EFFECTS OF Cryptolepis buchanani EXTRACTS ON INFLUENZA A VIRUS
REPLICATION
Nuttaporn Boonsert*, Jaturapon Sawangjaroen, Radeekorn Akkarawongsapat#
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, 10400,
Thailand
*e-mail: Nuttapornboonsert@gmail.com, #e-mail: radeekorn.akk@mahidol.ac.th
Abstract
Influenza A virus, a member of the Orthomyxoviridae family, is a significant
pathogen of respiratory illness or flu that causes high morbidity and mortality annually
worldwide. One of the major problems in fighting against influenza A virus infections
nowadays is the emergence of antiviral drug-resistant strains, suggesting that the search for
alternative antiviral agents is important in order to control the virus.
Cryptolepis buchanani Roem & Schult or “Thao en on” belongs to the family
Asclepiadaceae. It is a climbing tree that commonly used in traditional medicine recipes for
many decades. Several evidences have shown that C. buchanani possesses many
pharmacological properties including anti-inflammation, immunomodulatory activity, antibacterial, and nosocomial infections treatment. However, there is no report concerning the
antiviral activity of C. buchanani. Based on this correlation, it is of interest to evaluate the
anti-influenza activity of C. buchanani.
In this study, we demonstrated that C. buchanani extracts, VR20800, VR20804, and
VR20808, significantly inhibited infections of both A/Puerto Rico/8/34 (H1N1) (PR8) and
A/Hong Kong/8/68 (H3N2) subtypes of influenza A viruses in MDCK cells with 50%
effective concentrations (EC50) below 7 µg/ml. The three extracts possibly have similar
mechanism of inhibition since their results are parallel in all antiviral assays tested. Although
additional studies are needed, the results in this study raise the possibility of using C.
buchanani extracts as antiviral agents for influenza A virus.
Keywords: Cryptolepis buchanani, natural compounds, Influenza A virus, antiviral agents
Introduction
Influenza A virus, a causative agent of influenza or flu, causes annual epidemics and
several pandemics through the twentieth century (1). The symptoms of influenza A virus
infection are associated with the respiratory illness, including pulmonary complications that
raise the mortality rate (2). Although vaccine for influenza is available, the virus still
circulates, mutates and infects human, causing hospitalization, mortality, and economic
burden worldwide (3).
Influenza A virus belongs to the family Orthomyxoviridae. The genome is composed
of eight segments of single-stranded, negative-sense RNA (4). Influenza A virus is classified
into subtypes on the basis of the antigenic properties of the surface hemagglutinin (HA) and
neuraminidase (NA) proteins (5, 6). Currently, influenza A virus has 18 HA subtypes (H1H18) and 11 NA subtypes (N1-N11) (7).
Two classes of anti-influenza drugs are available. They are M2 inhibitors and
neuraminidase inhibitors which act at different steps in the viral replication cycle. However,
several evidences have shown the emergence of influenza viruses that are resistant to the
available antiviral drug (6, 8). Therefore, new antiviral compounds are needed as alternatives
to deal with influenza A virus infection.
Natural compounds have been served as traditional medicines for many decades;
hence, they could be good candidates for antiviral drug development. Cryptolepis buchanani
Roem. & Schult (Thao en on) is a climbing tree that belongs to the family Asclepiadaceae
(9). According to Ayurveda, C. buchanani has been known as Indian folk medicine that used
for treatment of diarrhea, inflammation, bacterial infection, ulcerative, coughing, lactation in
women, rickets in children, and blood purification (10). In Thailand, C. buchanani is
commonly used in folk medicine recipes for the treatment of inflammation conditions such as
arthritis, and bone and joint pain (11, 12). Regardless of the knowledge described by several
cultures, C. buchanani has been tested and shown to possess various pharmacological
properties including anti-inflammation, immunomodulatory activity, anti-bacterial, and
nosocomial infections treatment (9, 10, 13). Although various therapeutic properties are
reported, none has examined antiviral property, if any, of C. buchanani.
In this study, effects of C. buchanani extracts on influenza A virus replication are
investigated. We demonstrate that C. buchanani extracts, VR20800, VR20804, and
VR20808, exhibit inhibitory activities against both A/Puerto Rico/8/34 (H1N1) (PR8) and
A/Hong Kong/8/68 (H3N2) subtypes of influenza A viruses in MDCK cells. The effective
concentrations that show inhibition at least 50% (EC50) were determined. The results of this
study could provide information about novel function of C. buchanani as anti-influenza
agents and support the use of medicinal plants as alternative medicine.
Methodology
Cell line and virus
Madin-Darby canine kidney (MDCK) cells were cultured in minimal essential
medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and
antibiotics (100 U/ml Penicillin and 100 µg/ml Streptomycin) at 37ºC in 5% CO2 atmosphere.
Human influenza virus strains A/Puerto Rico/8/34 (H1N1) (PR8) and A/Hong
Kong/8/68 (H3N2) were propagated in MDCK cells.
Cryptolepis buchanani extracts
Three fractions of Cryptolepis buchanani extracts in ethyl acetate (EtOAc), named as
VR20800, VR20804, and VR20808, were kindly provided by Associate Professor Dr.
Patoomratana Tuchinda (Mahidol University, Thailand). All extracts were dissolved in
DMSO at a stock concentration of 20 mg/ml and stored at -20ºC.
Cytotoxicity Assay
Overnight culture of MDCK cells (2 × 104 cells/well) in 96-well plates were washed with
MEM supplemented with antibiotics and then treated with 2-fold serially diluted C.
buchanani extracts at the indicated concentrations or mock control (MEM with antibiotics) at
37ºC and 5% CO2 for overnight. Twenty µl of the CellTiter 96 Aqueous One Solution
Reagent (Promega, Madison, WI) was added to all tested wells and the plates were incubated
at 37ºC for 90 minutes. Then, optical density (OD) was measured at wavelength 492 nm by
ELISA plate reader (Tecan, Austria).
Antiviral assays
All antiviral assays were done by plaque reduction assay. Various concentrations of
C. buchanani extracts, VR20800 (0.78-25 µg/ml), VR20804 (1.56-25 µg/ml), and VR20808
(0.156-5 µg/ml) were tested in four antiviral assays: comprehensive antiviral assay, pretreatment assay, simultaneous assay, and post-entry assay. These four assays are different at
the time C. buchanani extracts were present, which are at all time of the assay, prior to
infection, during infection, and post infection, respectively.
Comprehensive antiviral assay
MDCK cells (6 × 105 cells/ml) in a 6-well plate for overnight were washed with
MEM supplemented with antibiotics and then treated with diluting media (MEM with 0.2%
BSA and antibiotics) alone or indicated concentrations of C. buchanani extracts for 1 hour at
37ºC. Then, the medium was discarded, and MDCK cells were infected with the influenza A
virus strain PR8 or H3N2 (40 PFU/well) in the presence of C. buchanani extracts at 37ºC for
1 hour. After infection, C. buchanani extract-virus mixture was discarded, and infected
MDCK cells were treated with plaque mixture solution (1.2% Avicel, 1X MEM, 0.22%
NaHCO3, 0.21% BSA, 0.01 M HEPES, 2 mM glutamine, 0.01% DEAE-dextran, (100 U/ml
Penicillin and 100 µg/ml Streptomycin), and 2 µg/ml TPCK-trypsin) in the presence of C.
buchanani extracts. After incubation at 37ºC for 2 days, the infected cells were washed with
PBS and treated with 3.7% formaldehyde for 1 hour at room temperature. To visualize
plaques, each well of infected cell monolayer was stained with 1.5 ml of 1.25% crystal violet
for 10 minutes at room temperature and washed with tap water. Numbers of plaque formation
were counted and plaque forming unit (PFU) was calculated.
Pre-treatment assay
In a 6-well plate, confluent MDCK cells were washed with MEM supplemented with
antibiotics and treated with diluting media alone or various concentrations of C. buchanani
extracts at 37ºC for 1 hour. Then, pre-treatment solution was removed and the cells were
washed with diluting medium and infected with PR8 or H3N2 (40 PFU/well) at 37ºC for 1
hour. After infection, C. buchanani extracts-virus mixture was discarded and MDCK cells
were treated with plaque mixture solution at 37ºC for 2 days. Plaques were visualized and
counted as described above.
Simultaneous assay
Confluent MDCK cells were washed once with washing media (MEM with
antibiotics). Then, the cells were infected with PR8 or H3N2 (40 PFU/well) in the presence
of various concentrations of C. buchanani extracts at 37ºC for 1 hour. Subsequently, C.
buchanani extracts-virus mixture was discarded and MDCK cells were treated with plaque
mixture solution at 37ºC for 2 days. Plaques were visualized and counted as described above.
Post-entry assay
Confluent MDCK cells were washed once with washing media. Then, the cells were
infected with PR8 or H3N2 (40 PFU/well) for 1 hour. Subsequently, the virus was discarded
and infected MDCK cells were treated with plaque mixture solution in the presence of
various concentrations of C. buchanani extracts at 37ºC for 2 days. Plaques were visualized
and counted as described above.
Results
Cryptolepis buchanani extracts inhibited both PR8 and H3N2 influenza A virus replication
Initially, eleven Cryptolepis buchanani extracts, named VR20799-20809, were
screened for their ability to inhibit influenza A virus strains A/Puerto Rico/8/34 (H1N1)
(PR8) and A/Hong Kong/8/68 (H3N2) replication in Madin-Darby canine kidney (MDCK)
cells by comprehensive antiviral assay. In this assay, various concentrations of C. buchanani
extracts (0.156 – 12.5 µg/ml) were present at all time of the assay so that cumulative effects
would be observed. Based on the ability to inhibit influenza A virus replication, VR20800,
VR20804, and VR 20808 were selected for further experiments.
In the screening comprehensive antiviral assay, cytotoxic effects on MDCK were
observed starting at concentrations of 6.25 µg/ml for VR20800, 12.5 µg/ml for VR20804, and
1.25 µg/ml for VR20808 (data not shown). As a consequence, the comprehensive assay was
rerun with concentrations of C. buchanani extracts lower than cytotoxic concentration. The
tested concentrations were adjusted to 0.78-6.25 µg/ml for VR20800, 1.56-12.5 µg/ml for
VR20804, and 0.156-1.25 µg/ml for VR20808. All three C. buchanani extracts significantly
reduced PR8 and H3N2 virus titers at the highest concentrations tested compared to no
extract control (Figure 2). The results showed that the C. buchanani extracts inhibited both
PR8 and H3N2 replication in a dose-dependent manner. The effective concentrations that
show inhibition at least 50% (EC50) were determined (Table 1). VR20808 is the most
effective, inhibiting both PR8 and H3N2 viruses with EC50 values of 0.625 µg/ml and 1.25
µg/ml, respectively.
Treatment of VR20800, VR20804, and VR20808 prior to infection did not protect cells from
infection
To determine whether VR20800, VR20804, and VR20808 provided cellular
protection, MDCK cells were pre-treated with various concentrations of the extracts for 1
hour and rinsed prior to infection with the influenza viruses. Pre-treatment of the cells with
all three extracts did not significantly reduce virus titers even at the concentrations higher
than that tested in the comprehensive assay (Figure 1). The results suggested that the C.
buchanani extracts were inactive when they were present prior to virus infection.
Treatment of VR20800, VR20804, and VR20808 during infection reduced viral replication
Simultaneous assay was performed to determine whether VR20800, VR20804, and
VR20808 could inhibit influenza A virus infection at the same time of infection. Various
concentrations of VR20800, VR20804, or VR20808 were incubated simultaneously with
either PR8 or H3N2 virus infection for 1 hour. The EC50 values are summarized in Table 1.
As shown in Figure 1, VR20800 reduced both PR8 and H3N2 viral titers by approximately
44% and 50%, respectively, at the highest concentration tested (25 µg/ml). Similar to
VR20800, VR20804 showed the concentration-dependent reduction of PR8 and H3N2 virus
replication, inhibiting approximately 50% at highest concentration tested (25 µg/ml). The
same tendency applied to VR20808, reducing PR8 and H3N2 virus titers by 16 and 36%,
respectively, at the highest concentration tested (5 µg/ml). These results suggest that all three
C. buchanani extracts had some effects at early event of virus infection.
Post-entry treatment of VR20800, VR20804, and VR20808 showed reduction of PR8 virus but
not H3N2 virus
To address whether the inhibitory effect seen was a result of post-entry event, various
concentrations of VR20800, VR20804, or VR20808 were added after removal of PR8 or
H3N2 virus from MDCK cell monolayer that had been infected for 1 hour. The extracts were
incubated further for 2 days. Due to the long incubation period, the extract concentrations
were prepared similar to that in the comprehensive assay. As shown in Figure 2, all three
extracts reduced PR8 virus titer in a dose-dependent manner. At the highest concentration
tested, post-entry treatment of infected MDCK cells with VR20800 (6.25 µg/ml), VR20804
(12.5 µg/ml) and VR20808 (1.25 µg/ml) significantly reduce viral titer by approximately
80%, 70%, and 65%, respectively. Interestingly, inhibition of H3N2 virus replication was not
seen when the infected cells were treated with the extracts after they had been infected for 1
hour. These results suggest that the inhibitory mechanisms of the C. buchanani extracts
against PR8 and H3N2 infection are different.
Discussion and Conclusion
Nowadays, natural compounds are well-known for their roles as prophylaxis and
therapeutic agents for many diseases. Cryptolepis buchanani or “Thao en on” is a natural
herbal plant that is shown to possess various pharmacological properties and thus has been
used as a remedy in folk medicine for many decades (10-12). However, little is known about
its antiviral property. Here, we demonstrate that C. buchanani extracts, VR20800, VR20804,
and VR20808, possess inhibitory activities against influenza A virus replication in MDCK
cells. This study provides the first available data for anti-influenza property in C. buchanani.
Dose-response studies illustrated that VR20800, VR20804, and VR20808 blocked
both A/Puerto Rico/8/34 (H1N1) (PR8) and A/Hong Kong/8/68 (H3N2) infection with EC50
values ranging from as low as 0.625 to 6.25 µg/ml when the extracts were present in the
entire assay period. In addition, when the extracts were present at some time points of the
assay (i.e. pre-treatment, simultaneous, and post-treatment assays), they displayed parallel
pattern of inhibitory activities, suggesting that the mechanism of inhibition among the
extracts could be similar. These results also indicate that the compound compositions in the
extracts may be analogous one another. However, further studies to find out active
components in the extracts are needed in order to confirm the presumption.
The fact that all three C. buchanani extracts have no effect on reducing PR8 and
H3N2 virus titers in the pre-treatment assay suggests that the extracts have no effect on the
cells. Therefore, using of C. buchanani extracts as prophylaxis agents may not be useful for
viral control. In contrast to the pre-treatment assay, results obtained from the simultaneous
assay showed the concentration-dependent reduction of PR8 and H3N2 virus titers. Although
VR20808 appeared to show slight inhibition of both virus subtypes compared to the results of
others, these inhibitory activities are not account for all antiviral activities since the EC 50
values are essentially higher than those observed in the comprehensive assay. These results
suggest that all three C. buchanani extracts had some effects at early event of virus infection.
The possibility that the extracts block infection at the attachment step is inconclusive as they
did not prevent infection when they were treated with the cells before infection and a specific
attachment assay is required.
Due to the long incubation period similar to that in the comprehensive assay, the same
concentrations of VR20800, VR20804, and VR20808 were tested in post-entry assay. Results
observed in the post-entry assay were unexpected. All three extracts inhibited PR8 by
reducing virus titer in a dose dependent manner. However, the extracts had no inhibitory
effect on H3N2 virus infection in the post-entry assay. These results suggest that the
inhibitory mechanisms of the C. buchanani extracts against PR8 and H3N2 infection are
different. It is possible that PR8 and H3N2 modulate different cellular signaling pathways to
successfully infect the host cell (14). Additional studies in order to determine the mechanism
of inhibition are required.
Plaque assay was used for quantification of the viral infectivity in all antiviral assays
tested in this study. Besides reduction in plaque number, reduction in plaque size is observed
as well (data not shown). Viral plaque size was significantly decreased for both PR8 and
H3N2 in a dose-dependent manner compared to no extract control in the assays with long
incubation period, i.e. post-entry assay and comprehensive antiviral assay (data not shown).
These suggest the role of C. buchanani extracts to interfere both PR8 and H3N2 virus
replication process when the extracts are present for a long time. Although the H3N2 virus
titer was not significantly inhibited in post-entry assay but the decrease of plaque size
formation is obviously observed. A possible explanation could be that the extracts interfere
the functions of viral proteins necessary for replication, resulting in reduction of the virus
replication rate. The differences in genetic background among PR8 and H3N2 virus may be
involved (15).
In summary, we demonstrate that C. buchanani extracts possess antiviral activities
against influenza A virus infection. The steps in infection that the extracts act remain to be
elucidated further. The fact that all three extracts tested in this study are crude extracts,
which contain a variety of components derived from extraction process, identification and
purification of the extracts would likely lead to the active compounds that could potentially
be developed as an alternative influenza A virus inhibitor.
PR8
H3N2
140
140
120
120
100
80
**
*
60
40
Virus Titer (%)
Virus Titer (%)
A)
100
*
*
80
*
60
40
20
20
0
0
0
3.125 6.25 12.5
25
Concentrations (µg/ml)
0
3.125 6.25 12.5
25
Concentrations (µg/ml)
B)
140
Virus Titer (%)
Virus Titer (%)
120
100
80
60
40
20
0
0
160
140
120
100
80
60
40
20
0
*
*
0
3.125 6.25 12.5
25
Concentrations (µg/ml)
**
3.125 6.25 12.5
25
Concentrations (µg/ml)
140
140
120
120
Virus Titer (%)
Virus Titer (%)
C)
100
80
60
40
20
*
100
80
**
60
40
20
0
0
0
0.625 1.25
2.5
Concentrations (µg/ml)
5
0
0.625 1.25
2.5
5
Concentrations (µg/ml)
Figure 1. The effect of VR20800 (A) , VR20804 (B) , and VR20808 (C) in pre-treatment assay (black bar)
in comparison with simultaneous assay (lined bar) on A/Puerto Rico/8/34 (H1N1) (PR8) and A/Hong
Kong/8/68 (H3N2) infection. In the pre-treatment assay, various concentrations of the C. buchanani extracts
were added to MDCK cells for 1 hour and rinsed before infection with the viruses for 1 hour. In the
simultaneous assay, the extracts were present during infection with the viruses for 1 hour. Virus titers were
determined by plaque assay. Each data point represents the mean from three independent experiments.
Error bars represent the standard deviations of the means (*, P < 0.05; **, P < 0.01).
PR8
H3N2
120
140
100
120
Virus Titer (%)
Virus Titer (%)
A)
80
60
**
40
**
**
20
*
80
60
**
40
20
**
0
0
100
0
0.78 1.56 3.125 6.25
Concentrations (µg/ml)
0
0.78 1.56 3.125 6.25
Concentrations (µg/ml)
B)
140
120
100
Virus Titer (%)
Virus Titer (%)
120
*
80
*
60
40
**
20
100
80
*
60
40
**
20
**
0
0
0
1.56 3.125 6.25 12.5
Concentrations (µg/ml)
0
1.56 3.125 6.25 12.5
Concentrations (µg/ml)
C)
100
*
80
60
*
**
40
20
**
0
0
0.156 0.3125 0.625 1.25
Concentrations (µg/ml)
Virus Titer (%)
Virus Titer (%)
120
160
140
120
100
80
60
40
20
0
*
0
0.156 0.3125 0.625 1.25
Concentrations (µg/ml)
Figure 2. The effect of VR20800 (A) , VR20804 (B) , and VR20808 (C) in post-entry assay (black bar) in
comparison with comprehensive antiviral assay (lined bar) on A/Puerto Rico/8/34 (H1N1) (PR8) and
A/Hong Kong/8/68 (H3N2) infection. In the post-entry assay, various concentrations of the C. buchanani
extracts were added to MDCK cells after being infected with the viruses for 1 hour and were incubated
further for 2 days. In the comprehensive assay, the extracts were present at all times of the assay. Virus
titers were determined by plaque assay. Each data point represents the mean from three independent
experiments. Error bars represent the standard deviations of the means (*, P < 0.05; **, P < 0.01).
Table 1. Summary of 50% effective concentrations (EC50) of VR20800, VR20804 and VR20808 in various
antiviral assays
EC50 (µg/ml)
Compounds
VR20800
VR20804
VR20808
Pre-treatment
assay
PR8
H3N2
> 25
> 25
> 25
> 25
>5
>5
Simultaneous assay
PR8
> 25
> 25
>5
H3N2
25
25
>5
Post-entry assay
PR8
6.25
12.5
1.25
H3N2
> 6.25
> 12.5
> 1.25
Comprehensive
antiviral assay
PR8
H3N2
1.56
6.25
6.25
6.25
0.625
1.25
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Neumann G, Noda T, Kawaoka Y. Emergence and pandemic potential of swine-origin H1N1 influenza
virus. Nature. 2009;459(7249):931-9. Epub 2009/06/16.
Dolin R. Influenza. In: D.L. Longo, A.S. Fauci, D.L. Kasper, S.L. Hauser, J.L. Jameson, Loscalzo J,
editors. Harrison's Principles of Internal Medicine. New York: McGraw-Hill; 2012. p. 9-15.
Ludwig S, Planz O, Pleschka S, Wolff T. Influenza-virus-induced signaling cascades: targets for antiviral
therapy? Trends in molecular medicine. 2003;9(2):46-52. Epub 2003/03/05.
Medina RA, Garcia-Sastre A. Influenza A viruses: new research developments. Nature reviews
Microbiology. 2011;9(8):590-603. Epub 2011/07/13.
Butel JS. Orthomyxoviruses (Influenza Viruses). In: Geo. F. Brooks KCC, Janet S. Butel, Stephen A.
Morse, Timothy A. Mietzner, editor. Jawetz, Melnick, & Adelberg's Medical Microbiology. 25 ed: The
McGraw-Hill Companies; 2010. p. 536-49.
Kawaoka Y, Neumann G. Influenza viruses: an introduction. Methods Mol Biol. 2012;865:1-9. Epub
2012/04/25.
Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza a
viruses. PLoS pathogens. 2013;9(10):e1003657. Epub 2013/10/17.
Monto AS. The role of antivirals in the control of influenza. Vaccine. 2003;21(16):1796-800. Epub
2003/04/11.
Laupattarakasem P, Wangsrimongkol T, Surarit R, Hahnvajanawong C. In vitro and in vivo antiinflammatory potential of Cryptolepis buchanani. Journal of ethnopharmacology. 2006;108(3):349-54.
Epub 2006/07/13.
Kaul A, Bani S, Zutshi U, Suri KA, Satti NK, Suri OP. Immunopotentiating properties of Cryptolepis
buchanani root extract. Phytotherapy research : PTR. 2003;17(10):1140-4. Epub 2003/12/12.
Laupattarakasem P, Houghton PJ, Hoult JR, Itharat A. An evaluation of the activity related to inflammation
of four plants used in Thailand to treat arthritis. Journal of ethnopharmacology. 2003;85(2-3):207-15. Epub
2003/03/18.
Panthong A, Kanjanapothi D, Taylor WC. Ethnobotanical review of medicinal plants from Thai traditional
books, Part I: Plants with anti-inflammatory, anti-asthmatic and antihypertensive properties. Journal of
ethnopharmacology. 1986;18(3):213-28. Epub 1986/12/01.
Sittiwet CaDP. Anti-bacterial activity of Cryptolepis buchanani aqueous extract. International Journal of
Biological Chemistry. 2009;3(2):90-4.
Liqian Zhu, Hinh Ly, Liang Y. PLC-gamma1 signaling plays a subtype-specific role 1 in post-binding cell
entry of Influenza A virus. J Virol. 2013. Epub 23 October 2013.
Huang SS, Banner D, Fang Y, Ng DC, Kanagasabai T, Kelvin DJ, et al. Comparative analyses of pandemic
H1N1 and seasonal H1N1, H3N2, and influenza B infections depict distinct clinical pictures in ferrets. PloS
one. 2011;6(11):e27512. Epub 2011/11/24.
Acknowledgements
We would like to thank Associate Professor Dr. Patoomratana Tuchinda (Mahidol
University, Thailand) for providing the C. buchanani extracts, Assistant Professor Arunee
Thitithanyanont (Mahidol University, Thailand) and Suwimon Wiboon-ut for great supports
throughout the research work.