Antiviral Properties of Clinoptilolite

Antiviral properties of clinoptilolite
Magdalena Grce *, Kresˇimir Pavelic´
‘‘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
Available online 8 December 2004
Abstract
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 were treated with MZ in concentrations ranging from 0.5 to 50mg/ml. The viral proliferation was evaluated by optical microscope as percentage of cytopathic effect (CPE). Human adenovirus 5, herpes simplex virus type 1 (HSV 1) and human enteroviruses (coxsackievirus B5 and echovirus 7) were used in the antiviral assay. Concentrations of 0.5 and 5mg/ml of MZ induced a very low antiviral effect or the antiviral was not observed at all, while concentrations of 12, 25 and 50mg/ml of MZ induced a significant inhibitory effect upon viral proliferation. MZ inhibited the viral proliferation of HSV 1, coxsackievirus B5 and echovirus 7 more efficiently than adenovirus 5. The antiviral effect of MZ seems to be non-specific and is more likely based on the incorporation of viral particles into
pores of MZ aggregates than ion exchange properties of clinoptilolite. Our preliminary results indicate a possibility of therapeutical application 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.

2004 Elsevier Inc. All rights reserved.
Keywords: Clinoptilolite;Micronized zeolite (MZ); Antiviral properties; Cytopathic effect (CPE)
1. Introduction
Clinoptilolite is a natural, non-toxic zeolite that has monoclinic crystal structure symmetry and strong
adsorptive and ion exchange capacity [1]. These properties have been largely exploited in industrial, agricultural,
environmental and biological technologies [2]. Zeolites also possess biological activities, either positive or negative. The best known and documented positive biological activity of natural clinoptilolite is its action as antidiarrheal drug [3]. Furthermore, some of them seem to have antibacterial property [4]. The clinoptilolite from Vranje, Serbia used in this study has antioxidative and immunostimulatory effects [5], and it has been used as an adjuvant to anticancer therapy [6–8].
Clinoptilolite administered by gastric intubation to mice injected with melanoma cells significantly reduced the number of melanoma metastases [2]. Clinoptilolite treatment of mice and dogs suffering from a variety of
tumor types led to improvement in the overall health status, prolongation of life span, and decrease in tumor size. Local application of clinoptilolite to skin cancers of some dogs effectively reduced tumor formation and
growth [6].
The major negative biological effect of clinoptilolite could be its toxicity in higher organisms (mammal) if the content of heavy metals (Pb, Cd, Zn, etc.) is high. Therefore, a classic acute, sub-chronic and chronic toxicity study of the clinoptilolite from Vranje, Serbia was performed on mice and rats [6,9]. Results clearly show that oral (in diet) administration of clinoptilolite to mice and rats for 6 and 12months, respectively, caused no changes that could be considered a toxic effect of treatment.
1387-1811/$ – see front matter
2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.micromeso.2004.10.039
* Corresponding author. Tel.: +385 1 4661111;fax: +385 1 4661010.
E-mail address: grce@irb.hr (M. Grce).
www.elsevier.com/locate/micromeso
Microporous and Mesoporous Materials 79 (2005) 165–169
Based on these results we assumed that the adsorbent qualities and ion exchange properties of clinoptilolite could be effective on viruses too. Herein, we tested a natural clinoptilolite (Vranje, Serbia) [6] on in vitro viral replication of adenoviruses, herpesviruses and enteroviruses (coxsackievirus, echovirus).
2. Experimental
2.1. Natural clinoptilolite
A fine powder of natural clinoptilolite, i.e., micronized zeolite (MZ), was obtained by tribomechanical micronization [6] of natural clinoptilolite from Vranje, Serbia. Chemical composition and characteristics of MZ have been described previously [5,6].
2.2. Cell lines
Human cervical carcinoma cells (HeLa;ATCC number: CCL-2) and African green monkey kidney epithelial
cells (BS-C-1;ATC C number: CCL-26) were used. Cells were propagated in Dulbecco
s modified eagle medium (MEM;Gibco BRL, USA) supplemented with 10% inactivated foetal bovine serum (FBS;Gibco BRL, USA), 1% L-glutamine and 0.3% sodium bicarbonate at 37C and 5% CO2.
2.3. Viruses
Adenovirus 5 (ATCC number: VR-5), herpesvirus type 1 (HSV 1;ATC C number: VR-733), and two enteroviruses,
coxsackievirus B5 virus (ATCC number: VR- 185) and echovirus 7 (ATCC number: VR-37) were included in this study. Adenovirus and herpesvirus were propagated on HeLa, while enterovirus were propagated on BS-C-1 confluent cell monolayers. The viral suspension consisted the cell-free supernatant collected after centrifugation (20 min, 4C, 5000 · g) of infected media (MEM supplemented with 2% FBS) collected at maximal viral proliferation, i.e. 100% cytophatic effect (CPE) of whole cell monolayer. Five different relative viral titres (V1–V
4) obtained by serial dilution of viral suspension (1:2 for adenovirus and herpesvirus and 1:10 for enteroviruses) were treated with MZ prior to antiviral assay.
2.4. MZ treatment
Due to sedimentation of clinoptilolite in its water suspension, it is not possible to treat a cell culture with MZ and further follow up morphological changes of cells upon viral infection. For this reason, different viral titres
(V1–V
4) and MEM supplemented with 2% FBS (negative control) were treated with MZ at concentrations ranging from 0.5 to 50mg/ml. After incubation (15 h, 4 C, constant rotation), the suspension (media and MZ) was centrifuged (10 min, 4C, 3000 · g) to separate the liquid from the solid phase (MZ).
2.5. Antiviral assays
HeLa and BS-C-1 cell were seeded at 2 · 104 cells per ml on 24-well flat-bottomed microtitre plates (Becton Dickinson, USA). The viral infection was performed on one-day-old confluent cell monolayers. The plates were incubated at 37C and 5% CO2 and the CPE were followed by optical microscopy every 24-h during 3– 4 days (depending on the type of virus). Each assay was done four times. The inhibitory effect of viral proliferation was evaluated as percentage of CPE and was compared to CPE of similar dilutions of viral suspension also incubated at 4C during 15-h but without MZ (positive control).
3. Results and discussion
Four different viruses were chosen on the basis of their morphology and biological characteristics: (a) with or without lipoprotein envelope acquired from the host cell, (b) DNA or RNA replicating viruses and (c) high infectivity and relatively rapid CPE in cell culture. The herpesviruses capsid is surrounded by a lipoprotein envelope, varying in size from 100–200nm in diameter and their genome consists of double-stranded linear DNA. Adenoviruses and enteroviruses (coxsackieviruses and echoviruses) are non-enveloped and are relatively small (65–80 and 22–30nm virion size, respectively) viruses, as compared to herpesviruses. The genome of adenoviruses consists of linear double-stranded DNA, while the one from enteroviruses consists of singlestranded RNA.
Enteroviruses are highly infective and specific CPE (cell lysis) appears in cell culture (BS-C-1) rapidly, within 24–48h depending of the viral titre (1:10 serial dilution). Adenoviruses and herpesviruses are less infective than enteroviruses and specific CPE (cell rounding) appears in cell culture (HeLa), within 24–72h depending of the viral titre (1:2 serial dilution). The CPE of adenovirus 5 and herpesvirus type 1 (HSV 1) was observed on HeLa cells, while CPE of coxsackievirus B5 and echovirus 7 on BS-C-1 cells. The influence of clinoptilolite on viral proliferation depends on both the concentration of MZ (CMZ, ranging from 0.5 to 50mg/ml) and the viral titre (ranging from V1 to V
4), i.e. antiviral effect (Figs. 1–4). The antiviral effect was highest with the highest concentration of clinoptilolite (50mg/ml) and the lowest viral titre (V
4). The observed percentages of antiviral effect also depended on the type of virus (Tables 1–4).
166 M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169
Viral suspensions, regardless of viral titre, treated with MZ at concentrations of 0.5 and 5mg/ml prior to antiviral assay induce very low (5.6–28.6%) inhibition of specific CPE, or inhibition of adenovirus 5, HSV 1 and echovirus 7, except for coxsackievirus B5 which was inhibited completely at viral titre V
4 (Tables 1–4).
Concentrations of 12mg/ml of MZ induced a maximum of 21.4% (V
4), 50% (V
4), 71.4% (V
3) and
100% (V
4) inhibition of CPE of adenovirus 5, echovirus 7, HSV 1 and coxsackievirus B5, respectively (Tables 1–4).
0
20
40
60
80
100
0 0.5 5 12 25 50
CMZ (mg/ml)
CPE (%)
Fig. 2. Influence of different concentrations of clinoptilolite (CMZ) on
adenovirus 5 proliferation on HeLa cell line (percentage of cytopathic
effect—CPE) for viral titre V1 (), V
1 (), V
2 (·), V
3 (m), V
4 (j)
and negative control—culture media without virus (
).
0
20
40
60
80
100
0 0.5 5 12 25 50
CMZ (mg/ml)
CPE (%)
Fig. 1. Influence of different concentrations of clinoptilolite (CMZ) on
HSV 1 proliferation on HeLa cell line (percentage of cytopathic
effect—CPE) for viral titre V1 (), V
1 (), V
2 (·), V
3 (m), V
4 (j)
and negative control—culture media without virus (
).
0
20
40
60
80
100
0 0.5 5 12 25 50
CMZ (mg/ml)
CPE (%)
Fig. 4. Influence of different concentrations of clinoptilolite (CMZ) on
echovirus 7 proliferation on BS-C-1 cell line (percentage of cytopathic
effect—CPE) for viral titre V1 (), V
1 (), V
2 (·), V
3 (m), V
4 (j)
and negative control—culture media without virus (
).
0
20
40
60
80
100
0 0.5 5 12 25 50
CMZ (mg/ml)
CPE (%)
Fig. 3. Influence of different concentrations of clinoptilolite (CMZ) on
coxsackievirus B5 proliferation on BS-C-1 cell line (percentage of
cytopathic effect—CPE) for viral titre V1 (), V
1 (), V
2 (·), V
3
(m), V
4 (j) and negative control—culture media without virus (
).
Table 1
Percentage of inhibition of HSV1 proliferation upon treatment with
MZ
MZ (mg/ml) Viral titre
V
4 V
3 V
2 V
1 V1
0 0 0 0 0 0
0.5 0 7.1 6.3 5.6 0
5 16.7 28.6 18.8 27.8 0
12 66.7 71.4 50 44.4 20
25 83.3 78.6 68.8 55.6 50
50 83.3 85.7 75 66.7 60
Table 2
Percentage of inhibition of adenovirus 5 proliferation upon treatment with MZ
MZ (mg/ml) Viral titre
V
4 V
3 V
2 V
1 V1
0 0 0 0 0 0
0.5 7.1 0 0 0 0
5 14.3 0 0 0 0
12 21.4 6.3 5.6 0 0
25 28.6 18.8 16.7 15 10
50 42.9 31.3 27.8 25 15
Table 3
Percentage of inhibition of coxsackievirus B5 proliferation upon treatment with MZ
MZ (mg/ml) Viral titre
V
4 V
3 V
2 V
1 V1
0 0 0 0 0 0
0.5 50 0 0 0 0
5 100 20 5.6 0 0
12 100 80 77.8 45 0
25 100 90 88.9 70 10
50 100 100 100 80 20
M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169 167
Concentrations of 25 and 50mg/ml of MZ induced a significantly higher inhibition of CPE of most treated viruses, except adenovirus 5 (Figs. 1–4). The maximum of 28.6% and 42.9% inhibition of CPE of adenovirus 5 was observed at the lowest viral titre (V
4) treated with 25 and 50mg/ml of MZ, respectively (Table 2).
Concentrations of 25 and 50mg/ml of MZ induced a high inhibitory effect of CPE of HSV 1 of 83.3% (V
4) and 85.7% (V
3), respectively (Table 1, Fig. 1). Similarly, concentrations of 25 and 50mg/ml of MZ induced the highest inhibitory effect of CPE, up to 100% (Tables 3 and 4) of coxsackievirus B5 (Fig. 3) and echovirus 7 (Fig. 4).
Our study indicates an inhibitory effect of MZ upon viral proliferation. The inhibitory effect was represented by the inhibition of specific viral CPE on cell culture compared to the same without treatment with MZ. As mentioned previously, the inhibitory effect of MZ depends on the concentration of MZ (0.5–50mg/ml), the type and the concentration of virus (viral titre ranging from V1 to V
4) (Figs. 1–4 and Tables 1–4). A significant inhibition of viral proliferation over 50% was observed with concentration of MZ over 12mg/ml.
Treatment of viral suspension of adenovirus 5 with MZ did not induce any significant inhibition of viral proliferation contrary to HSV 1, coxsackievirus B5 and echovirus 7. The inhibition of viral proliferation must probably be unspecific and independent of virion size, structure and genome type. As MZ consists of a mixture of particles of approximately 1lm in diameter and a internal pore size of 0.35nm, virions ranging from 20 to 200nm in size were probably incorporated within the mesoporous zeolite aggregate and/or adsorbed on the surface of their crystalline microstructure during the 15h treatment of virally infected culture media. This would be the most plausible explanation because a similar phenomenon is used in the method of viral concentration by capture on borosilicate glass powder although the particle size is much larger (100–200lm) [10]. Furthermore, MZ adsorb essential minerals and amino acids from culture media [11]. Inhibition of viral proliferation by capture and/or adsorption of virions onto
MZ crystalline microstructure requests further research (electron microscopy analysis, for instance). Another possible mechanism of action of MZ onto viral particles is its ion exchange capability that could destabilise morphology of viral particles;namely as lipoprotein structure (viral envelope) is less resistant to environment 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 proliferation
of enteroviruses (coxsackievirus B5 and echovirus 7), also non-enveloped virions were almost equally inhibited
by MZ as those of herpesviruses (HSV 1). Thus, in such impoverished culture media the viral viability and infectivity is reduced. The exact mechanism of action of MZ based on the ion exchange property of their interaction with viral particles in an aqueous solution (culture media), needs further investigation, extensive biochemical analysis of media and virion changes.
The mechanisms of action of MZ upon different types of viruses are probably non-specific which makes it more
interesting than conventional antiviral drug [12]. Such inactivation of viral particles by MZ would be extremely
interesting for viruses that infect the digestive tract such as enteroviruses and adenoviruses, and because MZ can
be orally administrated without toxicity [6] it could be used for therapeutic purposes. Beside that, MZ could be used as traditional natural antidiarrhoeal therapy such as clay and activated charcoal [12,13].
Herpesviruses are able to establish life-long latency after primary infection that can be reactivated, especially in immunocompromised transplant recipients and patients with AIDS. Generally, herpesvirus infections have been treated successfully with systemic administration of acyclovir [14]. However, drug resistance variants emerge after long-term treatment, which leads to treatment failures. This is why new efficient and inexpensive potential drugs such as MZ could be helpful to inhibit, if not eradicate, viral infections. Additionally, MZ could be administrated locally on skin as cream or gel in order to inhibit recurrent labial and genital herpesvirus infections that are often psychologically
and physically very painful.
4. Conclusion
Our preliminary results, indicate an antiviral property of clinoptilolite that open a possibility of therapeutical
application of MZ either locally (skin) against herpesvirus infections or orally in cases of adenovirus or enterovirus infections. However, the inhibitory effect of viral proliferation was observed with high concentration of MZ (over 12mg/ml) which makes the clinical applications and the dose-response effect difficult to establish. Fortunately, MZ could be used in purification of drinking water from different viral particles without concern of concentration of MZ for application.
Table 4
Percentage of inhibition echovirus 7 proliferation upon treatment with MZ
MZ (mg/ml) Viral titre
V
4 V
3 V
2 V
1 V1
0 0 0 0 0 0
0.5 0 12.5 0 0 0
5 0 25 12.5 0 0
12 50 37.5 25 10 0
25 50 75 93.8 55 20
50 100 87.5 87.5 80 40
168 M. Grce, K. Pavelic´ / Microporous and Mesoporous Materials 79 (2005) 165–169
Acknowledgments
We are grateful to Mihaela Alivojvodic´ for her technical assistance. The Croatian Ministry of Science and Technology supported this study (project No. 00981499).
References
[1] D.W.J. Breck, Chem. Educ. 41 (1964) 678.
[2] F.A. Mumpton, Proc. Natl. Acad. Sci. USA 96 (1999) 3463.
[3] G. Rodriguez-Fuentes, M.A. Barrios, A. Iraizoz, I. Perdomo, B.
Cedre, Zeolites 19 (1997) 441.
[4] T. Maeda, Y. Nose, Artif. Organs 23 (1999) 129.
[5] K. Pavelic´, M. Katic´, V. Sˇverko, T. Marotti, B. Bosˇnjak, T.
Balog, R. Stojkovic´, M. Radacˇic´, M. Cˇ olic´, M. Poljak-Blazˇi, J.
Cancer Res. Clin. Oncol. 128 (2002) 37.
[6] K. Pavelic´, M. Hadzˇija, L. Bedrica, J. Pavelic´, I. Ðikic´, M. Katic´,
M.;Kralj, M. Herak Bosnar, S. Kapitanovic´, M. Poljak-Blazˇi, S.
Krizˇanac, R. Stojkovic´, M. Jurin, B. Subotic´, M. Cˇ olic´, J. Mol.
Med. 78 (2001) 708.
[7] M. Poljak-Blazˇi, M. Katic´, M. Kralj, N. Zˇ arkovic´, T. Marotti, B.
Bosˇnjak, V. C ˇ verko, T. Balog, K. Pavelic´, Stud. Surf. Sci. Catal.
135 (2001) 170.
[8] K. Pavelic´, B. Subotic´, M. Cˇ olic´, Surf. Sci. Catal. 135 (2001)
374.
[9] I. Martin-Klein, Z. Flegar Mastric´, R. Zadro, D. Breljak, S.
Stanovic´ Janda, R. Stojkovic´, M. Marusˇic´, M. Radacˇic´, M.
Boranic´, Food Chem. Toxicol. 39 (2001) 717.
[10] D. C ˇ ecˇuk, M. Grce, Rev. Epide´m. Sante´ Publ. 40 (1992) 182.
[11] M. Katic´, Molecular mechanisms of clinoptilolite on tumor cells.
Ph.D. thesis, University of Zagreb, 2002.
[12] J.M. Hunter, Science 228 (1985) 1040.
[13] M.J. Rodman, R.N. 43 (1980) 58.
[14] E.J. De Clercq, Clin. Virol. 22 (2001) 73