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Doi:10.1016/j.micromeso.2004.10.039
Microporous and Mesoporous Materials 79 (2005) 165–169
‘‘Rudjer Boskovic’’ Institute, Division of Molecular Medicine, Bijenicka 54, HR-10002 Zagreb, Croatia
Received 14 August 2004;received in revised form 11 October 2004;accepted 26 October 2004
The aim of this study was to evaluate the antiviral properties of clinoptilolite, a natural non-toxic zeolite. Herein, a fine powder of
micronized zeolite (MZ) was obtained by tribomechanical micronization of natural clinoptilolite. Different viral suspensions weretreated with MZ in concentrations ranging from 0.5 to 50 mg/ml. The viral proliferation was evaluated by optical microscope aspercentage of cytopathic effect (CPE). Human adenovirus 5, herpes simplex virus type 1 (HSV 1) and human enteroviruses (coxsac-kievirus B5 and echovirus 7) were used in the antiviral assay. Concentrations of 0.5 and 5 mg/ml of MZ induced a very low antiviraleffect or the antiviral was not observed at all, while concentrations of 12, 25 and 50 mg/ml of MZ induced a significant inhibitoryeffect upon viral proliferation. MZ inhibited the viral proliferation of HSV 1, coxsackievirus B5 and echovirus 7 more efficiently thanadenovirus 5. The antiviral effect of MZ seems to be non-specific and is more likely based on the incorporation of viral particles intopores of MZ aggregates than ion exchange properties of clinoptilolite. Our preliminary results indicate a possibility of therapeuticalapplication of MZ, either locally (skin) against herpesvirus infections or orally in cases of adenovirus or enterovirus infections. Futhermore, MZ could also be used in purification of drinking water from different viruses. Ó 2004 Elsevier Inc. All rights reserved.
Keywords: Clinoptilolite;Micronized zeolite (MZ);Antiviral properties;Cytopathic effect (CPE)
Clinoptilolite administered by gastric intubation to
mice injected with melanoma cells significantly reduced
Clinoptilolite is a natural, non-toxic zeolite that has
the number of melanoma metastases Clinoptilolite
monoclinic crystal structure symmetry and strong
treatment of mice and dogs suffering from a variety of
adsorptive and ion exchange capacity . These proper-
tumor types led to improvement in the overall health
ties have been largely exploited in industrial, agricul-
status, prolongation of life span, and decrease in tumor
tural, environmental and biological technologies
size. Local application of clinoptilolite to skin cancers of
Zeolites also possess biological activities, either positive
some dogs effectively reduced tumor formation and
or negative. The best known and documented positive
biological activity of natural clinoptilolite is its action
The major negative biological effect of clinoptilolite
as antidiarrheal drug Furthermore, some of them
could be its toxicity in higher organisms (mammal) if
seem to have antibacterial property The clinoptilo-
the content of heavy metals (Pb, Cd, Zn, etc.) is high.
lite from Vranje, Serbia used in this study has antioxida-
Therefore, a classic acute, sub-chronic and chronic tox-
tive and immunostimulatory effects and it has been
icity study of the clinoptilolite from Vranje, Serbia was
used as an adjuvant to anticancer therapy
performed on mice and rats . Results clearly showthat oral (in diet) administration of clinoptilolite to miceand rats for 6 and 12 months, respectively, caused no
changes that could be considered a toxic effect of
Corresponding author. Tel.: +385 1 4661111;fax: +385 1 4661010.
1387-1811/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2004.10.039
M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169
Based on these results we assumed that the adsorbent
ranging from 0.5 to 50 mg/ml. After incubation (15 h,
qualities and ion exchange properties of clinoptilolite
4 °C, constant rotation), the suspension (media and
could be effective on viruses too. Herein, we tested a nat-
MZ) was centrifuged (10 min, 4 °C, 3000 · g) to separate
ural clinoptilolite (Vranje, Serbia) on in vitro viral
the liquid from the solid phase (MZ).
replication of adenoviruses, herpesviruses and enterovi-ruses (coxsackievirus, echovirus).
HeLa and BS-C-1 cell were seeded at 2 · 104 cells per
ml on 24-well flat-bottomed microtitre plates (BectonDickinson, USA). The viral infection was performed
on one-day-old confluent cell monolayers. The plateswere incubated at 37 °C and 5% CO2 and the CPE were
A fine powder of natural clinoptilolite, i.e., micron-
followed by optical microscopy every 24-h during 3–
ized zeolite (MZ), was obtained by tribomechanical
4 days (depending on the type of virus). Each assay
micronization of natural clinoptilolite from Vranje,
was done four times. The inhibitory effect of viral prolif-
Serbia. Chemical composition and characteristics of
eration was evaluated as percentage of CPE and was
compared to CPE of similar dilutions of viral suspensionalso incubated at 4 °C during 15-h but without MZ (po-
Human cervical carcinoma cells (HeLa;ATCC num-
ber: CCL-2) and African green monkey kidney epithelial
cells (BS-C-1;ATCC number: CCL-26) were used. Cellswere propagated in DulbeccoÕs modified eagle medium
Four different viruses were chosen on the basis of
(MEM;Gibco BRL, USA) supplemented with 10%
their morphology and biological characteristics: (a) with
inactivated foetal bovine serum (FBS;Gibco BRL,
or without lipoprotein envelope acquired from the host
USA), 1% L-glutamine and 0.3% sodium bicarbonate
cell, (b) DNA or RNA replicating viruses and (c) high
infectivity and relatively rapid CPE in cell culture. Theherpesviruses capsid is surrounded by a lipoprotein
envelope, varying in size from 100–200 nm in diameterand their genome consists of double-stranded linear
Adenovirus 5 (ATCC number: VR-5), herpesvirus
DNA. Adenoviruses and enteroviruses (coxsackieviruses
type 1 (HSV 1;ATCC number: VR-733), and two ente-
and echoviruses) are non-enveloped and are relatively
roviruses, coxsackievirus B5 virus (ATCC number: VR-
small (65–80 and 22–30 nm virion size, respectively)
185) and echovirus 7 (ATCC number: VR-37) were
viruses, as compared to herpesviruses. The genome of
included in this study. Adenovirus and herpesvirus were
adenoviruses consists of linear double-stranded DNA,
propagated on HeLa, while enterovirus were propagated
while the one from enteroviruses consists of single-
on BS-C-1 confluent cell monolayers. The viral suspen-
sion consisted the cell-free supernatant collected after
Enteroviruses are highly infective and specific CPE
centrifugation (20 min, 4 °C, 5000 · g) of infected media
(cell lysis) appears in cell culture (BS-C-1) rapidly, with-
(MEM supplemented with 2% FBS) collected at maxi-
in 24–48 h depending of the viral titre (1:10 serial dilu-
mal viral proliferation, i.e. 100% cytophatic effect
tion). Adenoviruses and herpesviruses are less infective
(CPE) of whole cell monolayer. Five different relative
than enteroviruses and specific CPE (cell rounding) ap-
viral titres (V1–VÀ4) obtained by serial dilution of viral
pears in cell culture (HeLa), within 24–72 h depending
suspension (1:2 for adenovirus and herpesvirus and
of the viral titre (1:2 serial dilution).
1:10 for enteroviruses) were treated with MZ prior to
The CPE of adenovirus 5 and herpesvirus type 1
(HSV 1) was observed on HeLa cells, while CPE of cox-sackievirus B5 and echovirus 7 on BS-C-1 cells.
The influence of clinoptilolite on viral proliferation
depends on both the concentration of MZ (CMZ, rang-
Due to sedimentation of clinoptilolite in its water sus-
ing from 0.5 to 50 mg/ml) and the viral titre (ranging
pension, it is not possible to treat a cell culture with MZ
from V1 to VÀ4), i.e. antiviral effect (The
and further follow up morphological changes of cells
antiviral effect was highest with the highest concentra-
upon viral infection. For this reason, different viral titres
tion of clinoptilolite (50 mg/ml) and the lowest viral titre
(V1–VÀ4) and MEM supplemented with 2% FBS (nega-
(VÀ4). The observed percentages of antiviral effect also
tive control) were treated with MZ at concentrations
M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169
CMZ (mg/ml) CMZ (mg/ml)
Fig. 1. Influence of different concentrations of clinoptilolite (C
Fig. 4. Influence of different concentrations of clinoptilolite (C
HSV 1 proliferation on HeLa cell line (percentage of cytopathic
echovirus 7 proliferation on BS-C-1 cell line (percentage of cytopathic
effect—CPE) for viral titre V1 (), VÀ1 (Ã), VÀ2 (·), VÀ3 (m), VÀ4 (j)
effect—CPE) for viral titre V1 (), VÀ1 (Ã), VÀ2 (·), VÀ3 (m), VÀ4 (j)
and negative control—culture media without virus (Ç).
and negative control—culture media without virus (Ç).
Table 1Percentage of inhibition of HSV1 proliferation upon treatment with
CMZ (mg/ml)
Fig. 2. Influence of different concentrations of clinoptilolite (CMZ) onadenovirus 5 proliferation on HeLa cell line (percentage of cytopathic
effect—CPE) for viral titre V1 (), VÀ1 (Ã), VÀ2 (·), VÀ3 (m), VÀ4 (j)
Percentage of inhibition of adenovirus 5 proliferation upon treatment
and negative control—culture media without virus (Ç). CMZ (mg/ml)
Percentage of inhibition of coxsackievirus B5 proliferation upontreatment with MZ
Fig. 3. Influence of different concentrations of clinoptilolite (CMZ) on
coxsackievirus B5 proliferation on BS-C-1 cell line (percentage ofcytopathic effect—CPE) for viral titre V1 (), VÀ1 (Ã), VÀ2 (·), VÀ3
(m), VÀ4 (j) and negative control—culture media without virus (Ç).
Viral suspensions, regardless of viral titre, treated
with MZ at concentrations of 0.5 and 5 mg/ml prior to
antiviral assay induce very low (5.6–28.6%) inhibition
of specific CPE, or inhibition of adenovirus 5, HSV 1and echovirus 7, except for coxsackievirus B5 whichwas inhibited completely at viral titre VÀ4
100% (VÀ4) inhibition of CPE of adenovirus 5, echovi-
Concentrations of 12 mg/ml of MZ induced a maxi-
rus 7, HSV 1 and coxsackievirus B5, respectively (
mum of 21.4% (VÀ4), 50% (VÀ4), 71.4% (VÀ3) and
M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169
Another possible mechanism of action of MZ onto
Percentage of inhibition echovirus 7 proliferation upon treatment with
viral particles is its ion exchange capability that could
destabilise morphology of viral particles;namely as lipo-
protein structure (viral envelope) is less resistant to envi-
ronment than protein (viral capside), this could explain
why herpesviruses (enveloped) were more destabilised
than adenoviruses (non-enveloped) by MZ. However,
this theory is not completely accurate because the prolif-
eration of enteroviruses (coxsackievirus B5 and echovirus
7), also non-enveloped virions were almost equally inhib-
ited by MZ as those of herpesviruses (HSV 1). Thus, insuch impoverished culture media the viral viability and
Concentrations of 25 and 50 mg/ml of MZ induced a
infectivity is reduced. The exact mechanism of action of
significantly higher inhibition of CPE of most treated
MZ based on the ion exchange property of their interac-
viruses, except adenovirus 5 ). The maximum
tion with viral particles in an aqueous solution (culture
of 28.6% and 42.9% inhibition of CPE of adenovirus 5
media), needs further investigation, extensive biochemi-
was observed at the lowest viral titre (VÀ4) treated with
cal analysis of media and virion changes.
The mechanisms of action of MZ upon different types
Concentrations of 25 and 50 mg/ml of MZ induced a
of viruses are probably non-specific which makes it more
high inhibitory effect of CPE of HSV 1 of 83.3% (VÀ4)
interesting than conventional antiviral drug Such
inactivation of viral particles by MZ would be extremely
larly, concentrations of 25 and 50 mg/ml of MZ induced
interesting for viruses that infect the digestive tract such
the highest inhibitory effect of CPE, up to 100% (
as enteroviruses and adenoviruses, and because MZ can
be orally administrated without toxicity it could be
used for therapeutic purposes. Beside that, MZ could
Our study indicates an inhibitory effect of MZ upon
be used as traditional natural antidiarrhoeal therapy
viral proliferation. The inhibitory effect was represented
by the inhibition of specific viral CPE on cell culture
Herpesviruses are able to establish life-long latency
compared to the same without treatment with MZ. As
after primary infection that can be reactivated, espe-
mentioned previously, the inhibitory effect of MZ de-
cially in immunocompromised transplant recipients
pends on the concentration of MZ (0.5–50 mg/ml), the
and patients with AIDS. Generally, herpesvirus infec-
type and the concentration of virus (viral titre ranging
tions have been treated successfully with systemic
administration of acyclovir However, drug resis-
cant inhibition of viral proliferation over 50% was ob-
tance variants emerge after long-term treatment, which
served with concentration of MZ over 12 mg/ml.
leads to treatment failures. This is why new efficient
Treatment of viral suspension of adenovirus 5 with
and inexpensive potential drugs such as MZ could be
MZ did not induce any significant inhibition of viral
helpful to inhibit, if not eradicate, viral infections. Addi-
proliferation contrary to HSV 1, coxsackievirus B5
tionally, MZ could be administrated locally on skin as
and echovirus 7. The inhibition of viral proliferation
cream or gel in order to inhibit recurrent labial and gen-
must probably be unspecific and independent of virion
ital herpesvirus infections that are often psychologically
size, structure and genome type. As MZ consists of a
mixture of particles of approximately 1 lm in diameterand a internal pore size of 0.35 nm, virions ranging from20 to 200 nm in size were probably incorporated within
the mesoporous zeolite aggregate and/or adsorbedon the surface of their crystalline microstructure during
Our preliminary results, indicate an antiviral prop-
the 15 h treatment of virally infected culture media. This
erty of clinoptilolite that open a possibility of therapeu-
would be the most plausible explanation because a sim-
tical application of MZ either locally (skin) against
ilar phenomenon is used in the method of viral concen-
herpesvirus infections or orally in cases of adenovirus
tration by capture on borosilicate glass powder although
or enterovirus infections. However, the inhibitory effect
the particle size is much larger (100–200 lm) Fur-
of viral proliferation was observed with high concentra-
thermore, MZ adsorb essential minerals and amino
tion of MZ (over 12 mg/ml) which makes the clinical
acids from culture media . Inhibition of viral prolif-
applications and the dose-response effect difficult to
eration by capture and/or adsorption of virions onto
establish. Fortunately, MZ could be used in purification
MZ crystalline microstructure requests further research
of drinking water from different viral particles without
(electron microscopy analysis, for instance).
concern of concentration of MZ for application.
M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169
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cal assistance. The Croatian Ministry of Science and
[7] M. Poljak-Blazˇi, M. Katic´, M. Kralj, N. Z
Technology supported this study (project No. 00981499).
ˇ verko, T. Balog, K. Pavelic´, Stud. Surf. Sci. Catal.
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