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Antihydatic and immunomodulatory effects of Algerian propolis ethanolic extract: In vitro and in vivo study

Objective: To evaluate the in vitro and in vivo effect of the Algerian propolis ethanolic extract (EEP) against Echinococcus granulosus (E. granulosus) infection. Methods: In vitro scolicidal activity of EEP was investigated on the protoscolices of hydatid cyst. This in vitro study was conducted by using an in vivo assay. BALB/c mice were inoculated with E. granulosus and treated with propolis for three months. Hydatid cysts development was assessed. Nitric oxide (NO), tumor necrosis factor alpha (TNF-α) production and inducible NO synthase, NF-κB, and TNF-α spleen expression were estimated by Griess method and immunofluorescence respectively. Results: Our study revealed that EEP has a high scolicidal activity against E. granulosus. Oral administration of EEP decreased TNF-α, NF-κB and inducible NO synthase expression in the spleen tissues in the CE+EEP group, in comparison with the CE group. Concomitantly, EEP treatment caused an important systemic decrease in NO and TNF-α levels. These findings are associated with the reduction of CE development. Conclusions: This is the first report demonstrating with interest the antihydatic and immunomodulatory effects of the Algerian EEP, suggesting its therapeutic potential for the hydatid disease treatment.


Introduction
Cystic echinococcosis (CE) is a major zoonotic infection that affects humans and domestic animals. It is caused by Echinococcus granulosus (E. granulosus) which is a metacestode belonging to the family of Taeniidae [1,2]. Cystic echinococcosis is still endemic and considered as a severe public health concern in many regions of the world, as is the case in Algeria [3]. This parasitic disease manifests frequently as unilocular hydatid cyst(s) which can develop in many organs including liver, lung, and occasionally, kidney, spleen, bone, heart and brain [4]. The clinical evolution of these cysts is slow and in some cases asymptomatic [1].
Duration and intensity of infection as well as the variety of immunological responses to parasitic epitopes are the major factors that influence the severity and variability of the clinical expression of CE [5][6][7].
Because of its high efficiency, surgery is still the preferred CE treatment method. However, in many cases, it is dangerous or sometimes impossible. It also increases the risk of scolices dissemination during the surgical operation which contributes to recurrence [1,8]. Chemotherapy using benzimidazole compounds such as albendazole and mebendazole indicated in inoperable cysts and complementarily with cyst puncture, aspiration, injection of chemicals as well as respiration, and surgery treatment were subsequently introduced [2]. However, due to their different side effects, the application of these drugs has become controversial and limited [9,10]. Therefore, enormous efforts and investments have been spent attempting to discover, especially from natural resources, alternative treatment strategies and new anti-hydatid compounds which have low or no adverse effects and high potency for hydatid cyst surgery [11][12][13].
In this context, we are interested in propolis which is a resinous hive product, composed of exudates of trees mixed by honey bees with enzymes, beeswax and pollen [14]. This substance is used by bees to fill gaps, cover hive walls and mummify the carcasses of external invader insects [15]. Its composition may vary qualitatively and quantitatively according to its harvest [16], the different plant sources and the collection season [17,18]. Basically, resins and balsams are the major constituents of crude propolis (50%). Other substances such as wax (30%), aromatic oils (10%), pollen (5%) and mineral and organic matter (5%) are found [19]. Till date, more than 300 chemical compounds have been identified in propolis, guaranteeing its curative effects. Among them are phenolic and flavonoic compounds [20].
In the present work, we investigated the antihydatic and immunomodulatory effects of Algerian propolis ethanolic extract (EEP) in an experimental cystic echinococcosis. In this sense, the in vitro effects of EEP on the protoscoleces of E. granulosus (PSCs) viability and the hydatid cysts development in BALB/c mice were evaluated. In addition, TNF-α, NF-κB and iNOS expression in the spleen was analyzed and NO and TNF-α levels were determined.

Preparation of ethanolic propolis extract
Propolis sample was harvested in May in El Bayadh (North-West of Algeria) by scraping it off from the frames of Apis mellifera hives.
Propolis was extracted following the protocol described by Baltas with some modifications [31].

Phenolic and flavonoid total contents
The phenolic total content in our propolis sample was determined based on the Folin-Ciocalteu assay as previously described by Zongo with slight changes [32]. Briefly, 0.

Evaluation of antioxidant capacity of EEP
The antioxidant capacity of our extract was evaluated using the 2,2diphenyl-1-picrylhydrazyl (DPPH) free radical method, as previously described by Wang, with slight modifications [34]. The ethanolic propolis extract was prepared at several concentrations (0.6 μg/mL to 500.0 μg/mL) in the methanol and mixed with the DPPH methanol solution (0.2 mM). The reaction mixture was gently homogenized with a vortex and incubated in the dark for 15 min. Using methanol as a blank, the optical density was measured at 517 nm. Quercetin and vitamin C were used as standards.
The scavenging rate was calculated according to the following equation: A 0 =the DPPH absorbance (without the EEP sample).
A 1 =the DPPH absorbance when the EEP sample is present.
Results were expressed as IC 50 value that was calculated using the relationship curve between the scavenging activities (%) and the respective sample concentrations.

Protoscolex collection
The

Acute toxicity study
Fifty healthy BALB/c female mice were split randomly into 5 groups (10 mice per group). All mice were given access to water while food was withheld for overnight prior to the treatment. They were given the ethanolic propolis extract at 1 000, 2 000, 3 000 and 4 000 mg/kg body weight concentrations while normal control group received hydroalcoholic solution at 20% used as gavage vehicle. The survival of mice as well as side effects were observed for 14 d.

Therapeutic trials
To estimate the Algerian EEP therapeutic effects during experimental echinococcosis, the animals were assigned randomly During this time period, food and water intakes were daily recorded.
A vehicle control (Ethanol at 2%) was confirmed to be not toxic.

Assessment of cyst development
At the treatment period's end, the weight of cysts taken from the peritoneal cavity was measured. The following formula was used to determine the inhibition rate and the larval growth: [(average cysts weight of CE group-average cysts weight of CE+EEP group)/average cysts weight of CE group]×100.

Serum collection and nitrite measurement
After 3 months of infection, all the mice were anesthetized. The blood samples collected via cardiac puncture and put in tubes without anticoagulant were centrifuged to obtain serum, which was used for measurement of nitrite.
Nitric oxide production in serum was evaluated via the measurement of nitrites using Griess reaction. In brief, a volume of 50 μL of serum was added to 50 μL of Griess reagent (5% sulfanilamide and 0.5% napthylethylenediamine dihydrochloride dissolved in 20% HCl) and incubated for 20 min in obscurity.
After incubation, the optical density was read at 543 nm and nitrite concentration was calculated using a standard curve performed with sodium nitrite [NaNO 2 ; (0-200) μmoL/mL].

Systemic cytokine levels measurement
The TNF-α levels in serum of mice were analyzed by using enzyme-linked immunosorbent assay (ELISA) kit (Sigma-Aldrich) according to the manufacturer's specifications.

Statistical analysis
To express the difference among the groups, our results were analyzed by one-way analysis of variance (ANOVA) or t-test.
GraphPad Prism 6.01 (GraphPad Inc.) and ImageJ softwares were used to perform statistical analyses and quantitative analyses of the immunofluorescence staining, respectively. Data are presented as means±SD and P values of less than 0.05 were accepted as statistically significant.

Amount of total phenolic and flavonoid contents
In order to determine the amount of total phenolic and flavonoid

DPPH scavenging activity measurement
Phenolic compounds such as flavonoid and phenolic acids, because of their hydroxyl groups, are known for their antioxidant activity [36].
Thus, the extracts activity was evaluated using the DPPH free radical scavenging test. Our propolis sample gave an IC 50

E. granulosus infection model
Three months after the intraperitoneal injection of protoscoleces, hydatid cysts were developed in all the mice with an average of 2.7 (1-7) cysts per mouse. These cysts were macroscopically visible and only located in the peritoneal cavity, primarily observed in the pancreas, liver or free in the peritonea. In our study, the used procedure permitted the infection of 100% of the mice, and a reliable and reproducible mouse infection model was therefore established.

Acute toxicity study
After 14 d of the study, the mice of both the normal control (vehicle) and the treated groups did not show any signs of toxicity and no death cases were observed. Indeed, the mice's appearance and behavior remained normal throughout the experiment duration.
These results indicated that our propolis extract is relatively harmless and its toxic dose could be more than 4 000 mg/kg body weight.

Effect of EPP on clinical parameters of secondary experimental echinococcosis
The weight of developed cysts was measured to assess the effect of propolis treatment at a dose of 100 mg/mL. Our data revealed that EEP intragastric administration to BALB/c mice for three months has a negative effect on the hydatid cysts development. Indeed, the cysts weight decreased significantly in the propolis treated group in comparison with the untreated control group (P=0.000 2).
Propolis treatment inhibited cyst growth by 73.75% in the CE+EEP group; weight (g) of cysts in untreated control water (CE) group and CE+EEP group were (11.280±6.008) g and (2.960±1.814) g respectively (Figure 3).

Effect of EEP on in vivo cytokine production
To investigate the effect of the EEP on proinflammatory cytokine production in vivo during CE, systemic TNF-α concentrations were determined in the serum of mice ( Figure 5). Our results showed that TNF-α levels were increased in CE group compared with the control

Effect of propolis on the expression of iNOS, TNFα and NF-κB in spleen tissue
The effect of propolis on the expression of spleen iNOS, TNF-α and NF-κB/p50, which is evaluated by immunofluerecscence, was shown in Figure 6 and Table 2 Table 2).

Discussion
There is a growing interest to find new agents such as medicinal plant extracts and natural products as alternative options to medicate CE, due to being easily available, inexpensive and safe (low side effects and toxicity) [37]. In this study, we focused on the antihydatic and immunomodulatory effect of EEP during cystic echinococcosis.
Our data demonstrated, for the first time, that administration of hydatid effect of Zataria multiflora [12].
The advantages of propolis versus other herbal scolicidal agents are its antioxidant, immunostimulatory and hepatoprotective properties [44,45]. Additionally, there is little concern regarding the toxic or intensive adverse effects of propolis. Our study of acute toxicity showed that the ethanolic propolis extract had an LD 50 higher than 4 000 mg/kg of mice. Indeed, all the animals had a similar behavior and appearance, and they didn't show any adverse changes throughout the acute toxicity test duration. These results indicated that EEP's safety margin was high as the mice EEP tolerance exceeded 4 000 mg/kg of extract given orally. Arvouet-Grand et al have shown in their study that propolis has a low toxicity in mice with an LD 50 ranging from 2 000 to 7 300 mg/kg [46]. In addition, Mohammadzadeh et al also reported that the use of 20 000 mg/kg dose of propolis extract did not cause any death or toxicity in Wistar rats [47].
In our present work, we also focused on the anti-inflammatory effect of EEP treatment during experimental hydatidosis. We Kupffer and hepatocytes cells of hepatic hydatid cysts of mice [48].
Remarkably It has been shown that propolis has known antioxidant properties and the extracts obtained from this natural product are able to scavenge RL and ROS like hydroxyl radicals and superoxide anions [58]. In addition, the literature reports that the antioxidant effect of propolis could be related to its flavonoids and phenolic content [59].
Our data showed that polyphenolics and flavonoids concentration was high in EPP of Algeria. It also showed a high DPPH free radical scavenging activity with an IC 50 value of 74.29 μg/mL. Several teams have reported a correlation between EEP radical scavenging activity and the concentration of polyphenolic compounds [60,61]. Our results indicated that the propolis obtained from El Bayadh region has higher total polyphenolic content (210.50 mg/g) compared with other propolis extracts from the Algerian north-western regions [62]. In fact, Benhanifia et al have found that total polyphenol contents of EEP collected from 5 regions of north-western Algeria varied between 9.99 mg/g and 46.63 mg/g. These differences may be attributed to the botanical origin, the harvest year and environmental hives [63].
Moreover, we explored the effect of ethanolic propolis extract on the hydatid cyst protoscoleces in various concentrations and over different time periods. Our study's results showed that the highest scolicidal activity was observed at the concentration of 25 mg/mL because 100% of mortality has been obtained in less time (10 min). In addition, all the protoscoleces were killed after 60 and 30 min of exposure to EPP at the concentrations of 5 mg/mL and 10 mg/mL, respectively.
Therefore, we notice that the increase in EEP concentrations induced higher protoscolicidal activity.
The protoscolicidal effect of many plants has been reported in several studies. Indeed, Moazeni et al demonstrated that 100% of mortality was obtained when protoscoleces were exposed to 25 mg/mL of garlic methanolic extract for 60 min [68]. Moreover, Sambucus ebulus extract with a concentration of 100 mg/mL killed 98.6% of the protoscolices after 60 min of application [69]. However, in our study, 100% of mortality was observed after only 10 min of EEP application at the concentration of 25 mg/mL. These results suggest that EEP can constitute a source of new compounds that could be used as an effective scolicidal agent. Thus, we can conclude that EEP could be considered as a safe and potent scolicidal agent used in hydatid cyst surgery.
Collectively, our study is the first report demonstrating with interest the antihydatic and immunomodulatory effects of the Algerian EEP, suggesting its possible therapeutic role in hydatid disease treatment. Additional research is needed to fully clarify the exact