雷公根與酸棗仁酒精萃取物於分化 PC12 細胞株中對類澱粉樣蛋白 β 胜肽產生神經毒性之保護

Abstract

阿茲海默症 (Alzheimer’s disease, AD) 是易發於 65 歲以上老年人之神經退化性疾病，使患者在記憶、語言與行為等多種認知功能出現退化，嚴重影響病人的生活品質與人際關係。其致病原因是聚合之類澱粉樣蛋白質 β (amyloid β, Aβ) 於腦部大量沉積，引發神經元累積活性氧分子 (reactive oxygen species, ROS)，產生氧化壓力與發炎反應，促使神經元受損。許多中草藥都具有良好的抗氧化與抗發炎能力，有潛力應用於治療或預防 Aβ 引起的氧化壓力上升或發炎反應。因此本研究目的為尋找具抗阿茲海默症之中草藥，並評估其可能作用機制，以開發預防阿茲海默症之保健食品。本研究第一部份，以 14 種可食用性中草藥，包含，茵陳、紅花、玫瑰、葛根、決明子、薑黃、百合、遠志、雷公根、咸豐草、丹蔘、芝麻、砂仁與酸棗仁為材料，篩選具高抗氧化力之中草藥萃取物。選出萃取物，再以 PC12 細胞為對象分析其細胞毒性與抗 Aβ 引起之細胞傷害。第二部分則針對篩選出抗 Aβ 毒性之雷公根與酸棗仁兩種中草藥萃取物，探討其可能對抗 Aβ 傷害之原因。 第一部分，14 種中草藥分別以 20、40、60、80 及 95% 濃度之酒精 (A、B、C、D 及E 組)，以及去離子水萃取 (F 組)。萃取物以四項體外抗氧化能力指標：總酚類含量、Trolox 等價抗氧化力 (trolox equivalent antioxidant capacity, TEAC)、還原力以及亞鐵螯合能力篩選。選出總酚類含量、TEAC 與還原力三項指標中最佳之玫瑰與茵陳 A、B、C 組萃取物；以及亞鐵螯合能力最佳之酸棗仁 A、B 組萃取物；另根據文獻指出雷公根具抗阿茲海默症效果，亦選擇雷公根各組萃取物，進行 PC12 細胞模式篩選。於 PC12 模式中得知，玫瑰與茵陳 A、B、C 組萃取物具高細胞毒性，不適合評估抗聚合 Aβ1-40 毒性傷害。而雷公根 A 組與 F 組萃取物以及酸棗仁 B 組萃取物，則對未分化與分化 PC12 細胞不具傷害性，且其 25、50 與 100 μg/mL 劑量皆有抗聚合 Aβ1-40 細胞毒性，維持細胞存活率，達到保護細胞之功效。但雷公根 A 組萃取物較 F 組萃取物保護時間長，故進一步探討雷公根 A 組與酸棗仁 B 組萃取物抗 Aβ 毒性之原因。 第二部分以分化 PC12 細胞模式探討雷公根 A 組萃取物與酸棗仁 B 組萃取物抗聚合 Aβ1-40 細胞毒性之原因。發現雷公根 A 組萃取物，係以降低 Aβ1-40 之聚合程度，提升體內抗氧化酵素包含觸媒 (catalase)、超氧化物岐化酶 (superoxide dismutase, SOD)、麩胱甘肽過氧化酶 (glutathione peroxidase, GPx) 與麩胱甘肽還原酶 (glutathione reductase, GR) 等多種酵素活性，增加抗氧化小分子麩胱甘肽 (glutathione, GSH) 含量，減低一氧化氮 (nitric oxide, NO) 含量，抑制由 Aβ1-40 引起之 ROS 累積與發炎反應，達到防止細胞損傷功能。酸棗仁 B 組萃取物則有絕佳的亞鐵螯合能力，可螯合亞鐵降低 Aβ1-40 與金屬離子結合產生之氧化物質。同時酸棗仁 B 組萃取物也具降低聚合 Aβ1-40 之聚合程度，提升體內抗氧化酵素包含 catalase、SOD、GPx、GR 等酵素活性，減低 NO 含量，而抑制由 Aβ1-40 引起之氧化與發炎反應，防止細胞損傷。綜合以上結果，雷公根與酸棗仁酒精萃取物具備提升細胞抗氧化防禦機制與降低 Aβ 聚合程度之功效，可以抑制由 Aβ1-40 引起之氧化與發炎反應，極具開發成預防 AD 保健產品或輔助治療藥劑之潛力。

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder mostly affecting the population of above 65 years of age. AD patients suffer from many cognitive abilities losses such as memory loss, language disability and behavior dysfunction, etc, and seriously affected their quality of life and social relationship. The most important pathology of AD is Amyloid β (Aβ) deposit, a condition which generates massive reactive oxygen species (ROS) in neurons and causes neurons damage. There are good antioxidant activities and anti-inflammation in many Chinese herbs that possibly protect neurons from AD induced oxidative stress and inflammation. Therefore, the purpose of this study is to search for high antioxidant activity Chinese herbs and assess the prevention mechanisms of these Chinese herbs. In the first section, the extracts of the 14 kinds of Chinese herbs, Aretemisia capillaries, Carthamus tinctorius, Rosa rugosa, Pueraria matsumura, Cassiae torae semen, Curcuma longa, Lilii bulbus, Polygala tenuifolia Willd, Centella asiatica, Bidens pilo, Salvia miltiorrhiza Bge, Sesamum indicum, Amomum villosum Lour and Ziziphi spinosae semen, were tested by antioxidant activity, then scanned by the PC12 cells model to analyze the cell toxicity and the protection ability against Aβ neurotoxicity. In the second section we studied the Aβ neurotoxicity prevention function of the Centella asiatica and the Ziziphi spinosae semen extracts. For the first section, 14 kinds of Chinese herbs were extracted with 20, 40, 60, 80 or 95% ethanol (A, B, C, D and E group) or were extracted with deionized water (F group). Then, all of the extracts were scanned by total polyphenols concentration, Trolox equivalent antioxidant capacity (TEAC), reducing power and Fe2+ chelating ability. We found that the Aretemisia capillaries and Rosa rugosa A, B and C group extracts had outstanding abilities in total polyphenols concentration, TEAC value and reducing power. The Ziziphi spinosae semen A and B group extracts had optimum capacity in Fe2+ chelating ability. In addition, the Centella asiatica extracts were selected because of previous studies had shown that the Centella asiatica extracts could prevent AD. In the aggregated Aβ1-40 neurotoxin model on PC12 cells, we found only the Centella asiatica A and F group extracts and the Ziziphi spinosae semen B group extract had lower toxicity than Aretemisia capillaries and Rosa rugosa A, B and C group extracts. Meanwhile the Centella asiatica A and F group extracts and the Ziziphi spinosae semen B group extracts at 25, 50 and 100 μg/mL dose could protect differentiated PC12 cells from aggregated Aβ1-40 neurotoxin. However, the Centella asiatica A group extracts showed longer protection effect than F group extracts. So we chose the Centella asiatica A group and Ziziphi spinosae semen B group extracts to study the reasond of the protection of differentiated PC12 cells againt Aβ1-40 neurotoxicity. In the second section, we further study the protection mechanism of the Centella asiatica A group and Ziziphi spinosae semen B group extracts. We discovered that the Centella asiatica A group extract could decrease Aβ1-40 aggregated levels and enhance the antioxidative system on the cell. The properties included activating catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and the antioxidant molecule, glutathione (GSH) and inhibited nitric oxide (NO) production. Therefore, the Centella asiatica A group extract could inhibit the ROS accumulated and inflammation induced by Aβ1-40. The Ziziphi spinosae semen B group extracts showed excellent Fe2+ chelating ability for block the reaction between Aβ1-40 and Fe2+ , which can induce massive ROS such as hydrogen peroxide. Furthermore, the medicinal property of the Ziziphi spinosae semen B group extract, like the Centella asiatica A group extract, cna decrease Aβ1-40 aggregated levels and enhance the antioxidative system on the cell. The properties included activating catalase, SOD, GPx and GR activities and decreasing NO production to inhibit the ROS accumulated and inflammation induced by Aβ1-40. In conclusion, the Centella asiatica A group and Ziziphi spinosae semen B group extracts have prevented AD function by inhibiting the Aβ induced oxidative stress and inflammation. Therefore, these extracts can have the potential to be developed into functional food for the prevention of AD or into adjuvant agent for AD therapy.