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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 [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. C We are grateful to Mihaela Alivojvodic´ for her techni- 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).
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