ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or 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, Unive rsity of Al-Qadisiya. College of Medicine,Unive rsity of Al-Qadisiya* Received in :2 August 2011 Accepte d in : 7 De cember 2011 Abstract The present study was designed to investigate the effect of R. stolonifer metabolic p roducts on some antioxidant defenses and some elements (Cu, Fe, Ca) . The exp eriment was p erformed 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 group s (five animals for each): control (treated orally with normal saline) and four treatment group s were drenched orally with four concentrations of R. stolonifer metabolic p roducts (15, 30, 60,120) μl/kg body weight. The animals were treated with one single dose of the p reviously described concentrations then left for 15 days. Animals of different group s were sacrificed under light ether anest hesia one day after the end of treatments and blood samp les were taken to determine some phy siological, biochemical p arameters. The results revealed that the treated group s showed significant decreases (P< 0.05) in serum vitamin C and Fe, Cu concentrations. The decrease was p rop ortional with t he increase the concentrations of t he metabolic products of R. stolonifer. On t he contrary Catalase activity increased significantly (P< 0.05), while Ca concentration didn’t differ significantly (P< 0.05) The findings of t his st udy may encourage other researcher for further study esp ecially that we find in our local market that there are many rotted vegetable and fruits which may cause serious p roblems if consumed for certain period. Key word: Fungi , Rhizop ns stolonifer, antioxidant VitaminC, Serum cop p er, Serum lron, Serum calcium Introduction Fungi, bein g ubiquitous in distribution, are highly successful in surviv al because of their great p lasticity and p hy siological versatility . Over the course of history fungi, have been a continuous source of great benefit and risk for human and animals l ife and sign ificant sourc e of exp erimental knowled ge, thus the importance of kingdom of fun gi relates t o its interactions on an equal basis with members of p lants, animals and prokaryotic microorganism [1] . In terms of abundance of sp ecies of fun gi with app roximately 1.5 million sp ecies r epresent the next major p lay er in the evolutionary drama of the planet [2] . Rhizopus belongs to most prominent orders of the Zygomycetes , M ucorales. The M ucorales are mostly sap rotrop hic and are abundant in soil, on dun g and on other or ganic matter in contact with t he soil. They may p lay an important role in the early colonization of subst rata. Sometimes, howev er, they can behav e ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 as weak pathogens of soft p lant tissues [3] . Several sp ecies of Rhizopus are p lant and human p athogens associated [4] . Sp ecies of Rhizopus are reported from human lesions, and these genera to gether with sp ecies of Absidia may also infect domestic animals. Rhizopus stolonifer is necrotrop hic p athogen,cause rots of fruit [3] can cause a rot of sweet p otatoes or fruits such as app les, t omatoes and st rawberries, such infections may cause sp oilage of food [5] . There are about 10 sp ecies which grow in soil [6] and on fruits, other foods and all kinds of d ecaying materials. Rhizopus stolonifer (sy n. R. nigricans) grows rapidly and it is often found on rip e fruits, esp ecially if these are incubated in a moist . M ould infection and mycotoxin contamination of cereal gr ains can occur in the field dur ing growin g, at harvest and dur ing st orage. As t he gr ains p rogress t hrough harvesting and storage, feed manufacture and delivery to farms, t herefore p ublic concern on health matters related to food has increased [7,8] and to our knowledge few st udies have been carried out to identify the direct relation between these fungi and animal health. T herefore the p resent study was conducted to investigate the effect of Rhizopus stolonifer metabolic p roducts on serum vitamin C, som e elements and Catalase in albino male r ats. Methods and Materials Rhizopus stolonifer Isolates of R.stolonifer cultured in nutrient broth media (from B.D.H) for 21 days. Aft er this p eriod the media was filtered by filter paper (what man no.1) [9] and maintained in clean st erile tubes until used in the exp eriment. Animals and Exp erimental Design The exp eriment was p erformed 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 group s (five animals for each): control (treated orally with normal saline) and four treatment group s were drenched orally with four concentrations of R. stolonifer metabolic p roducts (15, 30, 60,120) μl/kg body weight. The animals were treated with one single dose of the previously described concentrations t hen left for 15 day s. Blood Collection The animals were sacrificed under light ether anest hesia one day after the end of treatment. Blood samples were collected into clean dry centrifuge tubes and left to st and for 30 minutes at room temp erature, then centrifuged at 3000rp m for 10 minutes and serum samples were st ored in clean tubes at –20C t ill 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 enzy matic 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 A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 Determination of elements Serum iron (Fe), copp er (Cu), and calcium (Ca) levels were determined using flame atomic absorp tion sp ectrop hotometer (GBC 933 p lus) and fivefold dilution with de-ionized water. Iron was determined at a wavelength of 284.3 nm, copp er at (324.7) nm, and calcium at (319.9) nm. The concentrations of serum Fe, Cu, and Ca were calculated from the st andard curves of t hese elements [12] Statist ical Analysis The data were analyzed using F test taking P<0.05 as the lowest limit of significant difference and Duncan's M ultip le Range Test was used to identify group resp onsible for st atist ical difference through comp arison [13] . Results Table (1) summarized the results in normal and treated rats in. The treated group s show significant decreases (P< 0.05) in serum vitamin C Fe and Cu concentrations. T he decreasing was p rop ortional with the increasing of metabolic p roducts 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 st udied, to determine their modify ing effects on animal health [5] . M any st udies demonstrated different effects and mechanisms of action. Some authors have raised the theory that their action may be a result of disrup tion 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 p rimary antioxidant in p lasma and within cells and may contact with p lasma 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 hy droxy l radicals from sy st ems containing copp er ions and hy drogen p eroxide by a mechanism that involve the immediate att ack of vitamin C itself by free radicals, so t hey become no longer free in solution [16] . The elevation in serum Catalase activity observed in this st udy can be exp lained by the fact that t his enzy me considered as one of the enzy matic 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 st imulation of activity of the above mentioned enzy me and many other enzy mes which have related functions [19] . Trace elements are essential for human and animals. They are constituents of, or interact with, enzy mes and hormones that regulate the metabolism of much larger amounts of biochemical substrates [20] . Also they are found at t he active sites of many enzy mes and have the ability to facilitate the transfer of single electrons to molecular oxy gen to p roduce sup eroxide anion radical which in turn is converted to hy droxy l radical through Haber-Weiss reaction or Fenton reaction [21] . Decreasing the concentrations of serum Fe and Cu ions may exp lained on the basis of the roles of these metals, esp ecially their imp ortance in the activity of enzy mes [20] . Serum Ca concentration didn’t change comp ared with the other two elements. This may be exp lained by the fact that most of the calcium in the body is st ored in the bones [21] , thus any exp ected alteration in calcium st ate in the cell, bones may p rovide an extra amount of needed ions. ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 The results of the p resent st udy indicate that there are alterations in serum concentrations of some elements, vitamin C and catalase , suggesting that they may p lay a role in the p athop hy siology of the fungus. Re ferences 1. Webst er, J. & Webster, R.W.S. (2007). Introduction to Fungi. 3 ed . Cambridge University Press. 841. 2. Khachatatourians, G.G. & Arora, D.K. (2001). Applied My cology and Biotechnology , 1. Agricu lture and Food p roduction. 1 st ed. Elsevier Science B.V. Amst erdam, Netherland. 435. 3. Carlile, M .J., Watkinson, S.C. & Gooday , G.W. (2001). The Fungi. 2 nd ed. Acad emic Press. 565. 4. Rinaldi, M . G. (1989). Zy gomycosis. Infec. Dis. Clin. North Am., 3, 19-41. 5 . Samson, R. A., Hoekst ra, E. S., Frisvad, J. C. & Filtenbor g, O. (2002). Int roduction to Food-and Airborne Fungi, 6 th ed. Utrecht: Centraalbureau voor Schimmelcultures 6. Domsch, K. H., Gams, W. & Anderson, T . H. (1980). Comp endium of Soil Fun gi. London, Academic Press. 7. M artin, J.F.(1992). Secondary metabolite. In: App lied M olecular Genetics of Fil-amentous Fungi. Kinghorn, J.R. & Turner, G. (eds). Blackie Academic and Profrssional, London, pp : 214-252. 8. Sarig, P., Z ahvi, T., Zutkhi, Y., Yannai, S., Lisker, N. & Ben-Arie, R. (1996). Ozone for control of p ost-harvest decay of table grapes caused by Rhizopus stolonifer. Phy siol. M ol. Plant Pathol., 48, 403–415. 9. Colle, J.G., M armion, B.P., Fraser, A.G. & Simmons A. (1996). Practical M edical M icrobiology (M akie & M cCartne). 14 th ed. New York., Churchill Livin g Stone. . 131-150. 10. M cCormic, D.B. (1986). Vitamins. In: Te xtbook of Clinical Chemistry . Tietz, N.W.(ed). Philadelp hia, P.A. Saunders. 949. 11. Aebi, H.E. (1987). Catalase of enzy matic analysis. In: Enzy mes: Oxidoreductase, transferases. Bergmeyer, H.U. (ed). Weinhein, Germany . VCH Ver lags gesell & Schaft.: 273- 285. 12. Growenlock, A.H.; M cM urray , J.R. & M cLauchlan, D.M . (1988). Valery ' s Practical Biochemist ry . 6 th ed. Eds. Heineman medical books. London. 13. Schef ler, W.C. (1980). Statist ics for Biological Scien ce .2 nd ed. Addison Wesley , Pub. Co. London, Amsterdam.. 121. 14. Carr, A. C. & Frei, B. (1999). Toward a new recommended d ietary allowance for vitamin C based on antioxidant and health effects in humans. Am. J. Clin. Nutr., 69, 1086-1107. 15. M ay , J.M. (1999). Is ascorbic acid an antioxidant for p lasma membr ane?. FASEB. J., 13, 95-99. 16. Hughes, D. A. (2000). Dietary antioxidants and human immune function. Br. Nutr. Found. Nut r. Bull., 25, 35-41. 17. M ates, J.M . & Sanches-Jimenez, F.(1999). Antioxidant enzy mes and their imp lications in p athop hysiological processes. Front iers in Bioscience, 4, 339-345. 18. Harris, E.D. (1992).Regu lation of antioxidant enzy mes. FASEB. J., 6, 99-106. 19. Proctor, P.H. & Reynolds, E.S. (1984). Free radicals and d isease in man. Phy siological Chemistry and Phy sics and M edical NM R., 16, 175-195. 20. Burtis, C.A. & Ashwood, E.R. (1996). Tietz Fundamentals of Clinical Chemistry , 4 th ed. W.B. Saunders Company. Philadelp hia.. 272, 274. 21. Guy ton, A.C. & Hall, J.F.(1997). Textbook of M edical Phy siology . 5 th ed., W.B. Saunders Co. London,. 503-549. ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012 T able (1): Effect of Rhizopus stolon ifer met abolic p ro duct s on serum vit amin C , some elem ents and cat alase in albino male rat s treatm ent P aram et ers C Normal saline 15 μl/kg R. stolonifer 30 μl/kg R. stolonifer 60 μl/kg R. stolonifer 120 μl/kg R. stolonifer Vit amin 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 act ivit y (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 10 2±3.12 c 85.71 0.77± d Fe ( μg/dl) 14 0 ±4.00 a 138.30±1.0 5 a 131 2.10± a 11 2±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 represent ed averages ± st andard erro r. *Different let t ers m ean t hat t here are significan t differences (P <0.05). ةمجلة إبن الھیثم للعلوم الصرفة و التطبیقی 2012 السنة 25 المجلد 1 العدد Ibn A l-Hai tham Journal f or 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 فــي بعــض Rhizopus stoloniferاالیـضیة للفطــر صـممت الدراســة الحالیـة مــن اجـل التعــرف علـى تــأثیر المنتجــات مــن ذكـور الجــرذان، بمعـدل عمــر تــراوح 25تــم تقـسیم . مـضادات األكــسدة، والعناصـر، مثــل النحـاس، والحدیــد، والكالـسیوم 5كـل مجموعــة ضــمت ( مجــامیع متــساویة5غـرام، عــشوائیا إلـى ) 225-200(ًشــهرا ومعـدل وزن تــراوح بــین ) 3.5-3(بـین جموعـة الـسیطرة التـي جرعـت فمویـا بـالمحلول الملحـي الفـسیولوجي واربـع مجـامیع جرعـت فمویـا بأربعـة تراكیــز م): حیوانـات كیلــوغرام مــن وزن الجـسم ، وأعطیــت هـذه التراكیــز مــرة / مـایكرولیتر) 120، 60، 30، 15(مـن المنتجــات االیـضیة للفطــر امیع المختلفة بعد تخدیرها باالیثرعنـد نهایـة مـدة التجربـة بیـوم ثم قتلت الحیوانات في المج. یوما15واحدة فقط ثم تركت مدة . واحد وأخذت نماذج الدم لقیاس بعض المعاییر والحدیـد والنحــاس فـي المــصل مـع زیــادة تركیــز C أظهـرت النتــائج وجـود انخفــاض معنـوي فــي تركیـز كــل مـن فیتــامین 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 التـــي یمكــن أن تــسبب الكثیـــر مــن المـــشاكل كثیــر مــن أســـواقنا المحلیــة العدیــد مـــن الخــضراوات والفواكـــه المتعفنــة أو التالفــة . 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 A l-Hai tham Journal f or Pure and Applied Science No. 1 Vol. 25 Year 2012