title: Neuroprotection and spatial memory enhancement of four herbal mixture extract in HT22 hippocampal cells and a mouse model of focal cerebral ischemia.
abstract: BACKGROUND
  Four traditional Korean medicinal herbs which act in retarding the aging process, Polygonum multiflorum Thunb., Rehmannia glutinosa (Gaertn) Libosch., Polygala tenuifolia Willd., and Acorus gramineus Soland., were prepared by systematic investigation of Dongeuibogam (Treasured Mirror of Eastern Medicine), published in the early 17th century in Korea. This study was performed to evaluate beneficial effects of four herbal mixture extract (PMC-12) on hippocampal neuron and spatial memory.
  
  
  METHODS
  High performance liquid chromatography (HPLC) analysis was performed for standardization of PMC-12. Cell viability, lactate dehydrogenase, flow cytometry, reactive oxygen species (ROS), and Western blot assays were performed in HT22 hippocampal cells and immunohistochemistry and behavioral tests were performed in a mouse model of focal cerebral ischemia in order to observe alterations of hippocampal cell survival and subsequent memory function.
  
  
  RESULTS
  In the HPLC analysis, PMC-12 was standardized to contain 3.09% 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, 0.35% 3',6-disinapoyl sucrose, and 0.79% catalpol. In HT22 cells, pretreatment with PMC-12 resulted in significantly reduced glutamate-induced apoptotic cell death. Pretreatment with PMC-12 also resulted in suppression of ROS accumulation in connection with cellular Ca(2+) level after exposure to glutamate. Expression levels of phosphorylated p38 mitogen-activated protein kinases (MAPK) and dephosphorylated phosphatidylinositol-3 kinase (PI3K) by glutamate exposure were recovered by pretreatment with either PMC-12 or anti-oxidant N-acetyl-L-cysteine (NAC). Expression levels of mature brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element binding protein (CREB) were significantly enhanced by treatment with either PMC-12 or NAC. Combination treatment with PMC-12, NAC, and intracellular Ca(2+) inhibitor BAPTA showed similar expression levels. In a mouse model of focal cerebral ischemia, we observed higher expression of mature BDNF and phosphorylation of CREB in the hippocampus and further confirmed improved spatial memory by treatment with PMC-12.
  
  
  CONCLUSIONS
  Our results suggest that PMC-12 mainly exerted protective effects on hippocampal neurons through suppression of Ca(2+)-related ROS accumulation and regulation of signaling pathways of p38 MAPK and PI3K associated with mature BDNF expression and CREB phosphorylation and subsequently enhanced spatial memory.
authors.affiliation: Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. isdly@pusan.ac.kr.|Research Center for Anti-aging Technology Development, Pusan National University, Busan, 609-735, Republic of Korea. isdly@pusan.ac.kr.
authors.forename: Sung Min
authors.initials: SM
authors.lastname: Ahn
authors.affiliation: Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. sprout_ing@pusan.ac.kr.
authors.forename: Yu Ri
authors.initials: YR
authors.lastname: Kim
authors.affiliation: Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. haneui@pusan.ac.kr.
authors.forename: Ha Neui
authors.initials: HN
authors.lastname: Kim
authors.affiliation: Department of Horticultural Bioscience, College of Natural Resource and Life Science, Pusan National University, Miryang, 626-706, Republic of Korea. ywchoi@pusan.ac.kr.|Research Center for Anti-aging Technology Development, Pusan National University, Busan, 609-735, Republic of Korea. ywchoi@pusan.ac.kr.
authors.forename: Young Whan
authors.initials: YW
authors.lastname: Choi
authors.affiliation: Division of Pharmacy, College of Pharmacy, Pusan National University, Busan, 609-735, Republic of Korea. neuron@pusan.ac.kr.|Research Center for Anti-aging Technology Development, Pusan National University, Busan, 609-735, Republic of Korea. neuron@pusan.ac.kr.
authors.forename: Jae Won
authors.initials: JW
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, College of Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. kimcm@pusan.ac.kr.|Research Center for Anti-aging Technology Development, Pusan National University, Busan, 609-735, Republic of Korea. kimcm@pusan.ac.kr.
authors.forename: Cheol Min
authors.initials: CM
authors.lastname: Kim
authors.affiliation: Division of Humanities and Social Medicine, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. mukjagan@pusan.ac.kr.
authors.forename: Jin Ung
authors.initials: JU
authors.lastname: Baek
authors.affiliation: Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. julie@pusan.ac.kr.|Research Center for Anti-aging Technology Development, Pusan National University, Busan, 609-735, Republic of Korea. julie@pusan.ac.kr.
authors.forename: Hwa Kyoung
authors.initials: HK
authors.lastname: Shin
authors.affiliation: Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. choibt@pusan.ac.kr.|Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan, 626-870, Republic of Korea. choibt@pusan.ac.kr.|Research Center for Anti-aging Technology Development, Pusan National University, Busan, 609-735, Republic of Korea. choibt@pusan.ac.kr.
authors.forename: Byung Tae
authors.initials: BT
authors.lastname: Choi
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism*; D002460:Cell Line; D004195:Disease Models, Animal; D006624:Hippocampus / Q000166:cytology*; D051379:Mice; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010936:Plant Extracts / Q000494:pharmacology*; D065852:Spatial Memory / Q000187:drug effects*
chemical_list: D018696:Neuroprotective Agents; D010936:Plant Extracts