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Research Article
 

Administration of Zataria multiflora as a Novel Therapeutic Strategy in Destruction of the Germinal Layer of Hydatid Cyst



Ahmad Oryan, Mohammad Moazeni and Forough Zarei Kordshouli
 
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ABSTRACT

Background and Objective: Hydatid cyst is the larval stage of the tapeworm Echinococcus granulosus which causes one of the most important zoonotic helminthic diseases worldwide. Using scolicidal agents before surgery may prevent recurrence of infection, however many scolicidal agents may cause serious side effects. Previously it has been proven that methanolic extract of Zataria multiflora has scolicidal effect in vitro and in vivo. However, the exact mechanism by which Z. multiflora exerts its scolicidal effect is unknown. Methodology: Therefore, the destructive effect of total methanolic extract of Z. multifora on the germinal layer of hydatid cyst was investigated. The germinal layer together with laminated layer of hydatid cyst were collected from livers of sheep and were exposed to 10, 20 and 30 mg mL–1 concentrations of the methanolic extract of Z. multiflora for 20, 30 and 60 min. Viability of the protoscolices was confirmed by 0.1% eosin staining. Results: This therapeutic regimen resulted in discontinuity, destruction and irregularity of the cells in the germinal layer, degeneration and destruction of protoscolices, detachment of the germinal layer from the laminated layer and vacuolation of the laminated layer. Conclusion: The effects of methanolic extract of Z. multiflora were mainly related to its concentration but variation in time of exposure had slight effect on its effect on germinal layer and protoscoleces.

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Ahmad Oryan, Mohammad Moazeni and Forough Zarei Kordshouli, 2016. Administration of Zataria multiflora as a Novel Therapeutic Strategy in Destruction of the Germinal Layer of Hydatid Cyst. Research Journal of Parasitology, 11: 41-47.

DOI: 10.3923/jp.2016.41.47

URL: https://scialert.net/abstract/?doi=jp.2016.41.47
 
Received: April 24, 2016; Accepted: May 17, 2016; Published: September 15, 2016



INTRODUCTION

Cystic echinococcosis, caused by the larval stage of E. granulosus is considered a public health concern due to the fact that the disease has a global distribution. Moreover, it has the ability to infect humans as well as domestic livestock including cattle, sheep, horses and other herbivores (Eckert and Deplazes, 2004). The adult worm lives in the small intestine of carnivores, as definitive hosts, while the larval form, hydatid cyst is found in the internal organs of mammalian including human (Seimenis, 2003). The metacestode form is responsible for severe tissue damage, reduction in milk and meat and considerable economic loss due to condemnation of infected organs of the herbivorous animals (Oryan et al., 2012). The cyst usually lodges in liver and lungs; however it may develops in other organs such as muscles, kidneys, spleen, central nervous system and contributes to in these organs (Spotin et al., 2012). High prevalence of this zoonotic infection has been reported in most regions of the world involving Africa, South America, Eurasia, Australia and Middle East (Grosso et al., 2012). Hyper endemic incidence of cystic echinococcosis in Iran has been stated previously (Oryan et al., 1994; Sadjjadi, 2006).

Until now the therapeutic approach of hydatidosis includes surgery (Rajabi, 2009). However the utility and safety of surgery is limited due to complications including recurrence of echinococcosis and anaphylactic shock after dissemination of the protoscolex-rich fluid on peritoneum and visceral organ (Kilicoglu et al., 2008; Moro and Schantz, 2009). Previously different chemical agents have been tried to treat hydatidosis (Ahmadnia et al., 2013) however, the clinical use of such drugs are limited because they are severely nephrotoxic and/or hepatotoxic (Sahin et al., 2004; Caglar et al., 2008). Moreover, using benzimidazole carbamate derivatives, which are currently used for chemotherapeutic treatment of hydatidosis, is associated with adverse side effects because they must be applied in high doses for extended periods of time in human patients (Walker et al., 2004). Therefore, a new effective alternative treatment regime is extremely important in today’s climate, where species are becoming resistant to many traditional chemical treatment regimes and there is resurgence in the use of natural alternative therapies, instead of synthetic pharmaceuticals that often have severe side effects (Harris et al., 2000).

Zataria is one of the most reputable herbal medicines that gain much attention in recent years (Khalili and Vahidi, 2006). Former studies have revealed that Zataria multiflora has antioxidant (Sharififar et al., 2007), anti-inflammatory (Hosseinzadeh et al., 2000), antibacterial (Misaghi and Basti, 2007; Sharififar et al., 2007) antifungal (Gandomi et al., 2009) and antiprotozoal (Abdollahy et al., 2004) effects. Moazeni and Roozitalab (2012) have shown that methanolic extract of Z. multiflora has a high scolicidal effect against hydatid cyst in vitro (Moazeni and Roozitalab, 2012). The extract has also therapeutic and preventive effects on formation of the cysts (Moazeni et al., 2014a). However, this is not clear how the extract exert its scolicidal effect. This study has been devoted to evaluate the efficacy of the methanolic extract of Z. multiflora on destruction of the germinal layer of hydatid cyst in an in vitro condition.

MATERIALS AND METHODS

Extraction and preparation of different concentrations of Z. multiflora extract: The leaves of Z. multiflora were dried under shade and powdered mechanically, using a commercial electric blender. Hundred grams of the dry powder was added to 400 mL pure methanol and stirred gently for 1 h. The solution was left at room temperature for 24 h and then it was stirred again, filtered and the solvent was removed by evaporation in a rotary evaporator. The remaining semisolid material was then freeze dried and transferred to a sterile glass container which was stored at 4°C until its use. Three concentrations, 10, 20 and 30 mg mL–1, of Z. multiflora extract in normal saline were used in this study.

Collection and preparation of specimens: Unilocular hydatid cysts were obtained from the livers of 10 naturally infected sheep slaughtered at Shiraz and Marvdasht Slaughter houses in Fars Province, southern Iran. For viability test, the protoscolices were stained by 0.1% eosin and were observed by an ordinary light microscope (Olympus, Tokyo, Japan). The laminated and germinal layers of the cyst’s wall were carefully dissected, separated and sectioned to blocks of approximately 1 mm2.

Effectiveness of Z. multiflora extract on specimens: Three concentrations, 10, 20 and 30 mg mL–1 of Zataria multiflora extract in normal saline were used in this study. The specimens including the germinal layer, while attached to the laminated layer were exposed to different concentrations of Z. multiflora extract for 20, 30 and 60 min. The specimens of the control groups were exposed to the normal saline. All the specimens were then fixed in 10% neutral buffered formalin, dehydrated at graded ethanol, cleared by xylol, embedded in paraffin wax, sectioned at 4-5 μm, stained with haematoxylin and eosin and examined for histopathological changes by an ordinary light microscope (Olympus, Tokyo, Japan) by two pathologists unaware of the treatment protocols.

RESULTS

Efficacy of Z. multiflora extract on hydatid cysts was analyzed by investigation of discontinuity of the germinal layer, destruction of germinal layer, destruction of protoscolices and separation of the germinal layer and laminated layer. The scoring was performed so that the least changes received one and the highest changes received three score. The scoring results are shown in Table 1. The degree of discontinuity was higher in germinal layers exposed to higher concentration of Z. multiflora extract (Fig. 1). Themethanolic extracts of Z. multiflora at concentration of 30 mg mL–1 had a severe destructive effect on germinal layer. It was able to disorganize and disintegrate the cells lining this layer at 20, 30 and 60 min. The extract at concentrations of 10 and 20 mg mL–1 had mild and sever effect on irregularity of cells. At theses concentrations this effect was higher at 30 and 60 min compared to 20 min (Fig. 2). Two and three percent concentrations of Z. multiflora intensely affected the protoscolices after 30 and 60 min. Degradation of protoscolices was mild when exposed to 1% concentration of the methanolic extract (Fig. 3). The germinal layer was completely separated from the laminated layer when the hydatid cyst was exposed to 30 mg mL–1 concentration of Z. multiflora extract in all three periods as well as 20 mg mL–1 concentration at 60 min. Detachment of the germinal layer from laminated layer was moderate after 20 and 30 min at 20 mg mL–1 concentration and at all periods at 10 mg mL–1 concentration (Fig. 4). Severe vacuolation of the germinal layer exposed to 30 mg mL–1 concentration of Z. multiflora extract was seen in all three time points while 20 mg mL–1 extract had moderate to mild effect and 10 mg mL–1 concentration had mild to moderate effect (Fig. 5).

The 20 and 30 mg mL–1 concentrations of the methanolic extract of this plant had severe harmful effects on protoscoleces of hydatid cysts after 60 min and the changes represented as increase in degradation of these structures at longer durations and higher concentrations. The germinal layer was completely separated from the laminated layer when the hydatid cyst was exposed to 30 mg mL–1 concentration of Z. multiflora for 60 min.

DISCUSSION

Germinal layer has an important role in the growth and production of protoscolices and infectivity of hydatid cyst, hence destruction of this layer is an effective approach in treatment of the infection (Yalcin et al., 2010). Our experiment has revealed the interruption and disappearance of germinal layer as well as damage to protoscolices after exposure to methanolic extract of Z. multiflora. Such a result has been previously achieved by application of high-intensity focused ultrasound (Wang et al., 2007, 2009).

Table 1: Effects of the methanolic extract of Zataria multiflora on hydatid cyst
*Mild, **Moderate and ***Severe

Fig. 1:
Section from the germinal layer and protoscoleces of hydatid cyst exposed to 20 mg mL–1 concentration of the methanolic extract of Zataria multiflora for 30 min. Severe discontinuity of the germinal layer is evident in this section, scale bar: 200 µ, G: Germinal layer, P: Protoscoleces and L: Laminated layer

Fig. 2:
Section from the germinal layer and protoscoleces of hydatid cyst exposed to 30 mg mL–1 concentration of methanolic extract of Zataria multiflora for 60 min. Destruction and irregularity of the cells in the germinal layer is evident in this section, scale bar: 200 µ, G: Germinal layer, P: Protoscoleces and L: Laminated layer

The other effect of Z. multiflora on hydatid cyst was discontinuation of germinal layer.

Fig. 3:
Section from the germinal layer and protoscoleces of hydatid cyst exposed to 30 mg mL–1 concentration of the methanolic extract of Zataria multiflora for 60 min. Degeneration and destruction of protoscoleces is evident in this section, scale bar: 200 µ, G: Germinal layer, P: Protoscoleces and L: Laminated layer

Fig. 4:
Section from the germinal layer and protoscoleces of hydatid cyst exposed to 20 mg mL–1 concentration of the methanolic extract of Zataria multiflora for 30 min. Detachment of the germinal layer from the laminated layer is evident in this section, scale bar: 200 µ, G: Germinal layer, P: Protoscoleces and L: Laminated layer

Exposure of hydatid cyst layers to the methanolic extract of Z. multiflora resulted in detachment of the germinal layer of the hydatid cyst from the laminated layer.

Fig. 5:
Section from the germinal layer and protoscoleces of hydatid cyst exposed to 30 mg mL–1 concentration of the methanolic extract of Zataria multiflora for 30 min. Vacuolation of the laminated layer is evident in this section, scale bar: 200 µ, G: Germinal layer, P: Protoscoleces and L: Laminated layer

Since the laminated layer provides nutrients for germinal layer, detachment of these two layers may contribute in deprivation of germinal layer from such nutrients which was associated with other findings of the present experiment i.e., destruction and irregularity of the cells in the germinal layer. Such approaches have previously been used by Zeghir-Bouteldja et al. (2009) on human hydatid cyst. They have used nitric oxide in vitro to reach this approach; however the clinical use of nitric oxide was not studied (Zeghir-Bouteldja et al., 2009). Combination of albendazole sulfoxide and praziquantel has also led to stronger effect associated by changes in germinal layer including disappearance of cellular integrity as well as loss of cytoplasm and nucleus in cells lining of germinal layer (Palomares et al., 2006). Moreover, exposure of the cysts to the extract of Z. multiflora resulted in vacuolation of laminated layer. Since laminated layer prevents direct contact between host cell and germinal layer and activation of complement system (Ferreira et al., 2000) detachment of these layers and vacuolation of laminated layer as seen in the present study, may affect this prevention. Such changes in the germinal layer and laminated layer may exerted scolicidal effects so that the protoscolices exposed to 20 and 30 mg mL–1 concentrations of Z. multiflora extract were degenerated. Moazeni and Roozitalab (2012) have reported the scolicidal effect of the methanolic extract of Z. multiflora in vitro. Another study by Moazeni et al. (2014a) has shown that the methanolic extract of Z. multiflora has preventive and therapeutic effects on hydatidosis in Balb/C mice so that its preventive and therapeutic effects was very close to albendazol. Moreover, their scanning ultramicroscopic investigations have confirmed the destructive effect of Z. multiflora on germinal layer. Similar results have been obtained after administration of Z. multiflora aromatic water in Balb/C mice (Moazeni et al., 2014b). The effect of Z. multiflora extract on the germinal layer is comparable with that of other chemical therapeutic agents used in treatment and prevention of hydatid cyst. Albendazol and aqueous extract of huaier have been shown to be effective against the larval stage of Echinococcus granulosus by reducing in cell numbers in the germinal layer (Lv et al., 2013). Manouras et al. (2007) have used albendazole to detach the germinal layer from laminated layer before surgery. This procedure has resulted in safe removal of the cyst during surgery without any recurrence of infection.

The methanolic extract of Zataria multiflora contains phenolic compounds including quercetin, caffeic acid, catechin and gallic acid (Moazeni et al., 2014a). Phenolic compounds have antiseptic, antibacterial, anti-fungal, antiparasitic and anti-noceceptive activities (Hajhashemi et al., 2002). However, identification of the effective compounds, present in Z. multiflora on hydatid cyst was not the aim of our study. Our study has revealed that the effect of Z. multiflora extract on the germinal layer was mainly depended on concentration.

CONCLUSION

From this study it was concluded that the methanolic extract of Z. multiflora exerts scolicidal effects by affecting the germinal layer. It causes discontinuity of the germinal layer as well as destruction and irregularity of the cells in this layer and also detachment of the germinal layer from the laminated layer. The effects of Z. multiflora extracts on hydatid cysts, clearly correlated with concentration of the extract.

REFERENCES
1:  Ahmadnia, S., M. Moazeni, S. Mohammadi-Samani and A. Oryan, 2013. In vivo evaluation of the efficacy of albendazole sulfoxide and albendazole sulfoxide loaded solid lipid nanoparticles against hydatid cyst. Exp. Parasitol., 135: 314-319.
CrossRef  |  Direct Link  |  

2:  Abdollahy, F., H. Ziaei, B. Shabankhani and M. Azadbakht, 2004. Effect of essential oil and methanolic extract of Myrtus communis on Trichomonas vaginalis. Iran. J. Pharmaceut. Res., 3: 35-35.
Direct Link  |  

3:  Caglar, R., M.F. Yuzbasioglu, E. Bulbuloglu, M. Gul, F. Ezberci and I.T. Kale, 2008. In vitro effectiveness of different chemical agents on scolices of hydatid cyst. J. Invest. Surg., 21: 71-75.
CrossRef  |  Direct Link  |  

4:  Eckert, J. and P. Deplazes, 2004. Biological, epidemiological and clinical aspects of echinococcosis, a zoonosis of increasing concern. Clin. Microbiol. Rev., 17: 107-135.
CrossRef  |  PubMed  |  Direct Link  |  

5:  Ferreira, A.M., F. Irigoın, M. Breijo, R.B. Sim and A. Dıaz, 2000. How Echinococcus granulosus deals with complement. Parasitol. Today, 16: 168-172.
CrossRef  |  Direct Link  |  

6:  Gandomi, H., A. Misaghi, A.A. Basti, S. Bokaei, A. Khosravi, A. Abbasifar and A.J. Javan, 2009. Effect of Zataria multiflora Boiss. essential oil on growth and aflatoxin formation by Aspergillus flavus in culture media and cheese. Food Chem. Toxicol., 47: 2397-2400.
CrossRef  |  Direct Link  |  

7:  Grosso, G., S. Gruttadauria, A. Biondi, S. Marventano and A. Mistretta, 2012. Worldwide epidemiology of liver hydatidosis including the Mediterranean area. World J. Gastroenterol., 18: 1425-1437.
CrossRef  |  Direct Link  |  

8:  Hajhashemi, V., A. Ghannadi and S.K. Pezeshkian, 2002. Antinociceptive and anti-inflammatory effects of Satureja hortensis L. extracts and essential oil. J. Ethnopharmacol., 82: 83-87.
CrossRef  |  Direct Link  |  

9:  Harris, J.C., S. Plummer, M.P. Turner and D. Lloyd, 2000. The microaerophilic flagellate Giardia intestinalis: Allium sativum (garlic) is an effective antigiardial. Microbiology, 146: 3119-3127.
CrossRef  |  PubMed  |  Direct Link  |  

10:  Hosseinzadeh, H., M. Ramezani and G.A. Salmani, 2000. Antinociceptive, anti-inflammatory and acute toxicity effects of Zataria multiflora boiss extracts in mice and rats. J. Ethnopharmacol., 73: 379-385.
CrossRef  |  PubMed  |  Direct Link  |  

11:  Khalili, M.B. and A.R. Vahidi, 2006. The anti-microbial effect of Zataia multiflora drops on three bacterial species cause gastrointestinal disease. World J. Med. Sci., 2: 162-163.
Direct Link  |  

12:  Kilicoglu, B., K. Kismet, S.S. Kilicoglu, S. Erel and O. Gencay et al., 2008. Effects of honey as a scolicidal agent on the hepatobiliary system. World J. Gastroenterol., 14: 2085-2088.
CrossRef  |  Direct Link  |  

13:  Lv, H., Y. Jiang, M. Liao, H. Sun, S. Zhang and X. Peng, 2013. In vitro and in vivo treatments of Echinococcus granulosus with Huaier aqueous extract and albendazole liposome. Parasitol. Res., 112: 193-198.
CrossRef  |  Direct Link  |  

14:  Manouras, A., M. Genetzakis, E.E. Lagoudianakis, A. Papadima and C. Triantafillou et al., 2007. Intact germinal layer of liver hydatid cysts removed after administration of albendazole. Netherlands J. Med., 65: 112-116.
Direct Link  |  

15:  Misaghi, A. and A.A. Basti, 2007. Effects of Zataria multiflora Boiss. essential oil and nisin on Bacillus cereus ATCC 11778. Food Control, 18: 1043-1049.
CrossRef  |  Direct Link  |  

16:  Moazeni, M. and A. Roozitalab, 2012. High scolicidal effect of Zataria multiflora on protoccoleces of hydatid cyst: An in vitro study. Comp. Clin. Pathol., 21: 99-104.
CrossRef  |  Direct Link  |  

17:  Moazeni, M., S. Larki, A. Oryan and M.J. Saharkhiz, 2014. Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study. Vet. Parasitol., 205: 107-112.
CrossRef  |  Direct Link  |  

18:  Moazeni, M., S. Larki, M.J. Saharkhiz, A. Oryan, M.A. Lari and A.M. Alavi, 2014. In vivo study of the efficacy of the aromatic water of Zataria multiflora on hydatid cysts. Antimicrob. Agents Chemother., 58: 6003-6008.
CrossRef  |  Direct Link  |  

19:  Moro, P. and P.M. Schantz, 2009. Echinococcosis: A review. Int. J. Infect. Dis., 13: 125-133.
CrossRef  |  Direct Link  |  

20:  Oryan, A., N. Moghaddar and S.N.S. Gaur, 1994. Metacestodes of sheep with special reference to their epidemiological status, pathogenesis and economic implications in Fars province, Iran. Vet. Parasitol., 51: 231-240.
CrossRef  |  PubMed  |  Direct Link  |  

21:  Oryan, A., S. Goorgipour, M. Moazeni and S. Shirian, 2012. Abattoir prevalence, organ distribution, public health and economic importance of major metacestodes in sheep, goats and cattle in Fars, Southern Iran. Trop. Biomed., 29: 349-359.
Direct Link  |  

22:  Palomares, F., G. Palencia, J.R. Ambrosio, A. Ortiz and H. Jung-Cook, 2006. Evaluation of the efficacy of albendazole sulphoxide and praziquantel in combination on Taenia crassiceps cysts: In vitro studies. J. Antimicrob. Chemother., 57: 482-488.
CrossRef  |  Direct Link  |  

23:  Rajabi, M.A., 2009. Fatal reactions and methaemoglobinaemia after silver nitrate irrigation of hydatid cyst. Surg. Pract., 13: 2-7.
CrossRef  |  Direct Link  |  

24:  Sadjjadi, S.M., 2006. Present situation of echinococcosis in the Middle East and Arabic North Africa. Parasitol. Int., 55: S197-S202.
CrossRef  |  Direct Link  |  

25:  Sahin, M., R. Eryilmaz and E. Bulbuloglu, 2004. The effect of scolicidal agents on liver and biliary tree (experimental study). J. Invest. Surg., 17: 323-326.
CrossRef  |  Direct Link  |  

26:  Seimenis, A., 2003. Overview of the epidemiological situation on echinococcosis in the Mediterranean region. Acta Tropica, 85: 191-195.
CrossRef  |  Direct Link  |  

27:  Sharififar, F., M.H. Moshafi, S.H. Mansouri, M. Khodashenas and M. Khoshnoodi, 2007. In vitro evaluation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss. Food Control, 18: 800-805.
CrossRef  |  Direct Link  |  

28:  Spotin, A., M.M.A. Majdi, M. Sankian and A. Varasteh, 2012. The study of apoptotic bifunctional effects in relationship between host and parasite in cystic echinococcosis: A new approach to suppression and survival of hydatid cyst. Parasitol. Res., 110: 1979-1984.
CrossRef  |  Direct Link  |  

29:  Walker, M., J.F. Rossignol, P. Torgerson and A. Hemphill, 2004. In vitro effects of nitazoxanide on Echinococcus granulosus protoscoleces and metacestodes. J. Antimicrob. Chemother., 54: 609-616.
CrossRef  |  Direct Link  |  

30:  Wang, J.A., X.Y. Zou, B. Ye, C.W. Zhang and F.S. Zhao et al., 2009. High intensity focus ultrasound kills hydatid cysts of Echinococcus granulosus. Acta Academiae Medicinae Militaris Tertiae, 31: 1333-1336.
Direct Link  |  

31:  Wang, J.N., X.Y. Zou, B. Ye, C.W. Zhang and F.S. Zhao et al., 2007. The heat effect of irradiation with high intensity focused ultrasound on the hydatid cysts of Echinococcus granulosus. Chin. J. Zoonoses, 6: 576-579.
Direct Link  |  

32:  Yalcin, E., N. Kiper, C. Tan, U. Ozcelik and D. Dogru et al., 2010. The role of human leucocyte antigens in children with hydatid disease: Their association with clinical condition and prognosis. Parasitol. Res., 106: 795-800.
CrossRef  |  Direct Link  |  

33:  Zeghir-Bouteldja, R., M. Amri, S. Aitaissa, S. Bouaziz, D. Mezioug and C. Touil-Boukoffa, 2009. In vitro study of nitric oxide metabolites effects on human hydatid of Echinococcus granulosus. J. Parasitol. Res. 10.1155/2009/624919


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