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molecules
Article
Antiviral Effects of Houttuynia cordata
Polysaccharide Extract on Murine Norovirus-1
(MNV-1)—A Human Norovirus Surrogate
Dongqing Cheng 1, 2, †, Liang Sun 1, †, Songyan Zou 2, Jiang Chen 1, Haiyan Mao 1, Yanjun Zhang 1,
Ningbo Liao 1, 3, * and Ronghua Zhang 1, 2, *
1Department of Nutrition and Food Safety, Zhejiang Provincial Center for Disease Control and Prevention,
Hangzhou 310006, China; chengdq@zcmu.edu.cn (D.C.); lsun@cdc.zj.cn (L.S.); jchen@cdc.zj.cn (J.C.);
hymao@cdc.zj.cn (H.M.); yjzhang@cdc.zj.cn (Y.Z.)
2College of Medical Technology, Zhejiang Chinese Medical University, Hangzhou 310053, China;
zou_songyan@126.com
3School of Public Health, Division of Infectious Diseases and Vaccinology, University of California,
Berkeley, CA 94720, USA
*Correspondence: liaoningbo2010@126.com (N.L.); rhzhang@cdc.zj.cn (R.Z.);
Tel./Fax: +86-571-87115140 (N.L.); Tel./Fax: +86-571-87115214 (R.Z.)
†These authors contributed equally to this work.
Academic Editors: Cédric Delattre and Raphaël E. Duval
Received: 4 March 2019; Accepted: 9 May 2019; Published: 13 May 2019
Abstract:
Houttuynia cordata is an herbal plant rich in polysaccharides and with several pharmacological
activities. Human noroviruses (HuNoVs) are the most common cause of foodborne viral gastroenteritis
throughout the world. In this study, H. cordata polysaccharide (HP), with a molecular weight of ~43 kDa,
was purified from H. cordata water extract (HWE). The polysaccharide HP was composed predominantly
of galacturonic acid, galactose, glucose, and xylose in a molar ratio of 1.56:1.49:1.26:1.11. Methylation and
NMR analyses revealed that HP was a pectin-like acidic polysaccharide mainly consisting of
α
-1,4-linked
GalpA,
β
-1,4-linked Galp,
β
-1,4-linked Glcp, and
β
-1,4-linked Xylpresidues. To evaluate the antiviral
activity of H. cordata extracts, we compared the anti-norovirus potential of HP with HWE and ethanol
extract (HEE) from H. cordata by plaque assay (plaque forming units (PFU)/mL) for murine norovirus-1
(MNV-1), a surrogate of HuNoVs. Viruses at high (8.09 log10 PFU/mL) or low (4.38 log10 PFU/mL)
counts were mixed with 100, 250, and 500
µ
g/mL of HP, HWE or HEE and incubated for 30 min at room
temperature. H. cordata polysaccharide (HP) was more effective than HEE in reducing MNV-1 plaque
formation, but less effective than HWE. When MNV-1 was treated with 500
µ
g/mL HP, the infectivity of
MNV-1 decreased to an undetectable level. The selectivity indexes of each sample were 1.95 for HEE,
5.74 for HP, and 16.14 for HWE. The results of decimal reduction time and transmission electron microscopic
revealed that HP has anti-viral effects by deforming and inflating virus particles, thereby inhibiting the
penetration of viruses in target cells. These findings suggest that HP might have potential as an antiviral
agent in the treatment of viral diseases.
Keywords:
antiviral effects; Houttuynia cordata; polysaccharide; water extract; ethanol extract;
murine norovirus-1
1. Introduction
Houttuynia cordata Thunb. is a medicinal plant commonly found in Southeast Asia. In China,
the young roots and green leaves of H. cordata are popular vegetable products, being used in the
preparation of beverages by boiling decoction. Houttuynia cordata contains a wide range of compounds
including polysaccharides, fatty acids, polyphenols, flavonoids, and sterols, and has anti-viral,
Molecules 2019,24, 1835; doi:10.3390/molecules24091835 www.mdpi.com/journal/molecules
Molecules 2019,24, 1835 2 of 14
antifungal, detoxifying, and anti-bacterial properties [
1
]. Hayashi et al. [
2
] reported that H. cordata
extract reduces the infectivity (by >4 log) of several viruses, including the influenza virus and HIV.
Even though several biological activities of H. cordata have been studied [
3
], the effects of H. cordata
against human noroviruses (HuNoVs) and its mechanism of action remain unknown.
Human noroviruses, which belong to the Caliciviridae family, are the most common cause of
foodborne viral gastroenteritis throughout the world. Human noroviruses can be divided into five
major genogroups, genogroup I (GI) through genogroup V (GV); Genogroup II.4 (GII.4) HuNoVs
are closely related to most foodborne noroviral outbreaks. Human noroviruses infections mainly
occur via person-to-person transmission through fecal–oral route or by consuming contaminated food.
Human noroviruses are highly tolerant to environmental changes and have low infectious doses of
eight to 10 viral particles [
4
]. Most sources of foodborne disease caused by HuNoVs are foods prepared
with contaminated hands or cookware. Currently, there is no commercially available antiviral drug or
vaccine for the prevention of norovirus infections or outbreaks.
The inactivation of HuNoVs mainly relies on physical treatments including chemical methods
(e.g., titanium dioxide, sodium hypochlorite, and hydrogen peroxide), heat treatment, and ultraviolet
radiation. However, the use of such treatments is frequently inadequate in the food industry.
Therefore, it is of utmost importance to investigate safe anti-noroviral agents that can be consumed.
Several compounds isolated from plants (e.g., polysaccharides, polyphenols, and flavonoids) have
antimicrobial activity [
5
]. A recent review suggested that plant extracts containing polysaccharides
and polyphenols, such as Ganoderma lucidum polysaccharide (GLPS), Pericarpium granati extract
(PGE), and Pomegranate extract (PE), might prevent infection from NoV surrogates [
6
]. These natural
products are safe antiviral agents because they can be eaten, and their nutrition-promoting properties
have long been studied [7,8].
Even though it has been reported that HuNoVs may be cultured in B cells
in vitro
, there are
several disadvantages to this method for screening natural antiviral compounds [
4
]. Anti-noroviral
effects are evaluated from the reduction in infectivity of cultivatable HuNoV surrogates such as murine
norovirus (MNV-1), bacteriophage MS2, or other
in vitro
models of HuNoV infectivity detection
(in situ capture qRT-PCR) [
9
–
11
]. Among them, MNV-1, a genogroup V (GV) cultivable norovirus,
is currently recognized as the most suitable surrogate for HuNoVs [10].
In the study, we identified the anti-norovirus potential of crude water extract (HWE), purified
polysaccharide (HP), and ethanol extract (HEE) from H. cordata by plaque assay for MNV-1. Additionally,
we investigated the structure, chemical composition, and antiviral mechanism of HP.
2. Results
2.1. Chemical Analyses of HP, HWE, and HEE
Extraction yields of HP, HWE, and HEE from H. cordata were 5.7%, 16.3%, and 9.6%, respectively.
The chemical compositions of the extracts are shown in Table 1. Ethanol extract samples contained
25.42
±
2.31 gallic acid equivalents (GAE)/mg total phenolic, 19.76
±
3.73 retinol equivalent
(RE)/mg total flavonoid, and 2.37
±
1.45% neutral sugars. Crude water extract samples contained
14.59 ±2.41 GAE/mg
total phenolic, 10.62
±
1.43 RE/mg total flavonoid, 25.67
±
1.97% neutral sugars,
and 12.34
±
2.01% uronic acids. Compared to HEE, HWE contained more carbohydrate. The total
carbohydrate content (38.01%) of HWE was calculated by adding neutral sugar and uronic acid values.
Purified polysaccharide was isolated from HWE. The HPLC profile of HP revealed a single peak with
a molecular weight (Mw) of 43 kDa. The total carbohydrate content of HP was 81.12%. Monosaccharides
in HP were mainly galacturonic acid (GlaUA), xylose (Xyl), glucose (Glc), and galactose (Gal).
Minor amounts of glucuronic acid (GlcUA), arabinose (Ara), mannose (Man), and rhamnose (Rha)
were obtained. The protein content of HP was 0.89%. The sugar composition of HP was similar to the
H. cordata polysaccharide fractions (HCP and HCP-2) reported in past studies [12,13].
Molecules 2019,24, 1835 3 of 14
Table 1. Yields and chemical composition of Houttuynia cordata extracts HP, HWE, and HEE a.
Composition bExtract/Fraction
HP HWE HEE
Extraction yield (%) 5.73 ±2.16 16.31 ±1.64 9.63 ±2.31
Total phenolic (µg GAE/mg) ND c14.59 ±2.41 25.42 ±3.17
Total flavonoid (µg RE/mg) ND 10.62 ±1.43 19.76 ±3.73
Protein (%) 0.89 ±0.12 2.54 ±0.12 5.68 ±1.76
Total carbohydrate (%) 81.12 ±5.98 38.01 ±3.98 2.37 ±1.45
Neutral sugars (%) 56.33 ±4.37 26.58 ±2.54 2.37 ±1.45
Uronic acid (%) 23.77 ±3.42 12.34 ±2.13 ND
Molar ratio of monosaccharides d
GlaUA 1.56 1.37 ND
Gal 1.49 0.94 ND
Rha 0.83 ND ND
Ara 0.68 ND ND
GluA 0.31 ND ND
Glc 1.26 0.31 ND
Man 0.14 ND ND
Xyl 1.11 ND ND
a
HP, Houttuynia cordata polysaccharide; HWE, Houttuynia cordata water extract; HEE, Houttuynia cordata ethanol
extract.
b
GAE, gallic acid equivalents; RE, retinol equivalent.
c
Not detected.
d
GlaUA, galacturonic acid; Gal,
galactose; Rha, rhamnose; Ara, arabinose; GluA, glucuronic acid; Glc, glucose; Man, mannose; Xyl, xylose.
The IR spectrum of HP from H. cordata is shown in Figure 1. The band at 3406.0 cm
−1
is attributed
to O–H or N–H stretching vibration. The band at 2931.3 cm
−1
is attributed to sp
3
C–H. The peak
at 1644.8 cm
−1
might be due to amide C=O stretching vibration and/or C=C stretching vibration
and/or N–H [
14
]. The bands at 1644.8 and 1403.0 cm
−1
are attributed to the presence of proteins in HP.
Moreover, the characteristic absorption at 1200–1000 cm
−1
is attributed to the presence of glycosidic
linkages C–O and/or C–N in HP [15]. All these results suggest that HP is an acidic polysaccharide.
Molecules 2019, 24, x 3 of 15
Table 1. Yields and chemical composition of Houttuynia cordata extracts HP, HWE, and HEE a.
Composition b Extract/Fraction
HP HWE HEE
Extraction yield (%) 5.73 ± 2.16 16.31 ± 1.64 9.63 ± 2.31
Total phenolic (μg GAE/mg) ND c 14.59 ± 2.41 25.42 ± 3.17
Total flavonoid (μg RE/mg) ND 10.62 ± 1.43 19.76 ± 3.73
Protein (%) 0.89 ± 0.12 2.54 ± 0.12 5.68 ± 1.76
Total carbohydrate (%) 81.12 ± 5.98 38.01 ± 3.98 2.37 ± 1.45
Neutral sugars (%) 56.33 ± 4.37 26.58 ± 2.54 2.37 ± 1.45
Uronic acid (%) 23.77 ± 3.42 12.34 ± 2.13 ND
Molar ratio of monosaccharides d
GlaUA 1.56 1.37 ND
Gal 1.49 0.94 ND
Rha 0.83 ND ND
Ara 0.68 ND ND
GluA 0.31 ND ND
Glc 1.26 0.31 ND
Man 0.14 ND ND
Xyl 1.11 ND ND
a HP, Houttuynia cordata polysaccharide; HWE, Houttuynia cordata water extract; HEE, Houttuynia
cordata ethanol extract. b GAE, gallic acid equivalents; RE, retinol equivalent. c Not detected. d
GlaUA, galacturonic acid; Gal, galactose; Rha, rhamnose; Ara, arabinose; GluA, glucuronic acid;
Glc, glucose; Man, mannose; Xyl, xylose.
The IR spectrum of HP from H. cordata is shown in Figure 1. The band at 3406.0 cm−1 is
attributed to O–H or N–H stretching vibration. The band at 2931.3 cm−1 is attributed to sp3 C–H. The
peak at 1644.8 cm−1 might be due to amide C=O stretching vibration and/or C=C stretching vibration
and/or N–H [14]. The bands at 1644.8 and 1403.0 cm−1 are attributed to the presence of proteins in
HP. Moreover, the characteristic absorption at 1200–1000 cm−1 is attributed to the presence of
glycosidic linkages C–O and/or C–N in HP [15]. All these results suggest that HP is an acidic
polysaccharide.
Figure 1. FTIR spectra of HP in the frequency range 4000–500 cm−1.
The methylation analysis results of HP are summarized in Table 2. The polysaccharide HP
mainly consisted of 1,4-linked GalpA, 1,4-linked Galp, 1,4-linked Glcp, and 1,4-linked Xylp residues
in a molar ratio of 1.74:0.93:0.95:0.84. The high prevalence of Galp and GalpA residues revealed that
HP might be a pectin-like acidic polysaccharide with a 1,4-linked Galp core. In addition, other small
Figure 1. FTIR spectra of HP in the frequency range 4000–500 cm−1.
The methylation analysis results of HP are summarized in Table 2. The polysaccharide HP mainly
consisted of 1,4-linked GalpA, 1,4-linked Galp, 1,4-linked Glcp, and 1,4-linked Xylpresidues in a molar
ratio of 1.74:0.93:0.95:0.84. The high prevalence of Galpand GalpA residues revealed that HP might be
a pectin-like acidic polysaccharide with a 1,4-linked Galpcore. In addition, other small proportions of
residues 1,5-linked Araf, 1,2,4-linked Rhap, 1,6-linked Galp, 1,4,6-linked Galp, and terminal-linked
Galpand Glcp, which have been reported in the side chains, were detected.
Table 2. Methylation analysis of Houttuynia cordata polysaccharide HP.
Sugar aPartially O-methylated Alditol Acetates Molar Ratio Linkage d
GalpA2,3,6-Me3Gal-6-d2b1.74 →
4)-GalpA-(1
→
Galp2,3,6-Me3Gal 0.93 →4)-Galp-(1→
2,3,4-Me3Gal 0.14 →6)-Galp-(1→
Molecules 2019,24, 1835 4 of 14
Table 2. Cont.
Sugar aPartially O-methylated Alditol Acetates Molar Ratio Linkage d
2,3-Me2Gal 0.21 →
4,6)-Galp-(1
→
2,3,4,6-Me4Gal 0.35 Galp-(1→
Glcp2,3,6-Me3Glc 0.95 →4)-Glcp-(1→
2,3,4,6-Me4Glc c0.37 Glcp-(1→
Xylp2,3-Me2Xyl 0.84 →4)-Xylp-(1→
Rhap3-Me Rha 0.22 →
2,4)-Rhap-(1
→
Araf2,3-Me2Ara 0.17 →5)-Araf-(1→
a
GalpA, galactopyranosyluronic acid; Galp, galactopyranose; Glcp, glucopyranose; Xylp, xylopyranose; Rhap,
rhamnopyranose; Araf, arabinofuranose;
b
2,3,6-Me
3
Gal-6-d
2
=1,4,5-tri-O-acetyl-6,6-dideutero-2,3,6-tri-O-methyl-galactitol.
c2,3,4,6-Me4-Glc =1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol, etc. dBased on derived O-methylalditol acetates.
Most of the
β
-anomeric protons are in the
δ
4–5 ppm range while most of the
α
-anomeric protons
usually appear in the
δ
5–6 ppm region [
16
]. The resonances ranging from 4.8 to 5.24 ppm in the
1
H-NMR
spectrum of HP (Figure 2A) revealed that the sugar residues of HP might be connected by
β
- and
α
-glycosidic
bonds. The anomeric protons from each monosaccharide can give recognizable signals depending on their
β
- or
α
-configurations. The signal at
δ
5.04, 3.74, 3.97, 4.38, and 4.68 ppm was clearly assigned to H-1 to
H-5 of
α
-D-GalA residues, respectively. In a
13
C-NMR spectrum, the signals derived from
α
-anomeric
carbons will appear in the range
δ
95–101 ppm while most of the
β
-anomeric carbons usually appear in
the
δ
101–105 ppm region [
16
]. The
13
C-NMR spectrum of HP (Figure 2B) showed the anomeric peaks
were centralized between
δ
98.28 and
δ
105.06 ppm, indicating two kinds of anomeric configurations for
monosaccharide residues of HP. The
13
C-NMR spectrum showed characteristic anomeric signals at
δ
99.7,
103.1, and 104.4 ppm, which were due to the C1 resonances of 1,4-
α
-d-GalA residues, 1,4-
β
-d-Xylp residues,
and 1,4-
β
-d-Glcp residues, at
δ
105.5 ppm due to the C1 resonances of
β
-d-Galp residues (Supplementary
Materials Table S1). Other predominant signals at
δ
70.9, 71.4, 80.5, 73.4, and 176.4 ppm were related to C-2
to C-6 of
α
-d-GalA residues, respectively. In addition, the signals at the low field from
δ
160 to
δ
180 ppm in
the
13
C-NMR spectrum of HP (Figure 2B) illustrated that the polysaccharide contained uronic acid [
17
].
All the NMR chemical shifts were compared with previously reported values [18,19].
Molecules 2019, 24, x 5 of 15
Figure 2. 1H (A) and 13C (B) nuclear magnetic resonance (NMR) spectra of HP.
2.2. Cytotoxicity Assay
The cytotoxicity assay was determined by calculating CC50. The effects of H. cordata extracts on
the survival rate of the murine macrophage cell lines (RAW 264.7) are shown in Figure 3. Ethanol
extract, HWE, and HP had no severe cytotoxicity effects towards RAW 264.7 cells with CC50 values
> 500 μg/mL (Table 3). Therefore, at concentraetions ≤ 500 μg/mL, the extracts would not affect the
host cells in plaque assays.
Figure 2. 1H (A) and 13C (B) nuclear magnetic resonance (NMR) spectra of HP.
Molecules 2019,24, 1835 5 of 14
2.2. Cytotoxicity Assay
The cytotoxicity assay was determined by calculating CC
50
. The effects of H. cordata extracts on the
survival rate of the murine macrophage cell lines (RAW 264.7) are shown in Figure 3. Ethanol extract, HWE,
and HP had no severe cytotoxicity effects towards RAW 264.7 cells with CC
50
values >500
µ
g/mL (Table 3).
Therefore, at concentraetions ≤500 µg/mL, the extracts would not affect the host cells in plaque assays.
Molecules 2019, 24, x 6 of 15
Figure 3. The effect of Houttuynia cordata extracts (HP, HCWE, and HCEE) 250, 500, 750, and 1000
μg/mL and phosphate-buffered saline (PBS) control (0 μg/mL) on the viability of the RAW 264.7 cell
lines. Numbers are expressed as the % viability of bacteria cells and the murine macrophage cells
(RAW 264.7) remaining after 60 min incubation with Houttuynia cordata extracts. A higher number of
surviving reflects the concentrations of Houttuynia cordata extracts would not affect the ability to use
these host cells to determine anti-viral effects using plaque assays. Each experimental condition was
analyzed in triplicate. All data were expressed as mean ± standard deviation. Significant differences
with HEE group are designated as * p ≤ 0.05
Table 3. Antiviral activities of H. cordata extracts against murine norovirus in RAW 264.7 cells using
the mixed treatment assay.
H. cordata Extracts CC50 (μg/mL) a EC50 (μg/mL) b SI
c
HEE 2132.43 ± 426.17 1095.53 ± 113.43 1.95 ± 0.84
HWE 1237.52 ± 367.65 * 76.75 ± 17.89 * 16.14 ± 3.81 *
HP 1074.76 ± 187.31 * 187.24 ± 77.82 * 5.74 ± 1.96 *
a CC50: mean (50%) value of cytotoxic concentration. b EC50: mean (50%) value of effective
concentration. c SI: selectivity index, CC50/EC50. Each experimental condition was analyzed in
triplicate. Values are mean ± standard deviation. Significant differences with HEE group are
designated as * p ≤ 0.05
2.3. Anti-Viral Activity of HP, HWE, and HEE
We tested the dose-dependence of HP, HWE, and HEE on the reduction of viral titers (Table 4).
The anti-noroviral activities of HP, HWE, and HEE were assessed by plaque assay (plaque forming
units (PFU)/mL). H. cordata water extract (HWE) was assayed for its anti-viral activity on mouse
norovirus 1 (MNV-1) at high (8.09 log10 PFU/mL) and low (4.38 log10 PFU/mL) counts following
incubation with different concentrations (100, 250, and 500 μg/mL) of HWE for 30 min. When
MNV-1 was treated with 100 μg/mL HWE, the viral titer was reduced. The amount of MNV-1 titer
decreased by 1.38 log10 PFU/mL at high counts and by 1.87 log10 PFU/mL at low counts when
compared to the untreated controls. At HWE > 250 μg/mL, MNV-1 titers decreased to undetectable
level for low counts (4.38 log10 PFU/mL). Mouse norovirus 1 (MNV-1) was less sensitive to HP than
HWE. At low counts, we obtained a greater reduction in viral titers with HP than at high counts.
When MNV-1 was treated with 500 μg/mL HP, the infectivity of MNV-1 was decreased to
Figure 3.
The effect of Houttuynia cordata extracts (HP, HCWE, and HCEE) 250, 500, 750, and 1000
µ
g/mL and
phosphate-buffered saline (PBS) control (0
µ
g/mL) on the viability of the RAW 264.7 cell lines. Numbers are
expressed as the % viability of bacteria cells and the murine macrophage cells (RAW 264.7) remaining after
60 min incubation with Houttuynia cordata extracts. A higher number of surviving reflects the concentrations
of Houttuynia cordata extracts would not affect the ability to use these host cells to determine anti-viral effects
using plaque assays. Each experimental condition was analyzed in triplicate. All data were expressed as
mean ±standard deviation. Significant differences with HEE group are designated as * p≤0.05.
Table 3.
Antiviral activities of H. cordata extracts against murine norovirus in RAW 264.7 cells using the
mixed treatment assay.
H. cordata Extracts CC50 (µg/mL) aEC50 (µg/mL) bSI c
HEE 2132.43 ±426.17
1095.53
±
113.43
1.95 ±0.84
HWE
1237.52
±
367.65 *
76.75 ±17.89 * 16.14 ±3.81 *
HP
1074.76
±
187.31 *
187.24 ±77.82 * 5.74 ±1.96 *
a
CC
50
: mean (50%) value of cytotoxic concentration.
b
EC
50
: mean (50%) value of effective concentration.
c
SI: selectivity index, CC
50
/EC
50.
Each experimental condition was analyzed in triplicate. Values are mean
±
standard deviation. Significant differences with HEE group are designated as * p≤0.05.
2.3. Anti-Viral Activity of HP, HWE, and HEE
We tested the dose-dependence of HP, HWE, and HEE on the reduction of viral titers (Table 4).
The anti-noroviral activities of HP, HWE, and HEE were assessed by plaque assay (plaque forming
units (PFU)/mL). H. cordata water extract (HWE) was assayed for its anti-viral activity on mouse
norovirus 1 (MNV-1) at high (8.09 log10 PFU/mL) and low (4.38 log10 PFU/mL) counts following
incubation with different concentrations (100, 250, and 500
µ
g/mL) of HWE for 30 min. When MNV-1
was treated with 100
µ
g/mL HWE, the viral titer was reduced. The amount of MNV-1 titer decreased
by 1.38 log10 PFU/mL at high counts and by 1.87 log10 PFU/mL at low counts when compared to
Molecules 2019,24, 1835 6 of 14
the untreated controls. At HWE >250
µ
g/mL, MNV-1 titers decreased to undetectable level for low
counts (4.38 log10 PFU/mL). Mouse norovirus 1 (MNV-1) was less sensitive to HP than HWE. At low
counts, we obtained a greater reduction in viral titers with HP than at high counts. When MNV-1
was treated with 500
µ
g/mL HP, the infectivity of MNV-1 was decreased to undetectable levels in low
counts. Anti-viral activity was also observed with HEE. At 100, 250, and 500
µ
g/mL, HEE was effective
in reducing MNV-1 titers in a dose-dependent manner when compared to the untreated controls.
However, when HEE was used with high and low viral titers, we obtained less than 1 log10 PFU/mL
reduction in MNV-1 titer. The antiviral activity of H. cordata extracts was also evaluated by measuring
EC
50
(Table 3). Mean EC
50
values were 187
µ
g/mL for HP, 76
µ
g/mL for HWE, and 1095
µ
g/mL for
HEE. Mouse norovirus 1 (MNV-1) was more sensitive to HP and HWE than to HEE.
Table 4.
Effects of HP, HWE, and HEE with low concentrations (4.38 log10 PFU/mL) and high
concentrations (8.09 log10 PFU/mL) on mouse norovirus 1 (MNV-1) measured by plaque assay.
Extracts Concentration
MNV-1 (log10 PFU/mL)
Low Count (4.38 log10) High Count (8.09 log10)
Recovered Titer cReduction eRecovered Titer Reduction
PBS a0µg/mL 4.38 ±1.71 0 8.09 ±1.95 0
2TU b50 µM 2.50 ±0.83* 1.88 4.33 ±2.14 * 3.76
HP 100 µg/mL 3.77 ±1.04 0.61 7.49 ±2.53 0.60
250 µg/mL 2.62 ±1.03 * 1.76 6.86 ±1.81 * 1.23
500 µg/mL ND* d4.38 4.61 ±1.72 * 3.48
HWE 100 µg/mL 2.51 ±0.53 * 1.87 6.71 ±2.12 * 1.38
250 µg/mL ND * 4.38 6.26 ±1.32 * 1.83
500 µg/mL ND * 4.38 3.96 ±0.74 * 4.12
HEE 100 µg/mL 4.21 ±0.87 0.16 8.03 ±2.89 0.06
250 µg/mL 3.48 ±1.33 * 0.90 7.52 ±1.73 0.57
500 µg/mL 3.24 ±0.41 * 1.14 7.01 ±2.07 * 0.98
a,b
Phosphate-buffered saline (PBS) was used as untreated control and 2-thiouridine (2TU) as a positive control.
c
Values are mean
±
standard deviation.
d
Not detected.
e
Each titer was subtracted from the titer of the untreated
sample (PBS). Each experimental condition was analyzed in triplicate. Significant differences with untreated control
group are designated as * p≤0.05.
2.4. Time-Dependence Experiment of HP, HWE, and HEE on Viral Count Reduction
We tested the time-dependence of HP, HWE, and HEE on MNV-1 titer reduction. We incubated
250
µ
g/mL HP, HWE, or HEE with high counts of MNV-1 for up to 60 min and measured the viral titers.
As shown in Figure 4, MNV-1 titers in PBS (untreated control) did not appear to change during the
60-min exposure, while titers in 250
µ
g/mL HP or HWE decreased significantly. The antiviral action of
HP or HWE was rapid against MNV-1. Considerable changes (1.66 or 2.40 log10 PFU/mL) in MNV-1
titers were obtained in the first 10 min following HP or HWE addition. The counts continued to drop
by 1.74 log10 PFU/mL for HWE and 1.53 log10 PFU/mL for HP for the next 30 min and continued to
drop for the following 30 min. We obtained a total reduction in MNV-1 titers of 4.54 log10 PFU/mL for
HWE and 3.69 log10 PFU/mL for HP. A time-dependence of the anti-viral activity of HEE on high titer
MNV-1 was also observed. The viral titers of MNV-1 treated with HEE dropped slower in the first
30-40 min than HP and HWE and a total reduction in MNV-1 titers of 0.90 log10 PFU/mL for HEE
treatment were achieved. HWE and HP had stronger antiviral effects on MNV-1 than HEE.
Molecules 2019,24, 1835 7 of 14
Molecules 2019, 24, x 8 of 15
Figure 4. Change of MNV-1 titers versus incubation time after exposure to HP, HWE or HEE.
Two-hundred-and-fifty (μg/mL) of HP, HWE or HEE solutions were added to an equal volume of
MNV-1 at titer of ~8 log10 PFU/mL and incubated for up to 60 min at room temperature. The MNV-1
was recovered at 0, 10, 20, 30, 40, 50, and 60 min and assayed for its infectivity using standardized
plaque assay. Each experimental condition was analyzed in triplicate. All data were expressed as
mean ± standard deviation. Significant differences with control group are designated as * p ≤ 0.05.
2.5. Viral Inactivation Kinetics
Table 5 shows that there was a 4.34- and 18.34-fold decrease in D-values with 250 and 500
μg/mL HP, respectively, compared to 100 μg/mL HP. We obtained a 1.0-log reduction with 100
μg/mL HP-containing samples at 29.09 min and a 1.0-log reduction with 500 μg/mL HP-containing
samples at 1.59 min.
Table 5. The Effect of HP with different concentrations on the D-values of MNV-1.
HP (μg/mL) Weibull model parameters a
K ± SD b α ± SD D-value (min) ± SD RMSE R2
100 0.144 ± 0.65 0.581 ± 0.04 28.098 ± 2.44 0.03 0.97
250 0.213 ± 0.01 0.817 ± 0.26 6.647 ± 0.93 0.02 0.98
500 0.866 ± 0.08 0.308 ± 0.04 1.597 ± 0.31 0.04 0.95
a K is the characteristic time (h); α = shape parameter; D-value = storage time (day) required to
reduce MNV-1 or Escherichia coli by 90%; RMSE = correlation coefficient, a lower RMSE value
indicates a better fit to the data; R2 = correlation coefficient, a higher R2 value indicate a better fit to
the data. b Values are mean ± standard deviation. Each experimental condition was analyzed in
triplicate.
2.6. Effect of HP on MNV-1 Viral Particles
To further investigate the antiviral action of HP, the morphology of MNV-1 in low titer was
analyzed by transmission electron microscopic (TEM) before and after treatment with PBS (0
μg/mL) and HP (250 μg/mL). Before treatment, MNV-1 particles were spherical (Figure 5A) and the
size ranged from 30 to 35 nm as described previously [20]. After treatment with HP (250 μg/mL),
the size of the MNV-1 particles increased to 80–100 nm in diameter. Additionally, we observed
enlarged, denatured particles and disrupted particles (Figure 5B). The log10 reductions of MNV-1
Figure 4.
Change of MNV-1 titers versus incubation time after exposure to HP, HWE or HEE.
Two-hundred-and-fifty (
µ
g/mL) of HP, HWE or HEE solutions were added to an equal volume of
MNV-1 at titer of ~8 log10 PFU/mL and incubated for up to 60 min at room temperature. The MNV-1
was recovered at 0, 10, 20, 30, 40, 50, and 60 min and assayed for its infectivity using standardized
plaque assay. Each experimental condition was analyzed in triplicate. All data were expressed as mean
±standard deviation. Significant differences with control group are designated as * p≤0.05.
2.5. Viral Inactivation Kinetics
Table 5shows that there was a 4.34- and 18.34-fold decrease in D-values with 250 and 500
µ
g/mL HP,
respectively, compared to 100
µ
g/mL HP. We obtained a 1.0-log reduction with 100
µ
g/mL HP-containing
samples at 29.09 min and a 1.0-log reduction with 500 µg/mL HP-containing samples at 1.59 min.
Table 5. The Effect of HP with different concentrations on the D-values of MNV-1.
HP (µg/mL) Weibull Model Parameters a
K±SD bα±SD D-Value (min) ±SD RMSE R2
100 0.144 ±0.65 0.581 ±0.04 28.098 ±2.44 0.03 0.97
250 0.213 ±0.01 0.817 ±0.26 6.647 ±0.93 0.02 0.98
500 0.866 ±0.08 0.308 ±0.04 1.597 ±0.31 0.04 0.95
a
Kis the characteristic time (h);
α
=shape parameter; D-value =storage time (day) required to reduce MNV-1
or Escherichia coli by 90%; RMSE =correlation coefficient, a lower RMSE value indicates a better fit to the data;
R2=correlation coefficient
, a higher R
2
value indicate a better fit to the data.
b
Values are mean
±
standard deviation.
Each experimental condition was analyzed in triplicate.
2.6. Effect of HP on MNV-1 Viral Particles
To further investigate the antiviral action of HP, the morphology of MNV-1 in low titer was
analyzed by transmission electron microscopic (TEM) before and after treatment with PBS (0
µ
g/mL)
and HP (250
µ
g/mL). Before treatment, MNV-1 particles were spherical (Figure 5A) and the size ranged
from 30 to 35 nm as described previously [
20
]. After treatment with HP (250
µ
g/mL), the size of the
MNV-1 particles increased to 80–100 nm in diameter. Additionally, we observed enlarged, denatured
particles and disrupted particles (Figure 5B). The log10 reductions of MNV-1 under these conditions
were 2.75 with 250
µ
g/mL HP. These results suggest that HP denatures the viral capsid proteins,
thereby preventing viral adhesion.
Molecules 2019,24, 1835 8 of 14
Molecules 2019, 24, x 9 of 15
under these conditions were 2.75 with 250 μg/mL HP. These results suggest that HP denatures the
viral capsid proteins, thereby preventing viral adhesion.
Figure 5. Transmission electron microscopic (TEM) images of MNV-1 in the absence or presence of
HP. MNV-1 control (A) and MNV-1 treated with 250 μg/mL HP (B). Scale bars, 100 nm.
3. Discussion
Although most studies have focused on investigating the role of H. cordata extracts against
microorganisms [21,22], few studies have evaluated the effectiveness and mechanism of action of H.
cordata extracts against HuNoVs. In this study, H. cordata extracts prepared in water (HWE and HP)
and ethanol (HEE) exhibited various degrees of antiviral activity in a dose- and time-dependent
manner. The values of selectivity index (SI) were 1.95 for HEE, 5.74 for HP, and 16.14 for HWE.
Samples with high SIs had maximum antiviral activity, minimal cell toxicity, and a wider range of
applications [23]. Among the extracts, HWE, mainly composed of carbohydrates, exhibited the
strongest antiviral activity with the highest SI value. According to previous studies, carbohydrates
are the main active substances in H. cordata water extracts and exhibited potent anti-viral activities
[1,2,6,12,13]. In this study, we analyzed the H. cordata polysaccharide structures and their
anti-noroviral mechanisms.
A polysaccharide (HP), isolated and purified from HWE with a molecular weight < 50 kDa,
exhibited stronger anti-noroviral activity with higher SI value than HEE. Structural analysis
revealed that HP mainly consists of α-1,4-linked GalpA, β-1,4-linked Galp, β-1,4-linked Glcp, and
β-1,4-linked Xylp residues. The predominant number of Galp and GalpA residues suggests that HP
might be a pectin-like acidic polysaccharide with a 1,4-linked Galp core. In addition, 1, 6-linked
Galp, 1,4,6-linked Galp, 1,2,4-linked Rhap, 1,5-linked Araf, and terminal-linked Galp and Glcp in the
side chains were detected. It has been proposed that some carbohydrates consisting of 1,4-linked
Figure 5.
Transmission electron microscopic (TEM) images of MNV-1 in the absence or presence of HP.
MNV-1 control (A) and MNV-1 treated with 250 µg/mL HP (B). Scale bars, 100 nm.
3. Discussion
Although most studies have focused on investigating the role of H. cordata extracts against
microorganisms [
21
,
22
], few studies have evaluated the effectiveness and mechanism of action of
H. cordata extracts against HuNoVs. In this study, H. cordata extracts prepared in water (HWE and HP)
and ethanol (HEE) exhibited various degrees of antiviral activity in a dose- and time-dependent manner.
The values of selectivity index (SI) were 1.95 for HEE, 5.74 for HP, and 16.14 for HWE. Samples with
high SIs had maximum antiviral activity, minimal cell toxicity, and a wider range of applications [
23
].
Among the extracts, HWE, mainly composed of carbohydrates, exhibited the strongest antiviral
activity with the highest SI value. According to previous studies, carbohydrates are the main active
substances in H. cordata water extracts and exhibited potent anti-viral activities [
1
,
2
,
6
,
12
,
13
]. In this
study, we analyzed the H. cordata polysaccharide structures and their anti-noroviral mechanisms.
A polysaccharide (HP), isolated and purified from HWE with a molecular weight <50 kDa,
exhibited stronger anti-noroviral activity with higher SI value than HEE. Structural analysis revealed
that HP mainly consists of
α
-1,4-linked GalpA,
β
-1,4-linked Galp,
β
-1,4-linked Glcp, and
β
-1,4-linked
Xylpresidues. The predominant number of Galpand GalpA residues suggests that HP might be
a pectin-like acidic polysaccharide with a 1,4-linked Galpcore. In addition, 1, 6-linked Galp, 1,4,6-linked
Galp, 1,2,4-linked Rhap, 1,5-linked Araf, and terminal-linked Galpand Glcpin the side chains were
detected. It has been proposed that some carbohydrates consisting of 1,4-linked GalpA may prevent the
adhesion of some enteric pathogens such as protozoa, bacteria, and virus [
24
,
25
]. The polysaccharide
(HP) is mainly composed of carbohydrates consisting of 1,4-linked GalpA and 1,4-linked Galp.
Molecules 2019,24, 1835 9 of 14
The pectin-like acidic polysaccharide HP with a 1,4-linked Galp core might be the active substance in
HWE responsible for the antiviral activity against MNV-1.
The Weibull model was used to evaluate the antiviral behavior of HP during 60 min (Supplementary
Materials Figure S1). Based on the survival curves of MNV-1, the Weibull model showed the
best fit to HP treatment with the highest R
2
(>0.90) and lowest RMSE (<0.05) values. The kinetic
model for the inactivation of MNV-1 can be analyzed with the D-value, which represents the time
required to reduce the population of pathogens by 90% and can be measured from a time versus
log survivors’ curve [
26
,
27
]. In our study, D-values decreased with increasing HP concentration.
The D-values for MNV-1 inactivation were different (p
≤
0.05) between the low treatments (100
µ
g/mL)
and high treatments (250 and 500 µg/mL).
To further understand the antiviral mechanism of HP, we investigated the effects of HP on high-titer
MNV-1 (8.09 log10 PFU/mL) using dose- and time-dependence experiments. Additionally, we observed
the morphology of MNV-1 in low titer (4.38 log10 PFU/mL) with TEM under conditions that reduced its
infectivity by more than 3 log10 PFU/mL. It has been reported that some polysaccharides inhibit viral
infections by blocking the adsorption, entry, and/or cell-to-cell transmission of viruses. In addition,
polysaccharides bind to viral envelope glycoproteins and disrupt them with negatively charged
carboxylate groups, therefore minimizing/preventing viruses from penetrating target cells [
28
,
29
].
In our study, the changes in size and morphology of MNV-1 particles following HP treatment
demonstrated that the polysaccharide had anti-norovirus activity by denaturing and enlarging virus
particles to inhibit virus penetration. The morphological changes in MNV-1 following HP treatment
were similar to those obtained in bacteriophage T4 and rotavirus with flavonoid compound CJs
(cranberry juices) [
30
] and in MNV-1 treated with polyphenolic compound RCS-F1 (raspberry seed
extract fraction-1) [
31
]. These results suggest that these natural compounds inactivate viruses by
a similar mechanism.
In this study, we identified the anti-norovirus potential of HWE, HP, and HEE using a plaque assay
for MNV-1. The pectin-like acidic polysaccharide HP, with 1,4-linked Galpcore, might be the active
substance in HWE responsible for the antiviral activity against MNV-1. Currently, the only method
for preventing noroviral infections is hand washing. In this study, the antiviral effects of HP and
HWE reduced the residual MNV-1 infectivity following 10 min of incubation; therefore, these extracts
interact immediately with the virus. The use of these extracts entails no safety concerns because
H. cordata is a permitted food additive in China [
32
]. Our findings support the use of HP and HWE as
nontoxic agents. Zhu et al. [
33
] and Kumar et al. [
34
] have shown that H. cordata exerts strong effects
against a large number of enveloped and non-enveloped viruses. Therefore, HP and HWE might be
potential antiviral agents in the prevention of viral diseases. However, we used unpurified HWE in
this study. It is possible that total phenolics, proteins, and flavonoids in the extract may participate
in the inactivation of the MNV-1 in a synergistic manner. It has been reported that a new type of
flavonoid (Houttuynoids A–E (1–5)), from the whole plant of Houttuynia cordata, exhibited potent
anti-HSV (herpes simplex viruses) activity [
35
]. The determination of the phenolic content in HWE
through Folin–Ciocalteu solely corresponds to an estimation of the presence of reducing compounds
in this study. Thus, future studies should investigate the characterization of polyphenolic profiles in
HWE, and their mechanism of antiviral action.
4. Materials and Methods
4.1. Preparation of HP, HWE, and HEE
Houttuynia cordata was obtained from Zhejiang Chinese Medical University. Stems and leaves
were air-dried at room temperature and ground (particle size
≤
80 mesh) using a Thomas–Willey
milling machine (Thomas Willey Mills, Swedesboro, NJ, USA). To prepare HEE and HWE, we followed
the method reported by Sekita [
36
], with some modifications. To prepare HEE, we heated H. cordata
samples (50 g) in an electromagnetic cooker at 450 W for 10 min, wrapped them in aluminum foil,
Molecules 2019,24, 1835 10 of 14
and mixed them with 50 mL ethanol for 15 min. Following centrifugation at 2000
×
gfor 15 min,
the supernatant was recovered, lyophilized, and stored at 4
◦
C. To prepare HWE, H. cordata (50 g)
was kept overnight at 16
◦
C, immersed in 200 mL distilled water, and boiled under traditional
reflux for 90 min. After centrifugation at 2000
×
gfor 15 min, the supernatant was recovered and
concentrated under reduced pressure. Approximately 50 mL of concentrated extract was filtered,
lyophilized, and stored at 4
◦
C. H. cordata polysaccharide (HP) was purified from HWE as previously
reported [
37
]. Briefly, four volumes of 96% alcohol were added to HWE and stirred at room temperature.
The precipitate was centrifuged at 8000 rpm for 15 min, washed several times with ethanol and acetone,
and dried at room temperature. The purified polysaccharide obtained by DEAE chromatography
(
2.5 ×30 cm
, Bio-Rad, Richmond, CA, USA) was filtered through Sephacryl S-300 (1.6
×
100 cm).
The final fraction, labeled HP, was desalted and lyophilized. The homogeneity and molecular weight
of HP were determined by HPLC (Waters, Alliance 2695 pump, Milford, MA, USA) coupled with
a differential refractometer.
4.2. Chemical Analysis
The total carbohydrate content in H. cordata extracts was measured by the phenol–sulfuric acid
method [
37
]. Phenolic compounds were expressed as mg of gallic acid/g (dry wt) of extract and
tested by the Folin–Ciocalteu method. Protein was analyzed by the Lowry method. Uronic acid
was measured by the m-hydroxydiphenyl method using galacturonic acid as the standard [
38
].
Total flavonoids were determined by the aluminum chloride method and expressed as mg of rutin
equivalents (RE) per gram of extract [
39
]. In addition, monosaccharide composition was measured by
1-phenyl-3-methyl-5-pyrazolone (PMP)-HPLC [
40
]. Infrared spectra of HP were determined using KBr
disk method and recorded at 400–4000 cm−1.
Methylation analysis was performed as previously reported [
37
]. Hydroxyl groups were
methylated using lithium dimethylsulfonyl as anion and confirmed by FTIR spectroscopy. Methyl esters
of uronic acids were reduced by lithium triethylborodeuteride (Superdeuteride
®
, Aldrich, Milwaukee,
WI, USA) [
38
]. Methylated polysaccharides were subsequently hydrolyzed with 2 M trifluoroacetic
acid for 2 h at 120
◦
C. Prior to analysis, the derivatives were reduced by NaBD4 and acetylated with
acetic anhydride (Ac2O) and 1-methylimidazole (1-MeIm). Gas chromatography-mass spectrometry
(GC/MS) was performed using an HP-5890 system coupled to an OV1701 column (0.25 mm
×
30 m) and
a temperature program ranging from 140
◦
C to 280
◦
C at 3
◦
C/min. The quantification of alditol acetate
was carried out by the response factor and peak area of FID in GC. For nuclear magnetic resonance
(NMR) analysis, the purified HP (60 mg/mL) was deuterium-exchanged by freeze-drying three times
and then dissolved in 0.5 mL of 99% D
2
O. The spectra were recorded by a 500-MHz Bruker Avance 500
spectrometer for
1
H and 125 MHz for
13
C [
41
]. Signals at
δ
H 2.22 and
δ
C 31.1 for acetone were used
as external standards.
4.3. Propagation of Viruses
Murine norovirus-1 and RAW264.7 cells were obtained from American Type Culture Collection
(ATCC). Murine norovirus-1 propagation was performed by inoculation of confluent monolayers of
RAW 267.4 cells. The RAW 264.7 cells were grown at 37
◦
C in a 5% CO
2
atmosphere in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% heat inactivated 1
×
Anti-Anti (Gibco,
Grand Island, NY, USA) and fetal bovine serum (FBS). Stocks of MNV-1 were prepared and stored
at −70 ◦C [42,43].
4.4. Antiviral Effects of HP, HWE, and HEE
Ten-fold serial dilution of filter-sterilized H. cordata extracts (HP, HWE, and HEE) were used
to determine cytotoxicity. Cytopathic effects were assessed by visual inspection after 3 d of
incubation [
44
]. The cytotoxicity and virus-induced cytopathic effects were measured by MTT
(3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay [
45
]. The 50% cytotoxicity
Molecules 2019,24, 1835 11 of 14
concentration (CC
50
) value was determined following a 24-h treatment of RAW 264.7 cell monolayers
to different doses of H. cordata extracts [
18
,
45
]. H. cordata extracts (HP, HWE, and HEE) were
dissolved in sterile PBS at concentrations of 3000
µ
g/mL, sterilized by filtration (0.2-
µ
m pore-size
membrane), and diluted aseptically to 200, 500, 1000, 1500, and 2000
µ
g/mL in sterile PBS. To evaluate
dose dependence, an equal volume of HP, HWE or HEE was added to an equal volume of MNV-1
resulting in titers of 8.09 log10 PFU/mL or 4.38 log10 PFU/mL and incubated for 30 min at room
temperature. In this study, the 50% effective concentration (EC
50
) value was considered to be the
concentration of H. cordata extracts required to reach only 50% of cytopathogenic effects caused by the
MNV-1. Selectivity index was determined by the effectiveness at inhibiting MNV-1-induced cell death
(CC
50
/EC
50
) [
45
]. For an analysis of time dependence, a set concentration (250
µ
g/mL) of HP, HWE
or HEE was added to an equal volume of MNV-1 and incubated for 0, 10, 20, 30, 40, 50 or 60 min at
room temperature. 2-thiouridine was used as a positive control. For the untreated control, we used
individual viruses mixed with PBS. Following incubation, the reaction in each mixture was terminated
with the addition of PBS.
4.5. Plaque Assay
Infectivity of MNV-1 was determined by the standardized plaque assay [
10
]. The RAW 264.7 cells
were plated in 6-well plates at 2
×
10
6
cells per well and grown until reaching 85% to 90% confluency.
Serial ten-fold diluted viral strains were prepared in DMEM supplemented with 10% FBS. The viral
dilution (0.5 mL) was inoculated into each well after aspiration of the media and incubated at 37
◦
C for
2.5 h. Following viral adsorption, the supernatant was removed and replaced with fresh DMEM (2 mL)
containing 1% penicillin–streptomycin, 0.75% agarose, and 10% FBS. After 72 h of incubation, plates
were overlaid with media containing 0.02% neutral red. Finally, plaques were calculated following
incubation for 5 h at 37
◦
C. Titer reductions were measured by subtracting the titer of the samples from
the titer of the PBS control.
4.6. Transmission Electron Microscopy (TEM)
Three-microliter aliquots of MNV-1 suspensions with H. cordata polysaccharide HP were diluted
10-fold in water and placed on a carbon-coated electron microscopy (EM) grid. The viral samples were
stained with 3% aqueous uranyl acetate (3
µ
L) for approximately 1 min, air-dried, and observed under
a JEOL-1400 electron microscope (Jeol, Tokyo, Japan) at 80 kV. Images were captured with a Gatan
UltraScan 1000 XP camera (Gatan, Pleasanton, CA, USA) at a magnification of ×40,000 [46].
4.7. Viral Inactivation Kinetics
Viral inactivation kinetics were determined as previously described [
26
], with a slight modification.
Briefly, HP solutions were prepared in PBS at 0, 100, 250, and 500
µ
g/mL HP. The polysaccharide (100
µ
L)
was added to an equal volume of MNV-1. Following incubation for 0, 5, 10, 20, 30, and 60 min at room
temperature, viral counts were measured by calculating the PFU titers. All experiments were carried
out in triplicate. The decimal reduction time (D-value) was measured as previously reported [
27
] with
slight modifications. The non-linear model (Weibull) can be expressed as Equations (1) and (2) [27].
log10 N
N0!=−Ktα(1)
D=1
K1
α
(2)
where
α
is the shape parameter, Kis the characteristic time (min), and Dis the time required to reduce
the population of pathogens by 90%. The model-fitting ability was assessed by measuring the root
mean squared error (RMSE) and the coefficient of determination (R
2
). To estimate the viral inactivation
kinetics, the model was fitted using non-linear regression by Microsoft Excel 2013.
Molecules 2019,24, 1835 12 of 14
4.8. Statistical Analyses
Data were analyzed by Student’s t-test using the SPSS program. All experiments were carried out
three times. Data were expressed as mean ±SE. Statistical significance was set at p≤0.05.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions:
Conceptualization, N.L. and R.Z.; methodology, D.C.; software, L.S.; validation, H.M. and
S.Z.; formal analysis, J.C.; investigation, Y.Z. resources, Y.Z. data curation, S.Z.; writing—original draft preparation,
N.L.; writing—review and editing, N.L.; supervision, R.Z.; project administration, N.L.; funding acquisition, R.Z.
Funding:
This research was sponsored by the National Natural Science Foundation of China (# 31301715),
the National Science and Technology Major Project (#2018ZX10734401) and the Program of Health and Medicine
Science in Zhejiang Province (NO. 2017KY292).
Acknowledgments:
The authors express their thanks to Ran Cai for sample analysis. Technical assistance of
Xiuyu Lou and Liming Gong are also gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
HP Houttuynia cordata polysaccharide
HWE Houttuynia cordata water extract
HEE Houttuynia cordata ethanol extract
GlaUA galacturonic acid
Gal galactose
Rha rhamnose
Ara arabinose
GluA glucuronic acid
Glc glucose
Man mannose
Xyl xylose
HuNoV Human norovirus
MNV-1 murine norovirus-1
ATCC American Type Culture Collection
PFU Plaque forming units
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