHV is extremely infectious to colonies of structures share high degrees of similarity. In the MHV ex- 75
14 mice and causes hepatitis upon infection. SARS, MERS and periments, we also compared the potency of cinanserin hy- 76
15 MHV belong to the group 2 coronaviruses and are classified drochloride which has been proven to neutralize SARS 77
16 under the genus Betacoronavirus. In view of the relatedness of coronavirus in vitro. All the compounds were also tested for 78
17 these coronaviruses, MHV was selected in this study to act as a cytotoxicity in vitro, while the EA fraction was also tested for 79
18 surrogate model for SARS and MERS coronaviruses which acute oral toxicity in mice. 80
19 necessitate BSL-3 facilities, whereas MHV is considered a BSL- 81
20 2 pathogen. 2. Methods and materials 82
21 Belonging to the genus Flavivirus, dengue virus (DENV) 83
22 represents the most important mosquito-borne viral disease with 2.1. Plant material, ethanolic extraction, aqueous-EA 84
23 considerable resurgence in many parts of the world. There are fractionation, and flavonoids of H. cordata 85
24 four antigenically-related but distinct DENV serotypes, such that 86
25 infection with one serotype does not confer life-long immunity The aerial parts (fresh leaves) of H. cordata were collected 87
26 against the other serotypes. DENV infection causes dengue fe- from a farm in Johor, Malaysia and authenticated in the 88
27 ver, and occasionally the more serious conditions of dengue Department of Pharmacology, National University of Singapore. 89
28 hemorrhagic fever and dengue shock syndrome [4]. Antibody- The voucher specimen of H. cordata was deposited in the 90
29 dependent enhancement is thought to play a central role in Singapore Botanic Gardens and assigned the identification 91
30 dengue pathogenesis, with the risk of developing dengue hem- number SING2011-203. Prior to extraction, the herb (dry weight 92
31 orrhagic fever and/or dengue shock syndrome being greater in of 500 g) was washed with de-ionized water, homogenized to a 93
32 secondary infections with DENV-2 compared to other serotypes fine powder, and soaked overnight in 80% (v/v) ethanol. The 94
33 [5]. The different manifestations of dengue may also be attributed next day, the ethanolic extract was removed and stored. More 95
34 to DENV variants with varying degrees of virulence [6], while solvent was added to the blended herb, which was left to soak till 96
35 viral load is also a contributing factor in the development of exhaustion. The extract was filtered, concentrated with a rotary 97
36 potentially fatal complications [7]. In addition, being a highly evaporator (Buchi Rotavapor R-144) and freeze-dried to yield 98
37 prevalent serotype in tropical and subtropical regions 19.28 g of crude extract in powder form. The crude extract was 99
38 worldwide, DENV-2 was selected for this study. Although then dissolved in EA and de-ionized water. The water and EA 100
39 several dengue candidate vaccines are undergoing clinical trials, phases were separated with a separating funnel. The EA phase 101
40 there are currently no effective antiviral therapies which are was concentrated with a rotary evaporator, and the concentrated 102
41 urgently needed to control dengue. EA fraction was subsequently stored at −80
C overnight and 103
42 Belonging to the Saururaceae family, Houttuynia cordata freeze-dried. The flavonoids tested were quercetin dihydrate 104
43 (H. cordata) Thunb. is a perennial plant native to mountainous (98% HPLC), quercitrin, and rutin hydrate (95%), all pur- 105
44 regions of eastern Asia, with an indefinite spread as a creeping chased from Sigma–Aldrich. 106
45 rhizome in moist locations. This herb possesses very promising 107
46 antiviral properties especially against clinically important 2.2. Mouse hepatitis virus, cell culture, and 108
47 enveloped viruses such as herpes simplex virus-1 (HSV-1), neutralization test 109
48 influenza virus, and human immunodeficiency virus-1 in vitro. 110
49 Interestingly, the steam distillate of H. cordata can strikingly CCL9.1, a normal Mus musculus (mouse) liver epithelial cell 111
50 inactivate an enveloped virus but is incapable of inactivating a line, was used for propagation of MHV and for the MHV 112
51 non-enveloped virus [8–12]. neutralization test. Compounds that were tested against MHV 113
52 To support these observations, we and others have demon- were the EA fraction of H. cordata, its flavonoid components, 114
53 strated that the ethyl acetate (EA) fraction of H. cordata is and cinanserin hydrochloride (Tocris Bioscience). Each com- 115
54 effective in inhibiting the infectivity of enveloped viruses such pound was 2-fold serially diluted with the corresponding diluent, 116
55 as DENV [13]. Meng et al. discovered 11 common peaks in the and the dilutions were then added to wells of a 96-well micro- 117
56 HPLC-DAD MS fingerprint of fresh H. cordata [14]. In our titer plate. Next, 100 TCID50 of MHV was added to each diluted 118
57 previous project, we verified some of these peaks in the EA compound and incubated at 37
C for 1 h with 5% CO2. Also 119
58 extract as polyphenols or flavonoids (chlorogenic acid, tested was treatment with diluent only at various concentrations. 120
59 hyperoside, quercetin and quercetrin) and have investigated Virus-infected controls and uninfected cell controls were 121
60 their antiviral efficacy against DENV [13]. Flavonoids are a included in each batch of assays. Confluent CCL9.1 cells were 122
61 class of natural products with high pharmacological potency, cultured in DMEM supplemented with 10% horse serum, and 123
62 are ubiquitous in photosynthesizing cells and hence likely to 1 × 105 cells were seeded into each well of another 96-well 124

Please cite this article in press as: Chiow KH, et al., Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection,
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 APJTM148_proof ■ 26 December 2015 ■ 3/7

K.H. Chiow et al./Asian Pacific Journal of Tropical Medicine 2015; ▪(▪): 1–7 3

1 microtiter plate, and incubated at 37
C with 5% CO2 until the The inhibition rates were plotted against various sample con- 63
2 cells were 80% confluent. Cell culture fluid from each well was centrations to ascertain the concentration that causes 50% 64
3 discarded. Each virus-compound mixture and controls were cytotoxicity (CC50). The EA fraction and flavonoids (either 65
4 transferred to these wells in duplicate, followed by addition of individually or in combination) were tested at the same con- 66
5 DMEM with 10% (v/v) horse serum. The plate was sealed and centrations as those for neutralization tests. The assay included 67
6 incubated at 37
C with 5% CO2, and observed daily for 6 d. wells containing medium only as well as untreated control cells. 68
7 The highest dilution of compound that inhibited cytopathic ef- Each experiment was repeated, and the mean and standard de- 69
8 fects (CPE) was considered as the minimum inhibitory con- viation were calculated. 70
9 centration (MIC). 71
10 2.5. Acute oral toxicity testing 72
11 2.3. Dengue virus, cell culture, and plaque reduction 73
12 neutralization test This approach was adapted from the OECD Guideline for 74
13 Testing of Chemicals, 420: Acute Oral Toxicity – Fixed Dose 75
14 The New Guinea C strain of DENV-2 was propagated in the Procedure, and relied on the observation of clear signs of 76
15 C6/36 Aedes albopictus mosquito cell line and maintained in toxicity or even mortality. C57/BL6 nulliparous and non- 77
16 Leibovitz L15 medium supplemented with 3% (v/v) fetal pregnant female mice about 8-weeks old were obtained from 78
17 bovine serum at 28
C under 5% CO2 atmosphere. The anti- the Laboratory Animals Center, National University of 79
18 DENV-2 activity of H. cordata and its flavonoids were eval- Singapore. Upon arrival, they were kept in cages for 5 d prior to 80
19 uated by plaque reduction neutralization test or PRNT [18,19]. commencement of the study. On the actual dose-feeding day, the 81
20 BHK-21 (baby hamster kidney) fibroblasts were cultured to mice were fasted for about 4 h. Doses of 100, 500, 1 000 and 82
21 form cell monolayers in 24-well plates with RPMI-1640 sup- 2 000 mg/kg were prepared for administration using water as 83
22 plemented with 10% (v/v) fetal bovine serum at 37
C under vehicle to suspend the ethanolic extract of H. cordata at a 84
23 5% CO2. The test compounds were dissolved in the relevant constant volume of 1 mL/100 g of body weight. In total, the five 85
24 diluents, and 2-fold serial dilutions were prepared to obtain groups of mice (n = 2 each) were fed with a single dose of the 86
25 different concentrations. DENV-2 New Guinea C neutralizing extract (including the vehicle control) by oral gavage. A sighting 87
26 monoclonal antibody 3H5 from Chemicon served as the pos- study was then conducted frequently from the first 30 min, with 88
27 itive control [20]. Diluent only (at various concentrations), special attention to the first 4 h. Thereafter, the condition of each 89
28 virus, and cell controls were also included by adding the mouse was observed daily to detect abnormalities such as 90
29 corresponding diluent, virus suspension, and medium without changes in physical appearance, behavioral signs and body 91
30 the treatment compounds. Each experiment was performed in weight. All mice were finally euthanized after 14 d of obser- 92
31 duplicate. DENV-2 (90 plaque-forming units or PFU) were vation, and major organs (brain, heart, lungs, liver and kidneys) 93
32 incubated for 1 h with various concentrations of each com- were harvested for histopathological examination after staining 94
33 pound together with controls before adding to the cells. The with hematoxylin and eosin (H&E). 95
34 virus-sample mixtures were incubated with the cells at 37
C 96
35 under 5% CO2 for another hour with rocking at 15-min in- 3. Results 97
36 tervals before the cells were overlaid with 1% carboxymeth- 98
37 ylcellulose at 37
C under 5% CO2 for 6 d. The cells were then 3.1. Anti-MHV activities of EA fraction of H. cordata, 99
38 fixed with 20% formaldehyde and stained with 1% crystal quercetin and cinanserin 100
39 violet, and the number of plaques was counted. The percentage 101
40 plaque reduction of the compounds at every dilution was 102
The EA fraction of H. cordata was evaluated at concentra-
41 103
determined as follows: (mean number of plaques in virus tions of 3.91 mg/mL down to 0.24 mg/mL. Table 1 and Figure 1
42 control) − (average number of plaques in sample) × 100% 104
show that the EA fraction of H. cordata exhibited anti-MHV
43 105
divided by (mean number of plaques in virus control). The activity at a MIC of 0.98 mg/mL without any apparent cyto-
44 percentage plaque reduction was plotted against various con- 106
toxic effects on CCL9.1 cells.
45 107
centrations of the test agents to determine the concentration Tested at concentrations from 500.00 to 15.63 mg/mL, the
46 that causes 50% plaque reduction (IC50). 108
anti-MHV effect of quercetin was significantly less, with MIC
47 109
48 2.4. MTT cell viability and proliferation assay 110
49 111
50 Table 1 112
The MTT cell proliferation assay was performed to determine
51 IC50, CC50 and selectivity indices of H. cordata fraction, flavonoids, and 113
the cell viability following exposure to the test compounds.
52 cinanserin against MHV. 114
Various concentrations of each test compound were added to
53 wells containing cell monolayers and incubated at 37
C under Sample IC50 (mg/mL) CC50 (mg/mL) Selectivity index 115
54 5% CO2 for 48 h. After incubation, MTT reagent was added to EA fraction 0.98 NCa >4.00b 116
55 each well, and further incubated for 4 h or until purple pre- Quercetin 125.00 116.52 0.93 117
56 cipitates were visible under an inverted microscope. Then, Quercitrin NIL NCa NIL 118
57 Rutin NIL NCa NIL 119
100 mL of 100 g/L SDS in 0.01 mol/L HCl were added to each Cinanserin (1 dpi) 31.25 NCa >4.00c
58 well and incubated overnight in the dark at room temperature. 120
Cinanserin (2 dpi) 62.50 NCa >2.00c
59 The optical density (OD) at 570 nm was then read, and the cell a
121
60 NC: No cytotoxicity at all concentrations tested against CCL9.1 cells. 122
inhibition rate calculated from the formula: [1 − (OD of sample b
Highest selectivity index of EA fraction (i.e., lowest concentration for
61 with cells) − (OD of sample without cells) divided by (OD of 123
viral inhibition coupled with absence of cytotoxicity). c Selectivity index
62 solvent with cells) − (OD of medium without cells)] × 100%. with absence of cytotoxicity. 124

Please cite this article in press as: Chiow KH, et al., Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection,
Asian Pacific Journal of Tropical Medicine (2015), http://dx.doi.org/10.1016/j.apjtm.2015.12.002
 APJTM148_proof ■ 26 December 2015 ■ 4/7

4 K.H. Chiow et al./Asian Pacific Journal of Tropical Medicine 2015; ▪(▪): 1–7

1 63
2 64
3 65
4 66
5 67
6 68
Figure 1. Neutralization of MHV infectivity by the EA fraction of H. cordata on 2 dpi.
7 (A) Uninfected control of CCL9.1 cells. (B) MHV-infected CCL9.1 cells control. (C) MHV-infected CCL9.1 cells subjected to 0.98 mg/mL of EA fraction.
69
8 (D) MHV-infected CCL9.1 cells subjected to 0.49 mg/mL of EA fraction. Absence of obvious CPE in (C) indicates neutralization of MHV infectivity. 70
9 71
10 72
11 of 125.00 mg/mL (Figure 2), cytotoxicity CC50 value of MHV, no anti-DENV-2 activity was detected with rutin treat- 73
12 116.50 mg/mL, and selectivity index of 0.93. However, quer- ment (without cytotoxicity). 74
13 citrin and rutin did not exhibit antiviral activity on 2 days post- Given that quercetin and quercitrin individually exhibited 75
14 infection (dpi) at concentrations of 500.00 mg/mL and below anti-DENV-2 properties, we evaluated whether the combination 76
15 (Table 1). of both flavonoid compounds (at 1:1 ratio) could exert enhanced 77
16 Cinanserin hydrochloride was tested at concentrations ranging DENV-2 inhibition. Indeed, this combination of components 78
17 from 125.00 to 3.91 mg/mL. Table 1 indicates that cinanserin achieved a synergistic anti-DENV-2 effect and lower cytotox- 79
18 exerted anti-MHV activity at MIC of 31.25 mg/mL on 1 dpi and icity, with improved corresponding values of IC50 (158.21 mg/ 80
19 62.50 mg/mL on 2 dpi (Figure 3), with minimal or no cytotox- mL), CC50 (270.00 mg/mL), and selectivity index (1.71), as 81
20 icity. However, on 3 dpi, no viral inhibition was observed. shown in Table 2. 82
21 83
22 3.2. Anti-DENV-2 activities of EA fraction of H. cordata, 3.3. Absence of non-specific virus inhibition and 84
23 quercetin and/or quercetrin cytotoxicity of DMSO and aqueous alkali solvents 85
24 86
25 87
The EA fraction of H. cordata was tested starting with the The EA fraction, quercetrin and rutin were dissolved in 1%
26 88
highest concentration of 166.67 mg/mL followed by 2-fold di- DMSO. Quercetin was dissolved in aqueous alkali (0.06 mol/L
27 89
lutions down to 0.08 mg/mL. By means of PRNT, a distinct trend NaOH) as it was not very soluble in DMSO. Various concen-
28 90
of DENV-2 inhibition was observed at various concentrations of trations of EA fraction and compounds were subjected to the
29 91
the EA fraction without cytotoxicity to BHK cells, with the IC50 corresponding viral neutralization tests as well as the MTT
30 92
being 7.50 mg/mL. The efficacy of the EA fraction in inhibiting assay. As negative controls, DMSO and aqueous alkali were also
31 93
DENV-2 infection was clearly evident at concentrations of tested separately by DENV-2 PRNT, MHV neutralization test
32 94
20.83 mg/mL and above, at which complete viral inhibition was and the MTT assay which revealed absence of non-specific
33 95
achieved (Figure 4). DENV-2 and MHV inhibition as well as lack of cytotoxicity
34 96
Individual flavonoids were tested at concentrations of of these solvents (data not shown).
35 97
500.00 mg/mL down to 15.63 mg/mL. Table 2 indicates that
36 98
quercetin displayed anti-DENV-2 activity with an IC50 of 3.4. Absence of acute toxicity and pathology in mice
37 99
176.76 mg/mL, CC50 value of 155.38 mg/mL, and selectivity after oral feeding with H. cordata extract
38 100
index of 0.88. Although quercitrin showed less activity against
39 101
DENV-2 with an IC50 of 467.27 mg/mL, this flavonoid did not Throughout the 14-day period of observation, the control
40 102
cause cytotoxicity to BHK cells. Similar to experiments with group as well as the four groups of mice fed with varying doses
41 103
42 104
43 105
44 106
45 107
46 108
47 109
48 110
49 111
Figure 2. Neutralization of MHV infectivity by quercetin (dissolved in aqueous alkali) on 2 dpi.
50 112
(A) Uninfected control of CCL9.1 cells. (B) MHV-infected CCL9.1 cells control. (C) MHV-infected CCL9.1 cells subjected to 125 mg/mL of quercetin. (D)
51 MHV-infected CCL9.1 cells subjected to 62.5 mg/mL of quercetin. Absence of obvious CPE in (C) indicates neutralization of MHV infectivity. 113
52 114
53 115
54 116
55 117
56 118
57 119
58 120
59 121
60 Figure 3. Neutralization of MHV infectivity by cinanserin hydrochloride on 2 dpi. 122
(A) Uninfected control of CCL9.1 cells. (B) MHV-infected CCL9.1 cells control. (C) MHV-infected CCL9.1 cells treated with 31.25 mg/mL of cinanserin.
61 123
(D) MHV-infected CCL9.1 cells treated with 62.5 mg/mL of cinanserin. (E) MHV-infected CCL9.1 cells treated with 125 mg/mL of cinanserin. Absence of
62 obvious CPE in (D) and (E) indicates neutralization of MHV infectivity. 124

Please cite this article in press as: Chiow KH, et al., Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection,
Asian Pacific Journal of Tropical Medicine (2015), http://dx.doi.org/10.1016/j.apjtm.2015.12.002
 APJTM148_proof ■ 26 December 2015 ■ 5/7

K.H. Chiow et al./Asian Pacific Journal of Tropical Medicine 2015; ▪(▪): 1–7 5

1 4. Discussion 63
2 64
3 The experimental strategy in this study was to investigate the 65
4 prophylactic antiviral effects since the compounds were allowed 66
5 to interact with the viruses for 1 h before introduction into the 67
6 cells. This approach was employed as we previously found no 68
7 therapeutic effect when the compounds were added after prior 69
8 infection of cells with DENV [13]. 70
9 Overall, this study demonstrated that EA fraction of 71
10 H. cordata and its flavonoid component, quercetin, could inhibit 72
11 both MHV and DENV-2 in vitro. This was followed by quer- 73
12 Figure 4. The EA fraction of H. cordata inhibits DENV-2 infectivity but is
citrin which could inhibit DENV-2 but not MHV, whereas rutin 74
13 not cytotoxic to BHK-21 cells. 75
The EA fraction of H. cordata was tested at concentrations of 166.67 mg/
did not exert any inhibitory effect on both viruses. We and
14 others have provided evidence to show that different flavonoid 76
mL down to 0.08 mg/mL. The upper plot reveals a distinct trend of DENV-2
15 inhibition, with the IC50 being 7.5 mg/mL, and complete viral inhibition components in the EA fraction of H. cordata exert varying de- 77
16 being achieved at concentrations of 20.83 mg/mL and above. The lower plot grees of antiviral activity against different viruses. Our findings 78
17 shows that no cytotoxicity to BHK-21 cells was evident at various con- corroborate previous evidence that among the flavonoid com- 79
18 centrations of the EA fraction tested. ponents, quercetin is the most effective against DENV-2 [13]. 80
19 Wleklik et al. emphasized that there is a structural basis for 81
20 the distinct differences in antiviral activities of flavonoids [21]. 82
21 Table 2 Hence, it is noteworthy that being the most bioactive, 83
22 IC50, CC50 and selectivity indices of H. cordata fraction, flavonoids, and quercetin possesses the hydroxyl group at the R2 position 84
23 their combination against DENV-2. compared to the other two flavonoids tested, i.e., rhamnose in 85
24 quercitrin and rubinose in rutin [22]. 86
Sample IC50 (mg/ CC50 (mg/ Selectivity
25 mL) mL) index Quercetin is an aglycone present at high concentration in 87
26 onions. This compound has virucidal activity against enveloped 88
EA fraction 7.50 NCa >22.22b
27 Quercetin 176.76 155.38 0.88 viruses such as mengovirus, herpes simplex, parainfluenza type 89
28 Quercitrin 467.27 NCa >1.07c 3, pseudorabies, respiratory syncytial, and Sindbis viruses [23– 90
29 Quercetin + Quercitrin 158.21 270.00 1.71 26]. Quercetin is able to inhibit H+-ATPase of lysosomal 91
30 Rutin NIL NCa NIL membrane and thus prevent virus coat removal [15]. Moreover, 92
31 a
NC: No cytotoxicity at all concentrations tested against BHK-21 cells. quercetin exhibits significant inhibitory effects on the ATPase 93
32 b
Highest selectivity index of EA fraction (i.e., lowest concentration for of multidrug resistance-associated proteins, thus increasing the 94
33 viral inhibition coupled with absence of cytotoxicity). c Selectivity index bioavailability of anticancer and antiviral drugs in vivo [27]. 95
34 with absence of cytotoxicity. 96
Hence, quercetin can be considered for its potential efficacy in
35 antiviral drug therapies. 97
36 of H. cordata extract showed no mortality and no significant Quercitrin (quercetin-3-L-rhamnoside) and rutin (quercetin-3- 98
37 weight loss (data not shown). Moreover, all mice appeared to be rutinoside) occur as glycosides. Quercitrin appears to show the 99
38 active and well-groomed before euthanasia. From the histo- highest content in fingerprint analysis of H. cordata by HPLC 100
39 pathological sections of all major organs stained with H&E, [14]. Among the flavonoids, quercetin also exerts the highest 101
40 there was no significant difference between the control group activity on HSV-1, whilst rutin has no effect at all [21]. This 102
41 and all the test groups, as exemplified in Figure 5 which depicts characteristic implies that the substitution or addition of free 103
42 the control group compared with the group fed with the highest hydroxyl group at certain positions may culminate in a 104
43 dose of 2 000 mg/kg. decreased or even completely abolished antiviral effect. Such 105
44 106
45 107
46 108
47 109
48 110
49 111
50 112
51 113
52 114
53 115
54 116
55 117
56 118
57 119
58 120
59 Figure 5. The EA fraction of H. cordata does not cause acute oral toxicity in mice. 121
60 Representative histopathological sections of major organs stained with H&E for the vehicle control group (images A–E) and test group fed with 2 000 mg/kg 122
of EA fraction of H. cordata (images F–J). (A, F) Brain with (a) nuclei of neurons; (b) blood capillary. (B, G) Lung with (c) respiratory bronchiole; (d)
61 123
alveolar duct; (e) alveoli; (f) blood vessel. (C, H) Heart with (g) cardiac muscle. (D, I) Liver with (h) central vein; (i) hepatocytes. (E, J) Kidney with (j)
62 glomeruli. 124

Please cite this article in press as: Chiow KH, et al., Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection,
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1 structural-activity relationships can lead to the design and Conflict of interest statement 63
2 development of more active and less toxic flavonoids with 64
3 appropriate pharmacokinetic properties. Hence, they may pave The authors declare no conflict of interest with respect to the 65
4 the way for novel compounds that can match or even exceed the publication of this paper. 66
5 efficacies of existing antiviral drugs [26,28,29]. 67
6 Another interesting aspect was the enhancement of anti- Acknowledgments 68
7 DENV-2 activity of quercetin when combined with quercitrin. 69
8 Besides augmenting the antiviral effect, this combination also We thank Annie Hsu and S.H. Lau for laboratory assistance, 70
9 reduced the cytotoxicity relative to that induced by quercetin T. Narasaraju for helpful advice, and Mary Ng for providing the 71
10 alone. This characteristic reiterates that combinations of com- dengue virus strain. This study was supported by a research 72
11 ponents with greater anti-dengue efficacy but lower toxicity are grant from the National University of Singapore. Q3 73
12 potentially promising, as discovered previously using combi- 74
13 nations of quercetin, chlorogenic acid and/or hyperoside [13]. References 75
14 Indeed, several reports have already documented the 76
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Asian Pacific Journal of Tropical Medicine (2015), http://dx.doi.org/10.1016/j.apjtm.2015.12.002
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Please cite this article in press as: Chiow KH, et al., Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection,
Asian Pacific Journal of Tropical Medicine (2015), http://dx.doi.org/10.1016/j.apjtm.2015.12.002