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International Journal for Sciences and Technology

Volume 10. No. 2/ June 2015 / ISSN: 2305-9346

A Refereed Scientific Journal Since 2006

2006         Issued By: The International Centre for Advancement of Sciences and Technology In a cooperation with TSTC - Jordan

IJST contact Information: P.O. Box 2793 Amman 11953 Jordan Tel. +962796543469 E-mails: [email protected] / [email protected] URL: www.ijst-jo.com

EDITORIAL BOARD - 2015 Al- Shammari , Abdul- Jabbar N. (Editor-in- Chief) Professor of Microbiology / Dept. of Chemistry and Laboratory Medicine / Faculty of Sciences / AlBalqa' Applied University / Al- Salt / Jordan [email protected] Abbas, Jamal A. Professor of Plant Ecophysiology / Faculty of Agriculture / Kufa University / Iraq [email protected] Abdul- Ghani, Zaki G. Professor of Microbiology/ Faculty of Pharmaceutical Sciences / Amman Private University / Jordan [email protected] Abdul- Hameed, Hayder M. PhD in Environmental Engineering / Environmental Engineering Dept./ Faculty of Engineering/ University of Baghdad/ Iraq [email protected] Abdullah, Ahmed R. PhD in Cancer Immunology and Genetics /Biotechnology Research Centre / Al- Nahrain University / Baghdad / Iraq [email protected] Al- Daraji, Hazim J. Professor of Avian Reproduction and Physiology / Animal Resources Dept./ College of Agriculture / University of Baghdad / Iraq [email protected] Al- Douri, Atheer A. R PhD in Microbiology/Faculty of Veterinary Medicine/ University of Baghdad / Iraq [email protected] Al- Faris, Abdulbari A. Professor of Surgery / Dept. of Surgery and Obstetrics / College of Veterinary Medicine / University of Basrah / Iraq [email protected] Al- Jashami, Najim A. Professor of Nuclear Material Sciences / Dept. of Physics / College of Sciences / Kufa University / Iraq [email protected] Al- Mashaykhi, Akram Othman PhD in IT / Amman Arab University for Graduate Studies / Jordan [email protected] Al- Mathkhoury, Harith J F. Professor of Medical Microbiology / Dept. of Biology / College of Sciences / University of Baghdad/ Iraq [email protected]

Al- Murrani, Waleed K. Professor of Genetics and Biostatistics / University of Plymouth/ UK [email protected] Al- Noor, Nadia H. PhD. in Statistics, Mathematical Statistics / Dept. of Mathematics / College of Sciences / AlMustansiriya University / Baghdad / Iraq Al- Noor, Taghreed H. Professor of Chemistry / Dept. of Chemistry / College of Education – Ibn Al- Haitham / University of Baghdad / Iraq [email protected] Al- Saqur, Ihsan M. Professor of Parasitology/ Faculty of Sciences / University of Baghdad / Iraq [email protected] Al- Shamaony, Loai Professor of Biochemistry / Faculty of Pharmacy / Misr University for Sciences and Technology / Egypt [email protected] Al- Shebani, Abdullah S. PhD in Dairy Sciences and Technology / Food Sciences Dept./ Faculty of Agriculture / Kufa University / Iraq [email protected] Alwachi, Sabah N. Professor of Physiology / Biology Dept./ College of Sciences/ University of Baghdad / Iraq [email protected] Birasal, Narayan R. PhD in Zoology / KLE Society’s GH College Haveri / India [email protected] Khamas, Wael Professor of Anatomy and Histology / College of Vaterinary Medicine / Western University of Health Sciences / Ponoma -California/ USA [email protected] Mohammed, Ramadhan H. PhD in Geology / College of Sciences / Duhok University / Iraq [email protected] Sharma, Sunanda PhD in Veterinary Medicine / Dept. of Veterinary Obstetrics and Gynecology / College of Veterinary & Animal Science / Rajasthan University of Veterinary & Animal Sciences [email protected]

Editorial Board Secretary Pharmacist. Nansi Elian Amman- Jordan [email protected]

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

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FORWARD

Dear Collegues, Year after year, our steps become more strong, more steady towards converting the idea of a great significant Arabic scientific society into real thorugh our insistence to continue what we had started since 10 years ago, when our Journal IJST was created to existence. IJST was a fruitful effort issued by the International Centre for Sciences and Technology – ICAST, which tries to take part in both globalization and revolution in information and communication technologies, because S&T development becoming not only the key elements of economic growth and industrial competitiveness, but also essential for improving the social development, the quality of life and global environment. ICAST took then a decision to establish a scientific alliance with TSTC (Tharwa for scientific Training & Consultations) and this alliance comes to support the efforts towards publishing IJST. By this issue, IJST had been gifted with new Editorial Members and its my pleasure to welcome them in our committee, to Prof. Abdulbari Abbas Al- Faris from University of Basrah, and Prof. Harith Jabbar Al- Mathkhoury from University of Baghdad. On behalf of the Editorial Board Committee and Editorial Board Secretary, I would like to present them deep appreciations for being joined us in our journey. Today, we announce a new issue of our journal, that is the second issue from the tenth volume of IJST, June , 2015. As this issue is coming to you while Islamic world is celebrating Ramadan , I would like to present you all my deep wishes and faithful prays for peaceful and prosperity times and best movements. Finally, on behalf of the International centre, I would like to express my special thanking to the Editorial Board Secretary for her faithful efforts in managing the scientific, design, technical and administrative aspects of the Journal and for preparing this issue for final printing and publishing.

Editor-in-Chief IJST Abdul Jabbar Al- Shammari

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

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The Referees for this Issue * The referees and advisory group below are listed according to alphabetical order, with deep appreciation for all. Prof. Abdul- Jabbar N. Al- Shammari Dept. of Chemistry and Laboratory Medicine, Faculty of Sciences, Al- Balqa' Applied University , AlSalt . Jordan Dr. Abdullah Sh. M. Al- Shebani Dept. of food sciences, Faculty of Agriculture, Kufa University. Iraq Prof. Ahmed M. Abdul-Lettif College of Sciences, University of Karbala. Iraq Dr. Amer Al- Sarraj College of Pharmacy, Royal University for Medical Sciences (RUMS). Jordan Dr. Atheer AR. Al- Douri Faculty of Veterinary Medicine, University of Baghdad. Iraq Prof. Bashar R. Al- Shreidah National Centre for Agricultural Researches . Jordan Dr. Dawood S. Al- Azzawi College of Medicine, Diyala University. Iraq Prof. Harith F. Al- Mathkhouri College of Sciences, University of Baghdad. Iraq Prof. Jamal A. Abbas Faculty of Agriculture, Kufa University. Iraq Dr. Khalid Al- Azzawi Faculty of Pharmacy, Al- Isra University. Jordan Prof. Mahmoud M. Othman Matar College of Medicine, Al- Najah National University. Palestine Prof. Majed A. Al- Attar Ontario city, Canada ( professor of avian diseases , Veterinary Medicine) Dr. Mohammed Al- Bedri Faculty of Allied Medical Sciences, Royal University for Medical Sciences (RUMS). Jordan Dr. Ramadhan H. Mohammed College of Sciences , Duhok University . Iraq Prof. Taha H. Al- Samarrai College of Sciences, University of Samarra. Iraq Prof. Waleed Al- Murrani University of Plymouth , United Kingdom

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

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TABLE OF CONTENTS * Articles in this issue are listed below according to field specialties order, starting by English section and followed by Arabic section.

(I) ENGLISH SECTION AGRICULTURAL SCIENCES Effect of different explants and concentrations of plant growth regulators on micropropagation of carnation (Dianthus caryophyllus L.) Layla S. M. Al- Mizory, Sua'ad A.H. Yaseen & Ameena M. Hassan Effect of spraying nutrient solution "BASAK" and salicylic acid on some vegetative growth and flowering parameters of stock plant (Mathiola incana L.) Jamal A. Abbass, Mushtaq T. H. Al- Zurfy & Mustafa M. Ibrahim Feasibility of using brewers spent grain as a fertilizer in agriculture Amal A. Muhammed, Keith Thomas & Usama Bin- Hamed

7-15

16-21

22-30

ENVIRONMENT The composition of accumulated dust on bookshelves: analytical and molecular study Mohammed I. Khalil & Ghayda S. H. Al-Enezy

31-35

IMMUNOLOGY Immunohistochemical study of IL-17 expression in prostate tissue of a prostate cancer patients Hassan H. Zrage, Zainab F. Ashoor & Wasan A. Bakir

36-40

MEDICINE The menopausal age and associated factors in a sample of Iraqi postmenopausal women Dalya T. Ahmed & Ekhlas A. Hussein

41-46

MICROBIOLOGY Effect of silver nanoparticles prepared with pomegranate peel extract on antibiotic resistant Gram negative bacteria Salah S. Zain Al- Abdeen

47-51

MOLECULAR MICROBIOLOGY Molecular localization of envelope gene sequence of Human Mammary Tumor Virus in malignant endometrial and cervical tissues from Iraqi patients in Baghdad Saad H. M. Ali, Basim S. Ahmed & Sura D. Dawood

52-57

Partial purification and characterization of factors from Bacillus amyloliquefaciens strain that caused catenulate Vesicle like Sac formation of the germinating tube of Glomerella cingulata spores Abduljaleel H. Mohammed

58-63

PATHOLOGY A study on clinical signs and immunological parameters associated with experimental thyrotoxicosis in rats Khalil H. Al – Jeboori & Sattar R. Suhail Evaluation of some biochemical and thyroid hormones associated with experimental thyrotoxicosis in rats Khalil H. Al – Jeboori & Sattar R. Suhail

64-67

68-71

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

Continue>>>> English section

PHYSICS Radon emanation in granite and marble building materials that imported in Iraq Ramla D. Salman

72-78

VETERINARY MEDICINE Histopathological and microbiological study of avian respiratory infection in Iraq Methaq A.R. Abdul- Sammad, Bahaa A. Al- Sereah, Rasha M. Outhman & Wasan M. Shaker

79-84

Radiological and histopathological study of diabetes mellitus effect on bone healing in late stages in dogs Abdulbari A. Al- Faris, Alaa A. Sawad & Saad H. Zebon

85-92

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‫‪Vol. 10, No.2, June 2015‬‬

‫‪International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735‬‬

‫)‪ (II‬ﻗﺴﻡ ﺍﻝﺩﺭﺍﺴﺎﺕ ﻭﺍﻝﺒﺤﻭﺙ ﺍﻝﻌﺭﺒﻴﺔ – ‪ARABIC STUDIES AND RESEARCHES SECTION‬‬ ‫ﺍﻝﺘﺨﻁﻴﻁ ﻭﺍﻝﺘﻨﻤﻴﺔ‬ ‫ﺘﺨﻁﻴﻁ ﻭﺘﻨﻤﻴﺔ ﺍﻝﻘﻭﻯ ﺍﻝﻌﺎﻤﻠﺔ ﻭﺍﻝﻘﻁﺎﻋﺎﺕ ﺍﻻﻗﺘﺼﺎﺩﻴﺔ ﻓﻲ ﻤﺤﺎﻓﻅﺔ ﺩﻴﺎﻝﻰ‬

‫‪104 -94‬‬

‫ﺠﻭﺍﺩ ﻜﺎﻅﻡ ﺍﻝﺤﺴﻨﺎﻭﻱ‪ ،‬ﻭﺴﺎﻡ ﻭﻫﻴﺏ ﻤﻬﺩﻱ‪ ،‬ﺭﺸﻴﺩ ﺴﻌﺩﻭﻥ ﻤﺤﻤﺩ‬ ‫ﺍﻝﺘﻘﻨﻴـــﺎﺕ ﺍﻝﺤﻴﻭﻴـﺔ‬ ‫ﺇﻨﺘﺎﺝ ﻨﺒﺎﺘـﺎﺕ ﺍﻝﻁﻤـﺎﻁﻡ ‪ Lycopersicon esculetum‬ﺍﻝﻤﺤﻭﻝـﺔ ﻭﺭﺍﺜﻴـﺎ ﺒﺎﺴـﺘﺨﺩﺍﻡ ﺴـﻼﻝﺔ ‪ C58C1‬ﻤـﻥ‬ ‫ﺒﻜﺘﻴﺭﻴﺎ ‪Agrobacterium tumefaciens‬‬

‫‪110 -105‬‬

‫ﻨﻬﺎل ﻋﺯﺕ ﺍﻝﻁﺎﺌﻲ‪ ،‬ﺃﻤﺠﺩ ﻋﺒﺩ ﺍﻝﻬﺎﺩﻱ ﻤﺤﻤﺩ‪ ،‬ﺠﺎﺴﻡ ﻤﺤﻤﺩ ﻴﺎﺴﻴﻥ ﺃﺤﻤﺩ‬ ‫ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﺘﺜﺒﻴﻁﻲ ﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ‪ Ricinus communis‬ﻋﻠﻰ ﻨﻤﻭ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ ﻨﻭﻉ ‪HepG2‬‬

‫‪116 -111‬‬

‫ﺴﺭﺍﺏ ﺴﻠﻤﺎﻥ ﻜﺎﻅﻡ‪ ،‬ﺯﻴﻨﺏ ﻁﻼل ﺤﺴﻴﻥ ﺍﻝﺒﻴﺭﻗﺩﺍﺭ‪ ،‬ﻓﺎﺭﻭﻕ ﺇﺒﺭﺍﻫﻴﻡ ﻤﺤﻤﺩ‪ ،‬ﺤﺴﻥ ﻫﺎﻨﻲ ﺠﻭﺍﺩ‪،‬‬ ‫ﺃﺤﻤﺩ ﻤﺅﻴﺩ ﻤﺤﻤﺩ ﺼﺎﻝﺢ‬ ‫ﺍﻝﺼﻨﺎﻋـــﺎﺕ ﺍﻝﻐﺫﺍﺌﻴــــﺔ‬ ‫ﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺇﻀﺎﻓﺔ ﺒﺫﻭﺭ ﺍﻝﺸﻤﺭ ﺍﻝﻤﻁﺤﻭﻨﺔ ﻓﻲ ﺍﻝﺨـﻭﺍﺹ ﺍﻝﻨﻭﻋﻴـﺔ ﻝﻠﻜﻴـﻙ ﺍﻝﻤﻘـﺼﺭ ) ‪(Shortened cake‬‬ ‫ﺍﻝﻤﺼﻨﻊ ﻤﺨﺘﺒﺭﻴﺎ ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻝﻤﻭﺍﺩ ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﺼﻨﺎﻋﻴﺔ‬

‫ﺃﻭﺱ ﻫﻼل ﺠﺎﺴﻡ ﺍﻝﻌﺎﻨﻲ‬

‫‪122 -117‬‬

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

ENGLISH SECTION

Vol. 10, No.2, June 2015

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

Effect of different explants and concentrations of plant growth regulators on micropropagation of carnation (Dianthus caryophyllus L.) Layla S. M. Al- Mizory, Sua'ad A.H. Yaseen and Ameena M. Hassan Dept. of Horticulture / Faculty of Agriculture / University of Duhok / Kurdistan Region- Iraq E- mail: [email protected]

ABSTRACT The current study was conducted at Plant Tissue Culture Laboratory of the Horticulture Department, Faculty of Agriculture at University of Duhok, Iraq, during the period from April 2013 to November 2013. The aim of the study was to examine propagation Dianthus carryophyllus L. form shoots (10 cm) containing (shoot tips and node segments) which were excised and cultured on MS medium in the experience of the dates. The highest percentage of shoot response (100%) was recorded from node segments when cultured on MS medium, supplemented with 2mgl-1 in April and March and (100%) from shoot tips on the same medium in April and March. The highest number of shoots (4.30, 4.10 shoots/ explant) were recorded from node segments when cultured on MS medium supplemented with 2mgl-1 in March and April respectively, and (2.80 shoots/ explant) from shoot tips on the same medium in April. The lowest number of shoots (1.40 shoots/ explant) was recorded in February. On the other hand, the highest number of leaves (14.30 and 12.80 leaves/ explant) from node segment and shoot tips, respectively were found in April. At the multiplication stage, results had shown that the highest number of shoots/ explant observed on MS medium containing 3mgl-1 BA+ 0.1mgl-1 NAA (5.70) which was significantly different from other treatments container MS medium with BA and NAA alone and the highest number of leaves/ shoot (20.60) was formed on explants cultured on MS medium supplemented with (3 mgl-1 + 0.1 mgl-1 NAA). While The results showed that the highest node number /shoot (4.10) was formed on explants cultured on MS medium supplemented with 1 mgl-1BA + 0.1 mgl-1 NAA. As for shoot length/explant, the best result (3.91cm) was obtained when the explant was cultured on MS medium supplemented with 3mgl-1 + 0.1 mgl-1 NAA only, which was significantly higher than the other treatments container MS medium with BA and NAA alone 97.50% of shoots produced from multiplication stage were rooted when cultured on ¼ MS medium supplemented with 1.5 mgl-1NAA .Acclimatzed plants grow well under greenhouse condition. Keywords: Dianthus caryophyllus, micropropagation, plant growth regulators, shoot tips and node segments

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ‫ﺃﺠﺭﻴﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻓﻲ ﻤﺨﺘﺒﺭ ﺯﺭﺍﻋﺔ ﺍﻷﻨﺴﺠﺔ ﺍﻝﺘﺎﺒﻊ ﻝﻘﺴﻡ ﺍﻝﺒﺴﺘﻨﺔ ﻓﻲ ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ ﺒﺠﺎﻤﻌﺔ ﺩﻫﻭﻙ ﻓﻲ ﺠﻤﻬﻭﺭﻴﺔ ﺍﻝﻌﺭﺍﻕ ﺨﻼل ﺍﻝﻔﺘﺭﺓ ﻤﻥ ﺸﻬﺭ‬ ‫ ﻤﻥ ﺃﻓﺭﻉ‬Dianthus carryophyllus L ‫ ﻨﻭﻓﻤﺒﺭ ﻤﻥ ﺍﻝﻌﺎﻡ ﻨﻔﺴﻪ ﺒﻬﺩﻑ ﺇﻜﺜﺎﺭ ﻨﺒﺎﺕ ﺍﻝﻘﺭﻨﻔل‬/‫ ﺇﻝﻰ ﺸﻬﺭ ﺘﺸﺭﻴﻥ ﺍﻝﺜﺎﻨﻲ‬2013 ‫ ﺇﺒﺭﻴل ﻋﺎﻡ‬/‫ﻨﻴﺴﺎﻥ‬ (%100) ‫ ﻭﻗﺩ ﻜﺎﻨﺕ ﺃﻋﻠﻰ ﻨﺴﺒﺔ ﺍﺴﺘﺠﺎﺒﺔ ﻝﻸﻓﺭﻉ‬، MS ‫ ﺴﻡ ﺘﺤﺘﻭﻱ ﻋﻠﻰ )ﺍﻝﻌﻘﺩ ﺍﻝﻤﻔﺭﺩﺓ ﻭﺍﻝﻘﻤﻡ ﺍﻝﻨﺎﻤﻴﺔ( ﻭﺍﻝﺘﻲ ﺯﺭﻋﺕ ﻋﻠﻰ ﺍﻝﻭﺴﻁ ﺍﻝﻐﺫﺍﺌﻲ‬10 ‫ﺒﻁﻭل‬ ‫( ﻤﻥ ﺍﻝﻘﻤﻡ ﺍﻝﻨﺎﻤﻴﺔ ﻓﻲ ﻨﻔﺱ‬%100) ‫ ﻭ‬،‫ ﻝﺘﺭ ﻓﻲ ﺸﻬﺭﻱ ﺁﺫﺍﺭ ﻭﻨﻴﺴﺎﻥ‬/ ‫ ﻤﻠﻐﻡ‬2 ‫ ﺍﻝﻤﺯﻭﺩ ﺒـ‬MS ‫ﻤﻥ ﺍﻝﻌﻘﺩ ﺍﻝﻤﻔﺭﺩﺓ ﻋﻨﺩ ﺯﺭﺍﻋﺘﻬﺎ ﻋﻠﻰ ﺍﻝﻭﺴﻁ ﺍﻝﻐﺫﺍﺌﻲ‬ ‫ ﺍﻝﻤﺯﻭﺩ‬MS ‫ ﺠﺯﺀ ﻨﺒﺎﺘﻲ ﺘﻡ ﺇﻨﺘﺎﺠﻪ ﻤﻥ ﺍﻝﻌﻘﺩ ﺍﻝﻤﻔﺭﺩﺓ ﺍﻝﻤﺯﻭﺭﻋﺔ ﻋﻠﻰ ﺍﻝﻭﺴﻁ ﺍﻝﻐﺫﺍﺌﻲ‬/ ‫( ﻓﺭﻋﺎ‬4.10. ‫ ﻭ‬4.30) ‫ ﺒﻴﻨﻤﺎ ﺒﻠﻎ ﺃﻋﻠﻰ ﻋﺩﺩ ﻝﻸﻓﺭﻉ‬،‫ﺍﻝﻔﺘﺭﺓ‬ ‫ ﺠﺯﺀ ﻨﺒﺎﺘﻲ ﻤﻥ ﺍﻝﻘﻤﻡ ﺍﻝﻨﺎﻤﻴﺔ ﺍﻝﻤﺯﻭﺭﻋﺔ ﻋﻠﻰ ﻨﻔﺱ ﺍﻝﻭﺴﻁ ﻭﻝﻜﻥ ﻓﻲ ﺸﻬﺭ‬/ ‫( ﻓﺭﻋﺎ‬2.80) ‫ ﻭ‬،‫ﻝﺘﺭ ﻓﻲ ﺸﻬﺭﻱ ﺁﺫﺍﺭ ﻭﻨﻴﺴﺎﻥ ﻋﻠﻰ ﺍﻝﺘﻭﺍﻝﻲ‬/ ‫ ﻤﻠﻐﻡ‬2 ‫ﺒـ‬ ‫ ﺘﻡ ﺍﻝﺤﺼﻭل ﻋﻠﻰ ﺃﻋﻠﻰ ﻤﻌﺩل ﻝﻌﺩﺩ‬،‫ ﻭﻤﻥ ﺠﻬﺔ ﺃﺨﺭﻯ‬،‫ﺠﺯﺀ ﻨﺒﺎﺘﻲ ﻓﻲ ﺸﻬﺭ ﺸﺒﺎﻁ‬/ ‫ ﻓﺭﻋﺎ‬1.40 ‫ ﺒﻴﻨﻤﺎ ﻜﺎﻥ ﺃﻗل ﻤﻌﺩل ﻝﻌﺩﺩ ﺍﻷﻓﺭﻉ‬،‫ﺁﺫﺍﺭ ﻓﻘﻁ‬ .‫ ﺠﺯﺀ ﻨﺒﺎﺘﻲ ﻤﻥ ﺍﻝﻌﻘﺩ ﺍﻝﻤﻔﺭﺩﺓ ﻭﺍﻝﻘﻤﻡ ﺍﻝﻨﺎﻤﻴﺔ ﻓﻲ ﺸﻬﺭ ﻨﻴﺴﺎﻥ‬/ ‫( ﻭﺭﻗﺔ‬12.80 ‫ ﻭ‬14.30) ‫ﺍﻷﻭﺭﺍﻕ‬ ‫ ﻓﻲ ﻤﺭﺤﻠﺔ ﺍﻝﻤﻀﺎﻋﻔﺔ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ ﺃﻋﻠﻰ ﻤﻌﺩل ﻝﻌﺩﺩ ﺍﻷﻓﺭﻉ‬،‫ ﻋﻠﻰ ﺼﻔﺎﺕ ﺍﻝﻘﺭﻨﻔل‬NAA ‫ ﻭ‬BA ‫ﻭﺒﺩﺭﺍﺴﺔ ﺘﺎﺜﻴﺭ ﺇﻀﺎﻓﺔ ﻜل ﻤﻥ ﻤﺤﻔﺯ ﺍﻝﻨﻤﻭ‬ ‫ ﺤﻴﺙ ﻜﺎﻨﺕ ﺍﻝﺯﻴﺎﺩﺓ‬NAA ‫ ﻝﺘﺭ‬/ ‫ ﻤﻠﻐﻠﻡ‬0.1 + BA ‫ ﻝﺘﺭ‬/‫ﻤﻠﻐﻡ‬3 ‫ ﺍﻝﻤﺯﻭﺩ ﺒـ‬MS ‫ ﺠﺯﺀ ﻨﺒﺎﺘﻲ ﻋﻨﺩ ﺯﺭﺍﻋﺔ ﺍﻷﻓﺭﻉ ﻋﻠﻰ ﻭﺴﻁ‬/ ‫( ﻓﺭﻋﺎ‬5.70) ‫ﺒﻠﻎ‬ ‫ ﻋﻠﻰ ﺍﻷﻓﺭﻉ‬20.60 ‫ ﻭﻜﺎﻥ ﺃﻋﻠﻰ ﻤﻌﺩل ﻝﻌﺩﺩ ﺍﻷﻭﺭﺍﻕ ﻗﺩ ﺒﻠﻎ‬،‫ ﻋﻠﻰ ﺤﺩﺓ‬NAA ‫ ﻭ‬BA ‫ﻤﻌﻨﻭﻴﺔ ﻤﻘﺎﺭﻨﺔ ﺒﺒﻌﺽ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻝﻤﺤﺘﻭﻴﺔ ﻋﻠﻰ ﻜل ﻤﻥ‬ 4.10 ‫ ﺒﻴﻨﻤﺎ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ ﺃﻋﻠﻰ ﻤﻌﺩل ﻝﻌﺩﺩ ﺍﻝﻌﻘﺩ ﻗﺩ ﺒﻠﻎ‬، NAA ‫ ﻝﺘﺭ‬/ ‫ ﻤﻠﻐﻡ‬0.1 + BA ‫ ﻝﺘﺭ‬/‫ﻤﻠﻐﻡ‬3 ‫ ﺍﻝﻤﺯﻭﺩ ﺒـ‬MS ‫ﺍﻝﻤﺯﻭﺭﻋﺔ ﻋﻠﻰ ﻭﺴﻁ‬ ‫( ﺴﻡ‬3.91) ‫ ﻭ ﻜﺎﻨﺕ ﺃﻓﻀل ﺍﻝﻨﺘﺎﺌﺞ ﻝﻁﻭل ﺍﻷﻓﺭﻉ ﻫﻲ‬، NAA‫ ﻝﺘﺭ‬/ ‫ ﻤﻠﻐﻠﻡ‬0.1 + BA ‫ ﻝﺘﺭ‬/‫ﻤﻠﻐﻡ‬1 ‫ ﺍﻝﻤﺯﻭﺩ ﺒـ‬MS ‫ﻋﻨﺩ ﺯﺭﺍﻋﺔ ﺍﻷﻓﺭﻉ ﻋﻠﻰ ﻭﺴﻁ‬ ‫ ﻭﻜﺎﻨﺕ ﺍﻝﺯﻴﺎﺩﺓ ﻓﻲ ﺍﻝﻁﻭل ﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻤﻘﺎﺭﻨﺔ‬، NAA ‫ ﻝﺘﺭ‬/ ‫ ﻤﻠﻐﻡ‬0.1 + BA ‫ ﻝﺘﺭ‬/‫ﻤﻠﻐﻡ‬3 ‫ ﺍﻝﻤﺯﻭﺩ ﺒـ‬MS ‫ﻋﻠﻰ ﺍﻻﻓﺭﻉ ﺍﻝﻤﺯﻭﺭﻋﺔ ﻋﻠﻰ ﻭﺴﻁ‬ ‫ ﻤﻥ ﺍﻷﻓﺭﻉ ﺍﻝﻤﻨﺘﺠﺔ ﻓﻲ ﻤﺭﺤﻠﺔ ﺍﻝﺘﻀﺎﻋﻑ ﺠﺫﺭﺕ ﻋﻠﻰ‬%97.50 ‫ ﻭﻜﺎﻥ ﻤﺎ ﻨﺴﺒﺘﻪ‬،‫ ﻋﻠﻰ ﺤﺩﺓ‬NAA ‫ ﻭ‬BA ‫ﺒﺒﻌﺽ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻝﻤﺤﺘﻭﻴﺔ ﻋﻠﻰ ﻜل ﻤﻥ‬ .‫ ﻭﺘﻡ ﺃﻗﻠﻤﺔ ﺍﻝﻨﺒﺎﺘﺎﺕ ﺘﺤﺕ ﻅﺭﻭﻑ ﺍﻝﺒﻴﺕ ﺍﻝﺯﺠﺎﺠﻲ‬NAA ‫ ﻝﺘﺭ‬/ ‫ ﻤﻠﻐﻡ‬1.5 ‫ ﺒﺭﺒﻊ ﺍﻝﺘﺭﻜﻴﺯ ﻤﻥ ﻗﻭﺓ ﺍﻷﻤﻼﺡ ﺍﻝﻤﺯﻭﺩﺓ ﺒﻤﻘﺩﺍﺭ‬MS ‫ﻭﺴﻁ‬

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INTRODUCTION Carnation (Dianthus carryophyllus L.) is an important ornamental cut flower in the world due to its attractive and fragrance inflorescence. The plant belongs to family Carryopillaceae and covers more than 8 genera with 2000 species. The endemic origins ranged from southern Russia to Alpine Greece and the Auvergne mountains of France (1). The trend for monoxides type cultivar has improved the preferred characters and incorporated in existing cultivars, while the undesirable traits are swept out (2). The hastened genetic erosion of the plant has made great emphasis of preserving genetic resources for future need. In contrast from temperate region, the plant has limited life span in the tropics. The maintenance of huge genetic diversity for breeding to important characters in the field were then very laborious and constrained with some technical problems such as decrease in viability after long clonal propagation and accompanying risks of pest and disease attacks ,climatic perturbation and human error (3). Carnation flower is a beautiful accent to bunch and carnation home floral arrangement. It can be used as substitute for rose petals in making syrup. An essential oil is also obtained from its flowers, which is used in perfumery, where 500 Kg of flowers can produce 100 grams of oil carnation. The flower heads are dried and used in cosmetic and sachets (4). The most successful and most extensively used discipline of plant tissue culture technique is in vitro propagation, which indicates the propagation of plants by using meristem tip culture which is the transfer of apical buds and surrounding leaf primordial to sterile culture conditions (5-8). With the realization of foreseen advantages and unprecedented applications, this technique has received great importance all over the world. Thus, the current study was aimed to investigate how to optimize the culture conditions for micropropagation of Carnations (Dianthus caryophyllous L.).

MATERIALS AND METHODS Plant Materials: Active growing shoots, 10-15cm long were cut from one year old Dianthus caryophyllus L. grown in the greenhouse. Immediately after collection, the shoots were kept in polyethylene bags for later laboratory use. Explant Preparation: Shoots were stripped of their leaves and washed under tap water for 30 min. to remove soil and other superficial contaminations, followed by tap water and liquid soap for 20 min., followed by 3–5 min. rinses in sterile distilled water. Then they were cut into shorter sections (1.5 cm) long including the shoot tip and nodes segments (9).

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Explants Disinfestations: Shoot tips and nodes segments with axillary buds were surface sterilized with 70% ethanol for 30 seconds followed by 0.1% (HgCl2) Mercuric Chloride for 5 min. The disinfected explants were rinsed 3-4 times with sterilized distilled water, and the ends of explants were trimmed. The experiments were conducted with ten replicates and the explants were placed aseptically in 25x150 mm test tubes containing 15 ml of MS medium according to (10). Initiation Stage: Explants containing (Shoot tips and node segments) were taken in three different dates (15 February, 15 March and 15 April, 2013) and cultured on MS medium containing fixed concentration of BA at (2mgl-1) to reveal their effects on culture initiation. In each date (40) explants were planted in a test tube and (10) test tubes were used for each treatment. The rest sterile plantlets were kept for subsequent testing. Readings were taken after 6 weeks of culture under conditions of 16 hrs light and 8 hrs. darkness at light density (1000 Lux), which included: number of shoot development and average of shoots length (cm). Multiplication Stage: The produced shoots were moved to MS medium. Different concentrations of BA and NAA alone and BA concentrations combined with NAA concentrations were added to investigate their effects on multiplication stage. BA was tested at (0, 1, 3 and 5 mgl-1) and NAA at (0.1and 0.5 mgl-1). They were incubated at 25± 2°C under light conditions of 16h light and 8h darkness. After 4-6 weeks, the following data were recorded: number of shoot development, number of leaves per culture, number of node per culture and average of shoots length (cm). Rooting Stage Effect of Auxins on Shoots Rooting: The effects of IAA,IBA and NAA added to the culture medium on shoot rooting were studied by carrying out two separate experiments by adding IBA ,NAA and IAA with (0.0, 0.5,1 and 1.5) mgl-1. All these treatments were examined in half salt strength medium. As far as rooting phase is concerned, features such as percentage of rooted number of roots/ explant and root length (cm) were recorded for 6 weeks. Acclimatization Stage: After 6 weeks, Dianthus caryophyllus shoot rooting of several plantlets were selected from those that formed good vegetative and seed growth. They were washed under tap water to remove agar. They were then put in Benlate fungicide solution 0.1% (11) and then planted in plastic pots filled with a sterilized mixture of peat moss and sand garden (1:1). In order to keep high moisture in the culture environment, the pots were covered with a light plastic which permits light passing and has many pores to permit air circulation. Plants were watered and given a solution containing MS salts with 0.25 of it is

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

original power. The plastic cover was removed gradually, after two weeks from planting. After four weeks; the plastic cover was removed after the transplants being sprayed with Benlate fungicide (0.1%). Statistical Analysis: The investigations were organized by using Complete Randomized Design (CRD) using 10 replicates. Significant differences among mean values were separated using Duncan multiple range tests at P≤0.05 (12).

RESULTS AND DISCUSSION Initiation stage: Response percentage of Dianthus caryophyllus L.: At this stage, MS medium supplemented with 2mgl-1 BA was much affected in producing adventitious shoot from the explants when cultured at various months. Figure (1) shows the percentage of the shoot tips and node segments of D. caryophyllus at initiation stage after 4 weeks culture. The produced shoots were of highly shoot proliferation from shoot tips and node segments were significantly enhanced when cultured on modified MS medium. Differences among the treatments on shoots number per explants and shoot length were found to be highly significant. Since the highest percentage of shoot response (100%) was recorded from node segments when cultured on MS medium, supplemented with 2 mgl-1 in April and March and (100%) from shoot tips on the same medium in the March and April, while the lowest percentage of response (92%) was recorded from node segments and (80%) from shoot tips on the same medium in February.

Figure (1): Effect of (Data culture) months on the percentage of response of plantlets from shoot tip and node segments cultured on MS medium + 2 mgl-1 BA after 4 weeks.

Effect of explant taking date on the number shoots per explants, number of leaves shoot length: At this stage MS medium supplemented with 2 mgl-1 BA was very effective in producing adventitious shoots from the explants when cultured at various dates. The produced shoots were highly shoot proliferation from shoot tips and node

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segments were significantly enhanced when cultured on modified MS medium. Differences among the treatments on number of shoot per explants, number of leaves and shoot length were found to be highly significant (Figures 2- 6). Since the highest number of shoots (4.30, 4.10 shoots/ explant) were recorded from node segments when cultured on MS medium supplemented with 2mgl-1 BA in March and April respectively, and (2.80 shoots/ explant) from shoot tips on the same medium in April. While the lowest number of shoots (1.40 shoots/ explant) was recorded in February. On the other hand, the highest number of leaves (14.30 and 12.80 leaves/ explant) from node segment and shoot tips, respectively were found in April. The best results were found when node segments were cultured on MS medium supplemented with 2mgl-1 BA in March, which was superior significantly up on all treatments except shoot tips when cultured on the same date. Figure (7) shows that MS medium supplemented with 2mgl-1 BA gave the highest shoot length from nodal segments and shoot tips in April (3.05, 3.24 cm) respectively as shown in figure (6). While the lowest length of shoots (1.90 cm) were obtained from nodal segments in February. A study conducted by (13), reported that high concentration of cytokinin reduced the number of micropropagated shoots of Dianthus caryophyllus L. Similar results have already been found with Chrysanthemum by (14). In addition, the results are in consistent with the findings of (15) on Fragraia X ananassa Duch. Similar results were found on Tiliaplatyphyllos scop by (16). The developing shoots were elongated by culturing on the same concentration of plant growth regulators. Later on, the elongated shoots were excised and used for multiple shoot induction.

Figure (2): Effect of (Data culture) months on the number of Shoots of plantlets from shoot tip and node segments cultured on MS medium + 2 mgl-1 BA after 4 weeks.

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

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15 March Figure (3): in vitro shoot induction from shoot tips of Dianthis caryophyllus on MS medium containing 2 mgl-1 BAP after 4-6 weeks of inoculation.

15 March Figure (4): in vitro shoot induction from nodal segments of Dianthis caryophyllus on MS medium containing 2 mgl-1 BAP after 4-6 weeks of inoculation.

Figure (5): Effect of (Data cutlure) months on the number of leaves of plantlets from shoot tip and node segments cultured on MS medium + 2 mgl-1 BA after 4 weeks.

Figure (6): elongated Dianthis caryophyllus shoots from : A) shoot tips, B) node segments on MS containing 2 mgl-1 BAP after 4-6 weeks of inoculation

Figure (7): Effect of (Data culture) months on the length of plantlets from shoot tips and node segments cultured on MS medium + 2 mgl-1 BA after 4 weeks.

Multiplication Stage: Effect of BA and NAA on growth characteristics of Dianthus caryophyllus L.: Results in table (1) and figures (8 A, A1, B) showed that the shoots of Dianthus caryophyllus L. had grown on MS medium containing different concentrations of BA and NAA each alone and in combinations affect the growth of the explants where the effect of the increase the proliferation. The highest number of shoots/explant observed on MS medium containing 3mgl-1 + 0.1mgl-1 NAA (5.70) which was significantly different from other treatments container MS medium with BA and NAA alone, except the same treatment which was done with BA +NAA. In the same table showed the lowest number of shoots/explant (1.80) when used MS medium containing 0.1 mgl-1 NAA alone. As for shoot length/explant, the best result (3.91cm) was obtained when the explant was cultured on MS medium supplemented with 3mgl-1 + 0.1 mgl-1 NAA only, which was significantly higher than the other treatments container MS medium with BA and NAA alone. The lowest value of shoot length (1.55cm) was noticed when Dianthus caryophyllus L. explants were cultured on MS medium supplemented with free plant growth medium. Explants of Dianthus caryophyllus L. cultured on MS medium at different concentrations of NAA and BA showed significant variation in the frequency of number of leaves/ shoot. The results showed that the highest number of leaves/ shoot (20.60) was formed on explants cultured on MS medium supplemented with (3mgl-1 + 0.1 mgl-1 NAA); whereas the lowest number of leaves/shoot (10.30) was observed on explants cultured on without plant growth regulators MS medium. While The results showed that the highest node number/ shoot (4.10) was formed on explants cultured on MS medium supplemented with 1 mgl1 BA + 0.1 mgl-1 NAA; whereas the lowest node number/shoot (2.20) was observed on explants cultured on free plant growth regulators MS medium which the auxin/cytokinin balance is one of the factors with a determining effect on pattern of morphogenesis from explants. These combinations caused interactions that resulted in altered

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

morphogenetic responses. Carnation explants seemed to respond well to auxin. It is possible that endogenous, presumably cytokinins are synthesized in the shoots. The percentage between auxins and cytokinins is important with respect to morphogenesis in the culture system. Equally important is the concentration of these two groups of growth regulators (17). Addition of low level of auxin along with cytokinin proved to be excellent for shoot regeneration in carnation explants as observed by (18, 19).

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Although auxin may be essential, BA alone is highly effective in inducing shoot organogenesis. Thus, for each cultivar, the plant growth regulators should be optimized to maximize shoot regeneration. With higher levels of cytokinins, the number of shoots which form may be more but the growth of individuals may be arrested. A difference in adventitious shoot regeneration was observed between plant growth regulator combination of BA + IAA. Although a wider range and a higher levels of BA were used compared to previous studies on carnation species, significantly higher shoot regeneration from carnation explants was obtained with 3mgl-1BA. It had been observed that the combination of 0.02 mgl-1 NAA + 2 mgl-1Kin was the best combination for shoot regeneration in carnation cv. William Sim (20).

Table(1): Effect of different concentrations of BAP and NAA alone and BA combinations with NAA on in vitro shoot proliferation from Dianthus caryophyllus explants on MS medium after 6weeks of culture* PGRs mgL-1

No. of shoots/explant

No. of leaves/explant

No .of node/explant

Average length of shoots (cm)

0

1.60e

10.30d

2.20 e

1.55 d

1

2.60 de

16.50 abc

3.10 abcde

3.70 ab

3

4.10 bc 4.50 abc

21.30 a 20.10 ab

3.00 abcde 2.60 cde

3.09 abc 2.87 abc

0.1

1.80 e

14.10 bcd

2.50 de

2.03 cd

0.5

2.00 e

13.80 cd

2.80 bcde

2.24 cd

BA

5 NAA

BA+NAA 1+0.1

4.50 abc

21.30 a

4.10 a

3.62 ab

1+0.5

3.40 cd

16.10 abcd

3.60 abcd

3.59 ab

3+0.1

5.70 a

20.60 a

3.90 ab

3.91 a

3+0.5

4.70 ab

17.30 abc

3.40 abcd

3.67 ab

5+0.1

5.20 ab

15.10 abcd

3.70 abc

2.69 bc

5+0.5 4.50 abc 14.40 bcd 3.20 abcde 2.12 cd * Means followed by the same letter within each character (column) do not differ significantly (P≤0.05) according to Duncan's Multiple Range Test (12).

Effects of media type and different concentrations of NAA and IBA on rooting stage. 1. Rooting percentage: The percentage of root formation was significantly affected by the different treatments tested on Dianthus caryophyllus (Table 2 and Figure 8c). The highest percentage of root formation (75.31%) was observed as a result of amending the ¼MS medium compared with ½MS medium, while the percentage of root on (auxin types) NAA gave the better results on root percentage formation (73.75%) than IBA (73.50%). Data also showed that 1.5 mgl-1auxin concentrations gave significantly (90.00%) better results on root percentage formation than medium free auxin (36.88%).While the percentage of root in 0.5 and 1 mgl-1 auxin concentrations gave (83.13 and 82.50%) respectively.

Concerning the differences in rooting abilities of Dianthus caryophyllus, data in table (2) showed that NAA when added to the ¼ MS medium gave a higher rooting percentage (76.88%) compared with IBA when added to same medium (73.75%). However, IBA when added to ½MS medium gave a higher rooting percentage (71.25%) compared to NAA when added to the ½ MS medium (70.63%). A similar result was reported by (21). The MS medium containing 5 mgl-1 IBA induced Dianthus barbathus root growth from a 2 weeks-old culture with low rooting percentage (7%). Later, it was reported that the use of MS medium containing 0.5 mgl-1 NAA induced root growth (5.67 ± 1.15) and MS medium containing 1 mgl-1 IBA gave (2.15 ± 1.02) of Dianthus caryophyllus culture (22).

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Regarding the combination between types and concentration of auxins, 1.5 mgl-1 IBA produced the highest rooting percentage (91.25%). While the lowest percentage was produced (35.00%) when the explants were cultured in free hormone medium. On the other hand, the combination of ¼ MS medium supplemented with 1.5 mgl-1auxin produced the highest rooting percentage (95.00%), and in ¼ MS free hormone medium produced the lowest rooting percentage (35.00%). The effect of the three combinations (different media concentrations, different types and concentrations of auxin) indicated that rooting percentage could be obtained with the use of unmodified ¼ MS medium and ½ MS medium supplemented with NAA and IBA concentrations.

Vol. 10, No.2, June 2015

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The combination of explants from Dianthus caryophyllus cultured on ¼ MS medium supplemented with 1.5 mgl-1 NAA concentration produced an average of (97.50%) rooting compared with ½ MS medium supplemented with all concentration of NAA and IBA concentration. The highest percentage of roots/explant (97.50%) were recorded with ¼ MS supplemented with 1.5mgl-1 NAA. On the other hand, the lowest percentage of roots were formed on explants (35.00%) with ¼ MS and ½ MS medium free hormone. While, the highest percentage of roots /explant was obtained (92.50% and 90.00%) in ¼ MS and ½ MS medium supplemented with 1.5 mgl-1 IBA respectively.

Table (2): Effect of different types of auxin and different concentrations of auxin on roots response from Dianthus caryophyllus explants on (½, ¼) strength of MS medium and after 6weeks of culture*

Media

Auxin Conc. (mgl-1)

Types of Auxin 0.0

½MS ¼MS

NAA IBA NAA IBA

42.50c 35.00c 35.00c 35.00c

Media × Auxin Conc. (mgl-1)¼MS

½MS

Types of × Auxin Conc. (mgl-1) IBA

NAA

35.00c

0.5

1

85.00ab 82.50ab 85.00ab 82.50ab

77.50b 77.50b 90.00ab 85.00ab

38.75c

38.75b

35.00b

80.00ab 90.00ab 97.50a 92.500ab 77.50b

87.50ab 83.75a

82.50a

Mean effect of Auxin Conc. (mgl-1)

1.5

82.50b

83.75ab

85.00ab

83.75a 83.13a

Mean effect of Media

70.63a 71.25a 76.88a 73.75a

95.00a

81.25a 36.88b

Media × Auxin

82.50a

75.31a

Mean effect Types of Auxin

88.25a

91.25a

70.93b

73.75a 73.50a 90.00a

* Means within a column, row and their interactions followed with the same letters are not significantly different from each other according to Duncan,s multiple range test at 5% level

2. Roots number: Data in table (3) showed that there was a significant difference in the number of roots formed on shootlets of Dianthus caryophyllus as a result of tested treatments. The highest number of roots/ shootlet (5.55) was recorded with ¼MS medium compared with ½ MS medium (3.69) roots/ shootlet. While the lowest number of roots/ shootlet (4.20) was formed in medium containing NAA compared with the highest number of roots/ shootlet formed on shootlets (5.03), and the highest number of roots/shootlet (1.64) was formed on shootlets when 0.5 mgl-1 NAA was added to the medium. Combined of media with types of auxin significantly increased the number of roots (5.88 roots/ shootlet) which were formed on shootlets of Dianthus caryophyllus when cultured on ¼ MS supplemented with NAA compared with shootlets when cultured on ½ MS

supplemented with NAA and IBA (3.18, 4.20 roots/ shootlet) respectively. In this concern, auxin concentration combined with types of auxin had relatively the same result regardless of NAA and IBA levels used. The values recorded in this case were (5.90, 6.00 and 5.65 roots/ shootlet) when the medium was supplemented with (0.5, 1, 1.5) mgl-1 IBA respectively compared with medium was supplemented with all level of NAA. Data concerning the effect of auxin concentration treatments and its combinations with media type on Dianthus rooting showed that auxin concentration combined with ¼ MS medium has significant effect on this parameter comparing with auxin concentration with ½ MS medium treatments. The highest number of roots/shootlet (6.80) was recorded for shootlel with 1 mgl-1, while, the lowest value (2.60) was recorded with medium free hormone.

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Results concerning the effect of the three factors (different MS medium, type of auxin and auxin concentrations) indicated that the roots per shootlet could be obtained with the use of unmodified ½ MS medium and ¼ MS medium supplemented with NAA and IBA concentrations. Data also showed that IBA and NAA concentration when added to the ¼ MS has the pronounced and significant effect on this parameter. Shootlel form the combination of from Dianthus cultured on ¼ MS medium supplemented with concentration produced an average (7.10 roots/ shootlet) compared with ½ MS medium supplemented with IBA concentration (4.70 and 4.90 roots/shootlet) respectively. The highest number of roots /explant in B.×buttiana (7.10 roots/shootlet) was noticed in ¼ MS medium supplemented with (0.5 and 1) mgl-1 IBA. On the other hand, the lowest number of roots formed on Dianthus explants (2.60 roots/shootlet) was recorded with ½ and ¼ MS medium free hormone. While, the highest number of root /explants (4.90 roots/ shootlet) were recorded with ½ MS medium supplemented with 1 mgl-1 IBA.

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Results in this study are in agreement with those reported by (13). They explained that in order to improve in vitro adventitious rooting, the isolated plantlets were cultured on media containing 0.1, 1.0 and 10.0 mg/l IAA or NAA in various physical conditions. Optimum adventitious rooting and subsequent plant survival was obtained by culturing plantlets in medium containing 0.1 mg-1 NAA for 816 weeks prior to transplanting to soil (23). Date palm plants may be obtained by transferring individual plantlet to MS medium supplemented with 0.1 mg/l NAA to enhance rooting and 0.01mg/l BA to improve shoot system (24).

Table (3): Effect of different types of auxin and different concentrations of auxin on number root from Dianthus caryophyllus explants on (½, ¼) strength of MS medium and after 6weeks of culture*

Media

Auxin Conc. (mgl-1)

Types of Auxin 0.0

½MS

¼MS

0.5

1

NAA

2.60e

2.30e

3.60de

IBA

2.60e

4.70bcd

NAA

2.60e

IBA

2.60e

Media × Auxin Conc. (mgl-1)¼MS Types of × Auxin Conc. (mgl-1)

½MS

NAA

4.20d

3.18c

4.90bcd

4.60cd

4.20b

5.70abc

6.50a

6.10ab

5.23a

7.10a

7.10a

6.70a

5.88a

2.60c 2.60c

1.5

Media × Types of Auxin

6.40a 2.60c

3.50bc

4.25b

6.80a 4.00b

3.69b

5.55a

4.40b

Mean effect Types of Auxin

5.15a

4.20b

6.40a 5.05a

Mean effect of Media

IBA 2.60c 5.90a 6.00a 5.65a 5.03a * Means followed by the same letter within each character (column) do not differ significantly (P≤0.05) according to Duncan's Multiple Range Test (12).

3. Roots length (cm): Data in table (4) revealed that the highest mean of root lengths (4.39 cm) were formed due to amending the ¼ MS medium, while the shortest roots (3.64 cm) were formed on the ½ MS medium. The same table also showed that there were significant differences between IBA and NAA on the root lengths of Dianthus. The longest root (4.23 cm) was recorded in medium containing IBA compared with medium containing NAA (3.80 cm) and significant differences were observed between (0.5 and 1mgl-1) of auxin concentrations compared with medium containing 1.5 mgl-1 and it was significant differences with medium free hormone.

On the other hand, the highest length of roots/ shootlets (4.94, 4.87 cm) was recorded medium (0.5 and1mgl-1) respectively, and the shortest roots (2.10 cm) were obtained on medium free hormone. The effect of auxin treatments and its combinations with media different strength on Dianthus rooting showed that IBA combined with ¼ MS medium has significant effect on this parameter comparing with NAA and IBA with ½ MS medium treatments. The highest root length (4.64cm) was recorded for shootlets with IBA in ¼ MS medium. While, the lowest root lengths (3.44 cm) were recorded for shootlest treated with NAA in ½ MS. Similar results were obtained by (25) on Alnusnepalensis, and (26) on Khayaivorensis.

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

The effect of different media treatments and its combinations with different types of auxin on the four concentrations on Dianthus root length, that the type of auxin and concentration has no significant effect on this parameter. The combination of shootlets from Dianthus cultured on ¼ MS medium and supplemented with 0.5 mgl-1 IBA produced an average (6.55 cm) compared to ½ MS medium supplemented with 0.5 mgl-1 IBA (4.78 cm).

Vol. 10, No.2, June 2015

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The highest length of root /shootlet (6.55 cm) was recorded to ¼ MS plus with 0.5 mgl-1IBA. While the highest length of root/shootlet (5.12 cm) was recorded to ¼ MS plus with 0.5 mgl-1NAA compared with the highest length of root /shootlet (4.18 cm) was recorded to ½ MS plus with 0.5 mgl-1 NAA. On the other hand, the lowest length of roots wereformed (2.04 cm) with ½ MS and ¼ MS medium ½ MS and ¼ MS medium without auxin. The superiority of NAA over other auxins has also been reported inother plant species such as Caphaelisipecacuanha (27), Plantago ovate (28) and Rehumemodi (29).

Table (4): Effect of different types of auxin and different concentrations of auxin on root length (cm) from Dianthus caryophyllus explants on (½, ¼) strength of MS medium and after 6weeks of culture*

Media

0.0 ½MS

¼MS Media× Auxin Conc. (mgl-1)

Media × Auxin

Auxin Conc. (mgl-1)

Types of Auxin

0.5

1

1.5

4.10cde

NAA

2.04f

4.18bcde

3.45e

3.44c

IBA

2.04f

4.78abcd

4.94abc

3.57de

3.83bc

NAA

2.28f

5.12abc

5.00abc

4.24bcde

4.16ab

IBA

2.04f

5.66a

5.43ab

5.41ab

4.64a

¼MS Types of PGRs × Auxin Conc. (mgl-1) IBA

½MS

2.04d

2.16d NAA

5.39a 2.16c

2.04c

5.22a

4.48b

4.52b

5.21ab 4.65ab 5.19a

4.49ab

3.64b

4.39a

3.51c

Mean effect Types of Auxin

3.85c

3.80b

4.83ab 4.55ab

Mean effect of Media

4.23a

Mean effect of Auxin Conc. (mgl-1) 2.10c 4.94a 4.87a 4.17b *Means within a column, row and their interactions followed with the same letters are not significantly different from each other according to Duncan's multiple range test at 5% level

Figure (8: A, A1): cutting the explant for culturing in MS meium containing different concentrations of BA+ NAA from well developed in vitro shoots (8:B): multiple shoot formation from in vitro grown shoots of Dianthis caryophyllus on MS medium containing 1 mgl-1 BAP + 0.1 mgl-1 NAA. (8: C): extensive rooting of carnation shoots in vitro after 4-6 weeks inoculation.

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

REFERENCES 1. Jürgens A.; Witt T. and Gottsberger G.(2003). Flower scent composition in Dianthus and Saponaria species. Biochem. Sytemat. Ecol. 31: 345-357. 2. Ben-Yephet Y.; Shtienberg D.; Reuven M. and Mor Y. (2005). Response of carnation cultivars to Fusariumoxy sporumf.sp.dianthi in the field. Euro. J. Plant Phathol. 99 (1): 3-12. 3. Nugent G.; Wardley-Richardon T. and Lu CY.(1991). Plant regeneration from stem and petal of carnation (Dianthus caryophyllus L.). Plant Cell Reprod. 10: 477- 480. 4. Chopra RN.; Mayar SL. and Chopra IC.(1986). Glossary of Indian medicinal plants (including the supplement). Council of Scientific and Industrial Research, New Delhi. India. P. 22. 5. Ali A.; Munawar A. and Siddiqui FA. (2004). In vitro propagation of turmeric (Curcuma longa L.). Int. J. Biol. Biotech. 1(4): 511-518. 6. Mangal M.; Bhardwaj SV.; Kaur DR. and Mangal AK. (2002). Use of meristem tip culture to eliminate carnation latent virus from carnation plant. Ind. J. Exp. Biol. 40: 119-122. 7. Ioannov M. (1990). Production of carnation plants by shoot tip culture In vitro. Tech. Bull-Cyp. Agri. Res. Inst. 117: 1-8. 8. Villabobs AVM. (1986). Obtaining virus-free carnation by In vitro culture of meristems and apices. Tropic. Region. Amer. Soci. Hort. 616: 227230. 9. Mohammed AM. and Omer MS.(1990). Fundamental aspects of plant cell, tissue and organ culture. (In Arabic). Mosul University. Published by Ministry of Higher Education, Iraq. 10. Murashige I. and Skoog F. (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473-487. 11. Agrios G.(2005). Plant pathology. 2nd ed. Elsevier Academic Press. P. 922. 12. Duncan BD. (1955). Multiple range and Multiple F-tests. Biomet. 11:1-42. 13. Ali A.; Afrasiab H.; Naz S.; Rauf M. and Iqbal J.(2008). An efficient protocol for in vitro propagation of carnation (Dianthus Caryophyllus). Pak. J. Bot. 40 (1): 111-121. 14. Jevremovic S.; Trifunovic M.; Nikolic M.; Suboticand A. and Radojevic L. (2006). Clonal fidelity of Chysanthemum from long term cultures. Genetika. 38(3): 243- 249. 15. Sakila S.; Ahmed MB.; Roy UK.; Biswas MK.; Karim R.; Razvy MA.; Hossain M.; Islam R. and Hoque A. (2007). Micropropagatiom of swatberry (Fragraia X ananassa Duch): a newly introduced crop in Bangladesh. Am. Euras. J. Sci. Res. 2(2): 151-154. 16. Üçler AÖ. and MollamehmetoĀlu N. (2001). In vitro plantlet regeneration from mature of Linden (Tiliaplatyphyllosscop.) and multiplication of its bud . Turk. J. Agric. Forest. 25: 181 – 186. 17. Razdan MK. (2003). Introduction to plant tissue culture. 2nd ed. Published Science Publishers, Inc., India.

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18. Miller RM.; Kaul V.; Hutchinson JF. and Richards D. (1991). Adventitious shoot regeneration in carnation (Dianthus caryophyllus L.) from axillary bud explants. Ann. Bot. 67: 35- 42. 19. Earle ED. and Langhaus RW.(1975). Carnation propagation from shoot tips cultured in liquid medium. Hort. Sci. 10: 608-610. 20. Jalaska S. and Suitina R. (1977). Maintained culture of multiple plantlets from carnation shoot tips. Acta. Hort.(ISHS).78:333-340. 21. Simona L.; Cerasela P.; Giancarla V. and Maria B. (2012). In vitro culture initiation and phytohormonal influence on ornamental plants. J. Hort. Forest. Biotechnol. 16(2): 203-205. 22. Danial GH.; Yousif AN. and Omar MS. (2008). Clonal propagation of dianthus caryophyllus L. through tissues culture. The second Kurdistan Conference on Biological Sciences. J. Duhok. Univ. 12(1): (Special Issue). Pp: 91-95. 23. Tisserat B. (1982). Factors involved in the production of plantlets from date palm callus cultures. Euphytica. 31(1): 201-214. 24. Omar MS. (1988). Callus initiation, asexual embryogenesis and plant regeneration in phoenix dactylifera L. Date Palm J. 6:265-275. 25. Manisha T.; Sharma DR.; Kamlesh K.; Thakur M. and Kanwar K. (2001). Mass micropropagation of alnusnepalensis D. Don. Phytomorphol. 51 (2): 123-127. 26. Gad MMA.; El-Shihy OM. and Abd El-Dayem AM. (1999). In vitro high frequency plantlets production of khayaivorensis. The first International Conference in Egypt for Plant Tissue Culture and Its Application. pp. 161-174. 27. Jha S. and Jha TB. (1989). Micropropagation of caphaelisipecacuanha. Plant Cell Reprod. 8 : 437439. 28. La N. and Ahuja PS. (1989). Propagation of Indian rhubarb (Rheum emodi Wall.)using shoot tip and leaf explants culture. Plant Cell Reprod. 8 : 439496. 29. Wakhlu AK. and Barna KS. (1989). Callus initiation, growth and plant regeneration in plantagoovata Forsk. cv. Gl-2. Plant Cell Tiss. Organ Cult.17 : 235-241.

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

Effect of spraying nutrient solution "BASAK" and salicylic acid on some vegetative growth and flowering parameters of stock plant (Mathiola incana L.) Jamal A. Abbass, Mushtaq T. H. Al- Zurfy and Mustafa M. Ibrahim Dept. of Horticulture and Landscape Gardening / Faculty of Agriculture / University of Kufa / Republic of Iraq E –mail: [email protected]

ABSTRACT An experiment was conducted at the nursery of the faculty of agriculture at University of Kufa during growing season 2012 – 2013 to study the effect of spraying nutrient solution "BASAK" and salicylic acid on some vegetative and flowering parameters of stock plant. The experiment was adopted in Randomized Complete Block Design (R.C.B.D) with three replicates in two factors, first: three concentrations of nutrient solution "BASAK" (0 , 2 and 4) ml.L-1 and second three concentrations of salicylic acid (0, 50 and 100 ) mg.L-1 and their interactions , using Least Significant Difference (L.S.D) test to compare the means . Results showed that spraying nutrient solutions at concentration 4 ml.L-1 or salicylic acid at concentration 50 mg.L-1 increased significantly growth parameters (plant height , number of leaves , shoot dry weight , total content of chlorophyll, soluble carbohydrates , number of main roots , root dry weight , number of inflorescence and floret , diameter of floret and inflorescence dry weight) compared with control treatment, which gave the least values . In addition, results showed that spraying nutrient solution at concentration 4 ml.L-1 and Salicylic acid at concentration 50 mg.L-1 increased significantly vegetative and rooting growth parameters ( plant height , number of leaves , shoot dry weight , total content of chlorophyll and soluble carbohydrates , number of main roots , root dry weight ) and flowering parameters (number of inflorescence per plant and floret per inflorescence to (7.67) and (47.00 ) , diameter of floret to (4.17) cm and inflorescence dry weight to ( 2.27 gm ) , compared with control treatment which gave the least vales (2.67) inflorescence. Plant-1 , (30.67) floret. Inflorescence-1, (2.20) cm , (0.47) gm. Key words: Nutrient solution , Salicylic acid, stock plant ( Mathiola incana L. )

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ BASAK ‫ ﻝﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺍﻝﻤﺤﻠﻭل ﺍﻝﻤﻐﺫﻱ‬2013– 2012 ‫ﺃﺠﺭﻴﺕ ﺘﺠﺭﺒﺔ ﺤﻘﻠﻴﺔ ﻓﻲ ﻤﺸﺘل ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ ﻓﻲ ﺠﺎﻤﻌﺔ ﺍﻝﻜﻭﻓﺔ ﺨﻼل ﺍﻝﻤﻭﺴﻡ ﺍﻝﺯﺭﺍﻋﻲ‬ ‫( ﺒﺜﻼﺙ‬R.C.B.D ) ‫ ﻭﻗﺩ ﻨﻔﺫﺕ ﺍﻝﺘﺠﺭﺒﺔ ﺒﺘﺼﻤﻴﻡ ﺍﻝﻘﻁﺎﻋﺎﺕ ﺍﻝﻌﺸﻭﺍﺌﻴﺔ ﺍﻝﻜﺎﻤﻠﺔ‬، ‫ﻭﺤﺎﻤﺽ ﺍﻝﺴﺎﻝﺴﻠﻴﻙ ﻓﻲ ﻤﺅﺸﺭﺍﺕ ﺍﻝﻨﻤﻭ ﻭﺍﻹﺯﻫﺎﺭ ﻝﻨﺒﺎﺕ ﺍﻝﺸﺒﻭﻱ‬ ‫ ﺜﻼﺜﺔ ﺘﺭﺍﻜﻴﺯ ﻤﻥ ﺤﺎﻤﺽ ﺍﻝﺴﺎﻝﺴﻠﻴﻙ ﻫﻲ‬:‫ ﻭﺍﻝﺜﺎﻨﻲ‬1-‫ﻝﺘﺭ‬.‫ ( ﻤل‬4، 2 ،0) ‫ ﻫﻲ‬BASAK ‫ ﺜﻼﺜﺔ ﺘﺭﺍﻜﻴﺯ ﻤﻥ ﺍﻝﻤﺤﻠﻭل ﺍﻝﻤﻐﺫﻱ‬:‫ ﺍﻷﻭل‬،‫ﻤﻜﺭﺭﺍﺕ ﺒﻌﺎﻤﻠﻴﻥ‬ ‫ ( ﻭﻋﻨﺩ ﻤﺴﺘﻭﻯ‬L.S.D ) ‫ ﻭﻗﺩ ﻗﻭﺭﻨﺕ ﺍﻝﻤﺘﻭﺴﻁﺎﺕ ﺤﺴﺏ ﺍﺨﺘﺒﺎﺭ ﺃﻗل ﻓﺭﻕ ﻤﻌﻨﻭﻱ‬، ‫ ﻭﺘﻡ ﺩﺭﺍﺴﺔ ﺍﻝﺘﺩﺍﺨل ﻓﻴﻤﺎ ﺒﻴﻨﻬﻤﺎ‬1-‫ﻝﺘﺭ‬.‫ ( ﻤﻠﻐﻡ‬100 ، 50 ،0) .0.05 ‫ﺍﺤﺘﻤﺎل‬ ‫ ﻗﺩ ﺃﺩﻯ ﺇﻝﻰ ﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻤﺅﺸﺭﺍﺕ‬1-‫ﻝﺘﺭ‬.‫ ﻤﻠﻐﻡ‬50 ‫ ﻤﻊ ﺤﺎﻤﺽ ﺍﻝﺴﺎﻝﺴﻠﻴﻙ ﺒﺘﺭﻜﻴﺯ‬1-‫ﻝﺘﺭ‬.‫ ﻤل‬4 ‫ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ ﺭﺵ ﺍﻝﻤﺤﻠﻭل ﺍﻝﻤﻐﺫﻱ ﺒﺘﺭﻜﻴﺯ‬ ‫ ﻋﺩﺩ‬،‫ ﻤﺤﺘﻭﻯ ﺍﻷﻭﺭﺍﻕ ﻤﻥ ﺍﻝﻜﻠﻭﺭﻭﻓﻴل ﺍﻝﻜﻠﻲ ﻭﺍﻝﻜﺎﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﺍﻝﻜﻠﻴﺔ ﺍﻝﺫﺍﺌﺒﺔ‬،‫ ﺍﻝﻭﺯﻥ ﺍﻝﺠﺎﻑ ﻝﻠﻨﻤﻭ ﺍﻝﺨﻀﺭﻱ‬،‫ ﻋﺩﺩ ﺍﻷﻭﺭﺍﻕ‬،‫ﺍﻝﻨﻤﻭ ) ﺍﺭﺘﻔﺎﻉ ﺍﻝﻨﺒﺎﺕ‬ ‫ ﻗﻁﺭ ﺍﻝﺯﻫﻴﺭﺓ ﺍﻝﻭﺍﺤﺩﺓ ﻭﺍﻝﻭﺯﻥ ﺍﻝﺠﺎﻑ ﻝﻠﻨﻭﺭﺓ ﺍﻝﺯﻫﺭﻴﺔ‬،‫ ﻋﺩﺩ ﺍﻝﺯﻫﻴﺭﺍﺕ‬،‫ ﻋﺩﺩ ﺍﻝﻨﻭﺭﺍﺕ ﺍﻝﺯﻫﺭﻴﺔ‬،‫ ﺍﻝﻭﺯﻥ ﺍﻝﺠﺎﻑ ﻝﻠﻤﺠﻤﻭﻉ ﺍﻝﺠﺫﺭﻱ‬،‫ﺍﻝﺠﺫﻭﺭ ﺍﻝﺭﺌﻴﺴﺔ‬ . ‫ﻤﻘﺎﺭﻨﺔ ﺒﻨﺒﺎﺘﺎﺕ ﺍﻝﻤﻘﺎﺭﻨﺔ ) ﺍﻝﻤﺭﺸﻭﺸﺔ ﺒﺎﻝﻤﺎﺀ ﺍﻝﻤﻘﻁﺭ ﻓﻘﻁ ( ﻭﺍﻝﺘﻲ ﺃﻋﻁﺕ ﺃﻗل ﺍﻝﻘﻴﻡ‬ ‫ ﻗﺩ ﺃﺩﻯ ﺇﻝﻰ ﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ‬1-‫ﻝﺘﺭ‬.‫ ﻤﻠﻐﻡ‬50 ‫ ﻤﻊ ﺤﺎﻤﺽ ﺍﻝﺴﺎﻝﺴﻠﻴﻙ ﺒﺘﺭﻜﻴﺯ‬1-‫ﻝﺘﺭ‬.‫ ﻤل‬4 ‫ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻴﻀﺎ ﺃﻥ ﺭﺵ ﺍﻝﻨﺒﺎﺘﺎﺕ ﺒﺎﻝﻤﺤﻠﻭل ﺍﻝﻤﻐﺫﻱ ﺒﺘﺭﻜﻴﺯ‬ ‫ ﻤﺤﺘﻭﻯ ﺍﻷﻭﺭﺍﻕ ﻤﻥ ﺍﻝﻜﻠﻭﺭﻭﻓﻴـل ﺍﻝﻜﻠـﻲ‬،‫ ﺍﻝﻭﺯﻥ ﺍﻝﺠﺎﻑ ﻝﻠﻨﻤﻭ ﺍﻝﺨﻀﺭﻱ‬،‫ ﻋﺩﺩ ﺍﻷﻭﺭﺍﻕ‬،‫ﻓﻲ ﻤﺅﺸﺭﺍﺕ ﺍﻝﻨﻤﻭ ﺍﻝﺨﻀﺭﻱ ﻭﺍﻝﺠﺫﺭﻱ ) ﺍﺭﺘﻔﺎﻉ ﺍﻝﻨﺒﺎﺕ‬ ‫ ﻋـﺩﺩ ﺍﻝﻨـﻭﺭﺍﺕ ﺍﻝﺯﻫﺭﻴـﺔ ﻓـﻲ ﺍﻝﻨﺒـﺎﺕ ﺇﻝـﻰ‬،‫ ﺍﻝﻭﺯﻥ ﺍﻝﺠﺎﻑ ﻝﻠﻤﺠﻤﻭﻉ ﺍﻝﺠﺫﺭﻱ ﻭﺍﻝﺯﻫﺭﻱ‬،‫ ﻋﺩﺩ ﺍﻝﺠﺫﻭﺭ ﺍﻝﺭﺌﻴﺴﺔ‬،‫ﻭﺍﻝﻜﺎﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﺍﻝﻜﻠﻴﺔ ﺍﻝﺫﺍﺌﺒﺔ‬ ‫( ﺴﻡ ﻭﺍﻝﻭﺯﻥ ﺍﻝﺠﺎﻑ‬4.17) ‫ ﻭﻗﻁﺭ ﺍﻝﺯﻫﻴﺭﺓ ﺍﻝﻭﺍﺤﺩﺓ ﺇﻝﻰ‬،( 1-‫ ﻨﻭﺭﺓ‬.‫ ﺯﻫﻴﺭﺓ‬47.00) ‫ ﻋﺩﺩ ﺍﻝﺯﻫﻴﺭﺍﺕ ﻓﻲ ﺍﻝﻨﻭﺭﺓ ﺍﻝﻭﺍﺤﺩﺓ ﺇﻝﻰ‬،(1-‫ﻨﺒﺎﺕ‬. ‫ ﻨﻭﺭﺓ‬7.67) .‫ ﻨـﻭﺭﺓ‬2.67) ‫ ﻏﻡ( ﻤﻘﺎﺭﻨﺔ ﺒﻨﺒﺎﺘﺎﺕ ﺍﻝﻤﻘﺎﺭﻨﺔ ﻭﺍﻝﺘﻲ ﺃﻋﻁﺕ ﺃﻗل ﺍﻝﻘﻴﻡ ﻝﻤﺅﺸﺭﺍﺕ ﺍﻝﻨﻤﻭ ﺍﻝﺨﻀﺭﻱ ﻭﺍﻝﺠﺫﺭﻱ ﻭﺍﻝﺯﻫـﺭﻱ‬2.27) ‫ﻝﻠﻨﻭﺭﺓ ﺍﻝﺯﻫﺭﻴﺔ ﺇﻝﻰ‬ .(‫ ﻏﻡ‬0.47) ‫ ﺴﻡ( ﻭ‬2.20) ‫ ( ﻭ‬1-‫ ﻨﻭﺭﺓ‬.‫ ﺯﻫﻴﺭﺓ‬30.67) ‫ ( ﻭ‬1-‫ﻨﺒﺎﺕ‬

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INTRODUCTION Stock plant is one of the plant family members (Crucifera). It is a vertical plant with different height (20 - 25cm) and long term (70 - 75) cm ,with a woody stem at the base, and branching in the top , leaves take spear shapes with gray color, flower in spike inflorescences aromatic with different colors, white, yellow and gray and it is suitable for picking (1) . Its origin is Mediterranean and the Canary Islands. The plant is flowering during the spring and early summer and are grown in sunny locations (2). Stock plant is farmed in both general and private gardens and its aromatic oil can be extracted. Flowers are used in cosmetics (3). Stock plant requires good drainage and fertility soil in the beginning of the growth period (2). Several studies indicated the importance of foliar nutrition for plant when any obstacle to the process of absorption of nutrients through the roots can be occurred or as a result of unsuitable conditions (4), as in case of Iraqi soils (5), and the plants absorbs nutrients when leaves are sprayed in two mechanisms, i.e Symplast and Apoplast , so foliar nutrition has a good way to supply plant with nutrients, especially microelements in quick ways compared with ground fertilizations (6) . In 1986, Eibner had pointed out that foliar nutrition was a promising way of fertilization but it was not considered as an alternative of ground fertilization. However, both two ways, are integrated with each other (7). Nitrogen is an important element to plant growth, because it enters in the composition of many important organic compounds such as proteins, chlorophyll, coenzymes (i.e. NADPH, NADP and ATP), cell membranes, Mitochondria and chloroplasts (8). Also, phosphorus is involved in the composition of organic compounds in the information of nucleic acids, phospholipids and coenzymes. In addition, potassium is necessary for plant growth and development although it is not involved in compotation of any cellular components. It plays many important roles in the plant, such as activation of some important enzymes in the process of photosynthesis and respiration as well as in the circle in the construction of the starch and protein (9). Iron plays an important role in the life of the plant and it is the driving force for many metabolism processes in the plant. Zinc also is important for the plant growth due to its function in regulating sugar consumption and increasing the energy needed to produce chlorophyll and auxins formation (10). Al-Jubouri pointed out that spraying nutrient solution on Tegetes erecta L. had a significant increase in vegetative growth and flowering characteristics ( plant height, number branches and flowers and the diameter of the flower (11). Same results were obtained by (12) when spraying nutrient solution PRO.SOL" on the Pelargonium zonal L. led to a significant increase in plant " height and number of branches and leaves, leaf area, content leaves of chlorophyll and total dissolved carbohydrates, length of pindical, number of

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inflorescences and the first floret diameter in inflorescence. Salicylic acid is one of the derivatives of phenolic materials found widely in plant species. Salicylic derived from the Latin word Salix means gene name for Salix tree, (Salix helix Willow.). It was used by organ people of the American continent, for medical purposes. The chemical composition of Salicylic acid is C6H4(OH)COOH (13). Salicylic acid can be classified under plant hormones, and it has many important physiological roles in plant growth, flowering information, absorption of ions, movement of stomata and synthesis of ethylene, also it has an effect on Absicisic acid (ABA), In addition, it works to accelerate the formation of chlorophyll and Carotenoid pigments and plays a significant role in photosynthesis (14) . Martin-Mex et al. found that treatment Sinningia speciosa L. with salicylic acid at concentrations 10-8 M led to a significant increase in plant height, leaf area and number of leaves (15). In his study, Gharib observed that spraying Ocimum basilicum L. with salicylic acid at concentrations 10-3,10-4 and 10-5 M led to a significant increase in plant height, the number of total branches and leaves, leaf area and leaf content of total carbohydrates (16). For the importance of this plant, an experiment was conducted to study the effect of spraying nutrient solution " Basak " and Salicylic acid on growth and flowering parameters.

MATERIALS AND METHODS The experiment was conducted in the nursery at the faculty of agriculture /University of Kufa during the agricultural season 2012- 2013 to release the effect of spraying nutritional solution "Basak" and salicylic acid in vegetative and flowering growth parameters of Stock plant . Seeds were planted seeds spinach variety production by Euro Garden company on 01/09/2012, and seedlings were planted in pots after the emergence of four true leaves with a diameter 15 cm and a height of 20 cm and containing Silty clay soil (4 kg), which is composed of (4.8% Clay, 18.5% Silt and 76.7% Sand) Tables (1 and 2). Table (1): The chemical properties of the experimental soil. Chemical properties pH Electric conductivity (Ec) Calcium (Ca++) Potassium (K+) Magnesium (Mg++) Nitrogen(N) Organic matter

Values / units 6.35 2.83 Ds.M-1 22.8 Mm.L-1 1.85 Mm.L-1 15.8 Mm.L-1 30.2 Mg.g-1 8.3 g.kg-1

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Table (2): The contents of nutritional solution “Basak” Elements Total Nitrogen (N) Ureic Nitrate (NO3) Ammonium (NH4) Phosphoric Anhydride (P2O5) Potassium oxide (K2O) Boron (B) Copper (Cu) Iron (Fe) Manganese (Mn) Molybdenum (Mo) Zinc (Zn)

Values 4 V/W 2 V/W 2 V/W 6 V/W 8 V/W 0.01 V/W 0.01 V/W 0.02 V/W 0.01 V/W 0.002 V/W 4 V/W

The experiment was conducted in Randomized Complete Block Design (R.C. B. D) with three replicate in two factors; the first three concentration of nutritional solution (BASAK) (0, 2 and 4) ml.L-1; the second three concentrations of Salicylic acid (0, 50 and 100) mg. L-1. Spraying be done twice first after 20 days seedlings planting and second after 30 days from the first sprayer. Means were tested by least significant difference test (LSD) at probability level 0.05 (17). All operations services were conducted like irrigation and weeding when plants needed for all treatments. At the end of the experiment on 01/02/2013, the following parameters were measured: 1-Plant height (cm) 2- Number of leaves (leaf. Plant-1). 3- Shoot dry weight (g): Dry weight was calculated to shoot by cutting the whole plant after the end of the experiment then were placed in a well ventilated room for a period of 7-14 days (18). 4- Total chlorophyll content of leaves (mg.100g fresh weight): Determination of total chlorophyll according to (19). 5- leaves content of total soluble carbohydrate (mg. g-1 dry weight): estimated according to (20). 6- Number of main roots (Root. Plant-1): Pots were soaked in a large bowl filled with clear water until the separation of all soil around the roots for 24 hours, then measured it. 7- Root dry weight (g): Dry weight was calculated by cutting the root from the shoot and put it in an electric oven with the temperature 65o degrees for 72 hours. 8- Number of inflorescences ( Inflorescences.Plant1) 9- Number of florets (floret. Inflorescences-1) 10- Floret diameter (cm): Diameters were measured using (Vernier) . 11-florets dry weight (g): Dry weight was measured by putting flowers in a well ventilated room for a period of 7 -15 days.

RESULTS Results in table (3) showed that spraying nutritional solution "Basak" at concentration 4mg.L-1 increased significantly plant height, number of leaves, shoot dry weight, total chlorophyll content of leaves and

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leaves content of total soluble carbohydrates total to 17.68cm, 60.44 leaf. Plant -1, 5.56g, 49.23 mg . 100 gm-1 fresh weight and 5.60 mg. g -1 dry weight compared to the control, which gave the lowest values (12.94cm, 39.89 leaf. plant -1, 3.50g 44.84 mg. 100 gm-1fresh weight and 2.62 mg. g -1 dry weight) respectively. Results also showed that spraying of Salicylic acid at concentration 50 mg. L -1 increased significantly plant height, number of leaves, shoot dry weight, total chlorophyll content of leaves and leaves content of total soluble carbohydrates to 16.74cm, 3.78cm leaf .plant -1, 4.83g, 47.86 mg .100. g-1 fresh weight and 4.87 mg. g -1 dry weight compared to the control which gave the lowest values (14.60cm, 47.67cm leaf. plant -1, 4.37g, 46.17 mg100-1 fresh weight and 3.40 mg. g-1 dry weight, respectively. Results in table (3) also showed that spraying of nutritional solution "Basak" at concentration 4 mg. L-1and salicylic acid at concentration 50mg.L-1 increased significantly plant height, number of leaves, shoot dry weight, total chlorophyll content of leaves of and leaves content of total soluble carbohydrate to 21.67cm, 70.67 leaf. plant -1, 6.94g, 50.62 mg.100 g-1 fresh weight and 7.07 mg. g -1 dry weight compared to the plants spraying with distilled water only, which gave the lowest values (12.43cm, 35.00leaf.plant-1, 3.30g, 44.15 mg.100 g fresh weight and 2.20 mg. g -1dry weight, respectively. Results in table (4) showed the positive effect of spraying nutritional solution "Basak" at concentration 4 ml. L -1 and salicylic acid at concentration 50mg.L-1 that significant increased in the number of roots and root dry weight to 22.89 root. Plant-1, 1.83 g and 18.33 root. Plant-1, 1.48 g compared to the control group, which gave the lowest values (12.44 root .plant-1, 0.44g and 15.89. Plant-1, 1.42g) respectively. The interaction between spraying nutritional solution "Basak" at concentration 4mg.L-1 and salicylic acid at concentration 50 mg.L-1 increased significantly number of roots and the root dry weight to 27.00 root. Plant and 2.61g compared to the control that gave lowest values (10.67 root. Plant -1 and 0.41g) respectively. Results in table (5) showed that spraying nutritional solution "Basak" at concentration 4 ml.L-1 increased significantly number of inflorescences and florets, diameter of floret and the dry weight of florets and to 5.56 inflorescences. plant-1, 44.56 -1 floret.inflorescences , 2.94 cm and 1.30 g compared to the control treatment which gave the lowest values (3.00 inflorescences.plant-1, 34.67 floret. Inflorescences-1, 2.26 cm and 0.58g) respectively. Results also in table (5) showed that spraying with salicylic acid at concentration 50 mg. L-1 significantly increased number of inflorescences and florets, diameter of floret and the dry weight of the florets to 5.00 inflorescences. plant -1, 41.44. floret. inflorescences-1, 3.17cm and 1.31g compared to the control, which gave the lowest values (3.39 inflorescences. plant -1, 39.67 floret. Inflorescences-1 ,2.49 cm and 0.90g, respectively.

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Table (3): The effect of nutritional solution "Basak" and Salicylic and their interactions on vegetative growth parameters of Stock plant. Treatments 0 2 4

Nutrient solution (ml.L-1) L.S.D. 0.05

0 50 100

Salicylic acid (mg.L-1) L.S.D. 0.05

0 50 100 0 50 100 0 50 100

0 Nutrient solution × Salicylic acid

2

4 L.S.D. 0.05

Plant length (cm)

No. of leaves (leaf.plant-1)

12.94 15.52 17.68 1.305 14.60 16.74 14.80 1.305 12.43 12.90 13.50 14.50 15.67 16.40 16.87 21.67 14.50 3.529

39.89 52.00 60.44 3.392 47.67 53.78 50.89 3.392 35.00 40.10 44.67 48.00 50.67 57.33 60.00 70.67 50.67 6.381

Shoot dry weight (gm) 3.50 4.59 5.56 1.392 4.37 4.83 4.45 1.392 3.30 3.56 3.89 4.45 4.25 5.06 5.35 6.94 4.39 3.225

Chlorophyll (mg.100 g-1 fresh weight)

Carbohydrates (mg. g-1 dry weight)

44.84 46.69 49.23 1.421 46.17 47.86 46.74 1.421 44.15 44.79 45.58 45.92 46.77 47.37 48.43 50.62 48.65 3.541

2.62 3.74 5.60 1.003 3.40 4.87 3.70 1.003 2.20 2.37 3.30 3.13 3.87 4.23 4.87 7.07 4.87 3.206

Table (4): The effect of nutritional solution "Basak" and Salicylic and their interaction between us on root growth parameters of Stock plant No. of roots Roots.plant-1 12.44 16.22 22.89 2.142 15.89 18.23 17.33 2.142 10.67 12.43 14.33 14.57 15.65 18.33 22.33 27.00 19.33 5.226

Treatments 0 2 4

Nutrient solution (ml.L-1) L.S.D. 0.05

0 50 100

Salicylic acid (mg.L-1) L.S.D. 0.05

0 50 100 0 50 100 0 50 100

0 Nutrient solution × Salicylic acid

2

4 L.S.D. 0.05

Dry weight of roots Gm 0.44 1.49 1.83 0.627 1.42 1.48 0.86 0.627 0.41 0.25 0.64 1.36 1.57 1.54 2.48 2.61 0.41 0.998

. Table (5): the effect of Nutritional solution "Basak" and Salicylic and their interactions on flowering growth parameters of Stock plant Number of inflorescences (Inflorescences. Plant-1)

Treatments 0 2 4

Nutrient solution (ml.L-1) L.S.D. 0.05 Salicylic acid (mg.L-1)

0 50 100

L.S.D. 0.05 0 Nutrient solution × Salicylic acid

2

4 L.S.D. 0.05

0 50 100 0 50 100 0 50 100

3.00 4.22 5.56 1.552 3.89 5.00 3.89 1.552 2.67 3.00 3.33 3.67 4.33 4.67 5.33 7.67 3.67 3.629

Number of florets (floret. Inflorescences-1)

Floret diameter (cm)

florets dry weight (g)

34.67 43.78 44.56 1.829 39.67 41.44 41.89 1.829 30.67 34.33 39.00 41.33 45.33 44.67 47.00 44.67 42.00 4.062

2.26 2.89 2.94 0.267 2.49 3.17 2.43 0.267 2.20 2.23 2.33 2.37 3.10 3.20 2.90 4.17 1.77 0.529

0.58 1.13 1.30 0.214 0.90 1.31 0.81 0.214 0.47 0.41 0.86 0.84 1.24 1.31 1.39 2.27 0.25 0.599

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Spraying nutritional solution "Basak" at the concentration 4 ml. L -1 and salicylic at concentration 50 mg. L-1 significantly increased number of inflorescences and florets, diameters of floret and the dry weight of florets to7.67 inflorescences. Plant-1, 47.00 floret.inflorescences-1, 4.17cm and 2.27g compared to the plant spraying with distilled water (control), which gave the lowest values (2.67 inflorescences. Plant-1, 3.67 floret. inflorescences-1, 2.20cm and 0.47g) respectively.

DISCUSSION Results in tables (3, 4 and 5) showed that there was a significant increase when plant was sprayed with nutritional solution "Basak" in plant height, number of leaves, shoot dry weight and content of the leaves from total chlorophyll and total soluble carbohydrates, perhaps due to ingredients of the nutritional solution, which contains many important nutrients (table 1) necessary for plant growth, including nitrogen, which acts in the composition of chlorophyll, proteins and nucleic acids, enzymes and energy components, which all affect the growth and size of the plant (21) It is also important in the formation of cytokine, which stimulates the formation of shoots from new lateral buds (22). Phosphorus plays an important role in all biological processes such as cell division and transfer energy to all parts of the plant, and acts in the formation of the membrane chloroplasts and composition of amino and nucleic acids . Potassium plays a role in enhancing the formation of carbohydrates, proteins, and energy components “ATP”, which all affect to increase plant growth and its size (23). Also the presence of macro-nutrients, such as zinc that participates indirectly in the formation of chlorophyll through its direct effect in the formation of amino acids and energy components (24). All these results lead to improvement in the parameters of vegetative growth of plant. This conclusion was in consistent with (11) in marigold plant and (12). Regarding the significant increase in rooting parameters, they can be attributed to the role of nutrients found in combination of nutritional solution including nitrogen, phosphorus and potassium, which increases the growth of root by increasing the growth of the whole plant. Nitrogen also encourages the deepening and growth of roots (24), while phosphorus increases root growth and branches through increasing cell division (10). Potassium promotes maristeamic tissues than improving root growth (25), and finally development of root parameters. These results are similar to the results concluded to by (26) on Rosemary plant . Also spraying nutritional solution can increase significantly flowering growth parameters, and that may be attributed to the role of the nitrogen in increasing the shoot and root parameters (tables 3 and 4), leading to increase susceptibility of plants to absorb minerals that are probably reflected on the activation products of photosynthesis to increase food processing and thus transmission and

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accumulation in flowers, that improved flower growth (9,27), as well as potassium is important for plant growth and development as a cofactor in many operations include the formation of proteins, cell division and its relationship to represent the nucleic acids and food processing, which lead to improve flower growth (28). These results are similar to the results concluded to by (11) on marigold plant and (12) on Geranium plant. Salicylic acid also increases significantly growth parameters, perhaps due to its role as increasing protective levels of auxins and cytokine in plant tissues, which has active cell division and elongation in plant (29), and for its role to increase the power of the rooting system growth (14) as the increase of root system growth related with increasing level of cytokine, which plays an important role in increasing the cells division and break the apical merstim, as well as his role in the emergence of buds produced by cytokine and thus an increase in the efficiency of photosynthesis, leading to the provision of materials needed to build new tissue, and ultimately increase the growth of plants (30,31). These results are similar to the results concluded by (16) on Ocimum plant.

REFERENCES 1. Al- Sultan S M.; Al- Chalabi T M. and Al- Saraf MD. (1992). Ornamental plants. Ministry of Higher Education. Iraq. P. 463. 2. Al-Batal NN. (2005). Indoor ornamentals plant. Publications of Damascus University, Faculty of Agriculture. Syria. P.263. 3. Al-Degwy A. (2004). Encyclopedia of Ornamental plant . Press of Madbolia . Egypt. P. 400. 4. Abdel Hameid AF. and Al-Fouly M M. (1995). Economical uses of specialized foliar micro elements. Arabic. J. Fertil. 18:4-25. 5. Al- Mamouri AML. (1997). Effect of spraying liquid fertilizer and born on growth and yield of maize ( Zea mays L.) Ph. D. Thesis. Faculty of Agriculture, University of Baghdad. Iraq. 6. Al-Jawari AKS. (2002). Effect of spraying different nutrients on growth and yield of sweet pepper ( Capsicum annuum L. ). Master Thesis. Faculty of Agriculture, University of Baghdad. Iraq. 7. Eibner R. (1986). Foliar fertilization importance and prospect its crop production . In Foliar . fertilization . Proceeding of 1st symposium on foliar fertilization , Berlin: Germany. 8. Bidwell R. (1979). Plant physiology. 2nd ed. MacMillan Pub. Co. Inc., N.Y. USA. P. 466. 9. Al-Sahaf FHR. (1989). Applied nutrition plant. Ministry of Higher Education and Scientific Research. University of Baghdad. House of Wisdom. Iraq. P. 256. 10. Awad KM. (1987). Fertilization and soil fertility. University of Basra, Ministry of Higher Education and Scientific Research, Iraq. P.456. 11. Al-Jubouri RI. (2006). Effect of agrotonic fertilizer and magnetized water and planting date on vegetative and flowering growth and production of

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some carotinodes of marigold plant Tagests erecta L. Master Thesis. Faculty of Agriculture, University of Baghdad. Iraq. 12. Naser ZS. (2012). Effect of foliar application of "PRO.SOL" nutrient solution and liquorice extract on growth and flowering of Geranium (Pelargonium zonale L.). Master Thesis. College of Agriculture, University of Kufa. Iraq. 13. Lee HI.; Leon J. and Raskin I. (1995). Biosynthesis and metabolism of salicylic acid. PNAS. 92(10): 4076-4079. 14. Hegazi AM. and El-Shraiy AM. (2007). Impact of Salicylic acid and Paclobutrazol exogenous application on the growth, yield and nodule formation of common bean. Aust. J. Basic. Appl. Sci. 1(4): 834-840. 15. Martin-Mex R.; Villanueva-Couoh E.; UicabQuijano V. and Larqué-Saavedra A. (2003). Positive effect of salicylic acid on the flowering of gloxinia. Proceedings of the 31st Annual Meeting. Plant Growth Regulation Society of America. Vancouver, Canada. August,3-6: 149-151. 16. Gharib FAE. (2006). Effect of Salicylic acid on the growth, metabolic activities and oil content of Basil and Marjoram. Int. J. Agri. Biol. 8(4):485– 492. 17. Al- Rawi KM. and Khalaf-Alla A. (2000). Design and analysis of agricultural experiments. College of Agriculture and Forestry. University of Mosul – Iraq. P.487. 18. Ihsan SA. (1999). A study of some factors affecting the quantitative and qualitative attributes of essential oils in mint and menthe. PhD. Thesis. College of Agriculture, University of Baghdad. Iraq. 19. Goodwin TW. (1976). Chemistry and biochemistry of plant pigments. 2nd ed. Academic Press, USA. P. 373. 20. Duboies M.; Gilles KA.; Hamilton JK.; Robers RA. and Smith F. (1956). Colorimetric method for determination of agar and related substance. Anal. An. Chem. 28: 350-356. 21. Hassan NA. and Al-Dulaimi HY. and Al- Ithawi LA. (1990). Soil fertility and fertilizers. Dar AlHikma Press for Printing and Publishing. Ministry of Higher Education and Scientific Research. Baghdad University. Iraq. P. 336. 22. Al- Ani TA. (1987). Physiology of plant growth and composition. Ministry of Higher Education and Scientific Research. University of Baghdad. Iraq. 23. Al-Naimi SNA. (1985). Fertilizers and soil fertility. Ministry of Higher Education and Scientific Research. Technical Institutes Foundation. Baghdad, Iraq. P.778. 24. Abu Dhahi Y M. and Al- Younes MA. (1988). Guide on plant nutrition. Books store press and publishing, University of Baghdad. Ministry of Higher Education and Scientific Research. Iraq. P. 410. 25. Tisdale SL.; Nelson WL.; Berton JD. and Havlin JL. (1997). Soil fertility and fertilizers. PrenticeHall, New Delhi. India. 26. Al-Drickzle AEA. (2005). Effect of nitrogen and phosphorus fertilization on vegetative growth of rosemary plant Rosemarinus officinalis L. Master

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Thesis. Faculty of Agriculture. University of Baghdad. Iraq. 27. Isaac O. (1992). Die Ringelbum, Botonik, Chemical, pharmakology, Toxikology and therapeutic use. Wissenschaftlich Verlagsellschaft. Stuttgart. Germany. 28. Humble G. and Raschke H. (1971). Stomata opening quantity very related to potassium transport. J. Plant Physiol. 48: 447-453. 29. Shakirova FM.; Sakhabutdinova A R.; Bezrukova MV.; Fatkhutdinova RA. and Fatkhutdinova DR. (2003). Changes in the hormonal status of wheat seedlings induced by salicylic acid and salinity. Plant Sci. 164: 317–322. 30. Taiz L. and Zeiger E. (1998). Plant Physiology. 3rd Seminar Associates, INC., Publishers. Sunderland, Massachusetts. P. 110. 31. Khan W.; Prithviraj B. and Smith DL. (2003). Photosynthetic responses of corn and soybean to foliar application of Salicylates. J. Plant Physiol. 160: 485-492.

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Vol. 10, No.2, June 2015

Feasibility of using brewers spent grain as a fertilizer in agriculture Amal A. Muhammed (1), Keith Thomas (2) and Usama Bin- Hamed (2) (1) Dept. of Biology / College of Sciences for women / University of Baghdad /Republic of Iraq (2) Faculty of Applied Sciences / University of Sunderland / UK E –mail: [email protected]

ABSTRACT Brewers Spent Grain (BSG) is the main by-product of brewing industries. Although mostly used to supplement animal feed some is sent to landfill so incurring disposal costs. An alternative use of BSG as agricultural fertilizer has been proposed to reduce environmental pollution and broaden its availability. The study reported here conducted using cabbage and lettuce plants treated with different levels of BSG addition as a soil amendment enhanced plant growth when applied at high levels between 20-40% (v/v) and did not recycle plant diseases. The observed improvement in lettuce and cabbage plant growth with increasing level of BSG amendment reflects the improvement in soil fertility. The 40% level of BSG addition was the best for optimizing plant growth. The results indicated that BSG can be used as a fertilizer and is not associated with any visual symptoms of diseases. This supports the use of BSG in a wider range of applications than animal feed and may help to minimize its discard as a waste material. Keywords: Brewers Spent Grain (BSG), fertilizer

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ‫ ﻭﻋﻠﻰ ﺍﻝﺭﻏﻡ ﻤﻥ ﺃﻥ ﻤﻌﻅﻤﻬﺎ ﻴﺴﺘﺨﺩﻡ ﻜﻐﺫﺍﺀ ﻝﻠﺤﻴﻭﺍﻨﺎﺕ‬،‫( ﻤﻨﺘﺠﺎ ﺜﺎﻨﻭﻴﺎ ﻝﻤﺼﺎﻨﻊ ﺍﻝﺘﺨﻤﻴﺭ‬BSG) Barley Spent Grain ‫ﺘﺸﻜل ﺒﻘﺎﻴﺎ ﺤﺒﻭﺏ ﺍﻝﺸﻌﻴﺭ‬ ‫ ﻜﺒﺩﻴل ﻝﻸﺴﻤﺩﺓ ﻓﻲ ﺍﻝﺯﺭﺍﻋﺔ ﻤﻥ ﺇﺤﺩﻯ ﺍﻝﻭﺴﺎﺌل ﺍﻝﺘﻲ‬BSG ‫ ﻭﻴﻌﺘﺒﺭ ﺍﺴﺘﺨﺩﺍﻡ‬،‫ ﺤﻴﺙ ﺇﻥ ﻋﻤﻠﻴﺔ ﺍﻝﺘﺨﻠﺹ ﻤﻨﻬﺎ ﺘﻜﻠﻑ ﻜﺜﻴﺭﺍ‬،‫ﻭﻝﻜﻥ ﻴﺘﻡ ﻁﻤﺭ ﺍﻝﺒﻌﺽ ﻤﻨﻬﺎ‬ ‫ ﻭﻗﺩ ﺃﺠﺭﻴﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﺤﺎﻝﻴﺔ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻨﺒﺎﺘﺎﺕ ﺍﻝﻤﻠﻔﻭﻑ ﻭﺍﻝﺨﺱ ﺤﻴﺙ ﺘﻡ ﻤﻌﺎﻤﻠﺘﻬـﺎ ﺒﻤـﺴﺘﻭﻴﺎﺕ‬.‫ﺘﺴﻬﻡ ﻓﻲ ﺘﻘﻠﻴل ﺍﻝﺘﻠﻭﺙ ﺍﻝﺒﻴﺌﻲ ﻓﻬﻭ ﻤﺘﺎﺡ ﺒﺸﻜل ﻭﻓﻴﺭ‬ ‫( ﻭﻗﺩ‬V/V ٪40-20) ‫ ﺃﺩﻯ ﺇﻝﻰ ﺘﺤﺴﻴﻥ ﻨﻤﻭ ﺍﻝﻨﺒﺎﺘﺎﺕ ﻋﻨﺩ ﺍﺴﺘﺨﺩﺍﻤﻪ ﺒﺘﺭﺍﻜﻴﺯ ﻋﺎﻝﻴﺔ‬BSG ‫ ﻭﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ‬،‫ ﻜﻤﺨﺼﺏ ﻝﻠﺘﺭﺒﺔ‬BSG ‫ﻤﺨﺘﻠﻔﺔ ﻤﻥ‬ BSG ‫ ﺘﺸﻴﺭ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻴﻀﺎ ﺇﻝﻰ ﺃﻥ ﺍﻝﺘﻁﻭﺭ ﺍﻝﻤﻠﺤﻭﻅ ﻓﻲ ﻨﻤﻭ ﻨﺒﺎﺘﺎﺕ ﺍﻝﺨﺱ ﻭﺍﻝﻤﻠﻔﻭﻑ ﻤﻊ ﺘﺯﺍﻴﺩ ﻤﺴﺘﻭﻯ‬.‫ﺃﺩﻯ ﺇﻝﻰ ﻋﺩﻡ ﺇﻋﺎﺩﺓ ﺘﺩﻭﻴﺭ ﺍﻷﻤﺭﺍﺽ ﺍﻝﻨﺒﺎﺘﻴﺔ‬ ‫ ﻭﺘﺸﻴﺭ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻴﻀﺎ ﺇﻝـﻰ ﺇﻤﻜﺎﻨﻴـﺔ ﺍﺴـﺘﺨﺩﺍﻡ‬.‫ ﻫﻭ ﺍﻷﻓﻀل ﻝﺘﺤﺴﻴﻥ ﻨﻤﻭ ﺍﻝﻨﺒﺎﺕ‬BSG ‫ ﻤﻥ‬٪40 ‫ ﻭﻜﺎﻥ ﺍﻝﻤﺴﺘﻭﻯ‬،‫ﻴﻌﻜﺱ ﺍﻝﺘﺤﺴﻥ ﻓﻲ ﺨﺼﻭﺒﺔ ﺍﻝﺘﺭﺒﺔ‬ ‫ ﻓﻲ ﺘﻁﺒﻴﻘﺎﺕ ﻋﺩﻴﺩﺓ ﻜﺎﺴﺘﺨﺩﺍﻤﻬﺎ ﻋﻠﻔﺎ ﻝﻠﺤﻴﻭﺍﻨﺎﺕ ﻴﻤﻜﻥ ﺃﻥ‬BSG ‫ ﻭﺒﻬﺫﺍ ﻴﻤﻜﻥ ﺍﻝﻘﻭل ﺒﺄﻥ ﺍﺴﺘﺨﺩﺍﻡ‬،‫ ﻜﺴﻤﺎﺩ ﻭﺒﺩﻭﻥ ﻅﻬﻭﺭ ﺃﻱ ﺃﻋﺭﺍﺽ ﺇﻤﺭﺍﻀﻴﺔ‬BSG .‫ﻴﺴﺎﻋﺩ ﻓﻲ ﺘﻘﻠﻴل ﺍﻷﻀﺭﺍﺭ ﺍﻝﻨﺎﺠﻤﺔ ﻋﻥ ﺇﻫﻤﺎل ﺍﻝﻤﻭﺍﺩ ﺍﻝﻨﺎﺘﺠﺔ ﻤﻥ ﺒﻘﺎﻴﺎ ﻤﺼﺎﻨﻊ ﺍﻝﺘﺨﻤﻴﺭ‬

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INTRODUCTION

Brewers Spent Grain (BSG) is the main by-product of the brewing industry, representing around 85% of total by-products production (1). It is the insoluble remainder of malting barley resulting from the production of wort (2) and has high levels of cellulose and non-cellulosic polysaccharides which can be recycled (1). In addition to cellulose and noncellulosic polysaccharides, BSG contains arabinoxylans and lignin (3), which are all slow to digest in natural systems. On the other hand, BSG may have different compositions depending on barley variety, harvest time and inclusion of hops and other additional materials which are added during the brewing process (4). Moreover, BSG contains 1.5-2.5% mineral content (5) such as calcium, sodium, potassium, magnesium, aluminum, iron, barium, strontium, manganese, copper, zinc, phosphorus, sulfur, chromium and silicon (6). The UK Environment Agency estimated that the BSG production of UK brewers is more than half million tons of waste each year and about 3.5 million tons across Europe (7). In spite of large amounts of BSG produced each year, however, its uses are generally limited to animal feeding (3). The annual accumulation of brewer’s residues in large amounts results in environmental deterioration and pollution. In particular the structure of BSG makes it highly suitable for microbial growth due to its high moisture and nutritional content (8). Managing this byproduct is a major challenge for the brewing industries in addition to its difficult transport, storage and preservation. As a consequence rapid use of BSG is desirable, but not always achievable. Moreover, most of the available conservation methods can affect the nutritional quality of BSG (9). In order to decrease the pollution originating from industrial manufacture, especially in developed and underdeveloped countries, numerous efforts have been developed to modify or recycle these residues, particularly as BSG is available at low or no cost during the year and in large volumes from large and small breweries (3). Besides processing for removal BSG can be processed in order to produce a number of valuable products, for example by using physical, chemical and biological (enzymatic) pre-treatments (8). Storage of BSG has inherent limitations due to its large bulk. It has been found by (10) that the storage of fresh BSG at 20oC resulted in rapid microbial contamination combined with component losses. The same results occurred at refrigeration storage, but at lower levels. However, no changes in BSG structure occurred in frozen storage. Autoclaving of BSG caused solubilization of polysaccharides associated with phenolics related to the starch breakdown, arabinoxylan losses and decreases in Ferulic acid. A study conducted by (9), indicated that superheated steam drying is more acceptable than other drying methods since it removes aroma and flavor as well as conserving the nutritional value of BSG. There are other

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advantages to using superheated steam as indicated by (11) such as increases in efficiency, decreasing the hazard of fire and explosion, sterilization and deodorization in addition to faster drying rates. Another study conducted by (4) showed that oven drying gave the same results as freeze-drying at the lowest economic cost making it more suitable for BSG preservation. Because BSG contains high levels of protein and fiber, it can be used in human nutrition, for example in biscuits (12). It has also been used to produce ethylene and propylene which are derived from ethanol formed by fermentation of sugars from BSG cellulose, arabinoxylan and aromatic compounds (benzene, toluene and xylene) which are produced from BSG lignin (3). Furthermore, it is reported by (13) that the percentage of animal milk fat increases with the inclusion of BSG in the diet. Various nutrient components can be extracted from BSG and can be used in products such as proteins, sugar fractions and phenolic compounds (9). The composition of BSG samples is shown in table (1). Table (1): Approximate analysis of the BSG samples (14) Composition of BSG Crude Protein

Percentage (%) 23.20

Crude Fiber

12.85

Crude Fat

2.79

Moisture Content

6.14

Ash Content

16.99

Carbohydrate

51.39

Total Nitrogen

3.71

Nitrogen– Free Extract

38.03

Overall, the use of BSG in food processing is very limited and related to the deficiency of soluble proteins (2). A study conducted by (15) showed that the fungus Penicillium brasilianum produces arabinoxylandegrading enzymes when grown on BSG medium which have a role in degrading around 40% arabinoxylan in BSG. Additional studies conducted by (16) revealed that the mesophilic fungus Neurospora crassa produces ethanol when grown in BSG. In recent years, BSG has been utilized in biotechnological processes, such as in mushroom and Actinobacteria cultivation (3). The reason for the successful cultivation of mushroom Pleurotus ostreatus in BSG medium is related to its low pH (5.7) (17), high moisture content and physical parameters which make it suitable as a mushroom substrate. P. ostreatus fruit bodies cultivated on spent grain have a greater nutritional content amino acids and crude protein (18). Furthermore, BSG facilitates the isolation of Actinobacteria, particularly streptomyces from soil samples and improves its sporulation (19). This isolate of Actinobacteria, particularly Streptomyces, is able to produce xylanase as well as feruloyl esterase when grown on BSG substrate (20), (21). Other studies conducted by (22) showed that the bacterium Bacillus subtilis RB14 produces higher levels of iturin antibiotic and secondary metabolites like lipopeptide when grown in malt residue related to the nutrient

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

content. Furthermore, adding a neutralized acid extract of BSG to wort improves yeast performance and yields fermented products of the same quality as that fermented without spent grain. BSG can be also used in agriculture due to its high levels of organic matter. For example (23), found that BSG amended soils presented greater levels of NH4-N compared to NO3-N contents. Moreover, the exchangeable cations (Na+, Ca+2, Mg+2, K+) increase with BSG application. Another study conducted by (24), revealed that the compost from BSG can be used to improve plant growth such as geranium and tomato. In addition BSG contains minerals necessary for plant growth, (25) found that brewery compost addition had a role in decreasing the severity of root galling caused by the nematode Meloidogyne hapla infecting lettuce and its egg production and also enhanced lettuce growth. Furthermore, the addition of the brewery compost had a significant effect to decrease the incidence of root rot disease (26). Other advantages for BSG are its significant effect to decrease or dissolve gallstones in animals which can be attributed to increase the total bile acid concentration under BSG diet. On the other hand a BSG diet decreases the cholesterol concentration in both serum and bile. In addition to BSG effects on the metabolism of bile acids and cholesterol, it also has an effect on small intestinal mucosa morphology (27). Furthermore, BSG can be used as treatment of ulcerative colitis (3) and as a treatment of constipated patients resulting from its high contents of fiber, cellulose and hemicellulose (65.6%) (28). On the other hand, because of the low ash content and the high quantity of fibrous material (cellulose, non-cellulosic polysaccharides and lignin) of BSG, it can be utilized in building materials such as in brick production to provide a higher strength and porosity in addition to decreasing brick density after firing compared to standard production from clay (29). Furthermore, the fibres available in the largest particle fractions of BSG make it suitable to use as a raw material for paper production, paper towels and business cards (30). With so many potential uses, an increase in interest of BSG has been noted (3). Previous work has identified that output from small scale breweries has cost limitations on transport for animal feeding so making alternative and accessible uses desirable (31). The aim of this work is to investigate the feasibility of using BSG as a fertilizer in agriculture and to know whether BSG may cause transfer of spoilage microbes to new plants and so increase the possibility of disease. Tests with different levels of BSG were conducted to determine its effect on plant growth and to identify the best concentration of BSG to apply.

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MATERIALS AND METHODS Cabbage seedlings were cultivated on various concentrations of BSG in John Innes No 3 compost (0% as control, 10%, 20% and 40% v/v). Ten pots were used for each concentration with one cabbage plant in each pot. This experiment was carried out in Newcastle University Close House Farm UK. The soil samples and swabs from cabbage leaves (upper and lower leaf surfaces) were taken and re-suspended in 5 ml sterile saline. The soil samples and leaf swabs were analyzed to identify and monitor the microbial changes in the soil and cabbage leaves during the period of plant growth. For soil, all samples were prepared for microbial enumeration by dilution in sterile 0.85% sodium chloride. Enumeration was determined from growth on different media (malt agar, nutrient agar and MRS agar) with two repetitions for each media. For cabbage leaf samples, 4cm2 areas of leaf were swabbed, dispersed in 1ml of saline and 100µl aliquots spread on malt agar. Petri-dishes were incubated at 25oC for five days before enumeration and identification by phenotypic characteristics. To investigate the effect of BSG concentration on plant growth, cabbage height and the number of leaves was recorded regularly every two weeks, and at the end of growth, all cabbage plants were harvested, the leaves and roots weighed separately and all samples dried at 60oC for 6 days to constant weight. This experiment was repeated using cabbage and lettuce plants grown from seeds. Statistical analysis: Data were assessed for differences between group mean values using one-way and two-way ANOVA.

RESULTS Plant growth: Effect of different concentrations of BSG on Plant height: In the first experiment, the effect of different concentrations of BSG as malting residue on plant growth showed that after four weeks in the control treatment (0% BSG), the cabbage plants had better growth and taller plants in comparison with other levels of BSG treatments. However, two weeks later (six weeks of plant growth) this effect was reversed and the 40% BSG addition showed the tallest plants. This continued until the end of the test period while the control treatment remained the poorest. However, statistical analysis revealed that there were no significant differences between plants treated with different levels of BSG. In the second experiment using cabbage plants the effect of BSG is evident in plant height, but only at high levels (40%). Moreover, the effect of BSG on lettuce height was very clear and statistical analysis showed that the plant height increased gradually with increasing levels of BSG as shown in table (2) and figures (1- 4).

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Table (2): Plant growth (cm) with increasing concentrations (%v/v) of BSG amendment Cabbage (experiment 1) Time Control 10% 2 weeks 13.33 11.83 4 weeks 21.27 18.83 6 weeks 17.67 19.27 8 weeks 15.83 19.67 Cabbage (experiment 2) Time Control (a) 10% (a) 8 weeks 16.67 15.83 10 weeks 16.73 16.00 12 weeks 16.83 17.33 Lettuce Time Control (a) 10% (b) 6 weeks 6.5 13.00 8 weeks 6.67 13.37 10 weeks 9.00 15.33 Different letters indicate a significant difference between treatments

20% 12.33 19.67 18.90 20.00

40% 10 20.33 22.67 22.67

20% (a) 9.83 18.67 19.17

40% (b) 22.67 22.77 23.33

20% (b) 14.17 14.23 17.17

40% (c) 16.67 17.00 19.33

Figure (1): Effect of BSG on cabbage growth after 2 weeks, from right to left: control, 10%, 20%, and 40% BSG

Figure (3): Effect of BSG on lettuce growth, 2 columns on the left 20% and 2 columns on the right 40%.

Figure (2): Effect of BSG on cabbage growth after 6 weeks, from right to left: control, 10%, 20%, and 40%.

Figure (4): Effect of BSG on lettuce growth, 2 columns on the left control and the next 2 columns on the right 10%BSG

25

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Effect of different concentrations of BSG on leaf number: The effect of different levels of BSG on leaf number is evident in table (3) and figures (1- 4). These results showed the effect of BSG on the cabbage leaf number (experiment 1) initiated after six weeks and over. However, statistical analysis revealed that there are no significant differences between the control and other BSG levels. Different results were obtained with cabbage plants grown from seed in experiment 2 and in lettuce. Statistical analyses showed clear significant differences between the control and different levels of BSG all over the dates of assessments and in most cases the 40% BSG gave the greatest number of leaves. Effect of different concentrations of BSG on plant weight: Finally, at the end of this experiment, all cabbage and lettuce plants were harvested and transferred to the laboratory for analysis of wet and dry weight. The results of cabbage plant growth in experiment 1 revealed that the addition of BSG had a significant effect on the wet weight and dry weight of leaves. However, the addition of BSG on wet and dry weight of roots was higher at 40% BSG, but statistical analyses revealed that there were no significant differences between the control and BSG levels. The effect of BSG levels on cabbage weight in experiment 2 showed significant differences between BSG treatments on plant wet weight, but there were no significant differences between BSG on plant dry weight. Furthermore, the effect of BSG on lettuce plant weight is clearer in comparison with cabbage plants and statistical analyses revealed that the lettuce weight increased significantly with increasing levels of BSG as shown in table (4). Overall, at the end of this experiment the 40% addition of BSG consistently produced the best plant growth and was associated with large, green leaves, while the control treatment resulted in smaller plants with yellowish leaves. All the data indicated that the use of BSG to amend the soil resulted in an improved growth of cabbage and lettuce plants; however, high levels of 20-40% (v/v) are required. Microorganisms from soil samples and plants: To assess the extent of the microbial community in the soil and BSG; samples of soil and BSG were serially diluted and the number of bacteria colonies grown in different agar media are presented in table (5). The observations indicate that soil amended with BSG is colonized with more bacteria than the control soil in all agar media. During plant growth, the results of the bacteria count from all levels of BSG samples and for all scoring dates indicated that the number of bacteria colonies on nutrient and malt agar increased with increasing levels of BGS. On the other hand, the number of bacteria colonies on MRS fluctuated during the growth period with lower counts at four and six weeks and an increase at 8 weeks as shown in table (5). Investigation of fungal growth from soil on malt agar media revealed that the soil was colonized with

Vol. 10, No.2, June 2015

high levels of Monosporium fungus; however, high levels of Myceliasterlia with low levels of Monosporium were identified on nutrient agar. Identification of fungi from soil samples with different levels of BSG during cabbage seedling plant growth revealed that Mucor was the dominant fungus in all media through the test time, while levels of other fungi fluctuated within the same test period. These fungi included; Mucyliasterlia, Penicillium, Monosporium, and Streptomyces, as shown in tables (6-8). Initial identification of microorganisms from cabbage seedling leaves on malt agar revealed that the microbes which contaminated cabbage leaves were bacteria and Penicillium, Aspergillus, Mucor, Cladosorium and Stemphillium fungi. Identification of microorganisms from cabbage leaves during the growth period revealed that the microbes which contaminated leaves were predominantly bacteria and Myceliasterile, and were associated with low levels of other fungi as shown in table (6). Identification of microorganisms from lettuce leaves during the growth period showed that bacteria were also the most common contaminant, with low levels of fungi, particularly Monosporium and Myceliasteriie (Results not shown). Overall, the results showed that BSG can be used as a fertilizer to enhance plant growth and is not associated with any visual symptoms of diseases. Most of the microorganisms isolated from the plants leaves were bacteria and common fungi typical of plants and soil. Disease microorganisms were not observed in the BSG or on the plants grown on BSG. Table (3): Leaf number of plants grown with increasing concentrations (%v/v) of BSG amendment Cabbage (Experiment 1) Time Control 2 weeks 10.67 4 weeks 12.33 6 weeks 11.67 8 weeks 14.67 Cabbage (Experiment 2) Time Control (a) 8 weeks 5.67 10 weeks 6.33 12 weeks 6.00 Lettuce Time Control (a) 6 weeks 7.33 8 weeks 9.33 10 weeks 13.33

10% 10.67 13.67 16.67 17.00

20% 10.33 13.33 16.00 16.00

40% 9.67 13.67 15.67 19.67

10% (b) 8.33 8.00 10.33

20% (b) 6.33 10.33 11.33

40% (c) 11.67 13.00 14.33

10% (b) 10.33 15.33 18.33

20% (c) 11.33 17.00 24.00

40% (c) 12.00 19.67 25.67

Different letters indicate a significant difference between treatments

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Table (4): Wet and dry weight (gm) of plants grown in increasing concentrations (%v/v) of BSG as amendment

Vol. 10, No.2, June 2015

Table (5): Colony counts from soil samples with different levels of BSG on different agar media (cfu g-1) Nutrient agar.

Cabbage (experiment 1) Wet Dry Wet Dry W/leaves W/leaves W/roots W/roots 70.05 11.05 6.21 1.69 Control 80.45 11.40 6.92 1.63 10% 81.99 10.64 6.72 1.60 20% 134.74 15.41 10.93 2.58 40% Cabbage (experiment 2) Wet Dry Wet Dry W/leaves W/leaves W/roots W/roots 6.25 a 1.74 0.60 a 0.22 control 12.68 a 3.02 0.92 ab 0.35 10 13.15 a 2.02 0.95 ab 0.26 20% 23.04 b 4.67 1.24 b 0.55 40% Lettuce Wet Dry Wet Dry W/leaves W/leaves W/roots W/roots 3.93 a 0.46 a 0.69 a 0.13 a Control 28.28 b 2.98 b 2.91 b 0.71 b 10% 49.57 c 5.66 c 4.04 c 0.90 c 20% 65.47 d 5.27 c 4.55 c 0.99 c 40% Different letters indicate a significant difference between treatments

Control

2 weeks 1.52x109

4 weeks 2.10x108

6 weeks 1.01x1010

10%

1.48x109

4.65x108

1.55x1011

10

6.90x10 5.40x1010

9

2.93x10 2.50x109

11

1.23x10 1.75x1011

2.5x1012 2.30x1010 3.70x1012

3.85x109 5.50x109 2.20x108 5.83x109

6.50x107 4.05x108 7.90x108 1.96x109

5.50x109 8.80x1010 1.02x1011 1.94x1011

6.45x107 3.60x108 3.35x107 4.20x107

Control

0

0

0

2.47x107

10%

3.60x106

0

3.15x106

20%

6.50x106

0

0

40%

4.20x106

0

0

20% 40% Malt agar Control 10% 20% 40% MRS agar

8 weeks 2.05x1010

4.00x108 1.85x108 5.10x106

Table (6) : Fungal profiles from soil samples with increasing concentrations (%v/v) of BSG amendment grown on malt agar. (cfu g-1). Time

2 weeks

4 weeks

6 weeks

8 weeks

Concentration

Mucor

Mycliasterlia

Botryotrichium

Monosporium

Penicillium

Oidiodendron

Control

_

_

_

_

_

_

10%

11

1

_

_

_

_

20%

9

_

_

_

_

_

40%

15

_

_

_

_

_

Control

_

_

_

_

_

_

10%

4

1

_

_

_

_

20%

17

1

_

_

_

_

40%

19

_

_

_

_

_

Control

_

_

_

_

_

_

10%

1

_

5

1

20%

3

8

_

_

17

_

40%

11

_

3

_

_

_

_

Control

_

_

_

_

_

1

10%

9

_

4

_

_

2

20%

1

_

3

_

_

10

40%

1

2

_

_

_

7

27

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

28

Table (7) : Fungal profiles from soil samples on nutrient agar. (cfu g-1) Time

2 weeks

4 weeks

6 weeks

8 weeks

Concentrations

Mucor

Monosporium

Mycelia sterlia

Streptomyces

Control

_

_

_

_

10%

10

_

_

_

20%

5

1

_

_

40%

7

_

_

_

Control

_

2

_

_

10%

_

_

1

2

20%

20

_

_

11

40%

3

13

_

8

Control

_

_

_

4

10%

4

_

_

_ _

20%

_

_

_

40%

9

_

1

Control

_

_

_

1

10%

12

_

_

2

20%

_

_

_

40%

6

_

_

1

Table (8): Microbial profiles isolated from cabbage leaves (experiment 1) with increasing concentrations (%v/v) of BSG amendment grown on malt agar Time

Concentration control 10%

2 weeks

20% 40% control 10%

4 weeks 20% 40% control 10% 6 weeks 20% 40% control 10% 8 weeks 20% 40%

Level upper under upper under upper under upper

Penicilium _ 1 _ _ _ _ _

Myceliasterlia 4 1 _ _ 7

under upper under upper under upper under upper under upper under upper under upper under upper under upper under upper under upper under upper under

6

Cladosporium 2 _ 2 _ 10 1 1

Oidiodendron _ _ _ _ _ _ _

Geotrichum _ _ _ _ _ _ _

_

3

2

_

_

2 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

4

2 _ _ _ _ _ _ _ _ _ 2 _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ 3 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2 1 _ _ _ _ _ _

7 3 3 _ _ _ 3 1 1 _ 1 1 _ _ 1 _ _ 2 _ _ _ 1

Bacteria 23 84 51 4 8 9 33 6 100 23 40 1 9 2 183 114 8 186 59 1 59 2 32 1 60 3 102 1 12 1 61

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

DISCUSSION Much of the world’s soil is degraded by depletion of nutrients through typical growing of plants especially from losses of top soil. The use of compost is an effective and readily available way to support and recover soils. Moreover, with the pressure to reduce pollution emerging from industrial activities, many residues and byproduct are regarded as wastes. One way to decrease this pollution and residues is by recycling for another process. One example of a common by-product is BSG, which is available in large quantities. This material has high levels of organic matter and high nutritional value which makes it suitable for using in agriculture. As a byproduct, it is available at low or no cost through the year and may be associated with landfill costs if alternative uses are not readily available (3). The present study determined the feasibility of exploiting different levels of BSG with soil as a substrate for cultivation of cabbage and lettuce plants, to investigate its ability to enhance plant growth and to assess the potential of BSG to transmit or encourage plant diseases. The results showed the effect of BSG in enhancing the growth of cabbage and lettuce by increasing the number of leaves as shown in figures (2-4) and table (3). The effect of increasing levels of BSG on plant weight, especially at high levels (40%) is evident and showed significant differences between the high levels of BSG (20% and 40%) and the control or 10% BSG as shown in table (4). The effect of increasing levels of BSG in enhancing plant growth is evident in figure (2). The observed improvement in lettuce and cabbage plants with increasing level of BSG amendment reflects the improvement in soil fertility. The 40% level of BSG addition was the best for optimizing plant growth. This level in soils increased plant productivity possibly due to high contents of N, P, K, Ca, Mg and C, in addition to the low C: N ratio of this by product (32). The other advantages of BSG in addition to its high nutritional value are that its application leads to a reduction in loss of water, and decreases soil temperature related to high moisture content of BSG and physical features such as, particle size, volume weight, specific density, porosity and water holding capacity (18) which lead to enhanced plant growth. The results indicated that BSG is a good substrate material for plant cultivation. The greater number of leaves and the larger leaf area index associated with high BSG levels would lead to increased photosynthesis and enhanced plant growth. These results agree with (24) who revealed that BSG amended soil and enhanced the growth of geranium and tomato plants. The results also agree with (33) who indicated that the addition of wet spent malt to the soil has a positive effect on the growth rate of radish plant, but at high doses. Overall, it appears that soil amended with spent grain may have benefits for brewers and agriculture. Moreover, all plants grown in high levels of BSG seemed healthy and without any symptoms of

Vol. 10, No.2, June 2015

disease, had shiny green leaves of greater size compared with control plants. Furthermore, the present results showed that all soil samples had levels of bacteria which increased with increasing levels of BSG. Assessment of fungal numbers indicated that Mucor was the dominant fungus as shown in Table 8. These results are in agreement with (33) who indicated that BSG is rapidly colonized with microorganisms due to its nutrient levels and high moisture levels However, the addition of BSG caused no increase in plant diseases. These results encourage the use of BSG as a fertilizer in agriculture to enhance plant growth due to its low costs. Additional benefits for both agriculture and brewery industries are also evident in reducing the bulk of wastes and pollution.

CONCLUSION The use of BSG amendments to soil was found to increase growth of cabbage and lettuce plants when applied at 20% v/v and over. The fungus Mucor and various bacteria were the most common microorganisms in all soil samples. No pathogenic or disease fungi found. All the data indicated that the use of BSG at doses of 20 – 40% (v/v) to amend the soil resulted in an improved growth of cabbage and lettuce plants.

REFERENCES 1. Aliyu S. and Bala M. (2011). Brewer‟s spent grain: A review of its potentials and applications. Afri. J. Biotechnol. 10: 324-331. 2. Celus I.; Brijs K. and Delcour JA. (2007). Enzymatic hydrolysis of brewers spent grain proteins and technology properties of resulting hydrolysates. Agri. Food Chem. 55: 8703–8710. 3. Mussatto S I.; Dragone G. and Roberto I C. (2006). Brewers spent grain: Generation, characteristic and potential applications. J. Cereal Sci. 43: 1-14. 4. Santos M.; Jimenez J J.; Bartolome M.; GomezCordoves C. and del Nozal M J. (2003). Variability of brewers spent grain within a brewery. Food Chem. 80:17-21. 5. Quinde Z.; Ullrich SE. and Baik BK. (2004). Genotypicvariation in color and discolorationpotential of barley-based food products. Cereal Chem. 81:752– 758. 6. Mussatto S I. and Roberto I C. (2006). Chemical characterization and liberation of pentose sugars from brewer's spent grain. J. Chem. Technol. Biotechnol. 81: 268-274. 7. Jay A J.; Parker M L.; Faulks R.; Husband F.; Wilde P.; Smith A C.; Faulds C B. and Waldron K W. (2008). A systematic micro-dissection of brewers spent grain. J. Cereal Sci. 47: 357–364. 8. Nigam P S.; Gupta N. and Anthwal A. (2009). Biotechnology for agro-industrial residue utilization. Utilization of Agro-Residues. Springer Book. P. 469.

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9. Praveen J.; Jitendra P. and Stefan C. (2010). Issues with utilization of brewers' spent grain. Stewart Postharv. Rev. 6(4): 1-8. 10. Robertson J A.; Anson K J A.; Brocklehurst T F.; Faulds C B. and Waldron K W. (2010). Effect of storage conditions on the microbial ecology and biochemical stability of cell wall components in brewers spent grain. J. Agri. Food Chem. 58: 72667272. 11. Dilip D.; Andrew H. and Lise-Lott S. (2008). An evaluation of superheated steam drying of brewer's spent grain. Chemeca 2008: Towards a sustainable Australasia. [Barton, ACT]: Engineers Australia. 1144-1153. Conference Paper. 12. Sudha M L.; Vetrimani R. and Leelavathi K. (2007). Influence of fibre from different cereals on the rheological characteristics of wheat flour dough and on biscuit quality. Food Chem. 100: 4. 1365– 1370. 13. Miyazawa K.; Sultana H.; Hirata T.; Kanda S. and Itabashi H. (2007). Effect of brewer's grain on rumen fermentation, milk production and milk composition in lactating dairy cows. Anim. Sci. J. 78: 519-526. 14. Ajanaku K O.; Dawodu F A.; James O O.; Ogunniran K O.; Ajani O O. and Nwinyi O C. (2010). Histological studies of brewery spent grains in dietary protein formulation in Donryu rats. Senra Academic Publishers, Burnaby, British Columbia. 4: 1179-1185. 15. Panagiotou G.; Granouillet P. and Olsson L. (2006). Production and partial characterization of Arabinoxylan-degrading enzymes by Penicillium brasilianum under solid-state fermentation. Appl. Microbiol. Biotechnol. 72: 1117-1124. 16. Xiros C. (2008). Evaluation of Neurospora crassa as an enzyme factory for the bioconversion of brewer's spent grain. First European workshop on biotechnology for lignocellulose biorefineries. Copenhagen March 27-28. 17. Ozturk S.; Ozboy O.; Cavidoglu I. and Kokse H. (2002). Effect of brewers spent grain on the quality and dietary fibre content of cookies. J. Instit. brewing. 108. 1: 23-27. 18. Wang D.; Sakoda A. and Suzuki M. (2001). Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain. Bioresource Technol. 78: 293-300. 19. Szponar B.; Pawlik K J.; Gamian A. and Szwajcer Dey E. (2003). Protein fraction of barley spent grain as a new simple medium for growth and sporulation of soil Actinobacteria. Biotechnol. Lett. 25: 1717-1721. 20. Nascimento R P.; Coelho R R R.; Marques S.; Alves L.; Girio F M.; Bon E P S. and AmaralCollaco M T. (2002). Production and partial characterisation of xylanase from Streptomyces sp. strain AMT-3 isolated from Brazilian cerrado soil. Enz. Microb. Technol. 31: 549-555. 21. Bartolome B.; Gomez-Cordoves C.; Sancho A I.; Diez N.; Ferreira P.; Soliveri J. and Copa-Patino J L. (2003). Growth and release of hydroxycinnamic acids from brewers spent grain by Streptomyces

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avermitilis CECT 3339. Enz. Microb. Technol. 32: 140–144. 22. Khan A W.; Rahman M S. and Ano T. (2009). Application of malt residue in submerged fermentation of Bacillus subtilis. J. Environ. Sci. 21: 533–535. 23. Erdem N. and Ok S S. (2002). Effect of brewery sludge amendment on some chemical properties on acid soil in pot experiments. Bioresource Technol. 84: 271-273. 24. Stocks C.; Barker A J. and Guy S. (2002). The compositing of brewery sludge. J. Instit. Brewing. 108: 452-458. 25. Chen J.; Abawi G S. and Zuckerman B M. (2000). Efficacy of Bacillus thuringiensis, Paecilomyces marquandii, and Streptomyces costaricanus with and without organic amendments against Meloidogyne hapla infecting Lettuce. J. Nematol. 32: 70-77. 26. Abawi G S. and Widmer T L. (2000). Impact of soil health management practices on soilborne pathogens, nematodes and root diseases of vegetable crops. Appl. Soil Ecol. 15: 37-47. 27. Zhang J X.; Bergman F.; Hallmans G.; Johansson G.; Lundin E.; Stenling R.; Theander O. and Westerlund E. (1990). The influence of barley fibre on bile composition, gallstone formation, serum cholesterol and intestinal morphology in hamsters. APMIS. 98: 568-574. 28. Odes H S.; Madar Z.; Trop M.; Namir S.; Gross J. and Cohen T. (1986). Pilot study of the efficacy of spent grain dietary fiber in the treatment of constipation. Isr. Med. Sci. 22: 5-12. 29. Russ W.; Mortel H. and Meyer-Pittroff R. (2005). Application of spent grains to increase porosity in bricks. Construc. Build. Mat. 19:117-126. 30. Ishiwaki N.; Murayama H.; Awayama H.; Kanauchi O. and Sato T. (2000). Development of high value uses of spent grain by fractionation technology. MBAA Techn. Quart. 37: 261-265. 31. Ben-Hamed U. (2012). An analysis of the use of brewery spent grain: a case study of the UK brewing industry. PhD. Thesis. Faculty of Applied Sciences, University of Sunderland. UK. 32. Mbagwu J S C. and Ekwealor G C. (1990). Agronomic potential of brewers' spent grains. Biol. Wastes. 34: 335-347. 33. Thomas K R. and Rahman P K S. (2006). Brewery wastes. Strategies for sustainability. A Rev. Asp. Appl. Biol. 80:153-162.

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Vol. 10, No.2, June 2015

The composition of accumulated dust on bookshelves: analytical and molecular study Mohammed I. Khalil (1) and Ghayda S. H. Al-Enezy (2) (1) College of Environmental Sciences and Technology (2) College of Basic Education / University of Mosul / Republic of Iraq E –mail: [email protected]

ABSTRACT The presence of books on the shelves may take a long time depending on the users of the books. The book may remain for months and even years without use, especially in public libraries, making them prone to accumulation of dust, which may carry various harm effects on book users' health. The aim of this study was to assess the quality of the accumulated dust on the bookshelves in one of the largest libraries of University of Mosul "Central Library" where samples of dust were collected on books and characterized by difficulty handling because of dispersal with any movement. The samples were transferred to the molecular biology and ecology laboratories at the College of environmental science and technology to test the estimation of some heavy metals concentrations" Cu, Co, Fe, Zn, Pb and Cd" by using atomic absorption system. The results showed that these heavy metals were found in accumulated dust. This result could hide a hazardous impact especially if they become accumulated in the body, also PCR technique was used to investigate some microorganisms which affect public health. The results revealed the diagnosis of Alternariaalternata fungi, which causes many types of allergies that did not observed in direct plate culture as well as the existing of E. coli fecal bacteria, which caused many diseases. The PCR technique is very sensitive method in diagnosing a low level of microorganisms trace. The dust accumulation on bookshelves is unhealthy environment for workers and users by containing many pathogens and toxic metal elements if the user deal with them for a long time, and in the light of these results, it is recommended especially for the workers wear masks at work as well as removing the dust regularly and allergy tests to avoid the chronic allergy cases. Keywords: Book dust, heavy metals, allergens, PCR

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ‫ﺘﺨﺘﻠﻑ ﻓﺘﺭﺓ ﺒﻘﺎﺀ ﺍﻝﻜﺘﺏ ﻋﻠﻰ ﺍﻝﺭﻓﻭﻑ ﻓﻲ ﺍﻝﻤﻜﺘﺒﺎﺕ ﺒﺸﻜل ﻋﺎﻡ ﺒﺎﺨﺘﻼﻑ ﺍﺴﺘﺨﺩﺍﻤﻬﺎ ﺃﻭ ﺴﺤﺒﻬﺎ ﻤﻥ ﺘﻠﻙ ﺍﻝﺭﻓﻭﻑ ﺒﻭﺍﺴﻁﺔ ﻤﺭﺘﺎﺩﻱ ﺍﻝﻤﻜﺘﺒﺎﺕ‬ ‫ ﻤﻤﺎ ﻴﺠﻌﻠﻬﺎ ﻋﺭﻀﺔ ﻝﺘﺭﺍﻜﻡ ﻜﻤﻴﺎﺕ ﻜﺒﻴﺭﺓ ﻤﻥ‬،‫ ﻭﻗﺩ ﺘﺒﻘﻰ ﺍﻝﻜﺘﺏ ﺸﻬﻭﺭﺍ ﻭﺭﺒﻤﺎ ﺴﻨﻭﺍﺕ ﺩﻭﻥ ﺍﺴﺘﺨﺩﺍﻡ ﻭﺨﺎﺼﺔ ﻓﻲ ﺍﻝﻤﻜﺘﺒﺎﺕ ﺍﻝﻌﺎﻤﺔ‬،‫ﻭﺍﻝﻤﺴﺘﺨﺩﻤﻴﻥ‬ .‫ﺍﻷﺘﺭﺒﺔ ﺍﻝﻤﺤﻤﻠﺔ ﺒﻤﺨﺘﻠﻑ ﺍﻝﻌﻭﺍﻤل ﺍﻝﻤﺅﺜﺭﺓ ﻋﻠﻰ ﺍﻝﺼﺤﺔ ﺍﻝﻌﺎﻤﺔ ﻝﻤﺴﺘﺨﺩﻤﻲ ﺍﻝﻜﺘﺏ ﻭﺍﻝﻘﺎﺌﻤﻴﻥ ﻋﻠﻴﻬﺎ‬ ‫ﺠﺎﺀﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻝﺘﻘﻴﻴﻡ ﻨﻭﻋﻴﺔ ﺍﻝﻐﺒﺎﺭ ﺍﻝﻤﺘﺭﺍﻜﻡ ﻋﻠﻰ ﺭﻓﻭﻑ ﺍﻝﻜﺘﺏ ﻓﻲ ﺇﺤﺩﻯ ﺃﻜﺒﺭ ﻤﻜﺘﺒﺎﺕ ﺠﺎﻤﻌﺔ ﺍﻝﻤﻭﺼل "ﺍﻝﻤﻜﺘﺒﺔ ﺍﻝﻤﺭﻜﺯﻴﺔ" ﺤﻴﺙ ﺘﻡ ﺃﺨﺫ ﻋﻴﻨﺎﺕ‬ ‫ ﻭﻤﻥ ﺜﻡ ﻨﻘﻠﺕ ﺍﻝﻌﻴﻨﺎﺕ ﺇﻝﻰ ﻤﺨﺘﺒﺭﺍﺕ ﺍﻝﺒـﺎﻴﻭﻝﺠﻲ ﺍﻝﺠﺯﻴﺌـﻲ‬،‫ﻤﻥ ﺍﻝﻐﺒﺎﺭ ﺍﻝﻤﺘﺭﺍﻜﻡ ﻋﻠﻰ ﺍﻝﻜﺘﺏ ﺍﻝﺫﻱ ﺘﻤﻴﺯ ﺒﺼﻌﻭﺒﺔ ﺍﻝﺘﻌﺎﻤل ﻤﻌﻪ ﻝﺘﻁﺎﻴﺭﻩ ﻤﻊ ﺃﻱ ﺤﺭﻜﺔ‬ ‫ ﻭ‬Pb‫ ﻭ‬Zn ‫ ﻭ‬F‫ ﻭ‬Co‫ ﻭ‬Cu ‫ ﺤﻴﺙ ﻗﺩﺭﺕ ﺘﺭﺍﻜﻴﺯ ﺒﻌﺽ ﺍﻝﻤﻌـﺎﺩﻥ ﺍﻝﺜﻘﻴﻠـﺔ ﺍﻝﺘـﻲ ﺸـﻤﻠﺕ‬،‫ﻭﺍﻝﺒﻴﺌﺔ ﻓﻲ ﻜﻠﻴﺔ ﻋﻠﻭﻡ ﺍﻝﺒﻴﺌﺔ ﻭﺘﻘﺎﻨﺎﺘﻬﺎ ﻝﻐﺭﺽ ﺍﻝﻔﺤﺹ‬ ‫ ﻭﻗﺩ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻭﺠﻭﺩ ﻫﺫﻩ ﺍﻝﻤﻌﺎﺩﻥ ﻓﻲ ﺍﻝﻐﺒﺎﺭ ﺍﻝﻤﺘﺭﺍﻜﻡ ﻭﻫﻲ ﻨﺘﻴﺠﺔ ﺘﻌﻜﺱ ﻤﺩﻯ ﺨﻁﻭﺭﺓ ﺘﻠﻜﺎﻝﻤﻌﺎﺩﻥ ﻓﻲ‬، ‫ﺒﺎﺴﺘﺨﺩﺍﻡ ﺠﻬﺎﺯ ﺍﻻﻤﺘﺼﺎﺹ ﺍﻝﺫﺭﻱ‬Cd ‫ ﻓﻲ ﺍﻝﺘﺤﺭﻱ ﻋﻥ ﻭﺠﻭﺩ ﺒﻌﺽ ﺍﻷﺤﻴﺎﺀ ﺍﻝﻤﺠﻬﺭﻴﺔ ﺍﻝﺘﻲ ﺘﺸﻜل ﺨﻁﻭﺭﺓ ﻋﻠﻰ ﺍﻝﺼﺤﺔ‬PCR ‫ ﻜﻤﺎ ﺍﺴﺘﺨﺩﻤﺕ ﺘﻘﻨﻴﺔ ﺍﻝﺒﻠﻤﺭﺓ‬،‫ﺤﺎل ﺘﺭﺍﻜﻤﺕ ﻓﻲ ﺠﺴﻡ ﺍﻹﻨﺴﺎﻥ‬ ‫ ﺍﻝﻤﺴﺒﺏ ﻝﻠﻌﺩﻴﺩ ﻤﻥ ﺃﻨﻭﺍﻉ ﺍﻝﺤﺴﺎﺴﻴﺔ ﺍﻝﺫﻱ ﻝﻡ ﻴﻅﻬﺭ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻁﺭﻴﻘـﺔ ﺍﻝـﺯﺭﻉ‬Alternariaalternata ‫ ﻭﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺘﺸﺨﻴﺹ ﺍﻝﻔﻁﺭ‬،‫ﺍﻝﻌﺎﻤﺔ‬ ‫ ﺍﻝﺒﺭﺍﺯﻴﺔ ﻭﺍﻝﻤﺴﺒﺒﺔ ﻝﻠﻌﺩﻴﺩ ﻤﻥ ﺍﻷﻤﺭﺍﺽ ﺤﻴﺙ ﺘﻌﺩ ﻫﺫﻩ ﺍﻝﻁﺭﻴﻘﺔ ﺤﺴﺎﺴﺔ ﻝﺘﺸﺨﻴﺹ ﺃﻗل ﻤﺴﺘﻭﻯ ﻤﻥ‬E. coli ‫ ﻜﻤﺎ ﺒﻴﻨﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻭﺠﻭﺩ ﺒﻜﺘﻴﺭﻴﺎ‬،‫ﺍﻝﻤﺒﺎﺸﺭ‬ ‫ ﻓﺈﻥ ﺘﺭﺍﻜﻡ ﺍﻷﺘﺭﺒﺔ ﻋﻠﻰ ﺍﻝﻜﺘﺏ ﻴﻤﺜل ﺒﻴﺌﺔ ﻏﻴﺭ ﺼﺤﻴﺔ ﻝﻤﺎ ﺘﺤﺘﻭﻴﻪ ﻤﻥ ﻤﺴﺒﺒﺎﺕ ﻤﺭﻀﻴﺔ ﻭﻋﻨﺎﺼـﺭ ﻤﻌﺩﻨﻴـﺔ‬،‫ ﻭﺨﻼﺼﺔ ﺍﻝﻘﻭل‬،‫ﺘﻭﺍﺠﺩ ﺍﻷﺤﻴﺎﺀ ﺍﻝﻤﺠﺭﻴﺔ‬ ،‫ ﻭﻋﻠﻰ ﻀﻭﺀ ﻫﺫﻩ ﺍﻝﻨﺘﺎﺌﺞ ﻴﻨﺼﺢ ﻭﺨﺎﺼﺔ ﻝﻠﻌﺎﻤﻠﻴﻥ ﻓﻲ ﻫﺫﺍ ﺍﻝﻤﺠﺎل ﺍﺭﺘﺩﺍﺀ ﺍﻝﻜﻤﺎﻤﺎﺕ ﺃﺜﻨـﺎﺀ ﺍﻝﻌﻤـل‬،‫ﺴﺎﻤﺔ ﺍﺫﺍ ﻤﺎ ﺍﺴﺘﻤﺭ ﺍﻝﺘﻌﺎﻤل ﻤﻌﻬﺎ ﻝﻔﺘﺭﺍﺕ ﻁﻭﻴﻠﺔ‬ ‫ﻭﺒﺄﻫﻤﻴﺔ ﺍﻝﻤﺤﺎﻓﻅﺔ ﻋﻠﻰ ﺘﻨﻅﻴﻑ ﻭﺇﺯﺍﻝﺔ ﺍﻝﻐﺒﺎﺭ ﻭﺍﻷﺘﺭﺒﺔ ﻤﻥ ﻋﻠﻰ ﺍﻝﻜﺘﺏ ﻭﺍﻝﺭﻓﻭﻑ ﻓﻲ ﺍﻝﻤﻜﺘﺒﺎﺕ ﺒﺸﻜل ﻴﻭﻤﻲ ﻭﻋﻤل ﻓﺤﻭﺼـﺎﺕ ﺍﻝﺤـﺴﺎﺴﻴﺔ ﻝﺘﺠﻨـﺏ‬ .‫ﺍﻝﻭﺼﻭل ﺇﻝﻰ ﺍﻝﺤﺎﻝﺔ ﺍﻝﻤﺯﻤﻨﺔ ﻤﻥ ﺍﻝﺤﺴﺎﺴﻴﺔ‬

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INTRODUCTION Several studies had revealed that people spend 80% of their times inside buildings. This makes the study of indoor air quality very important for public health as it might contain heavy metals and microbes (1,2). Several types of pollutants in the air can be causative agents of carcinogenicity, such as heavy metals. Human can be exposed to heavy metals found in dust, where this dust might contain soil, metals and biomaterials (2). Workers can face several problems while they are working in the libraries, mainly accumulated dust on bookshelves. Any movement makes the accumulated dust spread which then may accidentally be inhaled by workers. Several studies indicated that workers in libraries and who deal with dusty books are exposed to several diseases ranging from lung cancer, heart attacks, chronic asthma, allergy problems, general weakness, neurological problems and problems in the outward appearance of the skin (3). The presence of microorganisms in dust is considered as a critical issue as many fungi and bacteria have the ability to produce a wide range of chemicals of internal toxins and volatile organic compounds (1). Inhaling the spores causes allergic reactions. Symptoms of allergies to fungi spores are most common from July to late summer. However, with fungi growing in so many places, allergic reactions can occur in year round (4). Not all microorganisms present in the dust are subjected to the same conditions and growth requirements, which makes the use of traditional methods in the diagnosis difficult and somehow inaccurate. The diagnosis of microorganisms needs to be quick, sensitive and including fungi that cannot be grown in the artificial medium (5). The polymerase chain reaction (PCR) technique is one of the rapid and accurate methods due to its dependence on preserved regions in DNA using specialized primers (6). The aim of this study was to evaluate and analyze the composition of the accumulated dust on the bookshelves throughout investigating the presence of some heavy metals and microorganisms.

MATERIALS AND METHODS Sampling: Accumulated dust samples were collected from books in the Central Library at the University of Mosul, by using a brush and a sterile nylon bag, then the samples were transported to the laboratory and kept at a temperature of 4‫ﹾ‬C while conducting experiments. Estimation of heavy metals in the dust: Six common heavy metals (copper, cobalt, iron, zinc, lead and cadmium) were estimated by using Atomic Absorption Spectrometry (AAS). Five grams (5 gms) of dust were weighted and placed in 250 ml beaker. An equal volume of concentrated nitric acid was added and heated inside the hood with stirring using a rod glass until all the acid evaporated completely and then distilled water was added and heated. Heavy metals were measured by

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using (AAS) after preparing the standard solutions from 1 to 7 mg / L of each metal (7). DNA extraction: A soil sample of 1g was introduced into a 30 ml centrifuge tube containing 10 ml extraction buffer (Tris HCL (pH 8.0), EDTA (pH 8.0) NaCl and SDS). The mixture was incubated under constant agitation at 150 rpm for 10-12 hrs. at 37 ºC. The sample was re-extracted in 1 ml of extraction buffer and the supernatant was collected by low speed centrifugation (5000 rpm for 10 minutes. Four ml of lysis buffer was added and incubated at 65º C for 2 hrs. After incubation, the sample was centrifuged at 10,000 rpm for 10 minutes 4ºC. After centrifugation, the upper aqueous layer was extracted with equal volume of phenol: Choloroform: isoamyl alcohol (25:24:1) at 10,000 rpm for 10 minutes at 4º C. After spun the upper aqueous phase was extracted with equal volume of choloform: isoamyl alcohol (24:1) at 10,000 rpm for 10 minutes at 4º C. Potassium acetate (7.5M) was added at the volume 1/10 and subsequently precipitated by adding 2 volumes chilled ethanol. DNA precipitate was collected by centrifugation at 10,000 rpm for 10 minutes and the pellet was air dried. The DNA was resuspended in 50 µl of sterile distilled water. The obtained DNA was subjected to 2% agarose gel electrophoresis to check the quality and spectrophotometric analysis was done to analyze the purity and yield of DNA (8). Isolation of fungi from dust using direct planting: About 0.5 grams of a book's dust were cultured directly on the PDA medium and dispersed on the surface of the middle and then incubated at a temperature‫ﹾ‬28 C for a week. Polymerase chain reaction PCR: Polymerase chain reaction with specialized primers was used for detection of allergen fungus Alternariaalternata for humans. Those primers were as follows: Aaltr F: GGC GGG CTG GAA CCT C Aaltr R: GCA ATT ACA AAA GGT TT (9) And the specialized primer for E. coli bacteriaare: P11P F: GAG GAA GGT GGG GAT GAC GT P13P R: AGG CCC GGG AAC GTA TTC AC (10). The PCR reaction included the following components in 50 µl as shown in table (1). Table (1): the components of polymerase chain reaction Components Taq Master mix Forward Reverse Template Double distilled water

concentration 2X 100mM 100 mM

63ng/µl

Volume µl 25 2.5 2.5 1 19

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Diagnosis of microorganisms:

Reaction components were distributed in three PCR tubes and placed in a Thermocycler. The conditions of PCR were as follows : initial denaturation of DNA at 94 ‫ ﹾ‬C for 3 minutes and then 35 cycles of three-step PCR amplifications consisting of denaturation at 94 ‫ ﹾ‬C for 1 min, primer annealing at 50 ± 10 ‫ ﹾ‬C for 1 min, and extension at 72 ‫ ﹾ‬C for 10 min at the end of the amplification cycles (8).

Using direct culture: All types of fungi were isolated by using PDA medium. However, after one week incubation a full plate with only big mass of Rhizopus sp. Grown in petri dish and no other species else. This mold can cause allergen (16).

Gel electrophoresis: After the end of the reaction time, 5 µl of each tube were loaded in 2 % agarose gel with DNA ladder 50 bp. Samples were run by electrical electrophoresis after staining by ethedium bromide for 15 minutes and then the bands were photographed using a Gel Documentation (11).

Using the PCR: After extracting DNA from the dust, the purity and concentration were measured by using a nano drop. The concentration was 510.6 ng / µl and the purity was 1.649 .As shown in figure (1) six pure bands (lines : 1, 2, 3, 4, 5, 10) of 10 replicated showed dust genomic DNA in 2% agarose gel.

RESULTS AND DISCUSSION Estimation of the heavy metals concentration: Table (2) show the concentrations of heavy metals measured in the accumulated dust on the books mg / Kg. Table (2): the concentrations of heavy metals in dust concentrations compared to acceptable values Heavy metal

Conc. (mg/kg)

Cu Co Fe Zn Pb Cd

20.08 8.85 21.16 117.84 16.01 2.61

Acceptable values (mg/kg) 50 50 30 300 100 3

The presence of many heavy metals in the dust sample (table 2) compared with the acceptable values did not exceed the normal values. Presence of heavy metal is considered a source of pollution, exposure to heavy metals continuously is very harmful to public health, since these metals can be accumulated in the body. It can be accumulated in the fat cells of the human body and affects the central nervous system, or transmitted through the circulatory system to enter into the internal organs (12). Some metals such as zinc and copper in low concentrations are also harmful , as well as cadmium and lead are toxic and carcinogenic (13). It is proposed that these metals at certain levels can interfere with some of nucleic acids such as DNA and gene expression (14). The presence of these metals in the dust comes from industrial waste that poses to the atmosphere ,cement factories, car exhaust fumes and electric generators that release many gases into the atmosphere , as well as desertification and lack of vegetation where allowed to air currents to carrying these metals into the cities and buildings , which lead to increase concentration in the atmosphere to fall out with the dust grains and settle on surfaces to be in touch with human life (15).

Figure (1): Genomic DNA in agarose gel extracted from dust, 1-10 genomic genomic DNA

After running the PCR product for allergen Alternariaalternatain agarose one band was appear in size 150 bp to each annealing temperature; except the third well was weak because increasing annealing temperature, these bands confirmed the presence of this fungus in the dust as shown in figure (2), while the direct method disable to diagnose this species and this may be due to low level of this fungi in dust or the dominance of Rhizopus in plate prevent the growth of other fungi. This result was in consistent with (9) who found that Alternariaalternata is most abundant species out of all allergen-producing fungi tested.

Figure (2): PCR product running at 2% of agarose gel for Alternariaalternate primer, M: Molecular weight marker, 13 different annealing temperatures (50 , 55 , 58 ) ‫ ﹾ‬C respectively

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The presence of fungus Alternariaalternata in the dust is a health problem because it causes different types of allergies, this species can be found in various places all over the world. Many of them are important pathogens for plants and cause important economical disease in wide range of hosts. Some of them live Saprophytic and are main part of fungi population in soil and dead or dying plant tissues. Polyphagia's nature and ability of Alternariain producing toxic and carcinogenic materials makes this fungi potentially hazardous to human , animals and plants health causing pathogens by moving with the dust grains to reach many areas (17). The results also showed the presence of E. coli in the dust by using PCR reaction and after running the gel a pure band in size 200 bp was appeared (Figure 3). The band was found in annealing temperature 55 ‫ ﹾ‬C and there are no bands in other annealing temperature. This result agrees with (10). The presence of the bacterium E. coli in the dust makes many health problems .Rosas et al. (1997) found that the presence of these bacteria in the air caused diarrhea for many children in Mexico(18), and attributed the presence of these bacteria in the dust to waste pollution fecal for humans , animals and transmitted with grains of sand by air to wander several places (19).

Figure (3): PCR product running at2% of agarose gel for E. coli primer, M: Molecular weight marker, 1-3 different annealing temperatures (50 , 55 , 58 ) ‫ ﹾ‬C respectively.

The use of PCR technique is considered as accurate method to detect microbes at lowest levels, where the classic methods cannot diagnose (20).

CONCLUSION Dust can be considered a very health effective factor, which can reach to everywhere and carry many biotic and non-biotic factors that cause several health problems and most people do not pay attention to it. In conclusion of the current study, the exposure to accumulated dust can be said to be one of the most important hidden pathogens and

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especially to those who work in libraries and patrons.

RECOMMENDATIONS Depending of results and conclusion obtained thoughout the current study, following recommendations are suggested for better protection of health against dust accumulated at bookshelves or any other places in libraries: 1. keep bookshelves cleaned day by day. 2. advice workers at libraries to wear face masks or at least keep a reasonable distance while they are organizing or cleaning books at shelves to prevent any possible contacts of dust and its hazardous contents.

REFERENCES 1. Yang S. and Heinsohn P.(2007). Sampling and analysis of indoor microorganisms. John Wiley and Sons. Inc. P.290. 2. Abdul Wahab N.; Darus F.; Isa N.; Sumari S. and Hanafi N. (2012). Heavy metal concentration of settled surface dust in residential bulding. Malays. J. Anal. Sci. 16(1):18 – 23. 3. Hassan B. (2003). Pay attention to books' deadly dust relationship of lung cancer and heart attack to library books' dust. The 6th edition of the Indoor Air Quality Meeting (IAQ2003). Padova , Italy. 4. Asthma and Allergy Foundation of America (AAFA) (2005). What are allergies? Available at: http://www.aafa.org/display.cfm?id=9&cont=78 5. Dhahi SJ.; Al-Assie AH. and Omear H A.(2011). Application of the randomly amplified polymorphic DNA (RAPD) markers to analyze the genetic variability in species of the fungus Alternaria. J. Raf. Sci. 22(1):11- 16. 6. Zhou G.; Whong WZ.; Ong T. and Chen B.(2000). Development of a fungus-specific PCR assay for detecting low-level fungi in an indoor environment. Mol. Cell Probes. 14(6):339-348. 7. Hassan S.(2012). Metal concentrations and distribution in the household, stairs and entryway dust of some Egyptian homes. Atm. Environ. 54: 207-215. 8. Rajesh S.; Siv J. and Jayalakhsmi S. (2013). Simple, rapid method for direct isolation of metagenomic DNA from Pichvarm Mangrove sediment. Int. J. Res. Biotechnol. Biochem. 3(1) : 19-21. 9. Lang-Yona N.; Dannemiller K.; Yamamoto N. ; Burshtein N.; Peccia YO. and Rudich Y. (2012). Annual distribution of allergenic fungal spores in atmospheric particulate matter in the Eastern Mediterranean; a comparative study between ergosterol and quantitative PCR analysis, Atm. Chem. Phys. 12: 2681-2690. 10. Millar B. ; Jirua X. ; John E. and Earle A. (2000). A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material. J. Microbiol. Method. 42 (2) :139-147.

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11. Mohini J. and Deshpande JD. (2010). Polymerase chain reaction: methods, principles and application. Int. J. Biomed. Res. Pp. 81-97. 12. Bocca B.; Alimonti A.; Petrucci F.; Violante N.; Sancesario G. and Forte G. (2004). Quantification of trace elements by sector field inductively coupled plasma spectrometry in urine, serum, blood and cerebrospinal fluid of patients with Parkinson’s disease. Spectrochim.Acta. B 59: 559- 566. 13. Willers S.; Gerhardsson L. and Lundh T. (2005). Environmental tobacco smoke (ETS) exposure in children with asthma-relation between lead and cadmium, and nicotine concentrations in urine. Resp. Med. 99:1521-15 27. 14. Menzie CA. ; Ziccardi LM.; Lowney YW.; Fairbrother A .; Shock SS.; Tsuji JS.; Hamai D.; Proctor D. et. al. (2009.) Importance of considering the framework principles in risk assessment for metals. Environ. Sci. Technol. 43:8478 – 8482. 15. Ibrahim IG.; Hassan M L.; Baban OS. and Fadhil SS. (2012). Effect of heavy metal content of some common spices available in local markets in Erbil city on human consumption. Raf. J. Sci. 23(3): 217 -225. 16. Esch ER.; Cecelia JH.; Rodger C. and Robert S J. (2001). Common allergenic pollens, fungi, animals, and arthropods. Clinical reviews in allergy and immunology. Humana Press Inc. 21: 295. 17. Kakvan N.; Hamidreza Z.; Bahar M.; Hossein T. And Shahab H.(2012). Study on pathogenic and genetic diversity of Alternariaalternata isolated from citrus hybrids of Iran, based on RAPD-PCR technique. Euro. J. Experim. Biol. 2 (3):570-576. 18. Rosas I.; Salinas E.;Yela A. and Calva E. (1997). Escherichia coli in settled-dust and air samples collected in residential environments in Mexico City. Appl. Environ. Microbiol. 63(10):4093-4095. 19. Orskov F. and Orskov I. (1975). Escherichia coli O:H serotypes isolated from human blood. Acta. Pathol. Microbiol. Scand. 838:595–600. 20. Bridge P.; Arora D.; Reddy C. and Elander R. (1998). Applications of PCR in Mycology. University Press, Cambridge,UK. p. 373. 21. Bernstein JA.; Alexis N.; Bacchus H.; Bernstein IL.; Fritiz P. et al. (2008). The health effects of nonindustrial indoor air pollution. J. Allerg. Immunol. 12(3): 585-590. .

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Vol. 10, No.2, June 2015

Immunohistochemical study of IL-17 expression in prostate tissue of a prostate cancer patients Hassan H. Zrage (1) Zainab F. Ashoor (2) and Wasan A. Bakir (3) (1) Dept. of laboratory/ Al- Emam Ali Hospital/ Hilla (2) Dept. of Microbiology / College of Medicine / Al- Mustansirya University (3) Dept. of cancer research/ Iraqi Center of Cancer and Medical Genetic Researches/ Baghdad /Republic of Iraq

E-mail: [email protected]

ABSTRACT The present study was designed to investigate a possible association between interleukin-17 (IL-17) expression and prostate cancer progression. The immunoexpression of (IL-17) was investigated as a pro- inflammatory cytokine in paraffin sections from (41) prostate cancer tissues, (46) benign prostatic hyperplasia tissue and (20) normal prostate tissues were selected as control group for comparison between groups involved regarding the study of IL-17 expression. All these samples were obtained from Baghdad hospital for specialist surgeries, AlYarmouk Teaching Hospital, Al- Hilla Teaching Hospital as well as cadaver. They were diagnosed surgically by consyultant pathologies. Works were conducted at Iraqi centre for cancer and medical genetics researches at AlMustansiryah University. Results showed that there was significant differences in mean level of IL-17 protein expression between each of poorly differentiated malignancy, moderately differentiated malignancy and benign prostatic hyperplasia group compared to healthy control subjects with P value (0.0001).

Keywords: interleukin-17 (IL-17), prostatic hyperplasia, moderate, benign, prostate cancer, malignancy.

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ‫ ﻭﻗﺩ ﺘﻡ ﺍﻝﺘﺤﺭﻱ ﻋﻥ‬،‫( ﻭﺘﻁﻭﺭ ﺴﺭﻁﺎﻥ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ‬IL-17) ‫ﻫﺩﻓﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﺤﺎﻝﻴﺔ ﺇﻝﻰ ﺍﻝﻜﺸﻑ ﻋﻥ ﺇﻤﻜﺎﻨﻴﺔ ﻭﺠﻭﺩ ﻋﻼﻗﺔ ﺒﻴﻥ ﺘﻌﺒﻴﺭ ﺍﻻﻨﺘﺭﻝﻭﻜﻴﻥ‬ (46)‫ ﻭ‬،‫( ﻋﻴﻨﺔ ﻨﺴﻴﺠﻴﺔ ﻝﺴﺭﻁﺎﻥ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ‬41) ‫ ﻭﺫﻝﻙ ﻓﻲ‬،‫ﺍﻝﺘﻌﺒﻴﺭ ﺍﻝﻤﻨﺎﻋﻲ ﻋﻠﻰ ﺸﻜل ﺴﺎﻴﺘﻭﻜﺎﻴﻨﻴﻥ ﻤﺤﻔﺯ ﻝﻼﻝﺘﻬﺎﺏ ﻭﺒﺎﺴﺘﺨﺩﺍﻡ ﻤﻘﺎﻁﻊ ﺸﻤﻊ ﺍﻝﺒﺎﺭﺍﻓﻴﻥ‬ ‫( ﻤﻥ ﻨﺴﻴﺞ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ ﺍﻝﻁﺒﻴﻌﻲ ﻜﻤﺠﻤﻭﻋﺔ ﻀﺎﺒﻁﺔ ﻻﺴﺘﺨﺩﺍﻤﻬﺎ ﻓﻲ ﺍﻝﻤﻘﺎﺭﻨﺔ ﺒﻴﻥ ﺍﻝﻤﺠﺎﻤﻴﻊ ﻓﻲ‬20) ‫ ﻭ‬،‫ﻋﻴﻨﺔ ﻨﺴﻴﺠﻴﺔ ﻤﺘﻀﺨﻤﺔ ﻤﻥ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ ﺍﻝﺤﻤﻴﺩ‬ ،‫ ﻭﻤﺴﺘﺸﻔﻰ ﺍﻝﺤﻠﺔ ﺍﻝﺘﻌﻠﻴﻤﻲ‬،‫ ﻭﻤﺴﺘﺸﻔﻰ ﺍﻝﻴﺭﻤﻭﻙ ﺍﻝﺘﻌﻠﻴﻤﻲ‬،‫ ﻭﻗﺩ ﺘﻡ ﺘﺠﻤﻴﻊ ﺍﻝﻌﻴﻨﺎﺕ ﻤﻥ ﻤﺴﺘﺸﻔﻰ ﺒﻐﺩﺍﺩ ﻝﻠﺠﺭﺍﺤﺔ ﺍﻝﺘﺨﺼﺼﻴﺔ‬، 17 ‫ﺩﺭﺍﺴﺔ ﺍﻻﻨﺘﺭﻝﻭﻜﻴﻥ‬ ‫ ﻭﻗﺩ ﻨﻔﺫﺕ ﻤﺭﺍﺤل ﺍﻝﺩﺭﺍﺴﺔ ﻓﻲ ﺍﻝﻤﺭﻜﺯ ﺍﻝﻌﺭﺍﻗﻲ‬،‫ ﻜﻤﺎ ﺠﺭﻯ ﺘﺸﺨﻴﺹ ﺍﻝﺤﺎﻻﺕ ﻋﻠﻰ ﻴﺩ ﺃﻁﺒﺎﺀ ﺍﻻﺨﺘﺼﺎﺹ‬،‫ﺒﺎﻹﻀﺎﻓﺔ ﺇﻝﻰ ﻤﺘﺒﺭﻋﻲ ﺍﻝﻁﺏ ﺍﻝﻌﺩﻝﻲ‬ .‫ﻝﺒﺤﻭﺙ ﺍﻝﺸﺭﻜﺎﻥ ﻭﺍﻝﻭﺭﺍﺜﺔ ﺍﻝﻁﺒﻴﺔ ﻓﻲ ﺍﻝﺠﺎﻤﻌﺔ ﺍﻝﻤﺴﺘﻨﺼﺭﻴﺔ‬ ‫ ﺒﻴﻥ ﻜل ﻤﻥ ﻤﺠﻤﻭﻋﺔ ﺴﺭﻁﺎﻥ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ ﻗﻠﻴﻠﺔ ﺍﻝﺘﻤﺎﻴﺯ ﻭﻤﺠﻤﻭﻋﺔ ﺴﺭﻁﺎﻥ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ‬17 ‫ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻭﺠﻭﺩ ﻓﺭﻭﻗﺎﺕ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﺘﻌﺒﻴﺭ ﺍﻨﺘﺭﻝﻭﻜﻴﻥ‬ (P< 0.0001) ‫ﻤﺘﻭﺴﻁﺔ ﺍﻝﺘﻤﺎﻴﺯ ﻭﻤﺠﻤﻭﻋﺔ ﺘﻀﺨﻡ ﺍﻝﺒﺭﻭﺴﺘﺎﺘﺎ ﺍﻝﺤﻤﻴﺩ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻀﺎﺒﻁﺔ ﺒﺩﺭﺠﺔ ﻤﻌﻨﻭﻴﺔ ﻭﺍﻀﺤﺔ ﻋﻨﺩ ﻗﻴﻤﺔ‬

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INTRODUCTION Prostate cancer is a form of cancer that develops in the prostate, a gland in the male reproductive system. The prostate is an exocrine gland that encloses the male urethra and its base is located at the bladder neck (1). The primary function of the prostate gland is to secrete a fluid that is added together with the spermatozoa from the seminal vesicles to constitute majority of semins. There are three distinct zones found in the prostate gland. These are: a) the peripheral zone, b) the transitional zone, and c) the central zone (2). The peripheral zone which approximately 70% of prostate is the most common origin of carcinoma chronic prostatitis, and post inflammatory atrophy. The central zone covers 25% of the prostate gland. The central zone contains approximately 1/3 of the ducts that secrete fluids that help creating semin. The transition zone makes up 5% volume of the gland and surrounds urethra. Prostate cancer is the second leading cause of cancer death in men exceeded only by lung cancer, according to American cancer society , 2015 (3). In the USA, prostate cancer is accounting for 29% of the newly diagnosed cancer cases (4). In Iraq, cancer of prostate is leading cause cancer in males accounting for 3.3% of newly dignosed cancer cases in males (5, 6). Most prostate cancer cases are slow growing. Prostate carcinoma begins when prostate gland cells multiply and grow out normal control. In the peripheral zone, prostatic carcinoma usually begins, where some clusters of cells are confined within the prostate gland. If this condition progresses, the uncontrol cells will form a tumor and may invade the stroma as well as extend to the seminal vesicle. Prostate tumors are usually slow growing and symptoms may not occur for many years (7). In the early stages, prostate cancer they are usually no symptoms. However, due to its location surrounding urethra, symptoms of the disease usually affects urination. It can also affect sexual functions, for example, difficulty in achieving eriction or painful ejacuation (5). If the cancer is advanced, it can spread to other organs causing bone pain in the velvis or ribs. The only well- established risk factors for prostate cancer are older age, family history, inerited genetic conditions and inflammation. About 56% of all prostate cancer cases are diagnosed in men 65 years of age and older (8), and 97% occur in men 50 years and older. Genetic studies suggested that strong familial predisposition may be responsible for 5% - 10% of prostate cancer. Studies suggested that a diet high in processed meat or dairy food may increase risk, and obesity increases the risk of aggressive prostate cancer (9). In addition, smoking is associated with prostate cancer death but not incidence (10). Inflammation of prostate area caused a novel putative prostate cancer precursor lesion called proliferative inflammatory atrophy (PIA), which shares some molecular traits with prostate intraepithelial neoplasia and PCa (11).

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During these transitions to PCa, a number of cytokinens and cytokines receptors display variation in their expressions. It is important to notice that cytokines are produced by a variety of cells, such as tumor-associated with proplasts, endothelial cells, or tumor infiltrating cells such as macrophages and / or lymphocytes have selected advantage of progression of prostate diseases. A key feature of PIA lesion is the presence of mononuclear and / or polymorphonuclear cells in both epithelial and stromal compartments (12). IL-17 is secreted by TH-17 cells and promotes the migration of endothelial cells and induces fibroplasts to upregulate pro angiogenic factors such as vascular endothelial growth factor, macrophage inflammatory proteins -2, prostaglandin and nitric oxide involved in angiogenesis, and in vivo growth of tumor cells (13). Furthermore, Steiner et al. (14) had shown that 58% of human malignant tissues had an increased level of IL-17 messenger ribonucleic acid and both prostate tumor cells and prostate stroma cells treated with IL-17 in vitro had an increase in messenger ribonucleic acid and protecin expression of both IL-6 and IL- 8 (14). These data suggested that IL-17 acts directly on prostate tumor cells and promote their growth and metastasis, or indirectly by elevating the level of inflammatory cytokines, and growth factors relased locally in the prostate. Aim of the study: To determine whether the expression of IL-17 is associated with grade of prostate tumor differentiation.

MATERIALS AND METHODS Immunohistochemical (IHC) detection of IL-17 protein expression in paraffin embedded tissue sections of prostate cancer: This technique is based on the detection of the product of the gene expression (protein) in malignant, benign, and normal cells using specific monoclonal antibodies (primary antibody for specific epitope – rabbit monoclonal Abs. for IL17). Then, it is bound to cytoplasmic or nuclear target protein. The bound primary antibody is then detected by secondary antibody, which contains a specific level. The secondary antibody is then deteted by detection system specific for the level 3,3- diaminobenzidine (DAB) in a chromogen solution. A positive reaction will result in a browncolored precipitate at the antigen site in the tested tissue. Then, the immunohisotchemistry is undergone according to (15). Statistical analysis: The significance of difference of different means (quantitative data) were tested using T- test for difference between two independent means or ANOVA test for difference among more than two independent means. The significance of difference of different percentages (qualitative data) were tested using Pearson's chi square test (X2 test).

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Statistical significance was considered whenever the P value for the test of significance was equal or less than 0.05

RESULTS This study included (87) Iraqi patients with prostate cancer and benign prostatic hyperplasia. They were divided into three groups, in which 16 (14.9%) were poorly differentiated malignancy, 25 (23.3%) were moderately differentiated malignancy, 46 (43%) were with benign prostatic hyperplasia, and there were 20 (18.6%) healthy patients as a control group. Distribution of age factor in studied cases and control group: The sampling procedure was designed to produce five groups, with approximately homogenized age groups (< 50 years, 50-59 years, 60-69 years, 70-79 years, and 80 > years) (Table 1). Table (1): Distribution of study subjects in five age groups Age groups

Poorly differentiated malignancy

< 50 years No % 50- 59 years No % 60-69 years No % 70- 79 years No % => 80 years No % Total No. %

Moderated differentiated malignancy

BPH

Normal

Total

1 0.6

3 0.12

2 0.4

20 100

26 24.2

1 9.1

1 9.1

9 81.1

0 0

11 10.2

4 25

6 24

20 43.4

0 0

30 28

6 37.5

12 48

14 30.4

0 0

32 30

4 25

3 12

1 0.2

0 0

8 0.7

16 14.9

25 23.3

46 43

20 18.6

107 100

Group (70-79 years)was the largest group among the total cases (30%), while the age group of (60-69 years) was the largest group among benign prostatic hyperplasia patients (43.4%) . in healthy control group, age group of 50 % No % Total No. %

Poorly differentiated malignancy

Moderated differentiated malignancy

BPH

Normal

Total

0 0

0 0

0 0

20 100

20 18.6

5 31.2

12 48

36 78.2

0 0

54 50.4

11 68.7

13 52

10 21.7

0 0

33 30.8

16 14.9

25 23.3

46 43

20 18.6

107 100

Statistical analysis and differences of IL-17 immunoexpression among different studie groups: There was a significant difference in mean level of Il-17 protein expression between each of poorly differentiated malignancy , moderately differentiated malignancy, and benign prostatic hyperplasia groups compared to healthy control group with P value (< 0.0001). also, there was a significant difference in mean level of IL-17 protein expression between each of poorly differentiated malignancy and moderately differentiated malignancy groups compared to benign prostatic hyperplasia group with Pvalue of (< 0.0001) for poorly differentiated malignancy and (0.007) for moderately differentiated malignancy groups. In comparing poorly differentaiated malignancy group to moderately differentiated malignancy group, there was significant differences in mean level of IL-17 protein expression with P value of (0.0001) (Table 3). Also there was a significant difference in mean level of IL-17 protein expression among all studied groups with P value of (0.0001) by using ANOVA test.

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Table (3): The statistical analysis and differences of IL-17 protein expression among studied groups IL-17% Average

Poorly Differentiated Malignancy 16 54.6±9.8 2.449

Moderately Differentiated Malignancy 25 45.8±9.7 1.942

BPH

Number 46 Mean±SD 38.0±11.8 Standard Error of 1.743 Mean Range 37.0-69.0 20.0-60.0 13.0-62.0 Percentile 05th 37.0 34.0 23.0 25th 44.5 39.0 31.0 50th (Median) 57.5 49.0 36.0 75th 62.5 52.0 43.0 95th 69.0 58.0 59.0 99th 69.0 60.0 62.0 P value compared to 0.0001* 0.0001* 0.0001* Normal P value compared to 0.0001* 0.007* BPH P value compared to Moderately 0.0001* Differentiated Malignancy . P value comparing all 0.0001# P value compared to 0.0001* 0.0001* 0.0001* Normal * Significant difference between two independent means using Students-t-test at 0.05 level # Significant difference among three independent means using ANOVA test at 0.05 level

DISCUSSION Association of age risk factor with prostate cancer: This study showed that the risk factor to prostate cancer was the older age, which was on consistent with other studies (9, 16). In this study, prostate carcinoma were more concentrated among the age of >70 years (30 %) as shown in table (1), this gave clear idea that there is a relationship between the disease and age, in which it was confirmed by a previous study, that found that in Baghdad city (40%) of patients with prostate cancer were in age group of more than 60 years (17). This is may be due to increase exposure to carcinogenic agent or may be due to that prostate cancer is asymptomatic tumor and slw growing , so it is diagnosed in late stage (18). Association of IL-17 with prostate cancer: IL-17 is a cytokine produced by Th17 cells, a T helper cell subset from an activated CD4+ T- cell (19). The prototypic family member has been identified as IL-17 A (20). However, in this study, when IL-17 protein was detected by using an immunohistochemical technique, IL-17 protein was overexpressed in prostate tissues of all studied groups, with strong immunostraining reaction being the most frequent score among both poorly differentiated malignancy group (68.7%) and moderately differentiated malignancy group (52.0%) (Table 2). The moderate immunostraining reaction being the most frequent score among benign prostatic hyperplasia group (78.2%), and weak immunostaining reaction was the most frequent score among healthy control group(100%)

Normal 20 7.6±0.8 0.186 6.0-9.0 6.2 7.0 7.6 8.0 9.0 9.0 -

-

(table 2). Also, there was significant difference in mean level of IL-17 protein expression between each of porly differentiated malignancy, moderately differentiated malignancy (table 3), with P value of 0.001. this explained that there was positive association between IL-17 expression and increase malignancy of tumor, this is due to the vast accumulation of IL-17 secreting cells in the tumor microenvironment (21) with a dual function of proinflammatory and regulation of local T cell function (22). This is achieved by blocking the entry of cytotoxic CD8 T- cells and the entry of myeloid derived suppressor cells (MDSCs), thus, modifying the local environment and subsequently reducing the local immune response towards the local tumor cells (23). These results were in agreement with Steiner et al. who concluded that IL-17 was found to be intensely expressed in prostate cancer tissue and weekly expressed in normal prostate tissue (14). Also tumor cells are able to perpetuate the local inflammatory response through the expression of CC chemokine ligand 2 (CCL2), which stimulates entry of IL-17 expressing monocytes which can contribute to the lcal inflammatory reaction. This is believed to be responsible for the tumor ability to maintain the pro-inflammatory reaction locally and also continue to stimulate tumor growth (24).

CONCLUSION In conclusion, the older age is appeared to be the most possible risk factor associated with prostate cancer. Moreover, there was a significant correlation between IL-17 immunoexpression and prostate cancer progression.

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REFERENCES 1. Blandy JP. and Lytton B. (1986). The prostate. London, Butter worths Publishers. P. 29-61. 2. Sommer FG.; Nghiem HV.; Herfkens R. and McNeal J. (1993). Gadoinium enhanced MRI of the abnormal prostate. Magnet. Resonan. Img. 11:941948. 3. American Cancer Society (2015). Cancer facts and figures. Atlanta , Ga. 4. Micheal M.J.; Jemal TA.; Siegel R.; Wared E.; Hao YB. and Jiaquan XT. (2008). Cancer statistics 2008. CA Cancer J. Clin. 58:71-96. 5. Espey DK.; Wu X. and Swan J. (2007). Annual report to the nation on the status of cancer. Cancer. 110: 2119-2152. 6. Jemal TA.; Siegel R.; Wared E.; Hao YB.; Xu J. and Thun MJ. (2009). Cancer statistics 2009. CA Cancer J. Clin. 59:225-249. 7. Galper SL.; Chen MH.; Catalona WJ.; Roehi KA.; Richie JP. and D'Amico AV. (2006). Evidence to support a continued stage migration and decrease in prostate cancer specific mortality. J. Urol. 175(3): 907-912. 8. Ries LAG.; Melbert D. and Krapako M. (2008). SEER cancer statistics review. 1975-2005, Section 16. 9. Kristal AR. and Gong Z. (2007). Obesity and prostate cancer mortality. Future. Oncol. 3(5): 557567. 10. Rohrmann S.;Genkinger JM.; Bruke A.; Helzsouer KJ.; Comstock GW.;Alberg AJ. and Platz EA. (2007). Smoking and risk of fatal prostate cancer in a prospective. US. Study. 6(4): 721-725. 11. Sciarra A.; Di SF.; Salciccia S.; Autran GAM. and Gentilucci A. (2007). Inflammation and chronic prostatic diseases: evidence for a link? Euro. Urol 52: 964- 972. 12. DeMarzo AM.; Marchi VL.; Epstein JI. and Nelson WG. (1999). Proliferative inflammatory atrophy of the prostate: implication for prostatic carcinogenesis. AMJ Pathol. 155: 1985-1992. 13. Numasaki M.; Fukushi J.; Ono M.; Narula SK.; Zavodny PJ.; Kudo T. et al. (2003). Interleukin-17 promotes angio-genesis and tumor growth. Blood. 101(7):2620–2627. 14. Steiner GE.; Newman ME.; Paikl D.; Stix U.; Memaran-Dagda N.; Lee C. and Marberger MJ. (2003). Expression and function of proinflammatory interleukin IL-17 and IL-17 receptor in normal, benign hyperplastic, and malignant prostate. Prostate. 56(3):171–182. 15. abcam (2011). IHC-Paraffin protocol (IHC-P). available at: www.abcam.com/technical 16. Walsks P. (2011). Guide to serving prostate cancer. Pprostate Cancer Foundation (PCF). P. 1319. 17. Khudur, K. (2012): Assessment of contributing risk factors for patients with prostate cancer in capital of Baghdad. Kufa. J. Nurs. Sci. 3(2): 101123. 18. Khan H. (2011). Determination of prostate cancer: The Birmingham Prostate Neoplasms Association Study . PhD. thesis , University of Birmingham , UK. 19. Korn T. ; Bettell E. ;Oukka M. and Kuchroo VK. (2009). IL-17 and Th17 cells. Ann. Rev. Immunol. 27: 485–517.

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20. Kolls JK. and Lindén A. (2004). Interleukin-17 family members and inflammation. Immun. 21(4):467–476. 21. Murugaiyan G. and Saha B. (2009). Protumorvs antitumor functions of IL-17. J. Immunol. 183(7): 4169–4175. 22. Kryczek I. ; Wu K. ; Zhao E.; Wei S.; Vatan L.; Szeliga W.; Huang E. et al. (2011). IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer. J. Immunol . 186(7): 4388–4395. 23. He D.; Li H. ; Yusuf N.; Elmets CA.; Li J.; Mountz JD. and Xu H. (2010). IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells. J. Immunol. 184(5): 2281–2288. 24. Vykhovanets EV.; MacLennan GT.; Vykhovanets OV. and Gupta S. (2011). IL-17 expression by macrophages is associated with proliferative inflammatory atrophy lesions in prostate cancer patients. Int. J. Clin. Exper. Pathol. 4 (6): 552–565.

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The menopausal age and associated factors in a sample of Iraqi postmenopausal women Dalya T. Ahmed and Ekhlas A. Hussein Dept. of Obstetric and Gynecology / College of Medicine / Al- Iraqia University / Republic of Iraq E –mail: [email protected]

ABSTRACT Menopause is a critical stage of women’s life which has symptoms and might cause some diseases that affect women’s health and their life. This study is performed to determine the average age of menopause in a sample of Iraqi women and to understand the factors that precede and may affect the timing of menopause. This descriptive cross sectional study has been performed on180 postmenopausal Iraqi women living in different areas of Baghdad city, all those 180 lady had undergo natural menopause and their last menstrual period at least one year in past , they were given informed consent then interviewing sheet was explained to them carefully , then after completing the all data, the information was analyzed by using SPSS program for finding the mean , median , mode and standard deviation and also using Z-value and P-value for comparing two means and finding significant relationship between different factors and menopausal age .The mean age of menopause is 49.01667 ±3.25, we find that the factors that had significant statistical relationship with age of menopause are those cause earlier menopause including smoking (1 year earlier) , tertiary education (8 months earlier) , working for a long period ( 7 months earlier ) , low fatty diet and malnutrition ( 1.5 year earlier ) and short menstrual cycle < 22 days (1 year earlier) , and factors that delayed menopause include obesity BMI > 31 ( 1year later ) , long duration menstrual cycle (1.2 year later ) , irregular cycle ( 1year later) , gradual cessation of the cycle ( 5 month later) than those with abrupt menopause , high gravidity and high parity ( 1 year later). Keywords: Menopause age , BMI , education , income , nutrition , obstetrical history and menstrual history

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ‫ ﻭﺭﺒﻤﺎ ﻴﺴﺒﺏ ﺒﻌـﺽ ﺍﻷﻤـﺭﺍﺽ‬، ‫ﺴﻥ ﺍﻝﻴﺄﺱ ﻫﻭ ﻤﺭﺤﻠﺔ ﺤﺭﺠﺔ ﻤﻥ ﺤﻴﺎﺓ ﺍﻝﻤﺭﺃﺓ ﻭﺒﺸﻜل ﺨﺎﺹ ﺍﺫﺍ ﻜﺎﻨﺕ ﺘﻌﺎﻨﻲ ﻤﻥ ﺃﻋﺭﺍﺽ ﻤﺎ ﺒﻌﺩ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ‬ ‫ ﻝﺫﺍ ﻨﻔﺫﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻝﺘﺤﺩﻴﺩ ﻤﺘﻭﺴﻁ ﺴﻥ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ ﻝﺩﻯ ﻋﻴﻨﺔ ﻤﻥ ﺍﻝﻨﺴﺎﺀ ﺍﻝﻌﺭﺍﻗﻴﺎﺕ ﻭﻓﻬﻡ ﺍﻝﻌﻭﺍﻤل ﺍﻝﺘـﻲ‬،‫ﺍﻝﺘﻲ ﺘﺅﺜﺭﻋﻠﻰ ﺼﺤﺔ ﺍﻝﻤﺭﺃﺓ ﻭﺤﻴﺎﺘﻬﺎ‬ ‫ ﺍﻤﺭﺃﺓ ﻋﺭﺍﻗﻴﺔ ﺒﻌﺩ ﺴﻥ ﺍﻝﻴﺄﺱ ﻴﻌﺸﻥ ﻓـﻲ‬180 ‫ ﻭﻗﺩ ﺘﻡ ﺘﻨﻔﻴﺫ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﻤﻘﻁﻌﻴﺔ ﺍﻝﻭﺼﻔﻴﺔ ﻋﻠﻰ‬،‫ﺘﺴﺒﻕ ﻭﻴﻤﻜﻥ ﺃﻥ ﺘﺅﺜﺭﻋﻠﻰ ﺘﻭﻗﻴﺕ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ‬ ‫ ﻭﻗﺩ ﻤﺭﺕ ﻜل ﻤﻨﻬﻥ ﺒﻤﺭﺤﻠﺔ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ ﺍﻝﻁﺒﻴﻌﻲ ﻭﻤﻭﻋﺩ ﺁﺨﺭ ﺩﻭﺭﺓ ﺸﻬﺭﻴﺔ ﻜﺎﻨﺕ ﺴﻨﺔ ﻭﺍﺤﺩﺓ ﻤـﻀﺕ ﻋﻠـﻰ‬،‫ﻤﻨﺎﻁﻕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﻤﺩﻴﻨﺔ ﺒﻐﺩﺍﺩ‬ ‫ ﺜﻡ ﺒﻌﺩ ﺍﻻﻨﺘﻬﺎﺀ ﻤﻥ ﺠﻤﻊ ﺍﻝﺒﻴﺎﻨـﺎﺕ‬،‫ ﻭﺘﻡ ﺸﺭﺤﻬﺎ ﻝﻬﻥ ﺒﻌﻨﺎﻴﺔ‬،‫ ﻭﺘﻡ ﺃﺨﺫ ﺍﻝﻤﻭﺍﻓﻘﺔ ﺍﻝﻤﺴﺒﻘﺔ ﻤﻥ ﺍﻝﺴﻴﺩﺍﺕ ﺍﻝﺨﺎﻀﻌﺎﺕ ﻝﻠﺩﺭﺍﺴﺔ ﻝﻠﻤﺸﺎﺭﻜﺔ ﺒﻬﺎ ﺨﻁﻴﺎ‬،‫ﺍﻷﻗل‬ ‫( ﻹﻴﺠﺎﺩ ﺍﻝﻭﺴﻁ ﺍﻝﺤﺴﺎﺒﻲ ﻭﺍﻻﻨﺤﺭﺍﻑ ﺍﻝﻤﻌﻴﺎﺭﻱ ﻭﺍﺴﺘﺨﺩﺍﻡ ﺍﻝﻘﻴﻡ ﺍﻹﺤﺼﺎﺌﻴﺔ ﻝﻠﻤﻘﺎﺭﻨﺔ ﺒﻴﻥ ﺍﺜﻨﻴﻥ ﻤـﻥ‬SPSS) ‫ﺘﻡ ﺘﺤﻠﻴل ﺍﻝﻤﻌﻠﻭﻤﺎﺕ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺒﺭﻨﺎﻤﺞ‬، ‫ ﻭﻗﺩ ﺒﻠﻎ ﻤﺘﻭﺴﻁ ﺴﻥ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ‬،‫ﺍﻝﻤﻌﺩﻻﺕ ﻭﺍﻝﻤﺘﻭﺴﻁﺎﺕ ﺍﻝﺤﺴﺎﺒﻴﺔ ﻭﺇﻴﺠﺎﺩ ﻋﻼﻗﺔ ﺫﺍﺕ ﺩﻻﻝﺔ ﺇﺤﺼﺎﺌﻴﺔ ﺒﻴﻥ ﺍﻝﻌﻭﺍﻤل ﺍﻝﻤﺨﺘﻠﻔﺔ ﻭﺴﻥ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ‬ ‫ ﻤﻨﻬﺎ ﻤﺎ ﻴﺠﻌﻠﻪ ﻤﺒﻜﺭﹰﺍ ﻤﺜل‬، ‫ ﻜﻤﺎ ﺘﻡ ﺘﺤﺩﻴﺩ ﺒﻌﺽ ﺍﻝﻌﻭﺍﻤل ﺍﻝﺘﺅﺜﺭ ﻋﻠﻰ ﺴﻥ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ‬، 3.25 ±49.01667 ‫ﻓﻲ ﻫﺫﻩ ﺍﻝﻌﻴﻨﺔ ﻤﻥ ﺍﻝﻨﺴﺎﺀ ﺍﻝﻌﺭﺍﻗﻴﺎﺕ‬ ‫ ﻨﻅﺎﻡ ﻏﺫﺍﺌﻲ ﻤﻨﺨﻔﺽ ﺍﻝـﺩﻫﻭﻥ‬،(‫ ﺃﺸﻬﺭ‬7 ‫ﻭﺍﻝﻌﻤل ﻝﻔﺘﺭﺓ ﻁﻭﻴﻠﺔ )ﻤﺒﻜﺭﹰﺍ‬، (‫ﺍﻝﺘﺩﺨﻴﻥ )ﻤﺒﻜﺭﹰﺍ ﺒﺴﻨﺔ ﻭﺍﺤﺩﺓ( ﻭﻤﺴﺘﻭﻯ ﺍﻝﺘﻌﻠﻴﻡ ﺍﻝﻌﺎﻝﻲ )ﻤﺒﻜﺭًﺃ ﺒﺜﻤﺎﻨﻴﺔ ﺃﺸﻬﺭ‬ ‫ ﺃﻤﺎ ﺍﻝﻌﻭﺍﻤل ﺍﻝﺘﻲ ﺘﺅﺩﻱ ﺇﻝﻰ ﺘﺄﺨﺭ ﺴـﻥ ﺍﻨﻘﻁـﺎﻉ‬،(‫ ﻴﻭﻤﺎ( )ﻤﺒﻜﺭﹰﺍ ﺒﺴﻨﺔ ﻭﺍﺤﺩﺓ‬22>) ‫ﻭﺴﻭﺀﺍﻝﺘﻐﺫﻴﺔ )ﻤﺒﻜﺭﹰﺍ ﺒﺴﻨﺔ ﻭﻨﺼﻑ( ﻭﺍﻝﺩﻭﺭﺓ ﺍﻝﺸﻬﺭﻴﺔ ﺍﻝﻘﺼﻴﺭﺓ‬ ‫ ﻭﻋﺩﻡ ﺍﻨﺘﻅﺎﻡ ﺍﻝـﺩﻭﺭﺓ ﺍﻝـﺸﻬﺭﻴﺔ‬، (‫ ﺍﻝﻤﺩﺓ ﺍﻝﻁﻭﻴﻠﺔ ﻝﻠﺩﻭﺭﺓ ﺍﻝﺸﻬﺭﻴﺔ ) ﻤﺘﺄﺨﺭﺓ ﺴﻨﺔ ﻭﺸﻬﺭﻴﻥ‬، (‫ﺍﻝﻁﻤﺙ ﻓﻬﻲ ﺯﻴﺎﺩﺓ ﻭﺯﻥ ﺍﻝﺠﺴﻡ ) ﻤﺘﺄﺨﺭﺓ ﺴﻨﺔ ﻭﺍﺤﺩﺓ‬ ‫ ﺃﺸﻬﺭ( ﻭﺯﻴﺎﺩﺓ ﻋﺩﺩ ﻤﺭﺍﺕ ﺍﻝﺤﻤل ﻭﺍﻝﻭﻻﺩﺓ )ﻤﺘﺄﺨﺭﺓ ﺴـﻨﺔ‬5 ‫ ﻭﺍﻻﻨﻘﻁﺎﻉ ﺍﻝﺘﺩﺭﻴﺠﻲ ﻝﻠﺩﻭﺭﺓ ﻓﻲ ﺴﻥ ﺍﻨﻘﻁﺎﻉ ﺍﻝﻁﻤﺙ ) ﻤﺘﺄﺨﺭﺓ‬،( ‫)ﻤﺘﺄﺨﺭﺓ ﺴﻨﺔ ﻭﺍﺤﺩﺓ‬ .(‫ﻭﺍﺤﺩﺓ‬

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

INTRODUCTION Menopause is a natural transition encompassing not only the biological changes but also the social and cultural changes associated with the aging process. It usually occurs sometime between 40 and 60 years and marks the end of the reproductive phase of a women's life (1-3). Natural menopause has been defined by World Health Organization (WHO) as at least 12 consecutive months of amenorrhea that is not associated with a pathologic cause (2,3). Menopause is a physiological event in the women’s life. It is caused by aging of ovaries which leads to decline in the production of Gonadotrophins, ovarian Estrogen and Progesterone hormones. The deficiency of these hormones elicits various somatic, vasomotor, sexual and psychological symptoms that impair the overall quality of life of women(2,4). While most women traverse the menopausal transition with little difficulty, others may undergo significant stress. With increasing age, emerging physical health problems can cause significant changes in the woman's lifestyle, leading to social withdrawal, avoidance and curtailment of physical activity (3). Most women, change points towards menopause are well aligned with proposed bleeding markers of the menopausal transition, but for some women they are not clearly associated. Increasing understanding of population differences in the transition experience may lead to new insights into ovarian aging (5). The mean age at natural menopause (ANM) is influenced by various determinants like genetic, demographic, socioeconomic, dietary, reproductive, and behavioral; of which some are modifiable like lifestyle and dietary (2). The lifestyle factors like current smoking, lower education, unemployment, divorced, widowed, vegetarian diet, high fat intake, and prior history of heart disease are independently associated with accelerated menopause; while others like parity, prior use of oral contraceptive pills, and higher body mass index (BMI) is associated with delays ANM. Recent studies have shown that women who carry breast cancer gene (BRCA) mutation have early onset menopause and heavy smoking compounds the risk (2,4). Many studies proved that current smokers reach menopause an average of 1.74 years earlier than nonsmokers (6-8). Although menopause is a universal phenomenon among women, the timing of the onset and the duration of the menopausal transition and the timing of the final menstrual period are not a universal phenomenon among women (9). Until recently, much of the knowledge about the timing of the natural final menstrual period has been affected by the nature of the samples of women studied and a number of other methodologic differences in the studies of this phenomenon, which must be considered in comparing and summarizing their results (9).

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The age at menopause in different societies depends on numerous factors such as development level, socio- economic status and race. The most typical age range for menopause in developing countries is between the ages of 41 and 47, and in developed countries is between the ages of 49 and 52 (10). No other factors appear to have an independent effect on age at menopause. There is no evidence of any secular trend in age at menopause. Evidence is accumulating that most physiological change associated with menopause either occurs or begins before the final menstrual period (11). Finding the factors related to the starting age of menopause, weather earlier or later than the average could help women to better understand this transition and prepare themselves to cope with the effect of menopause on their life. Hence, many researchers try to find causes contributing to the starting age of menopause e.g. menopausal age of mother and sisters, physical activity or socioeconomic status (8, 10, 12). In fact, the importance of menopause in family and society health is due to its complications in women and its health-threatening problems such as osteoporosis or cardiovascular diseases; however these complications are preventable. Considering to physical, emotional and psychiatric complications of early and delayed menopause, it is necessary to recognize associated factors of menopause to provide appropriate awareness and treatment before and after menopause (12).

PATIENTS AND METHODS Research design: A cross-sectional study was conducted to determine the average age of menopause in a sample of Iraqi women and to evaluate the factors that precede and may affect the timing of menopause by using descriptive design by using the (questionnaire). Patients: A convenient sample of 180 women at postmenopausal stages was recruited in the study between 2014 -2015, the study included only women who had undergone natural menopause and with their last menstrual bleeding at least one year in past, the data were collected from women in AlNoaman teaching hospital , employers of college of medicine and from private clinics. Tools: Interviewing sheet (questionnaire) was designed by the researchers and it includes data about women's socio demographic status including age , weight, height, BMI , education , income and type and quality of nutrition and also about menstrual history , obstetrical and gynecological history .also we used standard tools such as weight and height scales.

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Statistical analysis: Data including different factors such as age, demographic history , obstetrical and gynecological history was collected, coded, tabulated and analyzed, using the SPSS computer application for statistical analysis. Descriptive statistics was used to calculate percentages and frequencies and Z-value and P-value were used for comparing two means and finding significant relationship between different factors and menopausal age.

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RESULTS Results of statistical analysis indicated that the average age of menopause in the studied sample was 49.01667 ±3.25. For the variables that may play a role in determining the age of natural menopause, table (1) shows the role of demo-socio-economic factors and the time of menopausal age.

Table (1): Demo-socio-economic factors and the time of menopausal age Variables

No.

%

Mean ±SD

z-value

p-value significant 31

37

20.56

50.01±4.4

4.0995

< 0.0001

Yes

18

10

48.02±4.6

-4.1409

< 0.0001

No

162

90

49.07±1.5

0.2064

0.8365

7

3.89

49.03±3.4

0.0413

0.9671

173

96.11

49.2±4.2

0.7431

0.4574

Illiterate

56

31.11

49.05±6.2

0.1238

0.9014

Primary

61

33.89

49.02±3.4

0

> 0.9999

Secondary

15

8.33

48.9±3.5

-0.4954

0.6203

Tertiary

48

26.67

48.4±5.3

-2.5594

0.0105

43

23.89

49.3±3.2

1.1559

0.2477

BMI:

Smoking :

Marital status: Single Married Education:

Occupation: House wife Employer

32

17.78

48.5±6.3

-2.1466

0.0318

Retired

105

58.33

49.45±4.3

1.5687

0.1167

50

27.28

49.05±3.6

0.1238

0.9014

moderate

85

47.22

49.07±5.2

0.2064

0.8365

high

45

25

49.06±2.2

0.1651

0.8688

Income: low

Nutrition: Low

45

25

47.6±6.7

-5.8619

< 0.0001

Moderate

70

38.89

48.8±6.3

-0.9082

0.3638

Good

65

36.11

49.1±3.4

0.3303

0.7412

In table (1) above, it can noticed that increase body mass index (> 30) was associated with increase the age of menopause about 1 year ( p-value < 0.0001) , while active smoking was associated with decrease age of menopause by about 11 month ( p-value < 0.0001), tertiary education and working till menopausal age was associated with slightly decrease the age of menopause mean value (48.4),

(48.5) respectively (p-value= 0.0105 ,0.0318) respectively , and those who had low fatty diet had significantly had decrease age of menopause by about 1.4 year . Income, low , moderate or high , does not appear to affect the menopausal age significantly (p-value=0.9014).

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Table (2) reflects the relationship of menstrual history and the age of menopause, we see that there is significant decrease in age of menopause in those women who had short duration of menstrual cycle < 22 days ( mean age = 48.02±4.3) that’s mean about 1 year earlier (p-value=< 0.0001) , while those with long duration cycle >32 days had later menopause

Vol. 10, No.2, June 2015

by 1.2 year ( the mean age =50.42±3.5) and (pvalue=< 0.0001). Those with irregular menstrual period had also later age of menopause by 1.1 year ( the mean age = 50.11±4.2) and (p-value=< 0.0001), and those who had gradual cessation of the cycle also have slight increase in age of menopause (5 months later ) the mean age is (49.71±2.2) and (p-value=< 0.0001).

Table (2): menstrual history and menopausal age Variables

NO.

%

Mean ±SD

z-value

p-value significant 32 days

15

8.33

50.42±3.5

5.7794

< 0.0001

Regular

162

90

49.04±2.1

0.0826

0.9342

Irregular

18

10

50.11±4.2

4.4997

< 0.0001

Abrupt

57

31.67

49.35±3.2

1.3623

0.1731

Gradually

123

68.33

49.71±2.2

2.8484

0.0044

Regularity of period:

Pattern of menstrual cessation:

Table (3): obstetrical and gynecological history and menopausal age Variables

No.

%

Mean ±SD

z-value

p-valuesignificant 14

33

18.33

49.05±4.3

0.1238

Age at first pregnancy: < 20

95

52.78

49,3±5.1

1.1559

0.2477

20 – 30

62

34.44

49.07±4.3

0.2064

0.8365

> 30

23

12.78

49.1±4.2

0.3303

0.7412

< 20

12

6.67

49.09±2.5

0.289

0.7726

20 – 30

35

19.44

49.07±3.7

0.2064

0.8365

> 30

133

73.89

49.41±3.2

1.61

0.1074

0

20

11.11

48.81±3.2

-0.8669

0.386

1–4

126

70

49.32±2.2

1.2384

0.2156

5 & more

34

18.89

49.5±4.1

1.9815

0.0475

0

21

11.67

49.06±4.2

0.1651

0.8688

1–4

148

82.22

50.24±5.2

5.0363

< 0.0001

5& more

11

6.11

51.02±2.4

8.2563

< 0.0001

0

92

51.11

49.03±4.5

0.0413

0.9671

1–3

82

45.56

49.44±3.5

1.7338

0.083

4 & more

6

3.33

49.26±4.1

0.9908

0.3218

Yes

74

41.11

48.74±4.2

-1.1559

0.2477

No

106

58.89

49.05±5.1

0.1238

0.9014

Age at last pregnancy:

Gravidity:

Parity:

Abortion:

OCP consumption:

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Table (3) reflected the relationship between menopausal age and obstetrical and gynecological history. In the studied sample, it was found that there is no significant relationship between the age of menarche (< 12 or > 14) and the timing of menopause (all p-values > 0.05). For the age of first and last pregnancy, also results indicated that there is no significant relationship with menopausal age (all p-values > 0.05) . Multigravida women (gravida 5 and more) had significantly increased age of menopause (p-value= 0.0475) with mean age of menopause about (49.5±4.1) , for multiparas women, the later age of menopause found in those had parity (1-4) (mean age = 50.24±5.2) and (pvalue=< 0.0001) , while those with parity 5 7 more , (the mean age =51.02±2.4) and (p-value=< 0.0001). For those women who had previous abortion (one, two or recurrent abortions), there is no significant relationship with menopausal age (all p-values > 0.05), the same result for previous use of contraceptive pills.

DISCUSSION There is a great variation in the menopausal age between the western and the eastern countries, the mean age of natural menopause in European countries is 54 years (13). In the current study, the mean age of the menopause in Iraq was 49.01667±3.25, while the mean age at natural menopause among Saudi Arabian women was (47.9- 48.06) years and the median age was 49 years (14,15). In Iran, the mean menopause age was 47.6±4.45 years with the median age of 48 years (12), while in Egypt the mean age of the menopause is 46.7 years (14), in Turkey is 47 years old (16), in Jordan, the median age of menopause is 50 years (17), while in the far east world countries e.g. in Malaysia the mean age is 51.3 years old (18). For the factors that affect the age of menopause , it was found that the most affective factor that cause earlier menopause is poor nutrition and low fatty diet by about 1.4 year earlier (47.6±6.7). This result was in accordance with result of (10), who found that malnourished women had menopause 4 years earlier. This result did not agree with the result of a study done in Japan showing a later age at natural menopause associated with higher green and yellow vegetable intake (19), while another study found no association between the calories intake and the natural age of menopause (20). The second most affective factor is smoking, which was associated with earlier age of menopause by one year (48.02±4.6). This result was in agreement with most other studies (6-8). Also the same results were found by Harlowa (21) and Remez (22). The other affective factor is short duration of menstrual cycle < 22 days ( mean age = 48.02±4.3) associated with 1year earlier menopause, the same result was found by (21), that the length of menstrual cycle is significant factor in determining the age of natural menopause ,shorter cycle is

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associated with earlier menopause. Also the same result was found by (8, 10). For the education , it was found that women with higher education had later menopause by 8 months (mean age 48.4±5.3), while in another study, it was found that women with higher education had later menopause by two third of the year (23). Working till menopausal age also is associated with slightly decrease in the age of menopause about 7 months earlier (mean value 48.5±6.3). This result was different from results found by (20), who concluded that employment during follow-up is associated with later age of menopause. Factors that associated with delayed age of menopause include the multi-parity ( 5 and more), which are associated with 2 year later menopause (51.02±2.4) while multi-gravida ( 5 and more) are associated with slight delay in menopausal age about few months (49.5±4.1). The same result also was found in Iran as they found that women without parity had earlier menopause (12), and by (21), while in a large study done in USA, it was found that women who have ever given birth all experience a delay in reaching natural menopause relative to other women (22). The second most affective factors in delaying menopause is long duration of of menstrual cycle < 32 days, later by 1.2 year (50.42±3.5) , irregular menstrual cycle by 1 year (50.11±4.2) and gradual cessation of the cycle few month later (49.71±2.2) the same result found by Ellen(8,10) later natural menopause (mean = 0.8 year later) was observed in women with cycle lengths of 33 days or longer. Results revealed that the increase in BMI >31 is associated with increase in the mean age of menopause by 1 year ( 50.01±4.4) the same result was found in both (20, 24). The current study, results showed no significant relationship with level of income , or previous use of combined oral contraceptive pills (COCP) , while most other studies found that use of COCP was significantly associated with delayed age of menopause (20-22). Regarding previous studies conducted by (8, 10), it was found that the low income significantly associated with early menopause , this result did not agree with results of current study which found no significant relationship between the age of natural menopause with level of income.

CONCLUSION The mean age of menopause in a sample of Iraqi women was 49.01667 ±3.25 and this age is affected by different demographic (e.g., education and employment), menstrual and reproductive (e.g., duration and regularity of the cycle, gravidity and parity), and lifestyle (e.g., smoking, weight and diet) factors seem to be important determinants of the age, at which natural menopause occurs. It is recommended to do similar studies in other Iraqi governments to determine the factors affecting the

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time of occurrence of menopause earlier or later than the normal age, and determine the actual mean of age of menopause.

REFERENCES 1. Mohamed HAE.; Lamadah SM. and Al-Zamil LG. (2014). Quality of life among menopausal women. Int. J. Reprod. Contracept. Obstet. Gynecol. 3 : 552-561. 2. Sapre S. and Thakur R. (2014). Lifestyle and dietary factors determine age at natural menopause. J. Midlife Health. 5: 1-6. 3. Natarajan J.; Seshan D. and Muliira RS. (2013). Review literature on distress during the menopausal transition and their impact on the quality of lLife of women. IOSR J. Nurs. Health Sci. (IOSR-JNHS). 2(4):1-10. 4. Elsabagh EEM. and Abdullah ES. (2012). Menopausal symptoms and the quality of life among pre/post menopausal women from rural area in Zagazigcity .Life Sci. J. 9(2):283-291. 5. Bouzari Z.; Kotenaie MJ.; Darzi AA. and Hajian K.(2013). Menopausal symptoms can be influenced by various sociodemographic factors and quality of life (QoL) decreases after the menopause. World Appl. Sci. J. 23 (9): 1221-1230. 6. McKinlay SM.; Bifano NL. and McKinlay JB. (1985). Smoking and age at menopause in women. Annal. Inter. Med.103(3):350-356. 7. Hassa H1.; Tanir HM.; Tekin B.; Senses T.; Oge T. and Mutlu FS. (2006). Possible factors affecting the age at menopause among women in the central anatolian region of Turkey. Clin. Exp. Obstet. Gynecol. 33(1):59-60. 8. Gold EB.; Bromberger J.; Crawford S.; Samuels S.; Greendale GA.; Harlow SD. and Skurnicktors J. (2001). Associated with age at Natural menopause in a multiethnic sample of midlife women. Am. J. Epidemiol. 153(9):865-874. 9. Gold EB. (2012). The timing of the age at which natural menopause occurs. Obstet. Gynecol. Clin. N. Am. 38(3): 425–440. 10. Omidi A.; Bakht R.; Moghimbeigi A. and Balali Z. (2013). A study of age at menopause and related factors. Life Sci. J. 10(3):1295- 1299. 11. McKinlay SM. (1996). The normal menopause transition. Maturitas. 23(2): 137–145. 12. Abdollahi AA.; Qorbani M.; Asayesh H.; Rezapour A.; Noroozi M.; Mansourian M.; Soleimani MA. and Ansari H.(2013). The menopausal age and associated factors in Gorgan, Iran. Med. J. Islam. Repub. Iran (MJIRI). 27(2): 50– 56. 13. Palacios S.; HendersonVW.; Siseles N.; Tan D. and Villaseca P. (2010). Age of menopause and impact of climacteric symptoms by geographical region.Climact.13:419-428. 14. Al-Sejari MM. (2005). Age at natural menopause and menopausal symptoms among saudiarabian women in Al-Khobar. PhD thesis. Ohio State University.317-396.

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15. Al- Joharah M.; Al-Quaiz SA. and Tayel F A H. (2013). Assessment of symptoms of menopause and their severity among Saudi women in Riyadh. Ann. Saudi. Med. 33(1): 63-67. 16. Vehid S1.; Aran SN.; Köksal S.; Işiloglu H. and Senocak M. (2006). The prevalence and the age at the onset of menopause in Turkish women in rural area. Audi. Med. J. 27(9):1381-1386. 17. Shakhatreh FM1. and Mas'ad D. (2006). Menopausal symptoms and health problems of women aged 50-65 years in southern Jordan. Climact. 9(4):305-311. 18. Abdul Rahman SAS.; Zainudin SR. and Mun VLK. (2010). Assessment of menopausal symptoms using modified menopause rating scale (MRS) among middle age women in Kuching, Sarawak, Malaysia. Asia Pacif. Family Med. 9 (5):1–6. 19. Nagata C.; Takatsuka N. And Kawakami N. (2000). Association of diet with the onset of menopause in Japanese women. Am. J. Epidemiol. 152(9):863–867. 20. Gold EB.; Crawford SL.; Avis NE.; Crandall CJ.; Matthews KA. et. al. (2013). Factors related to age at natural menopause: longitudinal analyses from SWAN. Am. J. Epidemiol. 178(1): 70–83. 21. Harlowa BL. and Signorelloa LB. (2000). Factors associated with early menopause. Maturitas. 35(1):3–9. 22. Remez L. (2001). Multiple factors, including genetic and environmental components, influence when menopause begins family planning. Perspect. 33 (5): 234- 237. 23. Schoenaker DAJM.; Jackson CA.; Rowlands JV. and Mishra GD. (2014). Socioeconomic position, lifestyle factors and age at natural menopause: a systematic review and meta-analyses of studies across six continents. Int. J. Epidemiol. 43(5): 1542–1562. 24. Morris DH.; Jones ME.; Schoemaker MJ.; McFadden E.; Ashworth A. and Swerdlow AJ. (2012). Body mass index, exercise, and other lifestyle factors in relation to age at natural menopause: analyses from the break through generations study. Am. J. Epidemiol.175 (10) :998– 1005.

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Effect of silver nanoparticles prepared with pomegranate peel extract on antibiotic resistant Gram negative bacteria Salah S. Zain Al- Abdeen Dept. of Biology / College of Sciences / Kirkuk University / Republic of Iraq

E –mail: [email protected]

ABSTRACT The current study was conducted to investigate the activity of silver nanoparticles that were prepared by using the pomegranate peel extract against three multiple antibiotics resistant Gram negative pathogenic bacteria (Pseudomonas aeruginosa , Klebsiella pneumoniae and Escherichia coli). Results showed that the observed color of the mixed solution of extract and 1 mM silver nitrate changed to dark brown. The solution is considered to be an evidence of the nanoparticles formation, where the UV-VIS spectrophotometer showed the absorption peak 372 nm. However, the study of antibacterial activity of the particles prepared showed the antibacterial activity against tested species. Keywords: Antibiotics resistance ,silver nanoparticles , pomegranate peel extract

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ‫ﺘﻡ ﻓﻲ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﺘﺤﺭﻱ ﻋﻥ ﻓﻌﺎﻝﻴﺔ ﺩﻗﺎﺌﻕ ﺍﻝﻔﻀﺔ ﺍﻝﻨﺎﻨﻭﻴﺔ ﺍﻝﻤﺤﻀﺭﺓ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻤﺴﺘﺨﻠﺹ ﻗﺸﻭﺭ ﺍﻝﺭﻤﺎﻥ ﻀﺩ ﺜﻼﺜﺔ ﺃﻨﻭﺍﻉ ﻤﻥ ﺍﻝﺒﻜﺘﻴﺭﻴﺎ ﺍﻝﻤﺭﻀـﻴﺔ‬ ‫ ﻭﺍﻝﺘﻲ ﺘﺘﺼﻑ ﺒﺼﻔﺔ ﺍﻝﻤﻘﺎﻭﻤـﺔ‬،( Escherichia coli‫ ﻭ‬Klebsiella pneumonia‫ ﻭ‬Pseudomon asaeruginosa ) ‫ﺍﻝﺴﺎﻝﺒﺔ ﻝﺼﺒﻐﺔ ﺠﺭﺍﻡ‬ ‫ ﻭﺍﻝﺫﻱ ﻴﻌﺘﺒﺭ ﺩﻝﻴﻼ ﻋﻠﻰ ﺘﻜﻭﻥ‬،‫ ﻤﻥ ﻨﺘﺭﺍﺕ ﺍﻝﻔﻀﺔ ﺇﻝﻰ ﺍﻝﻠﻭﻥ ﺍﻝﺒﻨﻲ ﺍﻝﻐﺎﻤﻕ‬1Mm ‫ ﺇﺫ ﻝﻭﺤﻅ ﺘﻐﻴﺭ ﻝﻭﻥ ﻤﺯﻴﺞ ﺍﻝﻤﺴﺘﺨﻠﺹ‬،‫ﺍﻝﻤﺘﻌﺩﺩﺓ ﻝﻠﻤﻀﺎﺩﺍﺕ ﺍﻝﺤﻴﻭﻴﺔ‬ ‫ ﻤﻥ ﺠﺎﻨـﺏ‬. ‫ ﻨﺎﻨﻭﻤﺘﺭ‬372 ‫ ﺍﻝﻤﻁﻴﺎﻑ ﺍﻝﻀﻭﺌﻲ ﻭﻅﻬﺭﺕ ﻗﻤﺔ ﺍﻻﻤﺘﺼﺎﺹ ﻋﻨﺩ ﺍﻝﻁﻭل ﺍﻝﻤﻭﺠﻲ‬UV-VIS‫ ﻜﻤﺎ ﺘﻡ ﺍﺴﺘﺨﺩﺍﻡ ﺠﻬﺎﺯ‬، ‫ﺍﻝﺠﺯﻴﺌﺎﺕ ﺍﻝﻨﺎﻨﻭﻴﺔ‬ .‫ ﺘﺒﻴﻥ ﺃﻥ ﺍﻝﺠﺯﻴﺌﺎﺕ ﺍﻝﻤﺘﻜﻭﻨﺔ ﺘﺜﺒﻁ ﻨﻤﻭ ﺍﻝﻌﺯﻻﺕ ﺍﻝﺒﻜﺘﻴﺭﻴﺔ ﺍﻝﻤﺴﺘﺨﺩﻤﺔ ﻓﻲ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ‬،‫ ﻭﻋﻨﺩ ﺩﺭﺍﺴﺔ ﺍﻝﻔﻌﺎﻝﻴﺔ ﺍﻝﻤﻀﺎﺩﺓ ﻝﻠﺒﻜﺘﻴﺭﻴﺎ‬، ‫ﺁﺨﺭ‬

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INTRODUCTION

means of disc diffusion method (22), and the results were interpreted according to CLSI (23).

There are many antibiotics that being used to control bacterial infections. The emergence of new strains of bacteria appears resistant to the antibiotics. In the Gram negative bacteria, the resistance may be inheritor acquired as a result of the acquisition of resistance genes in (1,2). Natural product regards as good alternative to fight microorganisms that resist antibacterial agent such as antibiotics (3). The science of nanotechnology has been found to improve a new manufacturing materials (4). The silver compounds have activity against several microorganisms (5), when compared with other materials own highly toxic for microorganisms while it have low toxicity to mammalians (6). In spite, the silver nanoparticles are known since a long time, the researchers have begun to develop the inhibitors of microorganisms from it which has been prepared through using different methods (5,7,8,9,10). Chemical and physical methods are expensive with high energy consuming as well as each of them has a toxic effect to the environment (11). In addition, the used methods depending on plants extracts to prepare silver nano- particles with silver nitrate solution is more convenience to overcome the problems toxicity. The formation of nano particles has been confirmed through the reading of the absorption spectrum using UV-VIS spectrophotometer (12). Antibacterial activity of nanoparticles have been studied against humans pathogenic bacteria such as E.coli and Bacillus subtilis (13,14). Several studies were used certain plants in prepaeation of silver nanoparticles like leaves of green Artemisia pallens (15) and the, Murraya Koenigii and Zea mays in (12), bananas in (16) , lemon and black pepper in (17) and Myrtus communis in (18). In this study, the pomegranate peel extract was used to prepared the silver nanoparticles and examined the susceptibility of antibiotics against Gram negative bacteria.

3. Preparation pomegranate peel extract: Pomegranate peel extract has been prepared depending on (12). Washed Pomegranate peel with running water has been rewashed with distilled water. The Pomegranate peel then has been dried, cut into small pieces and crushed to be as a powder. 50 grams of the powder dissolved in 150 ml of distilled water has been left to boil for five minutes, at an interval of thirty seconds, then the extract was filtered through Whatman No.1 filter paper. (1mM) silver nitrate solution has been prepared. 10 ml of the extract added to 30 ml of silver nitrate solution and mixed well. The solution then has been exposed to sunlight until getting the dark color. Then it will be incubated in the dark place.

MATERIALS AND METHODS 1. Bacterial species: Three bacterial species, namely E.coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were used in this study .These species were isolated from clinical specimens belonging to patients suffering from bacterial infections in Kirkuk General Hospital. Characterization and identification methods were used according to previous studies (19-22). Confirm diagnosis was done by API 20 E system. 2. Antibiotics susceptibility: The susceptibility of the three bacterial species against 14 antimicrobial agents were determined by

4. Measuring the absorbance spectrum: The absorbance has been measured by UV-VIS spectrophotometer (JENWAY 7315) at the wavelengths between 250-600 nm (24). 5. Antibacterial activity: Agar well diffusion method has been used against different Gram negative multiple antibiotics resistant pathogenic bacteria (E.coli ,Klebsiella pneumoniae and Pseudomonas aeruginosa) (25) . Mueller Hinton agar prepared and poured in Petri dishes and left to solidified. The bacterial isolates concentrations has been determined by measuring optical density (OD) at 600 nm (0.1-0.08 OD600 corresponding to 108 CFU/mL) (26). The bacteria has been cultured on the surface of plates where wells of 6 mm diameter have been made on Mueller Hinton ager plates. Using micropipette, 60 µL of nanoparticle solution has been poured onto each well on all plates. After incubation at 37◦C for 24hrs, the diameter of zone of inhibition was measured in millimeter.

RESULTS AND DISCUSSION The biological preparation of nanoparticles is more competent than the physical and chemical techniques (17). Plant extracts have been considered good substitutes for preparation of nanoparticles (25) where the use of chemicals leads to the accumulation of some toxic substances on the surface of nanoparticles (27). When a plant extracts with [1mM] AgNO3 solution has been mixed, the color of the reaction medium changed rapidly from colorless to brown, which indicates the formation of silver nanoparticles during reduction of silver ion. In the same time, the control sample (AgNO3) solution didn’t change which agrees with the results in (28).

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In spite of some references show that the absorption spectrum is usually between (400-425) nm; the absorption spectrum of silver nanoparticles has been obtained 371 nm in (28). In figure (1), the absorption spectrum of the silver nano particles formed in presence pomegranate peel extract as a reductant which was 372 nm after half an hour. In addition, Singaravelan and Alwar (29) showed that the primary absorption spectrum was in the 398 nm and the secondary absorption spectrum was at 339 nm.

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The study of the antibacterial activity of silver nanoparticles which has been prepared using pomegranate peel extract showed that the existence of activity against three of multiple antibiotics resistant gram negative bacteria (E.coli , Pseudomonas aeruginosa and Klebseilla pneumonia) are 18, 17.5 and 19 mm respectively as shown in table (1). The results are in agreement with (18,3). Table (1) showed a comparison between the resistant bacterial isolates to antibiotics, which has emerged as a resistance to a number of antibiotics at the same time and bacterial growth inhibition using silver nanoparticles formed in existence of pomegranate peel extract. Figure (2) shows the presence of growth inhibition as an activity of the control sample of a solution of silver nitrate (1mM) according to presence of silver which characterized as an anti -bacterial (5, 31). On the other hand, many studies indicate the inhibitory effect of pomegranate peel on different types of bacteria (32-35). Therefore, the presence of activity of pomegranate peel extract, which has been used as a control it is not strange. As a conclusion, in spite of the activity of the AgNO3 solution and the activity of the extract, the silver nanoparticles, which has been prepared with pomegranate peel extract owns antibacterial activity.

Figure (1): Absorption spectrum of the silver nano particles

Table (1): Antibiotics resistant of tested bacteria and sensitivity to silver nanoparticles Isolates

AX25

AM10

PRL100

CFM5

ATM30

CAZ30

CTX30

CRO3

IPM10

AK30

GN10

TOB10

CIP5

E.coli R R R R R R R R S S R R S P.aeruginosa R R S R R R R S S S R S R K.pneumoniae R R R R R R R S S S R S R T=Tetracyclin, CIP=Ciprofloxacin, TOB=Tobromycin, GN=Gentamicin, AK=Amikacin, IPM=imepenam, CRO=Ceftriaxone, CTX=Cefotaxime, CAZ=Ceftazidime, ATM=Aztreonam, CFM=Cefixime,PRL=Pipracillin,AM=Ampicillin, AX= Amoxicillin, R=Resistance, S= Sensitive

Extract 20 18 16

AgNP

3 AgNO

T30 R R R

CONCLUSION

20

19 16

17.5 15

10

Figure (2): An antibacterial activity of silver nanoparticles

The activity of silver nanoparticles prepared through using the pomegranate peel extract against three multiple antibiotics resistant gram negative pathogenic bacteria (Pseudomonas aeruginosa , Klebsiella pneumonia and Escherichia coli).When a plant extracts has been mixed with [1mM] AgNO3 solution, the color of the reaction medium changed from colorless to dark brown which indicates the formation of silver nanoparticles during reduction of silver ion. The solution is considered to be an evidence of the nanoparticles formation where the UV-VIS spectrophotometer showed the absorption peak 372 nm. However, the study of antibacterial activity of the particles formed appears the inhibitory activity of the growth for the used bacteria.

Inhibition/ mm 18 17.5 19

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ACKNOWLEDGEMENT The author would like to thank Miss Shahla Jamal in chemistry department and Dr. Ahmad Hussein in computer department for their cooperation to achieve this work.

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antibacterial activity. Am. J. Advanced Drug Delivery. 2(2):174-182. 29. Singaravelan R. and Alwar B S.(2015). Electrochemical synthesis, characterisation and phytogenic properties of silver nanoparticles. Appl. Nanosci. 369: 10-16. 30. Ahmad HA.; Sakban S.; Dawood F. and Turki R.(2013). Evaluation of biosynthesis of nanoparticles using medicinal plant extract of its anti oxidant and anti microbial activities. Int. J. Sci. Technol. (IJST) 8(4): 91-95. 31. Guggenbichler J P.; Boswald M.,; Lujauer S. and Krall T.(1999). A new technology of microdispersed silver in polyurethane induces antimicrobial activity in central venous catheters. Infect. 27: S16-S73. 32. Khan J A. and Hance S. (2011). Antibacterial properties of Punica granatum peels . Int. J. Appl. Biol. Pharm. Technol. 2(3):23-27. 33. Nuamsetti T.; Dechayuenyong P. and Tantipaibulvut S. (2012). Antibacterial activity of pomegranate fruit peels and arils. Sci. Asia. 38: 319322. 34. Nikfallah F.; Venugopa A.; Tejani H. and Lakshmikantha H T. (2014). Evaluation of the antibacterial activity in pomegranate peels and arils by using ethanolic extract against S. Mutans and L. Acidophilus. Global J. Med. Res. 14(2): 29-36. 35. Al- Hazzani A A.; Shehata A I.; Moubayed NMS.; Al-Jafari A.; Ataya F.; Daoud M.; Al Houri H J .; Rizwana H. and Elgaaly G.(2013). Pomegranate (Punicagranatum) from ancient roots to modern life known with a potent antibacterial activity. Schol. Res. Libr. Annal. Biol. Res. 4 (5):75-87.

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Molecular localization of envelope gene sequence of Human Mammary Tumor Virus in malignant endometrial and cervical tissues from Iraqi patients in Baghdad Saad H. M. Ali (1) Basim S. Ahmed (2) and Sura D. Dawood (3) (1) Communicable Diseases Research Unit/ Baghdad Medical College (2) Dept. of Pathology/ College of Medicine/ Al-Mustansyria University (3) Dept. of Microbiology/ College of Medicine/AlMustansyria University / Republic of Iraq E –mail: [email protected]

ABSTRACT Previous studies have suggested a possible link between Human Mammary Tumor Virus (HMTV) infection and cancers affecting the hormonal –dependent tissues and among them endometrial and cervical carcinoma . This study in Iraq has used in situ hybridization to localized HMTV in tissue specimens from 70 hysterectomized patients diagnosed with malignant uterine tumors (30 cases), non-malignant uterine tumors (25 cases),and 15 cases as control tissues groups. Also the study has enrolled chronic cervicitis (40cases) and invasive cervical cancer from endometrial (6 cases) as well as 24 normal cervical tissues . In the cancer sites malignant uterine tumors group, HMTV Was detected in 5 cases(16.7%) ,and in2 cases (8%) non-malignant uterine tumors ,and 2 cases (13%)of control tissues group . Among cervical lesions , HMTV was detected in four cases(13%) with malignant uterine tumors and (16%) in four cases with non-malignant uterine tumors while in control cervix tissues , HMTV was detected in only one cases(6.7%). Although a relating low rates of HMTV infection were detected among different uterin as well as cervical lesion in this Iraqi study, it might mark or shade alight in the way for further researches in this field to exposure more aspects of this newly studied virus among other hormonal dependent malignancy. Keywords: Human Mammary Tumor Virus (HMTV), hysterectomized, gene sequence

VWXYZ[‫ ا‬V]^[_X `a^b[‫ا‬ ‫( ﻭﺍﻝﺴﺭﻁﺎﻨﺎﺕ ﺍﻝﺘـﻲ ﺘـﺅﺜﺭ ﻋﻠـﻰ ﺍﻷﻨـﺴﺠﺔ‬HMTV) ‫ﺃﺸﺎﺭﺕ ﺍﻝﺩﺭﺍﺴﺎﺕ ﺍﻝﺴﺎﺒﻘﺔ ﺇﻝﻰ ﺍﺤﺘﻤﺎل ﻭﺠﻭﺩ ﺼﻠﺔ ﺒﻴﻥ ﺍﻹﺼﺎﺒﺔ ﺒﺎﻝﻔﻴﺭﻭﺱ ﺍﻝﺒﺸﺭﻱ ﺍﻝﻠﺒﻨﻲ‬ .‫ ﻤﻥ ﺒﻴﻨﻬﺎ ﺴﺭﻁﺎﻥ ﺍﻝﺭﺤﻡ ﻭﻋﻨﻕ ﺍﻝﺭﺤﻡ‬،‫ﺍﻝﻬﺭﻤﻭﻨﻴﺔ‬ ‫ ﻤﺭﻴﻀﺔ ﺃﺠﺭﻴﺕ‬70 ‫ ﻓﻲ ﻋﻴﻨﺎﺕ ﺍﻷﻨﺴﺠﺔ ﺍﻝﻤﺄﺨﻭﺫﺓ ﻤﻥ‬HMTV‫ﺘﻡ ﺘﻨﻔﻴﺫ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﺤﺎﻝﻴﺔ ﻓﻲ ﺍﻝﻌﺭﺍﻕ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺘﻘﻨﻴﺔ ﺍﻝﺘﻬﺠﻴﻥ ﺍﻝﻤﻭﻀﻌﻲ ﻝﻔﻴﺭﻭﺱ‬ (‫ ﺤﺎﻝﺔ‬15 )‫ﺤﺎﻝﺔ( ﻀﻤﻥ ﺃﻭﺭﺍﻡ ﺍﻝﺭﺤﻡ ﻏﻴﺭ ﺍﻝﺨﺒﻴﺜﺔ ﻭ‬25)‫ ﺤﺎﻝﺔ( ﺸﺨﺼﺕ ﻀﻤﻥ ﺍﻷﻭﺭﺍﻡ ﺍﻝﺨﺒﻴﺜﺔ ﻝﻠﺭﺤﻡ ﻭ‬30) :‫ﻝﻜل ﻤﻨﻬﻥ ﻋﻤﻠﻴﺔ ﺍﺴﺌﺼﺎل ﺍﻝﺭﺤﻡ ﻤﻨﻬﺎ‬ ، ‫ ﻜﻤﺎ ﺘﻀﻤﻨﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻋﻴﻨﺎﺕ ﻝﻌﻨﻕ ﺍﻝﺭﺤﻡ ﻤﻥ ﻨﻔﺱ ﺍﻝﻤﺭﻴﻀﺎﺕ ﺍﻝﻼﺘﻲ ﺃﺠﺭﻴﺕ ﻝﻬﻥ ﻋﻤﻠﻴﺎﺕ ﺍﺴﺌﺼﺎل ﺍﻝﺭﺤﻡ‬،‫ﻜﻤﺠﻤﻭﻋﺔ ﺃﻨﺴﺠﺔ ﺍﻝﺴﻴﻁﺭﺓ‬ ‫ ﺃﻨﺴﺠﺔ ﻋﻨﻕ ﺍﻝﺭﺤﻡ‬24 ‫ ﻭﻜﺫﻝﻙ‬،‫ ﺤﺎﻻﺕ( ﺴﺭﻁﺎﻥ ﻋﻨﻕ ﺍﻝﺭﺤﻡ ﺍﻝﻐﺎﺯﻴﺔ ﻤﻥ ﺒﻁﺎﻨﺔ ﺍﻝﺭﺤﻡ‬6)‫ ﺍﻝﺘﻬﺎﺏ ﻋﻨﻕ ﺍﻝﺭﺤﻡ ﺍﻝﻤﺯﻤﻥ ﻭ‬، (‫ ﺤﺎﻝﺔ‬40) : ‫ﺤﻴﺙ ﺸﻤﻠﺕ‬ ‫( ﻤﻥ ﺃﻭﺭﺍﻡ ﺍﻝﺭﺤﻡ ﻏﻴﺭ‬% 8) ‫ ﻭﺤﺎﻝﺘﻴﻥ‬،(٪16.7) ‫ ﺤﺎﻻﺕ‬5 ‫ ﻓﻲ ﻤﺠﻤﻭﻋﺔ ﺃﻭﺭﺍﻡ ﺍﻝﺭﺤﻡ ﺍﻝﺨﺒﻴﺜﺔ‬HMTV ‫ ﻭﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻝﻜﺸﻑ ﻋﻥ‬،‫ﻁﺒﻴﻌﻴﺔ‬ ‫( ﻤﻥ‬%13) ‫ ﻓﻲ ﺃﺭﺒﻌﺔ ﺤﺎﻻﺕ‬HMTV ‫ ﻓﻘﺩ ﺘﻡ ﺍﻝﻜﺸﻑ ﻋﻥ‬،‫ ﺃﻤﺎ ﺒﻴﻥ ﺁﻓﺎﺕ ﻋﻨﻕ ﺍﻝﺭﺤﻡ‬،‫( ﻤﻥ ﻤﺠﻤﻭﻋﺔ ﺃﻨﺴﺠﺔ ﺍﻝﺴﻴﻁﺭﺓ‬٪13) ‫ ﻭﺤﺎﻝﺘﻴﻥ‬،‫ﺍﻝﺨﺒﻴﺜﺔ‬ ‫ ﺃﻤﺎ ﻓﻲ ﺃﻨﺴﺠﺔ ﻋﻨﻕ ﺍﻝﺭﺤﻡ ﻝﻤﺠﺎﻤﻴﻊ‬، ‫( ﻓﻲ ﺃﺭﺒﻊ ﺤﺎﻻﺕ ﻓﻲ ﻋﻨﻕ ﺍﻝﺭﺤﻡ ﻝﻠﻤﺼﺎﺒﺎﺕ ﺒﺄﻭﺭﺍﻡ ﺍﻝﺭﺤﻡ ﻏﻴﺭ ﺍﻝﺨﺒﻴﺜﺔ‬٪16) ‫ﻤﺠﻤﻭﻋﺔ ﺃﻭﺭﺍﻡ ﺍﻝﺭﺤﻡ ﺍﻝﺨﺒﻴﺜﺔ ﻭ‬ ‫ ﻭﻋﻠﻰ ﺍﻝﺭﻏﻡ ﻤﻥ ﻨﺴﺏ ﻤﻌﺩﻻﺕ ﺍﻹﺼﺎﺒﺔ ﺒﺎﻝﻔﻴﺭﻭﺱ ﺍﻝﺒﺸﺭﻱ ﺍﻝﻠﺒﻨﻲ ﺍﻝﻀﺌﻴﻠﺔ‬،(٪6.7) ‫ ﻓﻲ ﺤﺎﻝﺔ ﻭﺍﺤﺩﺓ ﻓﻘﻁ‬HMTV ‫ ﻓﻘﺩ ﺘﻡ ﺍﻝﻜﺸﻑ ﻋﻥ‬،‫ﺍﻝﺴﻴﻁﺭﺓ‬ ‫ ﻭﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺘﺴﻠﻁ ﺍﻝﻀﻭﺀ ﻝﻤﺯﻴﺩ ﻤﻥ ﺍﻷﺒﺤﺎﺙ ﻝﻬﺫﺍ‬،‫ﻭﺍﻝﺘﻲ ﺘﻡ ﺍﻝﻜﺸﻑ ﻋﻨﻬﺎ ﺒﻴﻥ ﺁﻓﺎﺕ ﺍﻝﺭﺤﻡ ﻭﻋﻨﻕ ﺍﻝﺭﺤﻡ ﺍﻝﻤﺨﺘﻠﻔﺔ ﻓﻲ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﻌﺭﺍﻗﻴﺔ‬ .‫ﺍﻝﻔﻴﺭﻭﺱ ﺍﻝﺠﺩﻴﺩ ﻀﻤﻥ ﺤﺎﻻﺕ ﺃﺨﺭﻯ ﻤﻥ ﺍﻝﺴﺭﻁﺎﻨﺎﺕ ﺍﻝﻤﺘﻌﻠﻘﺔ ﺒﺎﻷﻨﺴﺠﺔ ﺍﻝﻬﺭﻤﻭﻨﻴﺔ‬

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INTRODUCTION During 1970s and 1980s, several reports had localized the presence of Human Mammary Tumor Virus in breast cancers and absent in normal breast tissues counter part (1). In addition , the presence of viral particles with the morphological characteristics of beta retroviruses was first described in human milk by Feller and Chopra in 1969 (2), then in 1971, Moore et. al detected a beta retrovirus similar to Mouse Mammary Tumor Virus (MMTV) in the milk from 5% of American women with no familial history of breast cancers, 60% with familial breast cancers and 39% from Parsi women (3) .In the early 1990’s, researchers started to investigate this virus by PCR using primers for the Mouse Mammary Tumor Virus(MMTV) envelope gene (4), or they selected a common sequences of 250bp 600bp of fragments for the MMTV envelope gene to design probes for hybridization and detection of HMTV (5,6). Studies had reported 38% of breast cancers that are simply containing envelope gene sequences, which were 95-99% homologous to MMTV but with low 56% homology to Human Endogenous Retro Virus (HERV) , and designated as Human Mammary Tumor Virus (HMTV) (3). In human, HMTV has been described in (14% to 74%) of breast cancers (7) and detected in 16% in ovarian cancers, while 36% was reported in prostate cancers ,10% in endometrial cancers , and 9% in skin cancers (8). In 2003 , HMTV was characterized in patients with primary biliary cirrhosis (9), human lymphomas, hepatocellular carcinomas and various liver diseases (10,11). A significant association was observed between the presence the hormone receptors and detection of HMTV .Analysis of the sequencing data showed that HMTV contains hormone response elements (HRE) homologous to MMTV HRE (12). The present study and up to our knowledge is the first in Iraq to investigate the rates of the HMTV infection in endometrial as well as cervical cancers that were obtained from hysterectomized patients using in situ hybridization technique.

MATERIALS AND METHODS 1. Patients tissue samples: This retrospective study has enrolled seventy (70) cases represented by 158 selected formalin fixed paraffin embedded uterine tissues blocks were belonging to patients who had undergo hysterectomy. They were collected from the archives of histopathology laboratories at Teaching Laboratories in Medical City, Al-Yarmouk Teaching Hospital and private laboratories. These samples were related to the period from 2012 to 2014. The study tissues group comprised thirty cases represented by 66 malignant uterine tumor,25 non-malignant uterine tumors represented by (62 samples), and 15 control tissues group represented by 30 samples. These 70 cases have also enrolled 46

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accompanying cervical lesions involving chronic cervicitis (40cases) and cervical cancers that were invasive from endometrium cancers sites (6 cases). 2. Methods: Thick-tissue sections (4 mm) were prepared and stuck onto positively charged slides. This Kit (Zytofast Chromogenic in-situ-hybridization (CISH) implementation Kit AP-NBT/BCIP) use to detect DNA sequences of provirus of HMTV (integrated DNA sequences of HMTV) using biotinylated probes (Zytovision/ Germany). We design and request a probe for detection of HMTV by CISH test complementary to a sequence of 250bp size from MMTV envelope gene .Two probes, each of them have a length of 125bp, labeled with biotin and apply in a colometric in-situ hybridization on paraffin embedded tissues. The procedure of the (CISH) assay adopted by this study was carried out in accordance with the manufacturer company leaflet (zytovision/Germany) in the Research Laboratories at Communicable Disease Research Unit/Medical Baghdad College . Positive control were performed by replacing the probe with a biotinylated housekeeping gene probe while in the negative control, all reagents were added except the probe. The enzymatic reaction of NBT/BCIP leads to the formation of strong blue violet signals that can be visualized by light microscopy at (10-20x) dry lens. CISH signals were evaluated in at least 10 high power fields. Nuclear staining was considered a positive result for HMTV provirus. Positive CISH signal patterns was Punctuated (P) when as a distinct dot-like intra nuclear signals that are referring for an integrated patterns of HMTV in the examined tissues (13).

RESULTS 1. Molecular Detection of the HMTV infections in endometrial lesions: The enveloped gene DNA sequence of the HMTV were detected in tissue blocks from hysterectomized patients as blue signals discoloration at the sites of complementary sequences. The mean age of the patients with HMTV- positive endometrial lesion among hysterectomized patients was (48.9 ± 9.1) years with a range from (35to 63) years. The CISH-signals of integrated HMTV- DNA in the endometrial tissues were found in five cases (16.7%) of malignant uterine tumors ,two cases (8%) of non-malignant uterine tumors, and two cases(13.3%) of control tissue group. No significant statistical differences were reveald among the hysterectomized patients regarding their HMTV results (p>0.05) (table 1, figure 1).

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Table (1): Distribution of CISH-signal results of the HMTV-DNA integration in the endometrial carcenous tissues Endometrial CISH Results

CISH for HMTV-DNA integration

Positive Negative *P compared to NT *P compared to Con

Malignant uterine-tumor No % 5 16.7 25 83.3 0.771 0.337

Non malignant uterine tumor No % 2 8.0 23 92.0 0.419 -

Control uterine tissues No % 2 13.3 13 86.7 -

Significant difference between proportions using Pearson Chi-square test at 0.05 level . *P:p-value ,NT: Non-malignant uterine tumor ,Con: Control

Figure (1): Microphotographs of the CISH-signals of HMTV-integrated DNA in:(A,B,C)Adenocarcinoma , (D,E) Endothelial glands with uterine tumors and (F)Control uterine tissues .Blue signals are detected at complementarity sequence sites(yellow arrows: puntcate form)

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2. Molecular Detection of the HMTV infections in cervical lesions: Regarding the distribution of the results of CISHsignals of HMTV-integrated DNA in different cervical tissues (table 2 and figure 2). Four cases (13.3%) in cervical lesions among malignant uterine tumors group ,four cases (16%) in cervical

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lesions among non-malignant uterine tumors group, and one case (6.7%) in control tissues group were revealing positive signaling of CISH of HMTVintegrated DNA in their tissues. However, no significant differences were found among the study groups.

Table (2): Distribution of the CISH-signal results of integrated HMTV - DNA in the cervical tissues in the study groups Cervical CISH Results

Malignant Non malignant uterine uterine-tumor tumor No % No % HMTV-DNA Positive 4 13.3 4 16.0 CISH Results Negative 26 86.7 21 84.0 *P compared to NT 0.502 0.430 *P compared to Con 0.780 Significant difference between proportions using Pearson Chi-square test at 0.05 level. P:p-value ,NT: Non-malignant uterine tumor ,Con: Control

Control uterine tissues No % 1 6.7 14 93.3 -

Figure (2): Microphotographs of the HMTV-integrated DNA showing CISH-signals in the (A,B,C) Chronic cystic cervicitis ,(D) Tissue control.Blue signals are detected at complementarity sequence sites(yellow arrows: punctate form)

DISCUSSION In the present study ,HMTV was detected in the endometrial tissues from malignant uterine tumors in (16.7%) of cases .In the non-malignant uterine tumors HMTV was found in (8%) while in the control tissues this virus was revealed (13.3%) of cases (Table 1). Also, in the cervical sites of the malignant uterine tumors HMTV was detected

In (13.3%) of cases, in (16%) of non-malignant uterine tumors, while in control tissues group was found in (6.7%) of these cases (Table 2). These results are in agreement with the findings of (14), who detected HMTV in (23.7%) of endometrial carcinoma cases among Australian women using PCR and also with results of (8), who detect HMTV in (10%) of EC cases using PCR technique.

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A significant association was observed between the presence of hormone receptors and detection of HMTV in hormonal influenced tissues indicating that HMTV is not a breast cancer-specific virus and the tropism towards the glandular cells in these tissues could explain and support our results of HMTV among the studied tissues from our hysterectomized patients in this study. Here in ,HMTV has been detected in hormonally influenced tumors tissues such as (38-40%) in breast cancers (36%) in prostate cancers and (16%) in ovarian cancers (15). The present findings could support the suggestion that when the virus transmitted exogenously, first infects antigen-presenting (APCs)cells of the immune system which then displays the super antigen (SAg) with their major histocompatibility complex class II (MHCII) . Receptor on CD4+ Tcells identifies the antigen being presented on the APC MHCII. This recognition initiates an increase in the number of T-cells that are complimentary or cognates to the SAg on this virus, SAg-cognate CD4+ T-cells (16). In addition , this virus has the ability to down-regulate the immune response and to facilitate infection through an interaction with Toll-like receptor (TLR4), which is associated with release of interleukin 10 (IL-10) by B-cells (17). Later on, the lymphocytes will carry the virus to glandular tissues (9) and when the proviral DNA is integrated randomly into host chromosome, it’s expression is regulated by specific sequences in long terminal region (LTR) that resides a hormone response element(HRE) and cause increased in viral transcription in response to glucocorticoid and progesterone (12). Once the LTR contacts certain steroidal hormones in the hormonal influenced tissues (e.g; endometrium or cervix) an increase in the virion production that resulting in a greater numbers of infected cells and more random viral integrations into the genome. Because retroviral integration into the host chromosome occurs at random locations, the more viruses that are produced, the more likely it is that integration near cellular proto-oncogenes will occur and once virus expressed , will lead to alteration of the signaling pathway, and then development of the tumor since the large majority of virus integrations result in activation of proto-oncogenes that are not normally expressed (6,18). Several studies indicate that several oncogenes [Wnt1 (wingless family), Fgf3 (fibroblast growth factor family)], and Notch 4 (Notch family) are upregulated by MMTV introduction into the host cells and collaboration of these genes is important for tumor development (12,16). Also others have reported that binding of transcriptional factor (OTF-1) to a specific region within the LTR regulates the activity of progesterone in the presence of the MMTV promoter, and thus also regulates the MMTV’s ability to bind DNA to progesterone receptors and induction of tumor (19). The present findings are in consistent with results obtained from other studies

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and could suggest a similar proposed situation where might lead to regulation of the development of tumors that are among HMTV -positive selected endometrial as well as cervical carcinoma groups. More studies are to be recommended to explain more aspects of HMTV infections in these cervical and/or endometrial carcinomous tissues along with their impact on other hormonally influenced tissues, which have been infected with HMTV and their relations to the carcinogenesis of different anatomical locations and/or histopathological presentations.

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11. Johal H.; Scott GM.; Jones R.; Camaris C.; Riordan S. and Rawlinson WD. (2009). Mouse mammary tumor virus-like virus (MMTV-LV) is present within the liver in a wide range of hepatic disorders and unrelated to nuclear p53 expression or hepatocarcinogenesis. J. Hepatol. 50:548–554. 12. Curtis C.; DeLuca M.; Mullen K.; Smietana A.; Tsang S. and Angerio A. (2007). Viral etiology in breast cancer?. J. Health Sci. (GUJHS). 4(2): 11051110. 13. Zappacosta R.; ColasanteA.; Viola P.; Antuono TD.; Lattanzio G.; Capanna S.; Gatta DMP. and Rosini S. (2013). Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications. BioMed. Res. Int. published online. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC385 8005/ 14. Liane D.; Tania M.; Anna TL.; Polly E.; James F H.; Stella M. and Beatriz GTP. (2013). Human Mammary Tumor Virus (HMTV) in endometrial carcinoma. Int. J. Gynecol. Cancer. 23(8):1423– 1428. 15. Mazzanti CM.; Al Hamad M.; Fanelli G.; Scatena C.; Zammarchi F.; Zavaglia K.; Lessi F.; Pistello M.; Naccarato AG. and Bevilacqua G.(2011). A Mouse Mammary Tumor Virus envlike exogenous sequence is strictly related to progression of human sporadic breast carcinoma. Am. J. Pathol. 179 (4): 2083-2090. 16. Swanson I.; Jude BA.; Zhang AR.; Pucker A.; Smith ZE. and Golovkina TV. (2006). Sequences within the gag gene of mouse mammary tumor virus needed for mammary gland cell transformation. J Virol. 80(7):3215-3224. 17. Jude BA.; Pobezinskaya Y.; Bishop J.; Parke S.; Medzhitov RM. and Chervonsky AV.(2003). Subversion of the innate immune system by retrovirus. Nat. Immunol.4(6):573-578. 18. Hook LM .; Agafonova Y.; Ross SR.; Turner SJ. and Golovkina TV. (2000). Genetics of Mouse Mammary Tumor Virus-Induced Mammary Tumors: linkage of tumor induction to the gag Gene. JOV. 74(19): 8876–8883. 19. Stewart THM. and Sage RD. (2000). Breast cancer incidence highest in the range of one species of house mouse, mus domesticus. Brit. J. Cancer. 82: 446-451.

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Partial

purification

and

characterization

of

Vol. 10, No.2, June 2015

factors

from

Bacillus

amyloliquefaciens strain that caused catenulate Vesicle like Sac formation of the germinating tube of Glomerella cingulata spores Abduljaleel H. Mohammed College of Applied Sciences/ University of Samarra / Republic of Iraq E –mail: [email protected]

ABSTRACT Products, termed vesicle like sac formation factors (VLS-F) were present in a culture of Bacillus amyloliquefaciens grown in either a defined or simple medium. The factors inhibited appressorium development and caused the formation of vesicle like sac formation of various sizes of the germinating tubes of Glomerella cingulata spores. The factors were separated on size; a fraction less than 500 Da and another fraction between 3 and 5 KDa. Preliminary characterization showed the factors resistance to boiling. The fraction less than 500 Da was sensitive to Trypsin and Proteinase-K, whereas the fraction between 3 and 5 KDa demonstrated a resistance, to Trypsin and proteinase-K treatment.

Keywords: vesicle like sac formation factors (VLS-F), appressorium development, KDa, RPC; Tandem electrospray MS

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ ،‫ﻝﻭﺤﻅ ﻭﺠﻭﺩ ﻋﻭﺍﻤل ﺘﻜﻭﻴﻥ ﺍﻷﻜﻴﺎﺱ ﺍﻝﻤﺸﺎﺒﻬﺔ ﻝﻠﺤﻭﻴﺼﻼﺕ ﻓﻲ ﺍﻝﻭﺴﻁ ﺍﻝﺯﺭﻋﻲ ﻝﻌﺼﻲ ﺍﻤﻴﻠﻭﻝﻜﻴﻔﺸﻨﺱ ﺍﻝﺘﻲ ﺘﻨﻤﻭ ﻋﻠﻰ ﺃﻭﺴﺎﻁ ﻤﻌﺭﻭﻓﺔ ﺃﻭ ﺒـﺴﻴﻁﺔ‬ ‫ ﻭﻗﺩ ﺘﻡ ﻓﻲ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻓﺼل ﺍﻝﻌﻭﺍﻤل ﻋﻠﻰ ﺃﺴﺎﺱ ﺍﻝﺤﺠـﻡ‬، ‫ﻫﺫﻩ ﺍﻝﻌﻭﺍﻤل ﺘﺤﻭل ﺩﻭﻥ ﺘﻁﻭﻴﺭ ﺍﻷﺒﺭﺴﻭﺭﻴﻡ ﻭﺘﺘﺴﺒﺏ ﻓﻲ ﺘﺸﻜﻴل ﺤﻭﻴﺼﻠﺔ ﺘﺸﺒﻪ ﺍﻝﻜﻴﺱ‬ ‫ﻭﻝﻘﺩ ﻜﺎﻥ ﺍﻝﺠﺯﺀ ﺍﻷﻗـل‬،‫ ﺃﻅﻬﺭ ﺍﻝﺘﻭﺼﻑ ﺍﻷﻭﻝﻲ ﻤﻘﺎﻭﻤﺔ ﺍﻝﻌﻭﺍﻤل ﻝﻠﻐﻠﻴﺎﻥ‬.‫ ﻜﻴﻠﻭ ﺩﺍﻝﺘﻭﻥ‬5‫ ﻭ‬3 ‫ ﺩﺍﻝﺘﻭﻥ ﻭﺠﺯﺀ ﺁﺨﺭ ﺒﻴﻥ‬500 ‫ﻭﺍﻝﺫﻱ ﻗﺩﺭ ﺒﺠﺯﺀ ﺃﻗل ﻤﻥ‬ ‫ ﻜﻴﻠﻭ‬5–3 ‫ ﺒﻴﻨﻤﺎ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻤﻘﺎﻭﻤﺔ ﻝﻠﺘﺭﺒﺴﻴﻥ ﻭﺒﺭﻭﺘﻴﻨﻴﺯ – ﻜﻲ ﻭﺍﻝﺘﻲ ﺘﺭﺍﻭﺤﺕ ﺒﻴﻥ‬،‫ ﺩﺍﻝﺘﻭﻥ ﺤﺴﺎﺴﺎ ﻝﻠﺘﺭﺒﺴﻴﻥ ﻭﺍﻨﺯﻴﻡ ﺒﺭﻭﺘﻴﻨﻴﺯ – ﻜﻲ‬500 ‫ﻤﻥ‬ .‫ﺩﺍﻝﺘﻭﻥ‬

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

INTRODUCTION

Glomerella cingulata (Stoneman) is the sexual stage of the phytopathogen Colletotrichum gloeosporioides. Both this fungus, and the rice blast fungus, Magnaporthe grisea, enter their host by means of an infection structure, the appressorium. Penetration of the host tissue is achieved by means of turgor pressure generated within the appressorium and/or the secretion of wall degrading enzymes. Appressorium development is triggered when conidia are exposed to plant wax-coated hard surfaces. The conidium germinates and the emerging hypha stops elongating and differentiates to form a melanised appressorium from which the penetration plug (hypha) is forced into the plant tissue (1). The ascomyceteous yeasts and filamentous fungi produce both peptide and lipopeptide mating pheromones (2,3) to mediate conjugation of cells of opposite mating type. Heterothallic haploid Saccharomyces cerevisiae cell type-α or type-a. They secrete the corresponding mating pheromone (the K-mating factor peptide or the a-mating factor lipopeptide) and produce a plasma membrane-located receptor for the mating pheromone of the opposite type (2,4,5). Binding of the mating pheromone to its receptor causes a morphological change in the responding cell and transiently arrests growth. It has been shown that the S. cerevisiae K-mating factor can block the development of M. grisea appressorium (6-8) and that this fungus produces a peptide that inhibits appressorium development. However, the peptide sequence is not known (8). This study describes partial purification of factors from culture supernatant of Bacillus amyloliquefaciens that inhibited appressorium formation in G. cingulata conidia. Also, this fungus, enteritis their host by means of the appressorium (4,5). The role of factors in controlling the infection needs to be investigated.

MATERIALS AND METHODS 1. Fungal strain: Glomerella cingulata strain ICMP 11016, was maintained on potato dextrose agar. Conidia from G. cingulata need to be placed on ice bath until use. Bacterial strains Bacillus amyloliquefaciens, Bacillus subtilis subsp. Subtilis (Ehrenberg) Cohn ATCC 605l, Bacillus subtilis subsp. spizizenii ATCC 6633, and Bacillus subtilis ESR 151, were maintained on Nutrient agar (Difco). 2. Strain identification: B. amyloliquefaciens was identified according to the characteristics of morphology, physiology and biochemistry tests as described by Claus and Berkeley (9) and Gordon et al. (7, 10) and by the comparison of the16S rDNA sequence.

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3. Preparation of conidia: Following inoculation of potato dextrose agar overlaid with a sterile cellophane membrane production of Glomerella cingulata conidia were induced as described previously (11). Conidia were harvested, by repeated pipetting of sterile MQ water over the surface of the mycelium, and washed three times using sterile MQ water. The conidial concentration was determined using a haemocytometer. 4. Isolation of apple wax: Surface wax from 200 Granny Smith apples was extracted by submerging the fruit for 30s in 2 litres of chloroform containing 0.5% (v/v) methanol. The wax solution was concentrated by rotary evaporator to approximately 0.13 g ml −1, and the preparation stored at -20°C. 5. Bioassay for appressorium formation: Apple wax was smeared onto a glass microscope slide as described previously (11). Suspensions of fresh, washed conidia were mixed with samples to be tested for activity. Droplets (10 µl containing approximately 100 conidia) were applied to a waxcoated microscope slide and the slides were incubated in a humid atmosphere at 24°C for 18-38 hrs. 6. Preparation of factors: Stock cultures of B. Amyloliquefaciens (0.5 ml) were inoculated onto each of 10 plates of Nutrient agar. The plates were left open in the Laminar flow hood for 1-2 hrs to dry, and incubated at 26°C for 36 hrs. For inoculation of the culture medium, 0.5% glucose medium (2 ml) was added into each plate was collected by scrubbing with a sterile cotton swab and transferred to a Scott bottle containing either 1 litre of 0.5% glucose medium and or onto nitrogen base medium (Difco, Detroit, MI, USA) supplemented with 0.5% glucose in 1 l Scott bottle. The cultures were incubated at 26°C with shaking at 100 rpm. Cultures were sampled (1 ml) at different time intervals, centrifuged and the supernatant checked for the presence of Factors. After 2-3 days of incubation, supernatants were collected by centrifugation at 26000×g for 15 min and the resulting supernatant was boiled for 20 min and centrifuged at 40,000×g for 60 min to remove any precipitates. The final supernatants were aspirated and concentrated onto 20 ml under vacuum at 70°C. 7. Fractionation of the culture supernatant fluid (CSF): Fractionation of the concentrated supernatant was carried out by dialysis in 100-500 Da cut off dialysis membrane (Spectrum Laboratories Inc., CA, USA) against 600 ml of 20% ethanol at room temperature. The dialysate termed F-1 was concentrated to 10 ml under vacuum and kept on ice until use. The nondialysate was fractionated further by dialysis in 5 kDa cut-off membranes against 600 ml of 20%

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

ethanol at room temperature. The dialysates from 5 kDa cut-off membranes were dried under vacuum. Then the solid residues were dissolved in 20 ml of methanol. The methanol soluble materials were dried under vacuum and dissolved in 10 ml MQ water. Fractionation of this portion was carried out by dialysis in 3 kDa M W cut-off membranes against 600 ml of 20% ethanol for three days at ambient temperature. The non-dialysates termed F2. Both F-1 and F-2 were kept on ice bath until use. 8. Extraction of CSF, F-1 and F-2 with organic solvents: Samples from CSF, F-1 and F-2 were appropriately diluted with sterile MQ water to contain 1-2 units of factors (1-unit causes the development of vesicles like sac formation of approximately 98% of germinating spores). Each portion was divided into four parts (10 ml). The pH of the first part (untreated) was approximately 3.8, the second part was adjusted to pH 2 with hydrochloric acid, the third part to pH 6 and the fourth part to pH 10 with concentrated ammonium hydroxide. Each sample was aliquotted into four fractions one of which was kept as control and the remaining fractions were extracted with 10 ml of ethyl acetate, diethyl ether, or dichloromethane. The phases were separated by centrifugation. The solvent phases and the aqueous phases were separated and evaporated to dryness under vacuum. Each residue was re-dissolved in 10 ml of MQ water and bioassayed along with the unfractionated sample as control. 9. Stability studies: The temperature stability of the F-1 and F-2 were examined by exposure of 1 ml of F-1 and F-2 to 80°C, 100°C and 120°C for 10 min followed by cooling to room temperature and bioassay. F-1 and F-2 (200 µl) were a treated (2 h, and 18 h at 37°C) with two proteases, Trypsin and Proteinase K (1 mg ml −1 in 20 mmol l −1 phosphate at a pH of 6). The enzymes were inactivated by exposure of the mixture to 100°C for 10 min followed by cooling and bioassay. Samples of heat inactivated enzymes were used as a negative control. 10. Purification of F-1 and F-2: A) Fractionation of F-1 and F-2 by ion exchange chromatography: The pH of F-1 and F-2 were adjusted to pH 5 and 8 using KOH respectively and fractionated on HiTrap SP FF or HiTrap DEAE FF respectively (GE Health Auckland, New Zealand) and were eluted with a linear gradient of 1M NaCl at appropriate pH. B) Fractionation of F-1 by reversed-phase liquid chromatography (RPLC): Samples of F-1 in 0.1% trifluoroacetic acid (TFA) were subjected to RPLC by using a 1-ml HiTrap RPC column on AKTA Explorer high-performance liquid chromatography system (Amersham Pharmacia, Auckland, New Zealand). Separation was carried out according to the manufacturer’s operating specifications using solvent A (0.1% TFA) and B {(100% acetonitrile

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(ACN) in 0.01% TFA)} using a stepwise gradient from 0-95 % (v/v) ACN containing 0.1% (v/v) TFA. Fractions were dried under vacuum and dissolved in MQ water. AF-1 activity appeared in the flow through therefore a mixed-mode anion-cation exchange and hydrophilic interaction chromatography (ACE-HILIC) methods were employed as described previously (12). Determination of the biological activity was carried out as described above. C) Fractionation of F-2 by RPLC: Fractionation of the F-2 using a 1ml HiTrap RPC column and detection of the biological activity-was carried out as described above. The active fractions were pooled, and evaporated under vacuum. The solid material was dissolved in 1 ml 30% ACN containing 0.1% TFA and diluted with 2 ml of 0.1% TFA, and reapplied onto the column. Samples (1 ml) were injected onto the column at a flow rate of 0.1 ml/min and washed with the same solvent at 0.2 ml/min for a further 10 min. The solvent composition of the mobile phase was change as follows: The program consisted of 450 min with a constant mixing ratio (B=5%) from t=0 to 60 min, a linear gradient (B=5% to 40%) from t=60 to 140 min, a linear gradient (B=40 to 42 %) from t=60 to 200 min, a linear gradient (B=42to 44%) from t=250 to 300 min, a linear gradient (B=44 to 46%) from t=300 to 350 min a linear gradient (B=46 to 48%) from t=350 to 410 min, a linear gradient (B=48 to 55%) from t=410 to 450 min, a constant ratio (B=95%) from t=450 to 480 min and a downward gradient (B=95% to 0%) from t=480 to 490 min. Portions of the respective HPLC fractions were saved for Tandem electrospray MSMS measurements.

RESULTS AND DISCUSSION B. amyloliquefaciens strain was isolated from the root of a leguminous plant. Identification was carried out according to the characteristics of morphology, physiology, biochemistry tests and the comparison of a 16S rDNA sequence. B. amyloliquefaciens Strain showed: 100% sequence identities to B. amyloliquefaciens strain FZB 42, 99% sequence identity to B. subtilis subsp. subtilis str. SMY SMY_ctg6 and 93% sequence identity to B.subtilis subsp. subtilis str. SMY SMY_ctg6. 1. Kinetics of appearance of factors: The CSF of B. amyloliquefaciens strain from both a 0.5% glucose medium and nitrogen base medium was collected at various times and its absorbency measured (Figure 1). The activity appeared after 18 h of incubation. Maximal levels were reached after 7 days of incubation at 26 °C. The results indicated that the factors were produced and released early in the growth cycle. CSF from three strains of B. subtilis, and uninoculated medium showed no F-1 or F-2 activity.

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Figure (1): Time course of the appearance of factors formation by Bacillusamyloliquefaciens Strain grown in nitrogen base medium supplemented with 0.5% glucose. Incubation was carried out for 7 days at 26 ºC. () OD600, ( ) AF.

2. Preliminary characterization of factors: G. cingulata conidia germinated on apple waxcoated glass slides produced appressoria (Figures 2a and 3a). Addition of boiled undiluted CSF (1.6 mg/ml protein) of B. amyloliquefaciens strain to the conidia inhibited the germination, while dilution of ¼ of the CSF inhibited appressorium formation and resulted in formation of thick septated germinating tubes after 48 hrs incubation at 24°C (Figure 2b). Dilution of 1/8 – 1/16 CSF caused the formation of sac like catenulate vesicles of germinated tubes of conidia (Figure 2c). Ethyl acetate, diethyl ether, and dichloromethane extracts of the CSF, at three different pH’s, showed no activity. The aqueous phase from these extractions contained F-1 and F-2 activities comparable to the original culture supernatant.

Figure (2): Inhibition of appressorium development and formation of vesicles like sac of Glomerella cingulata germinated spores. Conidia were incubated without added CSF (A), or with diluted CSF (1/4) (B), diluted CSF (1/16) (C) on wax-coated glass slides for 18 hrs at 24 ºC. Bars represent 10 µm

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3. Partial purification of factors from CSF: Fractionation of the CSF was explained in the material and method. F-1 (63 µg/ml protein) at a dilution of 1/64-1/128 caused the development of vesicles like sac at the ends of germinated tube after overnight incubation at 24°C (Figure 3b). F-2 (4.8 mg/ml protein) at a dilution of 1/32 also caused the development of vesicles like sac at the ends of germinated tubes after overnight incubation at 24°C (Figure 3c). Dilution of 1/64 caused the development of catenulate vesicles like sac after 48 hrs incubation at 24°C. The first attempt to purify F-1 was carried out by employing SP and DEAE columns. The failure of the adsorption of the F-1 on cation and anion exchange resins is presumptive evidence that the F-1 molecules carry no positive and negative charges at appropriate pH. The second attempt was to bind F-1 onto HiTrap RPC column (see materials and methods). The F-1 did not bind onto the column and appeared at the flow through (similar as in Figure 3b). The failure of the adsorption of the F-1 onto the HiTrap RPC column is presumptive evidence that the F-1 molecules are small and very hydrophilic molecule. Since the separation selectivity provided by RP-HPLC has been limited to the hydrophobicity based resolution of relatively nonpolar sample components (polar compounds present a significant challenge for RPHPLC), a hydrophilic interaction chromatography (HILIC) was adapted. The F-1 did not bind onto the column and appeared in the flow through. Tandem electrospray MS MSMS showed a comprehensive list of hits from which the METLIN database was searched to come up with hits based on accurate mass data. The METLIN database search showed for any ion m/z mass, a number of hits, for example the ions at 198.0972 and 203.0526 are ammoniated and sodiated monosaccharide with the possible identities of one or mix of sugars. Also, the analysis showed the presence of phenolic compound derivatives. Thirteen short peptides (2-3 amino acid length) were present, but are likely to be anomalies. Synthesis of these peptides was carried out but none of the synthetic peptides had the activity or F-1 properties. No charge envelope representing larger peptides and proteins were detected at all. On other hand, F-2 was bound into RP-HPLC column and eluted at approximately 44%-50% ACN, (similar result as in Figure 3c) (see materials and methods). The activity was detected in 8 fractions and each fraction with variable strength of activity. Since the profile of the elution from reverse-phase chromatography was complex, the fractions were pooled and further fractionation was carried out by gradual changing the solvent composition of the mobile phase (see materials and methods). The F-2 activity was eluted at approximately 41-43% ACN. These fractions inhibited the germination of the spores (190 µg/ml protein). A dilution of 1/32-1/64 caused development of terminal vesicles like sac after 18 hr of incubation. Digestion with Trypsin or Proteinase K destroyed the inhibitory effect on germination of spores but not the formation of

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vesicles like sac activity. Based on that, the most pure preparations of F-2 have a complex elution profile from reverse-phase chromatography (Figure 4). Tandem electrospray MS revealed no results. Chymotrypsin digestion of the most active fraction followed by Tandem MSMS yielded several

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peptides but none of the synthetic peptides had the activity of vesicles like sac formation properties.

Figure (3): Vesicles like sac formation by Glomerella cingulata spores. Conidia were incubated; without F-1 or F-2 (A), or partially purified F-1 (B), partially purified F-2 (C) on wax-coated glass slides for 18 hrs at 24 ºC. Bars represent 10 µm

concentration 95%) also did not affect the activity. The proteases tested were effective in destroying the F-1 activity, suggesting that the F-1 activity could be due to one or more peptides containing the appropriate cleavage sit residues. On other hand proteases tested on F-2 destroyed the inhibitory effect on germinating of conidia but not the formation of vesicles like sac activity, suggesting that the F-2 activity was not due to a peptide.

Figure (4): HPLC reversed-phase chromatography of F-2. Samples of F-2were subjected to Reverse phase chromatography. A 1-ml HiTrap RPC column was eluted with a gradient of acetonitrile (see materials and methods). Milliabsorbance at 280 nm is indicated at left; F-2 activity is indicated by a line underneath the line of absorbency of eluants fractions

4. Stability studies on F-1 and F-2: The sensitivity of F-1 and F-2 to extremes of pH, proteases and elevated temperature was determined. Treatment at pH 2 or 10 for 2 hrs and 18 h failed to inactivate the F-1 and F-2 (Table 1). A temperature of 80 and 100°C did not inactivate the activity of the both F-1 and F-2. Conditions routinely employed during autoclaving (121°C, 10 min) did not destroy the activity. Ethanol and methanol (final

5. Estimation of the molecular weight of F-1 and F-2: To obtain an estimate of the molecular weight (MW) of the F-1 and F-2, a 10 ml sample of concentrated CSF was dialyzed in 100- 500 M W cut-off membrane (Spectra/Por; Spectrum Medical Industries, Houston, TX, USA) against sterile MQ water (200 ml of 20% ethanol) for overnight at 4°C. F-1 was observed in dialysates suggesting that the MW is below 100-500 Da. To obtain an estimate of the molecular weight of the F-2, a 10 ml retentate from 100-500 Da MW cut-off membrane, was dialysed in 5 KDa MW cut- off membrane against sterile MQ water (200 ml of 20% ethanol) for three days at ambient temperature. The F-2 stimulating activity was observed in dialysates. The dialysates were concentrated into 10 ml under vacuum and dialysed in 3 Kda MWCO membranes against sterile MQ water (200 ml of 20% ethanol) for three days at ambient temperature. F-2 stimulating activity was observed in retentate suggesting that the MW is below 5 kDa and above 3 kDa.

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Table (1): Stability of vesicles like sac formation factors to various treatments Treatment

F-1 %

F-2 %

Sterile medium

0.0

0.0

pH 2·0

91 ± 6·0

96±3.0

pH 10·0

92·0 ± 5·0

97±1.0

Trypsin

4 ± 0.5

88±6.0

Proteinase-K

0.0

91±4.0

121 ºC

93±4

94±5.0

ACKNOWLEDGEMENT This work was supported by the College of Education, The University of Samarra, Samarra, Iraq.

REFERENCES 1. Deising HB.; Werner S. and Wernitz M. (2000). The role of fungal appressoria in plant infection. Microb. Infect. 2: 1631-1641. 2. Duntze W.; Betz R. and Nientiedt M. (1994). Pheromones in yeasts. In: The Mycota: Growth, Differentiation and Sexuality (Wessels, J.G.H. and Meinhardt, F., Eds.). pp: 381-399. Springer, Berlin. 3. Caldwell GA.; Naider F. and Becker JM. (1995). Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation. Microbiol. Rev. 59:406-422. 4. Elion EA. (2000). Pheromone response, mating and cell biology. Curr. Opin. Microbiol. 3:573-581. 5. Zhang L.; Baasiri RA. and van Alfen NK. (1998). Viral repression of fungal pheromone precursor gene expression. Mol. Cell. Biol. 18: 953-959. 6. Shen WC.; Bobrowicz P. and Ebbole DJ. (1999). Isolation of pheromone precursor genes of Magnaporthe grisea. Fungal Genet. Biol. 27: 253263. 7. Poggeler S. (2000). Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora. Curr. Genet. 37: 403-411. 8. Beckerman JL.; Naider F. and Ebbole DJ. (1997). Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae K-factor. Science. 276:1116-1119. 9. Claus D. and Berkeley RCW. (1986). Genus Bacillus in Bergeys manual of systematic Bacteriology. The Williams and Wilkins, Baltimore. 10. Gordon RE.; Haynes WC. and Pang CH. (1973). The genus Bacillus. Agriculture Research Service, U.S. Department of Agriculture. 11. Al-Samarrai TH.; Sullivan PA.; Templeton MD. and Farley PC. (2002). Peptide inhibitors of appressorium development in Glomerella cingulata. FEMS Microbiol. Lett. 209: 203-207.

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12. Strege MA; Stevenson S. and Lawrence SM. (2000). Mixed-mode anion-cation exchange/hydrophilic interaction liquid chromatography-electrospray mass spectrometry as an alternative to reversed phase for small molecule drug discovery. Anal. Chem. 72: 4629-4633.

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A study on clinical signs and immunological parameters associated with experimental thyrotoxicosis in rats Khalil H. Al – Jeboori and Sattar R. Suhail Dept. of Pathology / College of Vaterinary Medicine / University of Baghdad/ Republic of Iraq E –mail: [email protected] ABSTRACT Thyrotoxicosis is an important disease in man and animals , for the importance of this disease , this study aimed to identify clinical signs and immunological parameters associated with experimental thyrotoxicosis in rats induced by thyroxin (T4) injection . Three groups of rats of both sexes ( 1-1.5 month age ) were S/C injected daily with 2.5 , 7.5 and 10 g/100 gm. b.wt. of rat, for 60 days the 4th group was S/C injected with phosphate buffer saline as a control group . At 40th , 50th days of T4 injection , both group of rats immunized S/C with Candida albicans Ag . The main clinical signs were dyspnea , polyuria , polyphagia , polydipsia , excessive sweating , weight loss and nervousness and heat production . Regarding the immunological findings , there is increase in the level of humoral immune response detected by passive hemagglutination test ( PHA) in different groups compared to control. Also there is significant increase in the level of cell- mediated immunity estimated by delayed type hypersensitivity ( DTH – skin test ) and increase in migration inhibition index using macrophage migration inhibition factor test ( MIF).

Keywords: DTH – skin test, thyrotoxicosis, rats

VWXYZ[‫ ا‬V]^[_X `a^b[‫ا‬ ‫ ﻓﻘﺩ ﺘﻡ ﺇﺠﺭﺍﺀ ﻫـﺫﻩ ﺍﻝﺩﺭﺍﺴـﺔ‬،‫ ﻭﻝﺫﻝﻙ‬،‫ ﻭﻫﻭ ﻤﻥ ﺍﻷﻤﺭﺍﺽ ﺍﻝﺘﻲ ﺘﺴﺘﺤﻕ ﺍﻝﺩﺭﺍﺴﺔ ﻭﺍﻝﺘﻌﻤﻕ‬،‫ﻤﺭﺽ ﺍﻻﻨﺴﻤﺎﻡ ﺍﻝﺩﺭﻗﻲ ﻤﺭﺽ ﻴﺼﻴﺏ ﺍﻹﻨﺴﺎﻥ ﻭﺍﻝﺤﻴﻭﺍﻥ‬ ‫ﺒﻬﺩﻑ ﺍﻝﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻝﻌﻼﻤﺎﺕ ﺍﻝﺴﺭﻴﺭﻴﺔ ﻭﺍﻝﻔﺤﻭﺼﺎﺕ ﺍﻝﻤﻨﺎﻋﻴﺔ ﺍﻝﻤﺭﺘﺒﻁﺔ ﺒﺎﻻﻨﺴﻤﺎﻡ ﺍﻝﺩﺭﻗﻲ ﺍﻝﺘﺠﺭﻴﺒﻲ ﺍﻝﻤﺴﺘﺤﺩﺙ ﻓﻲ ﺍﻝﻔﺌﺭﺍﻥ ﺒﻭﺍﺴﻁﺔ ﺤﻘﺕ ﻫﺭﻤـﻭﻥ‬ .(T4 ) ‫ﺍﻝﺜﻴﺭﻭﻜﺴﻴﻥ‬ ‫ ﻤﻴﻜﺭﻭﻏﺭﺍﻡ‬10 ،7.5 ،2.5) ‫ ﺸﻬﺭ( ﺘﺤﺕ ﺍﻝﺠﻠﺩ ﻴﻭﻤﻴﺎ ﺒﺜﻼﺙ ﺠﺭﻋﺎﺕ ﻤﺨﺘﻠﻔﺔ‬1.5 -1) ‫ﺘﻡ ﺤﻘﻥ ﺜﻼﺙ ﻤﺠﻤﻭﻋﺎﺕ ﻤﻥ ﺍﻝﻔﺌﺭﺍﻥ ﻤﻥ ﻜﻼ ﺍﻝﺠﻨﺴﻴﻥ ﺒﻌﻤﺭ‬ ‫ ﻭﻓﻲ ﺍﻝﻴﻭﻡ ﺍﻷﺭﺒﻌـﻴﻥ‬،‫( ﻜﻤﺠﻤﻭﻋﺔ ﺴﻴﻁﺭﺓ‬pbs) ‫ ﻓﻘﺩ ﺘﻡ ﺤﻘﻨﻬﺎ ﺘﺤﺕ ﺍﻝﺠﻠﺩ ﺒﻤﺎﺩﺓ‬،‫ ﺃﻤﺎ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﺭﺍﺒﻌﺔ‬،‫ ﻝﻤﺩﺓ ﺴﺘﻴﻥ ﻴﻭﻤﺎ‬،(‫ ﻤﻥ ﻭﺯﻥ ﺍﻝﺠﺴﻡ‬100 / .Candida albicans ‫ ﺘﻡ ﺘﻤﻨﻴﻊ ﺍﻝﻤﺠﻤﻭﻋﺎﺕ ﺒﻤﻀﺎﺩﺍﺕ‬T4 ‫ﻭﺍﻝﻴﻭﻡ ﺍﻝﺨﻤﺴﻴﻥ ﻤﻥ ﺤﻘﻥ‬ ،‫ ﻭﺍﻝﺘﻌـﺭﻕ ﺍﻝﻤﻔـﺭﻁ‬،‫ ﺍﻝﻌﻁﺵ‬،‫ ﺍﻝﻨﻬﺎﻡ‬،‫ ﻜﺜﺭﺓ ﺍﻝﺘﺒﻭل‬،‫ﺃﻅﻬﺭﺕ ﻨﺘﺎﺌﺞ ﺍﻝﺩﺭﺍﺴﺔ ﺃﻥ ﺃﻫﻡ ﺍﻝﻌﻼﻤﺎﺕ ﺍﻝﺴﺭﻴﺭﻴﺔ ﻝﻤﺭﺽ ﺍﻻﻨﺴﻤﺎﻡ ﺍﻝﺩﺭﻗﻲ ﻜﺎﻨﺕ ﻀﻴﻕ ﺍﻝﺘﻨﻔﺱ‬ ‫ ﺃﻤﺎ ﺍﻝﻨﺘﺎﺌﺞ ﺍﻝﻤﻨﺎﻋﻴﺔ ﻓﻘﺩ ﺃﻅﻬﺭﺕ ﺯﻴﺎﺩﺓ ﻓﻲ ﻤﺴﺘﻭﻯ ﺍﻻﺴﺘﺠﺎﺒﺔ ﺍﻝﻤﻨﺎﻋﻴﺔ ﺍﻝﺨﻠﻁﻴﺔ ﻭﺍﻝﺘﻲ ﺘﻡ ﺘﺤﺩﻴﺩﻫﺎ‬،‫ ﻭﺍﺭﺘﻔﺎﻉ ﺩﺭﺠﺔ ﺍﻝﺤﺭﺍﺭﺓ‬،‫ ﻭﺍﻝﻌﺼﺒﻴﺔ‬،‫ﻭﻓﻘﺩﺍﻥ ﺍﻝﻭﺯﻥ‬ ‫ ﻜﻤﺎ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺯﻴﺎﺩﺓ ﻤﻨﻭﻴﺔ ﻓﻲ ﺍﻻﺴﺘﺠﺎﺒﺔ ﺍﻝﻤﻨﺎﻋﻴﺔ ﺍﻝﺨﻠﻭﻴـﺔ‬،‫ ﺒﺎﻝﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺤﻴﻭﺍﻨﺎﺕ ﺍﻝﺴﻴﻁﺭﺓ‬passive hemaaglutination ‫ﺒﺈﺠﺭﺍﺀ ﺍﺨﺘﺒﺎﺭ‬ ‫( ﻭﺍﻝﺯﻴﺎﺩﺓ ﻓﻲ ﻤﺅﺸﺭ ﺘﺜﺒﻴﻁ ﻫﺠﺭﺓ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺘﺜﺒﻴﻁ ﻫﺠـﺭﺓ ﺍﻝﺨﻼﻴـﺎ‬DTH- Skin test) ‫ﻭﺍﻝﺘﻲ ﺘﻡ ﻗﻴﺎﺴﻬﺎ ﺒﻭﺍﺴﻁﺔ ﺍﺨﺘﺒﺎﺭ ﻓﺤﺹ ﺍﻝﺤﺴﺎﺴﻴﺔ ﺍﻝﻤﺘﺄﺨﺭﺓ‬ .(MIF) ‫ﺍﻝﺒﻠﻌﻤﻴﺔ‬

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INTRODUCTION Thyroid hormones ( T3, T4) secreted from thyroid gland , regulate growth , development and cellular metabolism (1). Thyroid hormones play a role in cardiovascular , nervous , immune and reproductive system (2). Thyroid hormones modulate the immune response , therefore , they play role in cellular and humoral immunity (3). Thyroid disorders is a general term representing several different diseases due to the disturbance in the thyroid hormones levels either increased or decreased resulted in to hyperthyroidism or hypothyroidism respectively (4). Through the importance of thyroid hormones in regulation growth , development and cellular metabolism and immunity . This study aimed to: 1- Study the clinical signs in rats following experimental thyroxin (T4) injection ( thyrotoxicosis ). 2- Study both the cellular and humoral immune response following experimental thyroxin (T4) injection ( thyrotoxicosis ).

MATERIALS AND METHODS Three groups of rats (1 – 1.5 months) , 100 gm body weight were taken. First group (10) 5 male and 5 female received 2.5 g/ 100 gm.b.wt thyroxin (T4) S/C for 60 days 2nd. Group (10) 5 female received g /100 b.wt. thyroxin (T4) S/C for 60 days 7.5 .Third group (10) 5 male and 5 female received 10 g/ 100 gm.b.wt thyroxin (T4) for 60 days. The fourth group (10) 5 male and 5 female received S/C phosphate buffer saline ( PBS) for 60 days . At 40th day of experiment, all the animals were immunized by inoculation 0.25 ml S/C of whole killed antigen of Candida albicans (9 x 108 CFU/ml) the booster dose was inoculated at 50th day of experiment with the dose of 0.5 ml of Candida albicansAg . At the 60th. day of experiment blood samples were taken for humoral immunity study using (5) method . For cellular immunity study macrophage migration inhibition factor (MIF) test were used according to (6) method and for Delayed type hypersensitivity (DTH – skin reaction ) test were used a method according to (7).

RESULTS 1. Clinical signs: The main clinical signs observed on male and female rats were nervousness, restlessness, polydipsia , polyuria , increase a petite , weight loss, dyspnea, rapid increase in heart beats, hyperactivity, excessive sweating , heat production, moist skin and death of some animals due to canabalism. These clinical signs were more prominent in group received high dose of thyroxin (T4), while the control group did not show any abnormal clinical signs.

2. Immunological study: Macrophage migration inhibition factor (MIF) Test : There are significant differences increase ( P < 0.05) in the index of migration inhibition areas between the different groups of animals , Also significant differences between the animals within the same group ( Table 1) , depending on the concentration of Ag used , so increase the index of migration inhibition with gradual increase of Ag concentration and vice versa. In the different groups of animals and within the same group of animal . For PHA there is no significant difference between groups of animals and animals within same group. Control (PBS) group , there is no migration inhibition area in all animals. Delayed type hypersensitivity (DTH- skin reaction ) Test: The results revealed that there is Significant differences increase (P‫ﺍﻝﻨﺒﺎﺘﻲ ﻝﻠﺘﻌﺭﻑ ﻋﻠﻰ ﺍﻝﻤﺠﺎﻤﻴﻊ ﺍﻝﻔﻌﺎﻝﺔ ﺍﻝﻤﻭﺠﻭﺩﺓ ﻓﻲ ﻜل ﻤﻨﻬﺎ‬ ‫ ﻓﻴﻤﺎ ﺍﺤﺘﻭﻯ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﻝﻬﺎ ﻋﻠﻰ ﺍﻝﻔﻼﻓﻭﻨﺎﺕ‬،‫ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺍﺤﺘﻭﺍﺀ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﻝﻸﻭﺭﺍﻕ ﻭﺍﻝﺴﻴﻘﺎﻥ ﻋﻠﻰ ﺍﻝﺼﺎﺒﻭﻨﻴﺎﺕ ﻭﺍﻝﺘﺎﻨﻴﻨﺎﺕ‬ ‫ ﻭﻗﺩ ﺘﻡ ﺍﻝﺘﺤﺭﻱ ﻋﻥ ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻲ ﺍﻝﺨﻠﻭﻱ ﻝﻠﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﺨﺎﻡ ﻷﻭﺭﺍﻕ ﻭﺴﻴﻘﺎﻥ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻋﻠﻰ ﺨﻁ ﺨﻼﻴﺎ ﺴﺭﻁﺎﻥ ﺍﻝﻜﺒﺩ ﺍﻝﺒﺸﺭﻱ‬.‫ﻭﺍﻝﺘﺎﻨﻴﻨﺎﺕ‬ ‫ ﻭﻗﺩ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ ﺃﻋﻠﻰ ﻨﺴﺒﺔ ﺘﺜﺒﻴﻁ ﻜﺎﻨﺕ‬،‫ ﺤﻴﺙ ﺘﻡ ﺘﺤﻀﻴﺭ ﺨﻤﺴﺔ ﺘﺨﺎﻓﻴﻑ ﻋﺸﺭﻴﺔ ﻤﻥ ﺍﻝﺘﺭﻜﻴﺯ ﺍﻷﺼﻠﻲ ﻝﻜل ﻤﺴﺘﺨﻠﺹ‬، (HepG2) ، 350µg/ml ‫ ﻴﻠﻴﻪ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺒﺎﺭﺩ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ‬، 81.69% ‫ ﻭﻜﺎﻨﺕ ﻗﻴﻤﺘﻬﺎ‬، 700µg/ml ‫ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺤﺎﺭ ﻝﻸﻭﺭﺍﻕ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ‬ ‫ ﻭﻜﺎﻥ ﺍﻝﺘﺭﻜﻴﺯ ﺍﻷﻤﺜل ﻷﻋﻠﻰ‬.65.00% ‫ ﺤﻴﺙ ﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻝﺘﺜﻴﻁ‬175 µg/ml ‫ ﺜﻡ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ‬، 75.85% ‫ﻭﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻝﺘﺜﺒﻴﻁ‬ ‫ ﻴﻠﻴﻪ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺤﺎﺭ ﻋﻨﺩ ﻨﻔﺱ‬، 81.47% ‫ ﻭﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻝﺘﺜﺒﻴﻁ‬1400µg/ml ‫ﺘﺜﺒﻴﻁ ﺒﺎﻝﻨﺴﺒﺔ ﻝﻠﺴﻴﻘﺎﻥ ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺒﺎﺭﺩ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ‬ ‫ ﻴﻤﻜﻥ ﺍﺴﺘﻨﺘﺎﺝ ﺃﻥ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ‬،‫ ﻭﺒﻬﺫﺍ‬.59.57% ‫ ﻭﺒﻨﺴﺒﺔ ﺘﺜﺒﻴﻁ‬700µg/ml ‫ ﺜﻡ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ‬، 75.85% ‫ﺍﻝﺘﺭﻜﻴﺯ ﺒﻨﺴﺒﺔ ﺘﺜﺒﻴﻁ‬ ‫ﻤﻥ ﺍﻝﻨﺒﺎﺘﺎﺕ ﺍﻝﻁﺒﻴﺔ ﺍﻝﻤﻬﻤﺔ ﻭﺍﻝﺘﻲ ﻤﻥ ﺍﻝﻤﻤﻜﻥ ﺍﺴﺘﺨﺩﺍﻤﻬﺎ ﻓﻲ ﻤﻌﺎﻝﺠﺔ ﺍﻝﺴﺭﻁﺎﻥ ﻤﻥ ﺨﻼل ﺘﺄﺜﻴﺭﻩ ﺍﻝﺘﺜﺒﻴﻁﻲ ﻭﺍﻝﺴﻤﻲ ﺍﻝﻘﺎﺘل ﻋﻠﻰ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ‬ .‫ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬ ‫ ﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬، cytotoxicity ‫ ﺍﻝﺴﻤﻴﺔ ﺍﻝﺨﻠﻭﻴﺔ‬، HepG2 ‫ﺨﻁ ﺨﻼﻴﺎ‬، ‫ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ‬:‫ﺍﻝﻜﻠﻤﺎﺕ ﺍﻝﻤﻔﺘﺎﺤﻴﺔ‬

Inhibition effect of Ricinus communis extracts on HepG2 cancer cell lines Sarab S. Kadhum(1) Zainab T. H. Al-Berikdar (1) Farooq I. Mohammad (2) Hassan H. Jawad (1) and Ahmad M. M. Salih (2) (1) Dept. of Biodetection /Pollutant Treatment Center/Environment and Water Directorate / Ministry of Sciences and Technology / Baghdad (2) Biotechnology Research Center/ Al Nahrain University / Republic of Iraq

ABSTRACT The aim of this study was to detect toxic effect for leaves and stems of castor plant crude extracts on HepG2 cancer cells lines and and determine the minimum inhibitory concentration for each extract. Plant samples were collected. The alcoholic and aqueous extracts for leaves and stems were prepared and undergone biochemical analysis to identify the active substances. Results showed that the aqueous extract of leaves and stems contained saponines, and tannins, while alcoholic extract of leaves and stems contained flavones, and tannins. Cytotoxicity of crude extracts for leaves and stems on hepatocyte cancer cell line (HepG2) had been investigated. Five diluents from each extract was prepared. Results showed that the highest percentage of inhibition of growth was of aqueous hot extracts of the leaves when the concentration 700µg/ml at the value of 81.69%, followed by aqueous cold extracts at concentration 350µg/ml inhibition rate was 78.85% then alcoholic extract at 175µg/ml at the value of 65.00% .The better concentration showed the highest inhibition for steam aqueous cold extracts was at 1400µg/ml concentration, the inhibition rate was 81.47 %, followed by aqueous hot extracts at concentration 1400µg/ml inhibition rate was 75.85 % then alcoholic extract at 700µg/ml at the value of 59.57%. It can be concluded that the castor plant medicinal plants, which could be used in the treatment of cancer through the inhibitory, toxic, cytostatic and deadly effects of HepG2 cancer cell line.

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‫‪Vol. 10, No.2, June 2015‬‬

‫‪International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735‬‬

‫ﺍﻝﻤﻘﺩﻤـــﺔ‬ ‫ﻨﺘﻴﺠﺔ ﻻﻨﺘﺸﺎﺭ ﺍﻝﻤﻠﻭﺜﺎﺕ ﺍﻝﺒﻴﺜﻴﺔ ﻭﻋﻼﻗﺘﻬﺎ ﺒﺎﻨﺘﺸﺎﺭ ﻭﻨﻤﻭ ﺍﻷﻭﺭﺍﻡ‬ ‫ﺍﻝﻤﺨﺘﻠﻔﺔ ﺴﻭﺀﺍ ﺍﻝﺤﻤﻴﺩﺓ ﺃﻭﺍﻝﺨﺒﻴﺜﺔ ﻓﻘﺩ ﺍﺘﺠﻬﺕ ﺍﻝﻜﺜﻴﺭ ﻤﻥ ﺍﻝﺩﺭﺍﺴﺎﺕ‬ ‫ﻹﻴﺠﺎﺩ ﻁﺭﻗﺎ ﻝﺘﻁﻭﻴﺭ ﻫﺫﺍ ﺍﻝﻨﻭﻉ ﻤﻥ ﺍﻝﺒﺤﻭﺙ ﻭﺍﺴﺘﺨﺩﺍﻤﻪ ﻓﻲ ﺍﻝﺘﻘﻠﻴل‬ ‫ﻤﻥ ﺘﺄﺜﻴﺭ ﻫﺫﻩ ﺍﻝﻤﻠﻭﺜﺎﺕ ‪ ،‬ﻭﻴﻌﺘﺒﺭ ﺍﺴﺘﺨﺩﺍﻡ ﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻨﺒﺎﺘﺎﺕ‬ ‫ﺍﻝﻌﺸﺒﻴﺔ ﻤﻥ ﺍﻝﻁﺭﻕ ﺍﻝﺒﺩﻴﻠﺔ ﺍﻝﻤﺴﺘﺨﺩﻤﺔ ﺤﺎﻝﻴﺎ ﻓﻲ ﻫﺫﺍ ﺍﻝﻤﺠﺎل ﻭﺫﻝﻙ‬ ‫ﻝﺴﻬﻭﻝﺔ ﺍﻝﺤﺼﻭل ﻋﻠﻴﻬﺎ ‪،‬ﻭﻝﻴﺱ ﻝﻬﺎ ﺘﺄﺜﻴﺭﺍﺕ ﺠﺎﻨﺒﻴﺔ ﻋﻠﻰ ﺍﻝﺨﻼﻴﺎ‬ ‫ﺍﻝﻁﺒﻴﻌﻴﺔ ﻜﻤﺎ ﻫﻭ ﺍﻝﺤﺎل ﺒﺎﻝﻨﺴﺒﺔ ﻝﻠﻤﻭﺍﺩ ﺍﻝﻜﻴﻤﺎﻭﻴﺔ )‪.(1‬‬ ‫ﺍﻝﺨﺭﻭﻉ ﻨﺒﺎﺕ ﺸﺠﺭﻱ ﻴﺘﺒﻊ ﺍﻝﻌﺎﺌﻠﺔ ﺍﻝﻔﺭﺒﻴﻭﻨﻴﺔ ‪، Euphorbiaceae‬‬ ‫ﺃﻭﺭﺍﻗﻪ ﺫﺍﺕ ﺨﻤﺴﺔ ﻓﺼﻭﺹ ﻓﻲ ﺸﻜل ﺭﺍﺤﺔ ﺍﻝﻴﺩ )ﺸﻜل ﺭﻗﻡ ‪،(1‬‬ ‫ﺍﻝﺨﺭﻭﻉ ﺃﻭ“ ﻜﺭﻨﻙ “ ﺒﺎﻝﻌﺭﺒﻴﺔ ﻴﺴﻤﻰ ﻋﻠﻤﻴﺎ ﺭﻴﺴﻴﻨﻭﺱ ﻜﻭﻤﻭﻨﻴﺱ‬ ‫‪ .Ricinus communis‬ﻭﻫﻭ ﺸﺠﺭﺓ ﺼﻐﻴﺭﺓ ﺨﺸﺒﻴﺔ ﻝﻴﻨﺔ ‪ ،‬ﻝﻬﺎ‬ ‫ﺍﻨﺘﺸﺎﺭ ﻭﺍﺴﻊ ﻓﻲ ﺠﻤﻴﻊ ﺃﻨﺤﺎﺀ ﺍﻝﻤﻨﺎﻁﻕ ﺍﻝﻤﺩﺍﺭﻴﺔ ﻭﺍﻝﻤﻨﺎﻁﻕ ﺫﺍﺕ‬ ‫ﺩﺭﺠﺎﺕ ﺍﻝﺤﺭﺍﺭﺓ ﺍﻝﺩﺍﻓﺌﺔ ‪ ،‬ﻭﺍﻝﻤﻭﻁﻥ ﺍﻷﺼﻠﻲ ﻝﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻫﻭ‬ ‫ﺍﻝﻬﻨﺩ ﻭﻴﺯﺭﻉ ﻓﻲ ﺠﻤﻴﻊ ﺃﻨﺤﺎﺀ ﺍﻝﺒﻼﺩ ﻓﻲ ﺍﻝﺤﺩﺍﺌﻕ ﻭﺍﻝﺤﻘﻭل ‪ ،‬ﻭﺘﻨﻤﻭ‬ ‫ﺃﻴﻀﺎ ﻓﻲ ﺍﻝﻤﻨﺎﻁﻕ ﺍﻝﺒﺭﻴﺔ )‪ ،(2‬ﻭﻴﺒﻠﻎ ﺍﺭﺘﻔﺎﻋﻬﺎ ﺒﻌﺽ ﺍﻷﻤﺘﺎﺭ‪،‬‬ ‫ﻤﺴﺘﺩﻴﻤﺔ ﺍﻝﺨﻀﺭﺓ‪ ،‬ﻭ ﺃﻭﺭﺍﻗﻬﺎ ﺒﺴﻴﻁﺔ ﻤﻔﺼﺼﺔ ﻜﺒﻴﺭﺓ‪ ،‬ﻤﻌﻨﻘﺔ‬ ‫ﺃﺯﻫﺎﺭﻫﺎ ﻭﺤﻴﺩﺓ ﺍﻝﺠﻨﺱ ﻝﻜﻨﻬﺎ ﻤﺠﺘﻤﻌﺔ ﻓﻲ ﻨﻔﺱ ﺍﻝﺸﺠﻴﺭﺓ‪ .‬ﺜﻤﺎﺭﻫﺎ‬ ‫ﻋﻠﺒﻴﺔ ﻭ ﻤﻜﺴﻭﺓ ﺒﺄﺸﻭﺍﻙ ‪ ،‬ﻭﺘﺤﺘﻭﻱ ﻋﻠﻰ ﻝﻭﺯﺓ ﺯﻴﺘﻴﺔ ﺘﻌﺘﺼﺭ‬ ‫ﻭﻴﺨﺭﺝ ﻤﻨﻬﺎ ﺯﻴﺕ ﻤﺸﻬﻭﺭ‪ ،‬ﻭﺘﺤﺘﻭﻱ ﺒﺫﺭﺓ ﺍﻝﺨﺭﻭﻉ ﻋﻠﻰ ﺤﻭﺍﻝﻲ‬ ‫‪ %50‬ﻤﻥ ﻭﺯﻨﻬﺎ ﺯﻴﺘﺎﹰ‪ ،‬ﻭﻫﺫﺍ ﺍﻝﺯﻴﺕ ﻫﻭ ﺍﻝﻤﺴﺘﺨﺩﻡ ﻁﺒﻴﺎ )‪.(3‬‬

‫ﺸﻜل ﺭﻗﻡ )‪ :(1‬ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ‬

‫ﻭﺘﻌﻤل ﺍﻝﻨﺒﺎﺘﺎﺕ ﺍﻝﻌﺸﺒﻴﺔ ﻋﻠﻰ ﺘﻘﻠﻴل ﺃﻭ ﺇﻴﻘﺎﻑ ﻨﻤﻭ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬ ‫ﺒﻁﺭﻴﻘﺘﻴﻥ‪ ،‬ﻓﺈﻤﺎ ﻤﻥ ﺨﻼل ﺘﺜﺒﻴﻁ ﻋﻤل ﺒﻌﺽ ﺍﻹﻨﺯﻴﻤﺎﺕ ﺍﻝﻤﻬﻤﺔ ﻓﻲ‬ ‫ﺘﺤﻔﻴﺯ ﻨﻤﻭ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ )‪ (4‬ﺃﻭ ﻤﻥ ﺨﻼل ﺇﺜﺎﺭﺓ ﺍﻝﺠﻴﻨﺎﺕ‬ ‫ﺍﻝﻤﺴﺌﻭﻝﺔ ﻋﻥ ﺒﺩﺀ ﻋﻤﻠﻴﺔ ﺍﻝﻤﻭﺕ ﺍﻝﻤﺒﺭﻤﺞ ‪ apoptosis‬ﻓﻲ ﺍﻝﺨﻼﻴﺎ‬ ‫ﺍﻝﻭﺭﻤﻴﺔ‪ ،‬ﺃﻭ ﺃﻥ ﻝﻬﺎ ﻗﺎﺒﻠﻴﺔ ﻝﻠﻌﻤل ﻜﻤﺅﺜﺭ ﻀﺩ ﻋﻤﻠﻴﺔ ﺍﻨﻘﺴﺎﻡ ﻭﺘﻀﺎﻋﻑ‬ ‫ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ )‪.anti-proliferation effect (1‬‬ ‫ﺇﻥ ﺒﻌﺽ ﺍﻷﻤﺭﺍﺽ ﺍﻝﻤﻌﻀﻠﺔ ﺍﻝﺘﻲ ﺘﺼﻴﺏ ﺍﻹﻨﺴﺎﻥ ﻤﺜل ﺍﻝﺴﺭﻁﺎﻥ‬ ‫ﻭﺍﻝﻌﻴﻭﺏ ﺍﻝﺨﻠﻘﻴﺔ ﺘﺤﺩﺙ ﻨﺘﻴﺠﺔ ﻻﻨﻘﺴﺎﻡ ﺍﻝﺨﻼﻴﺎ ﻭﺘﺨﺼﺼﻬﺎ ﻏﻴﺭ‬ ‫ﺍﻝﻁﺒﻴﻌﻴﻴﻥ‪ ،‬ﻭﺍﻝﻔﻬﻡ ﺍﻝﺠﻴﺩ ﻝﻠﻌﻤﻠﻴﺎﺕ ﺍﻝﺨﻠﻭﻴﺔ ﺴﻭﻑ ﻴﺴﺎﻋﺩ ﻋﻠﻰ ﺘﺤﺩﻴﺩ‬ ‫ﺍﻷﺴﺒﺎﺏ ﺍﻷﺴﺎﺴﻴﺔ ﻭﻤﻭﺍﻗﻊ ﺍﻝﺨﻁﺄ ﺍﻝﺘﻲ ﺘﺘﺴﺒﺏ ﻋﺎﺩﺓ ﻓﻲ ﺃﻤﺭﺍﺽ‬ ‫ﻤﻤﻴﺘﺔ‪.‬‬ ‫ﺘﺴﺘﺨﺩﻡ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ ﻝﺩﺭﺍﺴﺔ ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﺍﻝﺴﻤﻴﺔ ﺍﻝﺨﻠﻭﻴﺔ‬ ‫ﻝﻠﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻨﺒﺎﺘﻴﺔ‪ ،‬ﻭﻓﻲ ﺩﺭﺍﺴﺔ ﺃﺠﺭﻴﺕ ﻝﺩﺭﺍﺴﺔ ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﺍﻝﺴﻤﻴﺔ‬ ‫ﺍﻝﺨﻠﻭﻴﺔ ﻝﻠﻤﺴﺘﺨﻠﺼﻴﻥ ﺍﻝﻤﺎﺌﻲ ﻭﺍﻝﻜﺤﻭﻝﻲ ﺍﻝﺨﺎﻡ ﻝﻠﻌﺭﻫﻭﻥ‬ ‫‪ Agaricusbisporus‬ﻓﻲ ﺍﻝﺯﺠﺎﺝ ‪ in vitro‬ﻭﻓﻲ ﺍﻝﺠﺴﻡ ﺍﻝﺤﻲ ‪in‬‬ ‫‪ vivo‬ﻓﻲ ﺍﺜﻨﻴﻥ ﻤﻥ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‪ ،‬ﻫﻤﺎ ﺨﻁ ﺨﻼﻴﺎ‬ ‫ﺴﺭﻁﺎﻥ ﺍﻝﺤﻨﺠﺭﺓ ﺍﻝﺒﺸﺭﻱ )‪ (Hep-2‬ﻭﺨﻁ ﺨﻼﻴﺎ ﺴﺭﻁﺎﻥ ﺍﻝﻐﺩﺓ‬ ‫ﺍﻝﻠﺒﻨﻴﺔ ﺍﻝﻔﺄﺭﻱ )‪ ، (AMN3‬ﻭﻗﺩ ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻲ‬ ‫ﻝﻜﻼ ﺍﻝﻤﺴﺘﺨﻠﺼﻴﻥ ﺍﻝﺨﺎﻡ ﺍﻋﺘﻤﺩ ﻋﻠﻰ ﺍﻝﺘﺭﻜﻴﺯ ﺍﻝﻤﺴﺘﺨﺩﻡ ﻭﻤﺩﺓ‬ ‫ﺍﻝﺘﻌﺭﻴﺽ ‪.‬‬ ‫ﻭﻗﺩ ﻜﺸﻔﺕ ﺩﺭﺍﺴﺎﺕ ﻤﺴﺘﻔﻴﻀﺔ ﺃﻥ ﻋﺩﺩﺍ ﻤﻥ ﺍﻝﻨﺒﺎﺘﺎﺕ ﺘﺴﺘﻌﻤل ﻝﻠﻭﻗﺎﻴﺔ‬ ‫ﺍﻭ ﺍﻝﻌﻼﺝ ﻤﻥ ﺍﻝﺴﺭﻁﺎﻥ )‪ ، (5‬ﻭﻋﻠﻰ ﺴﺒﻴل ﺍﻝﻤﺜﺎل ﻭﺠﻭﺩ ﺍﻝﻠﻜﺘﻴﻥ‬ ‫ﻭﻫﻭ ﻨﻭﻉ ﻤﻥ ﺃﻨﻭﺍﻉ ﺍﻝﺒﺭﻭﺘﻴﻨﺎﺕ ﺍﻝﺘﻲ ﻝﻬﺎ ﻗﺩﺭﺓ ﺃﻭ ﻓﻌﺎﻝﻴﺔ ﻤﻀﺎﺩﺓ‬ ‫ﻝﻠﺠﺭﺍﺜﻴﻡ ‪ ، antibacterial‬ﻭﺍﻝﺴﺭﻁﺎﻥ )‪ ، (5) (anticancer‬ﻭﺇﻥ‬ ‫ﺍﻝﻠﻜﺘﻴﻥ ﺍﻝﻤﻭﺠﻭﺩ ﻓﻲ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻝﻪ ﻓﻌﺎﻝﻴﺔ ﻀﺩ ﻭﺭﻤﻴﺔ‬ ‫)‪ (antitumor‬ﻭﻴﻌﻤل ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻋﻤﻠﻴﺔ ﺍﻨﻘﺴﺎﻡ ﻭﺘﻘﺩﻡ ﺩﻭﺭﺓ ﺍﻝﺨﻠﻴﺔ‬ ‫)‪ ،(7 ،2‬ﻭﻴﻌﺘﺒﺭ ﺍﻝﺭﻴﺴﻴﻥ ﻭﺍﻻﺒﺭﻴﻥ ﻤﻭﺍﺩ ﺴﺎﻤﺔ ﺘﻡ ﻋﺯﻝﻬﺎ ﻤﻥ‬ ‫ﻤﺴﺘﺨﻠﺹ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻭﺍﻝﺘﻲ ﻴﻌﻭﺩ ﻝﻬﺎ ﺍﻝﻨﺸﺎﻁ ﺍﻝﻀﺩ ﻭﺭﻤﻲ‬ ‫ﻭﺍﻝﻨﺎﺘﺞ ﻤﻥ ﻗﺩﺭﺓ ﻫﺫﻩ ﺍﻝﻤﻭﺍﺩ ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻋﻤﻠﻴﺔ ﺘﺨﻠﻴﻕ ﺍﻝﺒﺭﻭﺘﻴﻥ )‪،8‬‬ ‫‪ ،(9‬ﻭﻗﺩ ﻅﻬﺭﺕ ﻓﻲ ﺍﻵﻭﻨﺔ ﺍﻻﺨﻴﺭﺓ ﺩﺭﺍﺴﺎﺕ ﻤﺴﺘﻔﻴﻀﺔ ﺤﻭل‬

‫ﺍﻝﻨﺒﺎﺘﺎﺕ ﺍﻝﻁﺒﻴﺔ ﻭﺃﻫﻤﻴﺘﻬﺎ‪ ،‬ﻭﻗﺩ ﺃﻭﻝﺕ ﻤﻨﻅﻤﺔ ﺍﻝﺼﺤﺔ ﺍﻝﻌﺎﻝﻤﻴﺔ‬ ‫)‪ (WFO‬ﺍﻫﺘﻤﺎﻤﺎ ﻓﻲ ﺘﻭﺴﻴﻊ ﺍﺴﺘﻌﻤﺎل ﺍﻷﺩﻭﻴﺔ ﺍﻝﻤﺼﻨﻌﺔ ﻤﻥ ﺍﻝﻨﺒﺎﺘﺎﺕ‬ ‫ﺍﻝﻁﺒﻴﺔ ﺒﺩل ﺍﻷﺩﻭﻴﺔ ﺍﻝﻤﺼﻨﻌﺔ ﻜﻴﻤﻴﺎﺌﻴﺎ‪.‬‬

‫ﺍﻝﻤﻭﺍﺩ ﻭﻁﺭﻕ ﺍﻝﻌﻤل‬ ‫ﺠﻤﻊ ﺍﻝﻨﻤﺎﺫﺝ‪:‬‬ ‫ﺠﻤﻌﺕ ﻨﻤﺎﺫﺝ ﺍﻝﻨﺒﺎﺕ ﻤﻥ ﺍﻝﺤﺩﺍﺌﻕ ﺍﻝﻌﺎﻤﺔ ﻓﻲ ﻤﺩﻴﻨﺔ ﺒﻐﺩﺍﺩ‪ ،‬ﺨﻼل‬ ‫ﺍﻝﻔﺘﺭﺓ ﻤﻥ ‪ 1/10/2013‬ﻝﻐﺎﻴﺔ ‪.1/2/2014‬‬ ‫ﺃﺨﺫﺕ ﺒﻌﺽ ﻤﻥ ﺍﻝﻨﻤﺎﺫﺝ ﺇﻝﻰ ﻤﺨﺘﺒﺭ ﺘﺼﻨﻴﻑ ﺍﻝﻨﺒﺎﺕ ﻓﻲ ﻜﻠﻴﺔ ﺍﻝﻌﻠﻭﻡ‬ ‫‪ ،‬ﻗﺴﻡ ﻋﻠﻭﻡ ﺍﻝﺤﻴﺎﺓ ﻓﻲ ﺠﺎﻤﻌﺔ ﺒﻐﺩﺍﺩ ﺍﻝﺘﺎﺒﻊ ﻝﻠﺩﻜﺘﻭﺭ ﻋﻠﻲ ﺍﻝﻤﻭﺴﻭﻱ‬ ‫ﻭﺘﻡ ﺘﺜﺒﻴﺕ ﺍﻻﺴﻡ ﺍﻝﻌﻠﻤﻲ ﻝﻠﻨﺒﺎﺕ ‪ ،‬ﻏﺴﻠﺕ ﺍﻷﻭﺭﺍﻕ ﻭﺍﻝﺴﻴﻘﺎﻥ ﺒﺎﻝﻤﺎﺀ‬ ‫ﻝﺘﻨﻅﻴﻔﻬﺎ ﻤﻥ ﺍﻝﻐﺒﺎﺭ ﺍﻝﻌﺎﻝﻕ ﺒﻬﺎ ‪ ،‬ﻭﺘﺭﻜﺕ ﻝﺘﺠﻑ ﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ‬ ‫ﺍﻝﻐﺭﻓﺔ‪ ،‬ﻭﺘﻡ ﺴﺤﻘﻬﺎ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻝﻁﺎﺤﻭﻨﺔ‪ ،‬ﺜﻡ ﺤﻔﻅﺕ ﺍﻝﻨﻤﺎﺫﺝ‬ ‫ﺍﻝﻤﻁﺤﻭﻨﺔ ﻝﻠﺴﻴﻘﺎﻥ ﻭﺍﻷﻭﺭﺍﻕ ﻜل ﻋﻠﻰ ﺍﻨﻔﺭﺍﺩ ﻓﻲ ﻗﻨﺎﻨﻲ ﻨﻅﻴﻔﺔ‬ ‫ﻭﻤﺤﻜﻤﺔ ﺍﻝﻐﻠﻕ ﻝﺤﻴﻥ ﺍﻻﺴﺘﺨﺩﺍﻡ‪.‬‬ ‫ﺤﻀﺭ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺒﺎﺭﺩ ﺒﻭﺯﻥ ‪ 15‬ﻏﻡ ﻤﻥ ﻤﺴﺤﻭﻕ ﺍﻝﻨﺒﺎﺕ‬ ‫ﻭﺃﻀﻴﻑ ﺍﻝﻴﻪ ‪ 100‬ﻤل ﻤﻥ ﺍﻝﻤﺎﺀ ﺍﻝﻤﻘﻁﺭ ﻭﻤﺯﺝ ﺠﻴﺩﺍ ﺒﺎﺴﺘﺨﺩﺍﻡ‬ ‫ﺍﻝﻬﺯﺍﺯ ﺍﻝﻤﻐﻨﺎﻁﻴﺴﻲ )‪ ،(magnetic shaker‬ﺜﻡ ﺃﻜﻤﻠﺕ ﺍﻝﺨﻁﻭﺍﺕ‬ ‫ﺤﺴﺏ ﻤﺎ ﻭﺭﺩ ﻓﻲ )‪ .(10‬ﺜﻡ ﺤﻀﺭ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺤﺎﺭ‬ ‫ﺒﺈﺫﺍﺒﺔ ‪ 15‬ﻏﻡ ﻤﻥ ﻤﺴﺤﻭﻕ ﺍﻝﻨﺒﺎﺕ ﻓﻲ‪ 100‬ﻤل ﻤﻥ ﺍﻝﻤﺎﺀ ﺍﻝﻤﻘﻁﺭ‬ ‫ﺍﻝﻤﻐﻠﻲ‪ .‬ﺒﻌﺩ ﺫﻝﻙ‪ ،‬ﻭﻀﻊ ﺍﻝﺨﻠﻴﻁ ﻓﻲ ﺤﺎﻀﻨﺔ ﻫﺯﺍﺯﺓ ﻝﻤﺩﺓ ‪ 30‬ﺩﻗﻴﻘﺔ‬ ‫ﻋﻨﺩ ‪ 37‬ﻡ ﻝﻠﺤﺼﻭل ﻋﻠﻰ ﻤﺴﺤﻭﻕ ﺠﺎﻑ‪ ،‬ﻭﺍﺘﺒﻌﺕ ﻨﻔﺱ ﺍﻝﺨﻁﻭﺍﺕ‬ ‫ﺍﻝﻤﺫﻜﻭﺭﺓ ﻓﻲ ﺘﺤﻀﻴﺭ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺒﺎﺭﺩ‪ ،‬ﻭﺍﺘﺒﻌﺕ ﻁﺭﻴﻘﺔ‬ ‫)‪ ،(12 ،11‬ﻭﺤﻀﺭ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﺍﻝﺒﺎﺭﺩ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻝﻤﻴﺜﺎﻨﻭل‬ ‫)‪ (70%‬ﺒﺩﻻ ﻤﻥ ﺍﻝﻤﺎﺀ ﺍﻝﻤﻘﻁﺭ ﻝﻠﺤﺼﻭل ﻋﻠﻰ ﻤﺴﺤﻭﻕ ﺠﺎﻑ‪.‬‬ ‫ﻭﺍﺴﺘﺨﺩﻤﺕ ﻨﻔﺱ ﺍﻝﺨﻁﻭﺍﺕ ﺍﻝﻤﺫﻜﻭﺭﺓ ﻓﻲ ﺘﺤﻀﻴﺭ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ‬ ‫ﺍﻝﺒﺎﺭﺩ ﻭﺤﺴﺏ ﻤﺎ ﻭﺭﺩ ﻓﻲ )‪ .(10‬ﺤﻀﺭﺕ ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﻝﻸﻭﺭﺍﻕ‬ ‫ﻭﺍﻝﺴﻴﻘﺎﻥ ﻜل ﻋﻠﻰ ﺍﻨﻔﺭﺍﺩ‪.‬‬ ‫ﺍﻝﺘﺤﻠﻴل ﺍﻝﻜﻴﻤﻴﺎﺌﻲ ﻝﻠﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻨﺒﺎﺘﻴﺔ ‪:‬‬ ‫ﺘﻡ ﺍﻝﻜﺸﻑ ﻋﻥ ﺍﻝﻤﺭﻜﺒﺎﺕ ﺍﻝﻔﻴﻨﻭﻝﻴﺔ ﻭﻋﻥ ﻭﺠﻭﺩ ﺍﻝﺼﺎﺒﻭﻨﻴﺎﺕ ﺒﺄﺴﺘﺨﺩﺍﻡ‬ ‫ﺍﻝﻁﺭﻴﻘﺔ ﺍﻝﻭﺍﺭﺩﺓ ﻓﻲ)‪ ،(13‬ﻭﺠﻭﺩ ﺍﻝﺘﺎﻨﻴﻥ ﻜﺸﻑ ﻋﻨﻪ ﺒﺎﺘﺒﺎﻉ ﻁﺭﻴﻘﺔ‬ ‫)‪ (14‬ﻭﺍﻝﻔﻼﻓﻭﻨﺎﺕ ﻭﻓﻘﺎ ﻝﻤﺎ ﻭﺭﺩ ﻓﻲ )‪ ، (15‬ﻭﺘﻡ ﺍﺘﺒﺎﻉ ﻁﺭﻴﻘﺘﻴﻥ‬ ‫ﻝﻠﻜﺸﻑ ﻋﻥ ﻭﺠﻭﺩ ﺍﻝﻘﻠﻭﻴﺩﺍﺕ ‪ .‬ﺘﻤﺕ ﺩﺭﺍﺴﺔ ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻲ ﺍﻝﻘﺎﺘل‬ ‫ﻝﻠﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻤﺎﺌﻴﺔ ﻭﺍﻝﻜﺤﻭﻝﻴﺔ ﻷﻭﺭﺍﻕ ﻭﺴﻴﻘﺎﻥ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻋﻠﻰ‬ ‫ﺍﻝﺨﻁ ﺍﻝﺨﻠﻭﻱ ﺍﻝﺴﺭﻁﺎﻨﻲ ‪.HepG2‬‬ ‫ﺍﺴﺘﺨﺩﻤﺕ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ ﻨﻭﻉ ‪ HepG2‬ﻭﺍﻝﺘﻲ ﺘﻡ ﺍﻝﺤﺼﻭل ﻋﻠﻴﻬﺎ‬ ‫ﻤﻥ ﻤﺭﻜﺯ ﺒﺤﻭﺙ ﺍﻝﺘﻘﻨﻴﺎﺕ ﺍﻻﺤﻴﺎﺌﻴﺔ ﻓﻲ ﺠﺎﻤﻌﺔ ﺍﻝﻨﻬﺭﻴﻥ‪ ،‬ﻭﺘﻤﺕ ﺇﺩﺍﻤﺔ‬ ‫ﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ ﻓﻲ ﺍﻝﻭﺴﻁ ﺍﻝﺯﺭﻋﻲ ‪ RPMI1640‬ﺍﻝﻤﺠﻬﺯ ﻋﻠﻰ‬ ‫ﺸﻜل ﺴﺎﺌل‪ ،‬ﻭﺤﻀﻨﺕ ﺍﻝﺨﻼﻴﺎ ﺒﺩﺭﺠﺔ ‪ 37o‬ﻓﻲ ﺠﻭ ﺭﻁﺏ ﻭﻤﺠﻬﺯ‬ ‫ﺒﻐﺎﺯ ﺜﺎﻨﻲ ﺃﻭﻜﺴﻴﺩ ﺍﻝﻜﺭﺒﻭﻥ ﺒﻨﺴﺒﺔ ‪.%5‬‬ ‫ﻗﻴﺎﺱ ﺍﻝﺴﻤﻴﺔ ‪:Cytotoxicity Assay‬‬ ‫ﺘﻡ ﺍﺨﺘﺒﺎﺭ ﺴﻤﻴﺔ ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻤﺤﻀﺭﺓ ﻋﻠﻰ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ ﻜل‬ ‫ﻋﻠﻰ ﺍﻨﻔﺭﺍﺩ‪ ،‬ﻭﺘﻡ ﺍﺴﺘﺨﺩﺍﻡ ﺍﻝﺨﻁ ﺍﻝﺨﻠﻭﻱ ﻝﺴﺭﻁﺎﻥ ﺍﻝﻜﺒﺩ ﺍﻝﻤﻌﺯﻭل ﻤﻥ‬ ‫ﺍﻹﻨﺴﺎﻥ ‪ ، HepG2‬ﺤﻴﺙ ﺇﻥ ﺍﻝﺨﻼﻴﺎ ﺘﻨﻤﻭ ﻋﻠﻰ ﺸﻜل ﻁﺒﻘﺎﺕ ﻤﺘﻌﺩﺩﺓ‬ ‫ﻭﺒﻜﺜﺎﻓﺔ ﻋﺎﻝﻴﺔ ﻭﺘﻜﻭﻥ ﺫﺍﺕ ﺸﻜل ﻤﻐﺯﻝﻲ‪ .‬ﺘﻤﺕ ﺯﺭﺍﻋﺔ ﺍﻝﺨﻼﻴﺎ ﻓﻲ‬ ‫ﺍﻝﻭﺴﻁ ﺍﻝﺯﺭﻋﻲ ‪ RPMI1640‬ﻭﺍﻝﻤﻀﺎﻑ ﺇﻝﻴﻪ ﻤﺼل ‪،٪F.C.S 10‬‬ ‫ﻭ‪ 50‬ﻤﻠﻐﻡ ‪ /‬ﻤل ﺴﺘﺭﺒﺘﻭﻤﻴﺴﻴﻥ ﻭﺍﻝﺒﻨﺴﻠﻴﻥ ‪ .1000 U / L‬ﻭﺤﻀﻨﺕ‬ ‫ﺒﺩﺭﺠﺔ ‪ 37o‬ﻓﻲ ﺠﻭ ﺭﻁﺏ ﻤﺠﻬﺯ ﺒﻐﺎﺯ ﺜﺎﻨﻲ ﺃﻭﻜﺴﻴﺩ ﺍﻝﻜﺭﺒﻭﻥ ﺒﻨﺴﺒﺔ‬ ‫‪ .%5‬ﻭﺃﺠﺭﻴﺕ ﺍﻝﺘﺠﺎﺭﺏ ﻋﻨﺩﻤﺎ ﻜﺎﻨﺕ ﺍﻝﺨﻼﻴﺎ ﻓﻲ ﻤﺭﺤﻠﺔ ﺍﻝﻨﻤﻭ‬ ‫ﺍﻝﻠﻭﻏﺎﺭﻴﺘﻤﻲ ﻭﻜﺎﻨﺕ ﺒﺼﺤﺔ ﺠﻴﺩﺓ‪ .‬ﺘﻡ ﺘﻭﻓﻴﺭ ﺍﻝﺨﻁ ﺍﻝﺨﻠﻭﻱ ﺍﻝﻜﺒﺩﻱ‬ ‫‪ HepG2‬ﻤﻥ ﻗﺒل ﻤﺨﺘﺒﺭ ﺯﺭﺍﻋﺔ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺤﻴﻭﺍﻨﻴﺔ ﻓﻲ ﻤﺭﻜﺯ ﺒﺤﻭﺙ‬ ‫ﺍﺍﻝﺘﻘﻨﻴﺎﺕ ﺍﻻﺤﻴﺎﺌﻴﺔ ﻓﻲ ﺠﺎﻤﻌﺔ ﺍﻝﻨﻬﺭﻴﻥ‪ .‬ﺘﻡ ﺇﺠﺭﺍﺀ ﺍﻝﻔﺤﺹ ﻭﻓﻘﺎ ﻝﻤﺎ‬ ‫ﻭﺭﺩ ﻓﻲ )‪ .(17‬ﺒﻌﺩﻫﺎ ﺤﺴﺒﺕ ﺍﻝﻨﺴﺒﺔ ﺍﻝﺤﻴﻭﻴﺔ ﻝﻠﺨﻼﻴﺎ ﻭﺍﻝﺘﻲ ﻴﺠﺏ ﺃﻥ‬ ‫ﺘﻜﻭﻥ ﺃﻜﺜﺭ ﻤﻥ ‪ . 95%‬ﻤﺯﺝ ﻋﺎﻝﻕ ﺍﻝﺨﻼﻴﺎ ﺠﻴﺩﺍ ﻭﻨﻘل ﻤﺎ ﻤﻘﺩﺍﺭﻩ‬ ‫‪ 200 µl‬ﻤﻥ ﺍﻝﻌﺎﻝﻕ ﻭﺒﻌﺩﺩ )‪ (104 × 5‬ﺨﻠﻴﺔ‪/‬ﺤﻔﺭﺓ ﺇﻝﻰ ﺍﻝﺼﻔﻴﺤﺔ‬ ‫ﺍﻝﻤﺎﻴﻜﺭﻭﻴﺔ ‪ Micro titer plate‬ﺘﺤﻭﻱ ﻋﻠﻰ )‪ (96‬ﺤﻔﺭﺓ ﻭﻤﻥ ﺜﻡ‬ ‫ﺃﻋﻴﺩ ﺇﻝﻰ ﺍﻝﺤﺎﻀﻨﺔ ﺒﺩﺭﺠﺔ ‪ 37o‬ﻓﻲ ﺠﻭ ﺭﻁﺏ ﻭﻤﺠﻬﺯ ﺒﻐﺎﺯ ﺜﺎﻨﻲ‬

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‫‪International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735‬‬

‫‪Vol. 10, No.2, June 2015‬‬

‫ﺃﻭﻜﺴﻴﺩ ﺍﻝﻜﺭﺒﻭﻥ ﺒﻨﺴﺒﺔ ‪ ، %5‬ﺜﻡ ﻓﺤﺼﺕ ﻓﻲ ﺍﻝﻴﻭﻡ ﺍﻝﺜﺎﻨﻲ ﻋﻥ‬ ‫ﻁﺭﻴﻕ ﺍﻝﻤﺠﻬﺭ ﺍﻝﻤﻌﻜﻭﺱ ﻝﻐﺭﺽ ﺍﻝﺘﺄﻜﺩ ﻤﻥ ﺍﻝﻨﻤﻭ ﻭﻋﺩﻡ ﻭﺠﻭﺩ‬ ‫ﺍﻝﺘﻠﻭﺙ‪ ،‬ﻭﺒﻌﺩﻫﺎ ﻋﺭﻀﺕ ﺍﻝﺨﻼﻴﺎ ﺇﻝﻰ ﺍﻝﺘﺭﺍﻜﻴﺯ ﺍﻝﻤﺨﺘﻠﻔﺔ ﻤﻥ‬ ‫ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﻭﺍﻝﻤﺤﻀﺭﺓ ﺒﺘﺨﺎﻓﻴﻑ ﻋﺸﺭﻴﺔ ﻤﻀﺎﻋﻔﺔ ﻤﻥ ﺍﻝﺘﺭﻜﻴﺯ‬ ‫ﺍﻻﺼﻠﻲ ﻭﺒﻭﺍﻗﻊ ﺜﻼﺙ ﻤﻜﺭﺭﺍﺕ )ﺜﻼﺙ ﺤﻔﺭ ﻤﻥ ﻜل ﺘﺭﻜﻴﺯ( ﻝﻜل‬ ‫ﻤﺴﺘﺨﻠﺹ‪ ،‬ﺃﻤﺎ ﻤﺠﻤﻭﻋﺔ ﺍﻝﺴﻴﻁﺭﺓ ﻓﺘﻤﺜﻠﺕ ﺒﺎﻝﺤﻔﺭ ﺍﻝﺤﺎﻭﻴﺔ ﻋﻠﻰ ﻋﺎﻝﻕ‬ ‫ﺍﻝﺨﻼﻴﺎ ﻤﻊ ﺍﻝﻭﺴﻁ ﺍﻝﺯﺭﻋﻲ ﻓﻘﻁ‪ .‬ﻭﺤﻀﻨﺕ ﺍﻝﺼﻔﻴﺤﺔ ﺍﻝﻤﺎﻴﻜﺭﻭﻴﺔ‬ ‫ﺒﻌﺩﻫﺎ ﻓﻲ ﺍﻝﺤﺎﻀﻨﺔ ﺒﺩﺭﺠﺔ ‪ 37o‬ﻓﻲ ﺠﻭ ﺭﻁﺏ ﻭﻤﺠﻬﺯ ﺒﻐﺎﺯ ﺜﺎﻨﻲ‬ ‫ﺃﻭﻜﺴﻴﺩ ﺍﻝﻜﺭﺒﻭﻥ ﺒﻨﺴﺒﺔ ‪ %5‬ﻝﻤﺩﺓ ‪ 72‬ﺴﺎﻋﺔ‪ .‬ﻭﺒﻌﺩ ﺍﻨﺘﻬﺎﺀ ﻤﺩﺓ‬ ‫ﺍﻝﺤﻀﻥ ﺘﻡ ﺍﻝﺘﺨﻠﺹ ﻤﻥ ﺍﻝﻭﺴﻁ ﺍﻝﺯﺭﻋﻲ ﻭﻏﺴﻠﺕ ﺍﻝﺨﻼﻴﺎ ﻭﺼﺒﻐﺕ‬ ‫ﺒﺼﺒﻐﺔ ‪ neutral red dye‬ﺒﻭﺍﻗﻊ ‪ 50 µl‬ﻝﻜل ﺤﻔﺭﺓ ﻭﻭﻀﻌﺕ ﻓﻲ‬ ‫ﺍﻝﺤﺎﻀﻨﺔ ﻝﻤﺩﺓ ﺴﺎﻋﺘﻴﻥ‪ .‬ﺒﻌﺩﻫﺎ ﺘﻡ ﺇﺯﺍﻝﺔ ﻤﺤﺘﻭﻴﺎﺕ ﺍﻝﺼﻔﻴﺤﺔ‬ ‫ﺍﻝﻤﻴﻜﺭﻭﻴﺔ ﺒﻐﺴﻠﻬﺎ ﺒﺩﺍﺭﺉ ﺍﻝﻔﻭﺴﻔﺎﺕ ﺍﻝﻤﻠﺤﻲ ﻝﺜﻼﺙ ﻤﺭﺍﺕ ﺜﻡ ﺃﻀﻴﻑ‬ ‫ﻤﺎ ﻤﻘﺩﺍﺭﻩ ‪ 100 µl‬ﻤﻥ ﺍﻝﻤﺤﻠﻭل ﺍﻝﻤﺨﻔﻑ )‪ PBS 1:1‬ﻭﺍﻴﺜﺎﻨﻭل(‬ ‫ﻝﻜل ﺤﻔﺭﺓ ﻝﻠﺘﺨﻠﺹ ﻤﻥ ﺍﻝﺼﺒﻐﺔ ﺍﻝﺯﺍﺌﺩﺓ ﻤﻥ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺤﻴﺔ‪ .‬ﻭﻗﺩ ﺠﺭﻯ‬ ‫ﻗﺭﺍﺀﺓ ﺍﻝﻜﺜﺎﻓﺔ ﺍﻝﻀﻭﺌﻴﺔ ﻋﻠﻰ ﺍﻝﻁﻭل ﺍﻝﻤﻭﺠﻲ ‪ 492 nm‬ﺒﺎﺴﺘﺨﺩﺍﻡ‬ ‫‪ ELISA reader‬ﻭﺤﺴﺒﺕ ﻨﺴﺒﺔ ﺍﻝﺘﺜﺒﻴﻁ ﻝﻠﺨﻼﻴﺎ ﺒﺘﻁﺒﻴﻕ ﺍﻝﻤﻌﺎﺩﻝﺔ‬ ‫ﺍﻝﻭﺍﺭﺩﺓ ﻓﻲ )‪ (18‬ﻭﻗﻭﺭﻨﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻤﻊ ﻗﺭﺍﺀﺓ ‪ OD‬ﻝﻠﺤﻔﺭ ﻏﻴﺭ‬ ‫ﺍﻝﻤﻌﺎﻤﻠﺔ ﺒﺎﻝﻤﺴﺘﺨﻠﺼﺎﺕ )ﻤﺠﻤﻭﻋﺔ ﺍﻝﺴﻴﻁﺭﺓ( ﻭﺍﻝﺘﻲ ﺘﻌﺘﺒﺭ ﻗﻴﻤﺔ‬ ‫ﺍﻻﻤﺘﺼﺎﺼﻴﺔ ﻝﻬﺎ ‪.%100‬‬ ‫ﺘﻡ ﺍﺤﺘﺴﺎﺏ ﻨﺴﺒﺔ ﺍﻝﺴﻤﻴﺔ ﻝﻠﺨﻼﻴﺎ ‪ cytotoxicity‬ﺒﺘﻁﺒﻴﻕ ﺍﻝﻤﻌﺎﺩﻝﺔ‬ ‫ﺍﻝﺘﺎﻝﻴﺔ‪:‬‬

‫‪Abs. at 492 nm of control – Abs. at 492 nm of test X 100‬‬ ‫‪Abs. at 492 nm of control‬‬

‫= ‪Inhibition rat IR‬‬

‫ﺍﺍﻝﻨﺘﺎﺌـــﺞ ﻭﺍﻝﻤﻨﺎﻗﺸــﺔ‬ ‫ﺤﻀﺭ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﻭﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﻷﻭﺭﺍﻕ ﻭﺴﻴﻘﺎﻥ‬ ‫ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻭﺘﻡ ﺘﺜﺒﻴﺕ ﺍﻝﻅﺭﻭﻑ ﺍﻝﻤﺜﻠﻰ ﻝﺘﺤﻀﻴﺭ ﻫﺫﻩ‬ ‫ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﻭﺘﺤﺩﻴﺩ ﺍﻝﻤﺠﺎﻤﻴﻊ ﺍﻝﻔﻌﺎﻝﺔ ﻝﻜل ﻤﻨﻬﺎ‪ ،‬ﻭﻴﺒﻴﻥ ﺍﻝﺠﺩﻭل ﺭﻗﻡ‬ ‫)‪ (1‬ﻨﺘﺎﺌﺞ ﺍﻝﻜﺸﻑ ﺍﻝﻨﻭﻋﻲ ﻝﻬﺫﻩ ﺍﻝﻤﺠﺎﻤﻴﻊ ﺍﻝﻔﻌﺎﻝﺔ ﻭﺍﻝﺘﻲ ﺘﻤﺜل ﻨﻭﺍﺘﺞ‬ ‫ﺃﻴﺽ ﺜﺎﻨﻭﻴﺔ ﻭﻝﻬﺎ ﺃﻫﻤﻴﺔ ﺩﻓﺎﻋﻴﺔ ﻝﻠﻨﺒﺎﺕ ﻭﺒﻨﻔﺱ ﺍﻝﻭﻗﺕ ﻴﺴﺘﻔﻴﺩ ﻤﻨﻬﺎ‬ ‫ﺍﻹﻨﺴﺎﻥ ﻭﻴﺴﺘﺨﺩﻤﻬﺎ ﻜﻌﻼﺝ ﻝﺒﻌﺽ ﺍﻷﻤﺭﺍﺽ )‪.(19‬‬ ‫ﺃﻭﻀﺤﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺃﻥ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﻭﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ‬ ‫ﻝﺴﻴﻘﺎﻥ ﻭﺃﻭﺭﺍﻕ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﺍﺸﺘﺭﻜﺎ ﺒﺎﺤﺘﻭﺍﺌﻬﻤﺎ ﺍﻝﻤﺠﻤﻭﻋﺎﺕ‬ ‫ﺍﻝﻔﻌﺎﻝﺔ ﺍﻝﻤﺘﻤﺜﻠﺔ ﺒﺎﻝﻔﻼﻓﻭﻨﺎﺕ ‪ Flavonoids‬ﻭﺍﻝﺘﺭﺒﻴﻨﺎﺕ ‪Terpens‬‬ ‫‪ ،‬ﻭﻫﺫﺍ ﻴﺘﻔﻕ ﻤﻊ ﻤﺎ ﺘﻭﺼل ﺍﻝﻴﻪ )‪ ، (2‬ﻭﻝﻠﻜﺸﻑ ﻋﻥ ﻓﻌﺎﻝﻴﺔ ﻫﺫﻩ‬ ‫ﺍﻝﻤﺠﺎﻤﻴﻊ ﺍﻝﻔﻌﺎﻝﺔ ﺍﻝﻤﻭﺠﻭﺩﺓ ﻓﻲ ﻤﺴﺘﺨﻠﺹ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﺘﻡ ﺘﻌﺭﻴﺽ‬ ‫ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ ﻝﺘﺭﺍﻜﻴﺯ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﻭﺩﺭﺱ ﺘﺄﺜﻴﺭﻫﺎ‬ ‫ﺍﻝﺘﺜﺒﻴﻁﻲ ﻋﻠﻰ ﻨﻤﻭ ﻫﺫﻩ ﺍﻝﺨﻼﻴﺎ‪.‬‬

‫ﺠﺩﻭل ﺭﻗﻡ )‪ :(1‬ﺍﻝﻜﺸﻑ ﺍﻝﻜﻴﻤﻴﺎﺌﻲ ﺍﻝﻨﻭﻋﻲ ﻋﻥ ﺍﻝﻤﺠﺎﻤﻴﻊ ﺍﻝﻔﻌﺎﻝﺔ ﺍﻝﺭﺌﻴﺴﻴﺔ ﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ‬ ‫‪Chemical‬‬ ‫‪compound‬‬

‫‪Methanol‬‬ ‫‪extract of‬‬ ‫‪stem‬‬

‫‪Cold water‬‬ ‫‪extract of‬‬ ‫‪stem‬‬

‫‪Hot water‬‬ ‫‪extract of‬‬ ‫‪stem‬‬

‫‪Methanol‬‬ ‫‪extract of‬‬ ‫‪leaves‬‬

‫‪Cold water‬‬ ‫‪extract of‬‬ ‫‪leaves‬‬

‫‪Hot water‬‬ ‫‪extract of‬‬ ‫‪leaves‬‬

‫‪Reagents‬‬

‫‪+‬‬

‫‪+‬‬

‫‪-‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫‪Ethanol+‬‬ ‫‪KOH‬‬

‫‪Flavonoids‬‬

‫‪+‬‬

‫‪-‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫‪Glacial acetic‬‬ ‫‪acid‬‬

‫‪Terpens‬‬

‫‪-‬‬

‫‪+‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪H2SO4‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪+‬‬

‫‪HgCl2‬‬

‫‪Saponins‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪Mayer's‬‬ ‫‪reagent‬‬

‫‪Alkaloids‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫‪+‬‬

‫)‪HCL(4%‬‬

‫‪Resins‬‬

‫‪+‬‬

‫‪-‬‬

‫‪-‬‬

‫‪-‬‬

‫‪+‬‬

‫‪CH3Coopb+‬‬ ‫‪FeCl2‬‬

‫‪Tannins‬‬

‫‪Steroids‬‬

‫‪-‬‬

‫ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻲ ﻝﻠﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻤﺤﻀﺭﺓ ﻋﻠﻰ ﻨﻤﻭ ﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ‬ ‫ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‪:‬‬ ‫ﺘﻡ ﺍﻝﺘﺤﺭﻱ ﻋﻥ ﺍﻝﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻲ ﺍﻝﺨﻠﻭﻱ ﻝﻠﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﺨﺎﻡ ﻷﻭﺭﺍﻕ‬ ‫ﻭﺴﻴﻘﺎﻥ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻋﻠﻰ ﺨﻁ ﺨﻼﻴﺎ ﺴﺭﻁﺎﻥ ﺍﻝﻜﺒﺩ ﺍﻝﺒﺸﺭﻱ‬ ‫)‪ ،(HepG2‬ﺇﺫ ﺘﻡ ﺘﺤﻀﻴﺭ ﺨﻤﺴﺔ ﺘﺨﺎﻓﻴﻑ ﻋﺸﺭﻴﺔ ﻤﻥ ﺍﻝﺘﺭﻜﻴﺯ‬ ‫ﺍﻻﺼﻠﻲ ﻝﻜل ﻤﺴﺘﺨﻠﺹ‪.‬‬ ‫ﻴﻭﻀﺢ ﺍﻝﺠﺩﻭل ﺭﻗﻡ )‪ (2‬ﻭﺠﻭﺩ ﻓﺭﻭﻗﺎﺕ ﻤﻌﻨﻭﻴﺔ )‪ (p≤0.05‬ﻝﻠﺘﺄﺜﻴﺭ‬ ‫ﺍﻝﺘﺜﺒﻴﻁﻲ ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﻷﻭﺭﺍﻕ ﺍﻝﻨﺒﺎﺕ ﻋﻨﺩ ﺍﻝﺘﺭﺍﻜﻴﺯ ﺍﻝﺨﻤﺴﺔ‬ ‫ﺍﻝﻤﻌﺘﻤﺩﺓ ‪ ،‬ﻭﻜﺎﻨﺕ ﺃﻋﻠﻰ ﻨﺴﺒﺔ ﺘﺜﺒﻴﻁ ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺤﺎﺭ‬ ‫ﻝﻸﻭﺭﺍﻕ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ ‪ 700µg/ml‬ﻭﻜﺎﻨﺕ ﻗﻴﻤﺘﻬﺎ ‪ 81.69%‬ﻴﻠﻴﻪ‬ ‫ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺒﺎﺭﺩ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ ‪ ، 350µg/ml‬ﻭﻜﺎﻨﺕ ﻨﺴﺒﺔ‬

‫ﺍﻝﺘﺜﺒﻴﻁ ‪ 75.85%‬ﺜﻡ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ‬ ‫‪ µg/ml‬ﻜﺎﻨﺕ ﻨﺴﺒﺔ ﺍﻝﺘﺜﻴﻁ ‪.65.00%‬‬ ‫ﻭﺍﻝﺠﺩﻭل ﺭﻗﻡ )‪ (3‬ﻴﻭﻀﺢ ﻭﺠـﻭﺩ ﻓﺭﻭﻗـﺎﺕ ﻤﻌﻨﻭﻴـﺔ )‪(p≤0.05‬‬ ‫ﻝﻠﺘﺄﺜﻴﺭ ﺍﻝﺘﺜﺒﻴﻁﻲ ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﻝﺴﻴﻘﺎﻥ ﺍﻝﻨﺒﺎﺕ ﻝﻠﺘﺭﺍﻜﻴﺯ ﺍﻝﺨﻤﺴﺔ‬ ‫ﺍﻝﻤﻌﺘﻤﺩﺓ ﻭﺍﻥ ﺍﻝﺘﺭﻜﻴﺯ ﺍﻻﻤﺜل ﻷﻋﻠﻰ ﺘﺜﺒﻴﻁ ﺒﺎﻝﻨﺴﺒﺔ ﻝﻠـﺴﻴﻘﺎﻥ ﻜـﺎﻥ‬ ‫ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺒﺎﺭﺩ ﻋﻨﺩ ﺍﻝﺘﺭﻜﻴﺯ ‪ 1400µg/ml‬ﻭﻜﺎﻨﺕ ﻨﺴﺒﺔ‬ ‫ﺍﻝﺘﺜﺒﻴﻁ ‪ 81.47%‬ﻴﻠﻴﻪ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ ﺍﻝﺤﺎﺭ ﻋﻨﺩ ﻨﻔﺱ ﺍﻝﺘﺭﻜﻴـﺯ‬ ‫ﺒﻨﺴﺒﺔ ﺘﺜﺒﻴﻁ ‪ 75.85‬ﺜـﻡ ﺍﻝﻤـﺴﺘﺨﻠﺹ ﺍﻝﻜﺤـﻭﻝﻲ ﻋﻨـﺩ ﺍﻝﺘﺭﻜﻴـﺯ‬ ‫‪ 700µg/ml‬ﻭﺒﻨﺴﺒﺔ ﺘﺜﺒﻴﻁ ‪.59.57%‬‬ ‫‪175‬‬

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

HepG2 ‫ ﻓﻌﺎﻝﻴﺔ ﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻷﻭﺭﺍﻕ ﻝﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻓﻲ ﺘﺜﺒﻴﻁ ﻨﻤﻭ ﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬:(2) ‫ﺠﺩﻭل ﺭﻗﻡ‬ Extract

Cold water extract of leaves

Concentration ( ìg/ml)

IR%

1400

58.42

700

78.42

350

78.85

175

50.14

87.5

20.14

1400

75.81

700

81.69

350

79.24

175

75.98

87.5

55.91

1400

35.5

700

19.75

350

27.95

175

65.00

87.5

58.66

Hot water extract of leaves

Methanol extract of leaves

IR =Inhibition Rate, LSD 0.05(Extraction solution, Concentration)=16.9, 21.9

HepG2 ‫ﻓﻌﺎﻝﻴﺔ ﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﺴﻴﻘﺎﻥ ﻝﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻓﻲ ﺘﺜﺒﻴﻁ ﻨﻤﻭ ﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬: (3) ‫ﺠﺩﻭل ﺭﻗﻡ‬ Extract

Cold water extract of stems

Hot water extract of stems

Methanol extract of stems

Concentration (ìg/ml)

IR%

1400

81.47

700

76.15

350

81.64

175

73.24

87.5

41.42

1400

75.85

700

51.42

350

38.57

175

34.42

87.5

27.71

1400

38.34

700

59.57

350

29.89

175

27.42

87.5

24.35

114

International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

human cancer cell lines. J. Biochem. Molec. Biol. 38(5): 526-532. 3. Weiss EA. (2000). Castor. In Oilseed Crops. 2nd ed. Blackwell Scientific Ltd., Oxford. Pp. 13-52. 4. Sharma DC. (2001). India favours acetic acid for early detection of cervical cancer. Lancet Oncol .2: 195. 5. Simone SGD.; Netto CC. and Silva FPJ. (2006). Simple affinity chromatographic procedure topurify β-galactoside binding lectins. J. Chromatog. B:838: 135-138. 6. Sylvestre M.; Pichette A.; Longtin A.; Nagau F. and Legault J. (2006). Essential oil anlysis and anticancer activity of leaf essential oil of Croton flavens L. From Guadeloupe. J. Ethnopharmacol. 103: 99-102. 7. Wei CH. and Koh C. (1978). Crystalline ricin D, atoxic anti-tumor lectin from seeds of Ricinus communis. J. Biol. Chem. 6:2061-2066. 8. Lin JY.; Kao WY.; Tserng KY.; Chen CC. and Tung TC. (1970). Cancer Res. 30: 2431 -2433. 9. Schep LJ.; Temple WA.; Butt GA. and Beasley MD. (2009). Ricin as a weapon of mass terror-separating fact from fiction. Environ. Int. 35 (8): 1267–1271. 10. Anesini C. and Perez C. (1993). Screening of plant used in argentine folk medicine for antimicrobial activity. J. Ethnopharmacol. 39(2): 119-128. 11. Harbon JB. (1984). Phytochemical methods. A guide to modern techniques of plant analysis. 2nd ed. Chapman and Hall. London. P: 288. 12. El- Fallal AA. and Kattan MH. (1997). Effect of plant extracts on the Mycelial growth of some cultivated mushrooms. Egypt. J. Microbiol. 32 (1): 41- 48. 13. Adeday O.; Anderson W.; Young M.; Sncickus V.; Patil P. and Kolawole D. (2001). Photochemistry and antibacterial activity of sennaalata flower. Pharma. Biol. 39: 1-5. 14. Jaffer HJ.; Mahmod MJ.; Jawad A M.; Naj A. and Al-Naib A. (1983). Phytochemical and biological screening of some Iraqi plant. Fito terapia Lix. P. 299. 15. Cai R.; Hettiarachy MG.; Jonson MA.; Janes MF. and Slavik MF. (2003). Preparation and evaluation of selected natural plant extracts as antimicrobial agents Lasteriamonocytogens, Salmonella typhimurium and E.coli 0157:H7. Session 29 F. Food microbiology control of foodborne microorganisms by antimicrobials.Chicago. 16. Harborn VB.(1973). Phrborn chemical methods. A guide to modern technique of plant analysis. London. 17. Freshney RI. (2000). Culture of animal cells : a manual for basic technique 4th ed . wiley – liss, John Wiley and Sons , INC. publication New York. 18. Freshney RI. (2001). Application of cell culture to toxicology. Cell Biol. Toxicol. 17:213-230. 19. Dubick AM. (1986). Historical perspectives on the use of Herbal preparations to promote health. J. Nat. 116: 1348-1354. 20. Lopus M. and Panda D. (2006). The enzophena thridine alkaloid sanguinarine perturbs micro-tubule

Vol. 10, No.2, June 2015

‫( ﺒﺄﻥ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﺨﺎﻡ ﻝﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ‬2،3) ‫ﺘﻅﻬﺭ ﻨﺘﺎﺌﺞ ﺍﻝﺠﺩﻭﻝﻴﻥ‬ ‫ﺴﺎﻡ ﻝﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ ﻝﻼﻨﺴﺎﻥ ﻭﻗﺩ ﺘﻜﻭﻥ ﻨﺘﻴﺠﺔ ﻝﺘﺤﻔﻴﺯ‬ ‫ﻋﻤﻠﻴﺔ ﺍﻝﻤﻭﺕ ﺍﻝﻤﺒﺭﻤﺞ ﻝﻠﺨﻼﻴﺎ ﻭﻫﺫﺍ ﻤﺎ ﺘﻭﺼل ﺇﻝﻴﻪ ﺒﺎﺤﺜﻭﻥ ﻤﻥ ﺃﻥ‬ ‫ﺍﺴﺘﺨﺩﺍﻡ ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﺨﺎﻡ ﻷﻭﺭﺍﻕ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﺩﻭﻥ ﻓﺼل‬ ‫ﺍﻝﻤﻭﺍﺩ ﺍﻝﻔﻌﺎﻝﺔ ﺒﻌﻀﻬﺎ ﻋﻥ ﺒﻌﺽ ﻭﻜﻤﺎ ﻫﻲ ﻤﻭﺠﻭﺩﺓ ﻓﻲ ﺍﻝﻁﺒﻴﻌﺔ‬ ‫ﺘﻌﻤل ﻜﻤﺤﻔﺯ ﻝﻌﻤﻠﻴﺔ ﺍﻝﻤﻭﺕ ﺍﻝﻤﺒﺭﻤﺞ ﻝﺨﻁﻭﻁ ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬ ‫ ﺃﻭ ﺃﻥ ﺍﻝﺘﺎﺜﻴﺭ ﺍﻝﺴﺎﻡ ﻝﻠﻨﺒﺎﺕ ﻨﺎﺘﺞ ﻤﻥ ﻭﺠﻭﺩ ﺍﻝﻠﻜﺘﻴﻥ ﺍﻝﺫﻱ ﻝﻪ‬، (20) ‫ ﻭﻴﻌﻤل ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻋﻤﻠﻴﺔ ﺍﻨﻘﺴﺎﻡ ﻭﺘﻘﺩﻡ ﺩﻭﺭﺓ‬،‫ﻓﻌﺎﻝﻴﺔ ﻀﺩ ﻭﺭﻤﻴﺔ‬ ‫ ﻭﺍﻻﺒﺭﻴﻥ ﻤﻭﺍﺩ ﺴﺎﻤﺔ ﺘﻡ ﻋﺯﻝﻬﺎ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﻨﺒﺎﺕ‬،(8 ،7) ‫ﺍﻝﺨﻠﻴﺔ‬ ‫ﺍﻝﺨﺭﻭﻉ ﻭﺍﻝﺘﻲ ﻴﻌﻭﺩ ﻝﻬﺎ ﺍﻝﻨﺸﺎﻁ ﺍﻝﻀﺩ ﻭﺭﻤﻲ ﻭﺍﻝﻨﺎﺘﺞ ﻤﻥ ﻗﺩﺭﺓ ﻫﺫﻩ‬ .(9 ،8) ‫ﺍﻝﻤﻭﺍﺩ ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻋﻤﻠﻴﺔ ﺘﺨﻠﻴﻕ ﺍﻝﺒﺭﻭﺘﻴﻥ‬ ‫ﻜﻤﺎ ﺃﻜﺩﺕ ﺩﺭﺍﺴﺔ ﺃﺨﺭﻯ ﺍﻝﻔﻌﺎﻝﻴﺔ ﻀﺩ ﺍﻝﻭﺭﻤﻴﺔ ﻝﻠﻤﺴﺘﺨﻠﺹ ﺍﻝﻤﺎﺌﻲ‬ (A375) ‫ﻷﻭﺭﺍﻕ ﺍﻝﺨﺭﻭﻉ ﻋﻠﻰ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ ﻝﺴﺭﻁﺎﻥ ﺍﻝﺠﻠﺩ‬ ‫ ﻭﻗﺩ ﻜﺸﻔﺕ ﺩﺭﺍﺴﺎﺕ ﻤﺴﺘﻔﻴﻀﺔ ﺃﻥ ﻋﺩﺩﺍ ﻤﻥ ﺍﻝﻨﺒﺎﺘﺎﺕ‬. (21) ‫ﺘﺴﺘﻌﻤل ﻝﻠﻭﻗﺎﻴﺔ ﺍﻭ ﺍﻝﻌﻼﺝ ﻤﻥ ﺍﻝﺴﺭﻁﺎﻥ ﻻﺤﺘﻭﺍﺌﻬﺎ ﻋﻠﻰ ﺍﻝﻤﺠﺎﻤﻴﻊ‬ ‫ ﻭﺍﻥ ﺍﻝﻠﻜﺘﻴﻥ ﺍﻝﻤﻭﺠﻭﺩ ﻓﻲ ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻝﻪ ﻓﻌﺎﻝﻴﺔ ﻀﺩ‬، (6) ‫ﺍﻝﻔﻌﺎﻝﺔ‬ ‫( ﻭﻴﻌﻤل ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻋﻤﻠﻴﺔ ﺍﻨﻘﺴﺎﻡ ﻭﺘﻘﺩﻡ ﺩﻭﺭﺓ‬antitumor) ‫ﻭﺭﻤﻴﺔ‬ . (7,8) ( antiproliferative activities) ‫ﺍﻝﺨﻠﻴﺔ‬ ‫ﺍﻥ ﻭﺠﻭﺩ ﺍﻝﻔﻼﻓﻭﻨﺎﺕ ﻓﻲ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻜﺤﻭﻝﻲ ﺘﻌﻁﻲ ﻝﻠﻤﺴﺘﺨﻠﺹ‬ ‫ﺨﺎﺼﻴﺔ ﺤﻤﺎﻴﺔ ﺍﻝﺨﻼﻴﺎ ﻤﻥ ﺍﻝﺘﻠﻑ ﺍﻭ ﺍﻝﺘﻀﺭﺭ ﺍﻝﻤﺘﺴﺒﺏ ﻤﻥ ﺍﻝﺠﺫﻭﺭ‬ ‫ ( ﻭ ﺍﻥ‬22) ‫ﺍﻝﺤﺭﺓ ﺤﻴﺙ ﺘﻌﺘﺒﺭ ﺍﻝﻔﻼﻓﻭﻨﺎﺕ ﻤﻭﺍﺩ ﻤﻀﺎﺩﺓ ﻝﻼﻜﺴﺩﺓ‬ ‫ﻝﺒﻌﺽ ﺍﻝﻤﻭﺍﺩ ﺍﻝﻜﻴﻤﻴﺎﻭﻴﺔ ﺍﻝﻤﻭﺠﻭﺩﺓ ﻓﻲ ﺍﻝﻨﺒﺎﺘﺎﺕ ﻝﻬﺎ ﺘﺄﺜﻴﺭ ﻋﻠﻰ ﺒﻌﺽ‬ ‫ﺍﻝﺨﻼﻴﺎ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ ﻻﺤﺘﻭﺍﺌﻬﺎ ﻋﻠﻰ ﻤﻭﺍﺩ ﺘﺅﺜﺭ ﻓﻲ ﺁﻝﻴﺔ ﺍﻨﻘﺴﺎﻡ ﺍﻝﺨﻼﻴﺎ‬ ‫ ﺍﻭ ﺍﺤﺩ ﺍﻻﻨﺯﻴﻤﺎﺕ ﺍﻝﻤﻬﻤﺔ‬DNA‫ﻤﻥ ﺨﻼل ﺘﺄﺜﻴﺭﻫﺎ ﻓﻲ ﺘﻀﺎﻋﻑ ﺍل‬ ‫ﻓﻲ ﺍﻝﺘﻀﺎﻋﻑ ﻤﺜل ﻤﺎﺩﺓ ﺍﻝﻜﻭﻝﺠﺴﻴﻥ ﺍﻝﻤﺴﺘﺨﻠﺼﺔ ﻤﻥ ﻨﺒﺎﺕ‬ ‫ ﺍﻝﺘﻲ ﺘﻤﻨﻊ ﺘﻜﻭﻴﻥ ﺍﻻﻨﻴﺒﻴﺒﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ‬Colchicum cwtumnale ‫ ﻤﻤﺎ‬tubulin ‫ﻝﺨﻴﻭﻁ ﺍﻝﻤﻐﺯل ﻻﻨﻘﺴﺎﻡ ﺍﻝﺨﻼﻴﺎ ﺍﺫ ﺘﻤﻨﻊ ﺘﻜﻭﻴﻥ ﺒﺭﻭﺘﻴﻥ‬ ‫ﻴﺅﺩﻱ ﺍﻝﻰ ﺘﻭﻗﻑ ﺍﻝﻜﺭﻭﻤﻭﺴﻭﻤﺎﺕ ﻓﻲ ﺍﻝﻁﻭﺭ ﺍﻻﺴﺘﻭﺍﺌﻲ ﻭﺒﺎﻝﺘﺎﻝﻲ‬ ‫( ﻜﺫﻝﻙ ﺍﻝﺤﺎل ﻤﻊ ﻤﺎﺩﺓ‬20) ‫ﻴﺅﺩﻱ ﺍﻝﻰ ﺘﻭﻗﻑ ﺍﻻﻨﻘﺴﺎﻡ ﺍﻝﺨﻠﻭﻱ‬ ‫ ﻭﻫﻲ ﻤﻥ ﺍﻝﻘﻠﻭﻴﺩﺍﺕ ﺍﻝﺘﻲ ﺘﻌﻤل ﻋﻠﻰ‬Benzo phenanthridin ‫ﺘﺜﺒﻴﻁ ﺍﻻﻨﻘﺴﺎﻤﺎﺕ ﺍﻝﻤﺘﻌﺩﺩﺓ ﻝﺒﻌﺽ ﺍﻨﻭﺍﻉ ﺨﻼﻴﺎ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬ ‫ﻝﻼﻨﺴﺎﻥ ﻭﺍﻝﻤﻘﺎﻭﻤﺔ ﻝﻠﻌﻘﺎﻗﻴﺭ ﺍﻝﻁﺒﻴﺔ ﻭﺩﻓﻌﻬﺎ ﺍﻝﻰ ﺍﻝﻤﻭﺕ ﺍﻝﻤﺒﺭﻤﺞ‬ ‫( ﻝﺫﺍ ﻋﺩﺕ ﺒﻌﺽ ﺍﻝﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻝﻨﺒﺎﺘﻴﺔ ﺍﻝﻁﺒﻴﺔ‬20) Apoptosis Anti- ‫ ﻭﺍﻝﺴﺭﻁﺎﻥ‬Anti-carcinogens ‫ﻜﻤﻀﺎﺩﺍﺕ ﻝﻠﺘﺴﺭﻁﻥ‬ .(23) cancer ‫ﺘﻭﻓﺭ ﺍﻝﻔﻼﻓﻭﻨﻭﻴﺩ ﺍﻝﻤﻭﺠﻭﺩﺓ ﻓﻲ ﺍﻝﻤﺴﺘﺨﻠﺹ ﺍﻝﻨﺒﺎﺘﻲ ﻝﻠﺨﺭﻭﻉ ﺍﻝﺤﻤﺎﻴﺔ‬ ‫ﻀﺩ ﺍﻝﺴﺭﻁﺎﻥ ﻭﺍﻝﺘﺴﺭﻁﻥ ﻋﻥ ﻁﺭﻴﻕ ﺘﺜﺒﻴﻁ ﺍﻝﻀﺭﺭ ﺍﻝﺘﺄﻜﺴﺩﻱ ﺤﻴﺙ‬ ‫( ﻭﺍﻥ ﻫﺫﻩ‬strong antioxidant) ‫ﺘﻌﺘﺒﺭ ﻤﻀﺎﺩ ﺍﻜﺴﺩﺓ ﻓﻌﺎل ﻭﻗﻭﻱ‬ ‫ ﻝﻬﺎ ﺘﺎﺜﻴﺭﺍﺕ ﻤﻀﺎﺩﺓ ﻝﻼﻜﺴﺩﺓ ﻤﻘﺎﺭﻨﺔ ﺒﻜل‬flavonoids ‫ﺍﻝﻤﺭﻜﺒﺎﺕ‬ ‫ ﻓﻬﻲ ﺘﺤﻤﻲ ﻭﺘﻤﻨﻊ ﺍﻝﻀﺭﺭ ﺍﻝﺫﻱ ﻴﻁﺭﺍ ﻋﻠﻰ‬E ‫ ﻭ‬C ‫ﻤﻥ ﻓﺘﺘﺎﻤﻴﻥ‬ ‫( ﻤﻥ ﺍﻝﺠﺫﻭﺭ ﺍﻝﺤﺭﺓ ﻨﺘﻴﺠﺔ ﻝﺤﺩﻭﺙ‬cellular damage) ‫ﺍﻝﺨﻼﻴﺎ‬ ،22) ‫ﺤﺎﻝﺔ ﻋﺩﻡ ﺘﻭﺍﺯﻥ ﺒﻴﻥ ﻋﻤﻠﻴﺔ ﺍﻻﻜﺴﺩﺓ ﻭﻨﺘﺎﺌﺞ ﺭﺩ ﺍﻝﻔﻌل ﻝﻼﻜﺴﺩﺓ‬ .(24

‫ﺍﻻﺴﺘﻨﺘﺎﺠﺎﺕ‬ ‫ﻨﺒﺎﺕ ﺍﻝﺨﺭﻭﻉ ﻤﻥ ﺍﻝﻨﺒﺎﺘﺎﺕ ﺍﻝﻁﺒﻴﺔ ﺍﻝﻤﻬﻤﺔ ﻭﺍﻝﺘﻲ ﻤﻥ ﺍﻝﻤﻤﻜﻥ‬ ‫ﺍﺴﺘﺨﺩﺍﻤﻬﺎ ﻓﻲ ﻤﻌﺎﻝﺠﺔ ﺍﻝﺴﺭﻁﺎﻥ ﻤﻥ ﺨﻼل ﺘﺄﺜﻴﺭﻩ ﺍﻝﺘﺜﺒﻴﻁﻲ ﻭﺍﻝﺴﻤﻲ‬ .‫ﺍﻝﻘﺎﺘل ﻋﻠﻰ ﺍﻝﺨﻁﻭﻁ ﺍﻝﺨﻠﻭﻴﺔ ﺍﻝﺴﺭﻁﺎﻨﻴﺔ‬

‫ﺍﻝﻤﺼــﺎﺩﺭ‬ 1. Sahu AN.; Damiki L.; Nilanjan G. and Dubey S. (2008). Phytopharmacological review of Boerhaaviadiffusa Linn. (Punarnava). Pharmacog. Rev .2: 14–22. 2. Dhuna V.; Bains JS.; Kamboj SS.; Singh J. and Shanmugavel SAK. (2005). Purification and characterization of a lectin from Arisaematortuosum Schott having in-vitro anticancer activity against

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assembly dynamic through tubulin binding. A possible mechanism for its antiproliferative activity. J.FEBSIO. 2139-2150. 21. Islam T.; Bakshi M. and Sharma M.(2011). Assessment of cytotoxic potential of aqueous extract of Ricinus communis leaves against melanoma cancer cell lines (A375). Inv. Impact. Ethnopharcol. 21: 212-216. 22. Omar BA.; Flores SC. and McCord JM. (1992). Superoxide dismutase pharmacological developments and applications. Adv. Pharmacol. 23: 109-161. 23. Gurib-Fakim A.(2005). Medicinal plants. Tradition of yesterday and drugs tomorrow. Molec. Asp. Med. 19: 136-139. 24. Ali M. (1998). Text Book of Pharmacognosy. CBS Publishers and Distributors, New Delhi. 2nd ed. Pp.151-153.

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International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735

Vol. 10, No.2, June 2015

(Shortened cake ) ‫ﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺇﻀﺎﻓﺔ ﺒﺫﻭﺭ ﺍﻝﺸﻤﺭ ﺍﻝﻤﻁﺤﻭﻨﺔ ﻓﻲ ﺍﻝﺨﻭﺍﺹ ﺍﻝﻨﻭﻋﻴﺔ ﻝﻠﻜﻴـﻙ ﺍﻝﻤﻘـﺼﺭ‬ ‫ﺍﻝﻤﺼﻨﻊ ﻤﺨﺘﺒﺭﻴﺎ ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻝﻤﻭﺍﺩ ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﺼﻨﺎﻋﻴﺔ‬ ‫ﺃﻭﺱ ﻫﻼل ﺠﺎﺴﻡ ﺍﻝﻌﺎﻨﻲ‬ ‫ ﺠﻤﻬﻭﺭﻴﺔ ﺍﻝﻌﺭﺍﻕ‬/ ‫ ﺒﻐﺩﺍﺩ‬/ ‫ ﻭﺯﺍﺭﺓ ﺍﻝﺘﻌﻠﻴﻡ ﺍﻝﻌﺎﻝﻲ ﻭﺍﻝﺒﺤﺙ ﺍﻝﻌﻠﻤﻲ‬/ ‫ﺩﺍﺌﺭﺓ ﺍﻝﺒﺤﺙ ﻭﺍﻝﺘﻁﻭﻴﺭ‬ [email protected] :‫ﺍﻝﺒﺭﻴﺩ ﺍﻻﻝﻴﻜﺘﺭﻭﻨﻲ‬

‫ﺍﻝﻤﻠﺨﺹ ﺒﺎﻝﻠﻐﺔ ﺍﻝﻌﺭﺒﻴﺔ‬ Shortened ‫( ﺍﻝﻤﻁﺤﻭﻨﺔ ﺒﻨﺴﺏ ﻤﺨﺘﻠﻔﺔ ﻓﻲ ﺍﻝﺨﻭﺍﺹ ﺍﻝﺤﺴﻴﺔ ﻭﺍﻝﻔﻴﺯﻴﺎﺌﻴﺔ ﻭﺍﻝﻤﻴﻜﺭﻭﺒﻴﺔ ﻝﻠﻜﻴﻙ ﺍﻝﻤﻘﺼﺭ‬Fv) Foeniculum vulgara ‫ﻫﺩﻓﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﺤﺎﻝﻴﺔ ﺇﻝﻰ ﻤﻌﺭﻓﺔ ﺘﺄﺜﻴﺭ ﺇﻀﺎﻓﺔ ﺒﺫﻭﺭ ﺍﻝﺸﻤﺭ‬ ‫ ﺃﻅﻬﺭﺕ ﻨﺘﺎﺌﺞ ﺍﻝﺘﻘﻴﻴﻡ ﺍﻝﺤﺴﻲ ﺃﻥ‬. ( (N) Nipagin ‫( ﻭﺍﻝﻨﻴﺒﺎﺠﻴﻥ‬Sb)Sodium benzoate ‫ ( ﺍﻝﻤﺼﻨﻊ ﻤﺨﺘﺒﺭﻴﺎ ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻝﻤﻭﺍﺩ ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﺼﻨﺎﻋﻴﺔ ) ﺒﻨﺯﻭﺍﺕ ﺍﻝﺼﻭﺩﻴﻭﻡ‬cake ) ‫ ﻜﻤﺎ ﻝﻭﺤﻅ ﻭﺠﻭﺩ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ‬. ‫ ) ﺍﻝﻤﻘﺎﺭﻨﺔ ﺒﺩﻭﻥ ﺍﻀﺎﻓﺔ ﻤﻭﺍﺩ ﺤﺎﻓﻅﺔ ( ﺤﺼﻠﺕ ﻋﻠﻰ ﺩﺭﺠﺔ ﺘﻘﻴﻴﻡ ﺃﻋﻠﻰ ﻤﻌﻨﻭﻴﺎ ﻤﻥ ﺒﺎﻗﻲ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻻﺨﺭﻯ ﻤﻥ ﺤﻴﺙ ﺼﻔﺔ ﺍﻝﺘﻘﺒل ﺍﻝﻌﺎﻡ‬A ‫ﺍﻝﻤﻌﺎﻤﻠﺔ‬ ‫ ( ﻋﻠﻰ ﻨﺴﺒﺔ ﻭﺯﻥ ﺍﻋﻠﻰ ﻤﻘﺎﺭﻨﺔ ﻤﻥ ﺒﺎﻗﻲ ﺍﻝﻤﻌﺎﻤﻼﺕ‬N ‫ ﻤﻥ‬% 0.2 ) C ‫ ﺤﻴﺙ ﺤﺼﻠﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ‬، ‫ ﻭﻤﻥ ﺤﻴﺙ ﻨﺴﺒﺔ ﺍﻝﻔﻘﺩ ﺒﺎﻝﻭﺯﻥ ﺒﻌﺩ ﺍﻝﺸﻭﻱ‬. ‫ﺍﻝﻤﻌﺎﻤﻼﺕ ﻤﻥ ﺤﻴﺙ ﺠﻤﻴﻊ ﺍﻝﺼﻔﺎﺕ ﺍﻝﺤﺴﻴﺔ‬ ‫ ﻭﺒﺨﺼﻭﺹ ﺍﻝﻔﺤﻭﺼﺎﺕ ﺍﻝﻤﻴﻜﺭﻭﺒﻴﺔ ﻤﻥ ﺤﻴﺙ ﺍﻝﻌﺩ ﺍﻝﻜﻠﻲ ﻝﻠﻤﺴﺘﻌﻤﺭﺍﺕ ﺍﻝﺒﻜﺘﺭﻴﺔ‬. ( Standing Height ) ‫ ﻭﻝﻡ ﺘﻅﻬﺭ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻤﻥ ﺤﻴﺙ ﺤﺠﻡ ﺍﻝﻜﻴﻙ‬. ‫ﺍﻻﺨﺭﻯ‬ 5 ‫ ﻭ‬3 ‫ ﻤﺩﺓ‬º‫ ﻡ‬30 ‫ ﺍﺴﺒﻭﻉ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬2 ‫ ﻭﺨﺯﻥ ﺍﻝﻜﻴﻙ ﻤﺩﺓ‬º‫ ﻡ‬14 ‫ ﺴﺎﻋﺔ ﻓﻲ‬3 ‫ ﻭﺍﻻﺫﺍﺒﺔ ﻤﺩﺓ‬º‫ ﻡ‬18 –‫ ﻝﻭﺤﻅ ﻭﺠﻭﺩ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻋﻨﺩ ﺨﺯﻥ ﺍﻝﻜﻴﻙ ﻓﻲ‬. ‫ﻭﺍﻝﻔﻁﺭﻴﺔ‬ º‫ ﻡ‬30 ‫ ﺍﺴﺒﻭﻉ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬2 ‫ﻤﺩﺓ‬º ‫ ﻡ‬19 – 14 ‫ ( ﻋﻨﺩ ﺨﺯﻥ ﺍﻝﻜﻴﻙ ﻓﻲ‬Fv ‫ ﻤﻥ‬%1 ) E ‫ ﺤﻴﺙ ﺒﻠﻐﺕ ﺒﺄﻋﻠﻰ ﺍﻋﺩﺍﺩﻫﺎ ﻓﻲ ﺍﻝﻤﻌﺎﻤﻠﺔ‬. ‫ ﺍﻴﺎﻡ ﻤﻥ ﺤﻴﺙ ﺍﻝﻌﺩ ﺍﻝﻜﻠﻲ ﻝﻠﻔﻁﺭﻴﺎﺕ‬5 ‫ ﺍﺴﺒﻭﻉ ﻤﺩﺓ‬1‫ﺃﻴﺎﻡ ﻭ‬ ‫ ﻭﻝﻭﺤﻅ ﺃﻥ‬. ‫ ﺍﻴﺎﻡ ﻤﻥ ﺤﻴﺙ ﺍﻝﻌﺩﺩ ﺍﻝﻜﻠﻲ ﻝﻠﻔﻁﺭﻴﺎﺕ‬3 ‫ ﻤﺩﺓ‬º‫ ﻡ‬30 ‫ ﺍﺴﺒﻭﻉ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬1 ‫ ﻤﺩﺓ‬º‫ ﻡ‬16 – 14 ‫ ﻭﻝﻡ ﺘﻅﻬﺭ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻋﻨﺩ ﺨﺯﻥ ﺍﻝﻜﻴﻙ ﻓﻲ‬. ‫ ﺃﻴﺎﻡ‬5 ‫ﻤﺩﺓ‬ ( Sb ‫ ﻤﻥ‬% 0.2 ) B ، A ‫ ( ﻭﻝﻡ ﺘﻅﻬﺭ ﻓﻲ ﺍﻝﻤﻌﺎﻤﻼﺕ‬Fv ‫ ﻤﻥ‬% 2) F ‫ ( ﻭﺍﻗﻠﻬﺎ ﻓﻲ ﺍﻝﻤﻌﺎﻤﻠﺔ‬Fv ‫ ﻤﻥ‬% 0.5) D ‫ﺃﻋﺩﺍﺩ ﺍﻝﻤﺴﺘﻌﻤﺭﺍﺕ ﺍﻝﺒﻜﺘﺭﻴﺔ ﺍﻝﻬﻭﺍﺌﻴﺔ ﺒﻠﻐﺕ ﺍﻋﻠﻰ ﻗﻴﻤﺔ ﻓﻲ ﺍﻝﻤﻌﺎﻤﻠﺔ‬ 18 - ‫ ﻭﻝﻡ ﺘﻅﻬﺭ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻓﻲ ﺍﻝﻜﻴﻙ ﺍﻝﻤﺨﺯﻥ ﻓﻲ‬. ‫ ﺴﺎﻋﺔ‬24 ‫ ﻤﺩﺓ‬º‫ ﻡ‬37 ‫ ﺴﺎﻋﺔ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬3 ‫ ﻤﺩﺓ‬º‫ ﻡ‬25 ‫ ﻭﺍﻻﺫﺍﺒﺔ ﻓﻲ‬º‫ ﻡ‬18 - ‫ ﻓﻲ ﺍﻝﻜﻴﻙ ﺍﻝﻤﺨﺯﻥ ﻓﻲ‬C ‫ﻭ‬ ‫ ﺒﻴﻨﻤﺎ ﻝﻡ ﺘﻅﻬﺭ ﺍﻝﺒﻜﺘﺭﻴﺎ ﻭﻝﻡ ﺘﻅﻬﺭ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻓﻲ ﺍﻝﻜﻴﻙ ﺍﻝﻤﺨﺯﻥ‬. Nutrient agar ‫ ﺴﺎﻋﺔ ﻋﻨﺩ ﺍﻝﺯﺭﻉ ﺒﺎﻝﻭﺴﻁ‬48 ‫ ﻤﺩﺓ‬º‫ ﻡ‬37 ‫ ﺴﺎﻋﺔ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬3 ‫ ﻭﺍﻻﺫﺍﺒﺔ ﻤﺩﺓ‬º‫ﻡ‬ ‫ ﻭﻜﺫﻝﻙ ﻝﻡ ﺘﻅﻬﺭ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻋﻨﺩ ﺨﺯﻥ‬. Macconkey agar ‫ ﺴﺎﻋﺔ ﻋﻨﺩ ﺍﻝﺯﺭﻉ ﺒﺎﻝﻭﺴﻁ‬48 ‫ ﻭ‬24 ‫ ﻤﺩﺓ‬º‫ ﻡ‬37 ‫ ﺴﺎﻋﺔ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬3 ‫ ﻭﺍﻻﺫﺍﺒﺔ ﻤﺩﺓ‬º‫ ﻡ‬18 - ‫ﻓﻲ‬ %3 ) G ‫ ﻭ‬C ‫ ﺒﻴﻨﻤﺎ ﻅﻬﺭﺕ ﺒﻜﺘﺭﻴﺎ ﻫﻭﺍﺌﻴﺔ ﺒﺄﻋﻠﻰ ﺃﻋﺩﺍﺩﻫﺎ ﻓﻲ ﺍﻝﻤﻌﺎﻤﻠﺔ‬. Macconkey agar ‫ ﺴﺎﻋﺔ ﻋﻨﺩ ﺍﻝﺯﺭﻉ ﺒﺎﻝﻭﺴﻁ‬48‫ ﻭ‬24 ‫ ﻤﺩﺓ‬º‫ ﻡ‬37 ‫ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬º‫ ﻡ‬27 – 25 ‫ﺍﻝﻜﻴﻙ ﻓﻲ‬ ‫ ﻭﻝﻭﺤﻅ ﻭﺠﻭﺩ ﻓﺭﻭﻕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ‬. Nutrient agar ‫ ﺴﺎﻋﺔ ﻋﻠﻰ ﺍﻝﺘﻭﺍﻝﻲ ﻋﻨﺩ ﺍﻝﺯﺭﻉ ﺒﺎﻝﻭﺴﻁ‬48 ‫ ﻭ‬24 ‫ ﻤﺩﺓ‬º‫ ﻡ‬37 ‫ ﺍﺴﺒﻭﻉ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬1 ‫ ﻤﺩﺓ‬º‫ ﻡ‬27 – 25 ‫ ( ﻋﻨﺩ ﺨﺯﻥ ﺍﻝﻜﻴﻙ ﻓﻲ‬Fv ‫ﻤﻥ‬ ‫ ﺤﻴﺙ ﺒﻠﻐﺕ‬.agar Macconkey ‫ ﻭ‬Nutrient agar ‫ ﺴﺎﻋﺔ ﻭﻋﻨﺩ ﺍﻝﺯﺭﻉ ﺒﺎﻝﻭﺴﻁﻴﻥ‬48 – 24 ‫ ﻤﺩﺓ‬º ‫ ﻡ‬37 ‫ ﺍﺴﺒﻭﻉ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬2 ‫ ﻤﺩﺓ‬º‫ ﻡ‬30 – 27 ‫ﺠﻤﻴﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﻋﻨﺩ ﺨﺯﻥ ﺍﻝﻜﻴﻙ ﻓﻲ‬ .‫ ﺴﺎﻋﺔ‬48 ‫ ﻤﺩﺓ‬º‫ ﻡ‬37 ‫ ﻭﺍﻝﺤﻀﻥ ﻓﻲ‬Macconkey agar ‫ ﻋﻨﺩ ﺍﻝﺯﺭﻉ ﺏ‬B ‫ﺍﻋﻠﻰ ﺃﻋﺩﺍﺩﻫﺎ ﻓﻲ ﺍﻝﻤﻌﺎﻤﻠﺔ‬ .‫ ﺒﺫﻭﺭ ﺍﻝﺸﻤﺭ‬،‫ ﺍﻝﻜﻴﻙ ﺍﻝﻤﻘﺼﺭ‬:‫ﺍﻝﻜﻠﻤﺎﺕ ﺍﻝﻤﻔﺘﺎﺤﻴﺔ‬

Study the effect of adding powder foeniculum vulgara L. seed on quality properties of laboratory shortened cake in comparison with industrial preservatives Aws H. J. Al- Ani Dept. of Research and Development / Ministry of Higher Education and Scientific Research /Baghdad / Republic of Iraq

ABSTRACT The aim of this study was to conducted the effect of addition of powder Foeniculum vulgar L. seed in different percentages on the physical ,sensory and microbial properties of the shortened cake in comparison with industrial preservatives (Sodium Benzoate) (Sb) and Nipagin (N). The results of sensory evaluation revealed that treatment A ( in comparison without adding preservatives ) were significantly gained higher score of overall acceptance than all other treatments . The results showed significant differences between all treatments in the sensory evaluation and in the weight loss percentage of cake. Results also showed that treatment C(0.2 % of N) were significantly gained higher score than all other treatments and did not show significant differences between all treatments in the size of cake ( Standing Height) . In regards with microbial tests of fungi and bacteria , It was found that there were significant differences between all treatments of storing cake at - 18ºC and dissolution for 3 hrs at 14ºC and storing cake for 2 weeks and incubation at 30ºC for 3 to 5 days and 1 week for 5 days , It was of the total fungi . It was found that the highest fungi number in treatment E (1% of Fv) when storing cake at 14 - 19ºC for 2 weeks and incubation at 30ºC for 5 days. Did not show significant differences between all treatments of storing cake at 14 – 16ºC for 1 week and incubation at 30ºC for 3 days; it was of the total fungi. And it was found that highest the aerobic bacterial number in treatment of D (0.5% of Fv) and the lowest in treatment F (2% of Fv) did not appeared in treatments A, B (0.2% of Sb) and C in storing Cake at – 18ºC and dissolution at 25ºC for 3 hours, and at 37ºC for 24 hours. And did Not show significant differences between all treatments of storing cake at - 18ºC and dissolution for 3 hours, and incubation at 37ºC for 48 hours when the culture in middle Nutrient agar. While did not show the bacteria and did not show significant differences between all treatments of storing cake at - 18ºC and dissolution for 3 hours, and incubation at 37ºC for 24 to 48 hours when the culture in middle Macconkey agar. And also did not show significant differences between all treatments of storing cake at 25 - 27ºC and incubation at 37ºC for 24 to 48 hours when the culture in middle Macconkey agar. while found that the highest aerobic bacterial number in treatment , C and G (3% of Fv) when storing cake at 25 27ºC for 1 week and incubation at 37ºC for 24 and 48 hours when the culture in middle Nutrient agar and found significant differences between all treatments of storing cake at 27 - 30ºC for 2 weeks and incubation at 37ºC for 24 - 48 hours when the culture in middles Nutrient agar and Macconkey agar . in addition, it was found that the highest numbers in treatment B were obtained when the culture in middle Macconkey agar and incubation at37ºC for 48 hours.

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‫‪Vol. 10, No.2, June 2015‬‬

‫‪International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735‬‬

‫ﺍﻝﻤﻘﺩﻤــــﺔ‬ ‫ﻴﻭﻓﺭ ﺍﺴﺘﺨﺩﺍﻡ ﺍﻝﻤـﻭﺍﺩ ﺍﻝﺤﺎﻓﻅـﺔ ‪ Preservatives‬ﻓـﻲ ﺍﻷﻏﺫﻴـﺔ‬ ‫ﺍﻝﻤﺼﻨﻌﺔ ﻝﻠﻤﺴﺘﻬﻠﻜﻴﻥ ﻏﺫﺍﺀ ﻤﺘﻨﻭﻋﺎ ﻭﻭﺍﺴﻌﺎ ﻭﻤﻨﺎﺴﺒﺎ ﻓﻲ ﺠﻤﻴﻊ ﺃﻭﻗﺎﺕ‬ ‫ﺍﻝﺴﻨﺔ ‪ ،‬ﺒﺎﻹﻀﺎﻓﺔ ﺇﻝﻰ ﺍﻝﻘﺩﺭﺓ ﻋﻠﻰ ﺇﻴﻘﺎﻑ ﺍﻝﻨﻤﻭ ﺍﻝﻤﻴﻜﺭﻭﺒﻲ ﻭﻴﻜـﻭﻥ‬ ‫ﺘﺄﺜﻴﺭﻫﺎ ﻋﻠﻰ ﺍﻝﺤﻔﺎﻅ ﻋﻠﻰ ﺍﻝﻘﻴﻤﺔ ﺍﻝﻐﺫﺍﺌﻴﺔ ﺃﻗل ﻤﺎ ﻴﻤﻜﻥ )‪ . (1‬ﻭﻤـﻥ‬ ‫ﻫﺫﻩ ﺍﻝﻤﻭﺍﺩ ﺍﻝﺤﺎﻓﻅﺔ ﺤﺴﺏ ﺍﻝﻤﺘﻭﺍﻓﺭ ﻓﻲ ﺍﻷﺴﻭﺍﻕ ﺍﻝﻤﺤﻠﻴﺔ ‪ :‬ﺒﻨﺯﻭﺍﺕ‬ ‫ﺍﻝﺼﻭﺩﻴﻭﻡ ‪ ، Sodium benzoate‬ﺍﻝـﺫﻱ ﻴـﺴﺘﺨﺩﻡ ﻓـﻲ ﺤﻔـﻅ‬ ‫ﺍﻝﻤﺸﺭﻭﺒﺎﺕ ﻭﺒﻌﺽ ﺍﻝﻤﻭﺍﺩ ﺍﻝﻐﺫﺍﺌﻴﺔ‪ ،‬ﻭﻝﻜﻥ ﻝﻴﺱ ﻝﻪ ﻗﻴﻤﺔ ﻏﺫﺍﺌﻴﺔ‪ ،‬ﻭﻫﻭ‬ ‫ﻤﻥ ﺍﻝﻤﻭﺍﺩ ﺍﻝﻤﻀﺎﻓﺔ ﻝﺤﻔﻅ ﺍﻷﻁﻌﻤﺔ ﻤﻥ ﺍﻝﺜﻠﻭﺙ ﻝﻔﺘﺭﺓ ﻤﻌﻴﻨﺔ ﺤـﺴﺏ‬ ‫ﻅﺭﻭﻑ ﺍﻝﺨﺯﻥ ﻭﻨﻭﻉ ﺍﻝﻐﺫﺍﺀ‪ ،‬ﻭﺘﺤﺩﺩ ﺍﻝﻜﻤﻴﺎﺕ ﺍﻝﻤﺴﻤﻭﺡ ﺒﻬﺎ ﺤـﺴﺏ‬ ‫ﻤﻨﻅﻤﺔ ﺍﻝﺼﺤﺔ ﺍﻝﻌﺎﻝﻤﻴﺔ ﻭﻤﻨﻅﻤﺔ ﺍﻷﻏﺫﻴﺔ ﻭﺍﻝﺯﺭﺍﻋﺔ ﺒﻤﺎ ﻤﻘـﺩﺍﺭﻩ ‪6‬‬ ‫ﻤﻠﻐﻡ ‪ /‬ﻜﻐﻡ ‪ ،‬ﺤﻴﺙ ﺇﻥ ﺍﻝﺠﺭﻋﺎﺕ ﺍﻝﻌﺎﻝﻴـﺔ ﺘـﺅﺩﻱ ﺇﻝـﻰ ﺘﻜـﻭﻴﻥ‬ ‫ﺍﻝــﺴﺭﻁﺎﻨﺎﺕ‪ ،‬ﻭﻜــﺫﻝﻙ ﺩﺍﺀ ﺍﻝﺭﺒــﻭ ﻋﻨــﺩ ﺍﻷﻁﻔــﺎل )‪. (2‬‬ ‫ﻭﺍﻝﻨﻴﺒﺎﺠﻴﻥ ‪ Nipagin‬ﺼﻴﻐﺘﻪ ﺍﻝﺘﺭﻜﻴﺒﻴﺔ ‪ ، C8H8O3‬ﻭﻝﻪ ﺃﺴـﻤﺎﺀ‬ ‫ﻋﺩﻴﺩﺓ‪ ،‬ﻤﻨﻬﺎ‪:‬‬ ‫‬‫‪Methyl paraben.‬‬ ‫‬‫‪Methyl p – hydroxyl benzoate‬‬ ‫‬‫‪Methyl parahydroxybenzoate‬‬ ‫‬‫‪Nipagin M‬‬ ‫‬‫‪E Number E 218 (3).‬‬ ‫ﻭﺘﻌﺩ ﻤﻥ ﺍﻝﻤﻭﺍﺩ ﺍﻝﻤﻀﺎﻓﺔ ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﺼﻨﺎﻋﻴﺔ ﻭﺍﻝﻤـﻀﺎﺩﺓ ﻝﻸﻜـﺴﺩﺓ‬ ‫ﻭﺍﻝﺒﻜﺘﻴﺭﻴﺎ ﻭﺍﻝﻔﻁﺭﻴﺎﺕ ﻓﻲ ﺍﻷﻏﺫﻴﺔ ﺍﻝﻤﺼﻨﻌﺔ‪ ،‬ﻭﻫـﻲ ﻤـﻭﺍﺩ ﺁﻤﻨـﺔ‬ ‫ﻝﻸﻏﺫﻴﺔ ﺍﻝﺘﻲ ﻴﺘﻡ ﺘﻨﺎﻭﻝﻬﺎ ﻋﻥ ﻁﺭﻴﻕ ﺍﻝﻔﻡ ﻭﺍﻝﺤﻘـﻥ ﻓـﻲ ﺤﻴﻭﺍﻨـﺎﺕ‬ ‫ﺍﻝﺘﺠﺎﺭﺏ )‪ . (4‬ﺃﻤﺎ ﺍﻝﻤﻭﺍﺩ ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﻁﺒﻴﻌﻴﺔ ﻓﻬﻲ ﻜﺜﻴﺭﺓ ﻭﻤﻨﻬﺎ ﺃﺤﺩ‬ ‫ﺍﻝﺘﻭﺍﺒل ﻭﻫﻭ ﺍﻝﺸﻤﺭ ﺍﻝﺫﻱ ﻴﻌﺩ ﻤﻥ ﺍﻝﻨﺒﺎﺘﺎﺕ ﺍﻝﻤﺸﻬﻭﺭﺓ ﻭﺍﻝﻤﻌﺭﻭﻓـﺔ‪،‬‬ ‫ﻭﻫﻭ ﻨﺒﺎﺕ ﻋﺸﺒﻲ ﻋﻁﺭﻱ ﻤﻌﻤﺭ ﻤﻥ ﺍﻝﻔﺼﻠﻴﺔ ﺍﻝﺨﻴﻤﻴﺔ )‪ ، ( 5‬ﻴﻨﺘﻤﻲ‬ ‫ﺍﻝﻰ ﻨﺒﺎﺕ ﺍﻝﺤﻭﺫﺍﻥ ﻭﻫﻭ ﻨﻭﻉ ﻤﻥ ﺃﻨﻭﺍﻉ ﺍﻝﺨﻀﺭﺍﻭﺍﺕ ﻤـﻥ ﻓـﺼﻠﻴﺔ‬ ‫ﺍﻝﺒﻘﺩﻭﻨﺱ )‪ ، (6‬ﺍﺴﻤﻪ ﺍﻝﻌﻠﻤﻲ ﻝﻪ‪ Foeniculum Vulgare‬ﻭﺍﻻﺴﻡ‬ ‫ﺍﻻﻨﺠﻠﻴﺯﻱ ‪ ، Fennel‬ﻜﻤﺎ ﻝﻪ ﺃﺴﻤﺎﺀ ﻋﺩﻴﺩﺓ ﻝﺩﻯ ﺸﻌﻭﺏ ﺍﻝﻌﺎﻝﻡ ﻭﻫﻲ‪:‬‬ ‫ﺍﻝﺸﻤﺭﺓ ﻓﻲ ﺍﻝﺸﺎﻡ‪ ،‬ﻭﺍﻝﺴﻨﻭﺕ ﻓﻲ ﺍﻝﻤﻐﺭﺏ ﺍﻝﻌﺭﺒﻲ ﻭﻤﺼﺭ ﻭﺍﻝﻴﻭﻨﺎﻥ‪،‬‬ ‫ﻭﺸﻤﺎﺭﻱ ﻝﺩﻯ ﺍﻝﻔﺭﺍﻋﻨﺔ ﻗﺩﻴﻤﺎ‪ ،‬ﻭﺸﺎﻤﺎﺭﻥ ﻭﺒﺴﺒﺎﺱ ﻭﺍﻝـﺴﻨﺎ ﻭﺍﻝﺤﺒـﺔ‬ ‫ﺍﻝﺤﻠﻭﺓ ﻭﺍﻝﺸﻤﺎﺭ ﻭﺤﺒﺔ ﺍﻝﺤﻼﻭﺓ ﺍﻝﻌﺭﺒﻴﺔ ﻭﻏﻴﺭﻫﺎ )‪ ، (7‬ﻜﻤـﺎ ﻴﻁﻠـﻕ‬ ‫ﻋﻠﻴﻪ ﻓﻲ ﺴﻭﺭﻴﺎ ﺸﻭﻤﺭ ﻭﻴﺤﺘﻭﻱ ﻋﻠﻰ ﻤﻭﺍﺩ ﻓﻌﺎﻝﺔ ﻭﻫـﻲ ﺍﻝﺯﻴـﺕ‬ ‫ﺍﻝﻁﻴﺎﺭ ﻭﻤﺎﺩﺓ ﺍﻷﻨﻴﺘﻭل ‪ Anethol‬ﻭﺍﻝﻔﺎﻨﺸﻭﻥ ‪.(8) Fenchon‬‬ ‫ﻭﺍﻝﺠﺯﺀ ﺍﻝﻤﺴﺘﺨﺩﻡ ﻤﻨﻪ ﻫﻲ ﺍﻝﺒﺫﻭﺭ ﺍﻝﺘﻲ ﺘﻜﻭﻥ ﻋﻠﻰ ﺸـﻜل ﺤﺒﻴﺒـﺎﺕ‬ ‫ﻁﻭﻝﻴﺔ ﺼﻔﺭﺍﺀ ﺭﻤﺎﺩﻴﺔ ﻤﺨﻁﻁﺔ )‪ ، (7‬ﻭﻤﻭﻁﻨﻪ ﺍﻷﺼـﻠﻲ ﺤـﻭﺽ‬ ‫ﺍﻝﺒﺤﺭ ﺍﻷﺒﻴﺽ ﺍﻝﻤﺘﻭﺴﻁ‪ ،‬ﻭﻗﺩ ﺍﻨﺘﺸﺭﺕ ﺯﺭﺍﻋﺘﻪ ﻓﻲ ﺠﻤﻴـﻊ ﺃﻨﺤـﺎﺀ‬ ‫ﺍﻝﻌﺎﻝﻡ ‪ ،‬ﻭﻴﻌﺩ ﺍﻝﺸﻤﺭ ﻤﻀﺎﺩﺍ ﻝﻠﻤﻴﻜﺭﻭﺒﺎﺕ ﻭﻤﻌﺎﻝﺠﺎ ﻝﻠﻐـﺎﺯﺍﺕ )‪، (9‬‬ ‫ﻭﻝﻴﺴﺕ ﻝﻪ ﺁﺜﺎﺭ ﺠﺎﻨﺒﻴﺔ ﻋﻨﺩ ﺘﻨﺎﻭﻝﻪ ﺒﺎﻋﺘﺩﺍل ﻤﺎ ﻋﺩﺍ ﺒﻌﺽ ﺍﻝﻤﺤـﺎﺫﻴﺭ‬ ‫ﻝﻠﻤـــﺭﺃﺓ ﺍﻝﺤﺎﻤـــل )‪ . (10‬ﻭﻴﺤﺘـــﻭﻱ ﺍﻝـــﺸﻤﺭ ﻋﻠـــﻰ‬ ‫ﻓﻴﺘﺎﻤﻴﻨﺎﺕ ‪ A‬ﻭ‪ B‬ﻭ ‪ C‬ﻭﺍﻝﻜﺎﻝﺴﻴﻭﻡ ﻭﺍﻝﻔﺴﻔﻭﺭ ﻭﺍﻝﺤﺩﻴﺩ ﻭﺍﻝﺒﻭﺘﺎﺴـﻴﻭﻡ‪،‬‬ ‫ﺇﻀﺎﻓﺔ ﺇﻝﻰ ﺍﻝﺯﻴﺕ ﺍﻝﻌﻁﺭﻱ )‪ ، (11‬ﻭﻴﺴﺘﺨﺩﻡ ﻤﺎﺩﺓ ﻤﻨﻜﻬﺔ ﻓﻲ ﺍﻝﻁﻌﺎﻡ‬ ‫)‪ ، (12‬ﻭﻴﻔﺘﺢ ﺍﻝﺸﻬﻴﺔ ﻭﻴﻌـﺎﻝﺞ ﺍﻀـﻁﺭﺍﺒﺎﺕ ﺍﻝﺠﻬـﺎﺯ ﺍﻝﻬـﻀﻤﻲ‬ ‫ﻭﺍﻹﻤﺴﺎﻙ ﻭﺍﻝﻤﻐﺹ ﻋﻨﺩ ﺍﻷﻁﻔﺎل )‪ ، (7‬ﻭﻴﺴﻬل ﺍﻝﻬﻀﻡ ﻝﻸﻁﻌﻤـﺔ‬ ‫ﺍﻝﺜﻘﻴﻠﺔ ﻤﺜل ﺍﻝﻔﺎﺼﻭﻝﻴﺎ ﻭﺍﻝﻔﻭل )‪ ، (13‬ﻭﻨﻅـﺭﺍ ﻷﻫﻤﻴﺘـﻪ ﺍﻝﻌﻼﺠﻴـﺔ‬ ‫ﻭﺍﻝﻐﺫﺍﺌﻴﺔ ‪ ،‬ﻓﻘﺩ ﻫـﺩﻓﺕ ﺍﻝﺩﺭﺍﺴـﺔ ﺍﻝﺤﺎﻝﻴـﺔ ﺇﻝـﻰ ﻤﻌﺭﻓـﺔ ﺘـﺄﺜﻴﺭ‬ ‫ﺇﻀﺎﻓﺔ ﺒﺫﻭﺭ ﺍﻝﺸﻤﺭ ﺍﻝﻤﻁﺤﻭﻨﺔ ﻓﻲ ﺍﻝﺨﻭﺍﺹ ﺍﻝﺤـﺴﻴﺔ ﻭﺍﻝﻔﻴﺯﻴﺎﺌﻴـﺔ‬ ‫ﻭﺍﻝﻤﻴﻜﺭﻭﺒﻴﺔ ﻝﻠﻜﻴﻙ ﺍﻝﻤﻘﺼﺭ ﺍﻝﻤﺼﻨﻊ ﻤﺨﺘﺒﺭﻴﺎ ﻤﻘﺎﺭﻨﺔ ﻤـﻊ ﺍﻝﻤـﻭﺍﺩ‬ ‫ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﺼﻨﺎﻋﻴﺔ ] ﺒﻨﺯﻭﺍﺕ ﺍﻝﺼﻭﺩﻴﻭﻡ ﻭﺍﻝﻨﻴﺒﺎﺠﻴﻥ [‪.‬‬

‫ﺍﻝﻤﻭﺍﺩ ﻭﻁﺭﻕ ﺍﻝﻌﻤـــل‬ ‫ﺘﻡ ﺼﻨﻊ ﺍﻝﻜﻴﻙ ﺒﺈﻀﺎﻓﺔ ﻤﻭﺍﺩ ﺤﺎﻓﻅﺔ ﺼﻨﺎﻋﻴﺔ ) ﺍﻝﻨﻴﺒﺎﺠﻴﻥ ‪ ،‬ﺒﻨﺯﻭﺍﺕ‬ ‫ﺍﻝﺼﻭﺩﻴﻭﻡ ( ﺒﻨﺴﺒﺔ ‪ % 0 .2‬ﻭﻤﻭﺍﺩ ﺤﺎﻓﻅﺔ ﻁﺒﻴﻌﻴﺔ ﻋﺭﻕ ﺍﻝـﺴﻭﺱ‬ ‫ﺒﺎﻝﻨﺴﺏ ‪ % 3 ، 2 ، 1 ، 0.5‬ﺒﻌﺩ ﺘﺤﻀﻴﺭ ﺍﻝﻌﺠﻴﻨﺔ ‪ ،‬ﻭﻜﺎﻥ ﻭﺯﻨﻬـﺎ‬ ‫‪ 842‬ﻏﻡ ‪ .‬ﻭﺤﺴﺏ ﺍﻝﻤﻘﺎﺩﻴﺭ ﺍﻵﺘﻴﺔ ‪ :‬ﺴﻜﺭ ‪ 201‬ﻏﻡ ‪ ،‬ﻁﺤﻴﻥ ‪290‬‬ ‫ﻏﻡ ‪ ،‬ﺯﻴـﺕ ‪ 111‬ﻏـﻡ ‪ ،‬ﺒﻴﻜـﻨﺞ ﺒـﺎﺩﻭﺭ ‪ 10‬ﻏـﻡ ‪ ،‬ﻓـﺎﻨﻴﻼ ‪5‬‬ ‫ﻏﻡ‪ ،‬ﺒﻴﺽ ‪ 110‬ﻏﻡ ﻭﺤﻠﻴﺏ ‪ 200‬ﻤل‪.‬‬

‫ﻁﺭﻴﻘﺔ ﺘﺤﻀﻴﺭ ﺍﻝﻜﻴﻙ‪:‬‬ ‫ﺘﻡ ﺨﻠﻁ ﺍﻝﺯﻴﺕ ﻭﺇﻀﺎﻓﺔ ﺍﻝﺴﻜﺭ ﻤﻊ ﺍﻝﺨﻠﻁ ﺍﻝﻤﺴﺘﻤﺭ ﻭﻤﻥ ﺜﻡ ﺍﻝﺒـﻴﺽ‬ ‫ﺍﻝﻤﺨﻔﻭﻕ ﻤﻊ ﺍﻝﺨﻠﻁ ﺍﻝﻤﺴﺘﻤﺭ ‪ ،‬ﺒﻌﺩ ﺫﻝﻙ ﺃﻀﻴﻑ ﻤﺯﻴﺞ ﻤﻥ ﺍﻝﻁﺤـﻴﻥ‬ ‫ﺍﻝﻤﻨﺨﻭل ﻭﺍﻝﻤﻠﺢ ﻭﺍﻝﺒﻴﻜﻨﺞ ﺒﺎﻭﺩﺭ ﻤﻊ ﺍﻝﺨﻠﻁ ﺍﻝﻤﺴﺘﻤﺭ ﺒﺎﻝﺘﻨـﺎﻭﺏ ﻤـﻊ‬ ‫ﺍﻝﺤﻠﻴﺏ ﺍﻝﻤﻀﺎﻑ ﺍﻝﻴﻪ ﺍﻝﻔﺎﻨﻴﻼ ‪ ،‬ﺜﻡ ﻭﻀﻊ ﺍﻝﺨﻠﻴﻁ ﻓﻲ ﻗﺎﻝﺏ ﺩﺍﺌـﺭﻱ‬ ‫ﻤﺩﻫﻭﻥ ‪ ،‬ﻗﻁﺭﻩ ‪ 12‬ﺴﻡ ﺒﻤﻌﺩل ‪ 8‬ﻜﻴﻜﺔ ﺒﻭﺯﻥ ‪ 100‬ﻏﻡ ﻝﻜل ﻤﻌﺎﻤﻠﺔ‬ ‫ﻋﻠﻰ ﺤﺩﺓ‪ ،‬ﺜﻡ ﺘﻡ ﺸﻭﺍﺅﻩ ﻓﻲ ﻓﺭﻥ ﺒﺩﺭﺠـﺔ ﺤـﺭﺍﺭﺓ ‪ 200‬ﻡ‪ º‬ﻝﻤـﺩﺓ‬ ‫‪ 45‬ﺩﻗﻴﻘﺔ‪.‬‬ ‫ﺍﻝﻔﺤﻭﺼﺎﺕ ﺍﻝﻔﻴﺯﻴﺎﺌﻴﺔ ﻝﻠﻜﻴﻙ ‪:‬‬ ‫ﺘﻡ ﻭﺯﻥ ﺍﻝﻌﺠﻴﻨﺔ ﻗﺒل ﻋﻤﻠﻴﺔ ﺍﻝﺸﻭﺍﺀ ﻭﺒﻌﺩﻩ ﻝﺤﺴﺎﺏ ﻨﺴﺒﺔ ﺍﻝﻔﻘﺩ ﺍﻝﻤﺌﻭﻴﺔ‬ ‫ﺒﺎﻝﻭﺯﻥ )ﻏﻡ( )‪ ، (14‬ﻭﺘـﻡ ﻗﻴـﺎﺱ ﺤﺠـﻡ ﺍﻝﻜﻴـﻙ ) ‪Standing‬‬ ‫‪ ( Height‬ﻭﺫﻝﻙ ﻋﻥ ﻁﺭﻴﻕ ﺍﺭﺘﻔﺎﻉ ﺍﻝﻘﺎﺌﻡ ﺤﺴﺏ ﻤﺎ ﺠﺎﺀ ﻓﻲ ﻗﺴﻡ‬ ‫ﺍﻝﻐﺫﺍﺀ ﻭﺍﻝﺘﻐﺫﻴﺔ ﺍﻝﺘﺎﺒﻊ ﻝﺠﺎﻤﻌﺔ ﻭﻻﻴﺔ ﻜﻨﺴﺎﺱ ﺍﻷﻤﺭﻴﻜﻴـﺔ ) ‪(1975‬‬ ‫)‪ ، (15‬ﺤﻴﺙ ﻗﻁﻌﺕ ﺸﺭﻴﺤﺔ ﻤﻥ ﻭﺴـﻁ ﺍﻝﻜﻴﻜـﺔ ﺴـﻤﻜﻬﺎ ‪ 2‬ﺴـﻡ‬ ‫ﻭﻭﻀﻌﺕ ﻋﻠﻰ ﻭﺭﻗﺔ ﻭﺤﺩﺩ ﺸﻜﻠﻬﺎ ﺒﻘﻠﻡ ﺍﻝﺭﺼﺎﺹ ﺒﻌـﺩﻫﺎ ﺭﺴـﻤﺕ‬ ‫ﺨﻤﺴﺔ ﺃﻋﻤﺩﺓ ﻓﻲ ﻭﺴﻁ ﺍﻝﺸﺭﻴﺤﺔ ﻭﺍﺜﻨﺎﻥ ﻋﻠﻰ ﻜـل ﻤـﻥ ﺍﻝﻨـﺼﻔﻴﻥ‬ ‫ﺍﻷﻴﻤﻥ ﻭﺍﻷﻴﺴﺭ ﻝﻠﺸﺭﻴﺤﺔ ﻭﻓﻲ ﻤﻭﺍﻀﻊ ﻤﺘﻨﺎﻅﺭﺓ ﺘﻘﺭﻴﺒﺎ ﻓـﻲ ﺩﺍﺨـل‬ ‫ﺸﻜل ﺍﻝﺸﺭﻴﺤﺔ ﺍﻝﻤﺭﺴﻭﻡ ﻋﻠﻰ ﺍﻝﻭﺭﻗﺔ‪ ،‬ﻭﻗﻴـﺴﺕ ﺃﻁـﻭﺍل ﺍﻷﻋﻤـﺩﺓ‬ ‫ﺍﻝﻤﺭﺴﻭﻤﺔ ﺒﺎﻝﻤﺴﻁﺭﺓ ﻭﺠﻤﻌﺕ ﺜﻡ ﻗﺴﻤﺕ ﻋﻠﻰ ﺍﻷﻋﻤـﺩﺓ ﻝﻠﺤـﺼﻭل‬ ‫ﻋﻠﻰ ﻤﻌﺩل ﺍﻷﺭﻗﺎﻡ ﺍﻝﺫﻱ ﻴﻤﺜل ﺍﻻﺭﺘﻔﺎﻉ ﺍﻝﻘﺎﺌﻡ‪.‬‬ ‫ﺍﻝﺘﻘﻴﻴﻡ ﺍﻝﺤﺴﻲ‪:‬‬ ‫ﺘﻡ ﺇﺠﺭﺍﺀ ﺍﻝﺘﻘﻴﻴﻡ ﺍﻝﺤﺴﻲ ﻝﻠﻜﻴﻙ ﺍﻝﻤﻘﺼﺭ ﻤﻥ ﻗﺒل ‪ 12‬ﻤﻘﻭﻤﺎ ﻤﻥ ﺫﻭﻱ‬ ‫ﺍﻻﺨﺘﺼﺎﺹ ﻭﻓﻕ ﺍﺴﺘﻤﺎﺭﺓ ﺍﻝﺘﻘﻴﻴﻡ ﺍﻝﺤﺴﻲ ﺍﻝﻤﻌﺘﻤﺩﺓ ﻤﻥ ﻗﺒـل )‪. (16‬‬ ‫ﻭﻗﺩ ﻗﻴﻡ ﺍﻝﻜﻴﻙ ﺍﻝﻤﻘﺼﺭ ﺒﻌﺩ ﺘﺠﻤﻴﺩﻩ ﻤﻥ ﺤﻴﺙ ﺨﻭﺍﺼﻪ ﺍﻝﺤﺴﻴﺔ ﺍﻝﺘـﻲ‬ ‫ﺘﺸﻤل ﺍﻝﻤﻅﻬﺭ ‪ ،‬ﺍﻝﻨﺴﺠﺔ‪ ،‬ﺍﻝﻁﺭﺍﻭﺓ ‪ ،‬ﺍﻝﻨﻜﻬﺔ )ﺍﻝﻁﻌـﻡ ﻭﺍﻝﺭﺍﺌﺤـﺔ (‬ ‫ﻭﺍﻝﺘﻘﺒل ﺍﻝﻌﺎﻡ‪.‬‬ ‫ﻁﺭﻴﻘﺔ ﺘﺨﺯﻴﻥ ﺍﻝﻜﻴﻙ‪:‬‬ ‫ﺘﻡ ﺘﺨﺯﻴﻥ ﻗﻁﻊ ﺍﻝﻜﻴﻙ ﻝﻠﻤﻌﺎﻤﻼﺕ ﺒﻌﺩ ﺘﻐﻠﻴﻔﻬﺎ ﺒﻭﺭﻕ ﺍﻷﻝﻤﻨﻴـﻭﻡ ﺜـﻡ‬ ‫ﻭﻀﻌﺕ ﻓﻲ ﺤﺎﻭﻴﺎﺕ ﺒﻼﺴﺘﻴﻜﻴﺔ ﻤﻌﻘﻤﺔ ‪ ،‬ﻭﺨﺯﻨﺕ ﻓﻲ ﺍﻝﺜﻼﺠﺔ ﻋﻨـﺩ‬ ‫ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ‪ 18 -‬ﻡ‪ º‬ﻝﺤﻴﻥ ﺇﺠﺭﺍﺀ ﺍﻝﻔﺤﺼﻭﺼﺎﺕ ﺍﻝﻤﻴﻜﺭﻭﺒﻴﺔ‪.‬‬ ‫ﺍﻝﻔﺤﻭﺼﺎﺕ ﺍﻝﻤﻴﻜﺭﻭﺒﻴﺔ‪:‬‬ ‫ﺨﺯﻨﺕ ﻨﻤﺎﺫﺝ ﺍﻝﻜﻴﻙ ﺍﻝﻤﺼﻨﻌﺔ ﻝﻤﺩﺓ ‪ 15‬ﻴﻭﻤﺎ ﻓﻲ ﺩﺭﺠﺔ ﺤـﺭﺍﺭﺓ ‪14‬‬ ‫– ‪ 19‬ﻡ‪ ، º‬ﻗﺩﺭ ﺨﻼﻝﻬﺎ ﺍﻝﻌﺩﺩ ﺍﻝﻜﻠﻲ ﻝﻠﻔﻁﺭﻴﺎﺕ ﺒﺎﺴـﺘﺨﺩﺍﻡ ﺍﻝﻭﺴـﻁ‬ ‫ﺍﻝﺯﺭﻋﻲ‪ . (17) Potato dextrose agar‬ﻭﺃﺠﺭﻱ ﺍﻝﺘﺨﻔﻴﻑ ﺍﻝﻼﺯﻡ‬ ‫‪ ، 1 10 x 1‬ﻭﺘﻤﺕ ﻤﺘﺎﺒﻌﺔ ﻋﺩﺩ ﺍﻝﻔﻁﺭﻴﺎﺕ ﺨـﻼل ﺍﻷﻴـﺎﻡ )ﺍﻷﻭل‬ ‫ﻭﺍﻝﺴﺎﺒﻊ ﻭﺍﻝﺨﺎﻤﺱ ﻋﺸﺭ( ﺒﻌﺩ ﺤﻀﻥ ﺍﻷﻁﺒﺎﻕ ﻓﻲ ‪ 30‬ﻡ‪ º‬ﻤﺩﺓ ‪ 3‬ﺃﻴﺎﻡ‬ ‫ﻭ ‪ 5‬ﺃﻴﺎﻡ ‪ .‬ﻭﻜﺫﻝﻙ ﺨﺯﻨﺕ ﻨﻤﺎﺫﺝ ﺍﻝﻜﻴﻙ ﺍﻝﻤﺼﻨﻌﺔ ﻝﻤﺩﺓ ‪ 15‬ﻴﻭﻤﺎ ﻓﻲ‬ ‫ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ‪ 29 - 25‬ﻡ‪ º‬ﻗﺩﺭ ﺨﻼﻝﻬﺎ ﺍﻝﻌـﺩﺩ ﺍﻝﻜﻠـﻲ ﻝﻠﺒﻜﺘﻴﺭﻴـﺎ‬ ‫ﺒﺎﺴـــــــــــــﺘﺨﺩﺍﻡ ﺍﻝﻭﺴـــــــــــــﻁ‬ ‫ـﺭﻱ‬ ‫ـﻲ ‪ . (18) Maccokey and Nutrient agar‬ﻭﺃﺠـ‬ ‫ﺍﻝﺯﺭﻋـ‬ ‫ﺍﻝﺘﺨﻔﻴﻑ ﺍﻝﻼﺯﻡ ‪ . 1 10x1‬ﻭﺘﻤﺕ ﻤﺘﺎﺒﻌﺔ ﻋﺩﺩ ﺍﻝﺒﻜﺘﻴﺭﻴﺎ ﺨﻼل ﺍﻻﻴﺎﻡ‬ ‫)ﺍﻷﻭل ﻭﺍﻝﺴﺎﺒﻊ ﻭﺍﻝﺨﺎﻤﺱ ﻋﺸﺭ( ﺒﻌﺩ ﺤﻀﻥ ﺍﻷﻁﺒﺎﻕ ﻓـﻲ ﺩﺭﺠـﺔ‬ ‫ﺤﺭﺍﺭﺓ ‪ 37‬ﻡ‪ º‬ﻝﻤﺩﺓ ﻴﻭﻡ ﺇﻝﻰ ﻴﻭﻤﻴﻥ )‪.(19‬‬ ‫ﺍﻝﺘﺤﻠﻴﻼﺕ ﺍﻹﺤﺼﺎﺌﻴﺔ‪:‬‬ ‫ﺘـﻡ ﺍﺴـﺘﻌﻤﺎل ﺍﻝﺒﺭﻨـﺎﻤﺞ ﺍﻹﺤـﺼﺎﺌﻲ ‪Statistical Analysis‬‬ ‫‪ (20) (2010) SAS- System‬ﻓــﻲ ﺍﻝﺘﺤﻠﻴــل ﺍﻹﺤــﺼﺎﺌﻲ ‪،‬‬ ‫ﻭﺍﺘﺒﻌﺕ ﻁﺭﻴﻘﺔ ﺍﻝﺘﺼﻤﻴﻡ ﺍﻝﻌﺸﻭﺍﺌﻲ ﺍﻝﻜﺎﻤـل ) ‪ ، ( CRD‬ﻝﺩﺭﺍﺴـﺔ‬ ‫ﺘﺄﺜﻴﺭ ﺍﻝﻌﻭﺍﻤل ﺍﻝﻤﺨﺘﻠﻔﺔ ﻓﻲ ﺍﻝﺼﻔﺎﺕ ﺍﻝﻤﺩﺭﻭﺴﺔ ‪ ،‬ﻭﻗﻭﺭﻨﺕ ﺍﻝﻔـﺭﻭﻕ‬ ‫ﺍﻝﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺍﻝﻤﺘﻭﺴﻁﺎﺕ ﺒﺎﺨﺘﻼﻑ ﺍﻗل ﻓﺭﻕ ﻤﻌﻨﻭﻱ )‪.( LSD‬‬

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‫‪International Journal for Sciences and Technology / ICV: 4.32 - SJIF: 3.735‬‬

‫‪Vol. 10, No.2, June 2015‬‬

‫ﺍﻝﻨﺘﺎﺌـــﺞ ﻭﺍﻝﻤﻨﺎﻗﺸـــﺔ‬ ‫ﺃﻅﻬﺭ ﺠﺩﻭل ﺭﻗﻡ )‪ (1‬ﺒﺄﻥ ﺍﻝﻤﻌﺎﻤﻠﺔ ‪ ) A‬ﺒﺩﻭﻥ ﺇﻀﺎﻓﺔ ﺍﻝﻤﻘﺎﺭﻨـﺔ (‬ ‫ﺤﺼﻠﺕ ﻋﻠﻰ ﺩﺭﺠﺔ ﺘﻘﻴﻴﻡ ﺃﻋﻠﻰ ﻤﻌﻨﻭﻴﺎ ﻤﻘﺎﺭﻨـﺔ ﻤـﻊ ﺍﻝﻤﻌـﺎﻤﻼﺕ‬ ‫ﺍﻷﺨﺭﻯ‪ ،‬ﻤﻥ ﺤﻴﺙ ﺼﻔﺔ ﺍﻝﻤﻅﻬﺭ ﻭﺍﻝﻨﻜﻬﺔ ﻭﺍﻝﺘﻘﺒل ﺍﻝﻌﺎﻡ ‪ .‬ﻭﻜـﺫﻝﻙ‬ ‫ﺤﺼﻠﺕ ﺍﻝﻤﻌﺎﻤﻠﺘﺎﻥ ‪ A‬ﻭ ‪ % 0.2) C‬ﻤﻥ ﺍﻝﻨﻴﺒﺎﺠﻴﻥ ( ﻋﻠﻰ ﺩﺭﺠـﺔ‬ ‫ﺘﻘﻴﻴﻡ ﺃﻋﻠﻰ ﻤﻌﻨﻭﻴﺎ ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻷﺨﺭﻯ ﻤﻥ ﺤﻴﺙ ﺼـﻔﺔ‬ ‫ﺍﻝﻨﺴﺠﺔ‪ .‬ﻜﻤﺎ ﺤﺼﻠﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ‪ C‬ﻋﻠﻰ ﺩﺭﺠﺔ ﺘﻘﻴﻴﻡ ﺃﻋﻠـﻰ ﻤﻌﻨﻭﻴـﺎ‬ ‫ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺒﺎﻗﻲ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻷﺨﺭﻯ ﻤﻥ ﺤﻴﺙ ﺼـﻔﺔ ﺍﻝﻁـﺭﺍﻭﺓ ‪.‬‬

‫ﻭﺤﺼﻠﺕ ﺍﻝﻤﻌﺎﻤﻠﺔ ‪ % 2 ) F‬ﻤﻥ ﺍﻝﺸﻤﺭ ( ﻋﻠﻰ ﺩﺭﺠﺔ ﺘﻘﻴـﻴﻡ ﺃﻗـل‬ ‫ﻤﻌﻨﻭﻴﺎ ﻤﻘﺎﺭﻨﺔ ﻤﻊ ﺒﺎﻗﻲ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻷﺨﺭﻯ ﻤﻥ ﺤﻴﺙ ﺠﻤﻴﻊ ﺍﻝﺼﻔﺎﺕ‬ ‫ﺍﻝﺤﺴﻴﺔ ﺍﻝﻤﺫﻜﻭﺭﺓ ﺃﻋﻼﻩ ‪ ،‬ﻭﺘﻌﻭﺩ ﺘﻠﻙ ﺍﻻﺨﺘﻼﻓﺎﺕ ﺇﻝﻰ ﻭﺠﻭﺩ ﺍﻝﻤـﻭﺍﺩ‬ ‫ﺍﻝﺤﺎﻓﻅﺔ ﺍﻝﺼﻨﺎﻋﻴﺔ ﻭﺍﻝﻁﺒﻴﻌﻴﺔ ﺍﻝﻤﺫﻜﻭﺭﺓ ﺃﻋﻼﻩ ﻭﺍﻝﺘﻲ ﺒﺩﻭﺭﻫﺎ ﺘـﺅﺜﺭ‬ ‫ﻋﻠﻰ ﺘﻘﺒل ﺍﻝﻤﺤﻜﻤﻴﻥ ﻭﺒﺎﻝﺘﺎﻝﻲ ﺘﺅﺜﺭ ﻋﻠﻰ ﺍﻝﺼﻔﺎﺕ ﺍﻝﺤـﺴﻴﺔ ﻝﻠﻜﻴـﻙ‬ ‫ﺍﻝﻤﺼﻨﻊ ﻭﻫﺫﺍ ﻴﺘﻔﻕ ﻤﻊ ﻤﺎ ﺫﻜﺭﻩ ) ‪ ، ( 1‬ﺒـﺄﻥ ﺍﻝﻤـﻭﺍﺩ ﺍﻝﺤﺎﻓﻅـﺔ‬ ‫ﺍﻝﺼﻨﺎﻋﻴﺔ ﻭﺍﻝﻁﺒﻴﻌﻴﺔ ﺍﻝﻤﻀﺎﻓﺔ ﺇﻝﻰ ﺍﻷﻏﺫﻴﺔ ﺘﺅﺜﺭ ﻓﻲ ﺼﻔﺎﺘﻬﺎ ﺍﻝﺤﺴﻴﺔ‪.‬‬

‫ﺠﺩﻭل ﺭﻗﻡ )‪ :(1‬ﺘﺄﺜﻴﺭ ﺍﻝﻤﻌﺎﻤﻼﺕ ﺍﻝﻤﺩﺭﻭﺴﺔ ﻓﻲ ﺍﻝﺘﻘﻴﻴﻡ ﺍﻝﺤﺴﻲ ﻝﻠﻜﻴﻙ ﺍﻝﻤﺼﻨﻊ ﻤﺨﺘﺒﺭﻴﺎ‬ ‫ﺍﻝﻤﺘﻭﺴﻁ ‪ ±‬ﺍﻝﺨﻁﺄ ﺍﻝﻘﻴﺎﺴﻲ‬ ‫ﺍﻝﻤﻌﺎﻤﻼﺕ‬ ‫‪ ) A‬ﺍﻝﻤﻘﺎﺭﻨﺔ ﺒﺩﻭﻥ ﺃﻀﺎﻓﺔ‬ ‫ﻤﻭﺍﺩ ﺤﺎﻓﻅﺔ (‬ ‫‪ % 0.2 ) B‬ﺒﻨﺯﻭﺍﺕ‬ ‫ﺍﻝﺼﻭﺩﻴﻭﻡ(‬ ‫‪ % 0.2 ) C‬ﺍﻝﻨﻴﺒﺎﺠﻴﻥ(‬ ‫‪ % 0.5 ) D‬ﺸﻤﺭ (‬ ‫‪ % 1 ) E‬ﺸﻤﺭ (‬ ‫‪ % 2 ) F‬ﺸﻤﺭ (‬ ‫‪ % 3 ) G‬ﺸﻤﺭ (‬ ‫ﻗﻴﻤﺔ ‪LSD‬‬ ‫* )‪.(P