THE EFFECT OF TWO FRUIT EXTRACTS AND DRUGS ON THE LIVER OF ALBINO MICE WITH INDUCED LEISHMANIASIS

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1 THE EFFECT OF TWO FRUIT EXTRCTS N RUGS ON THE LIVER OF LINO MICE WITH INUCE LEISHMNISIS Kawthar I.F. l-harmni*, Zohair I.F. Rahemo* and Hussain I.. l-khan** * University of Salahaddin, Erbil, Kurdistan, Iraq **epartment of iology, College of Science, University of Mosul, Mosul, Iraq *( zohair_rahemo@yahoo.com) STRCT mong 117 of the albino mice used in this study, 108 were intraperitionally infected with 100 X 106 promastigotes of Leishmania donovani (MHOM\ IQ\ 982\ RCI) strain, while the rest groups (9) were left without infection. The inoculation of albino mice caused the elevation of liver and spleen weight after 7-15 days and the treated mice with 20 mg/kg by Pentostam and llupurinol for 10 and 15 days caused elevation of liver and spleen weight, whereas the treated mice with 20 mg\kg for 20 days restored the liver and spleen weight near to normalization. The treated mice with mg\kg from Melia azedarach and mg\kg from Citrus colocynthis caused depletion of the average liver and spleen weight in comparison to the control positive. Histological examination of liver sections revealed that high concentration of two aqueous fruits extract returns the liver tissues towards the normalization. mong the most important histopathologcal results: In positive control scattered necrosis, lymphatic infiltration, proliferation of macrophages and with a variable number of leishman bodies were observed. In 20 mg/kg Pentostam-allupurinol for 10 days: multifocal necrosis, presence of Kupffer cells and macrophages colonized with leishman bodies, while in same concentration but for 20 days sinusoid dilataion, scattered multifocal necrosis were observed.in a concentration of M. azadarach 20 mg/kg : hyperplasia of phagocytic cells containing leishman bodies were seen, while in 40 mg/kg focal necrosis and inflammatory cells were revealed, but in mg/kg lymphatic infiltration, few mixed inflammatory cells infiltration were observed. In concentration of C. colocynthis 20 mg/kg central vein dilatation, multicemal necrosis, diffuse lymphatic infiltration and the presence leishman bodies can be distinguished, while in 40 mg/kg multifocal necrosis, diffuse lymphatic infiltration and the presence of leishman bodies were seen, in 60 mg/kg lymphatic infiltration only was distinguished, while in mg/kg liver return to normal histology. Key words: histopathology, liver, plants extracts. INTROUCTION Leishmaniasis is endemic in 88 countries with an estimated 350 million people at risk and 12 million people are affected (WHO, 2001; Tiuman et al., 2005; Singh, 2006; Mishra, et al., 2007). L. donovani complex causes visceral leishmaniasis(vl) or kala azar, while the L. tropica complex induces cutaneous leishmaniasis or oriental sore in the old world and L. mexicana complex causes cutaneous disease in Latin merica and L. braziliensis complex caused mucocutaneous disease in merica (Chan acab and Pena Rodriguez, 2001; Croft and Coombs, 2003).VL is the most severe clinical form of the disease (Singh, 2006).Pentavalent antimonials, the drug of choice have been successfully used for treatment of kala azar and has serious side effects, and requires a prolonged course of treatment it has been losing its efficacy in some regions due to increasing parasite resistance, and causes renal and cardiac toxicity (Croft and Yardley, 2002; Murray, 2004b; Ferreira et al., 2004; renzan et al., 2007). Pentamidine was used as a second choice for the treatment, which also may has serious side effects and acquired resistance even with prolonged dosage. llopurinol found to be more effective with little toxicity for treating leishmaniasis as used clinically as antigout drug, against promastigotes of L. tropica (Zgaeir, 1999). combination of llopurinol was more effective in leishmaniasis, combination was possible to reduce the duration of treatment and produces better results than antimony alone or allopurinol alone (as et al., 2001; Join er et al., 2005). Natural products have potential in search for new and selective agents for treatment of important tropical diseases.the plant kingdom is undoubtedly valuable as a source of new medicinal agents (Rocha et al., 2005), alkaloids, terpenes, quinones, chalcones, lactones and saponins are known as antiprotozoal products (Wright and Philipson, 1990).Crude extracts from lstonia macrophylla, Rhazya stricta, Triclisia patens showed strong activity against promastigote forms of L. donovani with.these extracts have stronger cytotoxic effect on the parasite than on mammalian cells (Camacho et al., 2003).The linalo rich essential oil from the leaves of Croton cajucara showed excellent inhibition of the growth of promastigote and amastigote forms of L. amazonensis (Rosa et al., 2003). Two extracts of two plants naturally cultivated in Iraq were selected to test their lethal effects on Leishmania donovani induced experimental albino L/C mice. These are the Chinaberry, M. azedarach and the bitter apple, Citrullus colocynthis as usually natural plants have less side effect compared to synthetic drugs. The aims of the present study: 1 etermining the effect of two aqueous fruit extracts M. azedarach and C. colocynthis on induced visceral leishmaniasis in L /c mice and comparing their effects with the combination of two standard drugs, namely Pentostam and llopurinol. 2 Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 16

2 etermining the effect of these two plant extracts on average liver and spleen weight. 3-Histopathological changes in the liver. MTERILS N METHOS strain of Leishmania donovani (MHOM / IQ / 1982 / RC1) provided l Nahreen University, aghdad. This stock culture was cultivated in iphasic medium (NNN).dult male L /c mice Mus musculus, gm, 6 8 weeks old were used for the pharmacological and histological studies. They were housed in controlled environment, the room temperature was maintained at 25 ± 2 C and 10 hr dark /14 light cycle with standard laboratory diet. Leishmania promastigotes were cultivated on NNN medium (Jawdat et al., 1985). NNN medium consists of two phases: solid phase, blood agar, and liquid phase Lock ś solution left out to cool off until antibiotics added (0.1mg / ml streptomycin, IU penicillin. One gram of dried extract fruits of M. azedarach and C. colocynthis, were dissolved in 5 ml.w. (total concentration 200 mg / ml as a stock culture).concentration 20, 40, 60, 80 and 100 mg / kg WT were prepared from the stock culture according to the procedure described by Riose et al. (1987). Sodium stibogluconate (Pentostam, Sb) prepared as aqueous solution: Each ml of the solution containing 100 mg pentostam, the stock solutions were freshly prepared and sterilized using millipore filter 0.22µ and injected either intramuscularly (IM) or intraperitoneally (IP). llopurinol (Urizol). Hpp.: Each tablet of allopurinol contains 100 mg allopurinol, dissolved one tablet in 100 ml. W. The concentration of the solution becomes 1mg / ml. The recommended dose of allopurinol for treatment of leishmaniasis was 20 mg / kg / day for days. One hundred and seventeen male L/c mice 6 to 8 weeks old were used in these experiments. 108 mice were intraperitoneally infected with promastigotes in stationary phase. The rest of mice were left without infection as control negative (con ve). The animals were divided into five groups as follows (Table 1): (1) The first group consists of 3 sub-groups : 3 animals for each sub group and were used to evaluate the therapeutic effect of Pentostam and allopurinol. Treatment with 20 mg / kg body weight / day was started 20days post infection. Each animal received 10 doses in the first sub group for 10 days. 15 doses in the second sub group for 15 days and 20 doses in the third sub group for 20 days, animals of these sub groups sacrificed 10, 15 and 20 days post infection, respectively. (2) The second group consists of 3 subgroups (15 animals for each sub group) were used to evaluate the plant extract of M. azedarach. Each animal received 10 doses in the first sub group, 15 doses in the second subgroup and 20 doses in the third subgroup, each sub groups received five different doses of the extract 20, 40, 60, 80 and 100mg / kg body weight orally.the animals of these sub groups were sacrificed 10, 15 and 20 days post infection, respectively. The post necropsy procedure was followed as in the first group (Table.3). (3) The third group was as the second group except that the plant extract was C. colocynthis used instead of M. azedarach. (4) The fourth group was consisted of noninfected untreated animals (n=9) which were left as a negative control (con ve)group and sacrificed at 10, 15 and 20 days later and subjected to the same procedure. (5) The fifth group was consisted of infected un treated animals (n = 9). This group served as positive control (con + ve). On the10 th, 15 th and 20 th day. t the end of the experimental period, after 10, 15 and 20 days the animals were sacrificed, various biochemical and haematological parameters were estimated. Table 1: No. of animals used, and the concentrations Melia azedarach and Citrullus colocynthis (20mg/kg 100mg/kg). Treatments (mg / kg bwt) uration of treatment(days) No. of animals Control ( ) Control (+) Pen/ll.20mg/kg Melia 20 mg/kg Melia 40 mg/kg Melia 60 mg/kg Melia 80 mg/kg Melia 100 mg/kg Citrullus20 mg/kg Citrullus40 mg/kg Citrullus60 mg/kg Citrullus80 mg/kg Citrullus100 mg/kg Total Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 17

3 For Histological studies: Liver tissues fixed in 10 % formaldehyde for 24 hrs for histopathological observation. The prepared 5 micron thick sections were stained with hematoxyline and eosin (H/E)( see Lee and Luna, 1960). RESULTS N ISCUSSION s shown in Table.2 and after Using the aqueous fruit extract of M. azedarach at the concentration 20mg / kg did not control the leishmanial growth for 10, 15, and 20 days, but using the aqueous fruit extracts at the concentration 40, 60, 80 and 100 mg / kg control the growth in infected mice for the same period of time, while the aqueous fruit extracts of C. colocynthis at the concentration 20 and 40 mg / kg did not control the leishmanial growth, but at the concentration 60, 80 and 100 mg / kg controlled the visceral leishmaniasis in liver and spleen of infected mice at the same period of time. This result indicates that the two aqueous fruit extracts of the two plants have inhibitory effect on the growth of VL in albino mice in vivo, and the con 40mg / kg Melia is equivalent to 60mg / kg of Citrullus extract. Table.2: The effect of different concentrations of M. azedarach and C. colocynthis extracts on the liver and spleen of infected mice with v. leishmaniasis in the inoculated cultures and comparison with pentostam and allopurinol, sacrified at 10, 15 and 20 days post infection. Treatments (mg / kg) Results of inoculated cultures Growth No. of animals Liver (days) Spleen (days) Cont (+) Pentostam and + + _ + + _ 9 llopurinol Melia 20 mg / kg Melia 40 mg / kg 9 Melia 60 mg / kg 9 Melia 80 mg / kg 9 Melia100 mg / kg 9 Citrullus20 mg /kg Citrullus40 mg /kg Citrullus60 mg / 9 kg Citrullus80 mg / 9 kg Citrullus100 9 mg/kg Total 108 ( + ) = Viable. ( ) = None viable. Table ( 3) showed the average weight of liver in infected mice (control positive, con + ve.).there was a significant variation in the average liver weight (P< 0.05) between the infected groups (con + ve.) for 10, 15 and 20 days were 1.79, 1.45 and 1.61g when compared with (con ve) were 1.29, 1.05 and 1.23 g respectively. These observations were supplemented with the presence of promastigotes in the inoculated cultures with the liver of infected mice after (5) days of inoculation. Our result also were confirmed with the histopathological examination of mice liver sections (Figs1, 2). The liver section in (con + ve.) with preserved general architecture, scattered necrosis, lymphocytic infiltration, proliferation of macrophages and clusters of macrophages often containing leishman bodies. (amastigote). This result revealed that, the average liver weight of infected mice with L. donovani promastigotes were increased in the first week then decreased in weight after this period of time. This result is in agreement with the confirmed previous experiments of Molan et al.(1989) who observed that the albino mice showed a remarkable increase in liver weight. This increasing was at its peak at the first seven days post injection, then the liver weight decreased after this Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 18

4 Table 3: The effect of different concentrations of aqueous fruit extracts of M. azedarach and C. colocynthis on liver weight (g) of mice infected with L.donovani in comparison with pentostam and allopurinol, sacrificed 10, 15 and 20 days post infection. uration (day) Treatments (mg/kg) Mean SE Cont ** Cont Ef * e * Pent /llo 20 mg/kg Cd * ** Melia 20 mg/kg G * Fg ** e Melia 40 mg/kg E *** Cde ** C Melia 60mg/kg d Melia 80 mg/kg d Melia 100 mg/kg ** Citrullus 20 mg/kg F * G E Citr. 40 mg/kg Ef Citr. 60 mg/kg d * d * Citr. 80 mg/kg d * Citr.100 mg/kg *Means with different letters have significant difference at p<0.05 according to uncan test. *Significant differences of the 10 day at p<0.05, ** at p<0.01 and *** at p< period of time. This means that the liver weight increased according to the parasite number, and also is in agreement with l Harmni (1988) who showed that the promastigotes of L. donovani in mice and golden hamsters was accompanied by marked enlargement of both spleen and liver. This result also agree with those of Grun and Stauber (1957) and Stauber et al. (1958) as they observed that primarily increasing in the number of parasites of VL then decreased, this difference may be due to the various strains of parasites and the stains of albino mice. This result also agree with l Shanawi (1975) who observed similar results. There were increasing average of liver weight of treated mice with combined drugs for 10 and 15 days 1.43, 1.33 g when compared with the normal groups (con ve ),1.29, 1.05 g. ut the treated mice for 20 days showed a decreased in the average liver weight to 0.85g in comparison with the normal group 1.23g and a significant variation (P < 0.01) between the mice treated with combined drugs for 20 days with these treated for 10 days. This indicates that the treatment with combined drugs for 10 and 15 days did not control the parasite. These observations were supplemented with the presence of promastigotes with the liver. However the number of promastigotes in cultures less than the numbers of promastigotes in inoculated cultures with the liver of (con + ) groups. The result also were supplemented with the histopathological examination (Fig.3), the liver section with multifocal necrosis, Kupffer cells and macrophages colonized by leishman body. ut the treated mice with the combined drugs for 20 days controlled the parasite completely. This result confirmed by the absence of promastigotes in inoculated cultures and the liver section with sinusoidal dilatation,scattered multifocal necrosis and without the leishman body (Fig.4). The treatment for a long time associated adverse side effects during a lengthy and painful treatment course (Reithinger et al., 2005: Sundar and Chatterjee, 2006: Colakoglu et al., 2006). ntileishmanial chemotherapy can have adverse effects and may fail to cure patients (Mosleh et al., 2008).It was well known that the result observed was in agreement with those of Goodwin (1945) who found that the experimental animals infected with VL and treated with low doses of Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 19

5 Pentostam increased the infection when compared with the (con + ve).similar results were obtained by l-khateeb et al.(1977). The result was also in agreement with Zgaeir ( 1999) who used llopurinol combined with Pentostam of the same concentrations on L. donovani and L. tropica in vitro, showed actually a synergistic effect which decreased the growth of parasite more than using llopurinol or Pentostam alone. The combination of llopurinol with antimony produced better results than each one alone for treatment of leishmaniasis (enerolle and ourdoiseau, 1999). The results observed in this study are in agreement with confirmed previous study, and also agree with the study reported by silian et al.(2004) Table (2) also showed the average liver weight in mice infected (I P) with 100X 10 6 promastigotes of L. donovani and treated with aqueous extract of M. azedarach in different concentrations 20, 40, 60, 80 and 100 mg / kg.there was a significant variation (P< 0.05) in the average liver weight of treated mice with 20 mg / kg from the extract were 2.1,1.6,1.7 g when compared with the average liver weight of (con ve ) group 1.29, 1.05 and 1.23 g for 10,15 and 20 days respectively. These observations were supplemented by the presence of promastigotes in the inoculated cultures with a piece of the liver of this group. ut the number of promastigotes in positive culture was less than the number of promastigotes in cultures inoculated with the liver of (con + ve) and the histopathological sections revealed the central vein dilatation, multifocal necrosis, diffuse lymphocytic infiltrations, hyperplasia of phagocytic cells containing leishman body (Fig.5)These results indicates a possible role of hepatocytes as a parasite reservoir during the infection (Gangneux et al., 2005). The liver weight of treated mice decreased with different concentration of aqueous Melia extracts 40, 60, 80 and 100 mg /kg for 10,15 and 20 days, then the decreased liver weight was restored towards the normalization and with nonsignificant variation when compared with the average liver weight of (con ve ).This result was supplemented by the absence of promastigotes in inoculated liver culture and the histopathological examination of liver sections with normal view. The liver of treated mice with 40 mg / kg of the Melia extract with few focal necrosis and inflammatory cells (Fig.6), but treated mice with 60 mg / kg show few piecemeal necrosis few mixed inflammatory cells infiltration (Fig.7), more wide spread necrosis and diffuse lymphocytic infiltration (Fig.8).The treated mice with 80 and 100mg / kg showed more diffuse necrosis and mixed inflammatory cells infiltration (Figs. 9,10). s shown in Table (3) there was a significant variation (P<0.05) in the average liver weight of treated mice with 20 and 40 mg / kg after applying the extract for 10, 15 and 20 days when compared with the average liver weight of (con ve). This result was supplemented by demonstration of promastigotes in the inoculated cultures with a piece of the liver of these groups. However the number of promastigotes in cultures was milder than the number of promastigotes in (con + ve) cultures. The histopathological sections include central vein dilatation, multifocal piecemeal necrosis, diffuse lymphatic infiltration and the presence of intracellular amastigotes (Figs.11, 12). In the treated mice with 60, 80, and100 mg / kg of the C. colocynthis extract the liver weight decreased and then restored towards the normalization, this result was confirmed by the absence of promastigotes in inoculated cultures and the histopathological examination of the liver sections with lymphocytic infiltration and appear with a normal view and no signs of amastigotes (Figs.13-16). The present results revealed that the combination of Pentostam and llopurinol in concentrations of 20 mg / kg/ for 20 days showed effectiveness against VL, but the treatment with lower doses show no encouraging results. Jha (2006) reported that these drugs are potentially toxic and often ineffective and have serious side effects and acquired resistance even with prolonged dosage when used as a monotherapy. The aqueous fruit extracts of M. azedarach and C. colocynthis were found to show a significant activity against VL and had stronger cytotoxic effects on the parasite than on mammalian cells which controlled the disease without any side effects on the host and did not result in any change in general behavior of the animals. s shown in Table (4) there was a significant variation (P<0.05) in the average spleen weight of infected mice (con + ve) for 10,15 and 20 days were 0.36, 0.33 and 0.31 g when compared with (con ve)were 0.15, 0.12 and 0.09 g, respectively. Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 20

6 Table(4) showed significant variation (P<0.05) of the average spleen weight of treated mice with combined drugs for 10, 15 days were 0.27 and 0.28 g compared with the (con ve) groups, but with non significant variation with the average spleen weight treated for 20 days 0.07 g. The treatment of albino mice with low doses of combined drugs did not completely control the parasite and failed to give protection to mice against L. donovani infection.this result was confirmed by the presence of promastigotes in inoculated spleen of these groups but no appearance in the inoculated spleen of treated mice with combined drugs for 20 days. Table (4) showed an average spleen weight of mice infected (IP) with promastigotes of VL and treated with different concentrations 20, 40, 60, 80 and 100 mg / kg from aqueous fruit extract of M. azedarach for 10, 15 and 20days post infection. There was a significant variation (P<0.05) of average spleen weight of treated mice with 20 mg / kg from M. azedarach extract for 10, 15 and 20 days were 0.41, 0.32 and 0.27 g when compared with non infected mice 0.15, 0.12 and 0.09 g. Table 4: The effect of different concentrations of aqueous fruit extracts of M. azedarach and C. colocynthis on spleen weight (g) of mice infected with L. donovani in comparison with pentostam and allopurinol, sacrificed at 10, 15 and 20 days post infection. ion (day) Mean SE Treatments (mg/kg) Cont b * Cont G * C Pent. / llo C Efg * Melia 20 mg/kg * G * Melia 40 mg/kg C ** c-f *** b Melia 60mg/kg bc b Melia 80 mg/kg b bc b Melia 100 mg/kg b b Citrullus 20 mg/kg Fg * Citr. 40 mg/kg C d-g * Citr. 60 mg/kg b-e Citr. 80 mg/kg Citr.100 mg/kg a-d bc * Means with different letters have significant difference at p<0.05 according to uncan test. * Significant differences of the 10 day at p<0.05, ** at p<0.01 and *** at p< b * b Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 21

7 C.V Figure 1: Liver section of mice (control negative), sacrificed 15days post infection (H +E 100X) C.V.: central vein. Figure 2: Liver section of mice (control positive) sacrificed 15 days post infection (H +E 1000X). L..: Leishman body S. Figure 3: Liver section of mice, treated with pentostam and allopurinol, sacrificed 10 days post infection (H +E 1000X). KC: Kupffer cell., L..: Leishman body. L. Figure 4: Liver section of mice, treated with pentostam and allopurinol, sacrificed 20 days post infection (H +E 400X).S..: Sinusoidal dilatation. Figure 4.5: Liver section of mice, treated with Melia azedarach 20mg / kg and sacrificed 15 days post infection (H +E 1000X). L..: Leishman body. Figure 4.6: Liver section of mice, treated with Melia azedarach 40mg / kg and sacrificed 15 days post infection (H +E 1000X). INF.c.: Inflammatory cells, N.: Necrosis. Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 22

8 Figure 7: Liver section of mice, treated with Melia azedarach 60mg / kg and sacrificed 15 days post infection (H +E 1000X). Figure 8: Liver section of mice, treated with Melia azedarach 60mg / kg and sacrificed 15 days post infection (H +E 100X). P.S.: Portal Space, Infl.C. :Inflammatory Cell, C.V.: Central Vein. Figure 9: Liver section of mice, treated with Melia azedarach 80mg / kg and sacrificed 20 days post infection (H +E 100X). P.S.: Portal space, C.V.: Central vein. Figure 10: Liver section of mice, treated with Melia azedarach 100mg / kg and sacrificed 15 days post infection (H +E 100X). INF.c.: Inflammatory cells. Figure 11: Liver section of mice, treated with Citrullus colocynthis 20mg /kg and sacrificed 15 days post infection (H +E 1000X). L..: Leishman body. Figure 12: Liver section of mice, treated with Citrullus colocynthis 40mg / kg and sacrificed 15 days post infection (H +E 1000X).L..: Leishman body. Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 23

9 C.V P.S Figure 13: Liver section of mice, treated with Citrullus colocynthis 60mg / kg and sacrificed 10 days post infection (H+E 400X). C.V..: Central vein, P.S.: Portal space. Figure14: Liver section of mice, treated with Citrullus colocynthis 60mg / kg and sacrificed 15 days post infection (H +E 100X). INF.c.: Inflammatory cells. Figure 15: Liver section of mice, treated with Citrullus colocynthis 80mg / kg and sacrificed 15 days post infection (H +E 100X). C.V.: Central vein, P.S.: Portal space. Figure16: Liver section of mice, treated with Citrullus colocynthis 100mg / kg and sacrificed 15 days post infection (H +E 100X). C.V.: Central vein, P.S.: Portal space. In addition to this, the positive cultures confirmed this. ut the spleen weight of treated mice with different concentration of M. azedarach 60, 80 and 100 mg / kg for 10,15 and 20 days showed no significant variations when compared with the average spleen weight of (con ve).this result indicated that the treatment of VL with M. azedarach were restored towards the normalization. In addition to this the absence of promastigotes in the cultures inoculated with the spleen of these groups. The previous result showed that the aqueous fruit extract of M. azedarach appears to offer a fruitful strategy for treatment of VL and also has a potential prophylactic and therapeutic efficacy against kala azar infection. Table (4) also showed the average spleen weight in infected albino mice treated with different concentrations 20,40,60,80 and 100 mg / kg of aqueous fruit extract of C. colocynthis.there was a significant variation (P< 0.05) in the average spleen weight of the treated mice with 20 and 40g / kg from the aqueous extract for 10,15 and 20 days when compared with the average spleen weight of (con ve). In addition to this, the positive cultures confirmed this, but with non-significant variation between the average spleen weight of treated mice with 60, 80 and 100 mg /kg for the same period of time when compared with the average spleen weight of (con ve).this result indicated that the treatment with C. colocynthis extract restore the spleen weight into normalization. The previous result showed that the infected mice with Kala azar and treated with 20mg / kg of combined drugs for 20 days consider as a good leishmanicidal agent. ut the two aqueous fruit extract of M. azedarach and C. colocynthis were more effective than the combined drugs for the treatment of VL in some concentrations mentioned before, the inoculated cultures of spleen and liver confirmed these observations. Such results are considered new for such kind of experimental study. Volume 1 Issue 3 (2012) ISSN: X (Print); (Online) 2012 M International. ll rights reserved. 24

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