Aeginetia Indica Roxb.)

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Aeginetia Indica Roxb.) STUDY OF IMMUNOTOXICOLOGICAL EFFECTS OF DOK DIN DAENG (Aeginetia indica Roxb.) MISS WIMOLNUT AUTTACHOAT A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Environmental Biology Suranaree University of Technology Academic Year 2003 ISBN 974-533-280-1 การศึกษาพิษวิทยาและผลตอระบบภูมิคุมกัน ของดอกดินแดง (Aeginetia indica Roxb.) นางสาววิมลณัฐ อัตโชติ วิทยานิพนธนี้เปนสวนหนึ่งของการศึกษาตามหลักสูตรปริญญาวิทยาศาสตรดุษฎีบัณฑิต สาขาวิชาชีววิทยาสิ่งแวดลอม มหาวิทยาลัยเทคโนโลยีสุรนารี ปการศึกษา 2546 ISBN 974-533-280-1 I วิมลณัฐ อัตโชติ : การศึกษาพิษวิทยาและผลตอระบบภูมิคุมกันของดอกดินแดง (Aeginetia indica Roxb.) (STUDY OF IMMUNOTOXICOLOGICAL EFFECTS OF DOK DIN DAENG (Aeginetia indica Roxb.)) อาจารยที่ปรึกษา: ผศ.ดร.เบญจมาศ จิตรสมบูรณ, 212 หนา. ISBN 974-533-280-1 การศึกษาพิษตอระบบภูมิคุมกันของดอกดินแดงใชสารสกัดจากพืชทั้งตนสกัดดวยเอธ ทานอล (DDDP) และ น้ํา (WDDDP) และ สวนเมล็ดสกัดดวยบิวทานอล (SDDD) ผลจากการ ทดลอง in vitro เมื่อใชเซลลของหนูเมาส C57BL6/j พบวาสารสกัดจากดอกดินแดง SDDD และ DDDP มีฤทธิ์กระตุนระบบภูมิคุมกัน สารสกัดจากตนสามารถกระตุนการตอบสนองของ T เซลล ตอ concanavalin A และ anti-CD3 แอนติบอดี และกระตุนการตอบสนองของ B เซลลตอ lipopolysaccharide ที่ความเขมขนระหวาง 1.25-500 µg/ml หนู B6C3F1 ที่ไดรับสารสกัด DDDP และ WDDDP เปนเวลา 28 วันไมเกิดการตายหรือแสดงอาการเปนพิษ สารสกัด DDDP และ WDDDP สามารถกระตุนการทํางานของ T lymphocytes ใน MLR และ CTL ในการ ทดลอง in vivo หรือใน in vitro อยางมีนัยสําคัญทางสถิติ แตไมมีผลกระทบตอการสราง แอนติบอดีของ B เซลล และ NK เซลล นอกจากนั้นเซลลมามจากหนู B6C3F1 ที่ไดรับสารสกัด DDDP ไมมีผลเปลี่ยนแปลงการสราง IL-2 และ IFN-γ ในการทดลอง in vitro อยางไรก็ตาม หนู B6C3F1 ที่ไดรับสารสกัด WDDDP เกิดการลดคารอยละและจํานวนรวมของ T เซลลชนิด CD4+CD25+ ในมามซึ่งมีหนาที่ควบคุมการกระตุนของ T เซลลอื่น ผลโดยรวมในการศึกษานี้ แสดงใหเห็นวาสารสกัดจากดอกดินแดงมีฤทธิ์กระตุนการตอบสนองของระบบภูมิคุมกัน สาขาวิชาชีววิทยา ลายมือชื่อนักศึกษา……………………….……. ปการศึกษา 2546 ลายมือชื่ออาจารยที่ปรึกษา.................................. ลายมือชื่ออาจารยที่ปรึกษารวม........................... ลายมือชื่ออาจารยที่ปรึกษารวม........................... II WIMOLNUT AUTTACHOAT : STUDY OF IMMUNOTOXICO- LOGICAL EFFECTS OF DOK DIN DAENG (Aeginetia indica Roxb.) THESIS ADVSIOR: ASST. PROF. BENJAMART CHITSOMBOON, Ph.D. 212 pp. ISBN 974-533-280-1 This study was focused on the immunotoxicological effects of a whole plant ethanol extracts (DDDP), water extracts (WDDDP) and seed extracts (SDDD) of Aeginetia indica Roxb. (DDD). Results from in vitro studies in C57BL6/j mice suggest that DDDP and SDDD possess immunostimulatory activity. Enhancement of T cell mitogenic responses to concanavalin A and anti-CD3 and B cell response to lipopolysaccharide were observed at 1.25-500 µg/ml of extracts. Exposure to DDDP and WDDDP in B6C3F1 mice for 28 days did not induce mortality or any overt toxicity. Significant increase in T cell functions including MLR and CTL activity were observed after DDDP and WDDDP treatment in either in vivo or in vitro. Nevertheless, the antibody forming cell response and NK activity were not affected. In addition, in vitro exposure of splenocytes to DDDP did not alter IL-2 and IFN-γ production. Finally, WDDDP exposed mice decreased both the percentage and absolute number of regulatory CD4+CD25+ T cells in the spleen. Overall, this study demonstrated that the DDD extracts have the potential to stimulate immune response. School of Biology Student’s Signature………………………………... Academic Year 2003 Advisor’s Signature……………………………....... Co-advisor’s Signature…………………………...... Co-advisor’s Signature ………………………......... ACKNOWLEDGEMENTS I would like to express my sincere and deepest gratitude to my kind advisor, Asst. Prof. Dr. Benjamart Chitsomboon for her guidance and valuable advices. She was never lacking in kindness and support in my work. Special and sincere appreciation is given to Prof. Dr. Kimber L. White, who has kindly provided me with valuable advices, supervision and encouragement throughout this study. I wish to express my appreciated to Prof. Dr. Masatoshi Mutsumura, Dr. Nakao Nomura, and Asst. Prof. Dr. Tai L. Guo for their valuable suggestion for immunological work. I am grateful to Dr. Jame F. Maxwell, Dr. Paul J. Grote and Mr. Chaiyasit for their helpful on plant collection and plant identification on this study. I wish to thank to Sakai Akiko, Hagio Maiko, and Ganghua Lang for Technical assistance at Biosystem Laboratory, University of Tsukuba (Japan). I would like to expressing appreciation to Vanessa L. Peachee, Ronnetta D. Brown, Deborah L. Musgrove, Zhang Xiu Ling, Julia Brantley, Aime´e Halphen, and Tammie Thompson at the Immunotoxicology Program, Department of Pharmacology and Toxicology Virginia Commonwealth University for their technical assistance. I am thankful to Karen H. Munsun for document preparation assistance, and my friends for their sympathy, sincere encouragement and great assistance in plant collection and extraction. They were always nice and friendly. IV I am particularly indebted to the Department of Environmental Biology, Science Institute at Suranaree University of Technology, Biosystem laboratory, University of Tsukuba and Immunotoxicology Program at Medical College of Virginia, Virginia Commonwealth University for providing to all laboratory facilities and financial support during this study. I am also grateful to the National Institute of Environmental Health Sciences (NIEHS contract no. ES-05454), The Royal Golden Jubilee Ph.D program grant, New Research Development grant for Ph.D. students from Thailand Research Fund, The Association of International Education, Japan (AIEJ) at University of Tsukuba, Ibaraki, Japan for financial support. Finally, I would like to take opportunity to express my deep gratitude to my parents, Mr. Thammanoon Auttachoat, Mrs. Sarunthorn Auttachoat, to my family and my dearest friend Sirawit Manheng for their love, support and understanding that helped me to overcome many difficult moments. Wimolnut Auttachoat CONTENTS Page Abstract in thai…………………………………………………........................... I Abstract in english………………………………………………………............. II Acknowledgements……………………………………………............................ III Contents…………………………………………………………………............. V List of tables ……………………………………………………………............. X List of figures ……………………………………………………………............ XIII List of abbreviations…………………………………………………….............. XIX CHAPTER Ⅰ INTRODUCTION……………………………………………........... 1 1.1 Objective of the study…………………………………….............. 6 1.2 Research hypothesis………………………………………............. 7 1.3 Scope and limitation of the study…………………………............ 7 1.4 Expected results……………………………………………........... 8 Ⅱ LITERATURE REVIEWS…………………………………............. 9 1. Aeginetia indica Roxb.…………………………………................ 9 1.1 Botanical information and distribution………………............... 9 1.2 Propagation……………………………………………............. 14 1.3 Chemical constituents………………………………................. 17 1.4 Ethnomedical usages…………………………………............... 19 1.5 Pharmacological studies………………………………............. 19 VI CONTENTS (Continued) Page 2. Immune system…………………………………………............... 21 2.1 Lymphocytes………………………………………….............. 21 2.1.1 B lymphocytes…………………………………............... 22 2.1.2 T lymphocytes…………………………………….......... 22 2.2 Antigen presenting cells………………………………............. 24 2.3 CD antigen……………………………………………............. 25 2.4 Cytokines……………………………………………............... 27 2.5 Organs in immune system…………………………….............. 28 2.5.1 Thymus…………………………………………............. 29 2.5.2 Spleen…………………………………………............... 30 3. Immunotoxicological studies……………………….................... 31 3.1 Tier testing approaches……………………………….............. 31 3.2 Approach to immunotoxicity assessment…………….............. 33 3.3 Target organ toxicity………………………………….............. 34 3.3.1 Kidneys………………………………………............... 35 3.3.2 Lung…………………………………………................ 35 3.3.3 Liver…………………………………………................ 36 3.4 Immunotoxicity in vitro……………………………................ 37 4. Immunomodulation substances………………………................ 38 4.1 Immunosuppressive agent…………………………….............. 38 4.2 Immunostimulation and adaptogen…………………................ 39 VII CONTENTS (Continued) Page 5. Immunostimulant agents………………………………............... 41 5.1 Resins………………………………………………................. 41 5.2 Flavoniods………………………………….............................. 43 5.3 Polysaccharides……………………………………….............. 43 5.4 Terpenoids……………………………………………............. 44 5.5 Phenolics……………………………………………................ 45 5.6 Glycoprotein………………………………………….............. 46 6. Plant extract procedures………………………………............... 46 6.1 Plant extraction and isolation of compound.…………............. 46 6.1.1 Soxhlet extraction……………………………................. 49 6.1.2 Liquid-liquid extraction………………………................ 49 6.2 Filtering and concentration of sample………………............... 50 7. Cell viability and cell proliferation method……………............. 51 7.1 3H-Thymidine incorporation…………………………............. 51 7.2 MTT colorimetric assay………………………………............ 52 7.3 Trypan blue dye exclusion……………………………............ 53 Ⅲ MATERIALS AND METHODS…………………………............... 54 3.1 Plant…………………………………………………................... 56 3.2 Methods…………………………………………………............. 60 3.2.1 Preparation of DDDP………………………………............ 60 3.2.2 Preparation of SDDD………………………………............ 63 VIII CONTENTS (Continued) Page 3.2.3 Preparation of WDDDP…………………………............... 64 3.2.4 Single spleen cell preparation…………………….............. 65 3.2.5 MTT colorimetric
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