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The biological (antiviral) activity of a dried purified extract of Stevia was evaluated in vitro. Tests were performed using Teschen disease virus, infectious rhinotracheitis virus, and human coronavirus.

S. A. Kedik,1 E. I. Yartsev,1 and I. E. Stanishevskaya1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 43, No. 4, pp. 19 – 20, April, 2009.
Original article submitted February 4, 2008.

Key words: Stevia, dried extract, diterpene glycosides, antiviral activity.
Extracts and isolated diterpene glycosides are currently
widely used in the food industry as sugar substitutes and
sweeteners, because of their unique organoleptic properties,
namely their sweetness and the virtual absence of bitterness
and adventitious flavors, as well as their extremely low calorific value [1].
At the same time, there is great interest in published data
on the antiviral and antibacterial activities of Stevia extracts.
Thus, in in vitro experiments, Stevia extracts effectively suppressed the activity of human retroviruses (HRV) by blocking the binding of virus with susceptible cells. Another study
presented data on the antibacterial activity of aqueous extracts of Stevia in relation to enterohemorrhagic Escherichia
coli [2 – 5].
The limited amount of information available on this
question led us to study the antiviral properties of a dried extract of Stevia.
EXPERIMENTAL SECTION
The biological activity of a dried, purified extract of
Stevia leaves was assessed using a modified method [6, 7] to
test antiviral activity in collaboration with the All-Russian
Science Research Institute of Veterinary Virology and Microbiology (VNIIBBiM).
VIRUSES
Tests were performed using the RNA-containing
Teschen disease virus (porcine teschovirus) and the
DNA-containing infectious rhinotracheitis (IRT) virus (bovine herpesvirus 1).
Experiments also used the RNA-containing human
coronavirus (human coronavirus (Hco V-229E)).
METHODS
Studies were performed using a model based on transformed green monkey kidney (Vero) cells. The virustatic and
virucidal actions of the dried extract were tested in three repeats on monolayer cultures of Vero cells, which were incubated at 37°C in an atmosphere of 5% CO2 using 96-well
plates (Costar, UK). The antiviral activity of the dried extract
from Stevia leaves was assessed using standard methods in
terms of the ability of the substance to prevent the cytopathic
effect of the virus on cells as compared with controls after
72 h of incubation [8]. Virus titers were measured as lg
TCD50 (50% tissue cytopathic doses) values as described by
Reid and Mench [9].
Controls consisted of cell cultures infected with virus at
the experimental dose without addition of test substance (virus controls) and intact cell cultures supplemented with Eagle-M maintenance medium instead of test solution (cell controls).
Virustatic (inhibitory) actions were determined by infecting Vero cell cultures with virus-containing material at a
multiplicity of infection of 0.0001 – 0.001 TCD50cell. After
virus infection, cells were incubated at 37°C for 1 – 1.5 h
(for virus adsorption), after which cell cultures were supplemented with solutions of the dried, purified Stevia extract at
different concentrations in an incubator at 37°C until the
clear appearance of the cytopathic effect (CPE) in the virus
control. After appearance of the CPE in the virus control, the
experimental and control samples were titrated in cell cultures. The virustatic action of the substance was assessed in
terms of the difference in virus titers in the experimental and
198
0091-150X/09/4304-0198 © 2009 Springer Science+Business Media, Inc.
Pharmaceutical Chemistry Journal Vol. 43, No. 4, 2009
1 M. V. Lomonosov Moscow State Academy of Fine Chemical Technology,
Moscow, Russia.
control tests, expressed in lg TCD50 (tissue cytopathic dose)
units.
The virucidal (inactivating) activity of solutions of the
dried, purified Stevia extract was assessed by mixing doses
of 5.0 – 5000.0 gml with equal volumes of virus-containing material and incubating at 37°C for 18 – 20 h. The maximum tolerable dose was 5000.0 gml. Controls consisted of
virus-containing material mixed with Eagle-M maintenance
medium instead of test substance solution and intact cell cultures. After contact, mixtures were titrated in parallel with
controls. Virucidal activity was determined in terms of the
difference in virus titers between the experimental and control tests and was expressed in lg TCD50 units. Results were
assessed after 72 – 144 h of incubation at 37°C, after the
clear appearance of the CPE in the virus controls.
RESULTS AND DISCUSSION
These studies showed that the dried, purified extract obtained from Stevia leaves at a dose of 2000 gml inhibited
the reproduction of Teschen disease virus by 0.5 lg TCD50,
IRT virus by 0.25 lg TCD50, and coronavirus by 0.33 lg
TCD50 (Table 1).
The dried, purified extract at a dose of 4000 gml inactivated Teschen disease virus by 0.75 lg TCD50, IRT virus by
0.5 lg TCD50, and coronavirus by 0.66 lg TCD50 (Table 2).
The commonly used antiviral agent remantadine is
known [10] to have high virustatic action but virtually no
virucidal activity, i.e., it does not suppress viruses in the
intercellular space or in blood vessels. Unlike remantadine,
solutions of the dried, purified Stevia extract had marked
virustatic and virucidal properties. Until recently, dried
Stevia extracts (steviosides) were regarded mainly as sweeteners. The demonstration of antiviral properties of Stevia extract makes it attractive for further investigation as a potential medicinal agent.
REFERENCES
1. G. T. Kroyer, Lebensm. Wiss. Technol., No. 32, 509 – 512
(1999).
2. E. Koyama, K. Kitazawa, Y. Ohori, et al., Food Chem. Toxicol.,
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Shigaku Zasshi, No. 9, 12 – 17 (1977).
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Mutat. Res., No. 169, 93 – 103 (1986).
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6. S. A. Kedik, E. I. Yartsev, I. E. Stanishevskaya, et al., Vestnik
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8. Handbook for Experimental (Preclinical) Studies of New Pharmacological Substances [in Russian], Moscow (2000).
9. G. N. Pershin, Methods in Experimental Chemotherapy [in Russian], Moscow (1971).
10. M. D. Mashkovskii, Medicines [in Russian], Minsk (1987),
Vol. 2.
Antiviral Activity of Dried Extract of Stevia 199
TABLE 1. Virustatic Activity of Dried, Purified Extract of Stevia
Leaves
Virus Dose,
gml
IRT virus Difference in
virus titers in
control and
experiment,
lg TCD50
experiment,
lg TCD50
control,
lg TCD50
Teschen disease virus
2000.0 3.25 3.75 0.5
Coronavirus 2000.0 3.00 3.33 0.33
Virus titer in: 2000.0 4.50 4.75 0.25
TABLE 2. Virucidal Activity of Dried, Purified Extract of Stevia
Leaves
Virus Dose,
gml
Virus titer in: Difference in
virus titers in
control
and experiment,
lg TCD50
experiment,
lg TCD50
control,
lg TCD50
Teschen disease virus
4000.0 3.00 3.75 0.75
Coronavirus 4000.0 2.66 3.33 0.66
IRT virus 4000.0 4.25 4.7