Schistosomicidal effect of curcumin

Mohamed M*., Abdel-Aziz**, Kadry A. Elbakry*, Elshahat A. Toson*; , and Ayman T. Abbas**

*Faculty of Science-Damietta;MansouraUniversity, Mansoura, Egypt. **Gastro-EnterologyCenter, MansouraUniversity, Mansoura, Egypt.

Correspondance:

Dr. Kadry A. Elbakry

Zoology Dep.,

Faculty of Science-Damietta,

Mansoura University,

Mansoura,

Egypt.

Tel.: +2057403866

+2057403980

e-mail:

Abstract

Schistosomiasis is one of the most widespread parasitic infection.The use of medicinal plants or their active components in the prevention and treatment of chronic diseases is based on experience from traditional systems of medicine from various ethnic societies.Curcumin isanimmunostimulant andantioxidant herbal plant. In the present study, the effects of curcumin on mice infected with 100 cercariae of Schistosomamansoni (S. mansoni) were studied.Mice were treated with PZQ and curcumin ( 250 mg/kgm body weight 3 times and single dose of 20 mg/kg body weight respectively ) after six weeks of infection. The number of worms recovered from mice infected with S. mansoni and treated were reduced significantly (P<0.0001) for both groups compared with the infected nontreated group. The percent of reduction was 84.61 % and 67.3 % after treatment with PZQ and curcumin respectively. Along with this reduction, the optical density ofS. mansoniantibodies were also decreased significantly after treatment with praziquantel(p<0.0001) and curcumin (P<0.05) as compared with the infected non-treated group (Control group). Gamma interferon and interleukin -2 (secreted from mouse T-helper1 lymphocytes) play an important role on the cellular immune response during schistosomal infection. The present study found that,the mean serum level of IL–2 was decreased significantly in PZQ and curcumin treated group (P<0.05). On the other hand, the level of gamma interferon did not affected by the treatment with PZQ while it decreased in curcumin treated group (P<0.05). Superoxide dismutase enzyme was improved only after treatment with PZQ. We also found that, serum activity of SGPT enzyme, serum levels of cholesterol , triglycerides and LDL were reduced significantly after curcumin treatment. However the level of HDL was significantly elevated. In conclusion, curcumin mayreduce S. mansoni infection and improve the cellular and humoral immunity to S. mansoni infection.

Key words: Schistosoma mansoni, curcumin , Lipogram and cytokins

Introduction

Schistosomiasis is one of the most widespread parasitic infection and schistosomal hepatic fibrosis is of primary importance among chronic liver diseases worldwide (Mahmoud et al., 2002). Eggs of the adult worms of Schistosoma mansoni can pass through the intestinal wall to be deposited with the feces or carried by the blood flow into the liver where they induce vigorous granulomatous response which is the first step of schistosomal liver fibrosis (Boros, 1989). Chemotherapy is the most widely methods to control schistosomiasis (Tsai et al., 2000). Praziquantel is the current drug of choice and the cornerstone of schistosomiasis control over the last decades, and well remain the strategy of choice in the near future (WHO, 1999). Different studies have been shown that, there is a degree of resistance to praziquantel in both mice and human ( Fallon and Doenhoff 1994, Ismael et al., 1996) as mentiond by Picquer et al., 1998; De Clereg et al., 1999). According to these studies , an alternative safe and effective antischstosomal drug is urgently needed ( Sheir et al., 2001). Curcuminis an extract of the yellow Indian spice. Curcumin is an excellent antioxidant andincreases the secretion of bile by stimulating the bile duct also protects the liver by detoxification, stimulating the gall bladder and scavenging free radicals and may also help break down fats and reduce cholesterol ( Charles and Charles 1992). Curcumin Plays an important role in stimulation of the immunity (Antony S, et al. 1999). Churchill, et al. (2000), found that, the intestine after ingestion of curcumin, contains CD4+ T-helper (that help in cellular and humoral immune response) and B lymphocyte cells (antibody secreting cells)in a greater number. In regard to its antioxidant acivities, curcumin enhances glutathione content and glutathion-S-transferase activity in liver;inhibits lipid peroxidation.and a potent antioxidant.Alsocultured endothelial cells from bovine aorta, when incubated with curcumin, expressed heme oxygenase, an enzyme that reacts to oxidative stress, produced the antioxidant biliverdin, and showed enhanced resistance to oxidative damage. Curcumin's ability to quench singlet oxygen in an aqueous system has recently been reported (Pendurthi UR, et al. 1997 and Kunchandy & RAO, 1990)). Rabbits fed an extract of curcumin and a high-cholesterol diet developed fewer fatty streak aortal lesions and showed less depletion of vitamin E and coenzyme Q10 (Cronin , 2003). To our knowledge, there is no work conducted to study the role of curcumin in S. mansoni infection and so the present is aneffort todetermine the effect of curcumin on schistosomal infection through determination of the antischistosomal antibodies, worm burden, levels of cellular immune cytokines (Gamma interferon and interleukin–2), activity of superoxide dismutase enzyme and lipogram in comparing with the already knownantischoisatosomal drug "praziquantel".

Material and Methods

Animals

Swiss albino femalemice intraperitonealy injectedwith 100 cercariae of S. mansoni(Egyptian strain) were purchased from Theodoure Bilhars Institute, Cairo, Egypt.

Praziquantel and curcumin treatment

After 6 week of infectionPraziquantel (Distocid, EPICO , 6th October City, Egypt ) was suspended in dist. water then mice were treated orally using stomach tube with 250 mg /kg body weight for 3 alternative days. Another group of mice were treated witha single dose of a curcumin extract at 20 mg / kg body weight using stomach tube. After two weeks of treatment,blood samples were collected and stored at -20 0C till used.

Mice perfusion

After blood collection, infected mice were perfused according to Smithers and terry (1979). Briefly,

Determination of antischistosomal antibodies

Polystyrene flat bottom microtiter plate was coated with 50 l / well of 25 g / ml from schistosome antigenic preparation (SWAP) at room temperature over night. After blocking , pooled sera from treated and non treated mice diluted in PBS–T20 were added ( 50 l / well ) and incubated at 37 oC for 2h . After washing with PBS–T20, 50 l/well of goat anti–mouse IgG Alkaline phosphatase conjugate diluted 1 : 500 in 0.2 % (w/v) non - fat dry milk in PBS – T20 was added and incubated at 37 0C for 1 h . After washing , 1mg / ml of P- nitrophenyl phosphate was used as a substrate , and the absorbance was read at 405 nm using micro plate auto reader( ).The level was expressed as optical density (OD).

Determination of serum IF N-  and IL – 2

A polystyrene flat bottom microtiter plate was coated with 50 l / well of treated & non treated sera diluted (1:100)in carbonate / bicarbonate buffer,( pH 9.6) overnight . After blocking, rat anti-mouse IFN - mAb and goat anti-mouse IL – 2 mAb ( Sigma ) diluted 1 : 1000 in PBS , PH 7.2 were added at 37 0C for 1h. After washing, 50 l /well of goat anti-rat IgG alkaline phosphatase conjugate (for IFN-) and rabbit anti goat IgG alkaline phosphatase cojugate for IL – 2 diluted 1 : 600 in PBS – T 20 were added at room temperature for 2h . 1 mg / ml P- nitro phenyl phosphate was used as Substrate and the absorbance was read at 405 nm using microtiter plate auto reader( ) . The levels was expressed as OD.

Determination of superoxide dismutase activity .

The method was done according to Nishikimi et al , ( 1972 ) , the assay depends up on the ability of the enzyme to inhibit the phenazine methosulphate – mediated reduction of nitroblue tetrazolium dye .

Determination of serum ALT activity

The activity of S. ALT of all groups was determined using ( ) and the method was done according to the manufacture Instruction .

Determination of serum cholesterol, triglycerides, HDL and LDL.

Serum cholesterol( ), triglycerides( ), HDL( ) and LDL)( ) were determined done according to the manufacture Instruction .

Statistical analysis

Student t- test was performed using the statistical program package , instate software , version 2.03 ( Graphpad , USA ) and on IBM PCIAT compatible computer . The degree of significance , P< 0.05 significant , P< 0.01 highly significant and P < 0.001 extremely significant .

Results

Worm burden:

Table (1) shows that the number of S. mansoniworm recovered from mice infected with S. mansoni and treated with PZQ and curcumin was reduced significantly after treatment (P<0.0001)

Serum level of anti-schistosomal antibodies:

Anti-schistosomal antibodies were decreased significantly (P<0.0001 ) in mice infected with 100 cercariae of S. mansoni and treated with praziquantel as well as mice infected and treated with curcumin (P<0.0001). This result this result was confirmed by the result that, there no difference between PZQ and curcumin ( table 1 )

Serumoptical densities of IFN- andIL – 2

Table (2) shows that , the level of IFN- was not decreased significantly after treatment with myrrh as well as after treatment with PZQ (P>0.05). However the level decreased non significantly in curcumin treated mice compared with the control group.

The level of IL – 2 was decreased in mice infected with S. mansoni and treated with PZQ and mice infected and treated with curcumin , (table 2).

Activity of superoxide dismutase and SGPT enzymes:

Table (3) shows that the activity SGPT was normalized after treatment with PZQ but , there is no improvement after treatment with myrrh . However, there is no improvement in the activity of dismutase after treatment with PZQ but not with curcumin.

Serum cholesterol, triglycerides, HDL and LDL:

No significant difference was observed after treatment with PZQ in comparing to the infected non treated control group. After treatment with curcumin, the levels of cholestrol and triglycerides were non-significantly decreased. However, HDL level was increased significantly(P<0.001) and LDL level decresed (P<0.05) after treatment with curcumin(table, 4).

Discussion

Schistosomiasis infects one in 30 people(Elliott,1996). A significant advance in the control of schistosomiasis chemotherapy was the introduction of praziquantel (Shekhar, 1991) but resistance to praziquantel is an emerging problem (Ismael et al., 1994,1996, Stelma et al., 1995, Xiao et al., 1985 and Sabah et al., 1986b). The use of medicinal plants or their active components in the prevention and treatment of chronic diseases is based on experience from traditional systems of medicine from various ethnic societies. During the past decade, a large number of natural products and dietary components have been evaluated as potential chemopreventive agents (Sharma et al., 1994). Curcumin (diferuoylmethane or 1,7-bis(4-hydroxy-3-methoxyphenol)-1,6-heptadiene-3,5-dione) is a compound found in tumeric that gives it its distinctive yellow colour (Gescher, et al., 1998). Recently it has been shown that curcumin has anticarcinogenic effects(Kelloff, et al.,1996) that may be linked to its antioxidant properties (Xu, et al., 1997). Studies have shown that curcumin can affect a number of cellular processes including: inhibition of platelet aggregation(Shah, et al.,1999 and Srivastava, et al.,1995), anti-intestinal parasites and inhibition of inflammatory cytokine production in macrophages (Abe et al., 1999). The present study, might through light on another role of curcumin on the infection with a blood parasite,S. mansoni. Mice infected with S. mansoni and treated with curcumin in a single dose of 20 mg / kg body weight after six weeks of infectionwas compared with the already known antischistosomal drug, PZQ.The worm recovered and anti-schistosomal antibodies were used to evaluate the reduction levels of infection (the effect on adult worm).The most important finding was that antischistososmal antibodies was decreased significantly after treatment with curcumin (P<0.05) and PZQ (P<0.0001).This result come in concomitant with our result of worm recovery of the same treated groups, in which the mean number of worms recovered after treatment with PZQ and curcumin was decreased significantly (P<0.0001). The respective roles of T-helper1(Th1) and T-helper2(Th2) cells in the immune response to S. mansoni infection has been the focus of intensive research ( Kaplan et al., 1998). Previous evidence suggested a role for both subsets , since the early stage of granuloma formation has the characteristic of Th1(IFN- and IL-2) cell response (Henderson et al., 1992 and Cook et al., 1993). The results of our study revealed that, treatment of mice infected with S. mansoni with curcumin can reduce the levels of both IFN-and IL-2 (Th1 response). Otherwise treatment with PZQ can improve interleukin -2 only. From these result one can conclude that curcumin can act in synergy with Gamma interferon and interleukin -2. Also we chose these cytokines because, at the time of dissecting their levels were dominant ( Boros et al., 1989) to give a clear difference after treatment. In the murine model of S. mansoni infection , the parasite ova settle in the liver , leading to the recruitment of various inflammatory cell types, primarily esinophils and to a lesser extent lymphocytes and macrophages to form granuloma which its function is to protect liver from the dissemination of toxic egg products ( Weinstock et al., 1992). McCormic et al.,1996 demonstrated that , granuloma associated eosinophils have the capacity to generate superoxide anion and hydrogen peroxidase( O2/H2O2). Unfortunately, the level of super oxide dismutase was not improved after treatment with a single dose of curcumin . This may be due to the low dose of treatment that may need to be repeated . This conclusion can explained by the finding that, liver enzyme (SGPT) was dcreased significantly after treatment with curcumin (P<0.0001). Another surprising findings is that, curcumin play an important role in improvement of the levels of serum cholesterol, triglycerides, HDL and LDL during the infection with S. mansoni unlike PZQ which cause no improvement in these characters as shown in table (4). Our results agree with the results of Rao et al., (1970) and Hussain and Chandrasekhara, (1994) which suggest that curcumin lowers serum cholesterol levels in rat. In mice, Soudamini et al. (1992) investigated the effect of oral administration of curcumin on serum cholesterol levels and on lipid peroxidation in the liver, lung, kidney, and brain of mice treated with carbon tetrachloride, paraquat, and cyclophosphamide. Oral administration of curcumin significantly lowered the increased peroxidation of lipids in these tissues produced by these chemicals. Administration of curcumin also significantly lowered the serum and tissue cholesterol levels in these animals, indicating that the use of curcumin helps in conditions associated with peroxide-induced injury such as liver damage and arterial diseases. Soni and Kuttan (1992.) examined the effect of curcumin administration in reducing the serum levels of cholesterol and lipid peroxides in 10 healthy human volunteers receiving 500 mg of curcumin per day for 7 days. A significant decrease in the level of serum lipid peroxides (33%), an increase in high-density lipoproteins (HDL) cholesterol (29%), and a decrease in total serum cholesterol (12%) were noted. The same results were obtained in the present study, in which , serum cholesterol level was reduced whereas the level of HDL was elevated significantly in mice infected with S. mansoni and treated with cucumin (20 mg/kg body weight). On the other hand the level of LDL was reduced significantly.Several studies suggest that curcumin inhibits oxidation of LDL (Quiles, et al., 1998 and Naidu and Thippeswamy, 2002).Curcumin at 10 mM produced 40 to 85% inhibition of LDL oxidation. Further, curcumin significantly inhibited both initiation and propagation phases of LDL oxidation. Ramirez-Tortosa et al., 1999). evaluated the effect of curcumin on LDL oxidation susceptibility and plasma lipids in atherosclerotic rabbits. Quiles et al. , (1998) evaluated the antioxidant capacity of a C. longa extract on the lipid peroxidation of liver mitochondria and microsome membranes in atherosclerotic rabbits. These findings suggest that active compounds in curcuma extract may be protectiveagainst lipoperoxidation of subcellular membranes in a dosage-dependent manner. The present study indicates that, the level of triglycerides was significantly reduced after treatment with curcumin in comparing to the infected non-treated control group (P<0.0001) as well as to the group of mice infected and treated with PZQ(P<0.05). Asai and Miyazawa (2001) examined the effect of curcumin on lipid metabolism in rats fed a control, moderately high-fat diet (15 g soybean oil/100 g diet) and those given supplements of 0.2 g curcuminoids/100 g diet. Liver triacylglycerol and cholesterol concentrations were significantly lower in rats fed curcumin than in control rats. Plasma triacylglycerols in the verylow- density lipoproteins fraction were also lower in curcumin-fed rats than in control (P < 0.05). Hepatic acyl-CoA oxidase activity of the curcumin group was significantly higher than that of the control. These results indicated that dietary curcuminoids have lipid-lowering potency in vivo, probably due to alterations in fatty acid metabolism. In conclusion, curcumin may play different roles during the infection with S. mansoni . It improve the status of liver enzymes, improve lipid metabolism, reduce the levels of Th1 cytokines that lower the pathogenesis of schistosomiasis. Surprisingly , curcumin reduce the intensity of S. mansoni infection by reducing the number and antibodies of S. mansoni worms.

Acknowledgement: The author thanks Prof. Dr. Farid A. Badria , Prof.,Mansoura faculty of pharmacy for providing the pure extract of curcumin .

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