Journal of Cardiology (2008) 52, 127���132 ORIGINAL ARTICLE Tropical fruit camu-camu (Myrciaria dubia) has anti-oxidative and anti-inflammatory properties Teruo Inoue (MD) a,���, Hiroshi Komoda (MS) a, Toshihiko Uchida (MD) b, Koichi Node (MD) a a Department of Cardiovascular and Renal Medicine, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan b Department of Cardiology, Koshigaya Hospital, Dokkyo Medical University, Koshigaya 343-8555, Japan Received 21 April 2008 received in revised form 12 June 2008 accepted 16 June 2008 Available online 29 July 2008 KEYWORDS Camu-camu Anti-oxidant Inflammation Vitamin C Smoking Summary Background: Oxidative stress as well as inflammation plays a pivotal role in the pathogenesis of atherosclerosis. Although, various anti-oxidative dietary supple- ments have been evaluated for their ability to prevent atherosclerosis, no effective ones have been determined at present. ������Camu-camu������ (Myrciaria dubia) is an Ama- zonian fruit that offers high vitamin C content. However, its anti-oxidative property has not been evaluated in vivo in humans. Methods: To assess the anti-oxidative and anti-inflammatory properties of camu- camu in humans, 20 male smoking volunteers, considered to have an accelerated oxidative stress state, were recruited and randomly assigned to take daily 70 ml of 100% camu-camu juice, corresponding to 1050 mg of vitamin C (camu-camu group n = 10) or 1050 mg of vitamin C tablets (vitamin C group n = 10) for 7 days. Results: After 7 days, oxidative stress markers such as the levels of urinary 8-hydroxy-deoxyguanosine (P 0.05) and total reactive oxygen species (P 0.01) and inflammatory markers such as serum levels of high sensitivity C reactive protein (P 0.05), interleukin (IL)-6 (P 0.05), and IL-8 (P 0.01) decreased sig- nificantly in the camu-camu group, while there was no change in the vitamin C group. Conclusions: Our results suggest that camu-camu juice may have powerful anti-oxidative and anti-inflammatory properties, compared to vitamin C tablets containing equivalent vitamin C content. These effects may be due to the exis- tence of unknown anti-oxidant substances besides vitamin C or unknown substances modulating in vivo vitamin C kinetics in camu-camu. �� 2008 Published by Elsevier Ireland Ltd on behalf of Japanese College of Cardiology. ��� Corresponding author. Tel.: +81 952 34 2364 fax: +81 952 34 2089. E-mail address: inouete@med.saga-u.ac.jp (T. Inoue). 0914-5087/$ ��� see front matter �� 2008 Published by Elsevier Ireland Ltd on behalf of Japanese College of Cardiology. doi:10.1016/j.jjcc.2008.06.004

128 T. Inoue et al. Introduction Accumulating evidence has demonstrated that oxidative stress (i.e., dysregulation of the cellular redox state) and inflammation play a pivotal role in the pathogenesis of atherosclerosis interacting with each other. Although, various anti-oxidative dietary supplements have been evaluated for their ability to prevent atherosclerosis, no effective ones have been determined at present. Vitamin C is not only the most important dietary anti-oxidant [1,2], but is also suggested to be a potent anti- inflammatory agent [3���5]. Several studies have found that dietary vitamin C or plasma vitamin C was associated with a protective effect against coronary artery disease [6���9]. ������Camu-camu������ is an Amazonian fruit that offers high vitamin C content ranging from 9 to 50 g/kg, which is twofold that of acelora [10]. Additionally, in vitro anti-oxidant activity of 100% camu-camu juice evaluated by DPPH method reaches 50-fold of that of 100% acelora juice (unpublished data), suggesting its potentiality as an effective dietary supplement to prevent atherosclerotic disease. However, the anti-oxidative property has not been evaluated in an in vivo human study. Furthermore, the effects of camu-camu on inflammation have not been elucidated. This study was designed to establish the anti- oxidative and anti-inflammatory properties of camu-camu in vivo in humans. Methods Study design Study subjects included 20 healthy male volun- teers, all of whom were habitual smokers, being considered to have an accelerated oxidative stress state. All of the participants had neither his- tory of atherosclerotic diseases such as coronary artery disease or cerebrovascular disease nor risk factors such as hypertension, diabetes, or hyperlipi- demia except smoking habit by the annual physical checkup. The participants were randomly assigned to take daily 1050 mg of vitamin C tablets (vitamin C group n = 10) or 70 ml of 100% camu-camu juice, containing 1050 mg of vitamin C (camu-camu group n = 10) as a dietary supplement for 7 days, and to continued smoking. The dose of 1050 mg/day of vitamin C in both groups was decided, based on pre- vious studies [11���16]. In all subjects, 10 ml of urine and 15 ml of peripheral blood were sampled at base- line before intake of these dietary supplements, 7 days after the intake, and 1 month after ceasing the intake as at a washout stage. The blood sam- ples were immediately centrifuged at 1500 �� g for 15 min at room temperature. The urine and serum were frozen and stored at ���80 ���C until analyzed. We measured urine 8-hydroxy-deoxyguanosine (8- OHdG) levels and serum total reactive oxygen species (ROS) levels as oxidative stress markers, and high sensitivity C-reactive protein (hsCRP) and multiple-cytokine levels as inflammatory markers. The local institutional review board approved the study protocol, and written informed consent was obtained from each participant. Baseline characteristics Prior to starting the intake of camu-camu or vitamin C, each participant was interviewed with regard to brand of the cigarette and daily number of cigarettes consumed and daily intake of tar and nicotine was calculated as the content of tar or nicotine for each cigarette brand �� daily number of cigarettes consumed. Blood pressure was measured to the nearest 2 mmHg in the same arm at base- line, using a mercury sphygmomanometer with an appropriately sized cuff. Measurements Urinary 8-OHdG levels were determined by compet- itive enzyme-linked immunosorbent assay (ELISA) using the commercially available kit (8-OHdG Check, Japan Institute for the Council of Aging, Fukuroi, Japan) [17]. Serum levels of total ROS were measured using a DEPPD reaction method by Hayashi et al. [18] applying the Fenton reaction on a multi-well plate. The hsCRP levels were measured by particle-enhanced technology on the Behring BN II nephrometer (Dade Behring Inc., Newark, DE, USA) [19]. This assay used monoclonal anti-CRP antibodies and a calibrator that was also traceable to World Health Organization reference materials. The Luminex micro-beads array system was used for a multiplex assay of simultaneous quantification of the following 10 cytokines: interleukin (IL)-1 , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, tumor necrosis factor- , granulocyte-macrophage colony stimulating factor, and -interferon. The assay was conducted as per the manufacturer���s instructions (Luminex Corp., Austin, TX, USA), using a commercially available kit (BioSource International, Inc., Camarillo, CA, USA) [20]. Statistical analysis Values are expressed as the mean �� S.D. for parametric data, and median values and interquar-