EGb761, a Ginkgo Biloba Extract, Is Effective Against Atherosclerosis In Vitro, and in a Rat Model of Type 2 Diabetes Soo Lim 1,2. , Ji Won Yoon 1,2. , Seon Mee Kang 1,2 , Sung Hee Choi 1,2 , Bong Jun Cho 1 , Min Kim 2 , Ho Seon Park 2 , Hyun Ju Cho 2 , Hayley Shin 3 , Young-Bum Kim 4 , Hyo Soo Kim 2 , Hak Chul Jang 1,2 , Kyong Soo Park 2 * 1 Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea, 2 Department of Internal Medicine, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Korea, 3 Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America, 4 Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America Abstract Background: EGb761, a standardized Ginkgo biloba extract, has antioxidant and antiplatelet aggregation and thus might protect against atherosclerosis. However, molecular and functional properties of EGb761 and its major subcomponents have not been well characterized. We investigated the effect of EGb761 and its major subcomponents (bilobalide, kaemferol, and quercetin) on preventing atherosclerosis in vitro, and in a rat model of type 2 diabetes. Methods and Results: EGb761 (100 and 200 mg/kg) or normal saline (control) were administered to Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, for 6 weeks (from 3 weeks before to 3 weeks after carotid artery injury). Immunohistochemical staining was performed to investigate cell proliferation and apoptosis in the injured arteries. Cell migration, caspase-3 activity and DNA fragmentation, monocyte adhesion, and ICAM-1/VCAM-1 levels were explored in vitro. Treatment with EGb761 dose-dependently reduced intima-media ratio, proliferation of vascular smooth muscle cells (VSMCs) and induced greater apoptosis than the controls. Proliferation and migration of VSMCs in vitro were also decreased by the treatment of EGb761. Glucose homeostasis and circulating adiponectin levels were improved, and plasma hsCRP concentrations were decreased in the treatment groups. Caspase-3 activity and DNA fragmentation increased while monocyte adhesion and ICAM-1/VCAM-1 levels decreased significantly. Among subcomponents of EGb761, kaemferol and quercetin reduced VSMC migration and increased caspase activity. Conclusions: EGb761 has a protective role in the development of atherosclerosis and is a potential therapeutic agent for preventing atherosclerosis. Citation: Lim S, Yoon JW, Kang SM, Choi SH, Cho BJ, et al. (2011) EGb761, a Ginkgo Biloba Extract, Is Effective Against Atherosclerosis In Vitro, and in a Rat Model of Type 2 Diabetes. PLoS ONE 6(6): e20301. doi:10.1371/journal.pone.0020301 Editor: Krisztian Stadler, Pennington Biomedical Research Center, United States of America Received November 8, 2010; Accepted April 29, 2011; Published June 2, 2011 Copyright: ß 2011 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Korean Society of Lipidology and Atherosclerosis (2008), the Korea Science and Engineering Foundation grants (M10642140004–06N4214–00410) and the Information Technology R&D program of Ministry of Information and Communication/Institute for Information Technology Advancement (2006-S075-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kspark@snu.ac.kr . These authors contributed equally to this work. Introduction Ginkgo biloba L. (Ginkgoaceae), known as the ‘maidenhair tree’, is the best-selling herbal remedy in the USA [1]. Traditionally, the fruits and seeds of Ginkgo have been used in Oriental medicine to improve chronic cough or enuresis [2]. Since the early 1990s, EGb761, a standardized extract of Ginkgo leaves, has become the most popularly used dietary supplement for treating vascular circulation problems and improving memory. There are two major fractions in this extract: flavonoids and terpenes. Interestingly, these two have different properties which are responsible for exerting unique and diverse pharmacological actions of EGb761. The flavonoid fraction has antioxidant effects resulting from direct attenuation of reactive oxygen species by chelating pro-oxidant transitional metal ions, and also by promoting the expression of antioxidant proteins which in turn increases antioxidant metabolites such as glutathione [3–5]. The chemical structure of flavonoids comprising of an aromatic ring and a double bond seem to react preferentially with hydroxyl radicals [2]. The terpene lactones include the ginkgolides A, B, C, J and M, and bilobalide [6]. These were found to reduce platelet activation and aggregation by antagonizing platelet activating factor [7,8]. This gives EGb761 the potential to improve blood circulation. In addition, bilobalide, a sesquiterpene trilactone, was shown to reduce cerebral edema, cortical infarct volume and ischemic damage in patients following a stroke [9]. EGb761 has also been shown to have various antiapoptotic properties[6] and to inhibit amyloid-beta aggregation [10]. PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20301