Joint Biotechnology Master Program Palestine Polytechnic University Bethlehem University Deanship of Higher Studies and Faculty of Science Scientific Research Ziziphus spina-christ i“Christ’s Thorn”: In Vitro Callus and Cell Culture, Qualitative Analysis of Secondary Metabolites and Bioassay By Ali Essa Abu Allan In Partial Fulfillment of the Requirements for the Degree Master of Science July, 2012 The undersigned hereby certify that they have read and recommend to the Faculty of Scientific Research and Higher Studies at the Palestine Polytechnic University and the Faculty of Science at Bethlehem University for acceptance a thesis entitled: Ziziphus spina-christi “Christ’s Thorn”: In Vitro Callus and Cell Culture, Qualitative Analysis of Secondary Metabolites and Bioassay By Ali Essa Abu Allan A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in biotechnology Graduate Advisory Committee: Committee Chair Name, University(typed) Date Committee Member Name, University(typed) Date External Committee Member Name, University(typed) Date Approved for the Faculties Dean of Faculty of Scientific Dean of Faculty of Science Research and Higher studies Bethlehem University Palestine Polytechnic University Date Date II ABSTRACT Ziziphus spina-christi “Christ’s Thorn”: In Vitro Callus and Cell Culture, Qualitative Analysis of Secondary Metabolites and Bioassay By Ali Essa Abu Allan Ziziphus spina-christi (L.) Desf. (Rhamnaceae) is a multipurpose tree distributed along the entire sahelian area from Senegal to Sudan and from middle to west Asia as well. In Palestine it occurs along the coast and in the Jordan Valley area. It has been used for different disease treatment in traditional medicine such as cholesterol reduction, treating eye inflammation and hair loss. Besides the traditional uses of this plant, recent studies reported that the extracts of every part of the plant has wide range of medical applications such as anti-diabetic, inhibition of hepatic carcinogenicity and antimicrobial activity. Moreover, important secondary metabolites were isolated from Z. spina-christi like Quercetin 3-O-b-D-galactoside and Kaempferol 3-O-rutinoside which are known for their antimicrobial and antifungal activity. This study was conducted to optimize a protocol for callus induction and secondary metabolites production from different tissues of Z. spina-christi . The highest callus induction (100%) was obtained from cotyledons and roots on MS media supplemented with 1.3 mg/l BA and 0.3 mg/l NAA with some variation in callus color and texture. Incubation in darkness had significant effects on callus induction time from cotyledons, leaves, and roots explants. Callus formation from cotyledons, leaves, and roots was observed after 8 days for all explants while in light incubation callus formed after 20, 30, 11 days respectively. Combination of 1.0mg/l BA + 2.0mg/l NAA significantly reduced the fresh weight of cotyledonary callus. It has been observed from growth curve of cotyledonary callus that callus is to be subcultured at the end of the third week of incubation for new media in both light and dark condition. Different callus lines of Z. spina-christi were successfully maintained for seven months on MS media supplemented with 1.3 mg/L BA and 0.3 mg /L NAA. Cell suspension was initiated on liquid MS media supplemented with 1.3 mg/l BA and 0.3 mg/l NAA, 26- day interval allowed the determination of a cell growth peak in both fresh and dry matter. TLC analysis using ethyl acetate: formic acid: glacial acetic acid: water (100:11:11:26) as solvent system detected the presence of flavoniod secondary III metabolite in different intensity from callus, in vitro, and ex vitro parts of Z . spina- christi. The bioassay of cotyledonary callus extract against breast cancer cell line (MCF-7) resulted at IC equals 95 µg/ml. 50 Keywords Ziziphus spina-christi , In Vitro culture, Callus induction, Secondary metabolites, Medicinal plant IV ﺔﯿﺑﺮﻌﻟﺎﺑ ﺺﺨﻠﻣ ﻲﻋﻮﻨﻟا ﻞﯿﻠﺤﺘﻟا ،ﺮﺒﺘﺨﻤﻟا ﻲﻓ ﺔﯿﺗﺎﺒﻨﻟا ﺎﯾﻼﺨﻟا و سﻮﻟﺎﻜﻟا جﺎﺘﻧا :"ﺢﯿﺴﻤﻟا ﺔﻛﻮﺷ"ﻲﺘﺴﯾﺮﻛ -ﻲﻛﻮﺸﻟا رﺪﺴﻟا ﻲﺟﻮﻟﻮﯿﺒﻟا ﺎھﺮﯿﺛﺄﺗ ﺔﺳارد و ﺔﯾﻮﻧﺎﺜﻟا تﺎﺒﻛﺮﻤﻠﻟ ﻞﺒﻗ ﻦﻣ نﻼﻋ ﻮﺑأ ﻰﺴﯿﻋ ﻲﻠﻋ ﺔ ﻋزﻮﻣ ، تﻻﺎﻤﻌﺘ ﺳﻻا و ضاﺮ ﻏﻷا ةدﺪ ﻌﺘﻣ ةﺮﺠ ﺷ ﻲ ھ (Rhamnaceae) (L.) Desf. ﻲﺘﺴﯾﺮﻛ-ﻲﻛﻮﺸﻟا رﺪﺴﻟا ﻦﯿﻄ ﺴﻠﻓ ﻲ ﻓ . ﺎ ﮭﺑﺮﻏ ﻰ ﻟإ ﺎﯿ ﺳأ ﻂ ﺳو ﻦ ﻣ ﺎ ﻀﯾأو نادﻮ ﺴﻟا ﻰ ﺘﺣ لﺎﻐﻨ ﺴﻟا ﻦ ﻣ ﺎ ﮭﻠﻤﻛﺄﺑ ﺔﯿﻠﺣﺎ ﺴﻟا ﺔ ﻘﻄﻨﻤﻟا لﻮﻃ ﻰﻠﻋ ،يﺪﯿﻠﻘﺘﻟا ﺐﻄﻟا ﻲﻓ ﺔﻔﻠﺘﺨﻣ ضاﺮﻣأ جﻼﻌﻟ ﮫﻣاﺪﺨﺘﺳا ﻢﺗ ﺪﻘﻟ .ندرﻷا يداو ﺔﻘﻄﻨﻣ ﻲﻓ و ،ﻞﺣﺎﺴﻟا لﻮﻃ ﻰﻠﻋ ةﺮﺸﺘﻨﻣ تﺮ ﻛذ ، تﺎ ﺒﻨﻟا اﺬ ﮭﻟ ﺔ ﯾﺪﯿﻠﻘﺘﻟا تﺎﻣاﺪﺨﺘ ﺳﻻا ﺐ ﻧﺎﺟ ﻰ ﻟإ . ﺮﻌ ﺸﻟا ناﺪ ﻘﻓ و ، ﻦﯿ ﻌﻟا بﺎ ﮭﺘﻟا ،لوﺮﺘﺴﻟﻮﻜﻟا ﺾﯿﻔﺨﺗ ﻞﺜﻣ ضﺮ ﻣ ﺔ ﺤﻓﺎﻜﻣ و ﻂﯿ ﺒﺜﺗ ﻞ ﺜﻣ ﺔﻌ ﺳاو ﺔ ﯿﺒﻃ تﺎ ﻘﯿﺒﻄﺗ ﺎﮭﯾﺪ ﻟ ﺔﯿﻟﻮﺤﻜﻟا و ﺔﯿﺋﺎﻤﻟا تﺎﺻﻼﺨﺘﺳﻻا نإ ﺔﺜﯾﺪﺤﻟا تﺎﺳارﺪﻟا لﺰ ﻋ ﻢ ﺗ ، ﻚ ﻟذ ﻰ ﻠﻋ ةوﻼ ﻋ . تﺎ ﺑوﺮﻜﯿﻤﻟا و تﺎ ﯾﺮﻄﻔﻟا ﺾﻌﺒ ﻟ دﺎ ﻀﻣ و ، ﺪ ﺒﻜﻟا تﺎﻧﺎﻃﺮ ﺳ ﺾ ﻌﺑ جﻼ ﻋ ، يﺮﻜ ﺴﻟا و Quercetin 3-O-b-D-galactoside ﻞ ﺜﻣ ، ﻲﺘ ﺴﯾﺮﻛ- ﻲﻛﻮ ﺸﻟا رﺪ ﺴﻟا ﻦ ﻣ ﺔ ﻤﮭﻤﻟا ﺔ ﯾﻮﻧﺎﺜﻟا تﺎ ﺒﻛﺮﻤﻟا ﺾ ﻌﺑ ﺔ ﺳارﺪﻟا هﺬ ھ ﺖ ﯾﺮﺟا ﺪ ﻗ و . تﺎ ﯾﺮﻄﻔﻟا و ﻢﯿﺛاﺮ ﺠﻠﻟ دﺎ ﻀﻤﻟا ﺎﮭﻃﺎﺸﻨﺑ ﺔﻓوﺮﻌﻤﻟا Kaempferol 3-O-rutinoside - ﻲﻛﻮ ﺸﻟا رﺪ ﺴﻟا ﺔ ﺘﺒﻧ ﻦ ﻣ ﺔ ﻔﻠﺘﺨﻣ ﺔﺠ ﺴﻧا ﻦ ﻣ ﺔ ﯾﻮﻧﺎﺜﻟا تﺎ ﺒﻛﺮﻤﻟا و سﻮ ﻠﻜﻟا جﺎ ﺘﻧإ ﺾﯾﺮ ﺤﺘﻟ لﻮ ﻛﻮﺗوﺮﺑ ﻊ ﺿﻮﻟ BA ﺔﻓﺎ ﺿﺈﺑ MS ﺔ ﺌﯿﺑ ﻰ ﻠﻋ روﺬ ﺠﻟا و تﺎ ﺘﺒﻨﻟا ﻦ ﻣ (%١٠٠) سﻮﻟﺎ ﻜﻠﻟ جﺎﺘﻧإ ﻞﻀﻓأ ﻰﻠﻋ لﻮﺼﺤﻟا ﻢﺗ .ﻲﺘﺴﯾﺮﻜﻟا نﺎﻛ .سﻮﻟﺎﻜﻠﻟ ﺲﻤﻠﻤﻟا و نﻮﻠﻟا ﻲﻓ فﻼﺘﺧا ﻊﻣ ،ﺮﺘﻟ/ﻢﻐﻠﻣ ٠,٣ ىﻮﺘﺴﻣ ﻰﻠﻋ NAA + ﺮﺘﻟ/ﻢﻐﻠﻣ ١,٣ ىﻮﺘﺴﻣ ﻰﻠﻋ نﺎ ﻛ . تﺎ ﺘﺒﻨﻠﻟ سﻮﻟﺎ ﻜﻟا نزو ﺾ ﻔﺧ ﻰ ﻠﻋ ﺮ ﯿﺒﻛ ﺮ ﺛا ﺮ ﺘﻟ/ﻢﻐﻠﻣ ٢ NAA + ﺮﺘﻟ/ﻢﻐﻠﻣ ١ ىﻮﺘﺴﻣ ﻰﻠﻋ BA ﺞﯾﺰﻤﻟ ﻞﯿﻜ ﺸﺗ ﻆﺣﻮ ﻟ ﺚ ﯿﺣ ، روﺬ ﺠﻟا و ،قاروﻻا ، تﺎ ﺘﺒﻨﻠﻟ سﻮﻟﺎ ﻜﻠﻟ ﺾﯾﺮ ﺤﺘﻟا ﻦ ﻣز ﻰ ﻠﻋ ﺮ ﯿﺒﻛ ﺮ ﺛا مﻼ ﻈﻟا ﻲ ﻓ ﺔﻧﺎ ﻀﺤﻠﻟ ﻰ ﻠﻋ ﺎ ﻣﻮﯾ ٣٠,٢٠,١١ ﺪ ﻌﺑ سﻮﻟﺎ ﻜﻟا ﻞﻜ ﺸﺗ ﻆﺣﻮ ﻟ ءﻮ ﻀﻟا ﻲﻓ ﺎﻤﻨﯿﺑ ءاﺰﺠﻟا ﻊﯿﻤﺠﻟ ﺔﻧﺎﻀﺤﻟا ﻦﻣ مﺎﯾا ٨ ﺪﻌﺑ ﺲﻟﺎﻜﻟا ﺐ ﺳﺎﻨﻤﻟا ﺖ ﻗﻮﻟا ﻮ ھ ﺔﻧﺎ ﻀﺤﻟا ﻦ ﻣ ﺚ ﻟﺎﺜﻟا عﻮﺒ ﺳﻻا ﺔ ﯾﺎﮭﻧ نا ﺪ ﺟو ، سﻮﻟﺎ ﻜﻟا تﺎ ﺘﺒﻨﻟ ﻮ ﻤﻨﻟا ﻰﻨﺤﻨﻤﻟ ﺔﺒﺴﻨﻟﺎﺑ .ﻲﻟاﻮﺘﻟا ةﺪ ﻤﻟ حﺎ ﺠﻨﺑ سﻮﻟﺎ ﻜﻟا ﻰ ﻠﻋ ﺔ ﻈﻓﺎﺤﻤﻟا ﻢ ﺗ . مﻼ ﻈﻟا و ءﻮ ﻀﻟا ﺔ ﻟﺎﺣ ﻲ ﻓ ءاﻮﺳ ﺪﺣ ﻰﻠﻋ ةﺪﯾﺪﺟ ﺔﺌﯿﺑ ﻰﻟا ﻞﻘﻨﻠﻟ سﻮﻟﺎﻜﻠﻟ ﻞ ﻜﻟ ﺮ ﺘﻟ/ﻢﻐﻠﻣ ٠,٣ ىﻮﺘﺴﻣ ﻰﻠﻋ NAA + ﺮﺘﻟ/ﻢﻐﻠﻣ ١,٣ ىﻮﺘﺴﻣ ﻰﻠﻋ BA ﺔﻓﺎﺿﺎﺑ MS ﺔﺌﯿﺑ ﻰﻠﻋ رﻮﮭﺷ ﺔﻌﺒﺳ ١,٣ ىﻮﺘ ﺴﻣ ﻰ ﻠﻋ BA ﺔﻓﺎ ﺿﺎﺑ MS ﺔ ﺌﯿﺑ ﻰ ﻠﻋ تﺎ ﺘﺒﻨﻠﻟ ﺎ ﯾﻼﺨﻟا ءﺎ ﺸﻧا أﺪ ﺑ .روﺬ ﺠﻟا و ،قاروﻻا ، تﺎ ﺘﺒﻨﻟا ﻦ ﻣ ﻰ ﻠﻋ ﺔﻧﺎ ﻀﺤﻟا ﺔﯾاﺪﺑ ﻦﻣ ﺎﻣﻮﯾ ٢٦ ﺪﻌﺑ ﺎﯾﻼﺨﻠﻟ ﻮﻤﻨﻟا ةورذ نﺎﻛ و ،ﺮﺘﻟ/ﻢﻐﻠﻣ ٠,٣ ىﻮﺘﺴﻣ ﻰﻠﻋ NAA + ﺮﺘﻟ/ﻢﻐﻠﻣ (١٠٠,١١,١١,٢٦) ﮫ ﺑاذا مﺎ ﻈﻧ ماﺪﺨﺘ ﺳﺎﺑ TLC ﺔﻄ ﺳاﻮﺑ ﻞ ﯿﻠﺤﺘﻟا .ﺔ ﻓﺎﺠﻟا و ﺔ ﺟزﺎﻄﻟا ﺎ ﯾﻼﺨﻠﻟ ﺔﺒ ﺴﻨﻟﺎﺑ ءاﻮ ﺳ ﺪ ﺣ تﺎﺟرﺪ ﺑ و ﺔ ﯾﺪﻧ اﻮ ﻠﻓ ﺔ ﯾﻮﻧﺎﺛ تﺎ ﺒﻛﺮﻣ دﻮ ﺟو ﺖ ﺒﺛا ﻞ ﯿﺜﯾﻻا تﻼ ﺧ: يﺪ ﯿﻠﺠﻟا ﻚ ﯿﻠﺨﻟا ﺾﻣﺎ ﺣ:ﻚ ﻣرﻮﻔﻟا ﺾ ﻤﺣ: ءﺎ ﻣ . ﺐ ﯿﺑﺎﻧﻻا ﻲ ﻓ ﺖ ﻤﻧ ﻲ ﺘﻟا و ﺔ ﻌﯿﺒﻄﻟا ﻦ ﻣ ﺖ ﻌﻤﺟ ﻲ ﺘﻟا قاروﻻا و روﺬ ﺠﻟا ﺎ ﻀﯾا و سﻮﻟﺎ ﻜﻟا عاﻮﻧا ﻊﯿﻤﺟ ﻲﻓ ﺔﻔﻠﺘﺨﻣ ﺚ ﯿﺣ ﺔﯿﻧﺎﻃﺮ ﺴﻟا ﺎ ﯾﻼﺨﻟا ﺪ ﺿ ﺮﯿﺛﺄ ﺗ ﻰ ﻄﻋأ MCF-7 ﺔ ﯿﻠﺨﻟا ﻂ ﺧ يﺪﺜﻟا نﺎﻃﺮﺳ ﺪﺿ تﺎﺘﺒﻨﻟا ﺔﺻﻼﺧ ﺮﯿﺛﺎﺗ ﺔﺳارد .ﻞﻣ /ماﺮﻏوﺮﻜﯿﻣ ٩٥ IC ﺔﻤﯿﻗ ﺖﻐﻠﺑ 50 ﮫﯿﺣﺎﺘﻔﻣ تﺎﻤﻠﻛ ﺔﯿﺒﻄﻟا تﺎﺗﺎﺒﻨﻟا ،ﺔﯾﻮﻧﺎﺜﻟا تﺎﺒﻛﺮﻤﻟا ،سﻮﻟﺎﻜﻟا ﺚﺣ ،ﺔﯾﺮﺒﺨﻤﻟا ﺐﯿﺑﺎﻧﻻا ﻲﻓ ﺔﻋارﺰﻟا ،ﻲﺘﺴﯾﺮﻛ-ﻲﻛﻮﺸﻟا رﺪﺴﻟا V DECLARATION I declare that the Master Thesis entitled " Ziziphus spina-christi “Christ’s Thorn”: In Vitro Callus and Cell Culture, Qualitative Analysis of Secondary Metabolites and Bioassay " is my own original work, and hereby certify that unless stated, all work contained within this thesis is my own independent research and has not been submitted for the award of any other degree at any institution, except where due acknowledgment is made in the text. Ali Essa Abu Allan Date Copyright © Ali Abu Allan,2012 All rights reserved VI STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for the joint master degree in biotechnology at Palestine Polytechnic University and Bethlehem University, I agree that the library shall make it available to borrowers under rules of the library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of the source is made. Permission for extensive quotation from, reproduction, or publication of this thesis may be granted by my main supervisor, or in his absence, by the Dean of Higher Studies when, in the opinion of either, the proposed use of the material is for scholarly purposes. Any copying or use of the material in this thesis for financial gain shall not be allowed without my written permission. Ali Essa Abu Allan Date VII DEDICATION I dedicate this great thesis to my children and wife for their love and encouragement. I also dedicate it for the soul of my dead father I won't also forget my brothers, sisters and mother, whom always stood by me during thick and thin, and providing me the moral support and resources to finish my work. Their unwavering faith in me has been a source of constant inspiration for me. VIII ACKNOWLEDGMENT I thank the almighty whose blessings have enabled me to accomplish my thesis work successfully. It is my privilege to express my sincere thanks and a sense of deep gratitude to my advisor Dr. Jawad Alzeer, Department of Biotechnology, for his valuable advice, splendid supervision, and immense patience due to which this work was able to take the shape in which it has been presented. It was his valuable discussions and constant encouragement through which I have gained a lot. I would like to thank my co- advisor Dr. Rami Arafeh for his advices that lead me the correct way during lab working. I wish to express my gratefulness to all the faculty members of the Department of Biotechnology research center at Palestine polytechnic university for their help and moral support during my project work. IX ABBREVIATIONS Abbreviation Word or Sentence % percent °C Degree centigrade Celsius 2,4 D 2,4-dichlorophenoxyacetatic acid ANOVA Analysis of variance BAP 6-Benzylaminopurine CRD Completely Randomized Design DMSO Dimethyl sulfoxide et al. And others g gram HPLC High Performance Liquid Chromatography HSV-1 Herpes simplex virus type1 IC Half Maximal Inhibitory Concentration 50 l liter LSD Least Significant Difference M molar m² Square meter MADICO Manasrah Development and Investment Company MCF-7 Michigan Cancer Foundation - 7 ml milliliter MS Murashige and Skooge NAA Naphthaleneacetic acid PGRs Plant Growth Regulators pH Potensial hydrogen R Retardation factor f rpm Round per minute sec. second TLC Thin layer chromatography USA United State of America UV Ultraviolet WHO World Health Organization X