ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Effect of Rhizopus Stolonifer Metabolic Products on Serum Vitamin C , some elements and catalase in albino male rats N. O. Al-Jubury , A.S. Sahood* , A.R. Rayshan* College of Agriculture, University of Al-Qadisiya. College of Medicine,University of Al-Qadisiya* Received in :2 August 2011 Accepted in : 7 December 2011 Abstract The present study was designed to investigate the effect of R. stolonifer metabolic products on some antioxidant defenses and some elements (Cu, Fe, Ca) . The experiment was performed using 25 mature male rats, their age average was about (3-3.5) months and their weight average was about (200-225) gm. The animals were randomly divided into 5 equal groups (five animals for each): control (treated orally with normal saline) and four treatment groups were drenched orally with four concentrations of R. stolonifer metabolic products (15, 30, 60,120) μl/kg body weight. The animals were treated with one single dose of the previously described concentrations then left for 15 days. Animals of different groups were sacrificed under light ether anesthesia one day after the end of treatments and blood samples were taken to determine some physiological, biochemical parameters. The results revealed that the treated groups showed significant decreases (P< 0.05) in serum vitamin C and Fe, Cu concentrations. The decrease was proportional with the increase the concentrations of the metabolic products of R. stolonifer. On the contrary Catalase activity increased significantly (P< 0.05), while Ca concentration didn’t differ significantly (P< 0.05) The findings of this study may encourage other researcher for further study especially that we find in our local market that there are many rotted vegetable and fruits which may cause serious problems if consumed for certain period. Key word: Fungi , Rhizopns stolonifer, antioxidant VitaminC, Serum copper, Serum lron, Serum calcium Introduction Fungi, being ubiquitous in distribution, are highly successful in survival because of their great plasticity and physiological versatility. Over the course of history fungi, have been a continuous source of great benefit and risk for human and animals life and significant source of experimental knowledge, thus the importance of kingdom of fungi relates to its interactions on an equal basis with members of p lants, animals and prokaryotic microorganism[1] . In terms of abundance of species of fungi with approximately 1.5 million species represent the next major player in the evolutionary drama of the planet [2]. Rhizopus belongs to most prominent orders of the Zygomycetes , Mucorales. The Mucorales are mostly saprotrophic and are abundant in soil, on dung and on other organic matter in contact with the soil. They may play an important role in the early colonization of substrata. Sometimes, however, they can behave ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 as weak pathogens of soft plant tissues [3] . Several species of Rhizopus are plant and human pathogens associated [4] . Species of Rhizopus are reported from human lesions, and these genera together with species of Absidia may also infect domestic animals. Rhizopus stolonifer is necrotrophic pathogen,cause rots of fruit [3] can cause a rot of sweet potatoes or fruits such as apples, tomatoes and strawberries, such infections may cause spoilage of food [5]. There are about 10 species which grow in soil [6] and on fruits, other foods and all kinds of decaying materials. Rhizopus stolonifer (syn. R. nigricans) grows rapidly and it is often found on ripe fruits, especially if these are incubated in a moist. Mould infection and mycotoxin contamination of cereal grains can occur in the field during growing, at harvest and during storage. As the grains progress through harvesting and storage, feed manufacture and delivery to farms, therefore public concern on health matters related to food has increased [7,8] and to our knowledge few studies have been carried out to identify the direct relation between these fungi and animal health. Therefore the present study was conducted to investigate the effect of Rhizopus stolonifer metabolic products on serum vitamin C, some elements and Catalase in albino male rats. Methods and Materials Rhizopus stolonifer Isolates of R.stolonifer cultured in nutrient broth media (from B.D.H) for 21 days. After this period the media was filtered by filter paper (what man no.1) [9] and maintained in clean sterile tubes until used in the experiment. Animals and Experimental Design The experiment was performed using 25 mature male rats, their age average was about (3-3.5) months and their weight average was about (200-225) gm. The animals were randomly divided into 5 equal groups (five animals for each): control (treated orally with normal saline) and four treatment groups were drenched orally with four concentrations of R. stolonifer metabolic products (15, 30, 60,120) μl/kg body weight. The animals were treated with one single dose of the previously described concentrations then left for 15 days. Blood Collection The animals were sacrificed under light ether anesthesia one day after the end of treatment. Blood samples were collected into clean dry centrifuge tubes and left to stand for 30 minutes at room temperature, then centrifuged at 3000rpm for 10 minutes and serum samples were stored in clean tubes at –20C till used for assays. Vitamin C concentration in serum Serum vitamin C concentration was analyzed by the method of M cCormic (1986) [10] Total Catalase Activity The total catalase enzymatic activity was determined manually according to the method described by Aebi (1987) [11] ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Determination of elements Serum iron (Fe), copper (Cu), and calcium (Ca) levels were determined using flame atomic absorption spectrophotometer (GBC 933 plus) and fivefold dilution with de-ionized water. Iron was determined at a wavelength of 284.3 nm, copper at (324.7) nm, and calcium at (319.9) nm. The concentrations of serum Fe, Cu, and Ca were calculated from the standard curves of these elements [12] Statistical Analysis The data were analyzed using F test taking P<0.05 as the lowest limit of significant difference and Duncan's Multiple Range Test was used to identify group responsible for statistical difference through comparison [13]. Results Table (1) summarized the results in normal and treated rats in. The treated groups show significant decreases (P< 0.05) in serum vitamin C Fe and Cu concentrations. The decreasing was proportional with the increasing of metabolic products concentrations of R. stolonifer. On the contrary Catalase activity increased significantly (P< 0.05), while Ca concentrations showed insignificant differcef significantly (P< 0.05). Discussion Fungi were extensively studied, to determine their modifying effects on animal health [5]. Many studies demonstrated different effects and mechanisms of action. Some authors have raised the theory that their action may be a result of disruption of the balance between oxidants and antioxidant. Serum vitamin C was regarded as one of the extracellular antioxidant which neutralizes the free radicals [14] .It is a primary antioxidant in plasma and within cells and may contact with plasma membrane [15]. Therefore, the decrease in serum vitamin C concentration may be due to that it acts as a sacrificial antioxidant, since it is able to inhibit the generation of hydroxyl radicals from systems containing copper ions and hydrogen peroxide by a mechanism that involve the immediate attack of vitamin C itself by free radicals, so they become no longer free in solution [16] . The elevation in serum Catalase activity observed in this study can be explained by the fact that this enzyme considered as one of the enzymatic antioxidants inside and outside of the cells [17, 18], thus foreign substances triggers the sequence of biochemical and cellular events associated with inflammation, which include stimulation of activity of the above mentioned enzyme and many other enzymes which have related functions [19] . Trace elements are essential for human and animals. They are constituents of, or interact with, enzymes and hormones that regulate the metabolism of much larger amounts of biochemical substrates [20]. Also they are found at the active sites of many enzymes and have the ability to facilitate the transfer of single electrons to molecular oxygen to produce superoxide anion radical which in turn is converted to hydroxyl radical through Haber-Weiss reaction or Fenton reaction [21]. Decreasing the concentrations of serum Fe and Cu ions may explained on the basis of the roles of these metals, especially their importance in the activity of enzymes [20]. Serum Ca concentration didn’t change compared with the other two elements. This may be explained by the fact that most of the calcium in the body is stored in the bones [21] , thus any expected alteration in calcium state in the cell, bones may provide an extra amount of needed ions. ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 The results of the present study indicate that there are alterations in serum concentrations of some elements, vitamin C and catalase , suggesting that they may play a role in the pathophysiology of the fungus. References 1. Webster, J. & Webster, R.W.S. (2007). Introduction to Fungi. 3ed. Cambridge University Press. 841. 2. Khachatatourians, G.G. & Arora, D.K. (2001). Applied Mycology and Biotechnology, 1. Agriculture and Food production. 1st ed. Elsevier Science B.V. Amsterdam, Netherland. 435. 3. Carlile, M.J., Watkinson, S.C. & Gooday, G.W. (2001). The Fungi. 2 nd ed. Academic Press. 565. 4. Rinaldi, M. G. (1989). Zygomycosis. Infec. Dis. Clin. North Am., 3, 19-41. 5 . Samson, R. A., Hoekstra, E. S., Frisvad, J. C. & Filtenborg, O. (2002). Introduction to Food-and Airborne Fungi, 6 th ed. Utrecht: Centraalbureau voor Schimmelcultures 6. Domsch, K. H., Gams, W. & Anderson, T. H. (1980). Compendium of Soil Fungi. London, Academic Press. 7. Martin, J.F.(1992). Secondary metabolite. In: Applied Molecular Genetics of Fil-amentous Fungi. Kinghorn, J.R. & Turner, G. (eds). Blackie Academic and Profrssional, London, pp: 214-252. 8. Sarig, P., Zahvi, T., Zutkhi, Y., Yannai, S., Lisker, N. & Ben-Arie, R. (1996). Ozone for control of post-harvest decay of table grapes caused by Rhizopus stolonifer. Physiol. Mol. Plant Pathol., 48, 403–415. 9. Colle, J.G., M armion, B.P., Fraser, A.G. & Simmons A. (1996). Practical Medical Microbiology (Makie & McCartne). 14 th ed. New York., Churchill Living Stone. . 131-150. 10. McCormic, D.B. (1986). Vitamins. In: Textbook of Clinical Chemistry. Tietz, N.W.(ed). Philadelphia, P.A. Saunders. 949. 11. Aebi, H.E. (1987). Catalase of enzymatic analysis. In: Enzymes: Oxidoreductase, transferases. Bergmeyer, H.U. (ed). Weinhein, Germany. VCH Verlagsgesell & Schaft.: 273- 285. 12. Growenlock, A.H.; McMurray, J.R. & McLauchlan, D.M . (1988). Valery 's Practical Biochemistry . 6th ed. Eds. Heineman medical books. London. 13. Schefler, W.C. (1980). Statistics for Biological Science .2 nd ed. Addison Wesley, Pub. Co. London, Amsterdam.. 121. 14. Carr, A. C. & Frei, B. (1999). Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am. J. Clin. Nutr., 69, 1086-1107. 15. May, J.M. (1999). Is ascorbic acid an antioxidant for plasma membrane?. FASEB. J., 13, 95-99. 16. Hughes, D. A. (2000). Dietary antioxidants and human immune function. Br. Nutr. Found. Nutr. Bull., 25, 35-41. 17. Mates, J.M. & Sanches-Jimenez, F.(1999). Antioxidant enzymes and their implications in pathophysiological processes. Frontiers in Bioscience, 4, 339-345. 18. Harris, E.D. (1992).Regulation of antioxidant enzymes. FASEB. J., 6, 99-106. 19. Proctor, P.H. & Reynolds, E.S. (1984). Free radicals and disease in man. Physiological Chemistry and Physics and Medical NMR., 16, 175-195. 20. Burtis, C.A. & Ashwood, E.R. (1996). Tietz Fundamentals of Clinical Chemistry, 4 th ed. W.B. Saunders Company. Philadelphia.. 272, 274. 21. Guyton, A.C. & Hall, J.F.(1997). Textbook of Medical Physiology. 5 th ed., W.B. Saunders Co. London,. 503-549. ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Table (1): Effect of Rhizopus stolonifer metabolic products on serum vitamin C , some elements and catalase in albino male rats treatment Parameters C Normal saline 15 μl/kg R. stolonifer 30 μl/kg R. stolonifer 60 μl/kg R. stolonifer 120 μl/kg R. stolonifer Vitamin C (ppm) 9.22 ±0.13 a 9.01±0. 18 a 8.79±0.07 a 7.50 0.19± b 6.68±0.18 c Catalase activity (u/l) 143.56±17. 72 a 146.7 17.7± a 166.28±4.9 7 b 183.40 15.28± c 185.72±14. 2 c Cu (μg/dl) 145.05±2.0 3 a 140. 00±0.9 a 126±1.00 b 102±3.12 c 85.71 0.77± d Fe (μg/dl) 140 ±4.00 a 138.30±1.0 5 a 131 2.10± a 112±4.65 b 95.00±4.07 c Ca (mg/dl) 9.95±0.63 e 9.87±0.23 e 9.53±0.71 e 8.86±1.00 e 8.83±1.30 e *values represented averages ± standard error. *Different let ters mean that there are significant differences (P<0.05). ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012 Cعلى تركیز فیتامین Rhizopus stolonifer تأثیر المنتجات االیضیة ل .البیضوبعض العناصر وفعالیة أنزیم الكاتالیز في مصل دم ذكور الجرذان *، احمد رحیم ریشان* نجالء عبیس الجبوري، أنوار صالح صیهود ،كلیة الزراعة ، جامعة القادسیة *كلیة الطب،جامعة القادسیة 2011 كانون االول 7: قبل البحث في 2011 أب 2:استلم البحث في الخالصة أثیر المنتجــات فــي بعــض Rhizopus stoloniferاالیـضیة للفطــر صـممت الدراســة الحالیـة مــن اجـل التعــرف علـى تــ مــن ذكـور الجــرذان، بمعـدل عمــر تــراوح 25تــم تقـسیم . مـضادات األكــسدة، والعناصـر، مثــل النحـاس، والحدیــد، والكالـسیوم ـهرا ومعـدل وزن تــراوح بــین ) 3.5-3(بـین ـامیع متــساویة5غـرام، عــشوائیا إلـى ) 225-200(ًشـ 5كـل مجموعــة ضــمت ( مجـ ة تراكیــز م): حیوانـات جموعـة الـسیطرة التـي جرعـت فمویـا بـالمحلول الملحـي الفـسیولوجي واربـع مجـامیع جرعـت فمویـا بأربعـ ـات االیـضیة للفطــر ایكرولیتر) 120، 60، 30، 15(مـن المنتجـ كیلــوغرام مــن وزن الجـسم ، وأعطیــت هـذه التراكیــز مــرة / مـ ة بیـوم ثم قتلت الحیوانات في المج. یوما15واحدة فقط ثم تركت مدة امیع المختلفة بعد تخدیرها باالیثرعنـد نهایـة مـدة التجربـ . واحد وأخذت نماذج الدم لقیاس بعض المعاییر ـامین والحدیـد والنحــاس فـي المــصل مـع زیــادة تركیــز C أظهـرت النتــائج وجـود انخفــاض معنـوي فــي تركیـز كــل مـن فیتـ نویـة فـي الحیوانـات المعاملـة فــي حـین لـم یتـأثر تركیـز الكالـسیوم فــي المعاملـة، بینمـا شـهدت فعالیـة إنـزیم الكاتــالیز زیـادة مع . المصل معنویا أن نتائج هذه الدراسة یمكن أن تشجع بـاحثین آخـرین لدراسـات أخـرى مستفیـضة فـي هـذا الموضـوع ال سـیما أننـا نجـد فـي ــي یمكــن أن تــسبب الكثیـــر مــن المـــشاكل كثیــر مــن أســـواقنا المحلیــة العدیــد مـــن الخــضراوات والفواكـــه المتعفنــة أو التالفــة التـ . الصحیة إذا استهلكت مدة من الزمن فطریات ،مضادات االكسدة ،فیتامین سي،سیروم كالسیوم،سیروم نحاس،سیروم حدید:الكلمات المفتاحیة ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 1 Vol. 25 Year 2012