創傷性腦損傷是由外力衝擊大腦所引起的腦損傷，主要是因為跌倒、交通事故、攻擊事件所造成，此種腦損傷可能會傷害腦神經，導致行為和認知功能受損。不幸地，目前尚無有效促進腦神經再生的治療方法，部分原因是因為中樞神經系統中神經的再生能力相當有限。為了找出能促進腦神經再生的有效方法，和辜嚴倬雲植物保種中心及高雄醫學大學天然藥物研究所合作，獲得了2,112種植物萃取物，我們篩選了此2,112種萃取物，並找到最有效的一種植物萃取物，是來自於拎藤龜背芋的植物萃取物。接著，從此植物萃取物中分離出水溶性和乙酸乙酯溶性的萃取物，透過神經培養和控制皮層撞擊模型研究了它們對於腦神經再生及大腦功能的影響。我們發現此植物的水溶性萃取物能促進受傷後的腦神經再生。另外也發現此植物萃取物能增加腦創傷周圍的神經數量及恢復行為功能。綜合以上研究的結果，指出拎藤龜背芋的水溶性萃取物中可能含有能促進腦神經再生的活性成分，具有發展成為治療TBI的有效藥物的潛力。

Traumatic brain injury (TBI) is defined as a brain injury caused by external mechanical forces which mainly results from falls, motor vehicle traffic crashes, struck by/against events, or assaults. Such brain injury can cause severe damage to brain and lead to impairment of motor, physical, and cognitive functions. Unfortunately, there is no effective treatment for neuronal regeneration currently in part due to the limited regenerative capacity of central nervous system (CNS). We thus would like to search for candidate treatment for promoting regeneration of injured brain neurons. We have screened 2,112 plant extracts provided from Dr. Cecilia Koo Botanic Conservation Center (KBCC) and the Graduate Institute of Natural Products at Kaohsiung Medical University (KMU) and identify the most promising one, Monstera epipremnoides extract (MeE). After fractionating the water-soluble and ethyl acetate-soluble extracts from MeE, their effects were investigated via in vitro primary neuronal culture and in vivo controlled cortical impact (CCI) model. Our results suggest that the active components should be in the water-soluble fraction of MeE, MeEw. MeEw as well as MeE can promote neurite re-growth of injured hippocampal and cortical neurons. Furthermore, MeE can increase neuron numbers around injured site and recover motor function. Together, data from this thesis demonstrate that active components within MeEw may provide therapeutic potential for TBI.

Abstract …………………………………………………………………………...……... i
摘要………………………………….………………………………................................... ii
致謝………………………………….……………………………….................................. iii
Table of Contents ………………………………….…………………………………....... vi
Introduction ……………………………………………………….…………………....…. 1
Materials and methods ……………………………………….….………………………. 10
Animals and ethics approval ….………………………….………………………... 10
Reagents and antibodies ………………………………………...…………………. 10
Plant material for screening …….…………………………..………………..……. 11
Preparation of extracts from Monstera epipremnoides ……....…………………... 11
Primary neuronal culture and injury ………………………………………………12
Treatment of plant extracts in vitro …………………….…………………………. 12
Immunofluorescence staining …..…………….………………………………….... 13
Quantification of neurite re-growth ……..……………………………………….... 13
Control cortical impact (CCI) model …………………….……………….………. 14
Intranasal administration …..…………….………………………………………... 14
Immunohistochemistry (IHC) staining ……..…………………………………...... 15
Analysis of fluorescence images of brain cryosections …………………….…........ 16
Cylinder test …..…………….………………………………………......................... 16
Horizontal bar test …………………………...…………………………………...... 17
Statistical analysis ………………………………………..……………………........ 17
Results ……………………………………………….………………………………….... 18
Screen plant extracts that promote regeneration of injured cortical neurons. …. 18
Monstera epipremnoides extract promotes regeneration of injured cortical and hippocampal neurons in vitro. ……………………………………………………... 19
Water-soluble extract isolated from MeE enhances regeneration of injured brain neurons in vitro. ………………………………………….......................................... 20
MeE increases neuron numbers and functional recovery in vivo. ……….…….... 21
Discussion ………………………………………………………………………………... 24
Expected effect of DMSO on neurite re-growth. …………………………………. 24
Dissolving MeEW in ddH2O on regeneration of injured brain neurons. ……….... 24
The effect of higher dosage of MeEW on functional assays upon TBI. ................... 25
Possible reasons for unexpected effect of MeEW after mouse brain injury. …....... 26
Effect of compounds with anti-inflammatory activity on recovery of TBI. …....... 28
Figures …………………………………………………………………………………..... 31
Figure 1. Experimental design of in vitro TBI model for screening 2,112 plant extracts. …………………………………………………………………………....... 31
Figure 2. Calculation of gap closure rate as a capacity of neuronal regeneration. ………………………………………………………………………... 32
Figure 3. Neuronal regenerative capacity of 2,112 plant extracts. ………............. 33
Figure 4. Neuronal regenerative capacity of fifteen plant extracts in the second screening. …………………………………………………………………….….….. 34
Figure 5. Experimental design of in vitro TBI model for evaluating the effects of MeE on injured cortical and hippocampal neurons. ………….…...………...…..... 35
Figure 6. MeE promotes regeneration of injured cortical and hippocampal neurons. …………………………………………………..…………………………. 36
Figure 7. Optimal dosages for MeE to enhance regeneration of injured cortical neurons. ………………………………..……………………………………………. 38
Figure 8. MeEW promotes neurite re-growth in injured cortical and hippocampal neurons. ……………………………………………………………………………... 40
Figure 9. Experimental design of in vivo TBI model. ……………………………... 41
Figure 10. The effect of MeE, MeEW or MeEEA on neuron numbers around injured sites. ……………………………………………………………………….………… 42
Figure 11. The effects of MeE, MeEW or MeEEA on expression of GFAP. ….......... 43
Figure 12. The number of rears of injured mice treated with MeE, MeEW or MeEEA. …………………………………………………………………….….……... 44
Figure 13. Dissolving MeEW in ddH2O enhances neurite re-growth in injured cortical and hippocampal neurons. ………….…..............................………...…..... 45
Figure 14. Experimental design of long-term treatment of in vivo TBI model. …………………………………………………..………………………….... 47
Figure 15. MeEW slightly increases neuron numbers around injured sites upon 14 days after brain injury. …………………………………………………………….. 48
Figure 16. MeEW slightly decreases P/D and P/U of GFAP expression upon 14 days after brain injury. …………………………………………………………………... 49
Figure 17. The number of rears of injured mice treated with MeEW. ………….... 50
Figure 18. The score of injured mice treated with MeEW from horizontal bar test. ……………….……………………………………………………….………… 51
Figure 19. Effect of antcin A and lucidone on neurite re-growth in injured cortical and hippocampal neurons. …………………………………………………………. 52
Figure 20. Effect of antcin A and lucidone on neuron numbers around injured sites. ……………………………………………………..…………………………... 54
Figure 21. Effect of antcin A and lucidone on P/D and P/U of GFAP expression. . 55
Figure 22. The number of rears of injured mice treated with antcin A and lucidone. ……………….………………………………………………….………… 56
Tables …………………………………………………………………………………….. 57
Table 1. ……………………………………………………………………………… 57
Reference ………………………………………………………………………………… 58

1. M. C. Dewan, A. Rattani, et al., Estimating the global incidence of traumatic brain injury. J Neurosurg 2019, 130, 1080-1097.
2. J. A. Langlois, W. R. Brown, et al., The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil 2006, 21, 375-378.
3. Y. C. Tung, H. P. Tu, et al., Increased incidence of herpes zoster and postherpetic neuralgia in adult patients following traumatic brain injury: a nationwide population-based study in Taiwan. PLoS ONE 2015, 10(6), e0129043.
4. C. A. Taylor, J. M. Bell, et al., Traumatic brain injury–related emergency department visits, hospitalizations, and deaths - United States, 2007 and 2013. MMWR Surveill Summ 2017, 66, 1-16.
5. A. E. Davis, Mechanisms of traumatic brain injury: biomechanical, structural and cellular considerations. Crit Care Nurs Q 2000, 23, 1-13.
6. I. Cernak, Animal models of head trauma. NeuroRx 2005, 2, 410-422.
7. Y. Xiong, A. Mahmood, et al., Animal models of traumatic brain injury. Nature Rev Neurosci 2013, 14, 128-142.
8. M. L. Pearn, I. R. Niesman, et al., Pathophysiology associated with traumatic brain injury: current treatments and potential novel therapeutics. Cell Mol Neurobiol 2016, 37(4), 571-585.
9. B. Fehily and M. Fitzgerald, Repeated mild traumatic brain injury: potential mechanisms of damage. Cell Transplant 2017, 26(7), 1131-1155.
10. E. A. Huebner and S. M. Strittmatter, Axon regeneration in the peripheral and central nervous systems. Results Probl Cell Differ 2010, 48, 339-351.
11. D. A. Morgenstern, R. A. Asher, et al., Chondroitin sulphate proteoglycans in the CNS injury response. Prog Brain Res 2002, 137, 313-332.
12. L. McKerracher, S. David, et al., Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron 1994, 13(4), 805-811.
13. V. Kottis, P. Thibault, et al., Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth. J Neurochem 2002, 82(6), 1566-1569.
14. M. S. Chen, A.B. Huber, et al., Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 2000, 403(6768), 434-439.
15. M. D. Benson, M. I. Romero, et al., Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc Natl Acad Sci U S A 2005, 102(30), 10694-10699.
16. F. Sun and Z. He, Neuronal intrinsic barriers for axon regeneration in the adult CNS. Curr Opin Neurobiol 2010, 20(4), 510-518.
17. N. Abe and V. Cavalli, Nerve injury signaling. Curr Opin Neurobiol 2008, 18(3), 276-283.
18. R. Seijffers, A. J. Allchorne, et al., The transcription factor ATF-3 promotes neurite outgrowth. Mol Cell Neurosci 2006, 32(1-2), 143-154.
19. T. Miao, D. Wu, et al., Suppressor of cytokine signaling-3 suppresses the ability of activated signal transducer and activator of transcription-3 to stimulate neurite growth in rat primary sensory neurons. J Neurosci 2006, 26(37), 9512-9519.
20. C. Y. Chang, M. Z. Liang, et al., WNT3A promotes neuronal regeneration upon traumatic brain injury. Int J Mol Sci 2020, 21(4), 1463.
21. P. K. Pandey, A. K. Sharma, et al., Blood brain barrier: An overview on strategies in drug delivery, realistic in vitro modeling and in vivo live tracking. Tissue Barriers 2016, 4, 1129476.
22. C. Katiyar, A. Gupta, et al., Drug discovery from plant sources: An integrated approach. Ayu 2012, 33(1), 10-19.
23. A. L. Harvey, Natural products in drug discovery. Drug Discovery Today 2008, 13(19-20), 894-901.
24. S. Sasidharan, Y. Chen, et al., Extraction, isolation and characterization of bioactive compounds from plants' extracts. Afr J Tradit Complement Altern Med 2011, 8(1), 1-10.
25. A. Jones, Chemistry: An introduction for medical and health sciences. John Wiley & Sons 2015, 5-6.
26. P. Patrignani and C. Patrono, Aspirin and cancer. Journal of the American College of Cardiology 2016, 68 (9), 967-976.
27. I. Carrera and R. Cacabelos, Current drugs and potential future neuroprotective compounds for Parkinson’s disease. Curr Neuropharmacol 2019, 17(3), 295-306.
28. T. O. Elufioye and T. I. Berida, Plants-derived neuroprotective agents: Cutting the cycle of cell death through multiple mechanisms. eCAM 2017, 2017, 3574012.
29. H. H. Chen, P. C. Chang, et al., Therapeutic effects of honokiol on motor impairment in hemiparkinsonian mice are associated with reversing neurodegeneration and targeting PPARγ regulation. Biomed Pharmacother 2018, 108, 254-262.
30. A. Woodbury, S. P. Yu, et al., Neuro-modulating effects of honokiol: A review. Front Neurol 2013, 4, 130.
31. J. Chen, R.J. Henny, et al., Aroids are important medicinal plants. Acta horticulturae 2007, 756(756), 347-353.
32. A. Camporese, M. J. Balick, et al., Screening of anti-bacterial activity of medicinal plants from Belize (Central America). J Ethnopharmacol 2003, 87, 103-107.
33. T. Khan, M. Ahmad, et al., Evaluation of phytomedicinal potentials of selected plants of Pakistan. Amer Lab 2006, 20-22.
34. T. Kato, B. Frei, et al., Antibacterial hydroperoxysterols from Xanthosoma robustum. Phytochemistry 1996, 41, 1191-1195.
35. H. R. El-Seedi, L. Bohlin, et al., Ecological active 2- octanoylcyclohexane-1,3-dione from Philodendron guttiferum. J Chem Ecol 2001, 27, 517-521.
36. C. O. Okoli, M. Pharm, et al., A pilot evaluation of the antiinflammatory activity of Culcasia scandens, a traditional antirheumatic agent. J Altern Complem Med 2000, 6, 423-427.
37. L. Juan, C. Keli, et al., Anti‐tumor activities of extracts from the medicinal plants Pinellia ternata and Pinellia pedatisecta. 3rd ICBBE 2009, 1- 4.
38. C. Y. Lee and Y. S. Wong, The illustrated medicinal plants of Malaysia, PK Herbal Research Centre 2004, 65.
39. Z. H. Liang, X. H. Cheng, et al., Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42. Neural Regen Res 2015, 10(8), 1292-1297.
40. S. Schröder, K. Beckmann, et al., Can medical herbs stimulate regeneration or neuroprotection and treat neuropathic pain in chemotherapy-induced peripheral neuropathy? eCAM 2013, 423713-423730.
41. B. E. Hammel, M. H. Grayum, et al., Manual de Plantas de Costa Rica. Missouri Botanical Garden Press 2003, 2, 1-694.
42. U. Quattrocchi, CRC World Dictionary of Plant Names. CRC Press 2000, 1723.
43. N.C. Cooperative Extension, Monstera epipremnoides. The North Carolina Extension Gardener Plant Toolbox 2020.
44. Plantophiles, Monstera Epipremnoides Care. Plant care 2020.
45. A. Quigley, Gardening 101: Monstera. Garden Design 101 2019, 3.
46. Z. Hussain, A. Waheed, et al., The effect of medicinal plants of Islamabad and Murree region of Pakistan on insulin secretion from INS-1 cells. Phytotherapy Research 2004, 18(1), 73-77.
47. B. Morrison 3rd, B. S. Elkin, et al., In vitro models of traumatic brain injury. Annu Rev Biomed Eng 2011, 13, 91-126.
48. J. Romine, X. Gao, et al., Controlled cortical impact model for traumatic brain injury. J Vis Exp 2014, 90, 51781.
49. R. G. Thorne, G. J. Pronk, et al., Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. Neuroscience 2004, 127(2), 127-481.
50. S. T. Fujimoto, L. Longhi, et al., Motor and cognitive function evaluation following experimental traumatic brain injury. Neurosci Biobehav Rev 2004, 28(4), 365-378.
51. K. L. Russell, K. M. Kutchko, et al., Sensorimotor behavioral tests for use in a juvenile rat model of traumatic brain injury: assessment of sex differences. J Neurosci Methods 2011, 199(2), 214-222.
52. Q. Ahkong, D. Fisher, et al., Mechanisms of cell fusion. Nature 1975, 253, 194-195.
53. T. J. Anchordoguy, J. F. Carpenter, et al., Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide. Biochim Biophys Acta 1992, 1104(1), 117-122.
54. Z. W. Yu and P. J. Quinn, The modulation of membrane structure and stability by dimethyl sulphoxide. Molecular Membrane Biology 1998, 15, 59-68.
55. B. WInckler, P. Forscher, et al. A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature 1999, 397, 698-701.
56. R. Shi, X. Qiao, et al., Dimethylsulfoxide enhances CNS neuronal plasma membrane resealing after injury in low temperature or low calcium. Journal of Neurocytology 2001, 30, 829-839.
57. Y. Yao, B. Zhang, et al., MG53 permeates through blood-brain barrier to protect ischemic brain injury. Oncotarget 2016, 7(16), 22474-22485.
58. B. J. Paleo, K. M. Madalena, et al., Enhancing membrane repair increases regeneration in a sciatic injury model. PLoS One 2020, 15(4), e0231194.
59. R. M. J. Deacon, Measuring motor coordination in mice. J Vis Exp 2013, 75, 2609.
60. F. Erdő, L. A. Bors, et al., Evaluation of intranasal delivery route of drug administration for brain targeting. Brain Res Bull 2018, 143, 155-170.
61. S. R. Mudshinge, A. B. Deore, et al., Nanoparticles: Emerging carriers for drug delivery. Saudi Pharmaceutical Journal 2011, 19(3), 129-141.
62. J. Yoo, C. Park, et al., Active targeting strategies using biological ligands for nanoparticle drug delivery systems. Cancers (Basel) 2019, 11(5), 640.
63. X. Yue, Q. Zhang, et al., Near-infrared light-activatable polymeric nanoformulations for combined therapy and imaging of cancer. Adv Drug Deliv Rev 2017, 115, 155-170.
64. S. Rao, R. Chen, et al., Remotely controlled chemomagnetic modulation of targeted neural circuits. Nature Nanotechnology 2019, 14, 967-973.
65. F. Talebpour, A. Ghahghaei, Effect of green synthesis of gold nanoparticles (AuNPs) from Hibiscus sabdariffa on the aggregation of α-lactalbumin. International Journal of Peptide Research and Therapeutics 2020, 1-10.
66. N. Gao, H. Sun, et al., Gold‐nanoparticle‐based multifunctional amyloid‐β inhibitor against Alzheimer’s disease. Chemistry-A European Journal 2015, 21, 829-835.
67. R. Prades, S. Guerrero, et al., Delivery of gold nanoparticles to the brain by conjugation with a peptide that recognizes the transferrin receptor. Biomaterials 2012, 33, 7194-7205.
68. N. R. Carlson and M. A. Birkett, Physiology of Behavior. 2010, 38-39.
69. R. Cabezas, M. Ávila, et al., Astrocytic modulation of blood brain barrier: perspectives on Parkinson’s disease. Front Cell Neurosci 2014, 8, 211.
70. J. W. Finnie, Neuroinflammation: beneficial and detrimental effects after traumatic brain injury. Inflammopharmacology 2013, 21(4), 309-320.
71. P. J. Bergold, Treatment of traumatic brain injury with anti-inflammatory drugs. Exp Neurol 2016, 275(3), 367-380.
72. R. Tehranian R, S. A. Jonsson, et al., Improved recovery and delayed cytokine induction after closed head injury in mice with central overexpression of the secreted isoform of the interleukin-1 receptor antagonist. J Neurotrauma 2002, 19(8), 939-951.
73. N. C. Jones, M. J. Prior, et al., Antagonism of the interleukin-1 receptor following traumatic brain injury in the mouse reduces the number of nitric oxide synthase-2-positive cells and improves anatomical and functional outcomes. Eur J Neurosci 2005, 22, 72-78.
74. H. Girgis, B. Palmier, et al., Effects of selective and non-selective cyclooxygenase inhibition against neurological deficit and brain oedema following closed head injury in mice. Brain Res 2013, 1491, 78-87.
75. P. G. Fine, The role of rofecoxib, a cyclooxygenase-2-specific inhibitor, for the treatment of non-cancer pain: A review. The Journal of Pain 2002, 3(4), 272-283.
76. M. A. Clond, B. S. Lee, et al., Reactive oxygen species-activated nanoprodrug of ibuprofen for targeting traumatic brain injury in mice. PLoS One 2013, 8, e61819.
77. T. Kunz, N. Marklund, et al., Effects of the selective cyclooxygenase-2 inhibitor rofecoxib on cell death following traumatic brain injury in the rat. Restor Neurol Neurosci 2006, 24,55-63.
78. Y. C. Chen, Y. L. Liu, et al., Antcin A, a steroid-like compound from Antrodia camphorata, exerts anti-inflammatory effect via mimicking glucocorticoids. Acta Pharmacol Sin 2011, 32(7), 904-911.
79. K. J. S. Kumar, H. L. Yang, et al., Lucidone protects human skin keratinocytes against free radical-induced oxidative damage and inflammation through the up-regulation of HO-1/Nrf2 antioxidant genes and down-regulation of NF-κB signaling pathway. Food and Chemical Toxicology 2013, 59, 55-66.