Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 100(7): 771-778, November 2005 771 Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni Manal A Hamed/+, Mona H Hetta* Departments of Medicinal Chemistry *Chemistry of Natural Compounds, National Research Centre, Dokki, Cairo, Egypt This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden. Key words: Citrus reticulata - Mirazid - schistosomiasis - enzymes, worm burden - ova count Schistosomiasis, a chronic and debilitating parasitic disease, affects approximately 200 million people in the developing world and imposes a substantial public health and economic impact, despite the continuous control efforts (Wang et al. 2004). Current control of the disease by chemotherapeutic agents is impractical because of the common occurrence of re-infection after treatment due to the relative resistance of the larval stages of Schistosoma mansoni to schistosomicide drugs (Silva et al. 2003). Praziquantel, the currently used drug for chemotherapeutic control, was reported to induce hemorrhage in the lung tissue of the host (Flisser & McLaren 1989) as well as abdominal pain and diarrhea (Kabatereine et al. 2003). The new trends nowadays is the use of natural plant extracts as new safe and effective drugs. In this study, Citrus reticulata root extract and Myrrh from Commiphora molmol tree (Mirazid) are evaluated for their antischistosomal activity. Extract of C. reticulata roots has been reported as anticancer (Manthey & Guthrie 2002), antibacterial (Tkachenko et al. 1999) as well as antioxidants activity (Hara et al. 2004). In addition, Withman et al. (2005) recorded the ability of citrus fruit-derived flavonoids to reduced plasma cholesterol concentration. Moreover, commiphora extract has been reported as a new safe and effective drug against S. mansoni and S. heamatobium (Abo-Madyan et al. 2004). The present study is a trial to clarify the antischistosomal effect of C. reticulata root extract compared to commiphora extract (Mirazid). Enzyme markers for different cell organelles were measured in liver of S. mansoni +Corresponding author. E-mail: Received 7 June 2005 Accepted 28 September 2005 infected mice; succinate dehydrogenase (SDH) for mitochondria; lactate dehydrogenase (LDH), and its isoenzymes for cytoplasm; glucose-6-phosphatase (G-6-Pase) for microsomes; acid phosphatase (AP) for lysosomes and 5'- nucleotidase for plasma membrane. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were also measured. Parasitological studies through ova count and worm burden will take into consideration. MATERIALS AND METHODS Chemicals - All chemicals used in the present study were of high analytical grade, products of Sigma (US), Merck (Germany), BDH (England). Mirazid (the oleo-resin extract from Myrrh of C. molmol tree, family: Burseraceae) is a product of Pharco Pharmaceutical Company, Egypt. Animals - The animals used were intact male Swiss albino mice of CDI strain of similar age (8 weeks) and weight (18-20 g). They were obtained from Theodor Bilharz Research Institute, Cairo, Egypt. Animals were kept in a controlled environment and were maintained on water and stock commercial pellet diet ad libitum. Plant material - C. reticulata (Family: Rutaceae) roots were collected from Modereyet El Tahrir, Behera, Egypt. It was authenticated by Dr Mohamed Abdel Ghaffar, Faculty of Agriculture, Al- Azhar University, Egypt. A voucher specimen is deposited at Chemistry of Natural Compounds Dept., National Research Center, Dokki, Cairo, Egypt. Extraction and isolation - Air dried powered roots of C. reticulata (0.85 kg) were extracted with 80% ethyl alcohol. The ethanolic extract was evaporated and the aqueous residue extracted sequentially thrice with equal volumes of n-hexane, ether, ethyl acetate, and n-butanol. The ethyl acetate extract was evaporated to dryness. The residue monitored by TLC using precoated silica gel 60 F254 aluminium sheets (0.2 mm thickness, Merk), was found to 772 C. reticulata and Mirazid in S. mansoni • MA Hamed, MH Hetta contain flavonoids. The phenolic residue was subjected to biochemical determinations. calculated by the method of Tendler et al. (1986) as follows : Doses and route of administration - Oral dose of 10 µg/ml/mice of the phenolic extract of C. reticulata root was given daily for three consecutive weeks which the more effective dose as described by (Nogata et al. 2001). Oral dose of Mirazid (600 mg/kg body weight) was given for three consecutive days on empty stomach, at least 1 h before meal regarding to the concentration of Mirazid illustrated in the brochure of the drug (Haridy et al. 2003). P = C - V/C × 100 Experimental design - The animals were divided into six groups each of eight mice. Group 1: normal healthy control. Group 2: received C. reticulata extract daily for three weeks, left one week and sacrificed. Group 3: received Mirazid daily for three days, left for 27 days and sacrificed. Group 4: S. mansoni infected mice with 100 cercariae of Egyptian strain with tail immersion technique (Oliver & Stirewalt 1952) and sacrificed after two months. Group 5: received C. reticulata extract daily for three weeks post two months of S. mansoni infection. Mice left for one week after treatment and sacrificed. Group 6: received Mirazid daily for three days after two months of S. mansoni infection. Animals left for 27 days after treatment and sacrificed. Preparation of tissue homogenates - Liver tissue was homogenized in 0.9N NaCl by a ratio 1:10 w/v for estimation of all enzymes under investigation, while it was homogenized in 0.01M tris-glycine buffer by a ratio 1:10 w/v for estimation of LDH isoenzymes, where 100 µg protein was applied to each gel. Parameter assays - Enzyme activities were evaluated using end point assay method. SHD: the reduction of FAD is coupled with (...truncated)