Herpes simplex virus (HSV) is a common human pathogen, widespread in
the human population, with two types, herpes simplex virus type 1 (HSV-1)
and herpes simplex virus type 2 (HSV-2). HSV-1 infects orofacial mucosal
surfaces whilst HSV-2 infects genital mucosal surfaces, resulting in clinical
symptoms such as herpes labialis, encephalitis and genital herpes. After
primary infection, HSV tends to persist in ganglia (Yang et al.,
2005). The virus can be reactivated, causing frequent recurrent infections
in some patients, while most people experience few recurrences (Tyring,
1998). Moreover, infections in immunocompromised patients are usually
more severe than in immunocompetent hosts (Schuhmacher et al.,
2003; Logu et al., 2000). Although, acyclovir (ACV), valaciclovir
and famciclovir have been used to treat herpes simplex virus infections,
the efficacy of these drugs is limited by side effects and increasing
resistance of the viruses at present. Antiviral-resistant HSV strains
can occur following mutation in either HSV thymidine kinase (TK) or DNA
polymerase. Moreover, several patients with frequent attacks may not be
able to afford the cost of long-term treatment, particularly immunocompromised
patients (Hammer and Inouye, 1997). Therefore, new antiviral agents from
natural products are demanded for prevention and treatment of HSV infection.
Anti-HSV activity has been studied on medicinal plants such as Camellia
sinensis Ktze. (Fukuchi et al., 1989; Sydiskia et al.,
1991), Nicotiana tabacum Linn. (Larsson et al., 1992), Glycyrrhiza
glabra Linn. (Ryu et al., 1993), Rhus javanica Linn.
(Kurokawa et al., 1995), Barleria lupulina Lindl.,
Clinacanthus nutans (Burm.f.) Lindau (Yoosook et al.,
1999), Mangifera indica Linn., Maclura cochinchinensis Cornor
(Yoosook et al., 2000), Scoparia dulcis Linn. (Galvis et
al., 2001), Dunbaria bella Prain (Akanitapichat et al.,
2006) and Eugenia caryophyllus (Spreng.) Bullock and S.G. Harrison
(Tragoolpua and Jatisatienr, 2007). In this study, we aimed to investigate
the anti-HSV activity of Cissus repanda Vahl. (Vitaceae). This
medicinal plant can be found in northern Thailand. The plant can be used
for treating rashes and detoxication (Chattawanith, 1997).
MATERIALS AND METHODS
Cissus repanda Vahl. (Vitaceae) plants were collected from Queen
Sirikit Botanic Garden during July-September, 2005 and were identified
by Associate Professor Dr. Chusie Trisonthi and Assistant Professor Paritat
Trisonthi, Department of Biology, Faculty of Science, Chiangmai University,
Voucher specimens (QSBG 12006, QSBG 12053) were deposited in the herbarium
at Queen Sirikit Botanic Garden, Chiang Mai Province, Thailand.
Cell Line and Viruses
Green monkey kidney cells (GMK) were grown in Minimum Essential Medium
(MEM) supplemented with 10% heat-inactivated bovine calf serum (Hyclone),
40 μg mL-1 gentamicin (BIO BASIC INC.), in a humidified
5% CO2 incubator at 37°C.
Herpes simplex virus types 1 (HSV-1; F strain) and 2 (HSV-2; G strain)
were used throughout the study. Quantitation of the virus was performed
in 24-well tissue culture plates, using a plaque titration assay. Plaques
were counted and the virus titers were expressed as plaque forming units
Dried plant leaves and climbers (500 g) were milled and soaked with
dichloromethane or methanol at room temperature for 3 days. Then, the
suspension was filtered and the solvent was evaporated, using a rotary
evaporator and dried with high vacuum. The dry material was dissolved
in dimethyl sulfoxide (DMSO) before investigation of anti-HSV activity.
Cytotoxicity Test by MTT Assay
Cytotoxicity tests were performed following previously published procedures
(Yu et al., 2004). Cell toxicity was quantified using a MTT
(3-[4,5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide assay).
GMK cells were seeded on 96-well microplates and incubated with the tested
plant extracts for 72 h. Media were removed before being incubated with
MTT solution for 4 h. Then, the blue formazan product was eluted from
cells by adding DMSO. The absorbance at 570 nm was determined using a
microplate reader. The concentration of the 50% cytotoxicity dose (CD50)
HSVs were treated with non-toxic concentrations of the extracts at
room temperature. Aliquots of the virus-extract mixture were taken and
diluted for residual virus assay at 1 h intervals for up to 4 h, using
the plaque titration assay. The residual virus titers were determined
from two independent experiments
Plaque Reduction Assay
The GMK cells were grown in 24-well tissue culture plates as a monolayer.
Approximately 100 PFU of HSV were added to the cells per well and were
incubated at room temperature for 1 h. Then, 200 μL of media, dichloromethane
or methanol extract of C. repanda at non toxic concentrations and
ACV at ED50 concentration, were applied into duplicate test
wells whereas, media were added into control wells. After that, 400 μL
of growth media, containing 2% sodium carboxymethyl cellulose, was added
to the cells. After 2-3 days incubation in the CO2 incubator,
the cells were stained with 0.1% crystal violet in 1% ethanol. The plaques
were counted and inhibitory activities of tested extracts were calculated
comparing to control. The 50% effective dose (ED50) was also
Effect of Plant Extract on Viral Attachment
The GMK cell monolayers were pre-chilled at 4°C for 1 h (Cheng et
al., 2002). The media were aspirated and the cell monolayers were
infected with HSV in the absence or presence of extracts and acyclovir.
Then, the infected cells were further incubation at 4°C for 3 h and
the cells were washed with PBS twice before overlaid with growth media,
containing 2% sodium carboxymethyl cellulose. After 2-3 days incubation,
the cells were stained with 0.1% crystal violet in 1% ethanol. The percentage
of inhibition for HSV attachment to GMK monolayer was calculated.
Effect of Plant Extract on Viral Penetration
GMK cell monolayers were pre-chilled at 4°C for 1 h (Cheng et al.,
2002). The media were aspirated and cell monolayers were infected by HSV.
Infected cells were further incubated at 4°C for 3 h and the extract
or acyclovir was added. After further incubation at 37°C for 10 min,
the infected cells were treated with PBS, pH 3 for 1 min and PBS, pH 11
were added immediately to neutralize acidic PBS. PBS was then removed
and cell monolayer was overlaid with growth media, containing 2% sodium
carboxymethyl cellulose and incubation for 2-3 days. The cells were stained
with 0.1% crystal violet in 1% ethanol and the percentage of inhibition
for HSV penetration was calculated.
Effect of Plant Extract on HSV Replication
GMK cells were grown as monolayers in 25 cm2 flasks. The cells
were infected with 1x106 PFU mL-1 of HSV. Virus
was allowed to adsorb to the cells for 60 min at room temperature. Then,
unadsorbed virus was discarded and the cells were washed twice with PBS.
The duplicated flasks of infected cells were maintained in media containing
crude extract of C. repanda at non-toxic concentration while only
media or media containing acyclovir at ED50 concentration were
added into control flasks. Infected cells were observed for cytopathic
effects (CPE) and were collected at 6, 12, 24 and 30 h after viral infection.
The cells were frozen and thawed twice. The supernatants containing viruses
were kept at -80°C and virus titers were determined using plaque titration
The CD50 values of C. repanda, extracted with dichloromethane
(CD) and methanol (CM) were 737±26 and 1876±0 μg mL-1,
respectively. Thus, non-toxic concentrations of C. repanda extract
were used to determine anti-HSV activity.
After incubation of viruses with the dichloromethane or methanol extract
of C. repanda at room temperature for 1 h intervals up to 4 h,
both HSV-1 and HSV-2 were directly inactivated (Table 1).
After incubation of HSV-1 and HSV-2 with CD extract for 2 h, the viruses
were directly inactivated to negligible amounts. Similar results were
observed when HSV-2 was incubated with CM extract, whereas HSV-1 was inactivated
after incubation with CM extract for only 1 h.
C. repanda extracts demonstrated anti-HSV activity against both
types of HSVs using plaque reduction assay. HSV-1 was inhibited by CM
and CD extracts with ED50 values of 310.6±50.0 and 249.2±40.7
μg mL-1 and selective index (SI) values of 6±1.2
and 2.9±0.4, respectively (Table 2). HSV-2 was
also inhibited by CM and CD extracts with ED50 values of 295.4±56.4
and 253.4±15.9 μg mL-1 and SI values of 6.3±1.6
and 2.9±0.2, respectively. Therefore, the SI of CM extract on HSV
was higher than CD extract. Moreover, ED50 values of ACV on
HSV-1 and HSV-2 were 2.31 and 14.92 μg mL-1, respectively.
||Direct inactivation of HSV-1 and HSV-2 by methanol (CM)
and dichloromethane (CD) extracts of C. repanda
|aValues are means of two independent experiments
||Anti-HSV activity of methanol (CM) and dichloromethane
(CD) extracts of C. repanda on GMK cells
|a,bValues are means±SD of two independent
experiments cCD50 ED50-1
||Effects of extracts and acyclovir on HSV attachment
and penetration to GMK cells
The effects of C. repanda extracts on HSV attachment are shown
in (Fig. 1). CM and CD extracts blocked HSV-1 attachment
with percentage of 55.3±11.1 and 51.5±0.8, respectively.
Similar results were observed when CM extract was applied to determine
the blocking of HSV-2 attachment. The efficacy of CM extract was better
than CD extract with percentages of 87.5±0.7 and 61.5±0.4,
Furthermore, CM and CD extracts inhibited HSV-1 penetration with percentages
of 30.6±1 and 31.0±0.8, respectively, while HSV-2 penetration
was blocked by CM and CD extracts with percentages of 51.8±5.9
and 42.1±6.4, respectively (Fig. 1).
||Inhibition of HSV-1 replication by dichloromethane (CD)
and methanol (CM) of C. repanda extract compared with ACV and
||Inhibition of HSV-2 replication by dichloromethane (CD)
and methanol (CM) of C. repanda extracts compared with ACV
To determine the effects of C. repanda extracts on HSV yield,
infected cells were maintained in the extract and were collected at 6,
12, 24 and 30 h. At 30 h after HSV-1 infection, log10 PFU mL-1
of virus control and ACV treated virus were 8.3±0.01 and 4.8±0.2,
respectively. An inhibition of HSV-1 replication was observed, since the
log10 PFU mL-1 of virus after treatment with CM
and CD extracts were 5.5±0.8 and 6.3±0.1, respectively (Fig.
2). HSV-2 replication was also inhibited after treatment with the
extracts. At 30 h after infection the log10 PFU mL-1
of HSV-2 were 2.7±0.4 and 5.2±0.8 after treatment with CM
and CD extracts compared with 8.5±0.4 of control virus and 5.6±0.9
of ACV treated virus (Fig. 3). Therefore, the inhibitory
activity of CM extract on HSV replication was higher than CD extract.
In this study, we investigated anti-HSV activity of C. repanda,
which is a medicinal plant species found in northern Thailand, used in
traditional remedies (Chattawanith, 1997). The toxicity on GMK cells of
dichloromethane extract was higher than methanol extract of C. repanda.
Anti-HSV activity was observed after treatment of HSV with non-toxic concentrations
of the extracts using the plaque reduction assay. ED50 values
of the CD extract were slightly lower than those obtained with the CM
extract, when HSV-1 and HSV-2 were tested with the extracts. However,
SI values (CD50 ED50-1) of CM extracts
were about 2-fold higher than treatment with CD extracts. Thus, CM extracts
showed higher anti-HSV efficacy than CD extracts. Moreover, the effects
of the extracts on various stages of HSV multiplication cycles were determined
to elucidate modes of action of the extracts. Both CM and CD extracts
had an ability to inhibit HSV particles. HSV-1 was directly inactivated
by CM and CD extracts to negligible amounts within 1 and 2 h, respectively,
whereas the inactivation of HSV-2 particles was observed after incubation
with CM and CD extracts for 2 h. Therefore, the efficiency of the CM extracts
on inactivation of HSV-1 particle was higher than that of the CD extracts.
Inhibition of HSV attachment and penetration was also observed. CM and
CD extracts inhibited HSV-1 attachment 1.8 and 1.7 fold higher than penetration
respectively. Similar results were obtained after treatment of HSV-2 with
the extracts, with inhibition of HSV-2 attachment being 1.7 and 1.5 fold
higher than viral penetration, after treatment with CM and CD extracts
Thirty hours after HSV-1 replication, the amounts of the virus were reduced
by 33.7 and 24.1%, compared with the control after treatment with CM and
CD extracts, respectively. Higher percentage inhibition of viral replication
was observed on HSV-2 after treatment with CM and CD extracts with the
reduction of viral amounts of 68.2 and 38.8%, respectively.
These results indicate that dichloromethane and methanol extracts of
C. repanda inhibit HSV at various stages in the viral multiplication
cycle i.e. attachment, penetration and replication. The most affected
stage was the attachment period. The highest anti-HSV activity was obtained
with methanolic extracts from C. repanda. Moreover, C. repanda
extracts inhibited replication of HSV-2 more than HSV-1. This is the
first report of anti-HSV activity of C. repanda, besides the activity
on HSV-1 by crude extract of endophytic fungi isolated from C. repanda
(Wiyakrutta et al., 2004). However, biologically active compounds
from C. repanda and their modes of action should be further studied.
This research was granted by the Commission on Higher Education granting
Ms. Jiraporn Nikomtat was supported forStrategic Scholarships Fellowships
Frontier Research Networks from the Commission on Higher Education. We
also thank Uttaradit Rajabhat University, Graduate school Chiang Mai University
and The Royal Project Grant number 3015-3396 for supporting this study
and Dr. Stephen Elliott for critically reading the manuscript.