Abstract: The effect of Schistosoma mansoni infection on the activities of aspartate and alanine aminotransferases, acid phosphatase, 5’nucleotidase, glucose-6-phosphatase, lactate dehydrogenase, alkaline phosphatase and succinate dehydrogenase were estimated as measures of hepatic metabolic function. The effect of infection, on free radical production in the form of lipid peroxides and on the levels of certain antioxidants namely, catalase, glutathione, vitamins C and E was also studied. The efficiency of administration of the chloroform extract of Ailanthus altissima stem bark and the alcoholic extract of Ziziphus spina christi roots as antischistosomal and hepatoprotective agents in infected mice was evaluated. In addition, the efficiency of the tested extracts on reducing the worm burden and ova counts in the infected mice was evaluated. The obtained data revealed that infection with S.mansoni increased lipid peroxides and decreased all antioxidant levels. On the other hand, the activities of acid phosphatase and 5`nucleotidase were higher while those of glucose-6-phosphatase, lactate dehydrogenase, alkaline phosphatase and succinate dehydrogenase were lower with respect to control .However treatment with both A.altissima and Z.spina christi ameliorated the disturbed lipid peroxides, antioxidants and enzymes’ levels to nearly the control values, the choloroform extract of A.altissima showing a more pronounced improving effect against liver damage caused by parasitic infection. This study confirms the recent approach of many researchers for the use of plant extracts for treatment of liver diseases as alternatives to the classical chemotherapeutic drugs since the latter may cause hazardous side effects, besides the achieved tolerance to prolonged drug administration.
Introduction
Schistosomiasis continues to plague populations living in endemic areas and exposure to infective cercariae results in more than 200 cases worldwide (Naples et al., 2005). Its rank follows malaria, at the second position of the world’s parasitic diseases in terms of the extent of endemic areas and the number of infected people. In Egypt, the disease remains a public health problem despite the continuous control efforts (El Baz et al., 2003). The disease causes tremendous disorders in liver tissue resulting in fibrosis due to spontaneous modulation of the egg granuloma (Farrah et al., 1998). Also, hepatic marker enzymes exhibit marked changes by schistosomal infections. There is yet no vaccine available and most of the antischistosomal drugs have deleterious side effects or low efficacy. However, the current main control is chemotherapy with the drug of choice, praziquantel. In view of concern about the development of tolerance and/or resistance to PZQ, there is a need for research and development of novel drugs for the prevention and cure of schistosomiasis (Utzinger et al., 2001). This approach, however, necessitates a search for new safe and effective drugs (Abo-Madyan et al., 2004).
In recent years, researchers have examined the effects of plants used traditionally by indigenous healers and herbalists to support liver function and treat diseases of the liver. In most cases, research has borne out the traditional experience by discovering the mechanisms and modes of action of these plants, as well as confirming the therapeutic effectiveness of certain plants or plant extracts in clinical studies.
Previously, the schistosomicidal properties of the ethanol and acetone extracts of Pavetta owariensis and Harrisonia abyssinieae were assessed in S. mansoni infected mice and showed promising effects on the spleen weight, number of adult worms and eggs and on size of granulomas (Balde et al., 1989). In addition, 23 plant species used popularly against schistosomiasis in Zimbabwe were screened for their antihelmintic effect (Molgaard et al., 2001). Later, Mahmoud et al.(2002) reported that Nigella sativa oil may play a role against the alterations caused by S. mansoni infection by improving the immunological and biochemical parameters in the host liver. Recently, Yousif and El-Regal (2004) showed that two C-glycosyl flavone O-glycosides isolated from Clerodendrum splendens G.Don leaves possessed strong hepatoprotective effects by improving the reduced levels of hepatic antioxidants in S. mansoni infected mice.
In the present study, the chloroform extract of Ailanthus altissima (Mill) stem bark and the alcoholic extract of Zizyphus spina roots were screened for their hepatoprotective effect since both these extracts were previously reported to possess a variety of biological activities. Thus, Buzina et al. (2001) showed that A. altissima possessed an in vitro antituberculosis activity. Moreover the methanolic extract of the stem barks of A. excelsa revealed fungistatic and fungicidal activities (Joshi et al., 2003). Also, extracts and isolated compounds from seedlings of A. altissima showed activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum (Okunade et al., 2003).
On the other hand, Zizyphus spina christi is one of the plants commonly used in Egyptian folk medicine for the treatment of different diseases. Thus, the butanol extract of Z. spina-christi leaves showed strong hypoglycemic and antihyperglycemic effects in diabetic rats (Glombitza et al.,2002).Also Adzu et al.,(2001) revealed an analgesic effect for Z. spina-christi root bark extract in mice and rats. In a related study, Shahat et al. (2001) investigated different extracts and fractions of the leaves, fruits and seeds of the plant grown in Egypt and reported that they possess antiviral, antifungal and antibacterial activities. In a more recent study by Adzu et al. (2003), the methanol extract of Z. spina-christi stem bark showed an antidiarrhoeal effect in rats.
Based on the previous information on the wide range of biological activities of both A. altissima and Z. spina christi, the present research aims to evaluate new biological activities for both these plants in a trial to open new areas of application of extracts of these plants as novel antischistosomal, antioxidant and hepatoprotective agents in folk medicine.
Materials and Methods
Animals
Forty-eight male albino mice weight range (20-25 g) were caged with a free
supply of food and water. After acclimization, they were randomly assigned into
six groups of eight mice each and were treated as follows:
| • | The 1st group was infected with cercariae of the Egyptian strain S. mansoni by the tail immersion method and were kept to develop liver granuloma for four months. |
| • | The 2nd and 3rd groups were infected with S. mansoni, left for three months, then treated with a total dose of 1/4 LD50 of both the chloroform extract of A. altissima stem bark (500 mg kg-1 b.w, using serial concentrations ranging from 100-4000 mg kg-1 b.w) and 70% ethanolic root bark extract of Z. spina christi (560 mg kg-1 b.w. according to Adzu et al., 2001) respectively five times weekly for one month |
| • | The 4th and 5th groups were treated as described above with the previous test extracts respectively after three months from the start of the experiment. |
| • | The 6th group was left uninfected untreated and served as control. |
Mice of all groups were sacrificed after four months. Appropriate anaesthetic and sacrifice procedures were followed ensuring that animals did not suffer at any stage of the experiments. Anaesthetic procedures are complied according to legal ethical guidelines approved by the Ethical Committee of the Federal Legislation and National Institute of Health Guidelines in USA. An overdose of ether was given gradually to mice and then the abdomen was opened by a mid-line incision and livers were separated. The livers of infected groups were perfused for worm (Smithers and Terry, 1965) and ova counts (Cheever and Anderson,1971)and then livers of all groups were subjected to the different antioxidant and hepatoprotective assays.
Antioxidant experiments were performed on fresh liver tissues whereas samples of liver tissues from the different mice groups were stored frozen at -80°C throughout a one week period for the hepatic marker enzyme activities.
Extraction of Plant Material
One kilogram of each of A. altissima stem bark and Z. spina christi
root bark were extracted with chloroform and 70% alcohol respectively in a continuous
extraction Soxhlet apparatus till exhaustion and the extracts were concentrated
under reduced pressure. The extracts were phytochemically screened and the results
showed the presence of alkaloids, sterols and/or triterpenes in both extracts
while saponins were also present in Z. spina christi extract.
Biochemical Assays
Antioxidant Evaluation
Lipid peroxides: one volume of saturated thiobarbituric acid in 10% perchloric
acid were added to three volumes of 20% trichloroacetic acid (working solution).
Malondialdehyde (MDA), the end product of lipid peroxidation is formed by reaction
of lipid peroxides present in 20% tissue homogenate with thiobarbituric acid
and boiling to give a pink coloured product that is read at 535 nm (Okhawa et
al., 1979). Malondialdehyde concentration was calculated using the extinction
coefficient value of 1.56<105 M-1cm-1 (Buege
and Aust, 1978).
Glutathione
GSH was estimated in 20% tissue homogenate by the method of Moron et
al. (1979) using 0.6mM dithiobisnitrobenzoic acid (DTNB) dissolved in 0.2
M phosphate buffer(pH 8). The developed colour in the supernatant after centrifugation
was read against blank at 412 nm within 5 min and glutathione was calculated
as mg g-1 tissue from a standard curve plotted for serial concentrations
of glutathione (5-100 μg).
Estimation of Vitamin C
Vitamin C was estimated by the method of Jagota and Dani (1982) by mixing
2M Folin-ciocalteu reagent and 10% TCA with 20% tissue homogenate. The developed
blue colour in the supernatant after centrifugation was read at 760 nm after
10 min. The amount of ascorbic acid was calculated from a standard curve of
vitamin C using 5-70 μg serial concentrations of the vitamin.
Estimation of Vitamin E
Vitamin E was measured by the colorimetric assay using the method of Angustin
et al. (1985).The method is based on the oxidation of xylene-extracted
tocopherols of the liver homogenate by ferric chloride and the pink complex
of ferrous ions with bathophenanthoroline is measured calorimetrically at 536
nm.
Estimation of Catalase
Catalase activity was assayed according to Lubinsky and Bewley (1979).The
reaction for assaying catalase activity was initiated by adding 20 μl 5%
liver homogenate to 2.53 mL of the reaction mixture (0.1M sodium potassium phosphate
buffer, pH 6.8 mixed with 120 mL H2O2). The disappearance
of hydrogen peroxide was monitored by following the decrease in absorbance at
230 nm using molar extinction coefficient for hydrogen peroxide of 62.4 (Nelson
and Kiesow, 1972).
Hepatic Marker Enzymes
Alanine and Aspartate Aminotransferases
ALT and ALT were measured by the Reitman and Frankel method (1957) using
the bioMerieux kit. The colorimetric determination of ALT and AST depends on
determining amounts of oxaloacetate and pyruvate formed from the 2,4-dinitrophenylhydrazine
of oxaloacetate and pyruvate, the colour of which is read at 520 nm.
Lactate Dehydrogenase
LDH was measured by monitoring the increasing intensity of NADH production
as a result of oxidation of lactate to pyruvate. Reduction of NAD is coupled
with reduction of tetrazolium salt (INT) with phenazine methosulphate as electron
carrier, the result is the formation of formazan of INT. The developed colour
is read at 503 nm (Babson and Babson, 1973).
Acid Phosphatase
The enzyme was estimated by the method of Wattiaux and De Duve (1956) using
Fiske-Subbarow reducing agent and sodium ß-glycerophosphate as substrate
in the presence of Triton-X-100. The liberated inorganic phosphate was read
at 660 nm.
5`Nucleotidase
The assay was carried out by the method of Bodansky and Schwartz (1963)
depending on the hydrolysis of 5’AMP to adenine and inorganic phosphate
in the presence of MgCl2 as a catalyst. The developed colour of liberated
inorganic phosphate is read calorimetrically at 660nm using Fiske-Subbarow as
the reducing agent.
Succinate Dehydrogenase
The enzyme catalyzes the oxidation of succinate to fumarate and reduction
of FAD coupled with reduction of tetrazolium salt (INT). 0.2M Tris buffer was
added to 0.5 M sodium succinate and 0.02 M INT. The produced formazan is measured
at 490 nm (Shelton and Rice, 1957).
Glucose-6-phosphatase
The enzyme is measured by the method of Swanson (1955) which depends on
measuring the liberated inorganic phosphate from glucose-6-phosphate as substrate
using Fiske-Subbarow as a reducing agent, 0.2 M tris buffer 0.001 M sodium sulphate
and 0.1 M substrate reagent. The developed colour is read at 660 nm.
Alkaline Phosphatase
The enzyme was estimated by the method of Belfield and Goldberg (1971) using
disodium phenyl phosphate and 0.05 M carbonate-bicarbonate bufferin a mixture
of 1:2 (pH 10). The liberated phenol is measured at 510 nm in the presence of
amino-4-antipyrene, sodium arsenate and potassium ferricyanide as the colour
reagent.
Antischistosomal Activity
Adult S. mansoni worms were recovered from the hepatic portal system
and the liver by the perfusion technique and the number of ova/g liver tissue
was calculated.
Estimation of Protein
Protein was estimated by the method of Bradford (1976). Bradford solution
was added to 5% liver homogenate of the different mice groups and the developed
blue colour was measured after 5 min at 595 nm in spectrometer (Novaspec, LKB,
Sweden) against a blank containing water instead of the homogenate. The amount
of protein was calculated from a standard curve using serial concentration of
bovine serum albumin (1-10 μg).
Statistical Analysis
Data were expressed as mean±SD. One way analysis of variance (ANOVA)
followed by LSD was used to show the statistical significance among the different
groups. The Statistical Package for the social Sciences(SPSS)version 8.0 was
used in data analysis. The probability level of significance (p) was determined
where p<0.005 was considered significant.
Results
The data obtained revealed that both extracts significantly ameliorated the elevated level of the lipid peroxides and the decreased levels of glutathione, vitamin C, vitamin E and catalase induced by infection. However, the chloroform extract of A. altissima caused a more pronounced improvement in the measured parameters (Table 1, Fig. 1 and 2).
In Table 2 and Fig. 3 and 4, results indicate that infection resulted in a significant increase in the activities of acid phosphatase and 5’nucleotidase and a significant decrease in the activities of glucose-phosphatase, lactate dehydrogenase and succinate dehydrogenase. Treatment with the two test extracts restored the activities of these enzymes to values near to normal.
| Table 1: | Effect of Ailanthus altissima and Ziziphus spina christi extracts on lipid peroxides and hepatic antioxidants in control and Schistosoma mansoni infected mice |
| Data are means±SD of six mice in each group. Values of Lipid peroxide are expressed as nm/mg protein, glutathione and Vitamin (C and E) are expressed as μg/mg protein and Catalase are expressed as μM/mg protein. ≤P is level of significance, where P<0.005 is significant. Analysis of data is carried out by one way (ANOVA) (analysis of variance) accompanied by post hoc (spss computer programme) | |
| Fig. 1: | Percentage change in lipid peroxides, glutathione and catalase in different mice groups as compared to control |
| Fig. 2: | Percentage change in vitamin C and vitamin E in different mice groups as compared to control |
| Table 2: | Effect of Ailanthus altissima and Ziziphus spina christi extracts on hepatic marker enzymes in control and Schistosoma mansoni infected mice |
| Data are means±SD of six mice in each group, Values of the enzymes are expressed as μmole/min/mg protein, p is level of significance, where p<0.005 is significant, Analysis of data is carried out by one way (ANOVA) (analysis of variance) accompanied by post hoc (spss computer programme) | |
| Fig. 3: | Percentage change in acid phosphatase (ACP) and 5` nucleotidase in mice livers of different groups as compared to control |
| Fig. 4: | Percentage change of G-6-pase, LDH and SDH in hepatic mice livers of different groups as compared to control |
| Table 3: | Effect of Ailanthus altissima and Ziziphus spina christi extracts on liver function enzymes (AST, ALT, ALP), egg count and worm burden in Schistosoma mansoni infected mice |
| Data are means±SD of six mice in each group, Values of the enzymes are expressed as μmole/min/mg protein, pis level of significance, where p<0.005 is significant, Analysis of data is carried out by one way (ANOVA) (analysis of variance) accompanied by post hoc (spss computer programme), Egg count are expressed/g tissue of liver | |
The efficiency of the two extracts under study as antischistosomal agents by reduction in worm burden and egg counts and also by the improvement of the liver function enzyme levels, AST, ALT and alkaline phosphatase is shown in Table 3, Fig. 5 and 6.
| Fig. 5: | Percentage change in AST, ALT and ALP in different mice groups as compared to control |
| Fig. 6: | Percentage change in egg count and worm burden in different mice groups as compared to infected group |
The results obtained demonstrated the potency of these extracts with A. altissima chloroform extract revealing a more pronounced effect on all parameters while Z. spina christi ethanolic extract showing a more normalizing effect on alkaline phosphatase.
Discussion
Previous studies have shown that the host’s response to Schistosoma mansoni infection involves the production of reactive oxygen species (El-Sokkary et al., 2002). In another study, Sayed and Williams (2004) reported that schistosomes use alternative electron donors and their variable resistance to over oxidation may reflect their presence in different cellular sites and emphasizes the significant differences in overall redox balance mechanisms between the parasite and the mammalian host. The data in the present work reveal that S. mansoni infection caused an elevation in lipid peroxides while glutathione, vitamin C, vitamin E and catalase activity were significantly reduced. These results suggest that infection with S. mansoni impairs the antioxidant system since the level of GSH depletion is used as an index of oxidative stress and a sign that hepatic cells are utilizing more antioxidant defenses (Ip et al., 2000). In a good agreement with the obtained data, Pascal et al. (2000) and Soliman et al. (2000) reported that oxidative stress due to schistosomiasis causes an elevation in lipid peroxides. On the other hand, Gharib et al. (1999) showed that liver GSH was drastically depleted in mice infected with S. mansoni due to increased cytotoxicity with H2O2 which is produced as a result of inhibition of glutathione reductase, that keeps glutathione in the reduced state.
With regards to vitamin C and coinciding with the present results, Frei et al. (1988) reported that peroxyl radicals are trapped by ascorbate and hence the level of the enzyme decreased during the free radical scavenging process. Also, the reduction in vitamin E after schistosomal infection occurs since the vitamin acts as a soluble antioxidant to protect biological membranes against oxidative stress which leads to disruption of cell function. In a related study, Sokol et al. (1998) reported that vitamin E protects hepatocytes against lipid peroxidation and toxic injury.
During oxidative stress, such as in case of schistosomal infection, peroxide dismutation yields H2O2, which is detoxified by catalase and thus results in decline in its activity as previously indicated by Gharib et al. (1999). Also Hahn et al. (2001) reported the participation of catalase in scavenging H2O2. In a recent study, Hanna et al. (2005) added that eosinophil peroxidase and its substrate H2O2 are released by inflammatory cells in the immediate vicinity of parasite eggs. Administration of both the chloroform and ethanolic extracts to non-infected mice revealed a non toxic effect since the levels of the measured parameters were nearly similar to normal values. However, administration of these extracts to infected mice ameliorated the levels of these parameters since lipid peroxides level was reduced while vitamin C, vitamin E and catalase activity were elevated with respect to the infected groups, although the obtained data did not reach the normal values.
In accordance with this, Rizk (1998) reported the protective effect of the alcoholic extract of Curcuma longa against oxidative stress in S. mansoni infected mice and showed that this extract improved the elevated lipid peroxides and reduced glutathione, vitamin C, vitamin E and catalase observed in the infected mice groups. Also, El-Sokkary et al. (2002) showed that melatonin reduces oxidative damage and increases survival of mice infected with S. mansoni and improved levels of lipid peroxides, glutathione vitamin E and superoxide dismutase. In a recent study, Yousif and El-Rigal (2004) studied the antioxidant activity of the aqueous and the flavone-containing fractions of the leaves of C. splendens G. Don and reported that the aqueous fraction showed a more antioxidant powerful activity, probably due to a synergistic effect of all its constituents including the flavone-containing fraction, to diminish the adverse effects of free radicals on hepatic cells.
The data obtained in the present study for the hepatic marker enzymes revealed a marked increase in the activity of acid phosphatase and 5’nucleotidase in S. mansoni infected mice while the activities of aspartate and alanine aminotransferases, glucose-6-phosphatase, lactate dehydrogenase and succinate dehydrogenase were reduced.
With respect to transaminases, a significant reduction was observed in both AST and ALT activities following schistosomal infection while treatment with the two test extracts induced a moderate increase. As previously mentioned, the free radicals are elaborated by schistosomal infection and this may cause irreversible damage to the mitochondrial membrane which may lead to discharge of its enzyme content. In this regard, Ozares et al. (2003) stated that these enzymes were decreased relative to the lowered liver protein content either due to their release to the blood stream or to decreased synthesis. Since aminotransferases are marker enzymes for cell toxicity, this gives an additional support on the liver injury induced by infection. This hepatocellular damage results from egg deposition resulting in cell fibrosis and/or increased cell permeability leading to enzyme discharge to the blood stream (El-Shazly et al., 2001).
Concerning acid phosphatase and 5`nucleotidase, the elevation in their activity may be due to tissue catabolism resulting from increased worm and egg toxins of infection and due to the deranged metabolic functions as a result of liver injury. In a related study, Rizk (1997) indicated that all lysosomal enzymes are activated in conditions of increased tissue catabolism leading to enhancement of phagocytic phenomenons. The results also confirmed the earlier studies by Rodrigues (1988) who observed changes in the lysosomal membrane of S. mansoni infected mice which are provoked by the catabolites excreted by immature or adult worms present in the portal venous system.
The depletion in hepatic microsomal glucose-6-phosphatase was previously reported by Rizk (1998) who described a marked decrease in glycogen content due to disturbance in glycogen synthesis with progressive decrease in glucose-6-phosphatase activity in cases of heavy schistosomal infections. Also the decrease in the enzyme activity resulted from hepatic hypoglycemia occurring during S. mansoni infection, resulting from the inability of hepatic cells to dephosphorylate glucose-6-phosphate into glucose (Metwally and Fahim, 1994).
Lactate dehydrogenase is a glycolytic enzyme located in the cytoplasm. In the present research the depletion in enzyme activity in the direction of lactate oxidation may be correlated with glycogen depletion confirming inhibition of aerobic glycolysis and stimulation of anaerobic glycolysis induced by the developing parasite. This aerobic-anaerobic switch was previously reported by Tielens (1994 and 1997) on the crabtree effect of schistosomes through which lactate is accumulated and glycogen is depleted.
Concerning mitochondrial SDH, the activity of the enzyme was reduced followed by infection. This was supported by Van Hellemond and Tielens(1994) reported that both SDH and Kreb`s cycle enzyme activities are repressed when limiting amounts of oxygen are present while Volpi et al. (1997) added that the increased NADH/NAD ratio, causes shift to the left in the equilibrium of the oxidoreductive couple malate-oxaloacetate resulting in depression of citric acid cycle and may contribute to SDH lowered activity.
It should be pointed out that treatment with the test extracts significantly ameliorated the levels of the hepatic marker enzymes and decreased the derangements in the different subcellular fractions. Previous research in the area of exploring new extracts from plant materials has proved a promising approach for the treatment of various diseases. Zaoui et al. (2002) studied the effect of Nigella sativa seed fixed oil on key hepatic enzymes in mice and supported the traditional use of this extract as a treatment of the dyslipedemia and hypoglycemia related abnormalities. A related study by Mahmoud et al. (2002) revealed that the same extract induced a hepatoprotective effect in liver damage caused by S. mansoni by ameliorating the liver function, the redox state and the liver granuloma.
In addition, El-Sokkary et al. (2002) measured alkaline phosphatase, total protein, albumin and other antioxidant parameters as evidences of liver damage in S. mansoni infected mice and concluded that melatonin administration prevented most of the changes that occurred. In a more recent study, Abo-Madyan et al. (2004) evaluated the efficacy of Mirazid (the resin extract from Myrrh of commiphora molmol tree on the treatment of both S. mansoni and S. hematobium and reported that the extract proved to be safe and very effective.
In good coincidence with the present data, Glombitza et al. (2002) reported that the butanol extract of Z. spina-christi leaves reduced the serum glucose level, liver phosphorylase and glucose-6-phosphatase activities in diabetic rats and also increased serum pyruvate level and glycogen content. The present research was further extended to investigate the antischistosomal activity of the tested extracts by measuring the reduction in the worm and egg counts. It was found that both extracts exerted significant effects, the chlororoform extract showing a more potent activity. Similar studies have revealed the efficiency of different plant extracts on reducing the number of developed worms and the egg count of mature parasites. Thus, Massoud et al. (2004) and Haridy et al. (2003) indicated that mirazid is an effective fasciolicidal drug and also reported its use in treatment of Schistosoma hematobium.
Conclusions
It could be concluded that Schistosoma parasite generates free radicals in the infected host and these disorders are ameliorated by treatment with the two natural extracts under study. Current estimates indicate that about 80% of people in developing countries still rely on traditional medicine based largely on various species of plants and animals for their primary healthcare. As a consequent, thirty per cent of the worldwide sales of drugs is based on natural products. Thus, the use of natural extracts as alternatives to the chemically synthesized formulations may prove a successful tool in drug technology for treatment of different diseases. Both the test extracts under study proved potent antioxidant and hepatoprotective activities and may be applied in folk medicine for improving liver disorders.