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| ORIGINAL ARTICLE |
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| Year : 2017 | Volume
: 6
| Issue : 1 | Page : 57-61 |
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Effect of Commiphora molmol extract (Mirazid) on the tegument of Schistosoma mansoni and serum acetylcholinesterase activity in infected mice
Mohammad Aziz Al-Kazzaz
Department of Parasitology, Medical Research Institute, Alexandria University, Alexandria, Egypt
| Date of Web Publication | 17-Jul-2017 |
Correspondence Address:
Mohammad Aziz Al-Kazzaz
Department of Parasitology, Medical Research Institute, Alexandria University, P. O. Box 21561, 165 El-Horreya Avenue, El-Hadara, Alexandria
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/sjhs.sjhs_72_16
Context: Scanning electron microscopy (SEM) has become a useful tool for the study of the ultrastructural changes of the surface of the Schistosoma worms in response to chemotherapy as well as evaluation of serum acetylcholinesterase (AChE) determine safety of the drug on cholinergic function. Aims: The aim was to study the effect of Mirazid (MZD) on the teguments of Schistosoma mansoni worms recovered from infected mice and its effect on the cholinergic function of treated animals compared to praziquantel (PZQ). Materials and Methods: Forty albino mice were enrolled in four groups of ten mice each: Group 1: normal group; Group II: infected control given only the vehicle; Group III: infected mice treated orally with MZD in a dose of 500 mg/kg for 5 days at 7-week postinfection; and Group IV: infected mice treated orally with PZQ 500 mg/kg for 2 days. Two-week posttreatment, mice were sacrificed and Schistosoma worms were examined by SEM and blood samples were taken for estimation of serum AChE. Statistical Analysis Used: The data of serum AChE level were analyzed using Student's t-test for comparison of means. Results: PZQ caused extensive tegumental damages in male and female worms in the form of rupture of tubercles and loss of spines in male worms and ulceration in female worms. MZD showed tegumental damages with shrinkage of the outer surface of male tubercles with marked loss of spines and mild ulceration in female worms. The two drugs showed nonsignificant effect on serum AChE compared to the normal mice. Conclusions: MZD showed potent antischistosomal activity but lower than that of PZQ and the two drugs had no effect on cholinergic function at the studied dose.
Keywords: Acetylcholineasterase, mice, microscopy, Mirazid, praziquantel, Schistosoma mansoni
How to cite this article:
Al-Kazzaz MA. Effect of Commiphora molmol extract (Mirazid) on the tegument of Schistosoma mansoni and serum acetylcholinesterase activity in infected mice. Saudi J Health Sci 2017;6:57-61 |
How to cite this URL:
Al-Kazzaz MA. Effect of Commiphora molmol extract (Mirazid) on the tegument of Schistosoma mansoni and serum acetylcholinesterase activity in infected mice. Saudi J Health Sci [serial online] 2017 [cited 2022 Jun 28];6:57-61. Available from: https://www.saudijhealthsci.org/text.asp?2017/6/1/57/210815 |
| Introduction | |  |
The tegument of schistosomes has been described as a living, anucleate, and cytoplasmic structure covering the outer surface of the worm.[1] The tegument has secretory functions, is involved in nutrient absorption, and shields schistosomes from the immune response by the infected host.[2],[3] Scanning electron microscopy (SEM) has become a useful tool for the study of the ultrastructural changes of the surface of the Schistosoma worms in response to chemotherapy by showing the effect on the tegumental structures (tubercles, spines, intertubercular ridges), oral and ventral suckers.[4] The ultrastructural changes are directly proportional to the potency of antischistosomal drugs [5] and may clarify the procedure of killing these worms.[6] It was suggested that focal tegumental damage induced by an antischistosomal drug might be repaired effectively over the course of 7–14 days after cessation of the drug while in case of severe tegumental damage, the host immune response might impact this repair process.[7] These morphological alterations are accompanied by an increased exposure of schistosome antigens (epitopes) at the parasite surface,[8] leading to disappearance of the immunological “disguise” of the worm and also inability to engulf food by oral and ventral suckers. This is believed to be of prime importance in causing death of the worms.[9],[10]
Cholinesterases are enzymes (protein in nature) present in cholinergic and noncholinergic tissues as well as blood and other body fluids. There are two isoforms according to their catalytic properties and specificity for substrates, sensitivity to inhibitors, and tissue distribution (butyrylcholinesterase [BChE] or pseudocholinesterase [PChE] and acetylcholinesterase [AChE] or true cholinesterase).[11] The two isoforms differ genetically, structurally, and for their kinetics.[12] AChE is synthesized mainly in the hepatocytes then released into the blood to its target sites. It is an enzyme participating in cholinergic neurotransmission as it breaks down acetylcholine (causes the stimulation of neurons) while cholinesterase causes the ending of stimulation by breaking down the acetylcholine into choline and acetic acid which terminates the neurotransmission process postsynaptically or through the neurosynaptic cleft in both the central and the peripheral nervous system (reaction necessary to allow a cholinergic neuron to return to its resting state after activation or stimulation).[13] The AChE is mainly found in the brain, muscles, erythrocytes, lymphocytes, and cholinergic neurons.[14],[15]
Inhibition of the AChE will cause high concentration of acetylcholine then accumulated leading to toxicity manifested by nicotinic, muscarinic, or central signs and symptoms according to the level of inhibition, and consequently, the receptors affected. The BChE is found in the intestine, liver, kidney, heart, lung, plasma, and serum. Butyrylcholine is not a physiological substrate in mammalian brain which makes the function of BChE of difficult interpretation with lower level than AChE. PChE has a broader range of esterase activity that can hydrolyze butyrylcholine, acetylcholine, and other aliphatic esters.[16],[17],[18],[19] Assay of AChE activity can serve for diagnosis after potential exposure to organophosphorus or carbamate pesticides and nerve agents.[20],[21] It can also be used for verification of treatment effectiveness, for example, for Alzheimer's disease therapy.[22]
| Materials and Methods | | |
Materials
Experimental animals
The study included 18 8-week-old female Swiss albino mice (Mus musculus) of the CD-1 strain.
Parasite strain
Laboratory-bred Biomphalaria alexandrina snails infected with miracidia of Egyptian (CD) strain of Schistosoma mansoni were obtained from the Schistosome Biological Supply Center, Theodor Bilharz Research Institute, Cairo, Egypt.
Drugs
Mirazid (MZD) capsules were obtained as free medical samples from Pharco Pharmaceuticals, Batch No: 296. Praziquantel (PZQ) as Biltricide tablets manufactured in Alexandria Company for Pharmaceutical and Chemical Industries, Batch No: 9118014.
Methods
Mice infection with Schistosoma mansoni cercariae
Infected B. alexandrina snails were washed with dechlorinated tap water and kept at an aquarium in an aerated (using electric pump) in a dark place under white fluorescent light for 30–60 min to release cercariae. Number of S. mansoni cercariae was counted and the average number per 1 ml was calculated according to Liang et al.[23] Mice were infected using paddling technique according to Smithers and Terry [24] with 100 cercariae/mouse. Stool examination was performed 45 days after cercarial infection to investigate the presence of S. mansoni eggs.[25]
Preparation of drug suspension
Preparation of praziquantel suspension
PZQ was prepared according to Bakr et al.[26] as fresh suspension was prepared by dissolving the tablet (600 mg) in 6 ml of 4% cremophor EL (4 ml cremophor EL + 96 ml sterile distilled water). Each mouse (20 g) requires 0.1 ml solution. A magnetic rod was placed into the flask, and then, the flask was put on a magnetic plate. The mixture was stirred for 30 min to ensure complete homogeneity of the drug suspension. The suspensions were dispensed into sterile labeled tubes with tight stoppers.
Preparation of Mirazid suspension
MZD was prepared according to Massoud et al.[27] as each capsule of the drug (300 mg) was evacuated in a flask containing 3 ml of 4% cremophore EL. Each mouse requires 0.1 ml solution.
Experimental design and treatment schedule
Forty albino mice were enrolled in four groups of ten mice each. Group 1: normal group and Group II: infected control given only the vehicle. Group III: infected mice treated orally with MZD in a dose of 500 mg/kg for 5 days at 7-week postinfection [27],[28] which is 4-fold the therapeutic dose in mice (125 mg/kg) based on the Food and Drug Administration guidelines. Group IV: infected mice treated orally with PZQ 500 mg/kg for 2 days [26] which is the therapeutic dose, but it should be given as a single dose.
Drug evaluation
Scanning electron microscopic study
Mice were sacrificed and perfused by the method of Smithers and Terry [24] to collect the worms from each group at 2-week posttreatment (WPT). Male worms were washed in saline then fixed in a fixative mixture containing 2.5% glutaraldehyde and formaldehyde in 0.15 M phosphate buffer (pH 7.2) at 4°C. Dehydration was carried out at room temperature through an ascending graded acetone series (30%–100%) and followed by critical point drying using liquid carbon dioxide (Samdri-PVT-3B, Tousimis, USA). Specimens were mounted on aluminum stubs with double-sided adhesive carbon and then coated with gold (fine coater). The specimens were examined in the Electron Microscope Unit, Faculty of Science, Alexandria University, using SEM model (Jeol-JSM-5300).[29]
Assessment of blood acetylcholinesterase activity
Blood samples were obtained before sacrifice of mice using capillary tubes introduced into the medial retroorbital venous plexus then centrifuged for serum collection. Evaluation of serum AChE level using clinical chemistry analyzer/spinLab (Spinreact, Spain) according to the colorimetric method of Ellman et al.[30]
Animal husbandry
Mice were housed in separate stainless steel wire mesh cages and received a standard well-balanced diet and water. Housing conditions were selected as controlled temperature (25°C ± 3°C) with a relative humidity of 50% ± 15% and a lighting conditions (12 h light/12 h dark cycle). Food as standard commercial diet pellets and distilled water were provided ad libitum. The mice were housed in accordance to the WHO and NRC guidelines.[31]
Ethical considerations
The study protocol was reviewed and approved by the Ethics Committee of the Medical Research Institute, University of Alexandria, Alexandria, Egypt.
Statistical analysis
The data of serum AChE level were analyzed using Student's t-test for comparison of means (Minitab version 14 statistical software, Minitab Ltd, State College, Pennsylvania, USA). Results were expressed as mean ± standard deviation and values of P > 0.05 were considered statistically nonsignificant while P< 0.05 was considered statistically significant.
| Results and Discussion | | |
In this work, PZQ caused pronounced tegument damage with rupture of tubercles and loss of spines in wide areas in male worms exposing the underlying muscle layers and ulcerations of female tegument [Figure 1]. These results were in accordance with several in vitro and in vivo studies that detected extensive tegumental damage in S. mansoni worms under the effect of PZQ.[9],[26],[32],[33],[34],[35],[36],[37],[38],[39],[40],[41] MZD resulted only in superficial tegumental damages in S. mansoni worms recovered at 2 WPT with shrinkage of the outer surface. It also resulted in marked loss of spines of male worms if present, loss of their sharpness with mild ulceration in female tegument. These results were agreement with previous reports [6],[26],[40] that showed exte nsive tegumental damages in S. mansoni worms eitherin vitro orin vivo under different concentrations or doses but disagreed with the results of Ramzy et al.[42] who reported that the dorsal and ventral surfaces of S. haematobium recovered from MZD-treated hamster (500 mg/kg for 3 days) were intact 3 months after treatment. | Figure 1: Scanning electron micrographs of the dorsal surface of female (right) and male (left) of Schistosoma mansoni worms recovered at 2 weeks posttreatment (normal worms [a1 and a2], Mirazid [b1 and b2] and praziquantel [c1 and c2])
Click here to view |
Infection of mice with S. mansoni resulted in a significant reduction of serum AChE activity in a rate 19.8% at 8-week postinfection (WPI) [Table 1] which is nearly similar to the results obtained by Sewify [43] (14% at 7 WPI) while Saba-El-rigal and Hetta [44] reported 56.1% inhibition in serum cholinesterase (SCE) activity in S. mansoni-infected mice at 8 WPI. It was attributed that the low SCE level is attributed to low serum total proteins which may be due to hepatocellular damage and consequently low serum proteins or may be due to secretion of toxins by the adult schistosomes inhibiting the enzyme activity. | Table 1: Independent samples' t-test comparing the mean difference of serum acetylcholinesterase level in normal and Schistosoma mansoni-infected mice treated with mirazid compared to praziquantel at 2 weeks posttreatment
Click here to view |
Treatment of infected mice with PZQ or MZD caused significant elevation in the depressed level of AChE. This effect was greater detected for PZQ (22.5%) than the observed level in MZD (16.2%) at 2 WPT compared to infected control mice which may be due to higher worm load reduction by PZQ than that achieved by MZD, but there is nonsignificant difference in proportions between PZQ and MZD which may be explained by the elevation effect of MZD on AChE level. Due to limited information concerning serum ACHE level in mice under the effect of PZQ, noncomparative data were noticed whereas it was noticed in MZD; Badria et al.[45] used the drug in dose of 500 mg/kg for 3 days for S. mansoni- infected mice and reported that death of adult worms may be due to paralysis and loss of musculature. Hassan et al.[6] and Sharaf [40] examined the muscle tension of S. mansoni worms under the effect of MZD in rising concentrations 100, 200, 300, and 400 μg/ml. The drug elicited somatic muscle contraction and reached highest response with the higher concentration. Massoud et al.[46] found that exposure of isolated rabbit duodenum to MZD in doses of 150–300 μg/ml induced inhibitory effect on motility but failed to evoke the contractile effect of acetylcholine (2 μg/ml). It was claimed that MZD is devoid of an effect on the muscarinic receptors. In normal noninfected mice, MZD proved to have highly significant stimulatory activity on angiotensin-converting enzyme level (14%).[44] It was claimed that MZD may act by stimulation of cholinesterase enzyme in the worms which leads to inhibition of acetylcholine and hence, muscle paralysis and finally death. Inhibition of acetylcholine in normal or inflamed muscle of rat or calcium antagonism was interestingly confirmed by the recent studies of Vissiennon et al.[47]
| Conclusions | | |
MZD showed potent antischistosomal activity but lower than that of PZQ and the two drugs have no effect on cholinergic function.
Acknowledgment
I would like to thank the staff members of the Electron Microscope Unit, Faculty of Science, Alexandria University, for their smart cooperation in the processing and examination of SEM pictures of this work.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1]
[Table 1]
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