Journal of Ethnopharmacology 118 (2008) 79–85

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Immunomodulatory and anti-SARS activities

of Houttuynia cordata
Kit-Man Lau a,1 , Kin-Ming Lee a,1 , Chi-Man Koon a,1 , Crystal Sao-Fong Cheung b , Ching-Po Lau a ,
Hei-Ming Ho b , Mavis Yuk-Ha Lee a , Shannon Wing-Ngor Au b , Christopher Hon-Ki Cheng b ,
Clara Bik-San Lau c , Stephen Kwok-Wing Tsui b , David Chi-Cheong Wan b ,
Mary Miu-Yee Waye b , Kam-Bo Wong b , Chun-Kwok Wong d , Christopher Wai-Kei Lam d ,
Ping-Chung Leung a , Kwok-Pui Fung a,b,∗
a
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
b
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
c
School of Pharmacy, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
d
Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China

a r t i c l e i n f o a b s t r a c t

Article history: Background: Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia caused by
Received 12 October 2007 SARS coronavirus (SARS-CoV). From late 2002 to mid 2003, it infected more than 8000 people worldwide,
Received in revised form 5 March 2008 of which a majority of cases were found in China. Owing to the absence of definitive therapeutic Western
Accepted 10 March 2008
medicines, Houttuynia cordata Thunb. (Saururaceae) (HC) was shortlisted by Chinese scientists to tackle
Available online 30 March 2008
SARS problem as it is conventionally used to treat pneumonia.
Aim of the study: The present study aimed to explore the SARS-preventing mechanisms of HC in the
Keywords:
immunological and anti-viral aspects.
Houttuynia cordata
Immunomodulation
Results: Results showed that HC water extract could stimulate the proliferation of mouse splenic lym-
SARS phocytes significantly and dose-dependently. By flow cytometry, it was revealed that HC increased the
Anti-viral proportion of CD4+ and CD8+ T cells. Moreover, it caused a significant increase in the secretion of IL-
3C-like protease 2 and IL-10 by mouse splenic lymphocytes. In the anti-viral aspect, HC exhibited significant inhibitory
RNA-dependent RNA polymerase effects on SARS-CoV 3C-like protease (3CLpro ) and RNA-dependent RNA polymerase (RdRp). On the other
hand, oral acute toxicity test demonstrated that HC was non-toxic to laboratory animals following oral
administration at 16 g/kg.
Conclusion: The results of this study provided scientific data to support the efficient and safe use of HC to
combat SARS.
© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 1997). Recently, several studies also provided scientific data to sup-
port and unveil its anti-inflammatory (Park et al., 2005; Lu et al.,
Houttuynia cordata Thunb. (Saururaceae) (HC) is a traditional 2006), anti-allergic (Li et al., 2005; Kim et al., 2007), virucidal
Chinese medicine (TCM) used for hundreds of year to relieve lung- (Hayashi et al., 1995; Chiang et al., 2003), anti-oxidative (Chen et
related symptoms such as lung abscess, phlegm, cough and dyspnea al., 2003; Cho et al., 2003; Ng et al., 2007) and anti-cancer (Chang
(State Pharmacopoeia Commission of People’s Republic of China, et al., 2001; Kim et al., 2001) activities.
2005) and is effective in treating pneumonia, infectious disease, During severe acute respiratory syndrome (SARS) outbreak
refractory hemoptysis as well as malignant pleural effusion (Anon, in late 2002 to mid 2003, there were more than 8000 probable
cases reported to WHO, of which over 7000 cases were found
in China (http://www.who.int/en/). Owing to the high infec-
tious rate and the absence of definitive therapeutic Western
∗ Corresponding author at: Institute of Chinese Medicine, Room E205 Science Cen- medicines, State Administration of Traditional Chinese Medicine
tre East Block, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, of the People’s Republic of China proposed six TCM formulae
China. Tel.: +852 3163 4370; fax: +852 2603 5248.
to general public as preventive measures on 24 April 2003
E-mail address: [email protected] (K.-P. Fung).
1
These authors contributed equally to this work.
(http://www.satcm.gov.cn/zhuanti/jbfz/20060901/100052.shtml).

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2008.03.018
80 K.-M. Lau et al. / Journal of Ethnopharmacology 118 (2008) 79–85

Houttuynia cordata was one of the component herbs in a heat- 2.4. 3 H-thymidine incorporation assay
removing and detoxifying formula. In order to reveal its underlying
mechanisms in preventing SARS, HC was investigated for its Water extract of HC was dissolved in complete RPMI medium
immunomodulatory effect in mouse splenic lymphocytes and and incubated with 10 ␮g/ml of polymyxin B sulfate (Sigma-Aldrich
inhibitory activity on SARS coronaviral 3C-like protease (3CLpro ) Co., St. Louis, MO, USA) at 37 ◦ C for 1 h to mask the effect of prob-
and RNA-dependent RNA polymerase (RdRp) in this work. able presence of endotoxin in the extract. Splenic lymphocytes
(5 × 105 cells/well) were seeded in a 96-well plate and incubated
with HC extract at 0, 50, 100, 200 or 400 ␮g/ml for 48 or 72 h.
2. Materials and methods Subsequently, 3 H-thymidine (0.5 ␮Ci per well) was added and the
plate was incubated at 37 ◦ C for 6 h. The cells were harvested with
2.1. Preparation of herbal extract Unifilter-96 Harvester (PerkinElmer Inc., Waltham, MA, USA) and
3 H-thymidine incorporation was then measured by TopCount NXT

Dried HC, harvested in SiChuan Province of China, was pur- liquid-scintillation counter (PerkinElmer Inc.).
chased from an herbal supplier in Hong Kong. It was authenticated
cpm of treated cells
by morphological characterizations and thin layer chromatography Stimulation index =
cpm of untreated cells
(TLC, Merck silica gel G, hexane:ethyl acetate = 9:1 as the solvent
system and visualized with staining solution of dinitrophenylhy-
2.5. T cell population determination
drazine TS) against methyl nonyl ketone (National Institute for
the Control Pharmaceuticals and Biological Products, Beijing, PRC)
Mouse splenic lymphocytes (5 × 106 cells) were treated for 24,
as the chemical marker in accordance with the Chinese Pharma-
48 or 72 h in 12-well plate at 37 ◦ C with HC water extract at 0,
copoeia (State Pharmacopoeia Commission of People’s Republic
100, 200 or 400 ␮g/ml that was pre-incubated with polymyxin
of China, 2005). A voucher specimen of HC was deposited in the
B sulfate as described above. Splenic lymphocytes were then
museum of Institute of Chinese Medicine, The Chinese University
washed with cold staining buffer (2% heat inactivated FBS and
of Hong Kong (Voucher specimen number: 2005-2606).
0.05% sodium azide in PBS) and blocked with mouse and rat
Water extract was prepared by boiling 300 g of HC in 3 l distilled
IgG. Splenic lymphocytes were incubated with fluorescein isoth-
water under reflux for 1 h twice. The extract was then centrifuged
iocyanate (FITC)-conjugated rat anti-mouse CD4 antibody and
and filtered to remove tiny debris. The filtrate was eventually
phycoerythrin (PE)-conjugated rat anti-mouse CD8 antibody (BD
lyophilized into dry powder for experiments. The extraction yield
Pharmingen, San Diego, CA, USA) on ice for 30 min. Cells were
was about 14%. HPLC profile of the extract for standardization was
then washed with staining buffer and fixed with 0.8 ml of 1%
developed with Beckman-high performance liquid chromatogra-
paraformaldehyde (Sigma) and analyzed by FACSort flow cytometer
phy system gold equipped with Beckman ultra-sphere ODS column
(Becton, Dickinson & Co., Franklin Lakes, NJ, USA).
(4.6 mm × 250 mm i.d., particle size 5 ␮m) and photodiode array
detector (Beckman 168 DAD detector) (Liu, 2007). The extract was 2.6. ELISA
dissolved in 30% ethanol to make a solution at concentration of
10 mg/ml and filtered with a 0.45-␮m membrane prior to injection. Cell culture supernatant from mouse splenic lymphocytes
Ten microliters of the solution was injected into the HPLC system treated with HC extract for 24, 48 or 72 h was tested for the pres-
with the detector set at 254 and 320 nm and eluted at a flow rate ence of cytokines including IL-2, IL-4, IL-10 and IFN-␥ by ELISA.
of 1 ml/min with a gradient profile of 0.1% formic acid in H2 O as It was carried out according to the manufacturer’s protocol (BD
solvent A and acetonitrile as solvent B (starting with 5% of B, from Pharmingen).
5% to 15% of B in 15 min; 15% to 25% of B in next 15 min; 25% to 55%
of B in next 20 min and 55% to 90% of B in next 5 min). 2.7. SARS-CoV 3C-like protease (3CLpro ) assay

2.7.1. Construction of His-3CLpro and protease substrate

2.2. Animals expression plasmids
DNA fragment encoding SARS-CoV main protease
Balb/c mice were supplied by Laboratory Animal Services Cen- was amplified by PCR using primers [5 -TAGCTAGAATTC-
ter, The Chinese University of Hong Kong. They were housed under GGATCCAGTGGTTTTAGGAAAATG-3 ] and [5 -TAACTAAAGCTTTC-
the conditions of 22–25 ◦ C and a 12-h light–dark cycle. They were ATTGGAAGGTAACACCAGA-3 ], followed by digestion by BamHI
supplied with standard animal chow (PicoLab Rodent Diet 20, PMI and HindIII. The DNA was ligated with digested pET3a plasmid,
Nutrition International, Inc., Brentwood, MO, USA) ad libitum with which contains the gene of maltose binding protein (MBP). The
free access to tap water. resulting plasmid encodes the protease with an N-terminal
His-MBP tag. For construction of the substrate, the primers [5 -
GCCGCCGAGCTCACCAGCGCGGTGCTGCAGAGCGGCTTTCGCAAGATG-
2.3. Splenic lymphocytes preparation 3 ] and [5 -TATGCAGGTACCTTACTTGTACAGCTCGTCCAT-3 ] were
used to produce DNA fragment of substrate sequence with yellow
Female Balb/c mice (6–8 weeks) were sacrificed by cervical dis- fluorescent protein (YFP). After digestion by SacI and KpnI, the
location and spleens were removed. Single cell suspension was DNA was ligated with linearized pET3a plasmid cloned with
obtained by pressing the spleens through a sterile wire sieve into cyan fluorescent protein (CFP) previously, so that the resulting
complete RPMI medium. The single cell suspension was carefully plasmid encodes a protein-containing substrate sequence cloned
layered onto the Ficoll-Paque Plus solution (Amersham Biosciences, in between CFP and YFP.
Piscataway, NJ, USA) with a density gradient of 1.077 g/ml. The
splenic lymphocytes were collected at the interface after centrifu- 2.7.2. Expression and purification of His-tagged SARS-CoV 3CLpro
gation at 660 × g for 20 min at room temperature. The splenic and 3CLpro substrate proteins
lymphocytes were washed with RPMI medium and then suspended Escherichia coli stain BL21 (DE3) (Novagen, EMD Chemicals
in complete RPMI medium to a concentration of 5 × 106 cells/ml. Inc., Darmstadt, Germany) was used to express the recombinant
 K.-M. Lau et al. / Journal of Ethnopharmacology 118 (2008) 79–85 81

pET-3a-3CLpro protein. BL21 (DE3) cells were transformed with The amplified DNA fragment was digested at BamHI and NotI sites
pET-3a expression vector containing the pET-3a-3CLpro insert, as and subcloned into pGEX-6p2 vector (Amersham Biosciences)
described above. The transformants were cultured in 500 ml of to form the pGEX-6p2–RdRp construct. At the 5 -end of the
LB medium containing 0.4% glucose, 1 mM MgSO4 , 100 ␮g/ml RdRp gene, a sequence encoding the GST protein was attached.
ampicillin and 50 ␮g/ml chloramphenicol at 37 ◦ C until Abs600 nm The open reading frame of the final construct and the encoding
reached 0.8. After that, they were induced with 0.4 mM isopropyl of SARS-CoV RdRp (residues 1–932) were confirmed by DNA
␤-d-thiogalactoside (IPTG) at 37 ◦ C for 4 h. The cells were then har- sequencing.
vested by centrifugation at 4 ◦ C.
For His-tagged SARS-CoV 3CLpro protein purification, the cell 2.8.2. Expression and purification of GST-fusion SARS-CoV RdRp
pellet was first resuspended in buffer A [20 mM Tris–HCl (pH full-length protein
7.8), 20 mM NaCl, 10 mM imidazole] and disrupted on ice by son- The pGEX-6p2–RdRp plasmid was transformed into Origami
ication. The lysate was subsequently centrifuged to collect the (DE3) E. coli strain (Novagen). A single colony was grown at 37 ◦ C
supernatant for loading onto a nickel-chelated (Ni-NTA) column. for 16 h in 10 ml of 2× YTG medium supplemented with ampicillin
The bound protein was eluted in a gradient of 0–100% buffer B (0.1 mg/ml). The cell culture was further grown at 37 ◦ C in 1L of 2×
[20 mM Tris–HCl (pH 7.8), 20 mM NaCl, 300 mM imidazole]. Purity LB medium containing ampicillin (0.1 mg/ml) until OD600 reached
of the isolated protein was examined by SDS-PAGE, followed by 0.6–0.8. Protein expression was induced with 0.4 mM IPTG at 28 ◦ C
Coomassie blue staining. Thereafter, the semi-purified 3CLpro pro- for 6 h before harvesting.
tein was pooled and dialyzed against buffer C [20 mM Tris–HCl Protein purification was carried out using affinity chro-
(pH 8.0), 50 mM NaCl, 2 mM CaCl2 , 10 mM 2-mercaptoethanol] matography with glutathione SepharoseTM Fast Flow (Amersham
overnight. The resulting protein samples were subjected to fac- Biosciences). The cell pellet was resuspended in buffer A (50 mM
tor Xa cleavage at room temperature for 10 h. The His-tags were Tris–HCl, pH 8.0, 500 mM NaCl, 2 mM DTT) containing 1 mM PMSF
later removed by the nickel hi-trap column. At the end, the pro- and 1 mM benzamidine and lysed on ice by sonication. The lysate
teins so obtained were further purified using the S75 gel filtration was centrifuged at 40,000 × g and 4 ◦ C for 1 h. The supernatant was
column [buffer D: 20 mM Tris–HCl (pH 7.8), 20 mM NaCl, 10 mM 2- loaded onto a glutathione Sepharose 4B column equilibrated with
mercaptoethanol]. The protein concentration was determined by the washing buffer (buffer A). The column was washed with buffer
Bradford method (Loffler and Kunze, 1989) for protease activity A and the bound protein was then eluted with five column vol-
assay. umes of buffer B (10 mM reduced glutathione, 50 mM Tris–HCl, pH
Similarly, the plasmid pET-3a-3CLpro substrate was transformed 8.0). The eluted fraction were pooled and concentrated. The con-
into the E. coli stain BL21 (DE5) (Novagen), and the protein was centrated proteins were dialyzed against buffer C (50 mM Tris–HCl,
expressed at 22 ◦ C for 16 h with 0.4 mM IPTG induction. His-tagged pH 8.0, 10% glycerol, 1 mM DTT) for 24 h. SDS-PAGE analysis were
SARS-CoV pET-3a-3CLpro substrate protein was isolated by means performed to check the purity and quality of the protein samples
of the nickel-chelating column chromatography. Briefly, the pellet at every stage of purification. The purified proteins were character-
was resuspended in buffer A [20 mM Tris–HCl (pH 7.8), 20 mM NaCl, ized by western blot, mass spectrometry and polymerase activity
10 mM imidazole] and disrupted on ice by sonication. The lysate assay. The protein concentration was determined by the Bradford
was centrifuged. The supernatant resulted was allowed to bind onto method.
a Ni-NTA column. Meanwhile, any unbound proteins were washed
away thoroughly with buffer A. Then, the His-3CLpro substrate was 2.8.3. Radiometric RdRp assay—filter-binding enzyme assay
eluted in a gradient of 0–100% buffer B [20 mM Tris–HCl (pH 7.8), The filter-binding polymerase assay was modified from that
20 mM NaCl, 300 mM imidazole]. The pure protein fractions were used in polymerase activity assay of hepatitis C virus (HCV) RdRp
pooled and dialyzed against buffer D [20 mM Tris–HCl (pH 7.8), (Yamashita et al., 1998). A total volume of 50 ␮l polymerase-
20 mM NaCl, 10 mM 2-mercaptoethanol] overnight. Again, the pro- containing reaction mixture included 50 mM Tris–HCl, pH 8.0,
tein concentration was determined by the Bradford method and it 7.5 mM KCl, 8 mM MgCl2 , 10 mM DTT, 1%BSA, 3.5 ␮l of 1 mM UTP,
was ready for the protease activity assay. 3.33 ␮Ci of [␣-32 P] UTP (Amersham Pharmacia Biotech, Sweden),
3.12 ␮g/ml of poly A (Roche Applied Science, Switzerland), 5 ␮g/ml
2.7.3. Fluorogenic SARS-CoV 3CLpro activity assay of RNA, 1 ␮g/ml of oligoU16 (Tech Dragon Ltd., Hong Kong, China),
The effect of HC water extract on SARS-CoV 3CLpro was studied 20 units of RNAase inhibitor (Promega Bioscience, Madison, USA)
as previously reported with minor modification (Kuo et al., 2004). and with or without HC water extract. The mixture was incu-
HC extract, at different concentrations in reaction buffer (150 mM bated at 37 ◦ C for 1 h and reaction was stopped by adding a cold
Tris, 150 mM NaCl, 10 mM 2-mercaptoethanol, pH 7.8), was incu- buffer solution containing 20 mM sodium pyrophosphate and 5%
bated with 80 ␮l of 3CLpro (final concentration: 2 ␮M) for 15 min at trichloroacetic acid. The solution was dotted on the Whatman GF/C
room temperature. After that, 80 ␮l of 3CLpro substrate (20 ␮M) was glass microfiber filter and washed with the cold buffer five times
added to the reaction mixture and fluorescence signals (Ex430 nm, and then 75% ethanol once. The incorporated triphosphate was
Em486 nm, Em530 nm) were measured immediately at 15-s inter- assayed by measuring 32 P using liquid scintillation counter (Beck-
val for 25 min by multilabel fluorescence reader (PerkinElmer 2101 man LS 6000SC).
EnVision, PerkinElmer Inc.).
2.9. Acute oral toxicity test
2.8. SARS-CoV RNA-dependent RNA polymerase (RdRp) assay
The acute oral toxicity test was carried out according to the Pro-
2.8.1. Construction of GST-RdRp full-length expression plasmid cedures and Methods for Toxicological Assessment on Food Safety,
A 2.5 kb DNA fragment corresponding to the full Peoples Republic of China. Balb/c mice (male and female) each
length of the SARS-CoV RdRp CUHK W-1 (NCBI accession weighed between 17 and 20 g were used. Twelve hours prior to
code: AAP13566) was amplified using the forward [5 - dosing, all food was removed to fast the animals before initiating
GTGGTGGGATCCTCTGCGCGGATGCATCAACGTTTTT-3 ] and reverse the test. On the day of the test, animals were identified and body
[5 -TATGCGGCCGCTCACTGCAAGACTGTATGTGGTGTGTA-3 ] primer weights recorded. The dosage to be administered was calculated
set. The two primers contain BamHI and NotI sites, respectively. based on the animal’s body weight. The dosage of HC water extract
82 K.-M. Lau et al. / Journal of Ethnopharmacology 118 (2008) 79–85

2.10. Statistical analysis

Data were analyzed using Mann–Whitney U test with SPSS 14.0

for Window. Statistical significance was accepted at p < 0.05.

3. Results

3.1. Proliferation of splenic lymphocytes

Splenic lymphocytes of Balb/c mice were incubated with HC

extract at a concentration of 0–400 ␮g/ml in the presence of
polymyxin B sulfate for 48 and 72 h. The proliferation response was
shown in Fig. 1. HC extract was found to significantly stimulate the
proliferation of mouse splenic lymphocytes in a dose-dependent
manner. However, the stimulation at 48 h is higher than that of
72 h.

Fig. 1. Lymphoproliferation activity of Houttuynia cordata extract on mouse splenic 3.2. Effect on CD4+ and CD8+ T cells in vitro
lymphocytes after 48 and 72 h treatment. The results are expressed as mean ± S.D.
of six values from one representative experiment. **p < 0.01 vs. control (0 ␮g/ml). The above data raised our interest to investigate whether HC
extract stimulates T cell population. Splenic lymphocytes were
cultured with various concentrations of HC extract in the pres-
was 16 g/kg body weight. The maximum volume is 0.4 ml/10 g body ence of polymyxin B sulfate for 24, 48 and 72 h. The numbers
weight. The control group was treated with same amount of dis- of CD4+ and CD8+ T cells were measured by flow cytometry.
tilled water. Animals were closely observed for gross toxicological It was found that HC extract increased the proportion of CD4+
effects at 1, 3 and 6 h immediately after a single dose administration T cells in a dose-dependent manner (Table 1). HC extract also
of the sample and then daily for a 7-day observation period. Test increased the proportion of CD8+ T cells after treatment (Table 2).
animals’ body weights, a sensitive indicator of toxic insult, were These results indicated that HC stimulates T cells proliferation
recorded during the observation period. in vitro.

Fig. 2. Effect of Houttuynia cordata on IL-2 (A), IL-10 (B), IL-4 (C) and IFN-␥ (D) production in mouse splenic lymphocytes. Mouse splenic lymphocytes were treated with HC
extract for 24, 48 and 72 h. Cytokine production was measured by ELISA. Data are expressed as mean ± S.D. of values from at least three independent experiments. *p < 0.05,
**p < 0.01 vs. control (0 ␮g/ml).
 K.-M. Lau et al. / Journal of Ethnopharmacology 118 (2008) 79–85 83

Table 1
Percentage of CD4+ T cells in mouse splenic lymphocytes after HC treatment

Concentration of HC extract (␮g/ml)

0 100 200 400

24 h 31.8 ± 0.9 37.3 ± 1.4* 36.8 ± 0.5* 37.4 ± 1.0*

48 h 31.8 ± 0.7 35.5 ± 0.9* 36.3 ± 0.4* 38.0 ± 0.5*
72 h 27.1 ± 2.1 31.8 ± 0.5* 32.8 ± 1.5* 35.1 ± 0.7*

Data are means ± S.D. of four values.

*
p < 0.05.

Table 2
Percentage of CD8+ T cells in mouse splenic lymphocytes after HC treatment

Concentration of HC extract (␮g/ml)

0 100 200 400

24 h 10.0 ± 0.3 11.2 ± 0.6* 10.9 ± 0.2* 10.2 ± 0.3

48 h 7.3 ± 0.4 9.1 ± 0.4* 9.2 ± 0.2* 9.2 ± 0.3* Fig. 4. Effect of Houttuynia cordata on the SARS-CoV RdRp activity using radiometric
72 h 4.8 ± 0.2 7.9 ± 0.2* 8.6 ± 0.6* 8.0 ± 0.2* RdRp assay by [␣-32P] UTP incorporation. Data are expressed as mean ± S.D. from
Data are means ± S.D. of four values. three individual experiments. *p < 0.05, **p < 0.01 vs. control.
*
p < 0.05.

3.5. Effect on RdRp activity

3.3. Effect on cytokine secretion
The RdRp activities of the protein samples were further con-
firmed by the conventional [␣-32 P] UTP incorporation using
In addition to examining the effect of HC on T cell activation, we
filter-binding polymerase assay modified from that used for HCV
investigated its effect on helper T cell differentiation by measuring
RdRp (Yamashita et al., 1998). During RNA synthesis, the [␣-32 P]
IL-2, IL-4, IL-10 and IFN-␥ secretion. After 48 and 72 h treatment,
UTP was incorporated into the newly synthesized RNA. Afterwards,
HC extract increased the level of IL-2 in a dose-dependent manner
the RNA binding on the filter was separated from the free unincor-
(Fig. 2A). HC extract also caused a significant increase in secretion of
porated nucleotides and the radioactivity was measured by liquid
IL-10 by mouse splenic lymphocytes (Fig. 2B). However, the levels
scintillation counting.
of IL-4 did not change (Fig. 2C) and the level of IFN-␥ decreased in
The purified GST-RdRp exhibited a dose-dependent RdRp activ-
a dose-dependent manner after treatment (Fig. 2D).
ity when polyA was used as templates (Data not shown). To study
the effect of HC on the polymerase activity, different concentrations
3.4. Effect on SARS-CoV 3CLpro activity of HC (50, 100, 200, 400 and 800 ␮g/ml) were tested. All concentra-
tions of HC showed a significant decrease in RdRp activity. At the
The effect of HC on SARS-CoV 3CLpro was evaluated by a protein- highest dose (800 ␮g/ml), the RdRp activity was decreased to 26%
based fluorogenic assay. HC, at concentration of 200 ␮g/ml or of control. This result indicated that HC exhibited a dose-dependent
above, could significantly inhibit the activity of 3CLpro (p < 0.05). inhibition on RdRp activity significantly (Fig. 4).
At the highest testing dose (1000 ␮g/ml), the 3CLpro activity was
decreased to 50% of control. This result indicated that HC exhibited
3.6. Acute oral toxicity test
a dose-dependent inhibition on 3CLpro activity significantly (Fig. 3).

Twenty Balb/c mice (10 male, 10 female) were administrated a

single oral dose of HC water extract at 16 g/kg. All animals appeared
normal throughout the 7-day observation period. There was no
difference of body weights between HC group and control group
(p > 0.05) (Table 3). According to Procedures and Methods for Toxi-
cological Assessment on Food Safety, Peoples Republic of China, HC
water extract was tested as specified and considered to be essen-
tially non-toxic to laboratory animals following oral administration
at 16 g/kg.

4. Discussion and conclusion

T cells are essential for adaptive immunity against viral infec-

tions. Anti-viral CD4+ helper T cells (Th) help the production of
virus-specific antibodies by B cells, while CD8+ cytotoxic T cells
(Tc) can kill virus-infected host cells. Lymphopenia was usually
observed in SARS patients at the initial phase of infection (Li et
al., 2003; Yu et al., 2003; He et al., 2005). Virus-induced apopto-
sis of lymphocytes has been suggested as a cause for lymphopenia
seen in SARS patients (O’Donnell et al., 2003). Since monocytes
Fig. 3. Effect of Houttuynia cordata on the SARS coronaviral 3CLpro activity. Data are
and T cells are involved in both the innate and adaptive immune
expressed as mean ± S.D. of values from four independent experiments. *p < 0.05 vs. response, destruction of these cells may result in a compromised
control (0 ␮g/ml). immune response and the development of disease. Our results
84 K.-M. Lau et al. / Journal of Ethnopharmacology 118 (2008) 79–85

Table 3
Change of body weights of animals in the oral acute toxicity study of Houttuynia cordata water extract

Group Number of animal Dose (g/kg) Mortality (%) Average body weight (g) Body weight gain (g) p-value vs. control

Initial Final

HC extract M 10 16 0 18.9 ± 0.5 20.5 ± 0.7 1.6 ± 0.5 >0.05

F 10 16 10 17.2 ± 0.6 17.9 ± 0.8 0.9 ± 0.6 >0.05

Control M 10 – 0 18.7 ± 0.6 20.8 ± 0.5 2.1 ± 0.4

F 10 – 0 19.0 ± 0.7 20.4 ± 1.0 1.4 ± 0.7

Data are means ± S.D.

demonstrated that HC extract could exhibit immunostimulatory contain additional viral and cellular proteins (Thiel et al., 2003).
effect (Fig. 1; Tables 1 and 2). It was found to stimulate the prolifer- Given the crucial role of RdRp in the virus life cycle and the suc-
ation of CD4+ helper T cells and CD8+ cytotoxic T cells, which may cess obtained with polymerase inhibitors in the treatment of viral
help to prevent SARS-CoV infection. infections including human immunodeficiency virus type 1 (HIV-
CD4+ helper T cells can be divided into different subtypes, based 1), human hepatitis B virus (HBV) (Korba et al., 2006), HCV and
on the specific profiles of cytokines that they release. Th1 cells have herpes virus, SARS-CoV RdRp is an attractive target for the devel-
been found to produce IL-2, IFN-␥ and lymphotoxin-␣, whereas opment of anti-SARS drugs. At present, there are no structural and
Th2 cells produce IL-4, IL-5, IL-9 and IL-13 (Romagnani, 2006). Th1 very limited biochemical data on coronavirus polymerase. Thus,
cells assist in activating macrophages and Tc cells, which are par- study on the polymerization mechanisms is likely to aid the devel-
ticularly useful for eliminating intracellular infections; Th2 cells opment of anti-SARS agent. It is reported that during purification,
mediate anti-helminth and allergic responses. Type 1 regulatory T the full-length enzyme is found to be hydrolytically cleaved into
(Tr1) cells are distinct from the classical Th1 or Th2 cells. They pro- three main fragments: an N-terminal p12 fragment, a middle p30
duce high levels of IL-10, with or without TGF-␤, IFN-␥ and IL-5, and fragment, and a C-terminal p64 fragment which comprises the
low or no IL-2 and IL-4 (Veldman et al., 2006). IL-10 has a key effect polymerase catalytic domain (Cheng et al., 2005). Currently, the
on the suppression of Th1 cell responses and has a role in host pro- recombinant SARS-RdRp expressed in E. coli is characterized by
tection against harmful effects of an exacerbated cellular immune radioactive assay (Al et al., 1998; Yamashita et al., 1998; Cheng
response during acute infection. In order to investigate how HC et al., 2005) and we demonstrated that radiolabeled nucleotides
extract affect the T cell differentiation, we measured the levels of incorporation increased with increasing SARS-CoV RdRp protein
IL-2, IL-4, IL-10 and IFN-␥ secreted by mouse splenic lymphocytes. (Data not shown). HC extract, at 50 ␮g/ml or above, could effectively
From our results, IFN-␥ and IL-4 that are secreted by Th1 and Th2, inhibit [␣-32 P] UTP incorporation, implying that it can inactivate
respectively, did not increase. On the contrary, IL-10 that is secreted the SARS-CoV RdRp activity.
by Tr1 increased significantly after treatment. We speculated that Taking all the results into account, the actions of HC extract on
HC extract caused the induction of Tr1. In the 3 H-thymidine incor- SARS may be biphasic. Before the invasion of SARS-CoV, HC extract
poration assay, the stimulatory response in 72 h is lower than that may activate the cell-mediated immunity to prevent viral infection.
of 48 h (Fig. 1). This may due to the negative feedback by Tr1 cells In case of infected, HC extract may slow down the viral replica-
that suppress Th cells response. Induction of Tr1 cells can assist in tion process by inhibiting the pivotal enzymes and trigger negative
turning off immune response after pathogen elimination and pre- feedback control in immune system.
vent harmful effect. However, further in-depth studies are needed
to confirm the induction of Tr1 by HC extract.
Acknowledgments
The SARS-CoV main protease, 3CLpro , is responsible for releas-
ing the key replicative enzymes such as RdRp and helicase from the
This work was supported by the Research Fund for the Control of
polyprotein precursors (Thiel et al., 2003). This functional impor-
Infectious Diseases (02040332), Health, Welfare and Food Bureau,
tance of 3CLpro in the life cycle of virus makes it a key target for the
The Government of the Hong Kong Special Administrative Region
development of drugs directed against SARS (Anand et al., 2003;
(HKSAR). We would also like to thank Mrs. Yeung Yeo G.Y. and Miss
Gan et al., 2006; Wu et al., 2006) and for screening the anti-SARS
Chong L.T. for their technical support.
effect of traditional Chinese medicines (Lin et al., 2005). In order
to study the effect of HC extract on SARS-CoV 3CLpro , a convenient
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