Efficacy of Garlic and Mirazid in Treatment of the Liver Granuloma in Mice Infected with Schistosoma mansoni

MATERIALS AND METHODS

Infection of mice: S. mansoni cercariae and clean CD1 male albino mice were supplied from Theodor Bilharz Research Institute, Imbaba, Giza, Egypt. Male CD1 albino mice were infected with 80±10 S. mansoni cercariae per mouse by the tail immersion method, modified by Olivier and Stirewalt (1952). The ethical obligations to experimental animals were followed. The experiments were carried out at Zoology Department, Faculty of Science, Fayoum University, Fayoum, Egypt.

Experimental design: 40 mice were divided into four groups of 10 mice each. Group I was infected with S. mansoni cercariae and non-treated. Group II was infected with S. mansoni cercariae and treated with oral dose of garlic, 150 mg kg^-1 (TOMEX, ATOS Pharma, Egypt); Group III was infected with S. mansoni cercariae and treated with oral dose of Mirazid (600 mg kg^-1 b.wt., the oleoresin extract from Myrrh of the C. molmol tree, family: Burseraceae) is a product of Pharco Pharmaceutical Company, Egypt. Mirazid was given for three consecutive days on the empty stomach, at least 1 h before a meal regarding to the concentration of Mirazid illustrated in the brochure of the drug (Haridy et al., 2003) and group IV was infected with S. mansoni cercariae and treated with garlic and mirazid together. All mice were sacrificed at the end of the 8th week of the experiment (starting from the day of infection).

Parasitological parameters
Worm burden: Perfusion of adult worms from the liver and portomesenteric system was performed 8 weeks after infection according to Duvall and DeWitt (1967) and Smithers and Terry (1965). The percentage reduction in worm numbers after treatment was calculated by the method of Tendler et al. (1986) as follows:

where, P = % of protection, C = Mean number of parasites recovered from infected animals and V = Mean number of parasites recovered from treated animals.

Tissue egg load: The number of eggs per gram tissue (liver) was studied according to the procedure by Cheever (1968). Worm counts were made at 10-weeks post-infection after perfusing the hepatic portomesenteric vessels of the animal (Duvall and DeWitt, 1967) according to the procedure outlined by Smithers and Terry (1965). The whole livers of both the treated and infected control mice were weighed and 0.5 g was removed, blotted between two filter papers and placed in a test tube containing 5 mL of 5% KOH solution at 37°C for 24 h until completely digested (Cheever, 1968).

Biochemical measurement: Blood was collected and serum was separated by centrifugation at 3000 rpm (10 min) for estimation of total bilirubin concentrations by colorimetrically measured (Shimadzu-CL 770 spectrophotometer), whereas aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were measured using the enzyme-kinetic method.

Histopathological study: Liver tissue specimens were fixed in 10% buffered formalin and then embedded with paraffin. Sections (4 μm thick) were stained with hematoxylin and eosin (H and E) to measure hepatic granulomas and to examine the associated histopathological changes. The size of Schistosoma granulomas at x10 was measured per section using an ocular micrometer. Only lobular granulomas containing an egg in the center and confluent were measured. The diameters of the 10 largest granulomas in each section were measured and mean granuloma diameter (±SD) calculated for each group of mice according to Lichtenberg (1962). Only granulomas appearing as circular in section were measured. Granulomas adjacent to areas of hepatocyte necrosis were excluded from diameter measurement.

Statistical analysis: Comparison was performed between the treated groups and untreated control using SPSS11 windows program (ANOVA test). The data were considered significant if p-values were less than 0.05.

RESULTS

Results in Table 1 showed the significant reduction (43, 51.5 and 70%) in the mean number of S. mansoni adult worms in the groups of infected mice treated with garlic, mirazid and mixture of garlic and mirazid, respectively when compared to the infected controls (p<0.01, 0.009, 0.005, respectively ) where, the number of worm loaded in infected mice with schistosoma was 25.6±0.55 which reduced into 14.39±0.43 in garlic treatment, 12.4±0.23 in mirazid treatment and 8.18±0.09 in garlic and mirazid treatment groups.

Table 1:	Effects of mirazid (C. molmol derivative) and garlic on worm load in mice infected with Schistosoma mansoni
The data presented as Mean±SD, *p<0.05 vs. untreated control

Table 2:	Effects of myrrh derivative (mirazid) and garlic on egg load in mice infected with Schistosoma mansoni
The data presented as Mean±SD, *p<0.05 versus untreated control

Table 3:	Effects of myrrh derivative (mirazid) and garlic on liver functions in mice infected with Schistosoma mansoni
The data presented as Mean±SD, *p<0.05 versus untreated control, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase

Moreover, in the Table 2, significant reduction in the mean number of ova/gram tissue of liver was detected in the groups treated with garlic (3920 ova), mirazid (2801 ova) and mixture of garlic and mirazid (1200 ova) compared to infected group (6856), (p<0.01, 0.007 and 0.005, respectively).

The Activities of Serum AST, ALT and total bilirubin of normal controls and treated groups are represented in Table 3. Serum ALT and AST activities in infected mice (group I) were significantly increased compared to group II (infected group received garlic) during the experiment. On the other hand, serum ALT and AST activities in the infected mice receiving mirazid (group III) were decreased and significantly decreased in the group IV (infected group received garlic and mirazid).

After 8 weeks post S. mansoni infection, typical hepatic bilharzial lesion is observed in sections of infected mouse liver. Schistosome eggs are surrounded by inflammatory cells and schistosome fibrotic changes are evident in this area. Histopathological examination of the liver of infected non-treated mice revealed chronic granulomatous inflammation characterized by inflammatory infiltrate and numerous granulomas. Granulomas were marked by concentric fibrosis with many fibroblasts encircled the trapped eggs. They were surrounded by a cuff of aggregated lymphocytes, epithelioid cells, eosinophils and collagenous fibers.

Fig. 1(a-f):

Hematoxylin and Eosin stain Photomicrographs of a liver section of S. mansoni-infected, (a, b and c): Non-treated mice showing formation of granuloma (G) with trapped central eggs (arrow) surrounded by concentric rings of epithelioid cells and lymphocytes, The disorganized hepatic strands, dilated sinusoids (*), some necrotic hepatocytes and mass of lymphocytes and fibrotic projection into the lumen of the portal vein (curved arrows). (d and e): Photomicrographs of a liver section of S. mansoni-infected and treated with Mirazid and garlic mice, respectively showing a reduced granuloma size, accumulation of inflammatory cells surrounding the granuloma (IC), vacuolated hepatocytes and some hypereosinophilic hepatocytes. (f): Photomicrograph of a liver of S. mansoni-infected, garlic and mirazid mixture treated mouse showing a remarkably reduced granuloma and ameliorated hepatocytes. However, there are some inflammatory cells (IC) and some dilated sinusoids (*)

Table 4:	Granuloma diameters (μm) in the liver of the treated and non-treated animal groups
The data presented as Mean±SD, *p<0.05 versus untreated control

Perigranulomatous hepatocytes revealed hypereosinophilia, vacuolar degeneration and necrotic foci characterized by nuclear pleomorphism. Portal veins were dilated and infiltrated with mass of inflammatory cells.

Examination of the liver of garlic, mirazid and a combination of garlic and mirazid treated mice showed a significant reduction in the number and size of granulomatous inflammation compared with infected non-treated ones. The hepatic lobular architecture restored its normal feature and most hepatocytes showed normal appearance. However, some sinusoids were still dilated and infiltrated with lymphocytes (Fig. 1). The mean diameter of the hepatic granuloma was significantly smaller in infected and treated mice than in infected and non-treated mice (p<0.05, Table 4).

DISCUSSION

During the past decades, the intensive use of chemical anti-helminthic (AHs) has unfortunately led to the widespread development of resistant in worm populations. Considerable attention has been given recently to bioactive plants which can be used as non-conventional AHs.

The search for bioactive plants which can be used as non-conventional antihelmintics (AHs) has received considerable attention in recent times because of the increasing, worldwide development of resistance to chemical anti-helminthic in worm populations. However, scientific evidence to validate the use of plants remains limited (Hoste et al., 2008). Thus, this study was oriented to evaluate the protective and curative capacity of garlic against S. mansoni through different means. The assessment of the influence of garlic administration on infected mice has been attained by comparing the results of treated infected mice to that of the corresponding Mirazid (Myrrh) treated-infected mice (control group).

The anti-schistosomal drug Mirazid has been available in the local Egyptian market since 2001. Studies on its anti-schistosomal efficacy, whether in experimental animals or in humans, has been reported by the discoverers of these properties and no independent confirmation has appeared so far of these anti-schistosomal effects (El-Baz et al., 2003). Furthermore, a multicenter investigation of the potential anti-schistosomal activity of different derivatives of the resin, including the commercial preparation Mirazid was tested in experimental animals. The drug was found toxic for mice at high doses and produced modest or no worm reduction at lower doses (Botros et al., 2004). In the current study, the efficacy of myrrh in treating S. mansoni infections was tested and was compared with garlic treatment.

However, the results obtained showed that garlic or mirazid administration caused a highly significant reduction on egg loads in liver tissue.

The effect of garlic and mirazid may not be on ovum itself, since no effective anti-schistosomal drug acts on the eggs themselves. Eggs continue their development in the tissue up to maturation for a period of 12 days and elimination in the stools usually display no effect on the ova themselves. And ova depositions by worms continue their development in the tissue up to maturation. The mature ova remain alive in the tissues for a period of 12 days until their death and elimination in the stools (Standen, 1953). Accordingly, the significant reduction of ova might be due to the effect of garlic and Mirazid administration on worm fecundity, where some drugs seem to act initially on the reproductive organs of the worms. The reduction of ova loaded was occurred.

Riad et al. (2007) reported a significant reduction in the egg load after treating infected mice with aqueous garlic extract in acute and early chronic stages. El Shenawy et al. (2008) stated that aqueous garlic extract impaired the development and maturity of Schistosoma eggs, as the treatment resulted in the appearance of high numbers of dead eggs in the oogram assessment. This is possibly due to a positive linear relationship between the egg output and the worm burden, where the reduction of the number of worms is correlated with the reduction in the ova count. This is also suggested by Gryseels and Polderman (1991). However, several other factors may explain such reduction in schistosomal egg count. Probably, these factors diminished the fecundity of the worm pairs and increased the rate of egg excretion due to the egg death. Also, the present results recorded improvement levels of all enzymes under investigation after treatment of S. mansoni infected mice with mirazid and garlic. This amelioration was confirmed by a significant reduction of worm burden and ova count after treatment. Present data is confirmed by the previous reports by Hamed and Hetta (2005). They concluded that mirazid is an effective drug for treatment of Fasciola hepatica, S. mansoni and S. haematobium through worm disruption, collapse of tubercles and reduction of worm burden as well as the reduction in ova count in urine and stool (Hamed and Hetta, 2005). Toxic substances and free radicals elaborated by worms consume antioxidants and may affect the capacity of the liver to detoxify or naturalize the effect of the toxic endogenous and exogenous compounds (Sheweita et al., 1998). This gives an additional support that the improvement pattern in liver enzyme after treatment with Mirazid resulted from its antioxidant capacity.

The liver plays an important role in the vital activities of the body where its hepatocytes show differences in the localization and concentration of some enzyme systems. Many of these enzymes served as marker enzymes for different cell organelles and any defect of them will be reflected to the enzyme activity itself (Hamed and Hetta, 2005). Hence, studying changes in these enzymatic activities could be helpful in evaluating the possible side effects of different treatments on different cell organelles after S. mansoni infection and the improvement occurring in such enzymes after treatment.

Serum AST activity is more useful in assessing the histological severity of liver disease probably because this cytosolic and mainly mitochondrial enzyme is present in larger quantities in the liver compared to the cytosol and thus more is released when tissue damage is more severe (Aranda-Michel and Sherman, 1998). These enzymes are commonly employed as biological markers for hepatic cell damage and impaired cell membrane permeability or due to heavy Schistosoma egg deposition. There was significant difference (p<0.05) between the effect of Aged Garlic Extract (AGE) on AST, ALT and ALP (El Shenawy et al., 2008). So, In the present study, serum total bilirubin, ALT and AST activities increased progressively in S. mansoni infected mice in group I, this attributed to greater destructive changes in liver compared with the group II, III and IV.

Results showed that garlic and Mirazid administration showed marked anti-inflammatory activity where it significantly reduced the volume of granuloma size by 38, 46 and 67% in groups treated with garlic, mirazid and mixture of both them respectively when compared with the infected untreated group. Also, the hepatic lobular architecture restored its normal organization and most hepatocytes showed normal appearance, even in the vicinity of the granuloma. However, some sinusoids were still dilated and infiltrated with lymphocytes and some Kupffer cells were still hypertrophied, where the more hepatic lobular architecture restored its normal organization increasingly in treatment with garlic, mirazid and combination of them.

To our knowledge, this study is the first in using the combination of garlic and Mirazid in schistosomal treatment in mice. Marked treatment results were in infected mice treated with the combination of garlic and Mirazid than mice treated with either mirazid or garlic alone.

In conclusion, garlic, mirazid and a combination of both have the anti-schistosomal activity in mice. It significantly reduced the production of eggs output in S. mansoni infected mice. In addition this study showed that garlic and mirazid clearly reduced liver granuloma size and this modulation of the liver granulomas was reflected on some degree of improvement in the status of the liver.