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(AGLAONEMA SIMPLEX BL.) FRUIT EXTRACT Ratana Kiatsongchai

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(AGLAONEMA SIMPLEX BL.) FRUIT EXTRACT Ratana Kiatsongchai BIOLOGICAL PROPERTIES AND TOXICITY OF WAN KHAN MAK (AGLAONEMA SIMPLEX BL.) FRUIT EXTRACT Ratana Kiatsongchai 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 2015 ฤทธิ์ทางชีวภาพและความเป็นพิษของสารสกัดจากผลว่านขันหมาก (Aglaonema simplex Bl.) นางสาวรัตนา เกียรติทรงชัย วิทยานิพนธ์นี้เป็นส่วนหนึ่งของการศึกษาตามหลกั สูตรปริญญาวทิ ยาศาสตรดุษฎบี ัณฑิต สาขาวิชาชีววิทยาสิ่งแวดล้อม มหาวทิ ยาลัยเทคโนโลยสี ุรนารี ปีการศึกษา 2558 ACKNOWLEDGEMENTS First, I would like to sincerely thanks to Asst. Prof. Benjamart Chitsomboon my thesis advisor for her kindness and helpful. She supports both works and financials. She lightens up my spirit and inspires me to want to be better person. She gave me a chance that leads me to this day. I extend many thanks to my co-advisor, Dr. Chuleratana Banchonglikitkul for her excellent guidance, valuable advices, and kindly let me have a great research experience in her laboratory at The Thailand Institute of Scientific and Technological Research (TISTR), Pathum Thani. I also would like to thank Asst. Prof. Dr. Supatra Porasuphatana, Asst. Prof. Dr. Wilairat Leeanansaksiri, and Assoc. Prof. Dr. Nooduan Muangsan who were willing to participate in my thesis committee. I would never have been able to finish my dissertation without the financial support both of The OROG Fellowship from SUT Institute of Research and Development Program and The Thailand Institute of Scientific and Technological Research (TISTR) and many thanks go to my colleagues and friends, especially members of Dr. Benjamart’ laboratories. They are my best friends who are always willing to help in every circumstance. Lastly, I would also like to thank my family for their love, supports and understanding that help me to overcome many difficult moments. Ratana Kiatsongchai CONTENTS Page ABSTRACT IN THAI ................................................................................................... I ABSTRACT IN ENGLISH ........................................................................................ III ACKNOWLEDGEMENTS .......................................................................................... V CONTENTS…… ........................................................................................................ VI LIST OF TABLES ...................................................................................................... XI LIST OF FIGURES ................................................................................................... XII LIST OF ABBREVIATIONS .................................................................................... XV CHAPTER I INTRODUCTION ............................................................................................. 1 1.1 Significance of the study ............................................................................... 1 1.2 Research objectives ....................................................................................... 5 1.3 Research hypotheses ..................................................................................... 5 1.4 Scope and limitations of the study ................................................................ 6 1.5 Expected results ............................................................................................. 8 II LITERATURE REVIEWS............................................................................... 9 2.1 Agloanema simplex Bl. .................................................................................. 9 2.2 Traditional use and phytochemical properties of Agloanema simplex Bl. .............................................................................. 10 2.3 Phytochemical composition......................................................................... 12 2.4 Free radicals and antioxidants ..................................................................... 16 VII CONTENTS (Continued) Page 2.5 Toxicity test ................................................................................................. 20 2.6 Inflammation ............................................................................................... 22 2.7 Hypersensitivity ........................................................................................... 31 III MATERIALS AND METHODS .................................................................... 34 3.1 Materials ...................................................................................................... 36 3.1.1 Chemicals and reagents ..................................................................... 36 3.1.2 Experimental animals ........................................................................ 37 3.1.2.1 ICR mice .............................................................................. 37 3.1.2.2 Wistar rat ............................................................................... 38 3.1.3 Cell lines ............................................................................................ 38 3.1.3.1 Maintenances of RAW264.7 cells ........................................ 38 3.1.3.2 Maintenances of RBL-2H3 cells ........................................... 39 3.1.4 Plant materials .................................................................................. 40 3.2 Methods ....................................................................................................... 41 3.2.1 Plant extract preparation ................................................................... 41 3.2.2 Determination of chemical constituents ........................................... 42 3.2.3 Determination of phytochemical compounds................................... 44 3.2.3.1 Total phenolic content (TPC)................................................ 44 3.2.3.2 Total flavonoids content (TFC) ............................................ 45 3.2.3.3 Proanthocyanidins content .................................................... 45 3.2.4 Determination of antioxidant properties .......................................... 46 VIII CONTENTS (Continued) Page 3.2.4.1 DPPH assay ........................................................................... 46 3.2.4.2 FRAP assay ........................................................................... 47 3.2.4.3 Cellular antioxidant activity (CAA) assay ............................ 47 3.2.5 In vivo toxicities ................................................................................ 48 3.2.5.1 Acute oral toxicity test .......................................................... 48 3.2.5.2 Acute systemic toxicity test .................................................. 48 3.2.5.3 In vivo mutagenicity test ....................................................... 49 3.2.6 Dose range-finding studies ................................................................ 50 3.2.6.1 MTT method ......................................................................... 51 3.2.6.1.1 Cytotoxic effect of WKM extract towards RAW264.7 .............................................. 51 3.2.6.1.2 Cytotoxic effect of WKM extract towards RBL-2H3 ................................................ 52 3.2.6.2 Trypan blue exclusion method .............................................. 53 3.2.6.2.1 Cytotoxic effect of WKM extract towards RAW264.7 .............................................. 53 3.2.6.2.2 Cytotoxic effect of WKM extract towards RBL-2H3 ................................................ 54 3.2.7 Anti-allergy method .......................................................................... 55 3.2.8 Anti-inflammation method ................................................................ 56 3.2.8.1 nitrite assay ........................................................................... 57 IX CONTENTS (Continued) Page 3.2.8.2 expression of nitric oxide synthase and COX-2 ................... 57 3.3 Location of research .................................................................................... 59 3.4 Statistical analysis ....................................................................................... 59 IV RESULTS ......................................................................................................... 60 4.1 Phytochemical profile .................................................................................. 60 4.1.1 Percent yield of the WKM extraction ................................................ 60 4.1.2 Proximate composition of Wan Khan Mak dried fruit ...................... 60 4.1.3 Total phenolic content (TPC) ............................................................ 61 4.1.4 Total flavonoids content (TFC) ......................................................... 62 4.1.5 Proanthocyanidins content (PA) ........................................................ 62 4.2 Antioxidant properties of extract from WKM fruits ................................... 63 4.2.1 DPPH radical scavenging activity ..................................................... 63 4.2.2 Ferric Reducing/ Antioxidant Power (FRAP) assay ......................... 66 4.2.3 Cellular antioxidant activity (CAA) .................................................. 66 4.3 In vivo toxicity ............................................................................................. 69 4.3.1 Acute toxicity .................................................................................... 69 4.3.2 Systemic toxicity test........................................................................
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