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

Abstract

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 finding effective natural alternatives.  The current study targeted to assess the potential therapeutic effects of Zingiber officinale and Cinnamomum zeylanicum in comparison to albendazole, a conventional therapy for treatment of trichinosis. Sixty mice were classified into five groups (12 mice each), non-infected control, infected control, combined albendazole and prednisolone, Zingiber officinale, and Cinnamomum zeylanicum treated groups. Mice sacrifice was performed on the 7^th and 35^th days post infection for intestinal and muscular phases respectively. Efficiency of the used preparations was assessed by parasitological, histopathological, immunohistochemical, biochemical studies in addition to ultrastructural evaluation using transmission electron microscopy. A significant reduction in the mean number of T. spiralis adults and larvae was observed in Zingiber officinale 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 officinale 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 officinale 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 (García et al. 2014). It is transmitted by ingestion of raw or insufficiently 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 (García et al. 2014). 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 officinale or ginger belongs to the family Zingiberaceae. It is used worldwide as a spice and flavoring agent. Also, it has an antioxidant effect which augments the immune response, allowing the body to naturally fight infections. Furthermore, ginger has the ability to increase digestive fluids 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). Cinnamon showed antiparasitic effect against Anisakis larvae (Trabelsi et al. 2019), Babesia and Theileria (Batiha et al. 2020). The present study was carried out to assess the parasiticidal and both pro and anti-inflammatory effects of Zingiber officinale and Cinnamomum zeylanicum extracts against T. spiralis in experimentally infected mice in enteral and parenteral phases.

Material And Methods

Animals and parasite

A total of sixty parasite free, laboratory-bred, male Swiss Albino mice 5 weeks old, weighing about 20-25gm each, were used. Mice were obtained from animal house of Theodor Bilharz Research Institute (Giza, Egypt) and maintained in accordance with institutional and national guidelines. Mice were infected orally with 200-250 T. spiralis L1 larvae per mouse (Dunn and Wright 1985).

Experimental design

Animals were divided into 5 groups (12 mice each) as following: G (1) Non-infected control group (healthy control), G (2): Infected untreated (infected control), G (3): Infected then treated with albendazole and prednisolone, G (4): Infected then treated with Zingiber officinale extract and G (5): Infected then treated with Cinnamomum zeylanicum extract.

On the 7^th day p.i., six mice from each group were sacrificed and blood samples were collected for determination of serum TNF-α. The abdominal skin was sterilized then incised and the peritoneum was opened to dissect the small intestine which is then preserved in formalin 10% for histopathological examination and immunohistochemical study. The rest of the intestine was used for T. spiralis adult worm counting. Adults were then preserved in fresh modified Karnovsky’s fixative to be examined later on under electron microscopy. On the 35^th d.p.i., 6 mice from each group were sacrificed. Blood samples were collected for the determination of serum TNF-α. The peritoneum was opened and the diaphragm was carefully dissected for histopathological and ultrastructure studies. The rest of the muscle samples were digested for total larval count.

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

Briefly, 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 every 3 days. The extract was filtrated and ethanol was removed via rotary evaporator at 50^o C under reduced pressure to obtain viscous residues of crude plant extracts. These residues were then subjected to lyophilization to afford 40 gm of powdered extract of the plant. The suspension of the lyophilized extract was prepared for oral administration using 0.5% Tween-80 (ADWIC, Egypt) in normal saline. The concentration was adjusted that each 0.1 ml of the prepared suspension contains 0.3 mg of the plant extract (Mahmoud et al. 2014). Extracts were orally administered at a dose of 100 mg/kg (Hsiang et al. 2015) and 50 µg/g body weight (Kwon et al. 2011) once daily for Zingiber officinale and Cinnamomum zeylanicum respectively starting from the 1^st day of infection till the day of sacrifice.

Parasitological study

Isolation and counting of adult worms

Mice were sacrificed 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 finally 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 sacrificed 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). Briefly, 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; fixation and dehydration. Initially, tissue samples were immersed in 10% buffered formalin for 48 hrs, after that the fixative 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 paraffin wax, then embedded and blocked out. Paraffin 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). Briefly, sections were mounted on positively charged glass slides (Biogenex, USA), and deparaffinized in xylene. After xylene was removed by absolute ethanol, slides were placed in an unsealed plastic container filled with sufficient 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 five fields 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 1000 cells) can be computed.

Determination of serum TNF-α

The concentrations of serum TNF-α were quantitatively determined in all groups, on days 7 and 35 p.i. using Quantikine®ELISA kits, Cat. No. MTA00B (R&D systems, USA). All ELISA techniques were performed according to the manufacturer’s instructions. The absorbance of the serum samples was read within 10 min at a wave length of 450 nm using an ELISA micro-titer plate reader.

Transmission electron microscopy

Adults

The collected adults were centrifuged at 7000 rpm for 1 min, and then the parasites were resuspended in fresh modified Karunovsky’s fixative and fixed at 4°C for 3 days. Fixative was then removed and the parasites were post-fixed 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 fixed 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 significance of differences between studied groups using Statistical Package for Social Sciences (SPSS), version 18.0. All statistical tests were considered significant at p < 0.05 and highly significant at p < 0.01.

Results

Histopathological findings

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 inflammatory cellular infiltrations. An alteration in archticture of the muscle fibres was clearly detected. The marked reduction in the number of deposited larvae with a significant decrease of inflammatory cellular infiltrate around the larvae was evident in Zingiber officinale group. Cinnamomum zeylanicum treated group showed moderate inflammation while albendazole and prednisolone treated group displayed mild to moderate inflammatory cellular infiltrate 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 officinale 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).

Discussion

Trichinellosis is a worldwide severe zoonotic disease that involves the activation of inflammatory cells. It is also accompanied by prominent expression of proinflammatory 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 officinale and Cinnamomum zeylanicum on Trichinella spiralis infection.

Concerning the anti-parasitic effect, the existing finding revealed a significant reduction in the adult worms and larvae counts in Zingiber officinale (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 efficacy 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). Our findings are in accordance with Attia et al. (2015) and Shalaby et al. (2010), who recorded reduction in adult count, nevertheless, the larval reduction rate was 26.4%, also, Shoheib et al. (2006) recorded decreased effectiveness of albendazole against the encysted larvae with a reduction rate of 65.2%. Interestingly, Zingiber officinale and Cinnamomum zeylanicum extracts displayed a higher parasiticidal effect against larvae compared with their effect on adults. Therefore, the use of the same concentration with increasing the duration of treatment may improve the outcome of treatment and increase the parasite mortality.

The parasiticidal effect of Zingiber officinale 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 El-houseny 2013). Similarly, El-Sayed (2017), stated antiparasitic effect of Zingiber officinale on Toxocara canis, and, Merawin et al. (2010) reported that Zingiber officinale rhizome extract was effective against Dirofilaria immitis.The antiparasitic effect of Cinnamomum zeylanicum could be elucidated by its abundant content of trans-cinnamaldehyde and proanthocyanidin tannins (Williams et al. 2015). In agreement with our finding was that of Trabelsi et al. (2019) who studied the anti-parasitic effect of cinnamon extract on Anisakis and reported its lethal effect on Anisakis larvae type 1 both in vivo and in vitro.

Regarding the histopathological studies of the small intestine, a significant decrease in the intensity of inflammation and reconstructed villi architecture were evident in Zingiber officinale group compared with the positive control group in which, a dense inflammatory cellular infiltrate was evident mainly in the core of the villi and lamina propria with shortening of the villi and flattening of their tips and with Cinnamomum zeylanicum treated group that showed moderate inflammation in the core of villi. Concerning histopathological examination of skeletal muscles, Zingiber officinale group displayed an obvious reduction in larvae deposition with significant reduction of inflammatory cellular infiltrate around them. However, in Cinnamomum zeylanicum group there was a reasonable decrease in muscle larvae count with moderate inflammation around the larvae compared to the positive control.

TGF-β1 is a cytokine that plays a role in pro-inflammatory as well as in anti-inflammatory responses. The concomitant presence of TGF-β1 and IL6 favors a pro-inflammatory 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 inflammatory 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 officinale 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 et al (2020) who studied the effect of zingerone on induced lung fibrosis and reported reduction of TGF-β1 in lung tissue. Abdi et al. (2020) found that Zingiber officinale downregulated expression of TGF-β1 genes in comparison to diabetic group. Also, Zheng et al. (2011) stated that cinnamaldehyde prevents diabetic nephritis by inhibition of oxidative damage and downregulation of TGF-β1.

As regard the biochemical studies, Zingiber officinale treated group displayed a significant reduction in the level of TNF-α on the 7th & 35th d.p.i. (50%, 58.4%) while Cinnamomum zeylanicum (33.5%, 41%), compared to albendazole & prednisolone treated group (51.5%, 51.9%).“Zingerone”, one of the components of Zingiber officinale could be responsible for the reduction of TNF-α level. Our findings are supported with that of Ueda et al. (2010) who investigated the effect of squeezed ginger extract on many cytokines including TNF-α and reported a significant reduction in its level in peritoneal cells. Rehman et al, (2019), reported that zingerone can inhibit mRNA expression of TNF-α, consequently interfered with cell signaling pathway and suppress TNF-α expression. Furthermore, Haniadka et al. (2013) attributed the anti-inflammatory effect of Zingiber officinale to its ability to inhibit COX-1 and COX-2 with subsequent prostaglandins synthesis suppression.

In Cinnamomum zeylanicum treated group, we supposed that the significant 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-inflammatory 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 clarifies 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 inflammatory zone, and separation of superficial layers of the cuticle of the larvae were detected. These changes were more evident in Zingiber officinale treated group. The Cinnamomum zeylanicum group also showed blebbing of superficial cuticular layers. We suppose that Zingiber officinale 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 officinale followed by Cinnamomum zeylanicum ethanolic extracts have a therapeutic and anti-inflammatory effects on T. spiralis infection. These extracts may be promising alternatives in treatment of trichinellosis. We are looking forward to assess the efficacy of the active ingredients of these remedies with evaluation of their effects on the genetic diversity after treatment.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

References

1. Abdi T, Mahmoudabady M, Marzouni HZ, Niazmand S, Khazaei M (2020) Ginger (Zingiber officinale roscoe) extract protects the heart against inflammation and fibrosis in diabetic rats. Can J Diabetes S1499-2671(20)30345-2
2. Abou Rayia DM, Saad AE, Ashour DS , Oreiby RM (2017) Implication of artemisinin nematocidal activity on experimental trichinellosis: In vitro and in vivo studies. Parasitol Int 66(2):56-63
3. Ashour DS , Elbakary RH (2011) Pathogenesis of restricted movements in trichinellosis: An experimental study. Exp Parasitol 128(4):414-418
4. Attia RA, Mahmoud AE, Farrag HMM, Makboul R, Mohamed ME, Ibraheim Z (2015) Effect of myrrh and thyme on Trichinella spiralis enteral and parenteral phases with inducible nitric oxide expression in mice. Mem Inst Oswaldo Cruz 110: 1035-1041
5. Azab KS, Mostafa AHA, Ali EM, Abdel-Aziz MA (2011) Cinnamon extract ameliorates ionizing radiation-induced cellular injury in rats. Ecotoxicol Environ Saf 74(8) 2324-2329
6. Bedoya SK, Lam B, Lau K, Larkin J (2013) Th17 cells in immunity and autoimmunity. Clin Dev Immunol.: 986789
7. Bancroft JD, Gamble M (Editors) (2008) Theory and practice of histological techniques (6th ed.).  New York: Churchill Livingstone/Elsevier 726 -744
8. Basyoni MM, El-Sabaa AAA (2013) Therapeutic potential of myrrh and ivermectin against experimental Trichinella spiralis infection in mice. Korean J Parasitol 51(3):297-304
9. Batiha GES, Beshbishy AM, Guswanto A et al. (2020) Phytochemical characterization and chemotherapeutic potential of Cinnamomum verum extracts on the multiplication of protozoan parasites in vitro and in vivo. Molecules 25(4): 996
10. Bhattacharya D, Dwivedi VP, Kumar S, Reddy MC, Van Kaer L, Moodley M, Das G (2014) Simultaneous inhibition of T helper 2 and T regulatory cell differentiation by small molecules enhances Bacillus Calmette-Guerin vaccine efficacy against tuberculosis, J Biol Chem 289 (48) (2014) 33404–33411
11. Caner A, Döşkaya M, Değirmenci A, Can H, Baykan Şet al. (2008) Comparison of the effects of Artemisia vulgaris and Artemisia absinthium growing in western Anatolia against trichinellosis (Trichinella spiralis) in rats. Exp Parasitol 119(1):173-179
12. Chen W,  Konkel JE (2010) TGF-beta and 'adaptive' Foxp3(+) regulatory T cells, J Mol Cell Biol 2 (1) 30–36
13. Dunn I and Wright K (1985) Cell injury caused by Trichinella spiralis in the mucosal epithelium of B10A mice. J Parasitol 757-766
14. El-Sayed NM )2017) Efficacy of Zingiber officinale ethanol extract on the viability, embryogenesis and infectivity of Toxocara canis eggs. J Parasit Dis 41)4(:1020-1027
15. García A, Leonardi D, Vasconi MD, Hinrichsen LI,Lamas MC (2014) Characterization of albendazole-randomly methylated-β-cyclodextrin inclusion complex and in vivo evaluation of its antihelmitic activity in a murine model of trichinellosis. PloS one 9(11):e113296
16. Ghosh AK, Banerjee S, Mullick HI, Banerjee J (2011) Zingiber officinale: A natural gold. Int J Pharm Biol Sci 2(1):283-294
17. Gottstein B, Pozio E, Nöckler K (2009) Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol 22(1):127-145
18. Grainger JR, Smith KA, Hewitson JP, McSorley HJ, Harcus Y et al. (2010) Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway. JExp Med 207, 2331–2341, https://doi.org/10.1084/jem.20101074
19. Gungor H, Ekici M, Onder Karayigit M. et al. (2020) Zingerone ameliorates oxidative stress and inflammation in bleomycin-induced pulmonary fibrosis: modulation of the expression of TGF-β1 and iNOS. Naunyn-Schmiedeberg's Arch Pharmacol 393, 1659–1670
20. Haniadka R, Saldanha E, Sunita V, Palatty PL, Fayad R, Baliga MS (2013) A review of the gastroprotective effects of ginger (Zingiber officinale Roscoe). Food Funct 4(6): 845-855
21. Hong JW, Yang GE, Kim YB, Eom SH, Lew JH, Kang H (2012) Anti-inflammatory activity of cinnamon water extract in vivo and in vitro LPS-induced models. BMC Complement Altern Med 12, 237
22. Hsiang CY, Cheng HM, Lo HY, Li CC, Chou PC et al. (2015) Ginger and zingerone ameliorate lipopolysaccharide-induced acute systemic inflammation in mice, assessed by nuclear factor-κB bioluminescent imaging. J Agric Food Chem 63(26), pp.6051-6058
23. Iqbal Z, Lateef M, Akhtar MS, Ghayur MN, Gilani AH (2006) In vivo anthelmintic activity of ginger against gastrointestinal nematodes of sheep. J Ethnopharmacol 106(2):285-287
24. Jafarzadeh A, Larussa T, Nemati M, Jalapour S (2018) T cell subsets play an important role in the determination of the clinical outcome of Helicobacter pylori infection. Microb Pathog 116:227-36
25. Joshi K, Awte S, Bhatnagar P, Walunj S, Gupta R, et al (2010) Cinnamomum zeylanicum extract inhibits proinflammatory cytokine TNF: in vitro and in vivo studies. Res Pharm Biotech 2(2):14-21
26. Karunovsky M (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27(137):1A-149A
27. Khalil A, El-houseny W (2013) Ginger (Zingiber officinale) an antiparasitic and its effect on health status of Clarias gariepinus infested with gill Monogenia. Egypt J Aquac 3(4):55-62
28. Kisiel K, Kaszuba A (2011) Alclometasone dipropionate: properties and clinical uses. Post Dermatol Alergol 28(2):107-119
29. Kwon HK, Hwang JS, Lee CG, So JS, Sahoo A, et al. (2011) Cinnamon extract suppresses experimental colitis through modulation of antigen-presenting cells. World journal of gastroenterology: WJG: 17(8):976
30. Liao JC, Deng JS, Chiu CS, Hou WC, Huang SS, Shie PH, Huang GJ (2012) Anti-inflammatory activities of Cinnamomum cassia constituents in vitro and in vivo. Evid Based Complement Alternat Med Article ID 429320. http://dx.doi.org/10.1155/2012/429320
31. Lin RJ, Chen CY, Chung LY, Yen CM (2010a) Larvicidal activities of ginger (Zingiber officinale) against Angiostrongylus cantonensis. Acta Trop 115(1-2):69-76
32. Lin RJ, Chen CY, Lee JD, Lu CM, Chung LYet al. (2010b) Larvicidal constituents of Zingiber officinale (ginger) against Anisakis simplex. Planta Med 76(16):1852-1858
33. Luft JH (1961) Improvements in epoxy resin embedding methods. J Cell Biol 9(2):409-414
34. Mahmoud A, Attia R, Said S, Ibraheim Z.(2014) Ginger and cinnamon: can this household remedy treat giardiasis? Parasitological and histopathological studies. Iran J Parasitol 9(4):530-540.
35. Manzur AY, Kuntzer T, Pike M, Swan A. (2008) Glucocorticoid corticosteroids for Duchenne muscular dystrophy. Cochrane Database Systematic Review 23(1): CD003725
36. Martínez-Gómez, F, Santiago-Rosales R, Bautista-Garfias, CR (2009) Effect of Lactobacillus casei Shirota strain intraperitoneal administration in CD1 mice on the establishment of Trichinella spiralis adult worms and on IgA anti-T. spiralis production. Vet parasitol, 162(1-2), 171-175
37. Merawin L, Arifah A, Sani R, Somchit M, Zuraini A et al. (2010) Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis. Res Vet Sci 88(1):142-147
38. Munoz-Carrillo JL, Munoz-Escobedo JJ, Maldonado-Tapia CH, Chavez-Ruvalcaba F and Moreno-Garcia MA (2017) Resiniferatoxin lowers TNF-alpha, NO and PGE2 in the intestinal phase and the parasite burden in the muscular phase of Trichinella spiralis infection. Parasit Immunol 39(1): e12393
39. Neal MJ (2012) Medical pharmacology at a glance, Blackwell Science, Oxford, pp. 88–89
40. Piccirilli GN, García A, Leonardi D, Mamprin ME, Bolmaro REet al. (2014) Chitosan microparticles: influence of the gelation process on the release profile and oral bioavailability of albendazole, a class II compound. Drug Dev Compound Ind Pharm 40(11):1476-1482
41. Qian D, Liu Z (1992) Pharmacologic studies of antimotion sickness actions of ginger. Chinese journal of integrated traditional and Western medicine 12(2):95-98
42. Rathi B, Bodhankar S, Mohan, Thakurdesai P (2013) Ameliorative effects of a polyphenolic fraction of Cinnamomum zeylanicum L. bark in animal models of inflammation and arthritis. Sci Pharm 81(2) 567-590
43. Rehman MU, Rashid SM, Rasool S, Shakeel S, Ahmad B et al. (2019) Zingerone (4-(4-hydroxy-3-methylphenyl) butan-2-one) ameliorates renal function via controlling oxidative burst and inflammation in experimental diabetic nephropathy. Arch Physiol Biochem 125(3):201-209
44. Roberts LS, Janovy J and Nadler S (2013) Nematodes: Trichinellida and Dioctophymatida, Enoplean Parasites. In: Schmidt G.D. & Roberts L.S.’s Foundations of Parasitology. 9th edition. McGraw-Hill, New York, USA. pp. 381-388
45. Shalaby MA, Moghazy FM, Shalaby HA, Nasr SM (2010) Effect of methanolic extract of Balanites aegyptiaca fruits on enteral and parenteral stages of Trichinella spiralis in rats. Parasitol Res 107(1):17-25
46. Shin K, Watts GF, Oettgen HC, Friend DS, Pemberton AD (2008) Mouse mast cell tryptase mMCP-6 is a critical link between adaptive and innate immunity in the chronic phase of Trichinella spiralis infection. J  Immunol 180(7), 4885-4891.
47. Shishehbor F, Rezaeyan Safar M, Rajaei E, Haghighizadeh MH (2018) Cinnamon consumption improves clinical symptoms and inflammatory markers in women with rheumatoid arthritis. J Am Coll Nutr 37, 685-690
48. Shoheib ZS, Shamloula MM, Abdin AA, El-Segai O (2006) Role of α-chymotrypsin and colchicine as adjuvant therapy in experimental muscular trichinellosis: parasitological, biochemical and immunohistochemical study. Egypt J Microbiol 15: 773-790
49. Sung YY, Yoon T, Jang JY, Park SJ, Jeong GH, Kim HK (2011) Inhibitory effects of Cinnamomum cassia extract on atopic dermatitis-like skin lesions induced by mite antigen in NC/Nga mice. J Ethnopharmacol 133(2) 621-628
50. Suvarna Kim S, Christopher Layton and Bancroft John D. (2013): Bancroft's Theory and Practice of Histological Techniques, 7th Edition.)
51. Trabelsi N, Nalbone L, Marotta S, Taamalli A, Abaza L, Giarratana F (2019) Effectiveness of five flavoured Tunisian olive oil on Anisakis larvae type 1: application of Cinnamon and Rosemary oil in industrial anchovy marinating process. J Sci Food Agric 99(10):4808-4815
52. Ueda H, Ippoushi K, Takeuchi A (2010) Repeated oral administration of a squeezed ginger (Zingiber officinale) extract augmented the serum corticosterone level and had anti-inflammatory properties. Biosci Biotechnol Biochem 74:2248-2252
53. Vinitha M, Ballal M (2008) In vitro anticandidal activity of Cinnamomum verum.J Med Sci 8
54. Williams AR, Ramsay A, Hansen TV et al. (2015) Anthelmintic activity of trans-cinnamaldehyde and A-and B-type proanthocyanidins derived from cinnamon (Cinnamomum verum). Sci Rep 5:14791.
55. Wilson NO, Hall RL, Montgomery SP, Jones JL (2015) Trichinellosis surveillance--United States, 2008-2012. Morbidity and mortality weekly report. Surveillance summaries (Washington, D.C. : 2002) 64(1):1-8
56. Xu F, Hou B, Zhu X, Liu Y, Shi Xet al. (2019) Vaccaria n-butanol extract lower the production of proinflammatory cytokines and the infection risk of T. spiralis in vivo. Acta Parasitol 64(3):520-527
57. Yadav AK, Temjenmongla (2012) Efficacy of Lasia spinosa leaf extract in treating mice infected with Trichinella spiralis. Parasitol Res 110(1):493-8
58. Zheng H, Whitman S A, Wu W, WondrakG T, Wong P K , et al. (2011). Therapeutic potential of Nrf2 activators in streptozotocin‐induced diabetic nephropathy. Diabetes 60(11),3055–3066