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Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study
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Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study Mohammad Moazeni a,∗ , Sara Larki b , Ahmad Oryan c , Mohammad Jamal Saharkhiz d a b c d
Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran School of Veterinary Medicine, Shiraz University, Shiraz, Iran Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Department of Horticulture Science, College of Agriculture, Shiraz, Iran
a r t i c l e
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Article history: Received 18 December 2013 Received in revised form 13 June 2014 Accepted 1 July 2014 Keywords: Preventive Therapeutic Methanolic extract Zataria multiflora Hydatid cyst In vivo
a b s t r a c t The phenolic compounds of Zataria multiflora extract, were identified by HPLC analysis. Gallic acid, catechin, caffeic acid, and quercetin were found to be the major phenolic compounds. Eighty healthy laboratory Balb/C mice were infected intraperitoneally by injection of 1500 viable protoscoleces and were divided into prevention (40 mice) and therapeutic (40 mice) groups. To prove the preventive effect of Z. multiflora extract on development of hydatid cyst, the 40 infected animals were allocated into three treatment groups including Z. multiflora (4 g/l in drinking water for 8 months), albendazole (150 mg/kg BW/day for 10 days) and untreated (control) group. To estimate the therapeutic effect of Z. multiflora extract on the hydatid cyst, after 8 months of infection, the infected mice were allocated into three experimental treatment groups including Z. multiflora (8 g/l in drinking water for 30 days), albendazole (300 mg/kg BW/day for 20 days) and untreated (control) group. At the end of the treatment period, all mice were euthanized and necropsied, the hydatid cysts were carefully removed, weighed and their size were recorded. Weight and size of the hydatid cysts significantly decreased (p < 0.05) upon the treatment with Z. multiflora extract in both prevention and therapeutic groups. The germinal layer of the hydatid cysts recovered from the treated mice, either from the prevention or therapeutic group, were completely damaged at ultrastructural level by scanning electron microscopy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Echinococcosis/hydatidosis is a zoonotic disease that occurs throughout the world and causes considerable economic losses and public health problems in many countries (Dalimi et al., 2002). Echinococcosis is mostly prevalent in Australia, South America, Middle East, South Africa, Eastern Europe, and the Mediterranean region (Zulfikaroglu
∗ Corresponding author. E-mail address: [email protected] (M. Moazeni).
et al., 2008) and immigration between continents, has led to the disease being prevalent in other countries as well (Sayek and Onat, 2001). Cystic echinococcosis (CE), which is considered as an emerging disease in various regions, is economically important and constitutes a threat to public health in many countries (Dinkela et al., 2004). Human infection with Echinococcus granulosus typically results in a slowly growing parasitic cystic disease most frequently seen in the liver. The cysts may be asymptomatic for many years, and occasionally spontaneous regression has been noted. More commonly the disease is slowly progressive, and symptoms and complications eventually arise. The
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Please cite this article in press as: Moazeni, M., et al., Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.006
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symptoms include pain from expansion or rupture, fever from pyogenic infection due to intrabiliary rupture and jaundice, or anaphylaxis from intrabiliary or extrahepatic rupture (Sielaff et al., 2001). Currently, there are three treatment options for hydatid disease of the liver. These treatment strategies include: surgery, percutaneous aspiration and medical treatment from which surgery has been known as the most efficient treatment option in humans (Adas et al., 2009). Chemotherapy is the preferred treatment where, surgeons are not available or the cysts are too numerous, and in inoperable cases, “chemotherapy” is the only option. Chemotherapy has also been used as an adjunct to surgery for prophylaxis against spillage of the cyst contents (Blanton et al., 1998; Spicher et al., 2008b). Few chemotherapeutic agents are available for the medical management of hydatid disease (Blanton et al., 1998). Benzimidazole carbamate derivatives, such as mebendazole and albendazole, are currently used for chemotherapeutic treatment of cystic echinococcosis. In human patients, benzimidazoles are commonly applied in high doses and for extended periods of time; these frequently results in adverse side effects (Walker et al., 2004). Therefore, a new effective alternative treatment regime is extremely important in today’s climate, where species are becoming resistant, 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). It has been shown that Zataria multiflora (Lamiaceae) has antibacterial (Sharififar et al., 2007; Misaghi and Akhondzadeh Basti, 2007), antifungal (Gandomi et al., 2009), antiprotozoal (Abdollahy et al., 2004) and scolicidal (Moazeni and Roozitalab, 2012) properties. The aim of the current experimental work was to evaluate the efficacy of the methanolic extract of Z. multiflora on the prevention and treatment of hydatid cyst in a murine model.
2.2. Identification of phenolic compounds For identification of the phenolic compounds from the Z. multiflora extract, HPLC analysis was carried out on a Agilent 1200 series (USA), equipped with a Zorbax Eclipse XDB-C18 column (10 cm × 5 m i.d.; ×150 mm film thickness, RP), and a photodiode array detector (PAD). For preparing the injectable extract, 0.02 g of the dried residue of the plant extract was dissolved in 2 ml of methanol–acetic acid (85:15) and the aliquots was filtered through a 0.2 m membrane millipore chromatographic filter and injected into the HPLC system. The elution was monitored at 280 and 320 nm. Gradient elution was selected to achieve the maximum separation and sensitivity. The elution was performed by varying the proportion of solvent A (formic acid 1% in deionized water) to solvent B (methanol (v/v)) as follows: methanol:formic acid 1% (10:90), at 0 min; methanol:formic acid 1% (25:75), at 10 min; methanol:formic acid 1% (60:40), at 20 min and finally, methanol:formic acid 1% (70:30), at 30 min. The total running time was 30 min. The column temperature was 30 ◦ C. 2.3. Collection of protoscoleces Protoscoleces of E. granulosus were collected aseptically from the hydatid cysts of the liver of the naturally infected sheep, slaughtered at Shiraz Slaughterhouse, Shiraz, southern Iran. The hydatid fluid of the cysts was aseptically transferred into glass cylinders and left to set for 30 min. The protoscoleces were settled at the bottom of the cylinders. The supernatant was then removed, and the yielded protoscoleces were washed several times with sterile 0.85% NaCl and stored in RPMI 1640 medium overnight at 37 ◦ C. The viability of the protoscoleces was confirmed from their motility characteristics under an ordinary light microscope after 0.1% eosin staining. They were finally transferred into a dark container containing normal saline and stored at 4 ◦ C for further use.
2. Materials and methods
2.4. Infection of mice
2.1. Extraction
Eighty healthy BALB/c male mice weighing 22–24 g were used in this study. All the mice were infected intraperitoneally by injection of 1500 protoscoleces per animal, dissolved in 0.5 ml of RPMI 1640 medium. The infected animals were allocated into two experimental treatment groups: (a) Prevention group, (b) Therapy group. All the animals in the study were fed ad libitum, and kept at 24–25 ◦ C.
Methanolic extract of Z. multiflora was prepared as described by Moazeni and Roozitalab (2012) with some modifications. Briefly, the leaves of Z. multiflora were dried under shade and powdered mechanically, using a commercial electric blender. A total of 2800 g of dried powder was extracted. One hundred grams of dry powder was added to 400 ml of pure methanol and gently mixed, using a shaking incubator, at 30–37 ◦ C for 1 h at 120–130 rpm. The obtained solution was left at room temperature for 24 h. The solution was stirred again and filtered, and the solvent was removed by evaporation in a rotary evaporator. The remaining semisolid material was then left at room temperature for 4 h and then freeze dried. The collected residue was transferred to a sterile glass container and stored in the refrigerator at 4 ◦ C before being used. 119 g of the dried extract was obtained from 2800 g of the dried powder of Z. multiflora.
2.5. Preventive trials To prove the preventive effect of Z. multiflora extract on the formation of hydatid cysts, the 40 infected animals were allocated into three experimental treatment groups: (a) Z. multiflora treatment group which were drunk with Z. multiflora extract (4 g/l) for 8 months (15 mice), (b) albendazole treatment group which received 150 mg/kg BW/day albendazole for 10 days (10 mice) and (c) untreated control group (15 mice). The remaining 25 mice were euthanized 8
Please cite this article in press as: Moazeni, M., et al., Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.006
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months after infection and necropsy was carried out immediately thereafter. 2.6. Therapeutic trials To evaluate the therapeutic effects of Z. multiflora extract on the hydatid cysts in the animals that were infected 8 months ago, the remaining 15 infected mice were allocated into three experimental treatment groups, five animal in each: (a) Z. multiflora treatment group which were drunk with 8 g/l of Z. multiflora extract for 30 days, (b) albendazole treatment group which received 300 mg/kg BW/day albendazole for 20 days and (c) untreated control group. All the mice were euthanized at the end of the treatment period and necropsy was carried out immediately thereafter. At necropsy, the peritoneal cavity was opened and the internal organs were observed for hydatid cyst. The hydatid cysts were carefully removed. Sizes of the cysts were measured using a scaled ruler and adobe Photoshop CS6. Each cyst’s weight was recorded using a digital scale (Kern & Sohn GmbH, D-72336 Balingen, Germany). The efficacy of Z. multiflora extract was evaluated by comparing the cysts numbers, sizes and weights and also according to the ultrastructural morphological changes between the three treatment groups. 2.7. Scanning electron microscopy The cysts were sectioned to blocks of approximately 1 mm2 for the scanning electron microscopic (SEM) studies. The specimens were fixed in 2.5% glutaraldehyde (10 ml), 4% paraformaldehyde (25 ml), in 0.1 M sodium cacodylate buffer, pH 7.2 (50 ml), for 4 h at room temperature, washed twice in 0.1 M sodium cacodylate buffer (each time 5 min) and postfixed in 2% osmium tetroxide (OsO4 ) for 2 h. The samples were then washed two times in distilled water (each time 5 min). Subsequently, the specimens were dehydrated in a graded series of ethanol. The dehydrated specimens were finally immersed in hexamethyldisilazane for 20 min and air dried under a fume hood. They were then sputter-coated with gold and inspected, using a scanning electron microscope (Cambridge, S360, UK) operating at 20 kV. 2.8. Statistical analysis Descriptive statistics were presented as mean and standard errors of means in each group. Differences between the weight and size of the cysts of different groups were compared by Kruskal–Wallis (nonparametric oneway ANOVA) and Mann–Whitney U tests in SPSS software (version 11.5). The p values less than 0.05 was regarded as statistically significant. 2.9. Ethics This study conformed to the guidelines for the care and use of laboratory animals established by the Ethics
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Committee of Shiraz University. Unnecessary animal suffering was avoided throughout the study. 3. Results HPLC analysis for identification of phenolic compounds from Z. multiflora extract, showed four phenolic compounds including gallic acid (1.1618 mg/g), catechin (2.808 mg/g), caffeic acid (5.531 mg/g), and quercetin (9.961 mg/g). A total of 80 mice were included in this experimental work, however, because of the long term of study, some animals died and were excluded from the study. The survived animals were chosen for statistical analysis. 3.1. Preventive trials Methanolic extract of Z. multiflora (4 g/l in drinking water for 8 months) showed preventive effect on the hydatid cyst formation and development in laboratory mice. The results presented in Table 1 show that hydatid cysts developed in only one mice out of 10 animals (10%), showing the preventive effect of Z. multiflora extract on hydatid cysts development. Furthermore, a significant decrease in the cysts’ weights and also cysts’ sizes compared to the untreated control group was obtained in the Z. multiflora treatment group (p < 0.05). The preventive effect of Z. multiflora extract on development of hydatid cysts was almost comparable with albendazole (150 mg/kg BW/day) and no statistical differences were observed in the cyst weights and also cyst sizes between treatment with Z. multiflora extract and albendazole (p > 0.05). The ultrastructural appearance of the germinal layer after scanning electron microscopic (SEM) analysis of the cysts recovered from the untreated and Z. multiflora treated mice are shown in Fig. 1. SEM analysis of the cysts recovered from the untreated control group revealed typical features of E. granulosus metacestodes, with distinct laminated and germinal layers without alterations (Fig. 1). In contrast, the germinal layer of the cysts recovered from the Z. multiflora extract treated mice was markedly altered and showed distorted morphology and partial lysis of the germinal layer-associated tissue (Fig. 1). 3.2. Therapeutic trials The methanolic extract of Z. multiflora (8 g/l in drinking water for 30 days) showed therapeutic effect on the hydatid cyst. The results presented in Table 2 show that Z. multiflora extract had a negative effect on the hydatid cysts development. A significant decrease on the cyst weights and also cyst sizes compared to the untreated control group was obtained in Z. multiflora treatment group (p < 0.05). The therapeutic effect of Z. multiflora extract on hydatid cysts was almost comparable with albendazole and no statistical differences were observed in the cysts’ weights and also cysts’ sizes between the cysts recovered from the mice treated with Z. multiflora extract and albendazole (p > 0.05). The ultrastructural appearance of the germinal layer after scanning electron microscopic (SEM) analysis of the cysts recovered from the untreated and treated with
Please cite this article in press as: Moazeni, M., et al., Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.006
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Table 1 The effect of the methanolic extract of Z. multiflora (4 g/l in drinking water for 8 months) and albendazole (150 mg/kg BW/day for 10 days) on the prevention of hydatid cyst formation in laboratory mice. Group Z. multiflora extract Albendazole Control *
No. of mice investigated 10 8 7
No. of infected mice 1 (10%) 0 (0%) 5 (71%)
Total number of cysts
Mean cysts weight (g) b*
1 0 13
0.009 0b 0.22 ± 0.10a
Mean cysts size (mm) 0.30 ± 0.30b 0b 5.81 ± 1.93a
Different letters show significant difference in each column.
Table 2 The effect of themethanolic extract of Z. multiflora (8 g/l in drinking water for 30 days) and albendazole (300 mg/kg BW/day for 20 days) on the treatment of hydatid cyst in laboratory mice. Group
No. of mice investigated
No. of infected mice
Total number of cysts
Mean cysts weight (g)
Mean cysts size (mm)
Z. multiflora extract Albendazole Control
5 5 5
4 4 5
8 8 12
0.04 ± 0.02b * 0.03 ± 0.03b 0.56 ± 0.06a
2.95 ± 1.07b 2.42 ± 0.65b 6.83 ± 1.04a
*
Different letters show significant difference in each column.
Z. multiflora are shown in Fig. 1. SEM analysis of the cysts recovered from the mice treated with albendazole and Z. multiflora extract showed a completely damaged germinal layer. 4. Discussion Benzimidazole compounds are the most therapeutic agents that have been used for chemical treatment of hydatidosis. Oxfendazole (Blanton et al., 1998), mebendazole (Anadol et al., 2001; Daniel-Mwuambete et al., 2003) and albendazole (Ingold et al., 1999; Falagas and Bliziotis, 2007) have been used for the treatment of hydatidosis. There are several limitations for the formulation of benzimidazole compounds including recurrence of parasite growth (Stettler et al., 2003). Clinically, echinococcosis is most commonly treated with albendazole. However, poor absorption in the intestinal tract, low levels of hepatic concentration, high cost, severe side effects such as liver toxicity, and resistance are some limitations for albendazole (Manterola et al., 2005; Polat et al., 2005; Abu-Eshy, 2006; Ma et al., 2007; Dziri et al., 2009). Combination of albendazole and other scolicidal agents have recently been tried against hydatid cyst, in several in vivo studies, and higher antiparasitic effect have been reported compared to the results with albendazole alone. Higher antiparasitic effect has been reported following the use of albendazole in combination with alkaloids from Sophora moorcroftiana seeds (Ma et al., 2007), dihydroartemisinin and artesunate (Spicher et al., 2008a), 2-methoxyestradiol (Spicher et al., 2008b) and Trametes robiniophila aqueous extract (Lv et al., 2013). We have previously demonstrated the in vitro scolicidal effect of several medicinal plant extract or essential oils and found that Z. multiflora was the most effective agent against protoscoleces of hydatid cyst in vitro (Moazeni and Nazer, 2010, 2011; Moazeni et al., 2012a,b; Moazeni and Mohseni, 2012; Moazeni and Roozitalab, 2012). In the present study, we investigated the effect of this extract on the germinal layer cells of hydatid cyst in a murine model. The results of our study showed that the methanolic extract of Z. multiflora (4 g/l in drinking water for 8 months) prevented formation and development of the
hydatid cyst in laboratory mice and this preventive effect was comparable with the preventive effect of albendazole (150 mg/kg BW/day for 10 days). Furthermore, the results of the present study also showed that the methanolic extract of Z. multiflora (8 g/l in drinking water for 30 days) has therapeutic effect on the hydatid cyst. The therapeutic effect of Z. multiflora extract on hydatid cysts was almost comparable with albendazole. In the present study, we found an obvious decrease in the weight and size of the hydatid cysts recovered from the infected mice following oral administration of Z. multiflora extract in the drinking water of these animals. Our findings are well correlated with the scanning electron microscopic analyses, confirming that in vivo exposure of hydatid cyst to Z. multiflora extract resulted in profound tissue alterations and loss of the characteristic multicellular structure of the germinal layer. The scanning ultramicrographs demonstrated that after administration of the methanolic extract of Z. multiflora, in both preventive and therapeutic trials, the germinal layer of the hydatid cysts was dramatically damaged. Stettler et al. (2003) reported vacuolization of the germinal layer, and separation of the laminated and germinal layers after administration of nitazoxanide, for alveolar hydatid cysts in SEM analysis in an in vitro study. Distorted morphology and partial lysis of the germinal layer-associated tissue have been reported by Spicher et al. (2008b) in SEM analysis of hydatid cysts recovered from the mice treated with 2 methoxyestradiol for 7 days. The killing effect of thymol, a phenolic compound, on the germinal layer of murine hydatid cysts has recently been reported, in an in vitro study by SEM analysis (Elissondo et al., 2013). In agreement with the previous studies (Stettler et al., 2003; Spicher et al., 2008b; Elissondo et al., 2013) we observed clear changes in the wall of hydatid cysts such as alterations in the germinal layer, massive signs of cellular destruction, detachment of germinal layer from the laminated layer, presence of large amount of cellular debris and complete absence of germinal layer in the cysts of the Z. multiflora extract treated animals. The germinal layer can contain viable protoscoleces (Adas et al., 2009). Moreover, the embryonic or stem cells, present in the germinal layer, have the potential to develop
Please cite this article in press as: Moazeni, M., et al., Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.006
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multiflora is an edible plant; therefore, it is well adapted to animals and human nature. Z. multiflora is safe and nonpathogenic on the fetus digestive system when used in pregnant BALB/c mice (Mokhberi et al., 2007). Furthermore, this herbal plant can remarkably stimulate innate and acquired immunity function in experimental animals and it may be used as an immunostimulatory agent (Shokri et al., 2006; Khosravi et al., 2007). The results of the present study revealed that this herbal plant which belongs to Lamiaceae or mint family, is rich in bioactive natural components such as quercetin, caffeic acid, catechin and gallic acid. These compounds may be responsible for its biological effects. It has also been reported that some plants belonging to the same family, such as Satureja khuzestanica (Moazeni et al., 2012b) and Origanum vulgare (Anthony et al., 2005) have shown strong anti parasitic activity. It has been shown that phenolic compounds have antiseptic, antibacterial, antifungal, antiparasitic as well as anti-noceceptive properties (Hajhashemi et al., 2002). The mode of action of the phenolic components has been studied in the literature. For example, Ultee et al. (2002) stated that the hydroxyl group and the presence of a system of delocalized electrons are important for the antimicrobial activity of phenolic compounds. Such a particular structure would allow the compounds to act as proton exchangers, thereby reducing the gradient across the cytoplasmic membrane. The resulting collapse of the proton motrice force and depletion of the ATP pool lead to eventual cell death. 5. Conclusions We found the methanolic extract of Z. multiflora is not only effective in the prevention of hydatid cyst formation and development, it is also effective in the treatment of hydatidosis in the infected mice. Z. multiflora is an edible plant and immunostimulatory agent, thus it may be safely used as an anti hydatid cyst agent. However, further studies will be necessary to determine the feasibility of developing an effective drug for the treatment of hydatidosis in human cases, using this traditional medicine. Acknowledgments
Fig. 1. Scanning electron micrographs showing the ultrastructure of the hydatid cysts recovered from the untreated (A) and treated (B and C) mice. Ultramicrograph (A) shows normal structure of the germinal layer. Ultramicrographs ((B) and (C)) show the ultrastructure of the hydatid cysts recovered from the mice treated with Zataria multiflora extract in the preventive (B) and therapeutic (C) groups, respectively. Note the distorted morphology and partial lysis of the germinal layer in ultramicrographs ((B) and (C)) (1000×, gl: germinal layer, ll: laminar layer).
new protoscoleces or brood capsules (Menezes da Silva, 2011). Therefore, to eliminate the hydatid cyst, the germinal layer should be destroyed. Our findings by scanning electron microscopy confirmed the destructive effect of Z. multiflora extract on the germinal layer of hydatid cyst. Z.
The authors would like to acknowledge the Research Councils of Shiraz University and NACE (Natural Antimicrobial Center of Excellence, Grant no. 87-GR-VT-24) for their financial supports. References Adas, G., Arikan, S., Kemik, O., Oner, A., Sahip, N., Karatepe, O., 2009. Use of albendazole sulfoxide albendazole sulfone, and combined solutions as scolicidal agents on hydatid cysts (in vitro study). World J. Gastroenterol. 15, 112–116. Abdollahy, F., Ziaei, H., Shabankhani, B., Azadbakht, M., 2004. Effect of essential oils of Artemisia aucheri Boiss. Zataria multiflora Boiss, and Myrtus communis L. on Trichomonas vaginalis. Iran. J. Pharm. Res. 2 (Suppl. 2), 35. Abu-Eshy, S.A., 2006. Clinical characteristics, diagnosis and surgical management of hydatid cysts. West Afr. J. Med. 25, 144–152. Anadol, D., Ozc¸elik, U., Kiper, N., Göc¸men, A., 2001. Treatment of hydatid disease. Pediatr. Drugs 3, 123–135.
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Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study Mohammad Moazeni a,∗ , Sara Larki b , Ahmad Oryan c , Mohammad Jamal Saharkhiz d a b c d
Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran School of Veterinary Medicine, Shiraz University, Shiraz, Iran Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Department of Horticulture Science, College of Agriculture, Shiraz, Iran
a r t i c l e
i n f o
Article history: Received 18 December 2013 Received in revised form 13 June 2014 Accepted 1 July 2014 Keywords: Preventive Therapeutic Methanolic extract Zataria multiflora Hydatid cyst In vivo
a b s t r a c t The phenolic compounds of Zataria multiflora extract, were identified by HPLC analysis. Gallic acid, catechin, caffeic acid, and quercetin were found to be the major phenolic compounds. Eighty healthy laboratory Balb/C mice were infected intraperitoneally by injection of 1500 viable protoscoleces and were divided into prevention (40 mice) and therapeutic (40 mice) groups. To prove the preventive effect of Z. multiflora extract on development of hydatid cyst, the 40 infected animals were allocated into three treatment groups including Z. multiflora (4 g/l in drinking water for 8 months), albendazole (150 mg/kg BW/day for 10 days) and untreated (control) group. To estimate the therapeutic effect of Z. multiflora extract on the hydatid cyst, after 8 months of infection, the infected mice were allocated into three experimental treatment groups including Z. multiflora (8 g/l in drinking water for 30 days), albendazole (300 mg/kg BW/day for 20 days) and untreated (control) group. At the end of the treatment period, all mice were euthanized and necropsied, the hydatid cysts were carefully removed, weighed and their size were recorded. Weight and size of the hydatid cysts significantly decreased (p < 0.05) upon the treatment with Z. multiflora extract in both prevention and therapeutic groups. The germinal layer of the hydatid cysts recovered from the treated mice, either from the prevention or therapeutic group, were completely damaged at ultrastructural level by scanning electron microscopy. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Echinococcosis/hydatidosis is a zoonotic disease that occurs throughout the world and causes considerable economic losses and public health problems in many countries (Dalimi et al., 2002). Echinococcosis is mostly prevalent in Australia, South America, Middle East, South Africa, Eastern Europe, and the Mediterranean region (Zulfikaroglu
∗ Corresponding author. E-mail address: [email protected] (M. Moazeni).
et al., 2008) and immigration between continents, has led to the disease being prevalent in other countries as well (Sayek and Onat, 2001). Cystic echinococcosis (CE), which is considered as an emerging disease in various regions, is economically important and constitutes a threat to public health in many countries (Dinkela et al., 2004). Human infection with Echinococcus granulosus typically results in a slowly growing parasitic cystic disease most frequently seen in the liver. The cysts may be asymptomatic for many years, and occasionally spontaneous regression has been noted. More commonly the disease is slowly progressive, and symptoms and complications eventually arise. The
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symptoms include pain from expansion or rupture, fever from pyogenic infection due to intrabiliary rupture and jaundice, or anaphylaxis from intrabiliary or extrahepatic rupture (Sielaff et al., 2001). Currently, there are three treatment options for hydatid disease of the liver. These treatment strategies include: surgery, percutaneous aspiration and medical treatment from which surgery has been known as the most efficient treatment option in humans (Adas et al., 2009). Chemotherapy is the preferred treatment where, surgeons are not available or the cysts are too numerous, and in inoperable cases, “chemotherapy” is the only option. Chemotherapy has also been used as an adjunct to surgery for prophylaxis against spillage of the cyst contents (Blanton et al., 1998; Spicher et al., 2008b). Few chemotherapeutic agents are available for the medical management of hydatid disease (Blanton et al., 1998). Benzimidazole carbamate derivatives, such as mebendazole and albendazole, are currently used for chemotherapeutic treatment of cystic echinococcosis. In human patients, benzimidazoles are commonly applied in high doses and for extended periods of time; these frequently results in adverse side effects (Walker et al., 2004). Therefore, a new effective alternative treatment regime is extremely important in today’s climate, where species are becoming resistant, 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). It has been shown that Zataria multiflora (Lamiaceae) has antibacterial (Sharififar et al., 2007; Misaghi and Akhondzadeh Basti, 2007), antifungal (Gandomi et al., 2009), antiprotozoal (Abdollahy et al., 2004) and scolicidal (Moazeni and Roozitalab, 2012) properties. The aim of the current experimental work was to evaluate the efficacy of the methanolic extract of Z. multiflora on the prevention and treatment of hydatid cyst in a murine model.
2.2. Identification of phenolic compounds For identification of the phenolic compounds from the Z. multiflora extract, HPLC analysis was carried out on a Agilent 1200 series (USA), equipped with a Zorbax Eclipse XDB-C18 column (10 cm × 5 m i.d.; ×150 mm film thickness, RP), and a photodiode array detector (PAD). For preparing the injectable extract, 0.02 g of the dried residue of the plant extract was dissolved in 2 ml of methanol–acetic acid (85:15) and the aliquots was filtered through a 0.2 m membrane millipore chromatographic filter and injected into the HPLC system. The elution was monitored at 280 and 320 nm. Gradient elution was selected to achieve the maximum separation and sensitivity. The elution was performed by varying the proportion of solvent A (formic acid 1% in deionized water) to solvent B (methanol (v/v)) as follows: methanol:formic acid 1% (10:90), at 0 min; methanol:formic acid 1% (25:75), at 10 min; methanol:formic acid 1% (60:40), at 20 min and finally, methanol:formic acid 1% (70:30), at 30 min. The total running time was 30 min. The column temperature was 30 ◦ C. 2.3. Collection of protoscoleces Protoscoleces of E. granulosus were collected aseptically from the hydatid cysts of the liver of the naturally infected sheep, slaughtered at Shiraz Slaughterhouse, Shiraz, southern Iran. The hydatid fluid of the cysts was aseptically transferred into glass cylinders and left to set for 30 min. The protoscoleces were settled at the bottom of the cylinders. The supernatant was then removed, and the yielded protoscoleces were washed several times with sterile 0.85% NaCl and stored in RPMI 1640 medium overnight at 37 ◦ C. The viability of the protoscoleces was confirmed from their motility characteristics under an ordinary light microscope after 0.1% eosin staining. They were finally transferred into a dark container containing normal saline and stored at 4 ◦ C for further use.
2. Materials and methods
2.4. Infection of mice
2.1. Extraction
Eighty healthy BALB/c male mice weighing 22–24 g were used in this study. All the mice were infected intraperitoneally by injection of 1500 protoscoleces per animal, dissolved in 0.5 ml of RPMI 1640 medium. The infected animals were allocated into two experimental treatment groups: (a) Prevention group, (b) Therapy group. All the animals in the study were fed ad libitum, and kept at 24–25 ◦ C.
Methanolic extract of Z. multiflora was prepared as described by Moazeni and Roozitalab (2012) with some modifications. Briefly, the leaves of Z. multiflora were dried under shade and powdered mechanically, using a commercial electric blender. A total of 2800 g of dried powder was extracted. One hundred grams of dry powder was added to 400 ml of pure methanol and gently mixed, using a shaking incubator, at 30–37 ◦ C for 1 h at 120–130 rpm. The obtained solution was left at room temperature for 24 h. The solution was stirred again and filtered, and the solvent was removed by evaporation in a rotary evaporator. The remaining semisolid material was then left at room temperature for 4 h and then freeze dried. The collected residue was transferred to a sterile glass container and stored in the refrigerator at 4 ◦ C before being used. 119 g of the dried extract was obtained from 2800 g of the dried powder of Z. multiflora.
2.5. Preventive trials To prove the preventive effect of Z. multiflora extract on the formation of hydatid cysts, the 40 infected animals were allocated into three experimental treatment groups: (a) Z. multiflora treatment group which were drunk with Z. multiflora extract (4 g/l) for 8 months (15 mice), (b) albendazole treatment group which received 150 mg/kg BW/day albendazole for 10 days (10 mice) and (c) untreated control group (15 mice). The remaining 25 mice were euthanized 8
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months after infection and necropsy was carried out immediately thereafter. 2.6. Therapeutic trials To evaluate the therapeutic effects of Z. multiflora extract on the hydatid cysts in the animals that were infected 8 months ago, the remaining 15 infected mice were allocated into three experimental treatment groups, five animal in each: (a) Z. multiflora treatment group which were drunk with 8 g/l of Z. multiflora extract for 30 days, (b) albendazole treatment group which received 300 mg/kg BW/day albendazole for 20 days and (c) untreated control group. All the mice were euthanized at the end of the treatment period and necropsy was carried out immediately thereafter. At necropsy, the peritoneal cavity was opened and the internal organs were observed for hydatid cyst. The hydatid cysts were carefully removed. Sizes of the cysts were measured using a scaled ruler and adobe Photoshop CS6. Each cyst’s weight was recorded using a digital scale (Kern & Sohn GmbH, D-72336 Balingen, Germany). The efficacy of Z. multiflora extract was evaluated by comparing the cysts numbers, sizes and weights and also according to the ultrastructural morphological changes between the three treatment groups. 2.7. Scanning electron microscopy The cysts were sectioned to blocks of approximately 1 mm2 for the scanning electron microscopic (SEM) studies. The specimens were fixed in 2.5% glutaraldehyde (10 ml), 4% paraformaldehyde (25 ml), in 0.1 M sodium cacodylate buffer, pH 7.2 (50 ml), for 4 h at room temperature, washed twice in 0.1 M sodium cacodylate buffer (each time 5 min) and postfixed in 2% osmium tetroxide (OsO4 ) for 2 h. The samples were then washed two times in distilled water (each time 5 min). Subsequently, the specimens were dehydrated in a graded series of ethanol. The dehydrated specimens were finally immersed in hexamethyldisilazane for 20 min and air dried under a fume hood. They were then sputter-coated with gold and inspected, using a scanning electron microscope (Cambridge, S360, UK) operating at 20 kV. 2.8. Statistical analysis Descriptive statistics were presented as mean and standard errors of means in each group. Differences between the weight and size of the cysts of different groups were compared by Kruskal–Wallis (nonparametric oneway ANOVA) and Mann–Whitney U tests in SPSS software (version 11.5). The p values less than 0.05 was regarded as statistically significant. 2.9. Ethics This study conformed to the guidelines for the care and use of laboratory animals established by the Ethics
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Committee of Shiraz University. Unnecessary animal suffering was avoided throughout the study. 3. Results HPLC analysis for identification of phenolic compounds from Z. multiflora extract, showed four phenolic compounds including gallic acid (1.1618 mg/g), catechin (2.808 mg/g), caffeic acid (5.531 mg/g), and quercetin (9.961 mg/g). A total of 80 mice were included in this experimental work, however, because of the long term of study, some animals died and were excluded from the study. The survived animals were chosen for statistical analysis. 3.1. Preventive trials Methanolic extract of Z. multiflora (4 g/l in drinking water for 8 months) showed preventive effect on the hydatid cyst formation and development in laboratory mice. The results presented in Table 1 show that hydatid cysts developed in only one mice out of 10 animals (10%), showing the preventive effect of Z. multiflora extract on hydatid cysts development. Furthermore, a significant decrease in the cysts’ weights and also cysts’ sizes compared to the untreated control group was obtained in the Z. multiflora treatment group (p < 0.05). The preventive effect of Z. multiflora extract on development of hydatid cysts was almost comparable with albendazole (150 mg/kg BW/day) and no statistical differences were observed in the cyst weights and also cyst sizes between treatment with Z. multiflora extract and albendazole (p > 0.05). The ultrastructural appearance of the germinal layer after scanning electron microscopic (SEM) analysis of the cysts recovered from the untreated and Z. multiflora treated mice are shown in Fig. 1. SEM analysis of the cysts recovered from the untreated control group revealed typical features of E. granulosus metacestodes, with distinct laminated and germinal layers without alterations (Fig. 1). In contrast, the germinal layer of the cysts recovered from the Z. multiflora extract treated mice was markedly altered and showed distorted morphology and partial lysis of the germinal layer-associated tissue (Fig. 1). 3.2. Therapeutic trials The methanolic extract of Z. multiflora (8 g/l in drinking water for 30 days) showed therapeutic effect on the hydatid cyst. The results presented in Table 2 show that Z. multiflora extract had a negative effect on the hydatid cysts development. A significant decrease on the cyst weights and also cyst sizes compared to the untreated control group was obtained in Z. multiflora treatment group (p < 0.05). The therapeutic effect of Z. multiflora extract on hydatid cysts was almost comparable with albendazole and no statistical differences were observed in the cysts’ weights and also cysts’ sizes between the cysts recovered from the mice treated with Z. multiflora extract and albendazole (p > 0.05). The ultrastructural appearance of the germinal layer after scanning electron microscopic (SEM) analysis of the cysts recovered from the untreated and treated with
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Table 1 The effect of the methanolic extract of Z. multiflora (4 g/l in drinking water for 8 months) and albendazole (150 mg/kg BW/day for 10 days) on the prevention of hydatid cyst formation in laboratory mice. Group Z. multiflora extract Albendazole Control *
No. of mice investigated 10 8 7
No. of infected mice 1 (10%) 0 (0%) 5 (71%)
Total number of cysts
Mean cysts weight (g) b*
1 0 13
0.009 0b 0.22 ± 0.10a
Mean cysts size (mm) 0.30 ± 0.30b 0b 5.81 ± 1.93a
Different letters show significant difference in each column.
Table 2 The effect of themethanolic extract of Z. multiflora (8 g/l in drinking water for 30 days) and albendazole (300 mg/kg BW/day for 20 days) on the treatment of hydatid cyst in laboratory mice. Group
No. of mice investigated
No. of infected mice
Total number of cysts
Mean cysts weight (g)
Mean cysts size (mm)
Z. multiflora extract Albendazole Control
5 5 5
4 4 5
8 8 12
0.04 ± 0.02b * 0.03 ± 0.03b 0.56 ± 0.06a
2.95 ± 1.07b 2.42 ± 0.65b 6.83 ± 1.04a
*
Different letters show significant difference in each column.
Z. multiflora are shown in Fig. 1. SEM analysis of the cysts recovered from the mice treated with albendazole and Z. multiflora extract showed a completely damaged germinal layer. 4. Discussion Benzimidazole compounds are the most therapeutic agents that have been used for chemical treatment of hydatidosis. Oxfendazole (Blanton et al., 1998), mebendazole (Anadol et al., 2001; Daniel-Mwuambete et al., 2003) and albendazole (Ingold et al., 1999; Falagas and Bliziotis, 2007) have been used for the treatment of hydatidosis. There are several limitations for the formulation of benzimidazole compounds including recurrence of parasite growth (Stettler et al., 2003). Clinically, echinococcosis is most commonly treated with albendazole. However, poor absorption in the intestinal tract, low levels of hepatic concentration, high cost, severe side effects such as liver toxicity, and resistance are some limitations for albendazole (Manterola et al., 2005; Polat et al., 2005; Abu-Eshy, 2006; Ma et al., 2007; Dziri et al., 2009). Combination of albendazole and other scolicidal agents have recently been tried against hydatid cyst, in several in vivo studies, and higher antiparasitic effect have been reported compared to the results with albendazole alone. Higher antiparasitic effect has been reported following the use of albendazole in combination with alkaloids from Sophora moorcroftiana seeds (Ma et al., 2007), dihydroartemisinin and artesunate (Spicher et al., 2008a), 2-methoxyestradiol (Spicher et al., 2008b) and Trametes robiniophila aqueous extract (Lv et al., 2013). We have previously demonstrated the in vitro scolicidal effect of several medicinal plant extract or essential oils and found that Z. multiflora was the most effective agent against protoscoleces of hydatid cyst in vitro (Moazeni and Nazer, 2010, 2011; Moazeni et al., 2012a,b; Moazeni and Mohseni, 2012; Moazeni and Roozitalab, 2012). In the present study, we investigated the effect of this extract on the germinal layer cells of hydatid cyst in a murine model. The results of our study showed that the methanolic extract of Z. multiflora (4 g/l in drinking water for 8 months) prevented formation and development of the
hydatid cyst in laboratory mice and this preventive effect was comparable with the preventive effect of albendazole (150 mg/kg BW/day for 10 days). Furthermore, the results of the present study also showed that the methanolic extract of Z. multiflora (8 g/l in drinking water for 30 days) has therapeutic effect on the hydatid cyst. The therapeutic effect of Z. multiflora extract on hydatid cysts was almost comparable with albendazole. In the present study, we found an obvious decrease in the weight and size of the hydatid cysts recovered from the infected mice following oral administration of Z. multiflora extract in the drinking water of these animals. Our findings are well correlated with the scanning electron microscopic analyses, confirming that in vivo exposure of hydatid cyst to Z. multiflora extract resulted in profound tissue alterations and loss of the characteristic multicellular structure of the germinal layer. The scanning ultramicrographs demonstrated that after administration of the methanolic extract of Z. multiflora, in both preventive and therapeutic trials, the germinal layer of the hydatid cysts was dramatically damaged. Stettler et al. (2003) reported vacuolization of the germinal layer, and separation of the laminated and germinal layers after administration of nitazoxanide, for alveolar hydatid cysts in SEM analysis in an in vitro study. Distorted morphology and partial lysis of the germinal layer-associated tissue have been reported by Spicher et al. (2008b) in SEM analysis of hydatid cysts recovered from the mice treated with 2 methoxyestradiol for 7 days. The killing effect of thymol, a phenolic compound, on the germinal layer of murine hydatid cysts has recently been reported, in an in vitro study by SEM analysis (Elissondo et al., 2013). In agreement with the previous studies (Stettler et al., 2003; Spicher et al., 2008b; Elissondo et al., 2013) we observed clear changes in the wall of hydatid cysts such as alterations in the germinal layer, massive signs of cellular destruction, detachment of germinal layer from the laminated layer, presence of large amount of cellular debris and complete absence of germinal layer in the cysts of the Z. multiflora extract treated animals. The germinal layer can contain viable protoscoleces (Adas et al., 2009). Moreover, the embryonic or stem cells, present in the germinal layer, have the potential to develop
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multiflora is an edible plant; therefore, it is well adapted to animals and human nature. Z. multiflora is safe and nonpathogenic on the fetus digestive system when used in pregnant BALB/c mice (Mokhberi et al., 2007). Furthermore, this herbal plant can remarkably stimulate innate and acquired immunity function in experimental animals and it may be used as an immunostimulatory agent (Shokri et al., 2006; Khosravi et al., 2007). The results of the present study revealed that this herbal plant which belongs to Lamiaceae or mint family, is rich in bioactive natural components such as quercetin, caffeic acid, catechin and gallic acid. These compounds may be responsible for its biological effects. It has also been reported that some plants belonging to the same family, such as Satureja khuzestanica (Moazeni et al., 2012b) and Origanum vulgare (Anthony et al., 2005) have shown strong anti parasitic activity. It has been shown that phenolic compounds have antiseptic, antibacterial, antifungal, antiparasitic as well as anti-noceceptive properties (Hajhashemi et al., 2002). The mode of action of the phenolic components has been studied in the literature. For example, Ultee et al. (2002) stated that the hydroxyl group and the presence of a system of delocalized electrons are important for the antimicrobial activity of phenolic compounds. Such a particular structure would allow the compounds to act as proton exchangers, thereby reducing the gradient across the cytoplasmic membrane. The resulting collapse of the proton motrice force and depletion of the ATP pool lead to eventual cell death. 5. Conclusions We found the methanolic extract of Z. multiflora is not only effective in the prevention of hydatid cyst formation and development, it is also effective in the treatment of hydatidosis in the infected mice. Z. multiflora is an edible plant and immunostimulatory agent, thus it may be safely used as an anti hydatid cyst agent. However, further studies will be necessary to determine the feasibility of developing an effective drug for the treatment of hydatidosis in human cases, using this traditional medicine. Acknowledgments
Fig. 1. Scanning electron micrographs showing the ultrastructure of the hydatid cysts recovered from the untreated (A) and treated (B and C) mice. Ultramicrograph (A) shows normal structure of the germinal layer. Ultramicrographs ((B) and (C)) show the ultrastructure of the hydatid cysts recovered from the mice treated with Zataria multiflora extract in the preventive (B) and therapeutic (C) groups, respectively. Note the distorted morphology and partial lysis of the germinal layer in ultramicrographs ((B) and (C)) (1000×, gl: germinal layer, ll: laminar layer).
new protoscoleces or brood capsules (Menezes da Silva, 2011). Therefore, to eliminate the hydatid cyst, the germinal layer should be destroyed. Our findings by scanning electron microscopy confirmed the destructive effect of Z. multiflora extract on the germinal layer of hydatid cyst. Z.
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Please cite this article in press as: Moazeni, M., et al., Preventive and therapeutic effects of Zataria multiflora methanolic extract on hydatid cyst: An in vivo study. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.07.006