Virucidal activity of a β-triketone-rich essential oil of Leptospermum scoparium (manuka oil) against HSV-1 and HSV-2 in cell culture

Article abstract of DOI:10.1055/s-2005-873175

The inhibitory activity of manuka oil against Herpes simplex virus type 1 (HSV-1) and Herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells (monkey kidney cells) using a plaque reduction assay. In order to determine the mode of antiviral

Full text of DOI:10.1055/s-2005-873175

Jürgen Reichling1
Christine Koch1
Virucidal Activity of a b-Triketone-Rich Essential Oil of
Leptospermum scoparium (Manuka Oil) Against HSV-1
and HSV-2in Cell Culture
Elisabeth Stahl-Biskup2
Cornelia Sojka2
Paul Schnitzler3
Abstract
respectively. On the other hand, pretreatment of host cells with
the essential oil before viral infection did not affect plaque for-
The inhibitory activity of manuka oil against Herpes simplex virus mation. After virus penetration into the host cells only replica-
type 1 (HSV-1) and Herpes simplex virus type 2 (HSV-2) was test- tion of HSV-1 particle was significantly inhibited to about 41%
ed in vitro on RC-37 cells (monkey kidney cells) using a plaque by manuka oil. Flavesone and leptospermone, two characteristic
reduction assay. In order to determine the mode of antiviral ac- û-triketones of manuka oil, inhibited the virulence of HSV-1 in
tion of the essential oil, manuka oil was added at different times the same manner as the essential oil itself. When added at non-
to the cells or viruses during the infection cycle. Both HSV types cytotoxic concentrations to the virus 1 h prior to cell infection,
were significantly inhibited when the viruses were pretreated plaque formation was reduced by 99.1%and 79.7%for flavesone
with manuka oil 1 h prior to cell infection. At non-cytotoxic con- and leptospermone, respectively.
centrations of the essential oil, plaque formation was significant-
ly reduced by 99.5%and 98.9%for HSV-1 and HSV-2, respective-
Key words
ly. The 50%inhibitory concentration (IC 50) of manuka oil for virus Leptospermum scoparium ´ Myrtaceae ´ manuka oil ´ flavesone ´
plaque formation was determined at 0.0001%v/v ( = 0.96 mg/ leptospermone ´ virucidal activity ´ HSV-1 ´ HSV-2
mL) and 0.00006%v/v ( = 0.58 mg/mL) for HSV-1 and HSV-2,
1123
Introduction
oesophagus, and the genital mucous membranes. The genital le-
sions (herpes genitalis) are caused by HSV-2 whereas the epider-
Infections with Herpes simplex viruses (HSV) are clearly at the mal lesions in the orofacial region (herpes labialis) are mainly
top of the list of viral infections in humans. The degree of infec- traced back to HSV-1, more rarely to HSV-2. Chemotherapy of
tion among the population of industrial countries reaches 90%to
nearly 100%for Herpes simplex virus type 1 (HSV-1), and 20 to nerve ganglia of the host and reactivates from time to time. Re-
0%for Herpes simplex virus type 2 (HSV-2) in non-risk groups currence commonly begins with a tingling, itching, and burning
HSV infections is difficult, since the viral DNA persists in the
3
and increases with age. Herpes simplex virus (HSV) is a human sensation. After about 3 days blisters have developed which are
pathogen which produces recurrent epidermal lesions mostly in filled with liquid. This liquid contains millions of viruses, which
and around the oral cavity, but also in the eye, nose, pharynx, are highly infectious. The healing phase takes 8 to 10 days,
Affiliation
1
Institute of Pharmacy and Molecular Biotechnology, Department of Biology, University of Heidelberg,
Heidelberg, Germany
2
Institute of Pharmacy, Department of Pharmaceutical Biology and Microbiology, University of Hamburg,
Hamburg, Germany
3
Hygiene Institute, Department of Virology, University of Heidelberg, Heidelberg, Germany
Correspondence
Prof. Dr. Jürgen Reichling ´ Institute of Pharmacy and Molecular Biotechnology ´ Department of Biology ´
University of Heidelberg ´ Im Neuenheimer Feld 364 ´ 69120 Heidelberg ´ Germany ´
E-mail: juergen.reichling@urz.uni-heidelberg.de
Received February 15, 2005 ´ Accepted June 10, 2005
Bibliography
Planta Med 2005; 71: 1123±1127 ´ ꢀ Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2005-873175 ´ Published online November 10, 2005
ISSN 0032-0943

whereby the blisters burst, become crusted and develop a scab Acyclovir
1], [2]. For topical treatment of herpes labialis people usually Acyclovir (ACV), purchased from GlaxoSmithKline, was dissolved
[
use creams containing acyclovir or penciclovir as the active com- in sterile water, and stored at ±208C.
pound. Both compounds are nucleoside analogues which inter-
fere with the viral DNA replication after activation by viral thy- Cell culture
midine kinase. Both nucleoside analogues are only moderately RC-37 cells (African green monkey kidney cells) were grown in
successful in the local treatment of herpes labialis. Furthermore, monolayer culture with Dulbecco's modified Eagle's medium
there has been an increase in acyclovir- and pencyclovir-resis- (DMEM) supplemented with 5%foetal calf serum (FCS), 100 mg/
tant strains in recent years [1], [2], [3].
mL penicillin and 100 mg/mL streptomycin. The monolayers
were removed from their plastic surfaces and serially passaged
Essential oils have been reported to exhibit an antiviral effect in whenever they became confluent. Cells were plated out onto
the treatment of herpes labialis. But most of the literature data 24-well and 6-well culture plates for cytotoxicity and antiviral
are based on anecdotal reports of patients. However, only few ex- assays, respectively, and propagated at 378C in an atmosphere
perimental data are available which substantiate these reports of 5%CO 2.
[4], [5]. We are investigating the antiherpetic potential of es-
sential oils traditionally used as anti-infective agents in more de- Viruses
tail [6], [7]. Manuka oil, a b-triketone-rich essential oil of HSV-1 strain KOS [12] and HSV-2 strain HG52 [13] were used for
Leptospermum scoparium (Myrtaceae), is commercially used as experiments. Virus was routinely grown on RC-37 cells as de-
an anti-infective agent in New Zealand. Recently, manuka oil scribed previously [14]. Herpes simplex virus stock cultures were
has been shown to possess antibacterial activities in vitro [1], prepared from supernatants of infected cells and stored at ±808C.
[8], [9], [10], [11]. Porter and Wilkins [8] described the b-trike- Infectivity titres were determined by a standard plaque assay on
tone fraction as the main active one. Hitherto, there is no report confluent RC-37 cells.
on the antiviral activity of manuka oil. Here we report studies on
the antiherpetic effects of New Zealand manuka oil as well as of Cytotoxicity assay
the b-triketones leptospermone and flavesone, two characteris- For cytotoxicity assays, cells were seeded into 24-well plates at a
4
tic components of manuka oil.
density of 5”10 cells per well and incubated for 24 h at 378C.
The medium was removed and fresh DMEM containing the ap-
propriate dilution of the different substances were added onto
subconfluent RC-37 cells in twelve replicates for each concentra-
tion of the drug. Wells containing 1 mL medium with 1%ethanol
but no drug were also included on each plate as controls. After 4
Materials and Methods
Manuka oil, leptospermone, flavesone
Manuka oil (density at 208C: r = 0.96) was purchased from days of incubation, the growth medium was removed and the
ALVA, Wallenhorst. The major substances of manuka oil, identi- viability of the drug-treated cells was determined in a standard
fied by a GC/GC-MS method, consisted of terpinene-4-ol (1.8%), neutral red dye uptake assay [15]. This assay quantifies the num-
isoleptospermone (3.9%), viridiflorene (4.4%), flavesone (4.5%), ber of viable cells after their exposure to toxicants by measuring
d-cadinene (6.1%), leptospermone (14.5%), and calamene (16.0%) the amount of neutral red dye taken up by the cells. Medium was
1
124
[9], [10]. Pure leptospermone (density at 158C: r = 1.05) and fla- replaced by 1 mL DMEM supplemented with neutral red at
vesone (density at 208C: r = 1.10) were synthesized: 0.12 mol 40 mg/mL and incubated for 3 h at 378C. The medium was re-
dried phloroglucin suspended in 80 mL CS were stirred for 10 moved and cells were rinsed with 1 mL of 0.5%formaldehyde in
2
min with 0.48 mol AlCl . After addition of 45 mL nitrobenzene 1%CaCl 2 for 1 min. Finally the cells were dissolved in 50%etha-
3
the liquid was brought to the boil and 0.12 mol isobutyryl chlo- nol with 1%acetic acid, incubated for 5 min on a shaker and neu-
ride (for flavesone) and 0.12 mL isovaleric acid chloride (for lep- tral red dye incorporated by the viable cells was eluted. The neu-
tospermone), each dissolved in 10 mL nitrobenzene, were added tral red dye uptake was determined by measuring the optical
followed by boiling under reflux for 40 min. The reaction mixture density (OD) of the eluted neutral red at 540 nm in a spectro-
was then cooled down to 408C and poured into 500 mL cold 4% photometer. The mean OD of the cell-control wells was assigned
HCl. The solvents were removed by steam evaporation. The a value of 100%. Uptake and accumulation of the supravital dye
intermediate products 2-methyl-1-(2,4,6-trihydroxyphenyl)- neutral red has been shown to be linear with cell numbers. The
propan-1-one or 3-methyl-1-(2,4,6-trihydroxyphenyl)-butan-1- maximum non-cytotoxic as well as the cytotoxic concentrations
one, respectively, could be separated in a separating funnel and of the drugs which reduced viable cell number by 50%(TC 50) of
were cleaned several times with boiling water. Two mmol of the control were determined from dose-response curves.
each intermediate product were heated with sodium methox-
ide/methyl iodide (0.3 g sodium/5 mL methanol/3 mL methyl io- Direct plaque assay
dide) under reflux for 2 h. The solvents and the precipitates were In order to determine the mode of antiviral action, cells were
removed (at 408C, 150 mbar and by filtering, respectively) to give pretreated with drugs before viral infection, viruses were incuba-
an oil residue which was acidified with 50 mL HCl to pH 1±2. ted with drugs before infection, cells and viruses were incubated
Leptospermone and flavesone, respectively, were then extracted together during adsorption and cells were incubated with
with ethyl acetate. Cleaning of the products was performed by drugs after penetration of the virus into the host cells, respec-
silica gel column chromatography as well as by fractionated tively. In view of the density of manuka oil, flavesone and leptos-
distillation. Manuka oil, leptospermone and flavesone are liquid permone the results in %(v/v) may be converted into mg/mL,
compounds.
which offers the opportunity to directly compare the anti-herpe-
Reichling J et al. Virucidal Activity of¼ Planta Med 2005; 71: 1123±1127

tic potential of these compounds with that of acyclovir. Manuka diluted in medium and aliquots of each dilution were adsorbed
oil and flavesone were always used at the non-cytotoxic concen- on cells for 1 h at 378C. Ethanol at a final concentration of 1%
tration of 0.003%v/v (28.8 mg/mL and 33.0 mg/mL, respectively), had no effect on virus titres. On the other hand, manuka oil in-
leptospermone was used at the non-cytotoxic concentration of hibited plaque formation of HSV-1 and HSV-2 in a dose-depen-
0
.001%v/v (10.5 mg/mL). Cell monolayers were pretreated with dent manner. The 50%inhibitory concentration (IC 50) of manuka
the substances prior to inoculation with virus by adding the oil was determined at 0.0001%v/v (0.96 mg/mL) and 0.00006%v/
drugs to the culture medium and by incubation for 1 h at 378C. v (0.58 mg/mL) for HSV-1 and HSV-2, respectively (Fig.1).
The compound was aspirated and the HSV inoculum was added.
For pre-treatment of Herpes simplex virus about 2”103 plaque The antiviral activity of acyclovir (ACV) was determined by add-
forming units (pfu) of HSV-1 and HSV-2 were incubated in medi- ing increasing concentrations of the drug after the adsorption
um containing 0.003%v/v (28.8 mg/mL) manuka oil, 0.003%v/v
period of the virus. At a concentration of 0.023 mg/mL ACV plaque
(
33.0 mg/mL) flavesone or 0.001%v/v (10.5 mg/mL) leptosper- formation was reduced by 37.9%, whereas at a concentration of
mone, respectively, for 1 h at room temperature prior to infection 22.5 mg/mL plaques were reduced by 90.2%(data not shown).
of RC-37 cells. For analysing the antiviral inhibition during the Therefore, a clearly dose-dependent effect could be observed.
adsorption period, the same amount of HSV-1 or HSV-2 was
mixed with the drugs and added to the cells immediately. After Herpes virus infection cycle is characterised by a cascade of coor-
1
h of adsorption at 378C, the inoculum was removed and cells dinately regulated events. The inhibitory effect of manuka oil
were overlaid with medium containing 0.5%methylcellulose. was determined by addition at different times during viral infec-
The effect of the drugs against HSV was also tested during the re- tion. To identify the step at which infection might be inhibited,
plication period by adding the test compounds after adsorption cells were infected with HSV after pre-incubation of the cells
to the overlay medium, as typically performed in antiviral sus- with manuka oil, pre-treatment of the virus with the essential
ceptibility studies. After incubation for 4 days at 378C, monolay- oil prior to infection, addition of the essential oil during adsorp-
ers were fixed with 10%formaldehyde. The cultures were stained
with 1%crystal violet and subsequently plaques were counted.
tion or after the adsorption period (Fig. 2). In all experiments un-
treated virus-infected cells were used as control. The percentage
The number of plaques of drug-treated cells and viruses were reduction was calculated relative to the amount of virus pro-
compared to solvent controls. By references to the number of duced in the absence of the compound. In all assays the non-cy-
plaques observed in virus control monolayers (solvent control), totoxic concentration of the essential oil (28.8 mg/mL) was used.
the concentration of test compounds which inhibited plaque Pretreatment of cells with the essential oil did not reduce virus
numbers by 50%(IC 50) of the control was determined from production. However, pretreatment of viruses with manuka oil
dose-response curves. All experiments were performed in paral- prior to infection caused a significant reduction of infectivity of
lel with acyclovir to show the suitability of the assay. Each assay 99.5%and 98.9%for HSV-1 and HSV-2, respectively. When man-
was repeated three-fold.
uka oil was added during the adsorption period, virus titres were
reduced by 40.5%for HSV-1 and by 54.2%for HSV-2. In contrast,
no significant effect on viral growth could be observed when the
essential oil was added to the cells after penetration of HSV-2
1125
Results
In order to determine the cytotoxicity of manuka oil, leptosper-
mone and flavesone, the compounds were dissolved in ethanol
and added to the nutrient medium at a final concentration of
1
%ethanol. Ethanol by itself did not exhibit any toxic effect on
RC 37 cells in this concentration. Monolayer cultures of RC-37
cells were grown in 0.0001±0.1%v/v (9.6±1100.0 mg/mL) drug-
containing medium and after 4 days of incubation, cell viability
was determined in a standard neutral red dye uptake assay. Man-
uka oil concentrations up to 0.003%v/v (28.8 mg/mL) were not
cytotoxic. Leptospermone and flavesone did not show any visible
changes in cell morphology and cell density at concentrations up
to 0.001%v/v (10.5 mg/mL) and 0.003%v/v (33.0 mg/mL), respec-
tively. The TC50 values of manuka oil, leptospermone and flave-
sone for RC-37 cells were 0.004%v/v (38.4 mg/mL), 0.01%v/v
(
105.0 mg/mL) and 0.1%v/v (1100 mg/mL), respectively. Acyclovir
was not cytotoxic at a concentration of 22.5 mg/mL.
To evaluate the virucidal effect of manuka oil against HSV the
viruses were exposed for 1 hour to various concentrations of the
oil ranging from 0.000001±0.01%v/v (0.0096 to 96.0 mg/mL) in
suspension assays. Since the initial dilution of the essential oil
was always performed in ethanol and all assays contained 1%
ethanol as final concentration, additional tubes containing virus
and 1%ethanol were used as controls. Briefly, the samples were
Fig. 1 Determination of 50% inhibitory concentration (IC ) of manu-
50
ka oil against HSV-1 and HSV-2. Viruses were incubated for 1 h at room
temperature with increasing concentrations of manuka oil and imme-
diately tested in a plaque reduction assay. All values were expressed as
mean  S.D. (n = 3).
Reichling J et al. Virucidal Activity of¼ Planta Med 2005; 71: 1123±1127

Fig. 2 Inhibitory effect of manuka oil against HSV-1 and HSV-2 by in-
cubation at different periods of time during cell infection. Manuka oil
was added at the non-cytotoxic concentration of 0.003% v/v (28.8 mg/
mL). Pretreatment virus = viruses were pretreated with manuka oil
prior to infection; adsorption = manuka oil was added during adsorp-
tion period; replication = manuka oil was added after penetration of
the virus into the host cells; pretreatment cells = host cells were pre-
treated with manuka oil prior to virus infection. All values were
expressed as mean  S.D. (n = 3).
Fig. 3 Antiviral effect of leptospermone and flavesone against HSV-1
by incubation at different periods of time during cell infection. The b-
triketones were added at non-cytotoxic concentrations of 0.001% v/v
(
10.5 mg/mL) and 0.003% v/v (33.0 mg/mL), respectively. The assay was
performed as described in Fig. 2. All values were expressed as mean 
S.D. (n = 3).
into the host cell. On the other hand, in the same assay, the pla- whereas a still non-toxic concentration of 0.003%(28.8 mg/mL)
que formation of HSV-1 was reduced significantly by about 41% of manuka oil could be determined repeatedly in cell culture.
(
see replication, Fig. 2).
Cells treated with manuka oil at this non-toxic concentration
never exhibited any sign of changes in morphology and viability.
All experiments were performed in parallel with acyclovir to This phenomenon of a very close range of non-toxic and toxic
show the suitability of the assay. Acyclovir achieved the highest concentrations has also been demonstrated for other essential
antiviral effect when applied during the replication period (data oils, e.g., tea tree oil [6] and peppermint oil [7]. Apparently the
not shown), plaques were reduced by 90.2%. When acyclovir was cytotoxic effect of manuka oil on host cells in vitro does not in-
added to the cells prior to infection, no antiviral effect could be crease linearly but rather exponentially with an increasing con-
observed. Pre-treatment of the virus or addition of the drug dur- centration of manuka oil. In order to determine the mode of anti-
ing the adsorption period reduced plaque formation by 79.8% viral action, either cells were pretreated before viral infection or
1
126
and 72.9%, respectively (data not shown).
viruses were incubated with non-cytotoxic concentrations of
ACV, manuka oil, leptospermone, or flavesone before infection,
The û-triketone fraction of manuka oil has been described to during adsorption or after penetration into the host cells. Pre-
contain the active components in antibacterial studies [9], [10], treatment of the cells with manuka oil, leptospermone, or flave-
[11]. Therefore, we tested leptospermone and flavesone, two sone had no significant effect on the production of infectious
characteristic compounds of the û-triketone fraction (see Fig. 4), viruses and plaque formation was not affected. However, pre-
against HSV-1 representatively. Pre-treatment of HSV-1 with treatment of HSV-1 and HSV-2, respectively, with manuka oil,
leptospermone and flavesone in non-cytotoxic concentrations leptospermone, and flavesone prior to infection resulted in a
reduced plaque formation by 79.7%and 99.1 %, respectively
concentration-dependent reduction of plaques. The IC50 of man-
(
Fig. 3). By addition of the drugs during the adsorption period uka oil was determined at 0.0001%v/v (0.96 mg/mL) for HSV-1
the plaque formation of HSV-1 was reduced by 11.7%and 25.5%
and 0.00006%v/v (0.58 mg/mL) for HSV-2, which revealed a
for leptospermone and flavesone, respectively. No significant an- much higher anti-herpes virus activity of manuka oil compared
tiviral effect could be observed when leptospermone and flave-
sone were added to the cells prior to infection or during the repli-
cation period.
Discussion
All experiments to assess the toxicity of manuka oil indicate a
moderate toxic behaviour in cell cultures. Its toxicity approached
5
0%(TC 50) at a concentration of 0.004%v/v (38.4 mg/mL) Fig. 4 Chemical structures of leptospermone and flavesone.
Reichling J et al. Virucidal Activity of¼ Planta Med 2005; 71: 1123±1127

to tea tree oil, eucalyptus oil [6], and peppermint oil [7]. When ment of HSV infections would be ideal, especially for those pa-
manuka oil was added during the adsorption period, the amount tients who experience frequent recurrences.
of plaques for HSV-1 and HSV-2 was reduced by 40.5%and
5
4.2%, respectively. A similar anti-adsorption effect was demon-
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