|Year : 2020 | Volume
| Issue : 1 | Page : 191-195
Anti-Toxocara effects of Cassia nodosa plant extract in experimentally infected mice
Nadia S El-Nahas1, Amira F Abd-Elatty1, Amany A Rady1, Bahaa El-Deen W. El-Aswad1, Amira M Attallah1, Aiat S Hemida2
1 Department of Medical Parasitology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||30-Jun-2017|
|Date of Decision||13-Aug-2017|
|Date of Acceptance||25-Aug-2017|
|Date of Web Publication||25-Mar-2020|
Amira M Attallah
Berket El-Saba, Menoufia
Source of Support: None, Conflict of Interest: None
The current study aimed to evaluate the parasitological and pathological effects of the ethanolic extract of Cassia nodosa in the treatment of murine toxocariasis.
Toxocariasis is mainly treated by albendazole (ALZ) and thiabendazole. C. nodosa is a plant which has been used in the traditional medicine.
Materials and methods
BALB/c mice (n = 50) were divided into five groups (10 mice each): group I (normal control), group II (infected untreated control), group III (infected and received ALZ), group IV (infected and received C. nodosa), and group V (infected and received both ALZ and C. nodosa). All the infected mice were orally given 1000 embryonated eggs of Toxocara canis.
All treatments revealed significant reduction of total T. canis larvae. C. nodosa achieved 69.45% reduction, which was better than ALZ (44%). The plant extract showed better control of pathology than ALZ. ALZ +C. nodosa revealed the highest reduction percentage with 93.2% (P < 0.001), in addition to decreasing the pathological lesions caused by T. canis in different organs.
C. nodosa reduced Toxocara larvae number and improved the pathological lesions, initiated by T. canis larvae, so C. nodosa extract can be a potential natural drug against this parasitic infection.
Keywords: albendazole, Cassia nodosa, mice, thiabendazole, toxocariasis
|How to cite this article:|
El-Nahas NS, Abd-Elatty AF, Rady AA, El-Aswad BE, Attallah AM, Hemida AS. Anti-Toxocara effects of Cassia nodosa plant extract in experimentally infected mice. Menoufia Med J 2020;33:191-5
|How to cite this URL:|
El-Nahas NS, Abd-Elatty AF, Rady AA, El-Aswad BE, Attallah AM, Hemida AS. Anti-Toxocara effects of Cassia nodosa plant extract in experimentally infected mice. Menoufia Med J [serial online] 2020 [cited 2020 Mar 30];33:191-5. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/191/281308
| Introduction|| |
Toxocariasis is caused by incidental ingestion of eggs of Toxocara canis or Toxocara cati roundworms, and this can occur through contaminated food or water. On ingestion, mature embryonated eggs hatch in the small intestine of humans, and the released larvae may migrate to several organs such as liver, lungs, eyes, or brain causing damage while inducing inflammatory responses,.
Human Toxocara spp. has a high prevalence among rats worldwide especially in tropical countries. The infection rate in Egypt ranges from 7.7 to 11.1%,. Children are at higher risk of toxocariasis than adults owing to lack of personal hygiene.
The infection may take the form of visceral larva migrans, ocular larva migrans, or covert toxocariasis. Toxocariasis induces mixed immunological responses with the predominance of thymic helper cell 2, in addition to production of specific anti-Toxocara spp. antibodies like immunoglobulins A, M, and G.
Until now, there is no satisfactory drug against toxocariasis; however, albendazole (ALZ), an anti-Toxocara drug, is commonly used. Cassia plant species have been proven to include many compounds of therapeutic efficacy like anthraquinones (1,2-dihydroxy-1,3-dihydroxy-6,8-dimethoxy-2-methylanthraquinone; and 1, 3, 5, 8-tetrahydroxy-6-methoxy-2-methylanthraquinone), phenols and flavonoid (rhamnoside, Javanine besides kaempferol, its 3- and 7-methyl ethers) in 10–40 mg/kg effective dose, but 210 mg/kg is toxic,, and this plant showed successful results experimentally in the treatment of some parasites, for instance Hymenolepis diminuta and Plasmodium berghei. The aims of present study were to assess parasitological and pathological effects of Cassia nodosa ethanolic extract against T. canis in experimentally infected mice.
| Materials and Methods|| |
Male BALB/C albino mice that were 3 months old and weighed 25–30 g were used in our study, and the mice were bred in the animal house of Faculty of Medicine, Menoufia University. The animals were fed a standard diet and water under environmentally suitable conditions. Treatment and handling of the mice were done following the internationally guidelines and ethical committee of Menoufia Faculty of Medicine.
Egg collection and embryonation
The pregnant T. canis females were collected from small intestine of the infected stray dogs. The intestines were put in clean petridishes containing 0.15 mol/l sodium chloride solution (EIPICO, Pharmaceutical Co., Tenth of Ramadan City, 1st Industrial Zone B1, Egypt) at 37°C and left for 24 h to induce passage of eggs from the gravid intestine. The eggs suspension was obtained by allowing passage through two layers of gauze, and then the eggs were incubated in 2% formalin (Epico Pharmaceutical Co.) at room temperature for 4–8 weeks to induce embryonation. After confirming maturation, the mature eggs were kept at 4°C until use. For infection, 1000 viable embryonated T. canis eggs were given for each mouse orally.
Plant extract preparation
The C. nodosa fresh leaflets obtained from Faculty of Agriculture, Menoufia University were washed with water and oven-dried at 50°C. Then the leaves were grinded using Soxhlet apparatus (Sigma-Aldrich, St Louis, Missouri, USA), and 150 g of powdered leaves were extracted in 1 l of 90% ethanol (Epico Pharmaceutical Co.). The crude extract was dissolved in PBS (Epico Pharmaceutical Co.) to form liquid solution.
Drugs and doses
ALZ (Alzental; Epico Pharmaceutical Co.) was given orally at a dose of 100 mg/kg diluted in 0.1 ml distilled water once daily for 5 consecutive days starting at the first day of infection.
C. nodosa extract was given orally in a dose of 20 mg/kg either alone or in combination with ALZ for 7 successive days beginning at the first day of infection.
The five mice groups (10 mice each) were established as the following:
- Group I: normal control
- Group II: infected untreated control (IU)
- Group III: infected and treated with ALZ
- Group IV: infected and treated with C. nodosa
- Group V: infected and treated with ALZ+C. nodosa.
All the mice were killed on 45th day after infection, and tissues specimens were collected for parasitological and histopathological studies.
T. canis larval recovery
Specimens from lung, liver, and muscle (0.5 g, each) were digested in 50-ml pepsin-HCl solution for 24 h at 37°C. After that, the mixture was centrifuged for 2 min, and the sediment was collected and examined for larval recovery using light microscope (Olympus, Tokyo, Japan). In case of brain, the tissue was squashed between two slides without digestion, and larvae were counted directly.
The reduction percentage of larvae after treatment was calculated according to the following formula: reduction (%)=(C − T)/C × 100, where C is the mean number of larvae recovered from infected untreated control mice (group II) and T is the mean number of larvae recovered from the treated mice group.
The tissues specimens derived from each mouse used in this study were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin stain according to the standard procedure.
The data were processed on an IBM-PC compatible computer using SPSS, version 18 (SPSS Inc., Chicago, Illinois, USA). Data were expressed as mean ± SD. Analysis of variance was performed using Student's t-test, and the data were considered significant if P value is less than or equal to 0.05.
| Results|| |
T. canis larvae were not found in lung, liver, or muscles in all the mice groups, and only the brain was found to have Toxocara larvae. The mice of group II revealed 11.8 ± 3.04 larvae. With introduction of all types of treatments, the larval number reduced, where ALZ+C. nodosa (group IV) achieved the highest significant reduction (93.2%; P < 0.001), followed by C. nodosa only (group V), which significantly (P < 0.001) reduced the larvae number by 69.49%, whereas ALZ (group III) significantly (P < 0.001) decreased the larvae count to 44% [Table 1].
|Table 1: Effects of Cassia plant alone or in combination with albendazole on number of Toxocara canis larvae in the brain of different infected mice groups|
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The mice of group I showed normal histological features without any Toxocara larvae.
Liver tissue of the infected mice (group II) had severe lobular inflammation, congestion of vessels, and degeneration of hepatocytes with microgranulomas but without larva. There were bridging necrosis and fibrosis. After treatment with ALZ (group III) or C. nodosa (group IV), there were mild portal inflammation, less degeneration, and moderate collection of mixture of acute and chronic inflammatory cells. Combined ALZ+C. nodosa treatment (group V) caused absence of inflammation [Figure 1].
|Figure 1: Liver tissue: (a) NC (group I) with normal liver histology (haemotoxylin and eosin, ×100), (b) IU (group II) shows sever lobular inflammation, congestion of vessels, and degeneration of hepatocytes (haemotoxylin and eosin, ×100), (c) ALZ (group III) showing moderate collection of mixture of acute and chronic inflammatory cells (haemotoxylin and eosin, ×200), (d) Cassia nodosa (group IV) has portal vein dilatation, mild portal inflammation, and lobular activity (haemotoxylin and eosin, ×100) and (e) group V (ALZ+C. nodosa) shows ballooning degeneration and absence of inflammation (haemotoxylin and eosin, ×200). ALZ, albendazole; IU, infected untreated; NC, normal control.|
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Lung tissue showed severe peribronchiolar collections of inflammatory cells with many collapsed alveoli and degenerated alveolar septae in IU mice (group II). ALZ (group III) or C. nodosa (group IV) improved the lung inflammation tissue with lesser degeneration of alveolar septae. However, ALZ+C. nodosa (group V) showed the best pathological improvement [Figure 2].
|Figure 2: Lung tissue: (a) NC (group I) with normal lung histology (haemotoxylin and eosin, ×200), (b) IU (group II) shows severe peribronchiolar collections of inflammatory cells) (haemotoxylin and eosin, ×100), (c) ALZ (group III) shows moderate peribronchiolar collections of inflammatory cells (haemotoxylin and eosin, ×100), (d) Cassia nodosa (group IV) shows mild peribronchiolar inflammation (haemotoxylin and eosin, ×100), and (e) ALZ+C. nodosa (group V) shows less inflammatory response (haemotoxylin and eosin, ×100). ALZ, albendazole; IU, infected untreated; NC, normal control.|
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In IU mice (group II), the muscle tissue revealed severe inflammation and degeneration with muscle fiber infiltration by inflammatory cells. ALZ (group III) caused mild muscle tissue infiltration by inflammatory cells. C. nodosa (group IV) and ALZ + C. nodosa (group V) led to no inflammation in most of the treated mice (70%) [Figure 3].
|Figure 3: Muscle tissue: (a) NC (group I) with normal muscle histology (haemotoxylin and eosin, ×100), (b) IU (group II) shows severe inflammation and degeneration (haemotoxylin and eosin, ×400), (c) ALZ (group III) reveals mild inflammation (haemotoxylin and eosin, ×100), and (d) Cassia nodosa (group IV) and (e) ALZ + C. nodosa (group V) show no inflammation (haemotoxylin and eosin, ×200). ALZ, albendazole; IU, infected untreated; NC, normal control.|
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Brain tissue of group II (IU) showed severe inflammation, scattered larvae without inflammatory collections. In all treated mice groups, larvae number decreased significantly in addition to decreasing degree of inflammations, reaching to its absence with C. nodosa treatment [Figure 4].
|Figure 4: Brain tissue: (a) NC (group I) with normal brain histology (haemotoxylin and eosin, ×100), (b) IU (group II) shows scattered larvae without inflammatory collections and inflammatory infiltrate in the form of lymphocytes (haemotoxylin and eosin, ×200), (c) ALZ (group III) reveals few Larvae with mild inflammation (haemotoxylin and eosin, ×200), (d) Cassia nodosa (group IV) shows mild inflammation (haemotoxylin and eosin, ×200), and (e) ALZ+C. nodosa (group V) shows no inflammation and normal appearance (haemotoxylin and eosin, ×100). ALZ, albendazole; IU, infected untreated; NC, normal control.|
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| Discussion|| |
ALZ is a member of benzimidazoles drugs that has a good effect against toxocariasis; however, this drug recorded many drawbacks, such as granulomatous hepatitis and pancytopenia, and this has led to the urgent issue of development of new anti-Toxocara drugs. The plant C. nodosa is one which has wide therapeutic activities including anti-inflammatory, antimicrobial, antifungal, analgesic, and hypoglycemic. Herein, we aimed to assess the efficacy of the ethanol extract of C. nodosa against T. canis in the experimentally infected mice regarding the parasitological effects and pathological alleviation.
The extract of C. nodosa achieved better larval reduction in comparison with ALZ (69.49 vs. 44%), whereas the combination of both drugs enhanced the antiparasitic efficacy reaching to 93.2%. Moreover, C. nodosa improved the pathological lesions resulting from the presence of the larvae in the examined tissues, and this efficacy was more increased when it was combined with ALZ. To the best of our updated knowledge, our study is the first study that examined the effect of C. nodosa extract against T. canis.
The reports that have evaluated efficacy of Cassia as antiparasitic agent are generally few. The hexane extract of Chenopodium ambrosioides reduced the inflammatory reaction produced by the infection of T. canis larvae owing to presence of flavonoids and tannins, with similar chemical components to Cassia plant. Moreover, the plant extract caused paralysis of Raillietina tape worm in in-vitro study. In another in-vitro study, Cassia alata extract caused rapid paralysis of Hetracus gallinarum worm.
Larvae were not completely eradicated from the brain in our study after treatment, which may be because of low-drug concentration, the ability of the larvae to escape from the lethal immune response initiated with that drugs, and larvae reaching the brain are no longer vulnerable to antihelminthic agents.
In our study, liver and lung were found to not have any larvae, and this could be attributable to introduction of the treatments concurrently with the infection, where T. canis larvae are in stage of hepatopulmonary migration as they are in contact with the treatment or because of continuous larvae migration, thereof leaving these organs after 45 days after infection.
Mice group revealed various pathological lesions with variable degrees of inflammation, owing to the parasite infection, and these results were in parallel to other workers who examined T. canis effects in the experimental animals,. These lesions caused by T. canis larvae might be attributed to proteolytic enzymes released from the larvae, shedding of active larval antigens, and the produced oxidative stress that generates toxic molecules having the ability to cause tissue damage like nitric oxide.
C. nodosa treatment (group IV) succeeded in controlling the inflammatory response caused by the infection, and this was better than the traditional ALZ drug. This further indicates the superiority of C. nodosa extract over ALZ. The combined treatment (group V) caused returning of the pathology into nearly histological appearance. These antipathological effects of the plant extract may be because of decrease in the number of larvae, and also, as the plant might possess many anti-inflammatory agents that combat the pathology.
ALZ caused obvious improvement of the histopathological lesions, and this was correlated with other works denoting that ALZ relieves inflammation in Toxocara spp.-infected hosts [21,,.
| Conclusion|| |
C. nodosa, a natural extract, reduced the larvae count and alleviated the pathological lesions caused by T. canis in experimentally infected mice, so it is recommended to involve C. nodosa as a potential treatment for Toxocara.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Brouke SV, Kanobana K, Polman K, Soentjens P, Vekeman SM, Theunissen C, et al
. Toxocariasis diagnosed in international travelers at the institute of tropical medicine, Antwerp, Belgium, from 2000 to 2013. PLoS Negl Trop Dis 2015; 6
Kong J, Won J, Yoon J, Lee U, Kim J, Huh S. Draft genome of Toxocara canis
, a pathogen responsible for visceral larva migrans. Korean J Parasitol 2016; 54
Kuenzli E, Neumayr A, Chaney A, Blum J. Toxocariasis-associated cardiac diseases-asystematic review of the literature. Acta Trop 2015; 154
El-Shazly AM, Abdel Baset SM, Kamal A, Mohammed KA, Sakrs TI, Hammad SM. Seroprevalence of human toxocariasis (visceral larva migrans). J Egypt Soc Parasitol 2009; 39
Faghly AM, Mohamed SM, Abdel-Rahman SA, Mohammed FE, El-Bahaie ES, El-shafey MA. The relation between the prevalence of soil transmitted parasites in the soil and among school children in Zagazig district, Sharkyia Governorate, Egypt. J Parasit Dis 2016; 40
Carvalho EA, Rocha RL. Visceral larva migrans syndromes associated with toxocariasis: epidemiology, clinical and laboratory aspects of human toxocariasis. Curr Trop Med Rep 2014; 1
Berenji F, Pouryousef A, Fata A, Mahmoudi M, Salehi M, Khoshnegah J. Seroepidemiological study of toxocariasis in the owners of domestic cats and dogs in Mashhad, Northeastern Iran. Iran J Parasitol 2016; 11
Resende MN, Gazzinelli-Guimarães PH, Barbosa FS, Oliveira ML, Nogueira DS, Gazzinelli-Guimarães AC, et al
. New insights into the immunopathology of early Toxocara canis infection in mice. Parasit Vectors 2015; 8
Kocięcki J, Kocięcka W, Dmitriew A. Toxocarosis of the organ of sight – the complex pathological and diagnostic problem. Acta Parasitol 2016; 61
Rychlicki W. Use of specific immunoglobulin G antibody avidity in the differential diagnosis of active and chronic Toxocara canis
infection. Wiad parazytol 2004; 50
Barrera MG, Leonardi D, Bolmaro RE, Echenique CG, Olivieri AC, Salomon CJ, et al
evaluation of albendazole microspheres for the treatment of Toxocara canis
larva migrans. Eur J Pharm Biopharm 2010; 75
Selim SA, Abdel Aziz MH, Mashait MS, Warrad MF. Antibacterial activities, chemical constitutes and acutetoxicity of Egyptian Origanum majorana L
., Peganum harmala L.
officinalis L. essential oils. Afr J Pharm Pharmacol 2013; 7
Kundu S, Roy S, Lyndem LM. Broad spectrum anthelmintic potential of Cassia
plants. Asian Pac J Trop Biomed 2014; 4
Abdulrazak N, Asiya UI, Usman NS, Unata IM, Farida A. Anti-plasmodial activity of ethanolic extract of root and stem back of Cassia sieberiana
DC on mice. J Intercult Ethnopharmacol 2014; 4
Barriga OO, Omar HM. Immunity to Toxocara vitulorum
repeated infections in arabbit model. Vet Immunol Immunopathol 1992; 33
Fan CK, Lin YH, Du WY, Su KE. Infectivity and pathogenicity of 14-month-cultured embryonated eggs of Toxocara canis
in mice. Vet Parasitol 2003; 113
Yarsan E, Altinsaat C, Aycicek H, Sahindokuyucu F, Kalkan F. Effects of albendazole treatment on haematological and biochemical parameters in healthy and Toxocara canis
infected mice. Turk J Vet Anim Sci 2003; 27
Chung LY, Fang BH, Chang JH, Chye SM, Yen CM. The infectivity and antigenicity of Toxocara canis
eggs can beretained after long-term preservation. Ann Trop Med Parasitol 2004; 98
Kessel RG. Techniques for the study of cells, tissues and organs. In: Kessel RG, editor. Medical histology
. New York, NY: Oxford University Press Inc.; 1998. pp. 265–266.
Zuluaga JI, Castro AE, Cadavid JC, Gutierrez JC. Albendazole-induced granulomatous hepatitis: a case report. J Med Case Rep 2013; 7
Reis M, Trinca A, Ferreira MJ, Monsalve-Puello AR, Gracio MA. Toxocara canis
: Potential activity of natural products against second-stage larvaein vitro
and in vivo.
Exp Parasitol 2010; 126
Durapandiyan V, Ayyanar M, Ignacimuthi S. Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC Complement Altern Med 2006; 6
Kundu S, Roy S, Lyndem LM. Cassia alata L
.: potential role as anthelmintic agent against Hymenolepis diminuta
. Parasitol Res 2012; 111
Samanta S, Ansari M. Embryonation of ova of Toxocara canis
(Werner, 1782) and migration of the larvae in mice. Indian J Anim Sci 1990; 60
Othman AA. Therapeutic battle against larval toxocariasis: are we still far behind? Acta Trop 2012; 124
Musa D, Senocak G, Borazan G, Atlas M, Ozgonual A, Sogut O, et al
. Effects of Nigella sativa
and albendazole alone and in combination in Toxocara canis
infected mice. J Pak Med Assoc 2011; 61
Lai SC, Jiang ST, Chen KM, Lee HH. Matrix metalloproteinases activity demonstrated in the infective stage of the nematodes, Angiostrongylus cantonensis
. Parasitol Res 2005; 97
Gargili A, Demirci C, Kandil A, Cetinkaya H, Atukeren P, Gumustas MK.In vivo
inhibition of inducible nitric oxide and evaluation of the brain tissue damage induced by Toxocara canis
larvae in experimentally infected mice. Chin J Physiol 2004; 47
Darakhshan S, Bidmeshki Pour A, Hosseinzadeh Colagar A, Sisakhtnezhad S. Thymoquinone and its therapeutic potentials. Pharmacol Res 2015; 95–96
Nassef NA, El-Kersh WM, El-Nahas NS, Shams El-Din SA, Oshiba SF, Nosseir MM. Parasitological, histopathological, and immunohistochemical assessment of nitric oxide synthase inhibitor: aminoguanidine versus albendazole in the treatment of experimental murine toxocariasis. Menoufia Med J 2014; 27
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