The Curative Effect of Zingiber Ocinale and Cinnamomum Zeylanicum Extracts on Experimental Trichinella Spiralis Infection

Trichinellosis is a re-emerging zoonotic disease that has become a public health concern since its reported human outbreaks in many countries. The traditional therapy has many adverse effects in addition to the developing resistance. So, this necessitates nding effective natural alternatives. The current study targeted to assess the potential therapeutic effects of Zingiber ocinale and Cinnamomum zeylanicum in comparison to albendazole, a conventional therapy for treatment of trichinosis. Sixty mice were classied into ve groups (12 mice each), non-infected control, infected control, combined albendazole and prednisolone, Zingiber ocinale, and Cinnamomum zeylanicum treated groups. Mice sacrice was performed on the 7 th and 35 th days post infection for intestinal and muscular phases respectively. Eciency of the used preparations was assessed by parasitological, histopathological, immunohistochemical, biochemical studies in addition to ultrastructural evaluation using transmission electron microscopy. A signicant reduction in the mean number of T. spiralis adults and larvae was observed in Zingiber ocinale and Cinnamomum zeylanicum treated groups, (64.5%, 50.8%) and (68%, 54.6%) respectively. Also, both extracts showed moderate cytoplasmic reactivity for TGF-β1, (69.3% & 67.8%) respectively. The highest reduction in serum TNF- α level was observed in Zingiber ocinale treated group during the muscle phase (58.4%) while in the intestinal phase was 50%. The ultrastructural study revealed degenerative effects on both adults and larvae in addition to obvious improvement of the histopathological changes in the small intestine and muscles. We concluded that these herbal extracts especially Zingiber ocinale can be considered a practical and successful alternative for the treatment of trichinellosis.


Introduction
Trichinellosis is a zoonotic disease caused mainly by the nematode parasite, Trichinella spiralis. It affects a wide range of hosts including man (Ashour and Elbakary 2011). Trichinellosis is distributed almost all over the world. It has a burden of approximately 10,000 people per year and 0.2% mortality rate ). It is transmitted by ingestion of raw or insu ciently cooked pork meat. The life cycle of T. spiralis consists of enteral and parenteral phases (Abou Rayia et al. 2017). Enteral or intestinal phase clinically presents by abdominal symptoms as diarrhea and abdominal pain (Wilson et al. 2015). Parenteral or muscular phase presents by periorbital edema, myalgia and muscle weakness (Gottstein et al. 2009).
Benzimidazoles as mebendazole and albendazole are commonly used for treatment of trichinellosis.
However, these drugs are not fully effective against T. spiralis larvae ). This may be attributed to poor bioavailability after oral administration which leads to poor oral absorption (Piccirilli et al. 2014). Moreover, some of these drugs are contraindicated in pregnancy and children under three years (Yadav and Temjenmongla 2012), other drugs are thought to be carcinogenic (Shalaby et al., 2010). Thus, there is an increasing need for safe and effective drugs, especially those derived from herbs (Yadav and Temjenmongla 2012), as they are less toxic and almost have no adverse effects (Basyoni and El-Sabaa, 2013).
Zingiber o cinale or ginger belongs to the family Zingiberaceae. It is used worldwide as a spice and avoring agent. Also, it has an antioxidant effect which augments the immune response, allowing the body to naturally ght infections. Furthermore, ginger has the ability to increase digestive uids and counteract toxins. These effects may help in parasite clearance (Ghosh et al. 2011). Few studies investigated the anthelmintic activity of ginger and its constituents (Iqbal et al. 2006). They showed a larvicidal agent against Angiostrongylus cantonensis (Lin et al. 2010a) and Anisakis simplex (Lin et al. 2010b). Cinnamomum zeylanicum (cinnamon) is derived from a Greek word that means sweet wood and comes from the inner bark of tropical cinnamon trees (Vinitha and Ballal 2008

Drugs
Albendazole was used as Alzental suspension (EIPICO), 20 mg/ml. Each mouse received a dose of 50 mg/kg orally for 3 consecutive days starting from the 3 rd day post infection according to Attia et al. (2015). Prednisolone was available as Predsol suspension, 5 mg/ml. It was given in a dose of 0.7 mg/kg orally for 3 successive days starting from the 3 rd day post infection (Manzur et al. 2008).
Plant material and preparation of extracts Brie y, 300 g of fresh ginger rhizome and 250 g of dried Cinnamomum zeylanicum bark were cut into small pieces, macerated into 90% ethyl alcohol and left for 10 days. Alcohol was replaced by fresh one

Parasitological study
Isolation and counting of adult worms Mice were sacri ced and the small intestine was taken off. After the intestine was washed with physiological saline, it was split longitudinally along its entire length and divided into 2-cm portions, then placed in a beaker full of physiological saline for 3-4 hrs at 37°C. The intestine was shaken well in the saline, rinsed in a similar amount of saline and nally removed and discarded. Adults were allowed to sediment by standing in the container for half an hour. The supernatant was removed; the sediment was reconstituted in 3-5 drops of physiological saline and poured into a petri-dish. The number of adults was counted under the dissecting microscope at X 20 power (Shin et al. 2008).

Total larval burden in muscles
Mice were sacri ced on the 35 th day p.i. Muscle larvae counts in whole carcasses were determined according to the method described by Martínez-Gómez et al (2009). Brie y, each mouse was skinned and eviscerated. Teeth, tail and ears were removed and the rest of the animal was cut into small pieces by scissors and digested in 1% pepsin-hydrochloride in 200 ml distilled water. Following incubation of the mixture at 37°C for 2 hrs with continuous stirring by an electromagnetic stirrer, encysted larvae were collected via the sedimentation technique and washed several times in distilled water. The number of larvae was counted microscopically using a McMaster counting chamber.

Histopathological study
The formol-saline preserved intestinal and muscle tissue specimens were processed in an automated tissue processor in two steps; xation and dehydration. Initially, tissue samples were immersed in 10% buffered formalin for 48 hrs, after that the xative was removed in distilled water for 30 minutes.
Dehydration process was then carried out by running the tissues through a graded series of alcohol (70%, 90 %and 100%). Tissue specimens were then cleared in several changes of xylene, then the samples were impregnated with molten para n wax, then embedded and blocked out. Para n sections (4-5 µm) were stained with hematoxylin and eosin (Suvarna et al. 2013).

Immunohistochemistry
The standard immunohistochemical methods were adopted with Bancroft and Gamble (2008). Brie y, sections were mounted on positively charged glass slides (Biogenex, USA), and depara nized in xylene.
After xylene was removed by absolute ethanol, slides were placed in an unsealed plastic container lled with su cient antigen retrieval solution (Citrate buffer solution, pH 6) and microwaved for 5 minutes at power 10. The container was removed and allowed to cool for 15 minutes and the slides were washed in deionized water for several times then placed in phosphate buffer saline (PBS) for 5 minutes. Tissue sections were incubated with an endogenous peroxidase blocking reagent containing hydrogen peroxide and sodium azide (DAKO peroxidase blocking reagent, Cat. No.S 2001). Excess buffer was blotted off, and the slides were allowed to dry except for the tissue section. The supersensitive primary monoclonal antibody against transforming growth factor-beta 1 (TGF-β1) was added to the sections and incubated for 60 min at room temperature then, the slides were rinsed in PBS, incubated with biotin-streptavidin (BSA) system. After that, 1-2 drops of the ready-to-use DAKO EnVision + system were applied for 20 minutes at room temperature and rinsed again with PBS. Diaminobenzidine (DAB) was used as a chromogen. The slides were mixed for 10-20 min until a desirable brown color was obtained then counterstained with Mayer's hematoxylin.
Quantitative scoring method TGF-β1 immuno-activity quantitation was made by digital image analysis through the image J analysis software on ve elds from each slide. This software could measure the total positive stained brown color/areas throughout the unstained cells. From this data an index (positively stained cells per a total of Transmission electron microscopy

Adults
The collected adults were centrifuged at 7000 rpm for 1 min, and then the parasites were resuspended in fresh modi ed Karunovsky's xative and xed at 4°C for 3 days. Fixative was then removed and the parasites were post-xed for 1 hour in 2% osmium tetroxide. Adults were then dehydrated in ethanol followed by acetone series solutions of increasing concentration (Karunovsky 1965). The material was embedded in the epoxy resin Epon 812 according to Luft's standard method (Luft 1961). After hardening, blocks were removed and sections were cut using an ultramicrotome. Ultrathin sections were examined using a JEM 2100 transmission electron microscope (TEM).
Muscle samples 1 mm³ diaphragm muscle samples were cut then xed within 5 minutes. Muscle samples were then prepared by the same steps as adults.

Ethical consideration
The study protocol was approved by the ethical committee of the Institutional Review Board (IRB) Unit, Faculty of Medicine, Zagazig University (approval number: 4002).

Statistical analysis
Quantitative values of the measured parameters were expressed as mean ± standard deviation (SD). Data were analyzed by one way-ANOVA to determine signi cance of differences between studied groups using Statistical Package for Social Sciences (SPSS), version 18.0. All statistical tests were considered signi cant at p < 0.05 and highly signi cant at p < 0.01.

Parasitological assessments
A signi cant reduction in the mean number of T. spiralis adults was observed among the treated groups compared with the infected untreated group. Mice treated with albendazole & prednisolone showed the highest reduction followed by Zingiber o cinale and Cinnamomum zeylanicum treated groups (93.5%, 64.5%, and 50.8%). Regarding larvae count, there was a signi cant decrease in mean number of total larvae in muscles in treated groups compared with the positive control group. The highest reduction was observed in albendazole & prednisolone treated group (90.6%), followed by Zingiber o cinale and Cinnamomum zeylanicum with reduction percentages of 68% and 54.6% respectively (Table 1). <0.001** p-value n = number of studied mice in each group, SD = standard deviation, F = ANOVA test, p value = probability, **Highly signi cant difference.

Small intestine
Dense in ammatory cellular in ltrate was observed in infected non-treated group predominantly in the core of the villi and extending into submucosa with attening of villi and sloughing of the villous tip. Cut sections in T. spiralis adults could also be noticed. On the other hand, a signi cant reduction in the intensity of the in ammation and prolonged villi was observed in Zingiber o cinale group. Cinnamomum zeylanicum treated group showed moderate in ammatory cells in ltration with improvement of architecture and length of the villi compared with the infected non-treated group. Mice treated with albendazole and prednisolone exhibited mild to moderate in ammation in the core of villi (Fig. 1).

Skeletal muscles
Histopathological examination of skeletal muscle of the infected control group showed encysted T. spiralis larvae within a nurse cell, collagenous capsule and dense in ammatory cellular in ltrations. An alteration in archticture of the muscle bres was clearly detected. The marked reduction in the number of deposited larvae with a signi cant decrease of in ammatory cellular in ltrate around the larvae was evident in Zingiber o cinale group. Cinnamomum zeylanicum treated group showed moderate in ammation while albendazole and prednisolone treated group displayed mild to moderate in ammatory cellular in ltrate around the deposited larvae (Fig. 2).

Immunohistochemical assessment
Investigated immune-stained sections from different segments of the small intestinal tract of the studied groups revealed a weak cytoplasmic reactivity for the used marker (TGF-β1) in negative control group; positive cells showed light brown-colored cytoplasmic contents. A strong cytoplasmic reactivity for the used marker was observed in infected control group and albendazole and prednisolone treated group, while there was moderate cytoplasmic reactivity for the used marker in Zingiber o cinale and Cinnamomum zeylanicum treated groups. Positive cells were encountered in the mucosal and submucosal stromal cells, vascular and microvascular endothelial cells (Fig. 3). Imaging analysis technique estimated the positive cells to be (25.56%, 73.5%, 76.1%, 69.3% & 67.8%) of the total cellular contents of the studied groups respectively (Table 2).

Transmission electron microscopy
Regarding the ultrastructural examination of T. spiralis adults, multiple cuticular depressions and disturbed continuity were evident in Zingiber o cinale group. Cinnamomum zeylanicum group showed loss of epicuticular waviness and lack of discrimination of cuticle layers (Fig.4). EM examination of the diaphragm in Zingiber o cinale group showed a marked reduction in the matrix and in ammatory zone and closer appearance of normal muscle with regular light and dark bands. Degenerative changes in the larvae and separation of super cial layers of the cuticle in a wide area were also detected. The Cinnamomum zeylanicum group also showed a marked reduction in the in ammatory zone together with separation and blebbing of super cial cuticular layers (Fig. 5).

Discussion
Trichinellosis is a worldwide severe zoonotic disease that involves the activation of in ammatory cells. It is also accompanied by prominent expression of proin ammatory cytokines in the affected host (Xu et al. 2019). Many experts are calling for the use of natural remedies, since most synthetic products have harmful effects and some of them have been found to be cancerous. Thus, an alternate, healthy, and effective herbal remedy is required to treat both enteral and parenteral phases of T. spiralis (Shalaby et al. 2010). For our knowledge, no study has been conducted assessing the effect of Zingiber o cinale and Cinnamomum zeylanicum on Trichinella spiralis infection.
Concerning the anti-parasitic effect, the existing nding revealed a signi cant reduction in the adult worms and larvae counts in Zingiber o cinale (64.5%, 68%) and Cinnamomum zeylanicum (50.8%, 54.6%) groups compared with the infected nontreated group. Albendazole and prednisolone treated group exhibited a lower e cacy on T. spiralis larvae than that on adults (90.6% vs 93.5%) due to the low bioavailability and water solubility after oral administration (Caner et al. 2008 The parasiticidal effect of Zingiber o cinale may be due to its anticholinergic effect (Qian and Liu 1992), since, acetylcholine is the neurotransmitter in nematode muscles (Neal 2012). Furthermore, its potent proteolytic enzyme "zingibain" could be responsible for its antiparasitic effect (Khalil and  Regarding the histopathological studies of the small intestine, a signi cant decrease in the intensity of in ammation and reconstructed villi architecture were evident in Zingiber o cinale group compared with the positive control group in which, a dense in ammatory cellular in ltrate was evident mainly in the core of the villi and lamina propria with shortening of the villi and attening of their tips and with Cinnamomum zeylanicum treated group that showed moderate in ammation in the core of villi. Concerning histopathological examination of skeletal muscles, Zingiber o cinale group displayed an obvious reduction in larvae deposition with signi cant reduction of in ammatory cellular in ltrate around them. However, in Cinnamomum zeylanicum group there was a reasonable decrease in muscle larvae count with moderate in ammation around the larvae compared to the positive control. TGF-β1 is a cytokine that plays a role in pro-in ammatory as well as in anti-in ammatory responses. The concomitant presence of TGF-β1 and IL6 favors a pro-in ammatory environment mediated by Th17 (Grainger et al. 2010). Th17 cells play a critical role in defense against some extracellular pathogens and are involved in numerous in ammatory and autoimmune diseases (Bedoya et al. 2013;Jafarzadeh et al. 2018). Production of TGF-β1 by antigen presenting cells leads to the differentiation of induced regulatory T cells (iTregs) (Chen et al. 2010) which inhibit the protective T cell responses of the host and contributes to disease progression. This fact highlights the importance of this cytokine during parasitic infections (Bhattacharya et al. 2014).
Our study revealed moderate cytoplasmic reactivity for TGF-β1 in Zingiber o cinale and Cinnamomum zeylanicum treated groups compared to a weak cytoplasmic reactivity in negative control group and strong cytoplasmic reactivity in infected control group and albendazole & prednisolone treated group. In agreement with our results was that of Gungor  In Cinnamomum zeylanicum treated group, we supposed that the signi cant decrease in TNF-α level could be linked to "cinnamaldehyde", the principal constituent of cinnamon as reported by Liao et al. (2012). Rathi et al. (2013), added that, the antioxidant components of cinnamon can inhibit Nuclear Factor kappa activation with the subsequent inhibition of pro-in ammatory cytokines including TNF-α. The administration of glucocorticoids for treatment of immediate hypersensitivity associated with trichinosis can inhibit cellular immune response (mainly lymphocytes) and reduce the cytokines production, including TNF-α as reported by Kisiel & Kaszuba (2011). This clari es the reduction of TNF-α in albendazole & prednisolone treated group.
Concerning the electron microscopy examination of both adult worms and muscle larvae samples, remarkable degenerative changes were observed in tested herbs groups compared with the control group. An obvious blunting of epicuticle, with a marked reduction in the in ammatory zone, and separation of super cial layers of the cuticle of the larvae were detected. These changes were more evident in Zingiber o cinale treated group. The Cinnamomum zeylanicum group also showed blebbing of super cial cuticular layers. We suppose that Zingiber o cinale potent proteolytic enzyme "Zingibain" is responsible for the resulted cuticular damage as reported by Khalil and El-houseny (2013). The Integrity of nematode cuticle is crucial for nutrition and defensive function as well as to maintain shape. Cuticular damage can extremely affect T. spiralis adults and larvae since it is considered as a safeguard against physical and immunological harm, and plays a role in osmoregulation (Roberts et al. 2013).
In conclusion, Zingiber o cinale followed by Cinnamomum zeylanicum ethanolic extracts have a therapeutic and anti-in ammatory effects on T. spiralis infection. These extracts may be promising alternatives in treatment of trichinellosis. We are looking forward to assess the e cacy of the active ingredients of these remedies with evaluation of their effects on the genetic diversity after treatment.

Con ict of interest
The authors declare that they have no con ict of interest.