Greeting Welcome to 6th International Niigata Symposium on Diet and Health (INSDH). It is my great pleasure as the chairman of INSDH to invite you all either new or repeated participants to Niigata and to construct this 6th meeting together to be full of substance. This symposium addresses and mainly focuses the translational aspect of food functions to establish the scientific base for the application of food function in disease prevention, amelioration and anti-aging, and thus the border field of food science and pharmacology is specifically interested. This makes this symposium characterized in collaboration with two established conferences as co-sponsor, the Oxygen Club of California (OCC) and the LPI conference on Diet and Optimum Health, both of which are know to cover more basic and micronutorients related field of food science, respectively. The issue of diet and health has been a basic interest of human being both in oriental and western societies since ancient times and the idea of food is medicine is more attractively revived in the current society. Current development of scientific technology facilitates rapid understanding of food functions and its application for better life of human. However, still we have many questions to be resolved such as the mode of function of food is the same as drugs, and how to apply and manage the food functions practically in health preservation of human in normal or Mibyou conditons, or in clinics. In addition to developing more basic studies on food functions, we need to perspective discussion for the future on these issues mentioned above. This symposium could be one of the opportunity to share the latest knowledge of food functional researches and develops perspective discussion on their practice. We, organizers hope all the participants will enjoy full of scientific and cultural exchanges with colleagues and contribute to the development of this exciting research area in this beautiful and nourish city, Niigata.

With my best regards,

Tetsuya Konishi Chairperson of 6th International Niigata Symposium on Diet and Health Founding organizer of INSDH

－1－ International Advisory Board Members

⃝Fabio Virgili National Research Institute for Food and Nutrition (INRAN), Italy ⃝Lester Packer University of Southern California, USA ⃝Roderick H. Dashwood Linus Pauling Institute, Oregon State University, USA ⃝Robert K. M. Ko Hong Kong University of Science & Technology, China ⃝Young-Joon Surh Seoul National University, South Korea ⃝Dilip Ghosh Soho Flordis International, Australia ⃝Chandan K. Sen Medical Center, The Ohio State University, USA ⃝Debasis Bagchi University of Houston College of Pharmacy, USA ⃝Marc Diederich Laboratoire de Biologie Moleculaire et cellulaire du Cancer, Luxembourg ⃝Balz Frei Linus Pauling Institute, Oregon State University, USA ⃝Shantanu Das Massey University, New Zealand ⃝P. K. Goyal University of Rajasthan, India ⃝Cesar G. Fraga University of Buenos Aires, Argentina ⃝Wang Xuejiang Capital Medical University, China

Domestic Advisory Board Members

⃝Soichi Arai Tokyo University of Agriculture, Japan ⃝Shinya Toyokuni Nagoya University, Japan ⃝Hiroyoshi Moriyama The Japanese Institute for Health Food Standards(JIHFS), Japan ⃝Yuji Naito Kyoto Prefectural University of Medicine, Japan ⃝Junji Terao The University of Tokushima, Japan ⃝Makoto Shimizu The University of Tokyo, Japan ⃝Kunio Miyanishi University of Niigata Prefecture, Japan ⃝Kenichi Watanabe Niigata Sake Brewers Association, Japan ⃝Masamichi Takagi NUPALS, Japan ⃝Masaji Ishiguro NUPALS, Japan ⃝Takumichi Sugihara NUPALS, Japan

－2－ Organizing Committee

Chairperson : Tetsuya Konishi NUPALS

Secretary General : Hiroshi Nishida NUPALS

Vice- Secretary General : Shinichi Asada NUPALS

Committee Member : Shinji Sato NUPALS Toru Shigematsu NUPALS Kenichi Watanabe NUPALS Tomoko Yamaguchi Niigata University Chigusa Tateyama University of Niigata Prefecture Hideyuki Sone University of Niigata Prefecture Masao Hirayama Bourbon Corporation, Japan Atsushi Kobayashi Echigoseika Co., Ltd

Secretariat

Hirokuni Mogi NUPALS, Liaison Center for R&D Promotion (LCRDP) Hanae Moriyama NUPALS, LCRDP

265-1 Higashijima, Akiha-ku, Niigata, 956-8603, Japan E-mail:[email protected] URL: http://shinsen.biz/INS6TH/

－3－ General Information

◆ Period October 16 and 17, 2012

◆ Venue Toki Messe Niigata Convention Center 6-1 Bandaijima, Chuo-ku, Niigata 950-0078, Japan

◆ Access to Toki Messe Niigata Convention Center and Hotel Nikko Niigata from Niigata Station There is public transportation (bus) available to Toki Messe. The bus station is located at 'Bandai exit' (North exit). Take the line #17 (bound to Sado-Kisen, departing from the bus stop #5) and get off at Toki Messe stop.

◆ On-site Secretariat 3F Room 303, Toki Messe Niigata Convention Center

◆ Welcome Party Date:October 15 (Mon), 17:30-19:00 Site:3F Foyer, Toki Messe Niigata Convention Center

◆ Banquet Date:October 16 (Tue), 19:00-21:00 Site:4F Toki, Hotel Nikko Niigata (adjacent to the conference venue)

*Participants will be guided to the site of banquet by conference staffs.

－4－ ◆ Lunch Conference lunch will be prepared for the sponsored seminars listed below: October 16 (Tue):11:40-12:40 Site:3F Room 301, Toki Messe Niigata Convention Center Research & Development on “Slow Calorie Confectionaries” – Focusing on quality of calories– (Bourbon Corporation Japan)

October 17 (Wed):12:00-13:00 Site:3F Room 301, Toki Messe Niigata Convention Center Novel Technologies to Reduce Allergenicity and Enrich Functional Components (Echigoseika Co., Ltd.)

Lunch will be distributed once participants are seated in the seminar room (3F Room 301).

◆ Exhibition 3F Foyer (In front of Room 301)

⃝Bourbon Corporation Japan ⃝Niigata Bio Research Park ⃝Niigata University of Pharmacy and Applied Life Sciences (NUPALS) (As of September 10, 2012)

－5－ Program at a Glance

October 15, Monday

Registration Room301 Room302A Room302B Time Program Time Program Time Program

16:00-18:00 17:30-19:00 Welcome Party (at 3F Foyer) Registration

October 16, Tuesday

Registration Room301 Room302A Room302B Time Program Time Program Time Program 8:30 40 8:30 50 9:00 9:00-9:10 Opening Remarks and 6th INSDH Perspective 10 8:30-10:00 Posting 20 30 Session 2 "Food Research & Development News 40 9:10-10:10 Session1 from Food Industry in Japan" 50 9:10-10:40 "Inflammatory Signaling as a Target for (Language: Japanese) 10:00 Dietary Prevention of Cancer and 10 Metabolic Disorders" 10:10-10:15 Coffee Break 20 30 40 10:40-10:45 Coffee Break Session 2 50 10:15-11:30 "Food Research & Development News 11:00 Session1 from Food Industry in Japan" "Inflammatory Signaling as a Target for (Language: Japanese) 10 10:45-11:35 Dietary Prevention of Cancer and 20 Metabolic Disorders" Poster 30 10:00-13:00 11:35-11:40 Coffee Break Viewing 40 50 Sponsored Seminar 12:00 “Research & development on “Slow Calorie Confectionaries” 10 11:40-12:40 – focusing on quality of calories–” 20 30 (Bourbon Corporation) 40 50 12:40-13:00 Group photo (at 2F Atrium) 13:00 10 20 8:30-18:30 Poster 30 Registration 13:00-14:00 Session 40 50 14:00 INSDH2012 for the Citizens of Niigata at 10 Extension Lecture 20 13:30-15:10 (Language : Japanese) 30 "Health and QOL for yourself, family, 40 children and grandchildren" Session 2 50 14:15-15:30 "Food Research & Development News 15:00 from Food Industry in Japan" 10 (Language: Japanese) 15:10-15:20 Coffee Break 20 30 15:30-15:35 Coffee Break Poster 40 14:15-17:00 Viewing 50 INSDH2012 for the Citizens of Niigata at 16:00 Extension Lecture Session 3 10 15:20-17:00 (Language : Japanese) 15:35-16:50 "Physiological and Pharmacological Function 20 "Health and QOL for yourself, family, of Natural Product and Food Factor" 30 children and grandchildren" 40 50 16:50-16:55 Coffee Break 17:00 10 20 Session 3 30 16:55-18:00 "Physiological and Pharmacological Function 40 of Natural Product and Food Factor" 50 18:00 10 20 30 40 50 19:00 10 20 30 40 50 20:00 19:00-21:00 Banquet (at Hotel Nikko Niigata 4F “Toki”) 10 20 30 40 50 21:00

－6－ October 17, Wednesday

Registration Room301 Room302A Room302B Time Program Time Program Time Program 8:30 40 8:30 50 9:00 10 Session 7 20 9:00-9:50 "Regional Trend in Functional Food 30 Session 4 Development in the World 40 9:00-10:15 "Standardized natural product: (Niigata Bio Research Park Co., Ltd. presents)" “Seed to Patient” Journey" 50 9:50-9:55 Coffee Break 10:00 10 Session 7 "Regional Trend in Functional Food 20 10:15-10:25 9:55-10:45 Coffee Break Development in the World 30 (Niigata Bio Research Park Co., Ltd. presents)" 40 Session 4 10:45-10:50 Coffee Break 50 10:25-11:15 "Standardized natural product: 11:00 “Seed to Patient” Journey" 10 20 11:15-11:25 Coffee Break 10:50-11:55 Session 8 "Food factors in health management and clinic" 30 Session 5 40 8:30-15:00 11:25-11:50 "Omic approach for understanding multitarget function of food and food factor" 9:00-14:30 Poster 50 Registration Viewing 11:50-12:00 Coffee Break 11:55-12:00 Coffee Break 12:00 10 Sponsored Seminar 20 “Novel technologies to reduce 30 12:00-13:00 allergenicity and enrich functional 40 components” 50 (Echigoseika Co., Ltd.) 13:00 13:00-13:05 Coffee Break 13:00-13:05 Coffee Break 10 20 Session 5 "Omic approach for understanding Session 8 30 13:05-13:55 13:05-13:55 multitarget function of food and food factor" "Food factors in health management and clinic" 40 50 13:55-14:00 Coffee Break 14:00 13:55-14:05 Coffee Break 10 20 14:00-14:50 Session 9 30 "Oriental philosophy and food functions" 14:05-15:10 Session 6 40 "Nobel function of lipophilic factors" 50 14:50-15:00 Coffee Break 15:00 10 15:10-15:20 Coffee Break 20 15:00-15:50 Session 9 30 "Oriental philosophy and food functions" 40 15:20-16:10 Session 6 50 "Nobel function of lipophilic factors" 16:00 10 16:10-16:20 Closing Remarks 20 30 40 50 17:00

－7－ Symposium Site Layout

－8－ Poster Sessions

Each poster is numbered (see the list of poster sessions). The presentation number (P-XX) will be found on the top corner of the display board. Schedule for the poster session is as follows:

Oct. 16 8:30-10:00 Posting 13:00-14:00 Poster Presentation (13:00-13:30 odd number) (13:30-14:00 even number) Oct. 17 14:30-17:30 Removal

*Poster presenters are requested to stand nearby their posters during above time period.

Posters that are not removed by 17:30 of Oct. 17 will be discarded by symposium committee personnel.

Room Layout (Room 302B)

Poster Size 20cm 90cm

No. 20cm

20cm

Poster Size 110㎝(width)×160㎝(height) 210cm

110cm

－9－ Program

October th ＿＿＿＿＿ ＿1＿6＿＿ Room 301 Opening Remark and 6th INSDH Perspective : 9:00-9:10 Tetsuya Konishi (NUPALS, Japan)

Session 1 : 9:00-11:35 “Inflammatory Signaling as a Target for Dietary Prevention of Cancer and Metabolic Disorders” Chairpersons: Young-Joon Surh (Seoul National University, South Korea) Tetsuya Konishi (NUPALS, Japan)

PL-1 9:10-9:50 “Redox modulation of pro-inflammatory and anti-inflammatory signaling by chemopreventive phytochemicals” Young-Joon Surh (Seoul National University, South Korea)

L-2 9:50-10:15 “Mechanisms of dietary energy balance effects on epithelial cancer development and progression” John DiGiovanni (University of Texas at Austin, Dell Pediatric Research Institute, USA)

L-3 10:15-10:40 “Using genomic technologies to help optimise an anti-inflammatory diet” Lynnette R. Ferguson (The University of Auckland, New Zealand)

Coffee Break : 10:40-10:45

L-4 10:45-11:10 “Omega-3 fatty acids and chronic disease prevention: learning from transgenic fat-1 mice” Jing X. Kang (Harvard Medical School, USA)

L-5 11:10-11:35 “Heme Oxygenase-1 induction inhibits intestinal inflammation; role of food factors” Yuji Naito (Kyoto Prefectural University of Medicine, Japan)

Coffee Break : 11:35-11:40

－10－ Sponsored Seminar : 11:40-12:40 Chairperson: Hiroshi Nishida (NUPALS, Japan) “Research & development on “Slow Calorie Confectionaries” -focusing on quality of calories-” Shigeru Mineo (Bourbon Institutes of Health, Bourbon Corporation, Japan) This seminar is sponsored by Bourbon Corporation, Japan

Group photo: 12:40-13:00

INSDH2012 for the Citizens of Niigata at Extension Lecture : 13:30-17:00 (Language : Japanese) “Health and QOL for yourself, family, children and grandchildren” Organizer : Kenichi Watanabe (NUPALS, Japan)

Room 302A

Session 2 : 9:10-15:30 (Language: Japanese) “Food Research & Development News from Food Industry in Japan”

Opening Remark of Session 2: 9:10-9:20 Masaji Ishiguro (NUPALS, Japan)

Chairpersons: Masaji Ishiguro (NUPALS, Japan) Yuji Nakai (University of Tokyo, Japan)

L-6 9:20-9:45 “High-pressure treatment for improvement of food functions” Toru Shigematsu (NUPALS, Japan)

L-7 9:45-10:10 “Continuities and changes of the biotechnology - create new ideas from old biotechnology !-” Makoto Kanauchi (Miyagi University, Japan)

Coffee Break : 10:10-10:15

－11－ Chairpersons: Makoto Kanauchi (Miyagi University, Japan) Toru Shigematsu (NUPALS, Japan)

L-8 10:15-10:40 “Clinical and basic studies from Niigata” Kenichi Watanabe (NUPALS, Japan)

L-9 10:40-11:05 “Dietary proanthocyanidins – occurrence, chemical structure, & health benefits –” Masao Hirayama (Bourbon Institutes of Health, Bourbon Corporation, Japan)

L-10 11:05-11:30 “Anti-obese activity of quercetin glycoside mediated by activation of Hormone-Sensitive Lipase (HSL)” Yoshinori Kitagawa (Institute for Health Care Science, SUNTORY WELLNESS Ltd., Japan)

Chairpersons: Yoshinori Kitagawa (Institute for Health Care Science, SUNTORY WELLNESS Ltd., Japan) Masao Hirayama (Bourbon Institutes of Health, Bourbon Corporation, Japan)

L-11 14:15-14:40 “Establishment of the new treatment and preventive method for lifestyle-related disease using Bio-Activating Advanced Nutrients (BAANs)” Susumu Satoh (Japan Advanced Institute of Science and Technology, Y’s Corporation, Japan)

L-12 14:40-15:05 “Immunomodulatory effects of mekabu fucoidan from brown alga Undaria pinnatifida” Keiko Yoshinaga (RIKEN VITAMIN CO., LTD., Japan)

L-13 15:05-15:30 “Current status and case studies in nutrigenomics” Yuji Nakai (The University of Tokyo, Japan)

Coffee Break : 15:30-15:35

－12－ Session 3 : 15:35-18:00 “Physiological and Pharmacological Function of Natural Product and Food Factor” Chairpersons: Marc Diedrich (Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Luxembourg) Tetsuya Konishi (NUPALS, Japan)

L-14 15:35-16:00 “Stress regulation by inhalation of linalool as seen from gene expression analysis” Akio Nakamura (R&D Center, T. Hasegawa Co., Ltd., Japan)

L-15 16:00-16:25 “Natural compounds as inhibitors of inflammation and activators of cell death mechanisms” Marc Diederich (Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Luxembourg)

L-16 16:25-16:50 “15-Hydroxyprostaglandin dehydrogenase as a potential molecular target for chemoprevention of inflammation-associated colon carcinogenesis by curcumin” Hye-Kyung Na (Sungshin Women's University, South Korea)

Coffee Break : 16:50-16:55

L-17 16:55-17:20 “Endophytic fungi as sources of new anti-cancer and antibiotic metabolites” Peter Proksch (Heinrich-Heine University, Germany)

PL-18 17:20-18:00 “Preventive and therapeutic management of cancer by natural products” Pradeep Kumar Goyal (University of Rajasthan, India)

Room 302B

Poster Session : 13:00-14:00 Chairperson: Toshiyuki Sakamaki (NUPALS, Japan)

Banquet (at Nikko Niigata 4F “Toki”) : 19:00-21:00

－13－ October th ＿＿＿＿＿ ＿1＿7＿＿ Room 301

Session 4 : 9:00-11:15 “Standardized Natural Product: “Seed to Patient” Journey” Chairpersons: Dilip Ghosh (Soho Flordis International, Australia) Hiroyoshi Moriyama (The Japanese Institute for Health Food Standards (JIHFS), Japan)

L-19 9:00-9:25 “Seed to patient: role of specifically clinically proven natural products” Dilip Ghosh (Soho Flordis International, Australia)

L-20 9:25-9:50 “The cellular and molecular basis of health benefits of grape seed proanthocyanidin extract” Debasis Bagchi (University of Houston College of Pharmacy, USA)

L-21 9:50-10:15 “Antioxidant beyond its natural definition” Pingfan Rao (Institute of Biotechnology, Fuzhou University, China)

Coffee Break : 10:15-10:25

L-22 10:25-10:50 “Role of inflammation in high-fat diet-induced tumor promotion” Jung Han Yoon Park (Hallym University, South Korea)

L-23 10:50-11:15 “The use of herbal medicine as therapeutics in dementia” Dennis Chang (University of Western Sydney, Australia)

Coffee Break : 11:15-11:25

－14－ Session 5 : 11:25-13:55 “Omic Approach for Understanding Multitarget Function of Food and Food Factor” Chairpersons: Fabio Virgili (National Research Institute for Food and Nutrition (INRAN), Italy) Toru Shigematsu (NUPALS, Japan)

L-24 11:25-11:50 “Tocotrienols: from antioxidants to receptor ligands” Fabio Virgili (National Research Institute for Food and Nutrition (INRAN), Italy)

Coffee Break : 11:50-12:00

Sponsored Seminar : 12:00-13:00 Chairperson: Toru Shigematsu (NUPALS, Japan) “Novel technologies to reduce allergenicity and enrich functional components” Kazutaka Yamamoto (National Food Research Institute National Agriculture and Food Research Organization Tsukuba, Japan) This seminar is sponsored by Echigoseika Co., Ltd., Japan

Coffee Break : 13:00-13:05

L-25 13:05-13:30 “Transcriptional biomarkers of aging and calorie restriction and modulation by dietary interventions” Jamie L. Barger (LifeGen Technologies, USA)

L-26 13:30-13:55 (Video Session) “Nutrient-genome interactions and prevention of cognitive impairment” Michael Fenech (CSIRO Food and Nutritional Sciences, Australia)

Coffee Break : 13:55-14:05

－15－ Session 6 : 14:05-16:10 “Nobel Function of Lipophilic Factors” Chairpersons: Teruo Miyazawa (Tohoku University, Japan) Takahiro Eitsuka (NUPALS, Japan)

PL-27 14:05-14:45 “The role of vitamin E in the brain, from adults to zebrafish” Maret G. Traber (Oregon State University, USA)

L-28 14:45-15:10 “Synergistic effect between GLA and gemcitabin on pancreatic carcinomas: mechanism of action"” Ruth Lupu (Breast Cancer Translational Research Laboratory, USA)

Coffee Break : 15:10-15:20

L-29 15:20-15:45 “Antioxidant action and multifunctionality of dietary carotenoids in humans” Teruo Miyazawa (Tohoku University, Japan)

L-30 15:45-16:10 “Early ADME properties for discovery of human in vivo probe” Yang Ling (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China)

Closing Remark : 16:10-16:20 Masaji Ishiguro (NUPALS, Japan)

Room 302A

Session 7 : 9:00-10:45 “Regional Trend in Functional Food Development in the World (Niigata Bio Research Park Co., Ltd., presents)” Chairpersons: Masaji Ishiguro (NUPALS, Japan) Hiroshi Nishida (NUPALS, Japan)

L-31 9:00-9:25 “Inflammation regulatory activities of fruit polyphenolics - opportunities for functional foods” Roger Hurst (The New Zealand Institute for Plant & Food Research Ltd., New Zealand) －16－ L-32 9:25-9:50 “The impact of food processing on the nutritional value of fruits: polyphenols and their antioxidant activity” Emira Mehinagic (Groupe Ecole Supérieure d'Agriculture d'Angers, France)

Coffee Break : 9:50-9:55

L-33 9:55-10:20 “Foodvalley and Wageningen UR: healthy and sustainable food innovation in the Netherlands” Christiaan Kalk (Food and Biobased Research Wageningen University and Research Centre, The Netherlands)

L-34 10:20-10:45 “Concentrations of radionuclides in agricultural crops in Japan: before and after the Fukushima daiichi nuclear power plant accident” Keiko Tagami (National Institute of Radiological Sciences, Japan)

Coffee Break : 10:45-10:50

Session 8 : 10:50-13:55 “Food Factors in Health Management and Clinic” Chairpersons: Debasis Bagchi (University of Houston College of Pharmacy, USA) Shinji Sato (NUPALS, Japan)

PL-35 10:50-11:30 “Flavonoids and the risk factors of metabolic syndrome” Cesar G. Fraga (University of Buenos Aires, Argentina)

L-36 11:30-11:55 “Green tea polyphenol EGCG sensing for cancer prevention” Hirofumi Tachibana (Kyushu University, Japan)

Coffee Break : 11:55-12:00

Coffee Break : 13:00-13:05

L-37 13:05-13:30 “Vitamin C and aging” Akihito Ishigami (Tokyo Metropolitan Institute of Gerontology (TMIG), Japan)

－17－ L-38 13:30-13:55 “Therapeutic efficacy and safety evalutation of water soluble undenatured type II collagen in moderately arthritic dogs” Manashi Bagchi (NutriToday LLC, USA)

Coffee Break : 13:55-14:00

Session 9 : 14:00-15:45 “Oriental Philosophy and Food Functions” Chairpersons: Robert K. M. Ko (Hong Kong University of Science & Technology, China) Kenichi Watanabe (NUPALS, Japan)

L-39 14:00-14:25 “Yin and Yang: a pharmacological perspective” Robert K. M. Ko (Hong Kong University of Science & Technology, China)

L-40 14:25-14:50 “Effect of mixture of Schisandra and sesame on antioxidation and liver function” Chin-Kun Wang (Chung Shan Medical University, Taiwan)

Coffee Break : 14:50-15:00

L-41 15:00-15:25 “Hyperglycemic effects of mangiferin hydrastis granuleinin GK rats with diabetes mellitus” Xiaobo Qu (Changchun University of Chinese Medicine, China)

L-42 15:25-15:50 “Protective potential of fruit extract of Emblica officinalis Linn. against radiation and lead induced hepatic lesions in Swiss albino mice” R. K. Purohit (Lecturer in Zoology Treasurer-ISRB, India)

－18－ Poster Presentations

Room 302B

Section 1 : Molecular bases of "Food as medicine"

P-1 Emerging trends in foods microbiome research : novel and future analytical approaches to investigate and control of various diseases Hemant K.Gautam (Institute of Genomics and Integrative Biology, India)

P-2 The antidiabetes constituents of yacon leaves Dou De-Qiang1, Xiang Zheng1 and Dong Feng2 (1Liaoning University of Traditional Chinese Medicine, 2Zhen-Ao Group Co., LTD., China)

P-3 Biochemical and chemical characteristics of “Yang-invigorating” action of Herba Cynomorii Jihang Chen and Kam Ming Ko (Hong Kong University of Science and Technology, Hong Kong S.A.R)

P-4 High-pressure induced generation of functional compounds in brown rice Shigeaki Ueno1, Toru Shigematsu2, Mayumi Hayashi2 and Tomoyuki Fujii1 (1Tohoku University, 2Niigata University of Pharmacy and Applied Life Sciences, NUPALS, Japan)

P-5 The effects of high-fat diet intake and aging on lipid metabolic system in mice Taro Honma and Tsuyoshi Tsuduki (Tohoku University, Japan)

P-6 Anti-fatty liver effect of 1-deoxynojirimycin extracted from mulberry Tsuyoshi Tsuduki and Taro Honma (Tohoku University, Japan)

P-7 Molecular mechanism of the anti-allergic effect of the emergency food Clethra barbinervis Tatsuo Katagiri and Tadashi Aradate (University of TOYAMA, Graduate School of Medicine and Pharmaceutical Sciences, Japan)

－19－ P-8 Systematic purification and analytical method of mucopolysaccharides in un-fractionated heparin Hiroshi Murata1,2, Akio Matsuhisa1, Keiichi Yamamoto1, Takao Toda1, Michio Muguruma2 and Satoshi Kawahara2 (1Fuso Pharmaceutical Industries, LTD., 2Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Japan)

P-9 The essential fatty acid ratio of the diet affect for the pathogenic progress in the NOD mice Yukiko Kagohashi1 and Hiroki Otani2 (1The University of Shimane, 2Shimane University Faculty of Medicine, Japan)

P-10 Suppression of postprandial hyperglycemia by odor precursor of garlic Tomomi Kobayashi1, Yuya Shirai1, Shoko Miyata1, Makoto Akao1, Hitoshi Kumagai2 and Hitomi Kumagai1 (1Nihon University, 2Kyoritsu Women’s University, Japan)

P-11 Evaluated the change in allergenicity of deamidated gliadin in a mouse model of wheat gliadin allergy Ryosuke Abe1, Misaki Ito1, Yuki Uchida1, Makoto Akao1, Hitoshi Kumagai2 and Hitomi Kumagai1 (1Nihon University, 2Kyoritsu Women’s University, Japan)

P-12 Suppressive effect of extracts from Lamiaceae plants on hepatic injury induced by carbon tetrachloride Sou Hironaka1, Makoto Akao1, Yoko Matsui2, Hideki Masuda2, Osamu Nishimura2 and Hitomi Kumagai1 (1Nihon University, 2Ogawa & Co., Ltd., Japan)

Section 2: Antioxidant and antiaging are the basic strategy for disease prevention

P-13 Dietary (-)-epicatechin ameliorated cardiac and renal oxidative modifications in an experimental model of metabolic syndrome Cesar G. Fraga1, Valeria Calabro1, Paula D. Prince1, Giovanna Aschettino1, Laura Fischerman1, Marcela A. Vazquez-Prieto2, Monica Galleano1 and Barbara Piotrkowski1 (1Physical Chemistry-Institute of Molecular Biochemistry and Medicine (IBIMOL), UBA- CONICET, 2Department of Pathology, School of Medicine, National University of Cuyo; and Laboratory of Cardiovascular Pathophysiology, Institute for Experimental Medical and Biological Research (IMBECU)-CONICET, Mendoza, Argentina, Argentina)

－20－ P-14 In vitro anti-diabetic and anti-diabetic complications activities of fucoxanthin with antioxidant activity Jae Sue Choi1, Nurul Islam1, Hyun Ah Jung2, Chan Mi Lee1 and Eon Ji Kim1 (1Pukyong National University, Busan 608-737, Republic of Korea, 2Chonbuk National University, Jeonju 561-756, Republic of Korea, Korea)

P-15 The decline of tyrosine hydroxylase phosphorylation and iron concentration cause the reduction of catecholamines in the brain of the SAMP10 Miki Miyajima, Takuya Numata, Moemi Minoshima, Masato Tanaka, Ryo Nishimura, Naoko Hishioka, Yukako Ueno, Tae Kawahara, Toshiyuki Hosokawa, Masaaki Kurasaki and Takeshi Saito (Hokkaido University, Japan)

P-16 Electrolyzed-reduced water alleviates oxidative stress induced by chronic heat exposure Tomoko Matsueda1, Motoi Kikusato1, Md. Abul Kalam Azad2 and Masaaki Toyomizu1 (1Graduate School of Agricultural Science, Tohoku University, 2Bangladesh Agricultural University, Japan)

P-17 Effect of UV irradiation on human hair treated with shampoo and treatment mixed catechin Hisakazu Okamura1, Yasuyuki Sugiyama1,2 and Masatoshi Ohta2 (1Hazel Tompson Inc., 2Niigata Univ., Japan)

P-18 Effects of consumption of green tea during lactation on epigenetic factors in female infants of pregnancy rats with low-protein diet Yongkun Sun1, Yuuka Mukai2, Shin Sato2, Takeshi Saito3 and Masaaki Kurasaki4 (1Graduate School of Environmental Science, Hokkaido University, 060-0810 Sapporo JAPAN, 2Graduate School of Health Sciences, Aomori University of Health and Welfare, 030-0841, Aomori, JAPAN, 3Faculty of Health Sciences, Hokkaido University, 060-0812 Sapporo JAPAN, 4Faculty of Environmental Earth Science, Hokkaido University, 060-0810 Sapporo JAPAN, China)

P-19 Comparison of the radical scavenging activity of the polyphenol fractions of Japanese and imported black teas Chigusa Tateyama1 and Kiharu Igarashi2 (1University of Niigata Prefecture, 2Faculty of Agriculture, Yamagata University, Japan)

P-20 Trans-fatty acids in blood and eating habits of teenagers Eri Oomi, Nanako Doro, Takako Baba and Mari Mori (Mukogawa Women’s University Institute for World Health Development, Nishinomiya, Japan)

－21－ P-21 Oxidative stability of rice-based diets may affect growth performance of broiler chickens Chiaki Ito, Fumika Nanto, Tomomi Kamizono, Tomoko Matsueda, Motoi Kikusato and Masaaki Toyomizu (Graduate School of Agricultural Science, Tohoku University, Japan)

Section 3: New role of food factors in complementary medicine

P-22 Effects of water-soluble undenatured type II collagen in collagen- induced arthritis mice Orie Yoshinari1, Yoshiaki Shiojima1, Hiroyoshi Moriyama1 and Manashi Bagchi2 (1Ryusendo Co., Ltd., 2NutriToday LLC., Japan)

P-23 Effects of liver hydrolysate on the blood glucose in metabolic syndrome model rats (SHR/NDmcr-cp) Naonori Inoue1, Shuji Hidaka2, Naoyoshi Miura1, Masahiro Fukahori1, Masugi Maruyama2, Satoshi Kawahara2, Kazuyoshi Ohta2 and Michio Muguruma2 (1Zeria Pharmaceutical Co., Ltd., 2University of Miyazaki, Japan)

P-24 The enzyme-treated asparagus offcinalis extract shows anti-stress effects in neural cells and prevents cognitive impairment in senescence- accelerated mice Takuya Sakurai1, Kentaro Kitadate2, Hiroshi Nishioka2, Koji Wakame2, Hajime Fujii2, Junetsu Ogasawara1, Takako Kizaki1, Shogo Sato1, Yoshinaga Ishibashi1, Tomonori Fujiwara1, Kimio Akagawa1, Kazuhiko Imaizumi3, Daizoh Saitoh4, Tetsuya Izawa5 and Hideki Ohno1 (1Kyorin University, School of Medicine, 2Amino Up Chemical Co., Ltd., 3Waseda University, 4National Defense Medical College Research Institute, 5Doshisha University, Japan)

P-25 Effect of brown rice extracts on immune response in mice Kayoko Kawakami1, Hitomi Mori2, Misugi Uraji1, Masayo Kimura1, Tadashi Hatanaka1 and Hideyuki Ito2 (1Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, 2Okayama University, JAPAN)

P-26 Therapeutic effect of petit vert on mice with acute DSS colitis Kohsuke Matsumoto1, Kenji Suzuki1, Yuki Nishizawa1, Masaki Yonezawa1, Yutaka Honda1, Junji Yokoyama1, Masaki Nagata2, Somasundaram Arumugam3, Rajarajan A. Thandavarayan3, Kenichi Watanabe3, Tetsuya Konishi3 and Yutaka Aoyagi1 (1Niigata University School of Medicine, 2Niigata University School of Dentistry, 3Niigata University of Pharmacy and Applied Life Sciences, Japan)

－22－ P-27 The protective effect of squalene on inflammatory responses in BALB/c mice Naoto Tatewaki, Hiroshi Nishida, Yurie Kuwabara, Yuuki Fuse, Takahiro Eitsuka and Tetsuya Konishi (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-28 Anti-allergic immune effects of Aspergillus oryzae and Lactobacillus sakei on an ovalbumin induced allergy mice Hideaki Suzuki1, Koji Kobayashi1, Koji Kobayashi1, Yuuko Kosuge1, Masami Igarashi1 and Tsuyoshi Sugiyama2 (1Kitasato Junior College of Health and Hygienic Sciences, 2OTO corporation, Japan)

Section 4: Chemoprevention by edible plants and the component

P-29 Antioxidant activity of ramie and perilla leaves grown in Niigata Tomoko Yamaguchi, Azusa Kawasaki and Jun-ichi Sakai (Niigata University, Japan)

Section 5: Calorie mimetic food factors in disease prevention and antiaging

P-30 Effect of quercetin intake during lactation on the AMPK activation in the hypothalamus of adult male rat offspring programmed by maternal protein restriction Masato Tanaka1, Mai Turuga1, Norihito Takahashi1, Aya Sasaki1, Miki Miyajima1, Ryo Nishimura1, Naoko Hishioka1, Yuuka Mukai2, Shin Sato2, Masaaki Kurasaki1, and Takeshi Saito1 (1Hokkaido University, 2Aomori University of Health and Welfare, Japan)

P-31 Isomaltulose used as a sugar substitute for rice cooking controls changes in blood glucose and insulin response Kiyoaki Miyasaka1, Yumiko Tezuka1, Kaori Araki2, Kazuki Mochizuki3, Masae Sakuma2, Hidekazu Arai2 and Yoko Ichikawa2 (1Mitsui sugar Co., Ltd., 2University of Shizuoka, 3University of Yamanashi, Japan)

P-32 Effect of quercetin intake during lactation on the AMP-activated protein kinase activity in the livers of adult male rat offspring programmed by maternal protein restriction Shin Sato and Yuuka Mukai (Aomori University of Health and Welfare, Japan)

－23－ Section 6: Others

P-33 Antihypertensive effect of boysenberry seed polyphenols on spontaneously hypertensive rats and identification of orally absorbable proanthocyanidins with vasorelaxant activity Ryo Furuuchi, Akito Matsushima, Yuta Nagakura, Tadayuki Yokoyama and Masao Hirayama (Bourbon Corporation, Japan)

P-34 Novel functions of Xylose to maintain slow proliferation and undifferentiated state of ES cells Sakiko Takizawa-Shirasawa1, Tadayuki Yokoyama1, Susumu Yoshie2, Ken Matsumoto3, Mika Nagai1 and Katsunori Sasaki2 (1Bourbon Corporation, 2Shinshu University of Medicine, 3Nissui Pharmaceutical Co. LTD., Japan)

P-35 Effect of Isomaltulose on liver and skeletal muscle glycogen accumulation in rats Katsumi Sasagawa1, Shigeru Mineo1, Hiroshi Nishida2, Shinji Sato2 and Masao Hirayama1 (1Bourbon Corporation, 2Niigata Univ. of Pharm. and Appl. Life Sci., Japan)

Section 7: MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2010-2012

P-36 Amadori-glycated phosphatidylethanolamine up-regulates telomerase activity -A link between diabetes and cancer- Takahiro Eitsuka1, Kiyotaka Nakagawa2, Hiroshi Nishida1, Tadao Kurata1 and Teruo Miyazawa2 (1Niigata University of Pharmacy and Applied Life Sciences, 2Tohoku University, Japan)

P-37 Involvement of AMPK-PKC-ζ-NF-κB signaling pathway and nitrosative stress on development of diabetic cardiomyopathy in streptozotocin- induced diabetic rats and its prevention by curcumin Vivian Soetikno, Arun Prasath Lakshmanan, Somasundaram Arumugam, Vigneshwaran Pitchaimani, Meilei Harima, Kenji Suzuki and Kenichi Watanabe (Niigata University of Pharmacy and Applied Life Sciences, Indonesia)

P-38 Mulberry leaf diet protects against progression of experimental autoimmune myocarditis to dilated cardiomyopathy via modulation of oxidative stress and adverse cardiac remodeling Somasundaram Arumugam, Rajarajan Amirthalingam Thandavarayan, Vivian Soetikno, Vigneshwaran Pitcahimani, Meilei Harima, Kenji Suzuki, Makoto Kodama and Kenichi Watanabe (Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan)

－24－ P-39 Dominant negative 14-3-3η promotes the myocardial apoptosis through the angiotensin-II type 1 receptor dependent-down-regulation of AMPK in the early stage of type 1 diabetes mellitus in mice Arun Prasath Lakshmanan, Flori Ratna Sari, Vivian Soetikno, Harima Meilei, Sayaka Mito, Somasundaram Arumugam, Vigneshwaran Pitchaimani and Kenichi Watanabe (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-40 The differential regulation of brain mitogen-activated protein kinase cascade in rats after the induction of diabetes mellitus and heart failure Vigneshwaran Pitchaimani, Arun Prasath Lakshmanan, Vivian Soetikno, Vijayakumar Sukumaran, Harima Meilei, Sayaka Mito, Somasundaram Arumugam and Kenichi Watanabe (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-41 New inhibitory effect of flavanones and isoflavones on cholesterol synthesis Saori Nakagawa, Mayuri Watanabe, Tomoko Tanaka and Susumu Yamato (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-42 Herbal extracts against fatty liver Shinichi Ichikawa, Tomohiro Takahashi, Yuya Takiguchi, Kento Takizawa, Wataru Sugawara and Michiyo Nagano-Ito (Niigata Univ. of Pharm. & Appl. Life Sci., Japan)

P-43 A vinegar production process using simultaneous fermentation by Saccharomyces cerevisiae and Acetobacter pasteurianus Toru Shigematsu1, Sakae Ikarashi1, Mayumi Hayashi1, Akinori Iguchi1 and Masao Hirayama2 (1Niigata Univ. Pharm. Appl. Life Sci. (NUPALS), 2Bourbon Corporation, Japan)

P-44 Enzymatic synthesis of a novel sialyl mimic molecules by use of marine bacterial sialyltransferase Tatsuo Miyazaki1, Toshiki Mine2, Takayuki Watanabe1, Hitomi Kajiwara2, Katsumi Ajisaka1 and Takeshi Yamamoto2 (1Niigata University of Pharmacy and Applied Life Sciences, 2Japan Tobacco Inc., Japan)

P-45 pH-sensing mechanism of sweet taste-modifying proteins Takayuki Okubo, Minoru Tamiya and Masaji Ishiguro (Niigata University of Pharmacy and Applied Life Sciences, Japan)

－25－ P-46 Biosynthesis of triterpene in higher plants Masaaki Shibuya (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-47 First total synthesis and revision of absolute configuration of (-)-1,3,4,5-tetragalloylapiitol Yutaka Nakamura, Masaru Kojima and Seiji Takeuchi (Niigata Univ. Pharmacy and Applied Life Sciences (NUPALS), Japan)

P-48 Future prospects of plant defensin Rs-AFP1 as a food preservative agent Hiroaki Takaku, Yoshifumi Oguro, Harutake Yamazaki and Masamichi Takagi (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-49 Prevention of metabolic syndrome and second meal effect Shinji Sato (Niigata Univ. of Pharm. & Appl. Life Sci., Japan)

P-50 Developing a self-pollinate buckwheat based on reverse genetics approach Jotaro Aii, Ayana Nakano, Shingo Sato, Taketo Funaki, Yuya Sato and Hiroshi Tanaka (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-51 Structural stability studies on various functional peptides Hiromu Yoshihara, Jun Saito, Hiroshi Hoshino, Yuuki Ishiguro, Kanako Iiyoshi and Toshinari Asakura (Niigata University of Pharmacy and Applied Life Sciences, Japan)

P-52 Survey on the use of supplements by questionnaire ～The difference of consciousness between some group of professional and citizen～ Noriaki Yohkoh, Manabu Abe and Mikio Saito (Niigata University of Pharmacy and Applied Life Sciences, Japan)

－26－ Symposium Session Abstract

Session1

PL1 Redox modulation of pro-inflammatory and anti-inflammatory signaling by chemopreventive phytochemicals

Young-Joon Surh

Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea

Oxidative stress has been implicated in various pathological conditions including cancer. However, the human body has an intrinsic ability to fight against oxidative stress. A wide array of antioxidant enzymes and cytoprotective proteins constitute a fundamental cellular defense system against oxidative and electrophilic insults. Transcriptional activation of genes encoding many antioxidant enzymes and cytoprotective proteins by NF-E2 related factor 2 (Nrf2), a member of the cap‘n’collar family of basic leucine zipper transcription factors, protect cells and tissues from oxidative/electrophilic insults. Numerous chemopreventive and chemoprotective phytochemicals have been found to activate this transcription factor, that functions as a master switch in turning on antioxidant/stress-response signaling. A new horizon in chemoprevention research is the recent discovery of molecular links between inflammation and cancer. Components of the cell signaling pathways, especially those that converge on redox-sensitive transcription factors, including nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) involved in mediating pro-inflammatory response, have been implicated in inflammation-associated carcinogenesis. A wide variety of chemopreventive and chemoprotective phytochemicals can also target NF-κB and STAT3. The modulation of cellular signaling by anti-inflammatory as well as antioxidant phytochemicals hence provides a rational and pragmatic strategy for molecular target–based chemoprevention.

－29－ Profile of Speaker

Prof. Young-Joon Surh currently serves as Director of Tumor Microenvironment Global Core Research Center of Seoul National University. He graduated from College of Pharmacy, Seoul National University with BS (Pharmacy) and MS (Biochemistry), and earned his PhD degree at the McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, USA. Professor Surh then had postdoctoral training at the Massachusetts Institute of Technology (MIT). In 1992, he was appointed as a tenure-track Assistant Professor at Yale University School of Medicine. Prof. Surh serves as an editorial board member for more than 25 international journals, including International Journal of Cancer, Molecular Carcinogenesis, Cancer Letters, Cancer Prevention Research, Mutation Research, Life Sciences, Molecular & Cellular Biochemistry, Free Radical Research, Food & Chemical Toxicology, Biofactors, Genes and Nutrition, Molecular Nutrition & Food Research, Journal of Clinical Biochemistry & Nutrition, etc. He is also editor of the following books: Oxidative Stress, Inflammation and Health (CRC Press), Molecular Targets and Therapeutic Use of Curcumin (Springer-Veralg), and Dietary Modulation of Cell Signaling Pathways (CRC Press). Prof. Surh has published more than 200 papers in international journals and more than 60 invited editorials, reviews and book chapters. The total number of citations of his publications is more than 8,900 (excluding self-citations) as of July, 2012. He received several awards including Elizabeth C. Miller and James A. Miller Distinguished Scholar Award from Rutgers University (2011), McCormic Science Institute Award from American Society for Nutrition (2009), the Merit Award from the International Society of Nutraceuticals and Functional Foods (2010).

－30－ L 2 Mechanisms of dietary energy balance effects on epithelial cancer development and progression

John DiGiovanni

Division of Pharmacology & Toxicology, College of Pharmacy and Department of Nutritional Sciences, College of Natural Sciences, The University of Texas at Austin, Austin, TX USA

Findings from both experimental and epidemiological studies suggest a chronic positive energy balance state, which leads to obesity, heightens the risk of developing multiple epithelial cancers, while a sustained negative energy balance state, as induced by calorie restriction (CR) decreases these risks. We have shown that dietary energy balance manipulation [across the spectrum of CR to diet-induced obesity (DIO)] modulates steady-state growth factor signaling pathways in multiple epithelial tissues of mice. We found that the DIO diet enhanced, while CR inhibited activation of Akt and mTOR, regardless of epithelial tissue examined or genetic background of the mice. Further analyses demonstrated that AMP-activated protein kinase (AMPK) activation was differentially modulated by dietary manipulation in liver but not in the epidermis or dorsolateral prostate, suggesting possible tissue differences in the regulation of AMPK. Western blot analyses also revealed reduced activation of both the epidermal growth factor receptor (EGFR) and IGF-1 receptors (IGF-1R) in several tissues of CR mice, compared to mice on the control or DIO diets. In two-stage skin carcinogenesis experiments, the effect of dietary energy balance manipulation on both the promotion and progression stages was evaluated. Tumors develop in this two-stage skin carcinogenesis model as a function of dietary energy balance status with the lowest tumor yields observed in the CR diet groups and the highest tumor yields in the overweight/obese diet group. Further mechanistic studies have demonstrated that CR reduced whereas DIO increased activation of both epidermal Akt and mTOR (assessed by Western blot analysis of phosphorylated proteins) both in the absence and presence of TPA treatment. Furthermore, activation of both the EGFr and IGF-1R was modulated in a similar manner by the different diets in epidermis of TPA treated mice. Finally, we observed significant changes in both positive (e.g., cyclin D1, c-myc) and negative cell cycle regulatory proteins (e.g., p21, p27) that correlated with diet effects on skin tumor promotion and epidermal proliferation. Thus, dietary energy balance modulated tumor promotion, in part, through modulation of critical signaling pathways downstream of the IGF-1R and EGFr leading to altered levels and/or activity of cell cycle regulatory proteins. Further mechanistic studies have also revealed that dietary energy balance modulates cross-talk between the IGF-1R and EGFR at least in part through reduction in circulating IGF-1. In additional studies, we have recently evaluated the ability of rapamycin (an mTORC1 inhibitor) and metformin (an AMPK

－31－ activator) to block skin tumor promotion by TPA in female FVB/N mice. Topical application of rapamycin (given 15 min before each TPA treatment) was extremely effective at blocking TPA promotion of skin tumors, even at doses as low as 5 nmol per mouse. At the doses used (5-200 nmol) rapamycin was capable of blocking mTORC1 downstream signaling and TPA-induced epidermal proliferation and skin inflammation. Studies with metformin in this model system will also be presented. Inhibition of mTORC1 may be an effective strategy for blocking tumor promotion during epithelial carcinogenesis and for offsetting the effects of obesity on tumor development.

Profile of Speaker

John DiGiovanni received his Ph.D in 1978 from the University of Washington in Seattle, WA under the mentoring of Drs. Mont Juchau and Thomas Slaga. He did his postdoctoral work from 1978-1980 with Dr. Roswell Boutwell at the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI. Dr. DiGiovanni is currently Professor and Coulter R. Sublett Endowed Chair in the Division of Pharmacology and Toxicology, College of Pharmacy with a joint appointment in the Department of Nutritional Sciences at the University of Texas at Austin. Prior to joining UT Austin, he was the Director of the MD Anderson Cancer Center, Science Park-Research Division from 1997-2009 and also Director of the NIEHS funded Center for Research on Environmental Disease for 15 years while at the MD Anderson Cancer Center. Dr DiGiovanni has a long-standing, internationally recognized research program in understanding mechanisms of multistage epithelial carcinogenesis and in identifying targets and mechanisms for cancer prevention. Dr. DiGiovanni’s CV lists over 250 primary research papers and he has had a continuous history of multiple NIH grants to support his research program. He is also Editor-in-Chief for the journal Molecular Carcinogenesis. He has served on numerous national scientific advisory boards and NIH grant review panels and has received a variety of awards. In 2006 he received the Faculty Achievement Award in Cancer Prevention from the MD Anderson Cancer Center. His talk today will be about his recent work on dietary energy balance and epithelial cancer.

－32－ L 3 Using genomic technologies to help optimise an anti-inflammatory diet

Lynnette R. Ferguson

Dept of Nutrition, Faculty of Medical & Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand

Long term adherence to a Mediterranean-style is known to have anti-inflammatory effects, and be beneficial in both cancer and cardiovascular disease, among others. However, previous studies in an Auckland population failed to show anti-inflammatory effects of this style of diet, using standard biomarkers and a six week dietary intervention. For subjects who are not highly committed to the necessary change in diet and lifestyle, being able to produce a demonstrable effect within 6 weeks is likely to encourage their maintenance to the regime. We modified a standard Mediterranean diet by adding several other dietary components, known from the literature to have anti-inflammatory effects, including ginger, curcumin and garlic. The study involved 30 people, in two arms. The low intervention group were provided with recipe books and daily meal suggestions, while the high intervention arm were also provided with most of the necessary food. Blood and urine samples were taken at the beginning and end of the study. There was good compliance, tested by using proteomic approaches. While a minimal effect of the intervention could be shown using standard biomarkers such as C-reactive protein, analysis of gene expression using Affymetrix primeview arrays showed strong modulation of a number of genes involved in inflammation, especially in the high intervention group.

－33－ Profile of Speaker MSc (hons), DPhil (Oxon.), DSc, QSO, FNZIFST Professor Lynnette Ferguson obtained her doctorate from Oxford University, working on the subject of DNA repair and mutagenesis. Her current research considers the interplay between genes and diet in the development of chronic disease, with particular focus on Inflammatory Bowel Disease, a cancer-prone condition, and also on prostate cancer. Presently, Professor Ferguson plays a dual role as a researcher with the Auckland Cancer Society Research Centre and as Head of the Discipline of Nutrition at the University of Auckland. As leader of the multidisciplinary, multi- organisation, Nutrigenomics New Zealand Programme, Professor Ferguson is also working with a range of other stakeholders to bring nutrigenomics tools and awareness to the New Zealand science scene.

－34－ L 4 Omega-3 fatty acids and chronic disease prevention: learning from transgenic fat-1 mice

Jing X. Kang, M.D., Ph.D.

Laboratory for Lipid Medicine and Technology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

The imbalance of omega-6 to omega-3 fatty acids in the human diet and body is now recognized as a modern nutritional problem important to human health. Whether balancing the omega-6/omega-3 fatty acid ratio by decreasing omega-6 and increasing omega-3 fatty acids can reduce risk of modern diseases needs to be addressed in well-qualified experimental models. The recent development of genetic approaches to balancing the omega-6/omega-3 fatty acid ratio by expressing the fat-1 gene in animals (encoding an omega-3 fatty acid desaturase that can convert omega-6 to omega-3 fatty acids) provides a new opportunity to address this issue. Using the transgenic fat-1 mouse model, we have performed a series of experiments in different disease conditions. Our data obtained so far support the notion that a reduced or balanced ratio of tissue omega-6 to omega-3 fatty acids can reduce risk of many chronic diseases, including inflammatory disorders, cancer, atherosclerosis, diabetes and neurodegenerative disease. This outcome is associated with alteration in lipid mediator formation and gene expression.

－35－ Profile of Speaker

Dr. Jing X. Kang is an Associate Professor of Medicine at Harvard Medical School and Director of the Laboratory for Lipid Medicine and Technology at Massachusetts General Hospital. He is one of the leading scientists in the field of omega-3 research and has been a pioneer in studying the role of omega-6/omega-3 fatty acid ratio in health and disease. His lab has made several pioneering discoveries, including the prevention and treatment of cardiac sudden death by omega-3 fatty acids and the creation of a biotechnology for producing the beneficial omega-3 fatty acids from omega-6 fatty acids in animals. His lab generated the world’s first transgenic mammal that is capable of converting omega-6 to omega-3 fatty acids. His studies were listed among “Top 100 Science Stories” by Discover magazine. He was named one of “The Best and Brightest 2007” by Esquire magazine. To date, he has published more than 120 scientific papers in high-impact scientific journals including Nature and Science, and has been invited to speak at more than 100 national and international meetings. He is currently the Secretary/Treasurer of the International Society of Nutrigenetics and Nutrigenomics (ISNN) and the Editor-in-Chief of the Journal of Nutrigenetics and Nutrigenomics (JNN).

－36－ L 5 Heme Oxygenase-1 induction inhibits intestinal inflammation; role of food factors

Yuji Naito, Tomohisa Takagi, Yasuki Higashimura, Hiromu Ohnogi, and Toshikazu Yoshikawa

Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme, followed by production of biliverdin, free iron and carbon monoxide (CO). HO-1 is a stress- responsive protein induced by various oxidative agents. Recent studies demonstrate that the expression of HO-1 in response to different inflammatory mediators may contribute to the resolution of inflammation and has protective effects in several organs against oxidative injury. Although the mechanism underlying the anti-inflammatory actions of HO-1 remains poorly defined, both CO and biliverdin/bilirubin have been implicated in this response. In the gastrointestinal tract, HO-1 is shown to be transcriptionally induced in response to oxidative stress, preconditioning and acute inflammation. Recent studies suggest that the induction of HO-1 expression plays a critical protective role in intestinal damage models induced by ischemia-reperfusion, indomethacin, lipopolysaccharide-associated sepsis, trinitrobenzene sulfonic acid (TNBS), and dextran sulfate sodium, indicating that activation of HO-1 may act as an endogenous defensive mechanism to reduce inflammation and tissue injury in the gastrointestinal tract. These in vitro and in vivo data suggest that HO-1 may be a novel therapeutic target in patients with gastrointestinal diseases. Agar is easily extracted from red algae and widely used as food and gelling agent in Asian countries. Agarose, which is the main component of polysaccharides in agar, is easily hydrolyzed to yield oligosaccharides, and the resultant oligosaccharides termed agaro- oligosaccharides (AGOs) have been widely investigated in structure and bioactivity. AGOs enhanced HO-1 expression in time- and concentration-dependent manners in RAW264 cells. AGOs administration inhibits TNBS-induced colitis in mice through the HO-1 induction in macrophages via phosphorylation of JNK. AGOs may be an additional therapeutic strategy for intestinal inflammation.

－37－ Profile of Speaker Research Associate, First Department of Medicine, Kyoto Prefectural University of Medicine, 1987-1998. Assistant Professor of Medicine, First Department of Medicine, Kyoto Prefectural University of Medicine, 1998-2000. Associate Professor of Medicine, First Department of Medicine, Kyoto Prefectural University of Medicine, 2000-2003 Visiting Professor, Department of Molecular and Cellular Physiology, LSU Health Sciences Center, 2001- Associate Professor, Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 2003-2005 Associate Professor, Medical Proteomics, Kyoto Prefectural University of Medicine, 2005, June 1st – Associate Professor, Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, 2008, Feb 1st –

Awards and Honors Young Investigator Award of the 6th Biennial Meeting of the Society for Free Radical Research International, Torino, 1992. Gastric Mucosal Bioregulation Research Award, Osaka, 2003 Lipid Peroxides and Free Radical Research Award, Tokyo, 2003 Astaxanthin Award, Sapporo, 2006 Scientific Societies Society for Free Radical Research International Japanese Society of Gastroenterology Japanese Society of Inflammation Japanese Society of Microcirculation Japanese Society of Pathophysiology Japanese Cancer Association Japanese Society of Ulcer Research Professional Activities Treasurer, Society for Free Radical Research (SFRR) Asia, 1992-1996. Vice Presidents and Secretary general, Society for Free Radical Research Japan, 2011- Secretary General, Society for Free Radical Research Asia, 2000- Member, Japanese Society of Gastroenterology, Council Members Member, Japanese Society of Ulcer Research, Council Members Chairman, Society for Free Radical Research International Meeting 2014 in Kyoto Editors Executive Editor of J Clinical Biochemistry and Nutrition (2006- ) Associate Editor of J Gastroenterology (2007- ) Editor of World J Gastroenterology (2004- ) Editorial Board Member of Annals of Nutrition and Metabolism (2009- ) Guest Editor of J Gastroenterology and Hepatology (2004- ) Reviewer of J Cardiovascular Research (2008- ) Editorial Board Member of International Journal of Inflammation (2009- )

－38－ Session2

L 6

高圧処理を利用した食品の機能性富化

重松 亨

新潟薬科大学 応用生命科学部

地球で最も深いとされる約10,000mの海底の水圧はおよそ100MPa（1,000気圧）である。この圧 力を超える数百MPaの高静水圧を食品あるいは食品素材に施すことで、風味や栄養成分を損なう ことなく非加熱で殺菌することが可能となる。最近では、高圧処理を単なる殺菌技術だけでなく、 食品素材に有用な質的変化を付与する加工技術として位置づける考え方が広まりつつある。 われわれは、高圧処理が引き起こす食品素材の構造変化や酵素反応を利用した新しい食品加工技 術、ならびに高圧処理を軸とした微生物制御技術の研究を進めている。概ね100〜400MPaの圧力 を細胞性の食品素材に施すと、脂質からなる細胞膜や細胞小器官の膜は損傷するが、タンパク質か らなる酵素の多くが活性を維持することが示されてきた。このことは、高圧処理を食品・食品素材 に施すことで有用な酵素反応を促進できる可能性を示すものと考えられる。 これまでの研究の結果、玄米における遊離アミノ酸やγ-アミノ酪酸(GABA)の生成反応、タマネ ギやパセリなどの野菜の抗酸化活性の増加といった有用な変化が高圧処理により引き起こされるこ とを明らにした。これらの成分変化の仕組みをさらに究明することで、高圧処理により食品ある いは食品素材中の代謝反応を制御し、機能性成分を富化させる、高圧処理の新しい利用法の確立に つながると考えている。また、ペクチナーゼ酵素処理と高圧処理を組み合わせることで、平均粒径 が非常に微細（20μm未満）でありながら、澱粉損傷度を5%未満に抑えた超微細米粉「スーパー米 粉」の開発に成功した。 発酵食品の製造プロセスにおいて、過発酵の防止や貯蔵期間の延長を目的として加熱や塩の添加 などの方法が用いられている。高圧処理を応用して発酵中の微生物制御ができれば、高品質の発酵 食品を作製することが可能となる。そのために、発酵微生物を圧力感受性にする微生物育種技術の 研究を進めている。発酵性能は変わらず圧力感受性を示す酵母変異株の取得に成功した。今後、圧 力耐性と遺伝形質との相関関係を究明することで、圧力感受性酵母を育種する技術の確立につなが る。高圧処理による発酵制御に圧力感受性微生物を応用することで発酵食品のさらなる高品質化に 結びつくことが期待される。 本研究は、新潟県地域結集型研究開発プログラム「食の高付加価値化に資する基盤技術の開発」に より実施した。

－39－ Profile of Speaker

Dr. Toru Shigematsu is a professor at Niigata University of Pharmacy and Applied Life Sciences (NUPALS). In 1995, he earned his PhD degree from the University of Tokyo under the guidance of Dr. Takeshi Uozumi. After the finishing his PhD course, he worked as a postdoctoral fellow at New Energy and Industrial Technology Development Organization (NEDO), subsequently at Marine Biotechnology Institute. Then he moved to Kumamoto University as an assistant professor, where he was engaged in studies on microbial communities related to methane fermentation and other environments. In 2006, he moved to NUPALS as an associate professor and got promoted to a professor in 2012. His recent main research themes are as follows: 1) Studies on food processing and fermentation technologies by using high hydrostatic pressure processes 2) Studies on uncultivated microorganisms.

－40－ L 7

不易流行のバイオテクノロジー ～オールドバイテクノロジーを新しい切り口で～

金内 誠

宮城大学 食産業学部

醸造は、古くから伝わるバイオテクノロジーであり、世界的に人々の生活や文化様式に密着して 発達した。それらの多くは化学などの学問分野が発達する前から、経験的な積み重なりにより発展 してきた。 たとえば、微生物の知識がなかった時代には経験的にブドウの搾汁液からワインを造り、酵素の 存在がまだ知られていない時代でさえ、麦芽からビール醸造してきた。さらに、日本では、麹造り に用いる種菌である「種麹」を木灰の使用により、汚染されることなく優先的に生育させる方法を 見つけ出した。このため室町時代には大きな同業者団体の「麹座」を形成させ、麹に関わる酒造や 味噌など調味料産業により莫大な利益をあげることが出来た。さらに15世紀に清酒を醸造していた 多門院という寺院には、パスツールの殺菌法発明（19世紀）以前に、清酒殺菌法が詳細に記載され ている。このように、経験値を主体に形成されてきた醸造は、まさにオールドバイオテクノロジー である。しかし、これは科学の進歩した現代でも進化し続け、まさに「不易流行；本質は変わらな いが、常に進化という意」と言えよう。今日、塩麹といわれる調味料がブームになっているのは、 その良い例である。 このような醸造技術の中には、「温故知新」できる技術が多く散見される。これらの多くは、現 代の科学で解明することにより十分に利用可能なものが多い。 そこで今回、中国古書「斉民要術」に記載されている醸造法を例とし、解説・報告する。「斉 民要術」は、紀元6世紀中ごろ、北魏の太守 賈思勰（かしきょう）によって編纂された。これは 「斉国」の農業技術をおさめた書物で、「酒」の醸造技術、特に数種の独特な酒造用麹の製造法が 記載されている。なかでも、植物の熱水抽出液を加えて製麹されている。筆者は、この添加意義を 検討した。モデル的に製麹試験をし、比較した結果、出来上がった麹は細菌が少なく、「酒」の香 気がよくなった。植物中の抗菌性物質が汚染菌を生育抑制していることを明らかにした。 このようなオールドバイオテクノロジーから、新しいヒントがみつかることもあり、新しいバイ オテクノロジーの発端につながる。従って、オールドバイオテクノロジーは不易流行的に発展して いくことになると確信している。

－41－ Profile of Speaker

Makoto Kanauchi is Associate Professor in Department of Food Management, Miyagi University, Sendai, Miyagi Japan. He graduated from the Tokyo University of Agriculture in Tokyo, Japan in March 1996 and received a Ph.D. in Bio-regulation Control from that university in March 1999. He worked in Prof. Charles Bamforth’s Laboratory in the Department of Food Science and Technology, University of California at Davis, California (Nov. 1999 – May 2003). Subsequently, he was employed at the Institute of Food Science in Fuji Oil Co. Ltd. in Moriya, Ibaraki, Japan as a Researcher (Aug 2003 – Mar 2005). Since April 2005, he has been at the Department of Food Management, Miyagi University. He has also been a Lecturer in enzymology and alcoholic beverages (mainly spirits and wine) at the Tokyo University of Agriculture and Tokyo College of Medico- pharmacotechnology.

－42－ L 8

臨床研究と基礎研究 －新潟から世界へ－

渡辺 賢一1, 平山 匡男2, 小林 隆司3, 後藤 博4

1新潟薬科大学 薬学部 臨床薬理学 教授, 2ブルボン健康科学研究所, 3東京大学大学院医学系研究科, 4新潟バイオリサーチパーク

高血圧・糖尿病・ストレス性胃腸炎などの増加が社会問題となっています。 新潟バイオリサー チパーク(株)を中心とした多施設共同の臨床研究と基礎研究を、ヒト臨床試験・病態モデル動物実 験の両面から下記のように紹介します。 [A]モデル動物実験。 ⑴ 心不全モデル・高血圧モデル・肥満モデル・糖尿病モデル・トランスジェニックモデルにお ける心機能・血圧・血糖・アポト－シス・酸化ストレス・遺伝子変化。 ⑵ 漢方薬・和漢薬。釣藤散・大柴胡湯・柴苓湯の自然発症高血圧ラット（SHR）への効果。 当帰芍薬散の妊娠中毒症モデル動物への効果。 ⑶ 機能性食品。プチベ－ルの潰瘍性大腸炎モデルへの効果。クルクミンの糖尿病性臓器障害へ の効果。桑葉の糖尿病への効果。 [B]ヒト臨床試験 ⑴ 漢方薬・和漢薬。黄連解毒湯のヒト血小板機能への効果 ⑵ 機能性食品。桑葉と糖尿病。GABAと高血圧。米タンパク質含有食品と低アルブミン血症。 胚芽米含有食品と血圧・血糖。BAANｓとメタボリックシンドロ－ム。 ⑶ 健康産業。温泉。サウナ。 興味のある方は、 我々の下記論文を参照してください(過去3年分)。 (1) Eur J Pharm Sci. 2012;47: 604-614. (2) Toxicology. 2012;291: 139-145. (3)Free Radic Res. 2012;46,154-163. (4)J Cell Mol Med. 2012; 16: 1-10. (5)Biochemical Pharmacology. 2012; 83: 653-660. (6)Int J Biol Sci. 2011; 7: 1077-1092. (7)Free Radic Res. 2011; 45, 1223- 1231. (8)Cell Physiol Biochem.27;487-496,2011. (9)Biol. Pharm. Bull. 34;974-979,2011. (10) J Med Food. 2011;14: 912-919. (11)Mol Nutr Food Res. 2011; 55: 1655-1665. (12)J Med Food. 2011 ;14:601-609. (13)Free Radic Res. 2011;45:788-95. (14)Free Radic Res. 2011, 45:575-84. (15)Pharmaceuticals. 2011, 4, 770-781. (16)Int J Biol.Sci. 2011; 7: 154-167. (17)Toxicology. 2011;279:91-99. (18)Inflammation & Allergy-Drug Targets, 2011, 10, 218- 225. (19)Eur J Pharm Sci. 2011;44: 627–634. (20)Pharmaceuticals 2011;4:551-566. (21) Eur J Pharmacol. 2011; 652:126-135. (22)Pathol Int. 2011;61:228-38. (23)Heart Vessels. 2011;26:81-90. (24)Cardiovasc. Pathol. 2011; 20: 281-290. (25)Free Radic. Biol. Med. 2010; 49:1422–1431. (26)Current Cardiology Reviews, 2010;6:280-290. (27)Drug Discovery Today 2010;15:826-841. (28)Cell Physiol Biochem. 2010;26:167-78. (29)J. Pharmacol. Sci. 2010;113:325-34. (30)Hypentens. Res. 2010;33:695-702. (31)Exp. Biol. Med. 2010;235:1338-

－43－ 1346. (32)Toxicology. 2010;274:18-26. (33)Free Radical Research 2010; 44:1369-1377. (34)Free Radical Research. 2010;44:1082-1090. (35)J Pharmacy and Pharmacology. 2010;62:1776-1783. (36)Pathol Int. 2010 ;60:93-101.

Profile of Speaker

Dr. Kenichi WATANABE is a professor of Niigata University of Applied Life Sciences (NUPALS). He obtained his MD in 1974 followed by a Medical PhD from Niigata Medical University Japan in 1985 and a Pharmaceutical PhD from Shizuoka Prefectural University Japan in 1995. After the finishing his PhD course, he worked at Niigata Kuwana Hospital and Tsubame-Rosai Hospital Japan as a head of the department of internal medicine and cardiology. He has done contributory works in the field of cardiology, heart failure, hypertension, diabetes mellitus, immunology and cell biology with Pool Wilson, head of National Heart- Lung Institution London. In 1997, he has joined NUPALS as a professor. Since then, he has been interested in the mechanisms of the cellular response against oxidative stress, cardiology, hypertension, diabetes mellitus, immunology. His mottos are “Understanding the Past, Predicting the Future” and “From Niigata Japan to the World”. Today’s talk will be a part of his work on basic and clinical studies using his 300 articles.

－44－ L 9

食品に含まれるプロアントシアニジン類 －起源、化学構造、および健康作用－

平山 匡男

ブルボン健康科学研究所 株式会社ブルボン 柏崎市、新潟、日本

縮合タンニンとも言われるプロアントシニジン類はフラバノールが重合したオリゴマーおよびポ リマーである。プロアントシニジン類は多くの食用植物中に広く分布していることが知られてい る。フラバノール間の結合は、C4位とC8位で結合している成分が最も多い。食品に含まれるプロ アントシニジン類は、その構造の多様性とともに健康作用を示す可能性が高いことから、注目も高 まっている。カカオやリンゴ、ブドウに含まれるプロアントシアニジン類は既に研究が進んでお り、それぞれが特有のフラバノール構造様式と重合度を持っていることが明らかになっている。こ れらの構造特性は、抗酸化、血管弛緩、抗菌、抗炎症活性などの生理活性と密接に関係していると 考えられている。 高血圧は心血管疾患を進行させる大きなリスク因子である。ポリフェノールを豊富に含む食事を 毎日摂ることは、このリスクを下げる重要かつ有効な方法となあることが明らかとなってきてい る。いろいろなポリフェノール類の中でも、プロアントシニジン類が心血管障害に有用な効果を示 すエビデンスが増えてきている。その先駆的な研究として、カカオやブドウに含まれるプロアント シニジン類により内皮依存性の血管弛緩作用を増強することがラット大動脈標本を用いたin vitro 試験で確認され、この作用は NO/cGMP 経路の NO 濃度を増加させることにより起こることが明 らかになっている。プロアントシニジン類の化学構造、生理作用および生体利用性は植物の種類や 部位、抽出法などで変動するが、これらの関係を明らかにするには更なる情報が必要になってい る。中でも、重合度は経口吸収性に影響するキー因子であることがわかっている。 本講演では、柿葉およびボイセンベリーのプロアントシニジン類の構造と血管弛緩作用について 紹介する。柿葉はエピガロカテキンガレートを含む4種類のフラバノール単位構造から構成される 特異なオリゴマーを含有している。その抽出物をWistarラットおよび自然発症高血圧ラット(SHR) に経口投与すると、それぞれ、血糖値および収縮期血圧値の上昇抑制作用が認められた。ボイセン ベリージュースおよび種子には2量体および3量体のみの短鎖プロアントシアニジンが含まれてお り、これらをSHRに経口投与すると吸収されて抗高血圧作用を示した。

－45－ Profile of Speaker

Dr. Masao Hirayama is a Labs Director of Bourbon Health Institutes of Health, Bourbon Corp, and a Professor Emeritus and a Guest Professor at Niigata University of Pharmacy and Applied life Sciences (NUPALS). Dr. Hirayama’s interests include research of functional food components and evaluation of the beneficial effects on human health. He has made important contribution to FOSHU (Foods of Specified Health Uses) to develop several functional components and the health claims, such as fructooligosaccarides (increase of intestinal bifidobacteria, maintenance of a healthful GI condition, and improvement of Ca absorption), guar gum hydrolysate (soluble and fermentable dietary fiber with low viscosity), casein phosphopeptides (improvement of Ca absorption), etc. His recent work focuses on the studies of beneficial effects of dietary polypnenols, such as improvement of visual functions by blackcurrant anthocyanins and vasodilative effects of persimmon and boysenberry proanthocyanidins. Dr. Hirayama graduated from postgraduate course (master’s degree of engineering) of Yokohama National University at 1968. From 1968 to 2004, he worked at Meiji Seika-Kaisha in Pharmaceutical Research (for 16 years) and in Functional Foods Research Labs (for 20 years). In 1983, he received his PhD in organic chemistry from Tokyo Institute of Technology. In 2004, he moved to NUPALS as a Professor of Department of Food Sciences, and after retirement in 2011, he has been in the present post.

－46－ L 10 Anti-obese activity of quercetin glycoside mediated by activation of Hormone-Sensitive Lipase (HSL)

Yoshinori Kitagawa

Institute for Health Care Science, SUNTORY WELLNESS Ltd., Osaka 618-8503, Japan

Obesity is one of the most important worldwide health problems. Previous studies have reported that consumption of flavonoids in the diet is inversely correlated to lower morbidity and mortality from coronary heart disease and many studies have shown that quercetin might possess beneficial effects on lipid and glucose metabolisms. These studies suggest that quercetin is promising as new pharmacologic agent for the treatment of obesity- related health problems such as metabolic syndrome. Thus, we investigated the impact of quercetin glycosides(QG), which are enzymatically trans-glycosylated isoquercitrin from natural sources (Sophora japonica) and have improved bioavailability, on visceral fat mass in diet-induced obese mice and the obese human. After 3months on the ad libitum feeding of QG (0.05, 0.25 and 1.25%) diet-induced obese C57BL/6J male mice resulted in reduced body weight gain dose-dependently, and significant reduction of adipose tissue mass (1.25% QG containing diet). Histological evaluation in epididymal adipose tissue shows that 0.25 and 1.25% QG diets clearly prevented the development of adipocyte hypertrophy, which was associated with the effects on body weight gain and the weight of visceral adipose tissue. Quercetin has been reported to have a lipolytic potency in rat primary adipocyte. Quercetin aglycone in the presence of 0.1μM noradrenaline accelerated the NEFA and glycerol release and phosphorylation of hormone-sensitive lipase (HSL) in dose-dependent manner in 3T3- L1 adipocytes. HSL is lipolytic enzyme and exists in cytoplasm in the dephosphorylated inactive form. When the cells are stimulated with some lipolytic signal, HSL is phosphorylated, activated and promotes hydrolysis of lipid droplets. Interestingly metabolites of quercetin such as glucuronide and sulfate conjugates also have almost same activities. These results suggest that quercetin and/or its metabolites activate some adrenergic lipolytic pathway in adipocytes. In a double-blind and placebo controlled clinical study, daily intake of 275mg QG containing beverage for 12weeks resulted in significant reduction of visceral fat mass in healthy obese people (BMI values were from 24 to 31) and they had experienced no adverse effects. QG containing beverage reduced total fat area compared with that of placebo and significant reduction was observed after 8 weeks ingestion. Similar data was obtained in visceral and subcutaneous fat area. Associated with the change in fat area, waist and hip circumference, and subcutaneous fat thickness in the back were significantly reduced compared with these of placebo. On the other hand, the body weight did not change significantly in this test period. All blood biochemical parameters were not influenced by QG ingestion. These data suggest that QG can reduce the abdominal body fat in human. This study suggested that the QG activate lipolytic pathway in adipose tissues and are beneficial to improve obesity in human.

－47－ Profile of Speaker

Dr. Yoshinori Kitagawa is a general manager of Institute for Health Care Science of SUNTORY Wellness Ltd. In 1990, he earned his PhD degree from Kobe University based on the research activities at the Institute for National Cancer Research Center, Tokyo Tsukiji, where he had been sent from SUNTORY Ltd. After coming back to SUNTORY, he worked at SUNTORY Institute for Biomedical Science, and he has done contributional works in the field of establishment of high-throughput screening system using recombinant cells expressing 5-HT receptors and a glucose transporter, GLUT4. In 2000, he had moved to Pharmaceutical Development Division and had been in charge of clinical pharmacological test for developing Memantine, which is unique pharmaceuticals blocking NMDA glutamate receptors for Alzheimer disease. In 2006, he has transferred to Institute for Health Care Science of SUNTORY Wellness Ltd. Since then, he has been in charge of developing functional foods and Foods for Specified Health Uses (FOSHU) beverages. Today’s talk will be a part of his work on development of functional foods.

－48－ L 11

Bio-Activating Advanced Nutrients (BAANs) を用いた 新しい生活習慣病の治療および予防方法の確立

佐藤 晋

北陸先端科学技術大学院大学, 株式会社ワイ’ズ

近年、先進国のみならず発展途上国でも糖尿病、高血圧、脂肪肝、脂質異常症、肥満などの生活 習慣病が増加してきており、国内では生活習慣病由来の心臓および脳血管障害による死亡率が癌に よる死亡率とほぼ同等になってきている。これらの生活習慣病は夜型のライフスタイルへの変化と 食事の西洋化のより引き起こされているといわれている。治療法として定期的な運動や食事の指導 が行われているが、いずれの方法も継続するのが難しく、薬剤による対症療法に頼らざるを得ない のが現状である。しかしながら、薬物療法は一生薬を飲み続けなければならず、QOLの面でも医 療経済学的にも大きな問題になりつつある。 古来より東洋医学では「医食同源」の言葉のように、天然の食材を摂取することで病気の治療や 予防が行われてきた。内臓脂肪は生活習慣病を引き起こす原因であることが知られており、我々は 「内臓脂肪を消費させ、細胞を活性化させるエネルギー源として用いる。」ことを目標に研究を開 始した。 先ず、天然の食品成分の中から生体を活性化させる成分を選び出す検討を始め、どのような組合 せが生活習慣病の改善に効果があるのかの検討をおこなった。 最終的にL-アルギニン、ω-3多価不飽和脂肪酸およびリボ核酸(RNA)の組合せが内臓脂肪を 分解し、細胞を活性化する成分として選び出され、これらの成分の組合せを『Bio-Activating Advanced Nutrients (BAANs) 』と名付けた。 BAANsの効果を検証するために、BAANs成分を含む代替食による三ヶ月の臨床試験を主に生 活習慣病患者を対象に行った。運動や食事の指導などない通常の生活で生活習慣病を改善すること を目標とし、運動および食事への介入は一切せずに、１日一回代替食を服用するのみのシンプルな 臨床試験で効果の確認を行った。 その結果、肝機能、血圧、血清脂質、血中グルコースなどの測定値において１ヶ月後から高い効 果が見られ、３ヶ月後にかけて低下した。この結果は、BAANsが生活習慣病の治療および改善に 対して有意に効果があることを示唆した。 特に注目される点は、肝機能の改善、アディポサイトカインの発現の正常化（アディポネクチン の発現の上昇、PAI-1の発現の減少）、および内臓脂肪量が大幅に減少したことであった。 一方、低カロリーダイエットで見うけられる好ましくない事象（体力や生理機能の低下、低栄養 性の脂肪肝、リバウンドなどの副作用）は試験期間中および試験後においても全ての被験者で見ら れなかった。 本シンポジウムでは、これまでに実施された臨床試験の結果について報告するとともに、今後の 進め方についても紹介したい。

－49－ Profile of Speaker

Entered in Fujisawa Pharmaceutical Co., Ltd, after completed the master's course of Tokyo Institute of Technology in 1979. In molecular biological division, human IGF-1 manufactured by recombinant E.coli had been marketed in 1983. Main theme was establishment of the drug evaluation system using recombinant cells and exploration of the innovative drug target gene related for stroke. After acquiring the doctor's degree in Tokyo Institute of Technology in 1992, studied in University of Pittsburgh Medical Center during one year for research of the molecule action mechanism of immunosuppressant FK506. After leaving Fujisawa Pharmaceutical in 2005, have established Y’s Corporation Co., Ltd. in 2007. It is taken up visiting professor and the university industrial adviser in Japan Advanced Institute of Science and Technology from 2011. My principal objective of the study is target research and development of products in preventive health care field. The product “Pollenon” for preventing pollenosis and “Dr. BAANs” for improvement lifestyle-related disease are already marketed. Now studying the development of the molecular introduction device which aimed at a gene therapy and regenerative medicine as the method of ultimate preventive health care collaborated with Ehime University.

－50－ L 12

海藻由来成分メカブフコイダンの免疫機能に及ぼす効果

吉永 恵子

理研ビタミン株式会社 ヘルスケア部

フコイダンは海藻の中でもわかめや昆布など褐藻類に含まれる、フコースを主とした硫酸化多糖 類の総称である。これまでの研究からフコイダンは免疫賦活作用を有することが明らかにされてい る。フコイダンは海藻ごとにその構造が異なり、機能性にも違いがあることが知られている。今回 はフコイダンの中でも、わかめの胞子葉であるメカブに含まれるメカブフコイダンについて、抗ウ イルス作用を中心についてこれまでに得られた知見を報告する。 メカブフコイダンは抗腫瘍効果、抗アレルギー作用などの免疫賦活作用を有することがこれまで のin vitro, in vivo試験より明らかとなっている。その機能性はマクロファージやNK細胞など免疫 細胞の活性化や、サイトカインの産生調節などにより誘導されることを報告している。また、イン フルエンザウイルスやヘルペスウイルスに対する抗ウイルス効果も明らかとされており、ウイルス 感染モデルマウスを用いた試験では、メカブフコイダンの経口投与により生体内でのウイルス増殖 抑制効果や、抗体産生増強作用などが認められている。さらに、特別養護老人ホームにおいて高齢 者を対象に実施したプラセボ対照二重盲検試験において、インフルエンザワクチン接種後の抗体産 生量が、メカブフコイダン食を摂取した群ではプラセボ群と比較して顕著に増加することも明らか となった。また、メカブフコイダン食を摂取後、摂取前と比較し有意にNK細胞活性が上昇するこ とも確認され、本試験からヒトにおいてもメカブフコイダンが免疫賦活作用を有することが証明さ れた。 より詳細にメカブフコイダンの免疫賦活作用メカニズムを探るべく、メカブフコイダンを7日間 にわたり経口投与したBalb/cマウスの腸管パイエル板におけるDNAマイクロアレイ解析を実施し た。その結果、メカブフコイダンを投与した群では、defense responseに関与する遺伝子群の発現 が亢進するなど、免疫機能を担う遺伝子群の興味深い発現変動が認められた。

本研究の一部は、（独）科学技術振興機構の研究成果最適展開支援プログラム(A-STEP)より支 援を受け実施した。

－51－ Profile of Speaker

Dr. Keiko Yoshinaga belongs to Health Care Department of Riken Vitamin Co., Ltd. She received her PhD from Graduate School of Agricultural and Life Sciences/ Faculty of Agriculture, the University of Tokyo in 2006, and joined Riken Vitamin Co., Ltd. in the same year. Since then, she has been developing functional foods.

－52－ L 13

ニュートリゲノミクス研究の今：最近のケーススタディより

中井 雄治

東京大学大学院農学生命科学研究科ILSI Japan 寄付講座

ニュートリゲノミクスは、食品に対する生体応答を遺伝子発現変動として捉え、網羅的に解析す ることで、様々な機能性食品成分の作用メカニズムを探ったり、食品や栄養条件をプローブとして 生体の恒常性調節メカニズムを探ったりする研究分野である。ゲノミクスという名前はついている が、その実体はトランスクリプトミクス、プロテオミクス、メタボロミクスといった網羅的解析手 法を包含する、統合オミクス研究である。中でも、DNAマイクロアレイを用いたトランスクリプ トミクスは、実験デバイスとしてもほぼ確立され、また、ここ数年でデータ解析手法も成熟してき ており、ニュートリゲノミクスの中心的手法となっている。当寄付講座は、実験デザイン、動物実 験の実施からDNAマイクロアレイデータの取得、そしてバイオインフォマティクスの最新の解析 手法を取り入れたデータ解析までを一貫して行なうことにより、成果をあげつつある1,2)。また、 当寄付講座の方針は多くの食品系企業に共同研究の形で取り入れられ、従来の方法論では見つける ことができなかった遺伝子発現の変化を解像度よく捉えることに成功している。本講演では、網羅 的解析のメリットを最大限に活かすための方針を、DNAマイクロアレイを用いた我々のグループ の最近の研究例を交えながらご紹介する予定である。

1) Ushiama et al., Biosci. Biotechnol. Biochem. 74 (2010) 1320-1323 2) Suyama et al., PLoS One 7 (2012) e29483

－53－ Profile of Speaker

Dr. Yuji Nakai is an associate professor of ILSI Japan-Endowed Chair of Functional Food Science and Nutrigenomics, Graduate School of Agricultural and Life Sciences, The University of Tokyo. In 1996, he earned his PhD degree from The University of Tokyo under the guidance of Prof. Soichi Arai. After the finishing his PhD course, he worked at National Institute of Health Sciences (Tokyo) and RIKEN (Tsukuba) as a postdoctoral fellow. In 2000, he has joined Faculty of Pharmaceutical Sciences, Kanazawa University as an assistant professor. He has been studied the mechanisms of phagocytic removal of apoptotic cells with Prof. Yoshinobu Nakanishi. In 2005, he joined Agricultural Bioinformatics Research Unit, The University of Tokyo as an associate professor. Since then, he has been interested in the field of nutrigenomics, especially in DNA microarray data analysis. In 2009 he took up the new post in ILSI Japan- Endowed Chair of Functional Food Science and Nutrigenomics. Using DNA microarray, he is studying changes in the gene expression response against alteration of nutritional condition. Today’s talk will be a part of his work on nutrigenomics.

－54－ Session3

L 14 Stress regulation by inhalation of linalool as seen from gene expression analysis

Akio Nakamura1, Satoshi Fujiwara1, Yuji Nakai2, Keiko Abe2

1R&D Center, T.Hasegawa Co.,Ltd., Kanagawa 211-0022, Japan 2Graduate School of Agricultural and Life Sciences, The University of Tokyo Tokyo 113-8657, Japan

Aromas may sometimes have an effect to relax our stress. In recent years, there has been more interest in the psycho-physiological effects elicited by pleasant aromas, because they are expected to contribute to health maintenance and promotion. Despite that, no molecular logic of this event has remained to be clarified. In our research of quantitatively analyzing psycho-physiological effects elicited by aromas, we profiled blood cells and gene expressions of the rats which inhaled (R)-(–)-linalool under a restraint stressed condition. In neutrophils and lymphocytes, significant changes in quantity caused by the restraint were repressed by exposure to the odorant. Moreover, inhalation of (R)-(–)-linalool induced significant changes in the stress-induced variations with respect to 115 gene expression levels of the whole blood. Of those, 109 genes were down-regulated1). Another study was carried out by gene expression profiling for a sample of hypothalamus as a stress response center. It resulted that inhalation of (R)-(–)-linalool under a restraint stress-added condition up-regulated a number of neuron differentiation-related genes toward activating the processes of neuronal maturation, and the inhalation also up-regulated restraint stress-inducible, heat shock protein-related genes that can be associated with the suppression of stress-caused apoptosis2). In conclusion, (R)- (–)-linalool inhalation returns stress-elevated levels of neutrophils and lymphocytes to near- normal levels. The inhalation also reduces the activity of more than 100 genes overdriven in stressful situations for the whole blood of the odorant-inhaling rats subjected to acute restraint stress. Our study is the first to reveal the possibility that olfactory input can modify the gene expressions in the hypothalamus neural network involved in feeding behaviors of animals, as well as their blood chemistry. Our finding has thus elucidated a physiological effect of an inhaled pleasant aroma, (R)-(–)-linalool in this case, by an in-depth analysis of gene expressions, and also could largely contribute as a new method for assessing effects caused by aromas on stress regulation.

－55－ 1)Nakamura, A.; Fujiwara, S.; Matsumoto, I.; Abe, K. Stress repression in restrained rats by (R)-(–)-linalool inhalation and gene expression profiling of their whole blood cells. J. Agric. Food Chem. 2009, 57, 5480-5485. 2)Nakamura, A.; Fujiwara, S.; Ishijima, T.; Okada, S.; Nakai, Y.; Matsumoto, I.; Misaka, T.; Abe, K. Neuron differentiation-related genes are up-regulated in the hypothalamus of odorant- inhaling rats subjected to acute restraint stress. J. Agric. Food Chem. 2010, 58, 7922-7929.

Profile of Speaker

Dr. Akio Nakamura is a food technologist of Technical Research Institute R&D Center, T.Hasegawa Co.,Ltd. After finishing his Master course in Chemistry and Biotechnology of Engineering, The University of Tokyo in 1999, he has joined T.Hasegawa Co.,Ltd till present. In 2010, he earned his PhD degree in Agricultural and Life Science from The University of Tokyo under the guidance of Dr. Keiko Abe. He joined ILSI Japan-Endowed Chair of Functional Food Science and Nutrigenomics, Graduate School of Agricultural and Life Sciences, The University of Tokyo under the guidance of Dr. Ichiro Matsumoto and Dr. Yuji Nakai, for studying “stress regulation by odorants” which was evaluated by gene expression analysis. Today’s talk will be a part of his work on stress regulation by inhaled odorants as seen from gene expression analysis.

－56－ L 15 Natural compounds as inhibitors of inflammation and activators of cell death mechanisms

Marc Diederich

Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9, rue Edward Steichen, L-2540 Luxembourg, Luxembourg

Programmed cell death plays a critical role in the cellular housekeeping, and aberrant cell death regulation leads to body malformations and to numerous diseases, including various autoimmune diseases, cancer, stroke, infarctations, and neurodegenerative diseases. Research in the field of cell death has evolved at a very rapid pace over the last fifty years. It is now generally accepted that programmed cell death can be subdivided into three different categories: apoptosis (programmed cell death type I), autophagy (programmed cell death type II, or lysosomal cell death), and oncosis (programmed cell death type III, also referred to as necrosis). Apoptosis, in particular, has been studied in great depth and autophagy has recently sparked an enormous research interest.

In the 1960’s to early 1980’s, at the same time as renowned biologists including Richard Lockshin and John Saunders, John Kerr, Alastair Currie, and Andrew Wyllie, and Sydney Brenner, Robert Horvitz, and John Sulston initiated their investigations into cell death, research groups lead by Paul Scheuer, John Faulkner, George Pettit, and other chemists pioneered the research field of natural products, with the aim to identify potent anti-cancer compounds amongst the toxins produced by marine organisms as a chemical defence mechanism against their predators. Since then, numerous marine natural products have been identified as modulators of cell death. Here, we discuss the mechanism of action of the major marine natural products reported as inducers of apoptosis, autophagy, or oncosis.

－57－ Profile of Speaker

Marc Diederich earned his PhD in molecular pharmacology in 1994 from the University Henri Poincaré Nancy 1, France. After training at the University of Cincinnati, USA, he focused his research on cancer and leukemia cell signaling pathways and gene expression mechanisms triggered by natural compounds with epigenetic-, anti-inflammatory- and cell death-inducing potential. He currently directs the Laboratory for molecular and cellular biology of cancer (LBMCC) at Kirchberg Hospital in Luxemburg. He was appointed associate Professor of Biochemistry at the College of Pharmacy of Seoul National University in 2012. Since 1998, he is the organizer of the “Signal Transduction” meetings in Luxembourg.

－58－ L 16 15-Hydroxyprostaglandin dehydrogenase as a potential molecular target for chemoprevention of inflammation-associated colon carcinogenesis by curcumin

Hye-Kyung Na

Department of Food and Nutrition, Sungshin Women’s University, Seoul 142-100, South Korea

Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is regulated not only by its biosynthesis, but also by the degradation process. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the first step in the inactivation of PGE2. 15-PGDH has been known to be a physiological antagonist of COX-2. In the present study, we have observed that expression of 15-PGDH is decreased in the colonic mucosa of dextran sodium sulfate (DSS)-treated mice, while expression of COX-2 increased. To determine whether 15-PGDH is negatively regulated by COX-2, we utilized a selective COX-2 inhibitor celecoxib. Oral administration of celecoxib increased the 15-PGDH expression while the same treatment decreased COX-2 expression in DSS-treated mouse colon. Moreover, 15-PGDH expression in colonic mucosa following treatment with AOM plus DSS was more prominent in COX- 2 knockout mice than that observed in COX-2 wild type animals. Likewise, levels of constitutively expressed 15-PGDH were higher in COX-2 knockout mice. We also observed that the mRNA levels and protein activity of 15-PGDH were upregulated by inhibition of COX- 2 using designed siRNA in colon cancer cell lines. In patients with colon tumors, the expression of 15-PGDH was markedly reduced in adenomas and carcinomas, compared with normal surrounding tissues. These finding suggest that expression of 15-PGDH is negatively regulated by COX-2, which may contribute to the inflammation-associated colon carcinogenesis. We also have found that curcumin, a yellow colouring agent present in the rhizome of Curcuma longa Linn (Zingiberaceae), induced expression of 15-PGDH in normal rat gastric mucosa cells (RGM-1) in a concentration and time dependent manner. The mRNA level of 15- PGDH was also increased by curcumin treatment. By using deletion constructs of 15-PGDH promoter, we found that activator protein-1 (AP-1) is the most essential transcription factor responsible for curcumin-induced upregulation of 15-PGDH expression. Curcumin enhanced the expression of c-Jun, c-Fos and CREB in the nuclear fraction of RGM-1 cells. In contrast, tetrahydrocurcumin which lacks the α,ß-unsaturated carbonyl group failed to induce expression 15-PGDH, suggesting that the electrophilic α,ß-unsaturated carbonyl group of curcumin plays a critical role in its induction of 15-PGDH expression in RGM-1 cells. These results suggest that curcumin-induced expression of 15-PGDH may be mediated by activation of AP-1 transcription factor.

－59－ Profile of Speaker

Dr. Hye-Kyung Na is an assistant professor of Sungshin Women’ s University, Department of Food & Nutrition from 2009. She obtained her PhD degree from the Department of Food & Nutrition of Chonnam National University, South Korea in 1996. After finishing her PhD course, she had a postdoctoral training at Department of Pediatrics and Human Development, Michigan State University in the James E. Trosko’ s Lab on gap junctional intercellular communication. After relocation to Korea in 2000, she joined the Prof. Young-Joon Surh’s research group in Seoul National University, College of Pharmacy and worked there until 2008 as Research Professor. Her work focuses on 1) upregulation of COX-2 expression causally linked to apoptosis induced by ET-18-O-CH3 2) Anti-inflammatory and anti-proliferative effects of diallyl trisulfide derived from garlic 3) Role of 15-deoxyΔ12,14-prostaglandin J2 in breast carcinogenesis 4) Anti-inflammatory and anti-oxidant effects of EGCG. 5) Role of the catechol estrogen 4-hydroxyestradiol in breast carcinogenesis. Currently she studies on the molecular mechanisms underlying regulation of 15-prostaglandin dehydrogenase in breast, colon, and stomach carcinogenesis.

－60－ L 17 Endophytic fungi as sources of new anti-cancer and antibiotic metabolites

Peter Proksch

Institut fuer Pharmazeutische Biologie, Heinrich-Heine-Universitaet Duesseldorf, Universitaetsstr. 1 Geb. 26.23, D-40225 Duesseldorf, Germany

This presentation gives an overview on our recent results on new bioactive compounds from endophytic fungi that inhabit higher plants and live with their hosts in a mutualistic relationship. Endopyhtes are known to produce anticancer metabolites such as paclitaxel, camptothecin and others that were hitherto only known from plants. In addition these fungi are prolific sources of other structurally new metabolites that are of interest due to their or anti-cancer properties and/or their antibiotic activity against multi drug resistant bacteria thus provoking strong interest in these compounds as lead structures for biodiscovery. In our group the focus is on endophytic fungi from extreme and less investigated habitats such as Mangrove swamps and medicinal plants. Fungi are selected for in depth investigations based on antibiotic and/or anti-cancer activities of their crude extracts followed by bioactivity guided isolation and structure elucidation of the active principles. Compounds isolated are structurally divers and include peptides and depsiteptides, alkaloids and polyketides such as dimeric and monomeric anthraquinones and xanthone derivatives. This presentation will focus on new pyrrocidine derivatives from the fungus Embellisia eureka that are potent inhibitors of various human cancer cell lines with IC50 values in the nanomolar range. Experiments on the mode of action suggest that inhibition of the nuclear factor Nf kappa B by the investigated pyrrocidines is important for the activity of the compounds. A further example covers dimeric anthracene derivatives that were isolated from the endophytic fungus Talaromyces wortmannii that inhibit the growth of multiresistant humanpathogenic bacteria.

－61－ Profile of Speaker

Dr. Peter Proksch is a full Professor of Heinrich Heine University at Duesseldorf, Germany. He earned his Ph D in 1980 from the University of Cologne (Germany) in the field of Biology. From 1980 – 1982 he was a Post Doctoral Fellow at the University of California, Irvine (USA). Upon returning to Germany he worked at the Universities of Cologne, Braunschweig and Wuerzburg as Assistant and Associate Professor in Pharmaceutical Biology. In 1999 he joined Heinrich Heine University in Duesseldorf where he presently working as a full Professor in the Institute of Pharmaceutical Biology and Biotechnology. His field of research is on isolation, identification and biological evaluation of new bioactive natural products from higher plants, marine organisms and fungi. His presentation will be on anti-cancer and antibiotic constituents from endophytic fungi.

－62－ PL 18 Preventive and therapeutic management of cancer by natural products

Pradeep Kumar Goyal

Radiation & Cancer Biology Laboratory, Department of Zoology University of Rajasthan, Jaipur (India)

Cancer remains one of the most feared diseases and approximately nine million cases of cancer occur in the world every year. Cancer is the second most common cause of death in the developed world, and a similar trend has also emerged in the developing countries. There are striking variations in the risk of different cancers by geographic area. Most of the international variation is due to exposure to known or suspected risk factors related to life style or environment, and provides a clear challenge to its prevention. Despite significant advances made in the cancer treatment strategies, there have been no significant reductions in the mortality rates for the common forms of cancer. This indicates that new approaches to control the cancer are critically needed. Cancer chemoprevention is an important strategy to reduce the cancer burden. It has the use of natural, synthetic, biological or chemical agents to reverse, suppress, or prevent carcinogenic progression. Recent evidences suggest that the new emphasis in the development of medical treatment of human diseases will be intimately connected to natural products. The use of natural products and nutraceuticals in modern medicine for the prevention or treatment of cancer is an important aspect.

The rich biodiversity available in Indian subcontinent has yielded several new drugs that find applications in modern medicine and there is like hood of discovering many more. The extracts of the various parts of some medicinal plant viz. Amla (Emblica officinalis), Rosmary (Rosemary officinalis), Methi (Trigonella foenum graecum), Sapthaparna (Alstonia scholaris), Bael (Aegle marmelos), Bhumi amla (Phyllanthus niruri), Jamun (Syzgium cumini), Gloe (Tinospora cordifolium), Kamrak (Averrhoa carambola), Linseed (Linum usitatssimum) have been trialed in this laboratory for their anti carcinogenic potential in skin, liver and stomach cancer models against chemical induced carcinogenesis. The results from the present investigation indicate that most of these plants extract have the potentiality to reduce the cancer burden in the form of tumor size, tumor weight, tumor number, tumor yield and by increasing the latent period of tumor appearance. Further, such plant extracts decrease the oxidative stress but increase the antioxidant enzymes (GST, CAT. GSH, SOD) in various biological tissues. It is concluded that the several medicinal plants may be used for preventative and therapeutic management of cancer.

－63－ Profile of Speaker

Dr. P. K. Goyal is serving as Professor in Department of Zoology, Chief of Radiation & Cancer Biology as well as Director, Life Sciences at University of Rajasthan, Jaipur. He is honored as the President of Indian Society for Radiation Biology and President of Indo Global Health Care Research Foundation. He has been elected as the Member of Advisory Committee of World Cancer Research Forum for plant based research on cancer management and Councilor of Asian Congress on Radiation Research. He is the recipient of several prestigious International/National awards. He has published more than 165 research papers, in various national and international scientific peer reviewed, and 15 text books.

－64－ Session4

L 19

“Seed to patient”: role of specifically clinically proven natural products

Dilip Ghosh and Andrea Zangara

SOHO Flordis International Sydney, New South Wales, Australia

Standardised extracts are processed products, where some specific and known components, recognised to contribute more than others to the therapeutic activity, are adjusted to a given amount, within the acceptable tolerance. Standardisation is achieved by adjustment of natural substance/herbal preparation with excipients or by blending batches of natural substance/herbal preparations. All the other components are still present in the extract, because the action of the plant may result from the synergistic activity of several constituents. Moreover, often all the constituents of a plant are not yet fully characterised. According to the concept of phytoequivalence, a chemical profile, such as a chromatographic fingerprint, for a natural product should be constructed and compare with the profile of a clinically proven reference product. It is important therefore to determine most of the phytochemical constituents of natural products in order to ensure the reliability and repeatability of pharmacological and clinical research. Plants are highly variable by nature and it is a common experience that batches of medicinal plants with similar specifications, as species and part of plant, may have quite different chemical compositions. The final product can be affected by: ◦The botanical variety and parts of plant used ◦The soil characteristic ◦Temperature and water availability in the growing season ◦Time of harvest ◦The processing of raw materials ◦Storage and transportation methods

For all the reasons above, it is clear that the “generic” concept which is valid for conventional medicines is mainly invalid for “Specifically Clinically Proven” natural medicines.

－65－ Profile of Speaker

Dilip has received his PhD in biomedical science from University of Calcutta, India. Previously, he held positions in Organon (India) Ltd., a division of Organon International, BV and AKZO-NOBEL, The Netherlands; HortResearch, New Zealand; USDA-ARS, HNRCA at Tufts University, Boston; The Smart Foods Centre, University of Wollongong, & Neptune Bio-Innovation Pty Ltd, Australia. He has been involved for a long time in drug-development (both synthetic and natural) and functional food research and development both in academic and industry domains. He is a fellow of American College of Nutrition and also in editorial board of several journals. Currently Dr. Ghosh is professionally involved with Soho- Flordis International, and Nutriconnect, Sydney, Australia; Honorary Ambassador, Global Harmonization Initiative (GHI). Dr. Ghosh has published more than 60 papers in peer reviewed journals, numerous articles in food and nutrition magazines and books. His recent book, “Biotechnology in functional foods and nutraceuticals”, CRC Press is published in 2010 and “Innovation in healthy and functional foods” is due in September 2012. Dr. Ghosh is Review Editor, Frontiers in Nutrigenomics; Editorial board member, The American Journal of Advanced Food Science and Technology, USA, Panacea E-Newsletter, India; Guest Columnist of NutraScope, India; Associate Editor & member, Toxicology Mechanisms and Methods, Taylor & Francis, USA (2006-2007). He can be reached at: dilip.ghosh@sfihealth.com, [email protected].

－66－ L 20 The cellular and molecular basis of health benefits of grape seed proanthocyanidin extract

Debasis Bagchi and Hiroyoshi Moriyama

University of Houston College of Pharmacy, Houston, TX, USA, and Iovate Health Sciences International Inc, Oakville, ON, Canada; Showa Pharmaceutical University, Tokyo, Japan

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose- dependent free radical scavenging ability of a grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical- induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco- induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl- X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical- mediated tissue injury.

－67－ Profile of Speaker

Debasis Bagchi, Ph.D., MACN, CNS, MAIChE, received his Ph.D. degree in Medicinal Chemistry in 1982. He is a Professor in the Department of Pharmacological and Pharmaceutical Sciences at University of Houston, Houston, TX, USA. Dr. Bagchi is also the Director of Innovation & Clinical Affairs at Iovate Health Research Sciences Inc., Oakville, ON L6M 0H4. Dr. Bagchi is currently the Vice- Chair of the International Society of Nutraceuticals and Functional Foods (ISNFF). Dr. Bagchi is the Immediate Past President of the American College of Nutrition, Clearwater, FL, and also serves as a Distinguished Advisor on the Japanese Institute for Health Food Standards, Tokyo, Japan, and Immediate Past Chairman of the Nutraceuticals and Functional Foods Division of the Institutes of Food Technologists, Chicago, IL. Dr. Bagchi received the Master of American College of Nutrition Award in October 2010. His research interests include free radicals, human diseases, carcinogenesis, pathophysiology, mechanistic aspects of cytoprotection by antioxidants, regulatory pathways in obesity, diabetes and gene expression. Dr. Bagchi has 288 papers in peer reviewed journals, 16 books and 16 patents. He has delivered invited lectures in various national and international scientific conferences, organized workshops, and group discussion sessions. Dr. Bagchi is a Fellow of the American College of Nutrition, Member of the Society of Toxicology, Member of the New York Academy of Sciences, Fellow of the Nutrition Research Academy, and Member of the TCE stakeholder Committee of the Wright Patterson Air Force Base, OH. Dr. Bagchi is a Member of the Study Section and Peer Review Committee of the National Institutes of Health, Bethesda, MD. Dr. Bagchi is the Associate Editor of the Journal of Functional Foods and Journal of the American College of Nutrition, and also serving as Editorial Board Members of numerous peer reviewed journals including Antioxidants and Redox Signaling, Cancer Letters, Toxicology Mechanisms and Methods, and other scientific and medical journals. He is also the Consulting Editor of CRC Press/Taylor & Francis. Dr. Bagchi received funding from various institutions and agencies including the U.S. Air Force Office of Scientific Research, Nebraska State Department of Health, Biomedical Research Support Grant from National Institute of Health (NIH), National Cancer Institute (NCI), Health Future Foundation, The Procter & Gamble Company and Abbott Laboratories.

－68－ L 22 Role of inflammation in high-fat diet-induced tumor promotion

Jung Han Yoon Park

Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea

Epidemiological evidence indicates that overweight and obesity increase risks of developing several types of cancers including colon, breast, liver, and melanoma. Over the past few years, we have examined roles of a high-fat diet (HFD) in tumor promotion and progression using several mouse tumor models. We observed that HFD feeding decreased the survival rate and increased tumor growth, tumor angiogenesis, tumor lymphangiogenesis, and liver, lung, or lymph node metastasis in the absence or presence of discernible obesity. Our results indicate that HFD feeding increases pro-inflammatory cytokines and chemoattractants in tumor tissues, adipose tissues and the blood, and these local and circulating factors induce the activation of various signaling molecules resulting in the activation of several transcription factors, which subsequently induces the expression of their target molecules involved in the stimulation of cell proliferation, inflammation, angiogenesis, and metastasis. To explore early changes in gene expression in the livers and lungs of the HFD-fed, tumor-bearing mice, total RNA from hepatic and pulmonary tissues was isolated for cDNA microarray analysis. Results from functional annotation and core network analyses revealed that HFD-induced alterations in deferentially expressed genes demonstrated the distinctive transcriptional features of “inflammation and cell cycle” in the metastasis target organs of HFD-fed mice suggesting that HFD promotes the formation of pro-inflammatory and pro-mitotic microenvironment in the target organs that are favorable for immune cells to infiltrate and differentiate, as well as metastatic tumor cells to infiltrate and proliferate. HFD feeding increased adipocytes and CD45+ leukocytes in tumor tissues and these leukocytes include CD68+ and F4/80+ macrophages. M2 macrophages were increased rather than M1 cells. In a 4T1 orthotopic model, co-injection of M2 macrophages with 4T1 mammary tumor cells increased tumor growth and metastasis. Interactions between 4T1 cells and M2 macrophages increased the proliferation of the tumor cells and infiltration of circulating leukocytes into tumor tissues. These interactions also induced HIF-1 alpha accumulation in the nucleus of tumor cells and the expression of VEGFs, resulting in increased tumor angiogenesis and lymphangiogenesis. In vitro results revealed that the migration of monocytes and the secretion of several cytokines and chemokines were increased when 4T1 cells were co-cultured with M2 macrophages. The blockade of MCP-1 or M-CSF expression in 4T1 cells by infecting shRNA markedly inhibited the infiltration of macrophages and their differentiation to M2 cells as well as tumor growth, metastasis, tumor angiogenesis and lung metastasis. Taken together, our results indicate that HFD feeding induces inflammatory changes in tumor tissues and target organs, which facilitates tumor angiogenesis and lymph angiogenesis, solid tumor growth and metastasis.

－69－ Profile of Speaker

Dr. Jung Han Yoon Park received her PhD in Nutrition from the University of Minnesota in St Paul, USA in 1982. After finishing her postdoctoral training at the University of Georgia, she started her research and academic career as an instructor at the University of Nebraska Medical Center in Omaha, Nebraska. In 1989 she accepted a faculty position at Creighton University in Omaha and was promoted to the rank of associate professor. Since 1994 she has been a professor at Department of Food Science and Nutrition in Hallym University, Korea. Dr. Park is the winners of many prestigious awards, including being selected as one of 12 women of achievement in Korea. She was elected as a regular member of the Korean Academy of Science and Technology, which is regarded as a highest honor for Korean Scientists. She was also awarded the first Mokwoon Life Science Award in 2010, which is given to the best researcher in the life science field. Dr. Park's research at Hallym University focuses on the role of dietary fat and bioactive components in the regulation of inflammation and the proliferation, apoptosis and metastatic potential of various cancers. She has published more than 140 research papers in several renowned SCI international journals in the areas of bioactive food components and cancer prevention (with more than 2300 citation numbers, H-index = 27). She has given more than 85 invited talks in both domestic and international workshops and conferences to professional audiences so far. She was the editor-in-chief of Journal of Medicinal Food and also serving on the editorial boards of the British Journal of Nutrition and Nutrition Research.

－70－ L 23 The use of herbal medicine as therapeutics in dementia

Dennis Chang

Centre for Complementary Medicine Research School of Science and Health, University of Western Sydney Sydney, New South Wales, Australia

Dementia is a progressive intellectual and functional impairment that affects memory and learning ability, activities of daily living, and quality of life. Dementia is a leading cause of mental and physical disability in the elderly and carries the highest disability weighting of all illnesses. Alzheimer’s disease (AD) is the most common form of dementia followed by vascular dementia (VaD) accounting for 80-85% and 15-20% of all dementia cases respectively. The pathogenesis of AD is heterogeneous and may include extracellular amyloid accumulation in the brain, formation of neurofibrillary tangles inside neurons, decreased central nervous system cholinergic function, genetic mutations (e.g., amyloid-precursor-protein gene), allelic variant of apolipoprotein-E (APOE), and lipid abnormalities. Several classes of pharmaceutical agents are currently used for the management of AD and VaD, among which cholinesterase inhibitors and glutamate receptor antagonists are suggested to produce best clinical outcomes. However, these improved clinical outcomes are often limited to modest symptomatic relief, meaning that a large proportion of the disease burden lingers. The use of complementary medicine has undergone a rapid expansion globally in recent decades. Herbal medicine, an important modality of complementary medicine has been used for the management of dementia-like symptoms over thousands of years. However the safety and the long-term therapeutic benefits of these interventions remain largely unvalidated. In this presentation, an overview of the existing evidence for the use of herbal medicine for dementia will be provided. As a case study, a series of pharmacological and clinical studies of a standard herbal formulation for the treatment of VaD will also be discussed. Combination therapies using multiple herbs are common in Chinese medicine. These complex chemical mixtures are believed to be able to enhance therapeutic efficacy through synergistic and multi- target mechanisms and are ideal for disorders such as AD and VaD that has multifactorial/ multisystem pathophysiological components. Based on this theory, our collaborative research teams in Xiyuan Hospital, China Academy of Chinese Medical Sciences and the Centre for Complementary Medicine Research, University of Western Sydney have employed conventional pharmaceutical techniques to develop a novel, standardised herbal formulation targeting VaD. Data from the preclinical studies have shown significant improvements in memory functions and in pathogenic biochemical parameters in various animal dementia models. Appropriate safety of the formulation has been shown in the acute and chronic toxicity and herb-drug interactions studies. A pilot clinical trial of a 4-month treatment has demonstrated a significant improvement in cognitive functions in patients with VaD.

－71－ Profile of Speaker

Dennis Chang is Associate Professor in Pharmacology and Director of Research in the School of Science and Health, University of Western Sydney (UWS), Australia. A/Prof Chang has established a strong track record in complementary medicine and anaesthesiology research. His recent research interests focus on clinical and pharmacological studies of herbal medicine for the management of cardiovascular (e.g. metabolic syndrome) and neurodegenerative (e.g. vascular dementia) diseases. A/Prof Chang has made significant contributions to the strategic planning and development of the Centre for Complementary Medicine Research, a designated University Centre of UWS.

－72－ Session5

L 24 Tocotrienols: from antioxidants to receptor ligands

Fabio Virgili, Ph.D.

National Research Institute for Food and Nutrition (INRAN), Rome, Italy

Nutrition is evidently much more than “fuel the engine”. Practically, all cellular processes in the flow of genetic information from gene expression to protein synthesis and degradation, have been demonstrated to be affected by lifestyle and in particular by the diet. The recognition that specific nutrients and bioactive food components can regulate the expression of specific genes, both at the transcriptional and at the post-transcriptional level, has opened the path to investigate the mechanisms of diet-dependent regulation of gene expression (molecular nutrition). Nutrigenomics, defined as the study of the interactions between dietary components and the genome, is challenging "traditional nutrition" to accept the limitations of the “classical” approach and learn how to take into account the potential of molecular nutrition research. As a paradigm of the application of “omics” approaches to nutritional sciences the case of Vitamin E will be reported. The term Vitamin E is frequently used to indicate 8 different vitamers, namely: α, β, γ and δ forms of tocopherol (TOC) and tocotrienol (TT). This term and the related activity was originally based on the capacity of countering foetal re-absorption in deficient rodents. In humans, Vitamin E activity was considered to be only related to the antioxidant properties of the tocolic chemical structure. In the last years, several reports have shown that TTs, but not TOCs, specifically inhibit proliferation and induce apoptosis in a large number of cancer cells, independently on the putative antioxidant properties. However, the molecular mechanism(s) involved in TTs action is still unclear. On the basis of a transcriptomic platform, we have recently demonstrated a novel mechanism for TTs activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicate a high affinity of specific forms of TTs for ERα , but not for ERβ. These data have been confirmed in human breast cultured cancer cells and in animals. Further experiments conducted on cell lines not expressing ERs showed that TTs induce apoptosis mediated by caspase 3 and independent of p53. The utilization of “high throughput” approaches indicated that an alternative pathway to cell death induced by TTs and shared by all cell lines exists, dependent on reticulum stress. Our studies confirm the potential of nutrigenomics approaches, indicating that specific forms of TTs have a distinct biological activity, significantly different from TOCs. We also propose that TTs should not be pooled together TOCs within the broad term “Vitamin E” on the solely base of their putative antioxidant properties.

－73－ Profile of Speaker

Fabio Virgili graduated in Biology from the University of Rome with first class honours, and completed his Ph.D. on “Cellular and tissue pathology” at the University of Modena and Reggio Emilia, Italy. He was visiting scientist at the Rowett Research Institute of Aberdeen, Scotland, at the Department of Pathology of the University of Modena, Italy and at the Department of Molecular and Cellular Biology of the University of California, (Berkeley, USA) in the laboratory directed by Prof. L. Packer. Fabio Virgili is Senior scientist, Director of the “Bioactive food components and Health” Unit and Head of “Nutritional Sciences” Department at the National Research Institute for Food and Nutrition (INRAN) in Rome, Italy. He was appointed Professor of Biochemistry at the University of Roma-Tre, in Rome from 2000 to 2010 and prtecipates to the board of teachers of different Master and Ph.D. courses. His research is focusing on the role of molecules of nutritional interest in gene expression and transcription factors activation. In particular, he is interested on the nutritional modulation of cellular response to different stimuli in the early stages of degenerative pathologies. His research is addressing the effect of molecules of nutritional interest and specific food items (wine, palm oil, herbal extracts) on the response of cultured cells and living animals to pro- pathogenic stimuli assessing the differential gene expression at transcriptomic and proteomic level and more recently on the effects of genetic variants on the risk of pathologies related to nutrition.

Fabio Virgili is editor in chief of the Journal “Genes and Nutrition” (http://www.springer. com/biomed/human+genetics/journal/12263)

－74－ L 25 Transcriptional biomarkers of aging and calorie restriction and modulation by dietary interventions

Jamie L. Barger, Ph.D.

LifeGen Technologies, LLC Wisconsin, U.S.A.

Studies using whole-genome transcriptional profiling have identified thousands of genes that are changed in expression with age and a calorie restricted (CR) diet. However, most of these age-related changes are not universal, but instead are specific to the genetic background of the organism being studied. Thus, there is great interest in identifying robust biomarkers of age and CR across multiple experimental models that are applicable to human aging.

We used gene expression profiling to identify transcripts that were consistently changed in expression with age or short-term CR. For aging studies, we compared gene expression patterns in old mice (28-30 months old) relative to young mice (5 months old); for short-term CR studies, we compared gene expression profiles of 5 month old mice that were either fed a normal diet or subjected to CR from 2-5 months of age. The effect of aging and short-term CR was measured in seven strains of mice and in four tissues (brain, heart, skeletal muscle and white adipose tissue). Following RTPCR confirmation of the microarray data, 10-15 genes were identified as robust transcriptional biomarkers (depending on the tissue). There was minimal overlap among tissues, suggesting that the effects of aging and CR at the individual gene level is tissue-specific. These panels of transcriptional biomarkers are being used as screening tools to determine the extent to which drugs and natural compounds can (1) oppose the aging process and/or (2) mimic the effect of a CR diet.

－75－ Profile of Speaker Jamie L. Barger, Ph.D. Chief Operating Officer, LifeGen Technologies, LLC

http://www.linkedin.com/in/jamiebarger

Dr. Barger received his Ph.D. in 2002 from the University of Alaska Fairbanks where he studied the molecular and physiological mechanisms regulating metabolism during hibernation. To expand his knowledge of how gene expression regulates whole-body metabolism, Dr. Barger applied for and received a Postdoctoral Fellowship from the National Institute of Health (NIH). This work was focused on understanding how adipose tissue gene expression influences the ability of a calorie restricted diet to slow the aging process. This work was conducted at the University of Wisconsin Madison with Dr. Richard Weindruch. In 2005, Dr. Barger joined LifeGen Technologies as a Scientist where he was responsible for analysis of gene expression and nutrigenomic data from a portfolio of international clients. Dr. Barger is currently the Chief Operating Officer at LifeGen Technologies , LLC in Madison, Wisconsin.

－76－ L 26 Nutrient-genome interactions and prevention of cognitive impairment

Michael Fenech

CSIRO Preventative Health Flagship, Australia. Email: [email protected]

There are various aspects of cognitive dysfunction (e.g. language, processing speed, eye- hand coordination, executive functioning, verbal memory and learning, visual memory and learning, visuoconstruction) which may result from neural deficits in specific brain regions (e.g. hippocampus, cerebellum, insula, thalamus) either due to inherited genetic defects, acquired genetic defects during foetal development or as a result of accelerated ageing. Such effects may also be mediated by accumulation and aggregation of toxic peptides such as α-synuclein or Aβ42 in the case of Parkinson’s and Alzheimer’s disease respectively. The propensity of accumulating genetic defects that impact the regenerative potential of neural cells (e.g. telomere shortening, DNA oxidation) and their mitochondrial function (e.g. mitochondrial DNA deletions, point mutations) is often affected by deficiency in nutrients required for genome maintenance (e.g. folate, vitamin B12) or excessive intake of minerals (e.g. iron, copper, manganese overload) that could exacerbate homeostatic imbalance in redox pathways. Furthermore the impacts of these dietary factors may depend on common polymorphisms in genes involved in the uptake and metabolism of micronutrients required for genome maintenance or appropriate processing of neurotoxic peptides. The presentation will provide an update on the most compelling examples of gene-nutrient interaction relevant to cognitive function and Alzheimer’s disease.

－77－ Profile of Speaker Name: Michael Fenech – Brief Biography (2012)

Title/Name: Professor / Michael Fenech

Affiliation: CSIRO Food and Nutritional Sciences

Email: [email protected]

Profile: Professor Michael Fenech is recognised internationally for his research in nutritional genomics and genetic toxicology and for developing the cytokinesis-block micronucleus (CBMN) assay which is now a gold standard method used internationally to measure DNA damage in human cells in vitro and in vivo. The CBMN assay has been endorsed by the International Atomic Energy Agency and the OECD for in vivo radiation biodosimetry and in vitro testing of genotoxins respectively. His key goal is to determine the nutritional and environmental requirements for DNA damage prevention using in vitro systems, epidemiology and placebo-controlled human intervention trials. In 2003-05, Dr Fenech proposed a novel disease prevention strategy based on personalised diagnosis and prevention of DNA damage by appropriate diet/life-style intervention, which has led to the Genome Health Clinic concept and its translation into practice (www.reach100.com.au). In 2003-2009 his laboratory further developed the CBMN assay into a 'cytome' assay consisting of six complementary biomarkers of DNA damage and cytotoxicity which is now published in Nature Protocols. He co-founded the ongoing HUMN and HUMN-XL projects on micronuclei in human populations (www. humn.org) and is a member of the coordinating group for the Micronutrients Genomics Project (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2989004/) which he has been leading since July 2011. His research is currently also focussed on the impact of nutrition on telomere length and function; he conceived and developed, together with other scientists in his group, a novel quantitative PCR method for measuring absolute telomere length. He was awarded the Flinders University’s Convocation Medal in 2007, the Alexander Hollaender Award (USA) in 2008 and the honorary titles of Adjunct Professor (University of South Australia) in 2009 and Visiting Professor (Taipei Medical University, Taiwan) in 2010 and Professorial Fellow (Flinders University) in 2011 for his leadership/contributions to environmental/public health sciences internationally. His 210 publications have been cited 9,000 times and his H-index is 48.

－78－ Session6

PL 27 The role of vitamin E in the brain, from adults to zebrafish

Maret G. Traber, Ph.D.

Professor, School of Biological & Population Health Sciences; Helen P. Rumbel Professor for Micronutrient Research, Linus Pauling Institute, Oregon State University; Corvallis, Oregon, USA

Vitamin E (α-tocopherol) was discovered almost 100 y ago, but its functions in the brain are still not understood. I will describe three different approaches my laboratory is taking to assess α-tocopherol’s roles in brain. The relationship between diet and brain have been evaluated in the Oregon Brain Aging Study cohort at Oregon Health Sciences University by using nutrient biomarker patterns as they relate to cognitive behavior and brain size measured using nuclear magnetic resonance imaging (MRI). Thirty plasma nutrition biomarkers were measured in the subjects (87±10 y, 62% female). Multivariate analysis constructed the nutrient biomarker patterns; regression models assessed the relationship of biomarker patterns with brain outcomes. Superior cognitive function and less brain shrinkage were associated with plasma patterns of high vitamins B (thiamin, riboflavin, niacin, and pyrodoxal 5-phoshate, folate, vitamin B12), C, D, and E or marine n-3 fatty acids. In contrast, a high trans-fat pattern was associated with worse cognitive behavior and less total brain volume. Thus, distinct nutrient patterns account for a significant degree of variance in both cognitive function and brain volume. α-Tocopherol was one of the key nutrients associated with better brain protection and function. Another key nutrient that is present in high concentrations in the brain is vitamin K, as menaquinone-4 (MK-4). Our model to study vitamin E metabolism in rats uses vitamin E injections (100 mg/kg body weight) to increase α-tocopherol metabolism. Importantly, we found that vitamin E injections decreased the high brain MK-4 concentrations by half, whether the vitamin was provided as dietary phylloquinone or menadione. Vitamin E intoxication altered, and perhaps impaired, the synthesis of MK-4 from the dietary forms, and thus altered regulation of brain vitamin K status. The hepatic α-tocopherol transfer protein (TTP) is required for optimal α-tocopherol bioavailability in humans and is expressed in human brain; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). α-Tocopherol is essential to prevent fetal resorption in rodents, but its precise physiological role in embryogenesis is unknown. We hypothesized that TTP is required to regulate delivery of α-tocopherol to critical target sites in the developing zebrafish embryo. TTP mRNA transcription increased >7-fold in embryos during the first 36 hours post-fertilization (hpf);

－79－ Ttpa transcripts localized to the developing brain, eyes and tail bud by 24 hpf. Severe head and eye malformations resulted from inhibiting TTP expression by injection of oligonucleotide morpholinos into one-cell stage embryos. Thus, TTP has a critical role in brain development, likely for delivery of α-tocopherol during a period corresponding to the first month of human pregnancy. Our studies have implicated α-tocopherol as a key nutrient for brain development and function, as well as maintaining brain size. Studies are ongoing to define the mechanisms for these actions.

Profile of Speaker

Dr. Maret Traber is Professor in the School of Biological & Population Health Sciences, College of Public Health and Human Sciences; Director of the Oxidative and Nitrosative Stress Core and the Helen P. Rumbel Professor for Micronutrient Research in the Linus Pauling Institute at Oregon State University (OSU), Corvallis, Oregon, USA. She received both undergraduate and doctoral degrees in Nutrition Science from the University of California (UC) at Berkeley. She worked with Dr. Herbert J. Kayden at New York University School of Medicine, New York, NY to pioneer the use of stable-isotope labeled vitamin E in humans to assess mechanisms of vitamin E transport and chiral discrimination. She returned to UC Berkeley in 1994 to collaborate with Dr. Lester Packer in studies of various antioxidants in models of oxidative stress. In 1998, she joined the LPI and the OSU faculty. With nearly 275 scientific publications, Dr. Traber is considered one of the world’s leading experts on vitamin E. Her research efforts are focused on human vitamin E kinetics and the factors that modulate human vitamin E requirements, especially bioavailability and metabolism. Dr. Traber is the President of the Oxygen Club of California (http://www.oxyclubcalifornia.org); she currently serves on the editorial board of Free Radical Biology & Medicine. In 2000, Dr. Traber served on the National Academy of Science’s, Institute of Medicine Panel on Dietary Antioxidants and Related Compounds that established the human dietary requirements for the antioxidant vitamins C and E, selenium and carotenoids.

－80－ L 29 Antioxidant action and multifunctionality of dietary carotenoids in humans

Teruo Miyazawa

Food and Biodynamic Chemistry Laboratory, Tohoku University, Sendai 981-7555, Japan

Oxidative stress is an important origin to the risk of chronic diseases. Dietary guidelines propose augmented consumption of fruits and vegetables to prevent the incidence of human diseases such as cancer, cardiovascular dysfunction, diabetes and age-related disease. Carotenoids are a family of antioxidant phytochemicals that mitigate the damage of oxidative stress. Red blood cells of Alzheimer’s disease (AD) patients are known to be in an excessively oxidative stress with a high accumulation of phospholipid hydroperoxides (PLOOH)). The RBC with high levels of lipid hydroperoxides may have a decreased ability to transport oxygen to the brain, which may impair blood rheology, thus facilitating dementia. We confirmed in vitro, in vivo murine, and in human studies that carotenoids can effectively inhibit the accumulation of PLOOH in the red blood cells. For the relationship between red blood cells carotenoids and PLOOH concentrations in AD patients, red blood cells carotenoids and PLOOH were evaluated in 28 normal control subjects (age: 74.1±1.3 years) and 28 patients with AD (age: 72.5±1.4 years). The concentrations of carotenoids, especially lutein, in AD patients were significantly lower than in control subjects. An inverse relationship was found between red blood cells carotenoids, especially lutein, and PLOOH concentrations in AD patients. These results suggest that lutein, in particular, contributes to suppress the PLOOH accumulation and lipid peroxidation in red blood cells of AD patients. Next, we conducted a randomized, double-blind, placebo-controlled human trial to assess the efficacy of 12 wk astaxanthin supplementation (6 or 12 mg/day) on both astaxanthin and PLOOH levels in RBC of 30 middle-aged and senior subjects. After 12 wk of treatment, RBC astaxanthin concentrations were higher in both the 6 and 12 mg astaxanthin groups than in the placebo group. In contrast, red blood cells PLOOH concentrations were lower in the astaxanthin groups than in the placebo group. In plasma, somewhat lower PLOOH levels were found after astaxanthin treatment. These results suggest that carotenoids especially xanthophylls such as lutein and astaxanthin supplementation results in improved red blood cells antioxidant status and decreased PLOOH levels, which may contribute to the prevention of dementia.

－81－ REFERENCES Takehiko Kiko, Kiyotaka Nakagawa, Tsuyoshi Tsuduki, Hiroyuki Arai, Teruo Miyazawa: Significance of lutein in red blood cells of Alzheimer’s disease patients. J. Alzheimer’s Disease, 28,593-600 (2012) Kiyotaka Nakagawa, Takehiko Kiko, T. Miyazawa:Antioxidant effect of lutein towards phospholipid hydroperoxidation in human erythrocytes. British J. Nutrition, 102:1280-1284(2009) Daigo Ibusuki, Kiyotaka Nakagawa, Akira Asai, Shinichi Oikawa, Yuichi Masuda, Toshihide Suzuki, T. Miyazawa: Preparation of pure lipid hydroperoxides. J. Lipid Res., 49, 2668-77 (2008)

Profile of Speaker

He received his Ph.D. in Lipid Chemistry from Tohoku University in 1982 where he developed a chemiluminescence detection-high performance liquid chromatography method for measuring lipid hydroperoxides in food and biological systems. After that, he carried out his research as a visiting scientist in the USDA Human Nutrition Research Center on Aging at Tufts University, Boston, to study oxidative challenges on the human brain in Alzheimer disease during his stay in 1995-1996. In 1998 he was appointed a professorship at the Graduate School of Agricultural Science (GSAS), Tohoku University, and is author and co-author of over 480 scientific papers, book chapters, books, and patents on the health functionalities of food lipids, lipid oxidations, vitamins, antioxidants and biofactors. He received Young Scientist Award of The Japan Bioscience, Biotechnology, and Agrochemistry Society at 1988； Excellent Thesis Award of The Japan Oil Industry Association 1990； The Japan Oil Chemists' Society Award 2000； Asahi Beer Life Science Award 2003； and JSNFS (Japan Society of Nutrition and Food Science) Excellent Research Award 2009. In 2010, he received the Ando Momofuku Prize and the Iijima Food Science Prize. Dr. Miyazawa has served as a Council member of Tohoku University. He serves now as President of JSNFS; President of the Japan Maillard Reaction Society; President-Elect of the International Maillard Reaction Society; President of the Japan Society on Lipid Peroxide Biology and Medicine; Director of the Vitamin Society of Japan; Director of the Japanese Society for Food Science and Technology; Executive council member of the Federation of Asian Nutrition Societies (FANS); Chair of the Organizing Committee of 12th Asian Congress of Nutrition 2015 Yokohama (Japan); and Member of the Science Council of Japan.

－82－ L 30 Early ADME properties for discovery of human in vivo probe

Ling Yang

Laboratory of Pharmaceutical Resource Discovery Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, China

The Pharmacokinetics (PK) is dedicated to the determination of the fate of substances administered externally to a living organism. Its purpose is to reveal the relationship of the processes of absorption, distribution, metabolism, and excretion (ADME) to the intensity and time course of pharmacological (therapeutic and toxicologic) effects of drugs and compounds. PK study is the most important method to determine the drug-like properties of a compound until now. Drug-like properties of a compound depend on both drug specific properties (DSP) and biological system specific properties (BSSP). Unfortunately, traditional PK study mainly focuses on the time course of blood/tissue concentrations of compounds, and both DSP and BSSP are generally neglected. However, both factors are the dominant factors resulting in the alteration of PK behavior in species or individuals of a certain compound. Mechanism- based PK, rather than the “black-box method” of traditional PK, can clarify the roles of these factors. Early ADME study (eADME) is to elucidate DSP and BSSP as well as their relation with compounds during the early stage of drug discovery and development. Physiologically based pharmacokinetic study (PBPK) based on early ADME can integrate the physiochemical, physiological and ADME properties into a system, and then accurately evaluate the ADME properties of compounds in individual human body. To obtain the physiological, biochemical and other key parameters as well as the action mode of human body on compounds, an in vivo probe is urgently needed. Among all these parameters, metabolic parameters exhibit the highest degree of inter- individual variability and species variability. As the most important PK determinants, CYP3A is responsible for the metabolic clearance majority of marketed drugs with different structure. The monitoring of CYP3A activity will be helpful for clarifying the individual responses of CYP3A substrates. The abundant sources of food and naturally products offer rich pickings for modern drug development. We have found an ideal in vivo probe of CYP3A from lignans in a traditional Chinese Medicine, Schisandra. The probe will be used to accurately quantify and predict the in vivo state of drug metabolized by CYP3A, to guide the rational use of drugs and traditional Chinese Medicine.

－83－ Profile of Speaker

Dr. Ling Yang is currently a Professor of Dalian Institute of Chemical Physics, the Chinese Academy of Sciences. He is also serving as Visiting Professor of Nankai University, Dalian Medical University, Inner Mongolia Medical College, and Liaoning Medical University. Dr. Ling Yang graduated from Zunyi Medical College in 1983. In 1990, he, awarded by the Japanese Government Scholarship, entered Graduate School, Toyama Medical and Pharmaceutical University. He obtained Ph. D (of Pharmaceutical Sciences) in 1996. In 2000, he was selected as one of the Outstanding Overseas Chinese Scientists (namely, One Hundred Talent Program) from the Chinese Academy of Sciences, a CAS initiative to recruit competitive professional, and joined DICP. Now Dr. Yang is serving as the editor board member for several international journals, including Xenobiotica, Pharmaceutical Biotechnology, Journal of Ginseng Research, International Journal of health and other famous journals. Dr. Yang has published more than 110 peer-reviewed papers and two books. He has filed more than 40 patents to date. His research interest is mainly focused on the research of early ADME/T properties of drugs and the industrial biotechnology with regard to drug R&D. Today’s talk will be a part of his work on early ADME.

－84－ Session7

L 31 Inflammation regulatory activities of fruit polyphenolics - opportunities for functional foods

Roger D. Hurst

Food and Wellness Group, The New Zealand Institute for Plant & Food Research Ltd., New Zealand

Fruits are rich sources of coloured polyphenolic compounds which are regarded as beneficial to human health and wellness. Some polyphenolic compounds possess strong antioxidant properties and it has been thought that their ability to assist in the prevention of common diseases and illnesses is via this activity. However, data from large-scale human intervention and meta-analysis studies question the relevance of the antioxidant ability of pigmented polyphenolic compounds for health. More recent studies are suggesting that polyphenolic compounds also possess inflammation-modulatory and immune-modulatory properties - the significance of which may be of even greater biological relevance for health promotion and disease mitigation.

At The New Zealand Institute for Plant & Food Research Ltd (PFR), we evaluate the targeted health benefits of fruit and vegetable polyphenolics: hence we have an interest in fruit polyphenolic inflammation modulatory activity. Our research investigates the health- promoting properties of fruit and vegetable varieties produced by New Zealand industries and from our breeding programs – we utilize well-defined compositional data and a range of cell-based screening and animal/human intervention trials. Our ultimate goal is the provision of appropriate information to enable plant breeders to select for scientifically proven health- promoting fruits and vegetables as whole fresh functional food varieties and/or functional food ingredients.

We have a research program evaluating airway inflammation as a model system to investigate the effects of fruit polyphenolic compounds on key inflammation responses. Observational studies have revealed a positive correlation between fruit nutrient intake and asthma, lung function or lung disease. Indeed, a reduction in antioxidants (particularly polyphenolic compounds) in the diet leading to an increased vulnerability of the pulmonary airways to inflammation has been proposed as a hypothesis to explain the rising prevalence of inflammatory lung conditions such as asthma. Furthermore, there is building evidence from cell studies, animal and human intervention trials to indicate benefits to lung health from fruit polyphenolics. We will present an overview of our research evaluating inflammation regulation. We will show our findings for how fruits high in polyphenolic compounds (particularly berryfruit) can modulate inflammation with a focus upon airway inflammation and an eye on future functional food opportunities.

－85－ Profile of Speaker

Dr Roger Hurst is the Science Group Leader for the Food & Wellness Group, Food Innovation Portfolio of The New Zealand Institute for Plant & Food Research Limited (Plant and Food Research). In 1989, he received his PhD doctorate in biochemistry from the UK and has experience in the biomedical health area through an academic career undertaking research at the University of Toronto, Canada ; the Babraham Research Institute, Cambridge, UK ; the Institute of Neurology, London, UK ; and the University of the West of England, Bristol, UK. Since joining Plant and Food Research in 2007 in has developed an interest in fruit compounds and their role in controlling oxidative stress, reducing inflammation, assisting the immune systems, and in aiding muscle recovery after strenuous exercise. Information from this research has supported the development of new food products and will be used in future developments of elite fruit cultivars and leading edge foods. Dr Hurst has published over 60 peer-reviewed science manuscripts and leads 2 large government and industry funded research programmes involving the evaluation of various commercial and new fruit cultivars created by Plant and Food Research scientists for targeted human health and wellness benefits. Today’s talk will overview some of this work.

－86－ L 32 The impact of food processing on the nutritional value of fruits: polyphenols and their antioxidant activity

Emira Mehinagic

UPSP GRAPPE, GROUPE ESA, France

Apples represent a major proportion of the fruit supply throughout the year in most European countries and The USA because of various factors: availability in the market, diversity of cultivars and variety of conditionings (fresh fruit, juice, purees). Many of the phytonutrients, such as polyphenols, found in apples are strong antioxidants. It has been estimated that apples could provide 20–25% of the per capita consumption of fruit polyphenols in the United States (1). The impact of these compounds on human health has been studied in recent years and they were recognised for their preventative effect on cardiovascular disease and some cancers (2, 3). The major classes of polyphenols in apples are flavonoids above which flavonols, flavanols, dihydrochalcones and anthocyanins, principally concentrated in fruit peel and seeds, whereas the major hydroxycinnamic acid (chlorogenic acid) concentrated in fruit flesh. Different classes of phenolic compounds are unevenly distributed within the apple fruit and demonstrate non-uniform behaviour during fruit development and in response to different factors such as fruit cultivar, origin, argonomic conditions, storage conditions, etc. Processing can also greatly affect apple phytochemicals. Thus the raw apple juice obtained by pulping and straight pressing or after pulp enzyming had an antioxidant activity that was only 10 and 3%, respectively, of the activity of the fresh apples (4). The levels of flavonoids and chlorogenic acid in the juice were reduced to between 50% (chlorogenic acid) and 3% (catechins). Most of the antioxidants were retained in the pomace rather than being transferred into the juice. The results indicate that processing can have a major impact on the partitioning of the polyphenolic content (4). The antioxidant activities of fruit juices have been estimated by measuring antioxidant status in human plasma in ten healthy men 25-26 years old (5). Within 30 minutes after consumption, apple juice has been shown to suppress reactive oxygen species generation in human plasma. This radical scavenging effect of fruit juices was maintained for up to 90 minutes post-consumption (5). Our latest research was oriented on preservation of native polyphenols and their antioxidant capacities in processed apple juices and apple-sauces. The influence of different processing operations on the antioxidant potential of native polyphenols in these products will be presented. In conclusion, future trends related to the multidisciplinary approach of health related effects of polyphenols in fruits will be underlined.

－87－ Cited references: 1. Vinson, J. A et al. Phenol antioxidant quantity and quality in foods: fruits. J. Agric. Food Chem. 2001, 49, 5315-5321 2. Boyer, J. et Liu, R.H.; Apple phytochemicals and their health benefits. Nutr. J. 2004, 3, 5. 3. Scalbert, A. et al. Dietary polypolyphenols and the prevention of deseases. Critical Rew. Food Sci. Nutr., 2005, 45 (4), 287-306. 4. Van der Sluis, A. A. et al. Activity and concentration of polyphenolic antioxidants in apple juice. 1. Effect of existing production methods. J. Agric. Food Chem., 2002; 50, 7211-7219. 5. Ko SH et al. Comparison of the antioxidant activities of nine different fruits in human plasma. JMed Food 2005; 8: 41–6.

Profile of Speaker

Dr. Emira Mehinagic is a research director in Groupe ESA, the institute of higher education for life sciences, in Angers (France). In 2004, she earned her PhD degree in Food science and technologies from University of Nantes under the guidance of Dr. Michel Demaimay. In 2004, she has joined Groupe ESA as an assistant professor. Since then, she has been interested in food processing and the preservation of phytonutrients in fruits during processing. In 2009 she was nominated director of the research unit GRAPPE. Thanks to this multidisciplinary combination, this unit developed an original research based on multiparametric approach of food quality and typicality in two specific models: apples and grapes. The mission of the team consists in providing to the professionals reliable indicators of changes in the quality of their products and tools to measure them. Quality is analysed from the sensory aspects, but also nutritional and technological points.

－88－ L 33 FoodValley and Wageningen UR: healthy and sustainable food innovation in the Netherlands

Christiaan Kalk

Food and Biobased Research Wageningen University and Research Centre, The Netherlands

Despite their small size, The Netherlands are second only to the United States as export nation of agri-food products. Part of this success is explained by a long tradition in international trade, as well as in logistics and transport. Another part is explained by an evolving, fine- maze research and development infrastructure, supported by diverse governmental, industrial, academic and contract research organizations (CRO’s) that work together in various ways. While FoodValley and Wageningen UR have become denominators for successful agri-food research and development in The Netherlands, the reality is not that monolithic. True, it started off with the foundation of an agricultural university in Wageningen in 1918. True, FoodValley is an effective binding factor and is successful in attracting industries, start-up companies and research centers to the greater Wageningen and Central/East Netherlands area. Yet, another part of the success is explained by the cooperation between governmental, industrial, academic and CRO partners on a pre-competitive basis in public-private partnership organizations like the Innovation Programme Food and Nutrition (which includes the Top Institute Food & Nutrition as well as Food and Nutrition Delta) and the recently formed Top Sectors Agri&Food and Tuinbouw (horticulture). Furthermore, many assignments are placed by industry in academic and CRO environments on a bilateral and competitive basis. Diversity, stimulation by governments, exchange of ideas and freedom of choice: all these factors contribute to innovation for health and sustainability in the Dutch agri-food sector. For developing a food R&D and technology cluster in Niigata, one should consider proven success factors like: strong traditions and simultaneous application of quick-win and long-term strategies towards uniqueness. One should start well, but also take time to let a fine-maze and diverse network evolve. Tentatively, areas like eukaryotic fermentation, or optimization of storage and distribution in (fresh) food chains seem to be worth further exploration. Interest of Japanese food companies to export to EU countries seems to be on the decline. This is a pity, as the Japanese diet has many healthy aspects, and as many highly relevant FOSHU and other functional foods have been developed in Japan. The EU Novel Food Regulation is often seen as a barrier by industry. Still, over 70% of Novel Food applications have proven successful. Also, some products may not be novel, while for other products a notification instead of a registration may suffice. Finally, the number of novel products that are admitted by acclamation of the Member States’ Competent Authorities is rising, making

－89－ evaluation by EFSA and approval by the European Commission is unnecessary. Regarding Health Claims, it is clear that any statement beyond the list that has now been accepted by EFSA and the European Commission will undergo strong scrutiny. But if a product is sufficiently characterized, if the relevance of the health effect is accepted within the context of the Regulation and if a cause and effect relationship can be significantly demonstrated in a relevant population, a claim can be made. Points to consider for human intervention studies in support of health claims will be given. Biomarkers for mild nutritional health effects that become manifest only after many years are often still lacking, as is a mechanistic explanation of observed effects. Initiatives are taken to improve both our methods and our knowledge. In order to demonstrate improvements, we may have to shift from single markers to patterns, applying –omics and systems biology. We may have to select responsive target populations, or create our own responsive group by applying a challenge and mildly disturbing homeostasis. As an example in the demanding area of dietary fibers and immunity, the newly started EU 7th Framework project FibeBiotics will be introduced. Dealing with the EU Regulation in a pro- active manner is likely to be more effective than waiting for higher standards for health claims to be set in non-EU countries as well.

Profile of Speaker

Christiaan Kalk, MSc is Business Development Manager, Healthy Foods and Senior Consultant at Wageningen University and Research Centre, Food and Biobased Research (WUR-FBR). In 1987, he graduated in Biology (Microbiology, Biochemistry, Environmental Law, Toxicology) at Groningen University under the guidance of Prof. Wim Harder. After a commercial career with New Brunswick Scientific Benelux BV (Nijmegen) he joined The Netherlands Organization for Applied Scientific Research (TNO) as Marketing Manager, Toxicology in 1990. In 1998, he became a part-time consultant in regulatory affairs, test strategy as well as nutritional and toxicological assessment. In 2002, he was appointed Business Development Manager and Senior Consultant, Food and Health at TNO. He has been involved in the development and registration of various food enzymes, additives, novel foods, food packaging materials, animal feed ingredients and additives, biocides, pesticides, industrial chemicals, household and personal care products, as well as (bio)pharmaceuticals. Also, he has contributed to the development of various health claims under the new EU Regulation. By joining WUR- FBR in 2012 he is able to combine the regulatory, safety and health fields with consumer sciences, (sustainable) food technology, (fresh) food chain management and distribution.

－90－ L 34 Concentrations of radionuclides in agricultural crops in japan: before and after the fukushima daiichi nuclear power plant accident

Keiko Tagami

Office of Biospheric Assessment for Waste Disposal National Institute of Radiological Sciences, Chiba, Japan

The magnitude-9.0 (Richter scale) earthquake on March 11, 2011 triggered a huge tsunami, and then it destroyed the water circulation systems of the Fukushima Daiichi Nuclear Power Plants (FDNPP). The FDNPP released volatile radionuclides to the environments next few days. The major released radionuclides were 132Te (half life = 77 h) and 131Te (24.8 min) and their respective progeny 132I (2.26 h) and 131I (8.02 d), as well as 134Cs (2.06 y) and 137Cs (30 yr). Several days after, rain events began in the Tohoku and Kanto regions, including Fukushima Prefecture; high radiation doses were reported in these regions due to these radionuclides deposited on the land surfaces. Consequently, vegetation in these regions received direct deposition which caused surface contamination of crops. The guidance level used at that time for 131I was 2000 Bq/kg of food materials, and 500 Bq/kg for total radiocesium (134Cs+137Cs); many types of leafy vegetables in these regions exceeded these guidance levels. The period of food contamination by direct deposition has almost ended in April 2011. Agricultural fields were ploughed again, and crops were replanted; then the concentration levels of radiocesium in most agricultural products has became lower than the detection limit due to the low soil-to-plant transfer factors of Cs. However, perennial plants, e.g. fruit trees, tea trees, and edible wild plants, showed higher concentrations of radiocesium than the guidance level. The mechanism of radiocesium transfer to edible part of agricultural crops is though to be absorption of radiocesium from above ground parts of plants and translocation to newly emerged plant tissues. Beginning on 1 April 2012, new standard limits for radionuclides in food have been employed in Japan, and the total radiocesium concentration in marketed raw food materials has been limited up to 100 Bq/kg by the Ministry of Health, Labour and Welfare (MHLW). Concentrations of radiocesium in most agricultural crops except above mentioned perennial crops has been lower than the limit level. Perennial plants still contain some radiocesium in their bodies, but the concentrations are decreasing due to dilution effect (body mass increase). The MHLW estimated daily radiocesium intake from foods as lower than 3.4 Bq/person, which was almost the same level that we used to have in 1960’s from global fallout. Food processing is also effective to decrease radiocesium from raw food materials.

－91－ Profile of Speaker

Dr. Keiko Tagami is a senior researcher of National Institute of Radiological Sciences (NIRS). After she graduated from Tsukuba University, Faculty of Agriculture and Forest in 1991, she joined NIRS as a research scientist. She have been studied radiochemistry and radioecology from then. In 1997, she received a PhD degree in Agriculture from Kyoto University; the title of her thesis was “Behavior of Technetium in Paddy Fields”. Technetium-99, a fission product from uranium-235 and plutonium-239, is her target radionuclide, and also, using the similar methodology, she extended her research to the fate of rhenium. Then, for the last ten years, she has been studied behaviors of radionuclides and trace elements in agricultural systems in Japan, considering deep underground disposal of radioactive waste management and the radionuclide transfer from the disposal sites to the surface “biosphere”. Due to her experiences, the fate of radiocesium in the environment, a main contaminant from the TEPCO’s Fukushima Daiichi Nuclear Power Plant (FDNPP) accident now, is of her interest as well. Today she will talk about radioactivities in agricultural foods before and after the FDNPP accident.

－92－ Session8

PL 35 Flavonoids and the risk factors of metabolic syndrome

Cesar G. Fraga

Physical Chemistry-IBIMOL(UBA-CONICET) University of Buenos Aires Buenos Aires, Argentina

Flavonoid-rich beverages, foods, and extracts, as well as pure flavonoids are currently studied for the prevention and/or amelioration of diseases, including metabolic syndrome (MS) and MS-associated risk factors. Increasing evidence support the existence of significant effects of flavonoids on MS. Mechanistically, for long time these health effects were associated with direct antioxidant actions, i.e. free radical scavenging and metal chelating. Current research is making evident that such antioxidant actions cannot by of relevance in most tissues, and/ or following a “normal” consumption of flavonoids. More specific biological interactions, inclusive some indirect antioxidant effects, can be “biologically efficient” at the concentrations flavonoids present in different tissues. There are convincing experimental evidence linking flavonoid consumption with the risk factors defining MS, i.e. obesity, hypertriglyceridemia, hypercholesterolemia, hypertension, and insulin resistance. Nevertheless a number of molecular mechanisms have been identified; the effects of flavonoids modifying major endpoints of MS are still inconclusive. These difficulties can be explained by: i) the complex relationships among the risk factors defining MS; ii) the multiple biological pathways/targets controlling these risk factors; and iii) the high number of flavonoids (including their metabolites) present in the diet and potentially responsible for the in vivo effects. In order to narrow done these complex matrix, we have studied the effects of a flavanol, (-)-epicatechin in two animal models, i.e. fructose overload, and L-NAME supplementation. We observed that the administration of (-)-epicatechin was associated with improving several physiological parameters, e.g. body weight, blood pressure, and insulin resistance. In addition, mayor changes in the regulation of oxidative stress, nitric oxide metabolism, and inflammation, were observed when these animals were supplemented with (-)-epicatechin. In summary, by integrating our results with other present in the literature it is possible to speculate that the balance between nitric oxide and superoxide anion, and concomitantly the regulation of nitric oxide synthases and NADPH-oxidases seems to be a central condition behind the action of (-)-epicatechin. These possibilities are well documented in the vascular system; it is feasible that other tissues and systems can share similar mechanisms. In addition, basic and clinical research is still necessary to better understand the role of flavonoids in the prevention and/or amelioration of MS and MS-associated diseases, and from there designing dietary and/or pharmacological strategies. This work was supported with grants from the University of Buenos Aires (UBACyT 20020090100111) and from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET) (PIP 112-201101-00612).

－93－ Profile of Speaker

Professor Fraga received his doctoral degree in Biological Chemistry from the University of Buenos Aires, in 1985. He held post- doctoral positions at the Department of Food Science and Technology at the University of California, Davis (1986-1988) and later at the Department of Biochemistry and Molecular Biology at the University of California, Berkeley (1988-1990). In 1990 he was appointed Professor of Physical Chemistry at the School of Pharmacy and Biochemistry, University of Buenos Aires and Researcher at the National Council for Scientific Research Argentina (CONICET), positions that he holds today. From 2004 Prof. Fraga also has a position of Research Professor at the Department of Nutrition, University of California, Davis. His original research interests in free radical and antioxidants in biological systems have developed in his current research centered on the biochemical mechanisms behind the effects of plant constituents on disease conditions, especially hypertension and digestive pathologies. Dr. Fraga has published more than 120 articles and given talks in more than 80 scientific conferences. Currently he acts as director of the Program on Free Radicals in Biochemistry (PRALIB) from UBA-CONICET, Argentina; Executive Vice President of the Oxygen Club of California, Treasurer of the Society for Free Radical Research International, and Associate Editor of the Food and Function (RSC) among other responsibilities. He was the editor of the book “Plant phenolics and human health: biochemistry, nutrition and pharmacology”, first volume of The Wiley-IUBMB Series on Biochemistry and Molecular Biology published in 2010.

－94－ L 36 Green tea polyphenol EGCG sensing for cancer prevention

Hirofumi Tachibana

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan. Bio-Architecture Center, Kyushu University, Fukuoka, Japan. Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka, Japan.

We have identified 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor that confers EGCG responsiveness to many cancer cells at physiological concentrations. Here we provide an overview of several pathways that sense and manage the cancer preventive activities of EGCG. EGCG has been shown to rescue mice from lipopolysaccharide (LPS)-induced lethal endotoxemia and downregulate inflammatory responses in macrophages. Anti-67LR antibody treatment or RNAi-mediated silencing of 67LR resulted in abrogation of the inhibitory action of EGCG on LPS-induced activation of downstream signaling pathways and target gene expressions in macrophages. EGCG induced a rapid upregulation of Tollip, a negative regulator of TLR-signaling, and this EGCG action was prevented by 67LR silencing or anti- 67LR antibody treatment. EGCG has been shown to be able to induce apoptotic cell death in multiple myeloma cells and primary patient myeloma cells through the 67LR, while having no significant effect on growth normal cells such as peripheral blood mononuclear cells (PBMCs). The expression of 67LR was significantly elevated in myeloma cell lines and patient samples compared to normal PBMCs. We found that the apoptosis-inducing activity of EGCG in multiple myeloma cells is attributable to the activation of acid sphingomyelinase (aSMase) that hydrolyzes sphingomyelin into ceramide as a lipid raft component and the lipid raft clustering through 67LR. We also found that EGCG induces aSMase translocation to the plasma membrane and protein kinase C delta (PKCδ□ phosphorylation at Ser664, which was necessary for aSMase/ ceramide signaling, via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell death pathway triggered by EGCG for the specific killing of multiple myeloma cells.

－95－ Profile of Speaker

Dr. Hirofumi Tachibana is distinguished professor of Kyushu University. He started his research career in 1987 with studies on the cellular immunology and antibody engineering in the Graduate School of Genetic Resources Technology at the Kyushu University, after graduating from the Faculty of Agriculture at the Kyushu University. He succeeded in the functional modification of human monoclonal antibodies via the light chain glycosylation. He obtained his Ph.D. in Agriculture Science from The Kyushu University in 1993, working on the human antibody engineering. He was promoted to assistant professor in 1991 and lecturer in 1994 at the Graduate School of Genetic Resources Technology Kyushu University. He was promoted to associate professor in 1996 and distinguished professor in 2012 at the Department of Food Science and Technology Kyushu University. He focused on the physiological mechanisms of the functional food factors at a molecular level. He identified some sensory systems that respond to the functional food factors. Especially, he succeeded in the discovery of green tea polyphenol EGCG receptor (2004). He was awarded The Japan Bioscience, Biotechnology and Agrochemistry Society Award for the Encouragement of Young Scientists in 1998, the Agricultural Sciences of Japan Award in 2004, the JSPS Prize in 2006, and the Japanese Association for Food Immunology Award in 2010.

－96－ L 37 Vitamin C and aging

Akihito Ishigami

Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology (TMIG), Tokyo 173-0015 JAPAN

Vitamin C (L-ascorbic acid) has a well-documented, strong anti-oxidant function, evident as its ability to scavenge reactive oxygen species (ROS) in cells and blood. Additionally, an anti-aging effect has been attributed to vitamin C stemming from its deletion of ROS; however, no scientific evidence has yet proven this assertion. In 1991, Senescence Marker Protein-30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases androgen-independently with aging. Recently, we identified SMP30 as the lactone-hydrolyzing enzyme gluconolactonases (GNL) of animal species (1). GNL is a key enzyme which involve in vitamin C biosynthesis, and the essential role of SMP30 in this synthetic process was verified by a nutritional study. These knockout mice fed a vitamin C-deficient diet did not thrive; i.e., they displayed symptoms of scurvy such as bone fracture and rachitic rosary, then died by 135 days after the start of receiving the deficient diet. Thus SMP30 knockout mice developed symptoms of scurvy when fed a vitamin C-deficient diet, verifying the pivotal role of SMP30 in vitamin C biosynthesis. It cannot be said that the vitamin C deficiency promoted aging, because SMP30/GNL knockout mice died of scurvy, when fed a vitamin C-deficient diet. A most important point is that scurvy is a disease, whereas aging is not. Aging refers to a slow gradual decrease of physiological functions of the body as time passes. In the early experiment, we observed that these SMP30/GNL knockout mice were shorter in life span than the wild type when fed autoclaved mouse chow contained ~55 mg/kg of vitamin C. We now know that this amount of vitamin C is too small to maintain normal levels in tissues; in fact, each mice was taken about 2.5% a day of vitamin C. However, we can say that aging progressed ~four times faster than normal in these knockout mice, because they received too little vitamin C over a long period of time. Thus, these results indicate that a shortage of vitamin C accelerated aging. Until now, there was no scientific evidence for this conclusion despite the conventional wisdom that vitamin C has an anti-aging influence. This, then, is the first report of research that used SMP30/GNL knockout mice to show in a scientific manner that the shortage of vitamin C decreased life.

1) Kondo Y., et al. : Proc. Nat. Acad. Sci. USA, 103: 5723-5728 (2006)

－97－ Profile of Speaker

Dr. Akihito Ishigami is a head of Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology (TMIG). In 1990, he earned his PhD degree from Toho University under the guidance of Dr. Sataro Goto. After the finishing his PhD course, he worked at National Institute on Aging/ National Institute of Health (Baltimore) as a postdoctoral fellow, and he has done contributional works in the field of aging and signal transduction with Dr. George Roth. In 1994, he joined TMIG as a research scientist and then he became a senior scientist in 2005. In 2008, he moved to Toho University as an associate professor and then backed to TMIG as a head of Molecular Regulation of Aging. He is studying about aging and vitamin C to elucidate the mechanism of aging throughout. Today’s talk will be a part of his work on the relationships between aging and vitamin C.

－98－ L 38 Therapeutic efficacy and safety evaluation of water soluble undenatured type Ⅱ collagen in moderately arthritic dogs

Baly Barlow1, Ramesh C. Gupta1, Hiroyoshi Moriyama2, Orie Yoshinari3, Yoshiaki Shiojima3 and Manashi Bagchi4

1Murray State University, Hopkinsville, KY, USA 2Showa Pharmaceutical University, Tokyo, Japan 3Ryusendo & Co., Ltd., Tokyo, Japan 4NutriToday LLC, Boston, MA

Osteoarthritis is a crippling chronic bone degenerative disease that commonly afflicts aging humans, dogs and horses. Currently, one out of every five to seven dogs suffer from arthritis, especially large breed dogs. The purpose of this study was to evaluate the safety and therapeutic efficacy of water soluble undenatured type II collagen (NATUC-II) in client- owned moderately arthritic dogs. A broad spectrum of safety studies including acute oral and dermal toxicity, primary dermal and eye irritation studies, Ames’ bacterial reverse mutation assay and mouse lymphoma assay confirmed the safety of NATUC-II. In another set of studies, moderately arthritic dogs received either placebo or 10 mg active NATUC-II once daily, orally for 150 consecutive days. On a monthly basis, dogs were evaluated for overall pain, pain upon limb manipulation, and pain after physical exertion, using veterinary numerical scales. In addition, dogs were evaluated for physical examination. Using biochemical markers, these dogs were also examined for hepatic (ALP, ALT, and bilirubin), renal (creatinine and BUN), and heart (CK) functions. NATUC-II treated dogs exhibited a significant (p<0.05) reduction in overall pain (53.45%), pain after limb manipulation (65.22%), and pain after physical exertion (62.5%) as compared to the control dogs. Serum chemistry analysis indicated that the dogs did not have any significant adverse side effects related to kidney, liver or heart functions. None of the treated dogs experienced any negative changes in body weight, heart rate, respiration rate or temperature. In conclusion, NATUC-II is safe and significantly ameliorated arthritis associated pain in moderate arthritic dogs. Also, arthritic dogs tolerated NAUC-II without any adverse event over a period of 150 days.

－99－ Profile of Speaker

Manashi Bagchi, Ph.D., FACN, received her Ph.D. degree in Chemistry in 1984. Dr. Bagchi is currently the Chief Scientific Officer of NutriToday LLC, Boston, MA. Dr. Bagchi served as Associate Professor in the Creighton University School of Pharmacy & Allied Health Profession, Omaha, NE, from Sept 1990 to Aug 1999. Later, she served as the Director of Research at InterHealth Nutraceuticals, Benicia, CA, from Sept 1999 to July 2009. Dr. Bagchi is a Member of the Study Section and Peer Review Committee of the National Institutes of Health (NIH), Bethesda, MD. Her research interests include free radicals, human diseases, carcinogenesis, anti-ageing and anti-inflammatory pathophysiology, mechanistic aspects of cytoprotection by antioxidants and chemoproteactants, regulatory pathways in obesity and gene expression. She is a Member of Society of Toxicology (Reston, VA), New York Academy of Sciences (New York, NY) and Institutes of Food Technologists (Chicago, IL). She is a Fellow and currently Board Member of the American College of Nutrition (Clearwater, FL). Dr. Bagchi has 205 papers in peer reviewed journals and 2 books. She has delivered invited lectures in various national and international scientific conferences, organized workshops, and group discussion sessions. Dr. Bagchi is serving as Editorial Board Member of the Journal of the American College of Nutrition. Dr. Bagchi received funding from various institutions and agencies including the U.S. Air Force Office of Scientific Research, Nebraska State Department of Health, and Cancer Society of Nebraska.

Manashi Bagchi, Ph.D., FACN Chief Scientific Officer NutriToday, Boston, MA Email: [email protected]

－100－ Session9

L 39 Yin and Yang: a pharmacological perspective

Robert K. M. Ko

Hong Kong University of Science & Technology, China

The Yin-Yang theory is an ancient Chinese philosophy which believes that everything has an opposing Yin and Yang aspect. These aspects are mutually controlled and inhibited each other, resulting in a state of dynamic equilibrium. In this talk, I will share with you our research work on Yin and Yang Chinese tonic herbs in an effort to establish the pharmacological basis of their health-promoting action. In particular, the effect of a Yang-invigorating Chinese tonic herb, Herba Cistanches, on cellular glutathione redox cycling will be discussed.

－101－ Profile of Speaker

Dr. Robert Ko Kam-Ming is currently Professor in the Division of Life Science at the Hong Kong University of Science & Technology. After graduating from the Chinese University of Hong Kong, he went on to Canada and obtained his Ph.D. in pharmacology at the University of British Columbia in 1990. Since then Dr. Ko returned to Hong Kong to pursue his research work on Chinese herbal medicine. Dr. Ko researches on the antioxidant and immunomodulatory properties in Chinese tonic herbs in establishing their scientific basis in terms of modern medicine, and has so far edited two books and published more than 150 scientific papers and book chapters on related topics. Dr. Ko is also a pioneer in developing proprietary Chinese herb- based health products and skincare products in Hong Kong.

－102－ L 40 Effect of mixture of Schisandra and sesame on antioxidation and liver function

Chin-Kun Wang

Chung Shan Medical University, Taichung, Taiwan

Schisandra and sesame exhibited perfect antioxidation. In vitro study showed that the mixture of Schisandra fruit extract and sesamin had synergistic scavenging effect on free radicals. The combination of Schisandra fruit extract and sesamin was used to evaluate the actual antioxidant performance and the improvement on liver function in human beings. This study used a randomized, parallel and placebo-controlled of human clinical trial. Forty subjects were divided into a test group and a placebo group. The study was focused on the potential effects of a mixture of Schisandra fruit extract and sesamin (hereinafter called ‘SCH’) in the subjects with borderline high levels (40-60 U/L) of alanine aminotransferase (ALT) or aspartate aminotransferase (AST). Twenty subjects taking SCH (four tablets per day) and 20 subjects taking placebo (four tablets per day) were studied. The effects of SCH on ALT, AST, total bilirubin, direct bilirubin, free radical levels, total antioxidant status, glutathione peroxidase, glutathione reductase, and the lag time for low-density lipoprotein oxidation were determined. The total test period was 5 months. Intervention of SCH clearly reduced the levels of ALT and AST, but it made no change in the total bilirubin and direct bilirubin. Intake of SCH also greatly increased the antioxidant capacity and decreased the values of thiobarbituric acid reactive substances, total free radicals, and superoxide anion radicals in the plasma. The activities of glutathione peroxidase and reductase in the erythrocytes were significantly increased. In addition, the lag time for low-density lipoprotein oxidation, an inflammatory marker, was evidently increased. Fatty liver was found to have been significantly improved in this study. SCH proved to have the effects of antioxidation and improving liver function.

－103－ Profile of Speaker

Dr. Chin-Kun Wang is a distinguished professor in Chung Shan Medical University, the President of Nutrition Society of Taiwan, council member of FANS, and executive member of ISNFF. He got his Ph.D. degree from National Taiwan University and worked at Chung Shan Medical University in 1993. In 1996, he promoted as a full professor, and then took the positions of chair, dean, vice president and president in Chung Shan Medical University. His research work is focused on human clinical trials and human metabolism of medicine, nutraceuticals, functional foods and herbs. He got the National Award of Biomedicine for his great contribution to the medical education in 2008. He was also honored as 2012 Who’s who in the world, Who’s who in Asia, 2011 Cambridge certificate for outstanding medical achievement, and 2009-2010 2011-2012 Who’s who in Medicine and Healthcare.

－104－ L 41 Hyperglycemic effects of mangiferin hydrastis granuleinin GK rats with diabetes mellitus

Xiaobo Qu , Zhe Lin, Na Li, He Lin

Pharmacology Laboratory of Chinese Drugs, Research and Development Center, Changchun University of Chinese Medicine, Changchun 130117, China

Mangiferin hydrastis granulein（MHG）is composes of mangiferin and berberine. Mangiferin and berberine display an extensive spectrum of pharmacological properties, including antioxidant,antivirus, antiinflammatory and as well as antilipid activity. Our early studies have demonstrated that MHG significantly decreased the level of blood glucose and blood fat of STZ-induced diabetic rats and mouses.These results suggest that MHG probability is a composition with exploitation potential of drug or functional food.The aim of this study was to investigate the effects of mangiferin hydrasis granule (MHG) on blood glucose, blood fat and insulin in Goto-Kakizaki (GK) model rats and their mechanism. Thirty-six male GK rats were randomly divided into control group, rosiglitazone group and MHG (20.0, 40.0 mg•kg-1) groups (n=9). The rats were administered by intragastric administration for 4 weeks . The change of body weight was observed and the blood glucose, blood fat , insulin in serum were detected. The body weight rats in control group was significantly decreased, and continuely droped during administration; compared with control group, the body weights of rats in rosiglitazone group and MHG 20.0, 40.0 mg•kg-1groups were increased (P< 0.05). Compared with control group , the contents of blood glucose in rosiglitazone group and MHG (40.0 mg•kg-1)group were decreased (P<0.05 ). There was no significant difference of the insulin content between various groups (P>0.05). Compared with control group, the contents of triglyceride (TG) and total cholesterol (TC)in rosiglitazone group were decreased (P< 0.05 ); the content of total cholesterol ( TC ) in MHG(40.0 mg•kg-1)group was decreased (P<0.05). This study provides that MHG can reduce the risk of diabetes mellitus by down-regulating the contents of blood glucose and blood fat in GK rats. Key words: mangiferin hydrasis granule; diabetes mellitus; blood glucose; blood fat; insulin; GK rats

－105－ Profile of Speaker

Dr. Xiaobo Qu is a professor of Changchun University of Chinese Medicine. She earned her PhD degree from Northeast Normal University.Recently she is mainly engaged in chinese herbs biological activity, action mechanism, structure-activity relationship and side effect,especially in basic and development research of animal drugs.

－106－ L 42 Protective potential of fruit extract of Emblica officinalis Linn. against radiation and lead induced hepatic lesions in Swiss albino mice

R.K.Purohit, Aruna Chakrawarti and Manisha Agarwal

Radiation Biology Laboratory Department of Zoology, Govt. Dungar College, Bikaner (Rajasthan), India. 334003 [email protected]

Research in medicinal plant has gained a renewed focus recently. The prime reason is that other system of medicine although effective come with a number of side effects that often lead to serious complications. Plant based system of medicine being natural does not pose the serious problems. Though Emblica officinalis has various medicinal applications, but it is the need of hour to explore its medicinal values at molecular level with help of various biotechnological tools and techniques. In light of the above, the present study was aimed to evaluate the protective effect of Emblica against radiation and lead induced histopathological and biochemical alterations in the liver of Swiss albino mice. The animals were exposed to 3.0 Gy and 6.0Gy of gamma rays with or without lead acetate treatment. The Emblica was administered seven days prior to irradiation or lead acetate treatment. The animals from all the experimental groups were sacrificed by cervical dislocation at each post-treatment intervals of 1,2,4,7,14 and 28 days. After sacrificing the animals, pieces of the liver were taken out and some of them were kept in the Bouin’s fluid for histopathological studies and remaining pieces were kept at -20℃ for different biochemical parameters. The value of total proteins, glycogen, acid phosphatase activity, alkaline phosphatase activity and RNA increased up to day-14 in non drug treated groups and day-7 in the Emblica treated groups, thereafter value declined up to day-28 without reaching to the normal. The value of cholesterol and DNA declined up to day-14 in non drug treated groups and day-7 in the drug treated groups, thereafter value elevated up to day-28. The histopathological changes included cytoplasmic degranulation, vacuolation, nuclear pycnosis, crenation, karyolysis and karyrrhexis etc. After combined treatment of radiation and lead synergistic effects were observed. The changes were found dose dependent. The liver of Emblica treated animals exhibited less severe damage as compared to non-drug treated animals at all the corresponding intervals. An early and fast recovery was also noticed in Emblica pretreated animals. Thus, it appears that Emblica is potent enough to check lead and radiation induced hepatic lesions in Swiss albino mice.

－107－ Profile of Speaker Dr. Rajendra Kumar Purohit Govt. Dungar College, Bikaner (India) 334001

Dr. R.K.Purohit had completed his M.Sc. (1989), M.Phil. (1990) and Ph.D.(1997) from the Govt. Dungar College, Bikaner (Rajasthan), India. He has been serving Govt. Dungar College, since1991 as Professor of Zoology. He has 22 years of teaching and research experience. He has attended more than 30 National and International Conferences and presented his papers. He has published more than 50 research papers in national and International Journals. Dr. Purohit has recently organized an International Conference on Emerging Frontiers and Challenges in Radiation Biology on January 24-25, 2012 at Bikaner. He has been honored by ISHEER award, Young Scientist Award at Baroda (India) in 1992, District Administration award and various other awards by national and international agencies for his contribution to science. At present he is also working as Treasurer of prestigious Indian Society for Radiation Biology.

－108－ Cancelled Presentation The effect of oxidative stress on hepatic oval cells malignant transformation and the intervention of traditional chinese medicine

Wang Xue-jiang1, Xue Xiao-wei1, Feng Ping1, Shi Zheng-ming2 and Wang Peng-yan1

1Capital Medical University, Beijing 100069 2Beijing Jishuitan hospital,Beijing 100035 China

【AIM】To determine the whether reactive oxygen species involve in the malignant transformation of hepatic oval cells. 【METHODS】Cultured rat liver oval cell line WB-F344 (referred to as WB cells), were divided into complete medium group, free-serum medium group and hydrogen peroxide stimulation group. The normal WB cells could be transformed by stimulation of 7×10-7M H2O2 under cultured condition of free serum medium. 【RESULTS】In morphology, the cells became anomalous and different in size, the ratio of nuclear to cytoplasm increased, and the cells growth polarities disappeared. In methylcellulose medium culture, the cells could form clone in the medium.In flow cytometry, G1 phase cells decreased, S phase cells increased and the proportion of aneuploid cells increased (P<0.05). The results of western blot showed that AFP expression was increased (P <0.05) and OGG1 was decreased. Both MP and MA inhibited the proliferation of the malignant WB cells in a time and dose-dependence manner with MTT assay, while with trypan blue exclusion test as quality control, we found that after the treatment of MP 5 g/L and MA 12 g/L for 72h, cell survival rate was 80%. The results of flow cytometry showed that MA and MP reduced the number of S-phase cells , increased G1 phase cells and decreased intracellular ROS (P <0.05). The inhibited ability of hydroxyl radicals was increased in cell supernatants (P <0.05). MA and MP could enhance the expression levels of p-P53 and P21, inhibited the tumor marker AFP and cyclin E (P<0.05). 【CONCLUSION】Microenvironment impacts the differentiation and proliferation of hepatic oval cells. Minidose hydrogen peroxide stimulated the WB cells for some times, the level of intracellular ROS could increase, then causing cell cycle-related proteins and DNA repair damage enzymes express abnormally, finally, leading to the hepatic oval cells malignant transformation.It was indicated that ROS, which regulated the hepatic oval cell proliferation and differentiation, could make an important part in the hepatic oval cells malignant transformation. MP and MA can inhibit the proliferation of the malignant cells, and induce these cells differentiating from the immature to mature in the same times. * Corresponding author to WANG Xue-jiang

－109－ Profile of Speaker

Wang Xuejiang is a pathophysiological professor and the vice director of school of basic medical sciences of Capital Medical University (CMU) in China. From1994, worked at the teaching and research section of pathology in the basic science department in the traditional chinese medicine (TCM ) and Pharmacy college of Beijing Union University as a lecturer and a vice-professor. In 1998, designated to Niigata Pharmacy college, Japan as a researcher of post –doctorate. From 2000 to now, as a professor work at the School of Basic Medical Sciences of Capital Medical University in China. Research: Pharmacology and molecular structure research into Chinese herb obstructing hepatic pre- cancerous pathological changes

－110－ Sponsored Seminar

Sponsored Seminar

Research & development on “slow calorie confectionaries” – Focusing on quality of calories–

Shigeru Mineo

Bourbon Institutes of Health, Bourbon Corporation Kashiwazaki, Niigata, Japan

Confectionary products are considered to be snack between meals and are loved by all people, young and old. They play an important role in not only supplying nutrients as useful supplementary food but also making happy as a communication tool with smile. However, the lifestyle-related diseases, such as obesity and diabetes, often known to be caused by overeating and an unbalanced diet, have had a serious impact on our dietary life, especially of young people. Some consumers have tended to hesitate to have confectionary products with some sort of guilt feeling because there’s been highlighted the negative images including risk factors of high calories, hyperglycemia, and caries of tooth.

Several foods highlighting calorie reduction, such as candies made by non-digestive saccharides, snacks adjusting edible serving size and beverages labeled zero- or low- carbohydrate with the artificial sweeteners, were commercially accepted in the present market. But I think those would be incomplete and remained improvement for better health. I have developed various tasty confectionaries at Bourbon Corp. in Niigata. I’ve been interested in isomaltulose (Palatinose®) having characteristics for good taste and favorable textures. Now, I’m going to forward with developing new products containing isomaltulose, what we call is “Slow Calorie confectionaries”.

Isomaltulose is a disaccharide composed with glucose and fructose (a structural isomer of sucrose) naturally found in honey. Digestibility of isomaltulose in vitro is 5-times slower and its sweetness is about 50% down compared with that of sucrose. Some studies showed that intake of isomaltulose-containing diets suppressed rapid increases of blood glucose and insulin levels and that they led to maintaining satiety and good concentration and continuous fat burning after exercise. Furthermore, we revealed a second meal effect which is lowering the blood glucose level after a later diet and an enhancing effect of glycogen synthesis in liver and skeletal muscle using a middle meal model experiment rats administering isomaltulose.

From these results, we proposed a concept called “Slow Calorie®” which meant to be delayed digestion and absorption of saccharides. “Slow Calorie Chocolates” and “Slow Calorie Cookies” using isomaltulose have been already produced in Bourbon Corp.. I’d like to introduce these products along with consideration about quality of calories.

－113－ Profile of Speaker

Mr. Shigeru Mineo is a manager of research section at Bourbon Institutes of Health (BIH), Bourbon Corporation. After he graduated the Department of Industrial Chemistry (Present-day Applied Chemistry), Faculty of Science and Engineering, Chuo University in 1989, he has been consistently belonged to R&D Division of confectioneries at Bourbon Corp. He transferred to BIH established in 2001, and he has being interested in beneficial effects of diets on health, He is engaged in researching and developing functional food materials and healthy diets for preventing lifestyle- related disease through coordination between the industry, the academic, and the government authorities. He is also FOSHU advisory staff.

－114－ Sponsored Seminar

Novel technologies to reduce allergenicity and enrich functional components

Kazutaka Yamamoto

National Food Research Institute National Agriculture and Food Research Organization Tsukuba, Ibaraki, Japan

In recent years, technologies to reduce allergenic symptoms or to enrich functional/ nutritional components in plant have been developed. The reduction of allergenic symptoms is above all important, since severe symptoms including anaphylaxis may lead to death of the patients. One of the novel technologies to reduce allergenic proteins in crops is high hydrostatic pressure (HHP) treatment. Before HHP treatment, rice allergenic proteins were enzymatically reduced and the allergen-reduced rice was commercialized by Shiseido Company Ltd. as Fine Rice® in 1991 and was approved in 1993 as the first FOSHU (Foods for specified health uses) product in the world. Thereafter, Echigoseika Company has commercialized A-cut Rice® whose albumin and globulin were by 95 % reduced by HHP treatment. After the successful application of HHP treatment to rice grain, the technology was applied to wheat for the production of A-cut Wheat Bread®, Cookie®, and Cracker® whose allergenic proteins were by 95 % removed. HHP treatment can be used to enrich functional food components such as GABA (gamma-amino butyric acid) An alternative to tackle allergenic diseases is to develop transgenic plants. Transgenic rice seed-based oral vaccines as allergen-specific immunotherapies have been developed in Japan. Plant-based vaccines have advantages over conventional vaccines in terms of scalability, lack of requirement for cold chain logistics, stability, safety, cost-effectiveness, and needle- free administration. For example, about 25 % of Japanese population is suffering from Japanese cedar pollen allergy from February to May, and “Rice-Seed-Based Japanese Cedar Pollen Allergy Vaccine” was developed for the pollinosis by a transgenic method. In addition, transgenic approach has been carried out to enrich functional / nutritional components in crops. Golden Rice® is enriched with vitamin A. GABA-enriched rice line has been developed, influencing a decrease in blood pressure in spontaneously hypertensive rats. For developing “novel” foods and feed, safety evaluation is indispensable. Since HHP treatment is a simple physical, namely “non-chemical”, treatment, it is widely accepted and commercialized in the world. As for the transgenic methods, Codex Alimentarius Commission established a Codex Ad Hoc Intergovernmental Task Force on Foods derived from Biotechnology, which has developed standards, guidelines, or recommendations for safety assessment of the technologies. In addition, the Taskforce for the Safety of Novel Foods and Feeds in the Organisation for Economic Co-operation and Development (OECD) has been

－115－ working on “Consensus Documents” which enable comparative approach to evaluate safety of transgenic crops such as rice, soybean, tomato, and cotton since 1999.

In this talk, the novel technologies will be briefly introduced with some topics, while touching upon the safety aspects of the technologies.

Profile of Speaker

Dr. Kazutaka YAMAMOTO is the research leader (head) of Food Piezotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization. In 1994, he received his Ph.D. from the University of Tokyo. Then he joined National Food Research Institute as a research scientist and worked on starch science and food processing. From 1997 to 1999, he worked at Swiss Federal Institute of Technology Zurich as a postdoctoral fellow and started to work on high hydrostatic pressure treatment. From 2000 to 2002, he was appointed to a research coordinator at the Ministry of Agriculture, Forestry and Fisheries and then started his double career as a food safety coordinator: representative of Japanese government at Codex Alimentarius Commission and OECD Taskforce; lecturer on food safety risk analysis. Currently, he works on high pressure food processing as a research scientist and on food safety administration as a food safety coordinator, especially as a vice chair of OECD Taskforce for the Safety of Novel Foods and Feeds.

－116－ Poster Session Abstract

S ection 1 P-1 Emerging trends in foods microbiome research : novel and future analytical approaches to investigate and control of various diseases

Hemant K. Gautam

Institute of Genomics and Integrative Biology, India

Life style disease (Non Communicable diseases) like diabetes, heart attack, arthritis and even cancer are associated with choice of food and human body microbes. This is further strengthen by the Human microbiome project which proved that number of diseases are associated with microbial population of human body and their interaction of microbes present in the food. Fruit and vegetable contains a large number of microbes and is a major source of disease fighting chemicals. These microbial communities, however, remain largely unstudied, leaving almost entirely unknown their influence upon disease and a source of new bioactive molecules. Traditional microbiology has focused on the study of individual species as isolated units. However many microbial species have never been successfully isolated as viable specimens for analysis, presumably because their growth is dependent upon a specific microenvironment inside the fruit. Advances in DNA sequencing technologies have created a new field of research, called fruit metagenomics, allowing comprehensive examination of microbial communities, even those comprised of uncultivable organisms. Instead of examining the genome of an individual bacterial strain that has been grown in a laboratory, the metagenomic approach allows analysis of genetic material derived from complete microbial communities harvested from natural environments. In the past fruit is never been used as source of novel bioactive molecules but now with the development of new technologies like natural-product extract libraries with high-throughput screening, recent advances in separation technology and structure elucidation have revived the implication of fruit and fruit products in search of novel drug like molecules. Now with the development of fruit metabolomics which will provide better understanding of various metabolic pathways in identification of novel food or fruit-derived biologically active compounds and genes involved in their biosynthesis. The combination of metagenomics and metabolomics will lead to the discovery of novel foods to control various diseases. The development of prebiotic and probiotic are undergone various developments but food will play a major role as it is a source of common man. Keeping in view different strategies will be discussed to study the microbial flora from different environments, food, fruits and vegetables and their management for various diseases.

－119－

P-2 The antidiabetes constituents of yacon leaves

Dou De-Qiang1, Xiang Zheng1 and Dong Feng2

1Liaoning University of Traditional Chinese Medicine, 2Zhen-Ao Group Co. LTD., China

Yacon, Smallanthus sonchifolius (Peoepp. & Endl.) H. Robinson, which was originally cultivated in the Andean highlands of South America, was introduced into China via Japan in the 1990s. It has been reported that the tubers of yacon contain a high concentration of oligofructans1 and polyphenols2, and that its leaf extract has potent antidiabetic effects3. Recently, yacon has become popular as a healthy, functional food in Japan, Peru and other countries. Chemical investigations of yacon have revealed that yacon leaf contains monoterpenes, sesquiterpenes and diterpenes, which have a physiological role in the pest- resistant and antimicrobial activities of this plant4, 5. At present yacon has been cultivated in China at a large scale. To determine the antidiabetic components of the yacon leaves, 16 compounds were isolated form water and ethanolic extracts of yacon leaves by various kinds of chromatography methods, such as silica gel, ODS, Sephadex-LH20 column and preparative HPLC. Eight new compounds were characterized as smaditerpenic acid A (1), smaditerpenic acid B (2), smaditerpenic acid C (3), smaditerpenic acid D (4), ent-kaurane-3β, 16 β, 17, 19-tertol (5), ent- kaurane-16β, 17, 18, 19-tertol (6), 4-[(1E)-3-butoxybut-1-en-1-yl]-3, 5, 5-trimethylcyclohex-3- en-1-yl β-D-glucopyranoside (7) and 5, 8-dihydroxyl-(5H, 8H)-β-ionol (8). Other ingredients were all firstly isolated from the title genus and they were: ent-kaurane-3β,16β,17-triol (9), ent- kaurane-16β,17-diol-19-oic acid (10), ent-kauran- 16β, 17, 18-triol (11), 1-pentacosanol (12), octacosanol (13), 3', 4', 5-trihydroxy-3, 7-dimethoxyflavone (14), 3, 4-dihydroxybenzaldehyde (15) and isorhamnetin (16). The α-glucosidase inhibition activity of smaditerpenic acid-type and kaurane-type compounds were examined, indicating the inhibition activity of smaditerpenic acid-type compounds are stronger and their intensity is similar to Acarbose. The contents of chlorogenic acid and smaditerpenic acid A in yacon leaves cultivated in China, Korea, Japan and Peru was compared, indicating the smaditerpenic acid A in yacon leaves increased concurrently as the latitude. In addition, the anti-diabetes activity of extracts prepared with different extraction methods was compared.

－120－

P-3 Biochemical and chemical characteristics of “Yang-invigorating” action of Herba Cynomorii

Jihang Chen and Kam Ming Ko

Hong Kong University of Science and Technology, Hong Kong S.A.R

Herba Cynomorii (Suo Yang), which transliterally means ‘locking the Yang’ in Chinese, is one of the most potent “Yang-invigorating” Chinese tonic herbs to supplement the primordial “Yang essence” for both men and women in China. This herb is also frequently added into teas and consumed by local people as invigorant or food. Recent studies in our laboratory have demonstrated that Herba Cynomorii could increase mitochondrial ATP generation capacity (ATP-GC), presumably by enhancing mitochondrial electron transport, in both H9c2 cardiomyocytes and rat hearts, which is characteristic of “Yang-invigoration” in Chinese medicine. In order to investigate the biochemical mechanism as well as the chemical basis underlying the “Yang-invigorating” action, the ethanol extract of Herba Cynomorii, which was found to be active in stimulating mitochondrial ATP-GC, was subjected to the bioactivity- guided fractionation, in which the measurement of ATP-GC in H9c2 cells were utilized as an activity monitor. The results showed that HCY2, which was the most abundant fraction, possessed the most potent activity in stimulating ATP-GC in H9c2 cells. This fraction was chemically characterized by LC-UV and LC-MS sepectrometry, and ursolic acid was found to be the main component of HCY2. Pretreatment with HCY2 or ursolic acid could not only stimulate state-3 respiration, but also caused an increase in state-4 respiration rate in H9c2 cells, with the resultant decrease in mitochondrial coupling efficiency. The result therefore suggests the involvement of mitochondrial uncoupling in the “Yang- invigoration” produced by Herba Cynomorii. In addition, pretreatment with HCY2 or ursolic acid also induced a time-driven cyclic variation of cellular reduced glutathione (GSH) level in H9c2 cells, indicative of up-regulation of mitochondrial glutathione redox cycling. The biochemical mechanism underlying the antioxidant action of Herba Cynomorii involves a sustained and low level of mitochondrial reactive oxygen species production, which is secondary to the increased activity of the electron transport chain, with the possible involvement of mitochondrial uncoupling. Both HCY2 and ursolic acid pretreatments were found to significantly protect against myocardial ischemia/reperfusion (I/R) injury. The cardioprotection afforded by HCY2 and ursolic acid pretreatments was accompanied with the enhancement in mitochondrial glutathione redox status, presumably by ameliorating the I/R-induced depletion in mitochondrial GSH level. The enhancement of glutathione redox status was associated with increases in mitochondrial glutathione reductase (GR) and isocitrate dehydrogenase (ICDH) activities under oxidative stress condition. As regards the functional ability of mitochondria, HCY2 and ursolic acid pretreatments reduced tissue ATP level in myocardial tissue under non-I/R condition, indicative of mitochondrial uncoupling. Under the I/R condition, both HCY2 and ursolic acid pretreatments decreased the extent of ATP depletion and increased the ATP-GC, when compared with the untreated I/R control. Taken together, “Yang-invigoration” produced by Herba Cynomorii, which can up- regulate celluar/mitochondrial antioxidant response and functional ability, as well as the induction of mitochondrial uncoupling, was found to protect against oxidant-induced tissue injury. Ursolic acid seems to be an “Yang-invigorating” ingredient of Herba Cynomorii..

－121－

P-4 High-pressure induced generation of functional compounds in brown rice

Shigeaki Ueno1, Toru Shigematsu2, Mayumi Hayashi2 and Tomoyuki Fujii1

1Tohoku University, 2Niigata University of Pharmacy and Applied Life Sciences, NUPALS, Japan

High-pressure treatment (HP) can preserve small molecules such as vitamins and free amino acids in foods and agroproducts. HP significantly modifies structures of proteins and other macromolecules by affecting non-covalent bonds. On the other hand, HP at 100 - 400 MPa induces various changes in agroproducts by damaging internal cell structures and membranes, leading to mass transfer of materials within cells. Since certain enzymes are still active even at 600 MPa, several biochemical reactions can often occur after HP. Therefore, if HP would give the suitable condition for certain biochemical reaction to an inside of agroproduct, the agroproduct could behave as a bioreactor. The biochemical reactions related to proteins and peptides are predicted to change the free amino acid composition in pressurized biological materials. In previous papers, we reported that HP treatment at 200 MPa resulted in high accumulation of free amino acids and GABA in water-soaked soybean (Glycine max) [1] and brown rice [2]. These findings could be explained by the effect of high-pressure induce transformation that HP enhanced the apparent activities of enzymes, glutamate decarboxylase (GAD) and/or proteases. HP treatment of water-soaked samples could also modify the mass transfer inside and apparent activities of enzymes, resulting in HP-dependent change of distribution of free amino acids. In this study, we supplied Glu into brown rice grains and allowed an enzymatic conversion from Glu to GABA induced by HP treatment. The brown rice grains acted as a bioreactor with a metabolic function for proteolysis and Glu conversion to GABA. The production level of GABA was affected by the initial Glu concentration, which could be controlled by the Glu concentration in the soaking solution. Moreover, the initial GABA production rate was accelerated by HP treatment at 200 MPa for 10 min. These results provide feasibility for a novel use of HP technology to alter the metabolic pathways in a cellular biological material and to accumulate useful metabolites, leading to an insight into a new technology for functional foods processing.

[1] Ueno S, Shigematsu T, Watanabe T, Nakajima K, Murakami M, Hayashi M, Fujii T, Generation of free amino acids and γ-aminobutyric acid in water-soaked soybean by high- hydrostatic pressure processing. J. Agric. Food Chem., 58, 1208-1213 (2010). [2] Shigematsu T, Murakami M, Nakajima K, Uno Y, Sakano A, Narahara Y, Hayashi M, Ueno S, Fujii T. Bioconverion of glutamic acid to γ-aminobutyric acid (GABA) in rice grain induced by high pressure treatment, Jpn. J. Food Eng., 11(4) 189-199(2010).

－122－

P-5 The effects of high-fat diet intake and aging on lipid metabolic system in mice

Taro Honma and Tsuyoshi Tsuduki

Tohoku University, Japan

The population of elderly persons has increased worldwide and this trend is expected to continue, with a concurrent increase in age-related diseases. Studies of aging are important for prevention of these diseases. In our previous study, we showed that senescence-accelerated mouse (SAM) P10 mice fed a standard diet exhibited age-dependent increases of liver hydroxysteroid 11-beta dehydrogenase 1 (Hsd11b1) mRNA level, which promoted insulin secretion and insulin resistance and caused hyperinsulinemia and lipid accumulation in liver and white adipose tissue. Since progression of senescence changes is influenced by energy intake, we hypothesized that a high-energy diet would promote hyperinsulinemia and fatty liver with aging. Therefore, it is interesting to examine the effects of a high-energy diet associated with aging. Although it is known well that the calorie restricted diet delays senescence, the age- related changes in lipid metabolic system on excessive energy intake are unclear. In this study, we examined age-dependent changes in lipid metabolism in SAMP10 mice fed a standard diet or a high-fat diet. SAMP10 mice (3 months old) whose the lifespan are about one year were bred with a standard diet (343.1kcal/100g) or a high-fat diet (405.5kcal/100g). Growth and biological parameters in plasma and liver of young mice (6 months old) and senescent mice (12 months old) were measured. These mice showed marked increases in liver triacylglycerol and plasma insulin levels with intake of a high-fat diet and aging. In addition, to evaluate the liver and pancreatic morphology, hepatocytes and pancreatic islets were observed. Lipid accumulation in hepatocytes and morphological aberrations and hypertrophy in pancreatic islets were promoted by a high-fat diet and aging. To investigate the mechanisms of age- dependent changes in lipid metabolism in SAMP10 mice fed a high-fat diet, the activities and mRNA levels for enzymes associated with lipid metabolism in liver were measured. The results indicated that the lipid metabolic system was activated by a high-fat diet and aging. In addition, the liver mRNA level of Hsd11b1 was measured, since this gene shows an age-dependent increase and promotes insulin secretion. As a result, it was significantly increased by high-fat diet intake and aging. Overall, our results show that intake of a high-fat diet accelerated aging, increased the expression level of Hsd11b1, increased insulin secretion, and promoted lipid accumulation in the liver of SAMP10 mice. Since the expression of Hsd11b1 increases with aging, a long-term approach to repression of the genes is required for prevention of metabolic syndrome and retardation of senescence, and this might be achieved through control of food intake.

－123－

P-6 Anti-fatty liver effect of 1-deoxynojirimycin extracted from mulberry

Tsuyoshi Tsuduki and Taro Honma

Tohoku University, Japan

Aza sugars (also referred to as imino sugars) including 1-deoxynojirimycin (DNJ) are an important class of glycosidase inhibitors that are of interest as potential therapeutic agents. Among the imino sugars, miglitol (Glyset) has been approved as a drug (a second-generation α-glucosidase inhibitor) for type 2 diabetes, and N-butyl-DNJ (Zavesca) has been used to treat patients with type 1 Gaucher disease. Despite the excellent in vitro α-glucosidase inhibitory activity of DNJ, its efficacy in vivo is only moderate. Therefore, we consider DNJ to be suitable for use as a “functional food” rather than a drug. For this reason, we recently produced a food-grade mulberry extract enriched with DNJ and conducted a study in which we showed that administration of this extract suppressed the rise of postprandial blood glucose in humans. These findings are of interest, but other physiological functions of DNJ are unknown. In this study, we examined the effects of oral administration of DNJ (1 mg / kg of body weight / day) or mulberry extracts enriched in DNJ (meDNJ; 100 or 200 mg extract / kg of body weight / day, equivalent to 0.53 or 1.06 mg DNJ / kg of body weight / day) in male SD rats for four weeks. Intake of DNJ and meDNJ strongly suppressed liver triacylglycerol levels. Since activation of the fatty acid β-oxidation system in the liver suppresses lipid accumulation, the activities of hepatic fatty acid β-oxidation enzymes (carnitine palmitoyltransferase and acyl CoA oxidase) were measured. The activities and mRNA expression levels of these enzymes were increased significantly by DNJ and meDNJ. The fatty acid β-oxidation system is also regulated by PPARα and AMPK, and therefore the mRNA expression levels were determined for these proteins. These results showed that AMPK mRNA expression was increased by DNJ and meDNJ. Since AMPK is regulated by the anti-obesity hormone adiponectin, the levels of mRNA expression for adiponectin were measured in plasma and white adipose tissue. These levels were both increased by DNJ and meDNJ. Collectively, these results suggest that DNJ enhances expression of adiponectin mRNA in white adipose tissue and increases the plasma adiponectin level. In turn, this enhances expression of AMPK mRNA, activates the fatty acid β-oxidation system, and suppresses lipid accumulation in the liver. In addition, intake of DNJ and meDNJ did not cause hepatic dysfunction and led to a reduction of oxidative stress. These results indicate the efficacy and safety of DNJ and meDNJ.

－124－

P-7 Molecular mechanism of the anti-allergic effect of the emergency food Clethra barbinervis

Tatsuo Katagiri and Tadashi Aradate

University of TOYAMA, Graduate School of Medicine and Pharmaceutical Sciences, Japan

Clethra barbinervis is a large, upright, deciduous shrub or small tree which typically grows 3-7 meter tall and features horizontally drooping, terminal racemes of pleasantly fragrant white flowers which bloom in mid to late summer and serrated, glossy, dark green leaves which turn bright yellow (sometimes red) in autumn. Japanese clethra is not popular as the food now, but it is known as emergency food for a long time at least in Japan and the rice with Clethra barbinervis (Ryoubu-meshi) was well known during in the Heian era to Edo era. Ekken Kaibara who is the one of most famous naturalist in Japan also introduced the Ryoubu-meshi in his report. The efficacy of the Clethra barbinervis as the Japanese tradition medicine deletes a spasm of pain and leaves the heat, and it is said that it functions as a supplement. Our original purpose was to find new natural anti-allergic reagents that affect to signal transduction pathway initiating allergic reactions on intracellular level. We examined more than 300 kinds of plant extracts to affect the Fc epsilon RI induced degranulation without direct harmful effect against RBL-2H3 cells. Finally, we found that Clethra barbinervis leaf extract (CBLE) did not affect cell viability itself, but significantly decreased degranulation in RBL- 2H3. In other words it was an accidental result that we found the antiallergic active molecular mechanism of the Clethra barbinervis. β-Hexosaminidase activity of RBL-2H3 was almost half level compare with positive control by CBLE treatment. To clarify how RBL-2H3 degranulation was inhibited by CBLE, we examined RBL-2H3 intracellular signaling by immunobiochemical methods. As the result, we found that CBLE reduce the level of total protein tyrosine phosphorylation signals and calcium response induced by antigen-IgE via Fc epsilon RI. Furthermore, MAPK response was suppressed. Interestingly, CBLE inhibited Lyn kinase activity in vivo and in vitro. All our results indicates that CBLE suppress the start point of FcεRI–mediated signals, especially Lyn, and finally degranulation was suppressed. Recently, we isolated two kinds of flavonoid from CBLE. One was (-)epicatechin, and the other was the one of glucosilated flavanone. It is well established that (-)epigallocatechin 3-O-(4-O-methyl)-gallate and its derivatives inhibit RBL-2H3 degranulation. However, both flavonoids from CBLE did not inhibit degranulation themselves. On the other hand, the aglycon of the glucosilated flavanone suppressed degranulation with the dose dependent manner. These results indicate the aglycon of the glucosilated flavanone is a candidate of the inhibitive function against degranulation by CBLE. Addition to the above data, the anti-allergy activity was resistance for heat treatment or acid processing that likes in the case of human digestion. All the results suggest that Clethra barbinervis leaf is not only emergency food but it could function as the health supplement that had anti-allergic activity.

－125－

P-8 Systematic purification and analytical method of mucopolysaccharides in un-fractionated heparin

Hiroshi Murata1,2, Akio Matsuhisa1, Keiichi Yamamoto1, Takao Toda1, Michio Muguruma2 and Satoshi Kawahara2

1Fuso Pharmaceutical Industries, LTD., 2Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki., Japan

Mucopolysaccharides (MSs) are mainly isolated from animal tissues. Heparin (HP), one of MSs whose molecular weight is approximately 15kDa is mainly isolated from porcine intestinal mucosa and available as an anticoagulant. HP is widely used in almost every hospital around the world as an injectable anticoagulant for a long time. In addition, it has been demonstrated that MSs would play important roles in cell proliferation, differentiation, and adhesion between cells and matrices. Therefore MSs have also come into widespread use as health foods and cosmetics. With increasing usage of MSs products, it is demanded to develop test methods for determination and purification of MS ingredients to satisfy consumer’s requirements for those safety. Un-fractionated heparin (UFH) is consisted of heparin and other MSs as chondroitin sulfate family (CSF). In early 2008, the over-sulfated chondroitin sulfate (OSCS) was found in some UFH products, and in many cases the contamination of OSCS was considered as the cause for the severe anaphylactoid reaction that occur after intravenous of HP in which finally leads to death. It is known that OSCS and CSF (type A～E) were resistant for de-polymerization with nitrous acid. Additionally, the individual solubility of MSs in organic solvent is different. So we developed an analytical method for MSs and a simple method for removing MSs impurities from UFH preparations by using those characteristics of the MSs. Molecular weight distribution of degraded products with nitrous acid was analyzed by the gel permeation HPLC after de-polymerization of OSCS standard (OSCS-STD), UFH sodium salt containing OSCS and other MSs (OSHP-SH), OSCS reference standards containing heparin (CSMS-CE1 and CSMS-CE2), and various UFH products. The molecular weight distribution of OSCS-STD did not differ between before and after nitrous acid de- polymerization. The molecular weight distribution of OSCS-STD was similar to that of de-polymerized OSHP-SH with nitrous acid. On the other hand, the molecular weight distributions of CSMS-CE1 and CSMS-CE2 were similar to that of chondroitin sulfate B (CS- B) standard. Then, UFH sodium salt and some drug substances containing heparin and other MSs were purified by ethanol precipitation. The gel permeation HPLC after nitrous acid de- polymerization and 1H-NMR spectroscopy revealed that resistant MSs to nitrous acid de- polymerization were fractionated into the supernatant and that HP concentrated in the colloidal precipitate. The predominant MS existed in UFH was CS-B. The combined method of ethanol precipitation and nitrous acid de-polymerization technique could be applied on the UFH to analyze the purity of HP and other MSs. In other word, these results suggested that the purification and analytical methods is applicable to not only the safety of medical agent but also the safety of cosmetics and health foods.

－126－

P-9 The essential fatty acid ratio of the diet affect for the pathogenic progress in the NOD mice

Yukiko Kagohashi1 and Hiroki Otani2

1The University of Shimane, 2Shimane University Faculty of Medicine, Japan

Maternal environment including maternal dietary nutrition has been implicated in the development of type 1 diabetes. In this study, to investigate the effect of maternal nutrition, in particular the essential fatty acid (EFA) ratio (n-6/n-3) and composition, on the development of type 1 diabetes in the offspring, we prepared different kinds of chows with different n-6/ n-3 ratio (n-6/n-3: High-n-3 < n-3 < control < Low-n-3), and provided them to pregnant and lactating mothers and post-weaning female offspring of non-obese diabetic (NOD) mice, a type 1 diabetes model. The n-3A chow included docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and n-3B chow was the diets which they did not include. From 6 weeks after birth, severity of insulitis was significantly higher in the High-n-3 and Low-n-3 chow-fed offspring, and lower in the n-3A and n-3B chow-fed offspring than in the control-chow-fed offspring. The incidence of overt diabetes was not suppressed in the Low-n-3 (70%) and n-3B chow-fed offspring (60%), but it was suppressed in the High-n-3 (45%) and n-3A chow-fed offspring (15%) than in the control chow-fed offspring (70%). The present findings suggested that the dietary n-6/n-3 ratio and composition modified immune response against islet beta cells in the offspring, which might further affect the development of overt diabetes.

－127－

P-10 Suppression of postprandial hyperglycemia by odor precursor of garlic

Tomomi Kobayashi1, Yuya Shirai1, Shoko Miyata1, Makoto Akao1, Hitoshi Kumagai2 and Hitomi Kumagai1

1Nihon University, 2Kyoritsu Women’s University, Japan

Garlic has been consumed around the world not only as a seasoning but also as a medicine, and is known to have various physiological functions such as anti-thrombotic and anti-cancer activities. The active components for these physiological functions are reported to be mainly sulfides such as diallyl disulfide (DADS), diallyl trisulfide (DATS) and methyl allyl trisulfide (MATS) produced enzymatically from S-allyl-L-cysteine sulfoxide (ACSO) and S-methyl-L-cysteine sulfoxide (MCSO) by C-S lyase when garlic is cut or crushed. As ACSO is much more abundant than MCSO, DADS and DATS are principally formed by the reaction. However, DADS and DATS are volatile lipophilic compounds having strong garlic odor, which limits their applicability in food. On the other hand, ACSO is odorless water-soluble compound, and can be widely used in various foods as a chemopreventive agent if it is metabolized to an active compound in vivo. Our previous findings showed that ACSO was converted to active compounds after absorption from the small intestine, and exerted the inhibitory activity against platelet aggregation and the preventive activity against hepatic injury. However, ACSO itself may have some other physiological function. Therefore, the investigation was conducted to examine the suppressive activity of ACSO against postprandial hyperglycemia. In in vivo experiment, an oral starch-tolerance test was performed by using Wistar male rats. First, ACSO dissolved in water was orally administered to the rats for 7 consecutive days. The same amount of water instead of ACSO solution was administered to the rats of control group. Then, starch was given to the rats together with ACSO on the 7th day. After the administration of starch and ACSO, the blood was drawn from the tail vein, and the levels of glucose and insulin were measured. As a result, the postprandial elevation of blood glucose level was significantly suppressed by the oral administration of ACSO. In addition, the oral administration of ACSO lowered the secretion of insulin that was triggered by the increase in blood glucose level. In in vitro experiment, the inhibitory activity of ACSO against α-amylase was compared with that of MCSO, S-ethyl-L-cysteine sulfoxide (ECSO) and S-allyl-L-cysteine (ACS) at pH values between 5 and 8. Interestingly, ACSO inhibited the α-amylase activity at pH values lower than 6. This might be one of the reasons for the suppression of postprandial hyperglycemia by the administration of ACSO. On the other hand, MCSO and ECSO inhibited α-amylase activity only at pH 5, and ACS did not show any inhibitory activity against α-amylase. This result indicates that the allyl group and the sulfoxide structure in ACSO are essential for its inhibitory activity against α-amylase.

－128－

P-11 Evaluated the change in allergenicity of deamidated gliadin in a mouse model of wheat gliadin allergy

Ryosuke Abe1, Misaki Ito1, Yuki Uchida1, Makoto Akao1, Hitoshi Kumagai2 and Hitomi Kumagai1

1Nihon University, 2Kyoritsu Women’s University, Japan

Gliadins and glutenins, the major proteins of wheat flour, are sometimes involved in the development of wheat allergy. In particular, wheat ω-5 gliadin and a high-molecular-weight glutenin subunit have been reported as the major allergens in wheat-dependent exercise- induced anaphylaxis (WDEIA). Deamidation could be used to reduce wheat allergenicity because some tandem sequence repeats having glutamine residues constitute the primary structure of IgE-binding epitopes in wheat gliadin and glutenin. Our previous findings showed that deamidation of wheat gliadin increased its solubility in water, and reduced its allergenicity during digestion both in vitro and in vivo. However, the allergic reaction occurs after the allergens are absorbed from the small intestine into the blood. Therefore, in the present study, the intestinal permeability of untreated and deamidated wheat-gliadin was compared by using a mouse model of wheat-gliadin allergy. In addition, as the allergic reaction is mediated through FcεRI, the high-affinity receptor for the Fc region of immunoglobulin E (IgE), on mast cells releasing histamine, the histamine and gliadin-specific IgE levels in plasma were measured. Male BALB/c mice were sensitized twice at 2-week intervals by intraperitoneal injection of wheat gliadin in the presence of aluminum hydroxide as an adjuvant. Two weeks after systemic priming, mice were repeatedly given untreated or deamidated wheat gliadin dissolved in water with an intragastric feeding needle 7 times per two weeks. Forty minutes after the 7th oral challenge of untreated or deamidated wheat gliadin, mice were anesthetized by barbital and injected with Tyrode buffer into the peritoneal cavity. Then, the peritoneal cavity was carefully opened, and the fluid containing peritoneal cells were aspirated and collected. After collection of the cells, whole blood was drawn and plasma was prepared. In addition, the small intestine was harvested to evaluate the permeability of untreated and deamidated wheat gliadin. Peritoneal cavity cells were stained with FITC-conjugated anti-mouse FcεRI and PE-conjugated anti-mouse c-kit. The cells were acquired on a flow cytometer, and data were analyzed with FlowJo software. The levels of histamine and gliadin-specific IgE in plasma were measured by ELISA, and intestinal permeability was determined by a closed loop experiment. The administration of untreated wheat gliadin to the sensitized mice enhanced the intestinal permeability, while that of deamidated wheat gliadin did not change it. In addition, the level of epitope peptides was much higher for mice administered untreated wheat gliadin than for those administered deamidated one. Oral challenge of deamidated wheat gliadin down- regulated the surface expression of FcεRI on peritoneal mast cells in mice of wheat gliadin allergy. Moreover, the levels of histamine and gliadin-specific IgE in plasma were lower for mice administered deamidated gliadin than for those administered untreated one. These results indicate that oral challenge of deamidated wheat gliadin decreased allergenicity in mice of wheat-gliadin allergy.

－129－

P-12 Suppressive effect of extracts from Lamiaceae plants on hepatic injury induced by carbon tetrachloride

Sou Hironaka1, Makoto Akao1, Yoko Matsui2, Hideki Masuda2, Osamu Nishimura2 and Hitomi Kumagai1

1Nihon University, 2Ogawa & Co., Ltd., Japan

Antioxidants in plants are often effective to scavenge reactive oxygen species (ROS) in the body and exert various physiological functions such as antiaging and anticancer activities. In our previous study, we examined the antioxidant capacity of 80 kinds of spices including Lamiaceae, Apiaceae, and Myrtaceae plants by the ORAC method, and found that some of the Lamiaceae plants have high antioxidant capacity. The components with high antioxidant capacity may scavenge free radicals and prevent various diseases in vivo. Therefore, the present study aimed to examine that suppressive effect of extracts from peppermint (PE), lemon balm (LB) and rosemary (RM) in Lamiaceae on hepatic injury induced by carbon tetrachloride (CCl4). The dried leaves and stems of PE, LB, and RM were mixed with 20 volumes of 50% ethanol and the mixture was filtered to obtain the ethanol-soluble extract. After volatile components were removed by column chromatography, the eluate was concentrated with an evaporator and lyophilized. An animal experiment was conducted to examine the preventive effect of 50% ethanol- soluble extracts from PE, LB, and RM on hepatic injury. Male Sprague-Dawley rats aged six weeks were orally administered with each sample dissolved in water for five consecutive days. One hour after the final administration of sample solution, 50% CCl4 in olive oil was intraperitoneally injected, and acute liver damage was induced. Then, 24 hours after CCl4 injection, the animals were anesthetized, and the blood samples were collected to determine the specific activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) as markers for hepatic injury. Immediately after the blood- sample collection, the animals were sacrificed and the liver samples were obtained to determine the level of thiobarbituric-acid reactive substances (TBARS), the specific activities of detoxification enzymes such as cytochrome P450 2E1 (CYP2E1) and glutathione S-transferase (GST), and those of antioxidant enzymes such as superoxide dismutase (SOD). CCl4 treatment caused significant elevation in the activities of AST, ALT and LDH in serum and the level of TBARS in liver tissue. Furthermore, the specific activities of CYP2E1, GST and SOD in liver tissue were significantly decreased probably by denaturation caused by trichloromethyl radicals. However, pretreatment with extracts from PE, LB and RM significantly suppressed the elevation in serum AST, ALT and LDH activities, and hepatic TBARS level, and the reduction in hepatic GST and SOD activities although they could not restore the CYP2E1 activity. These results indicate that PE, LB and RM possess suppressive effect on hepatic injury induced by CCl4. Their hepatoprotective effect would be attributed to the antioxidant activities. Antioxidants in them would have eliminated active oxygen and free radicals formed by CCl4, which led to the reduction in lipid peroxidation and oxidative stress.

－130－ Section 2 P-13 Dietary (-)-epicatechin ameliorated cardiac and renal oxidative modifications in an experimental model of metabolic syndrome

Cesar G. Fraga1, Valeria Calabro1, Paula D. Prince1, Giovanna Aschettino1, Laura Fischerman1, Marcela A. Vazquez-Prieto2, Monica Galleano1 and Barbara Piotrkowski1

1Physical Chemistry-Institute of Molecular Biochemistry and Medicine (IBIMOL), UBA- CONICET, 2Department of Pathology, School of Medicine, National University of Cuyo; and Laboratory of Cardiovascular Pathophysiology, Institute for Experimental Medical and Biological Research (IMBECU)-CONICET, Mendoza, Argentina, Argentina

Increasing evidence indicates that several mechanisms associated or not with antioxidant actions, are involved in the effects of flavonoids on health. Flavonoid-rich beverages, foods, and extracts, as well as pure flavonoids are studied for the prevention and/or amelioration of metabolic syndrome (MS) and MS-associated diseases, such as cardiovascular and renal diseases. Chocolate and cocoa products are rich in flavanols, a particular flavonoid subfamily, essentially in monomers such as (-)-epicatechin (EC) and (+)-catechin. The aim of this work was to evaluate the effect of one of those specific compounds, EC, on cardiovascular and renal alterations present in an experimental model of MS in rats. Male Sprague-Dawley rats were divided into three groups and received for 8 w: i) control diet and tap water (Control Group: C); ii) control diet and 10% (w/v) fructose in the drinking water (Fructose Group: F), and iii) 10% (w/v) fructose in the drinking water, and the diet supplemented with EC (20 mg/kg BW/ d) (Fructose + (-)-epicatechin Group: FEC). Fructose overload leads to increased systolic blood pressure, and EC prevented that increase (C=130±4; F=142±3*; FEC=133±3 mmHg; *p<0.05 with respect to C and FEC). Proteinuria, creatinine and urea clearances were evaluated as indicators of renal function. Proteinuria was significantly higher in F and FEC groups respect to C (*p<0.05). Parameters associated to oxidative metabolism were measured in heart and renal cortex. In both organs superoxide oxide production (measured as SOD inhibitable lucigenin emmision) and content of thiobarbituric reactive substances were increased in the F group and that increased was prevented by EC administration. Superoxide dismutase and glutathione peroxidase activities were decreased in hearts from F group compared to C group (41% and 48%, respectively) and EC treatment avoided the decrease in both enzymes activities. As opposite, superoxide dismutase and glutathione peroxidase activities were increased in renal cortex of F group (33 and 27 % respectively), but EC treatment also prevented this alteration. In addition, in renal cortex inflammatory markers (inducible nitric oxide and TNFalpha expression) were significantly increased in F group, while in FEC group values were not different to the C group. In summary, these results show that EC supplementation prevented the effects associated with fructose-overload treatment in heart and kidney, and this prevention could be related with the attenuation of the increased oxidant production related to MS. Additional studies will be necessary to understand the organ specific response. Supported by UBACYT 20020100300012, 20020090100111 and 20020100100659 and CONICET (PIP 112-201101-00612. BP, MVP, MG and CGF are members of the Scientific Investigator Career of CONICET, Argentina. VC and PDP hold CONICET fellowships.

－131－ P-14 In vitro anti-diabetic and anti-diabetic complications activities of fucoxanthin with antioxidant activity

Jae Sue Choi1, Nurul Islam1, Hyun Ah Jung2, Chan Mi Lee1 and Eon Ji Kim1

1Pukyong National University, Busan 608-737, Republic of Korea, 2Chonbuk National University, Jeonju 561-756, Republic of Korea, Korea

Fucoxanthin, which was first isolated from marine algae, is a xanthophyll of carotenoid group. Raw Laminaria japonica included the highest content of fucoxanthin, while raw Undaria pinnatifida, Eisenia bicycles, Sargassum fulvellum, Hizikia fusiformis are also rich sources of fucoxanthin. Dry algae contain xanthophylls, but the amount is lower than fresh algae indicating the process of drying decomposed fucoxanthin, since fucoxanthin is known to be sensitive to oxidation. In the present study we investigated the anti-diabetic and anti-diabetic complications potential of fucoxanthin via the inhibitory effects on rat lens aldose reductase (RLAR), human recombinant aldose reductase (HRAR), advanced glycation end products (AGEs) formation, protein tyrosine phosphatase 1B (PTP1B), as well as α-glucosidase. And we also studied the antioxidant potential of fucoxanthin via in vitro scavenging activities against 2,2’-axino-bis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS), and peroxynitrite (ONOO-). Fucoxanthin possessed six-times higher inhibitory activity with an IC50 value of 86.48μM against AGE formation as compared to the positive control, aminoguanidine with an IC50 value of 530.37μM . It also exhibited inhibitory activity against both HRAR and RLAR with corresponding IC50 values of 108.31 and 264.67μM compared to quercetin with IC50 values of 2.28 and 1.26μM, respectively, a positive control used in both assays. In addition, fucoxanthin showed potent inhibitory activity against PTP1B with an IC50 value of 4.80μM compared to the positive control ursolic acid with an IC50 value of 2.56μM. However, it did not show α-glucosidase inhibitory activity up to the concentration of 200μM. Moreover, kinetic study revealed that fucoxanthin showed competitive inhibition against RLAR while it showed mixed type inhibition against PTP1B. Fucoxanthin possessed dose-dependent ABTS scavenging activities with an IC50 value of 85.94μM, compared to L-ascorbic acid, and trolox with IC50 values of 12.05 and 17.33μM, respectively, positive controls used in this assay. Fucoxanthin’s trolox eqivanlent (TE) value is 0.2 whereas TE value of L-ascorbic acid is 1.44 . In addition L-ascorbic acid equivalent (AE) value of fucoxanthin is 0.14 compared to trolox with AE value of 0.70. Also fucoxanthin possessed dose-dependent ONOO- scavenging activity with an IC50 value of 205.44μM, compared to the positive control penicillamine with an IC50 value of 11.98μM. The results of the present study clearly demonstrated the potential of anti-diabetic, anti- diabetic complications and antioxidant activities of fucoxanthin. Therefore, our results can be used to develop fucoxanthin as a therapeutic agent for the treatment of type 2 diabetes mellitus as well as diabetes related complications.

－132－ P-15 The decline of tyrosine hydroxylase phosphorylation and iron concentration cause the reduction of catecholamines in the brain of the SAMP10

Miki Miyajima, Takuya Numata, Moemi Minoshima, Masato Tanaka, Ryo Nishimura, Naoko Hishioka, Yukako Ueno, Tae Kawahara, Toshiyuki Hosokawa, Masaaki Kurasaki and Takeshi Saito

Hokkaido University, Japan

Deficiencies of nutrients that affect brain development and function have been estimated to shift the world’s IQ potential negatively by at least 10 points. Iron deficiency (ID) is the most common of these nutrient deficiencies, affecting an estimated 2 billion people worldwide according to the WHO, including 20–30% of pregnant women and their offspring. And the learning and memory deficits occur while the infants are iron deficient and persist despite iron repletion. Previous studies show that iron is risk-factors for cognitive decline and brain atrophy. Catecholamine is one of the neurotransmitters including dopamine (DA), norepinephrine (NE) and epinephrine. It was implicated in ability of learning and memory. And, it was indicated that catecholamines were correlated with aging of brain. In order to understand the alteration of catecholamine metabolism, and the contribution of dietary intake of iron to catecholamine metabolism in the brain, we investigate a rate- limiting enzyme tyrosine hydroxylase in the synthesis of catecholamines, and fluctuation of iron metabolism that is involved in enzyme activity of the senescence-accelerated mouse prone 10 (SAMP10). Catecholamines and their metabolites in the cerebral cortex were measured by HPLC- ECD. Metal concentrations were determined by ICP-MS. The metal binding protein and metallo-enzymes such as ferritin, divalent metal transporter-1(DMT-1), hepcidin, transferrin, transferring receptor and Superoxide dismutase were determined by western blot analysis. DA and NE levels in SAMP10 were significantly lower than those in control animals, but no significant differences were detected in concentrations of DA and NE metabolites. No significant difference was observed in the levels of TH. However the level of TH phosphorylation at Ser40 in SAMP10 was significantly lower than that in control mice. And the lower values of iron and ferritin were detected in the cerebral cortex of SAMP10. But DMT-1 and hepcidin were no significant differences between SAMP10 and control mice. The decline in DA and NE concentrations was observed in the cerebral cortex of SAMP10 with aging, and this decrease of catecholamine levels was caused by impairment of their synthetic pathway. These impairments are considered to be caused by downregulation of TH phosphorylation at Ser40. Furthermore, the present study demonstrated that iron levels in SAMP10 were significantly lower than those in control animals. TH requires iron for enzyme activity, and converts tyrosine to DOPA, which is further processed to DA. These results suggest that the reduction of catecholamines may be caused by the decline of TH phosphorylation and iron concentration in the brain of the SAMP10.

－133－ P-16 Electrolyzed-reduced water alleviates oxidative stress induced by chronic heat exposure

Tomoko Matsueda1, Motoi Kikusato1, Md. Abul Kalam Azad2 and Masaaki Toyomizu1

1Graduate School of Agricultural Science, Tohoku University, 2Bangladesh Agricultural University, Japan

【Introduction】High-temperature environments could have serious consequences for animal production and health. To this extent, heat stress in acute or chronic forms induces the production of excess mitochondrial reactive oxygen species (ROS), resulting in increased oxidative stress and reduced growth performance of broiler chickens (Kikusato et al., 2010; Azad et al., 2010). As such, the down-regulation ROS production could reduce oxidative stress and thereby improve the growth performance of chickens exposed to heat stress. Electrolyzed-reduced water (ERW) contains a heightened level of molecular hydrogen that scavenges ROS and inhibits ROS-induced DNA damage (Shirahata et al., 1997). It has been recently discovered that molecular hydrogen selectively reduced levels of the hydroxyl radical (OH), one of the most cytotoxic forms of ROS, and reduced the extent of brain injury arising from ischemia–reperfusion oxidative damage in the rat (Ohta et al., 2007). Based on these reports, it could be hypothesized that ERW might alleviate oxidative damage induced by heat exposure. The present study was therefore conducted to investigate the effects of ERW on oxidative stress and growth performance in broiler chickens under conditions of chronic heat stress. 【Materials and methods】Sixteen-day-old chickens (n=6-8) were allowed ad libitum access to food and dechlorinated water or ERW for 6 d. Thereafter, over a 6-d period, half of the birds given dechlorinated water were maintained at thermoneutral temperature (24℃) while the other birds and ERW-given birds were exposed to a constant 32-33℃ The malondialdehyde (MDA) content of muscle and liver tissue as a marker of lipid peroxidation was assayed colorimetrically in terms of the production of 2-thiobarbituric acid- reactive substances (TBARS). The villus and crypt morphology in intestinal samples stained with hematoxylin and eosin was examined, with the villus height and the associated crypt depth measured microscopically by an operator blinded to the experimental treatment received by birds. To detect alterations in the mRNA expression of proteolysis-related genes (μ-calpain and ubiquitin) in the pectoralis muscle, real-time RT-PCR analysis was performed using the iCycler real-time detection system. 【Results】On exposure to chronic heat stress, body weight gain and feed consumption significantly decreased in birds provided wirh dechlorinated water. This decrease in growth performance due to chronic heat stress was limited to some extent in birds provided with ERW. Pectoralis muscle and liver MDA levels in birds provided control water were increased by chronic heat stress, whereas these increases were suppressed in birds given ERW. On exposure to chronic heat stress, the duodenal villus height and the ratio of villus height to crypt depth significantly decreased in birds provided control water. These decreases in duodenal morphology due to chronic heat stress were significantly improved in birds given ERW. μ-Calpain and ubiquitin gene transcript levels in the pectoralis muscles of birds given control water were slightly increased by chronic heat stress, while levels of these transcripts were suppressed in heat-stressed birds provided with ERW. From this study, it can be concluded that ERW alleviates lipid peroxidation in the pectoralis muscles and livers of broiler chickens exposed to chronic heat stress, resulting in improved growth performance.

－134－ P-17 Effect of UV irradiation on human hair treated with shampoo and treatment mixed catechin

Hisakazu Okamura1, Yasuyuki Sugiyama1,2 and Masatoshi Ohta2

1Hazel Tompson Inc., 2Niigata Univ., Japan

UV brings a lot of undesirable change to the hair; the dryness, the lowering of strength, the coarseness of the surface, the loss of the pigment and gloss, and so on. The melanin in hair is known to absorb UV, resulting in the formation of melanin radical. The amino acids; cysteine, tyrosine, phenylalanine, and tryptophan, which compose the protein in the hair, are also known to absorb UV, resulting in the damage to hair keratin. A green tea contains polyphenol, of which main components are four kinds of catechins; epicatechin, epigallocatechin, epicatechingallate, epigallocatechingallate. Catechin has flavan framework with several hydroxyl groups and is able to eliminate free radical such as active oxygen and so on, and then it is known to eliminate singlet oxygen (1O2) and superoxide (O2-) which are aging components in vivo. In this study, we are prepared shampoo and treatment mixed catechin in order to reduce the damage of the hair by UV irradiation. The effect of UV irradiation on the human hair treated the shampoo and treatment mixed catechin is investigated by using ESR. Used hair is Japanese black hair. The used catechins were a green tea (GT) powder and an epigallocatechingallate (EGCG) powder. The shampoo and treatment mixed GT and EGCG, which were referred to as ST-GT and ST-EGCG, respectively, were prepared by adding GT powder and EGCG powder of a various concentration, respectively. The surface condition of hair was observed by using with a microscope (Nikon, ECLIPSE ME600). UV irradiation was used with xenon lamp (Ushio Denki, Optical Module X, 0.5 kW). Radicals formed in hair by UV irradiation were measured with an X-band ESR spectrometer with a 100 kHz magnetic field modulator and a phase-sensitive detector (JEOL, JES-RE3X). The g value was calibrated by the signals of Mn2+ at g = 2.034 and 1.981. ESR signal intensity was evaluated by the area of the integral curve of the ESR spectrum. No change was observed in the surface shape of human hair treated with and without ST- GT or ST-EGCG. We investigated about the radical formed in GT powder and EGCG powder by UV irradiation. In the case of GT powder, ESR signal had a sharp peak at a near g = 2 and a broad peak at a larger g value than 2 before UV irradiation, and then only ESR signal peak intensity of a near g = 2 of GT powder increased by UV irradiation. In the case of EGCG powder, ESR signal had only a sharp peak at a near g = 2 before UV irradiation and then its ESR signal peak intensity increased by UV irradiation. Furthermore, we also investigated about radical formed in the hair treated with ST-GT or ST-EGCG by UV irradiation.

－135－ P-18 Effects of consumption of green tea during lactation on epigenetic factors in female infants of pregnancy rats with low-protein diet

Yongkun Sun1, Yuuka Mukai2, Shin Sato2, Takeshi Saito3 and Masaaki Kurasaki4

1Graduate School of Environmental Science, Hokkaido University, 060-0810 Sapporo JAPAN, 2Graduate School of Health Sciences, Aomori University of Health and Welfare, 030-0841, Aomori, JAPAN, 3Faculty of Health Sciences, Hokkaido University, 060-0812 Sapporo JAPAN, 4Faculty of Environmental Earth Science, Hokkaido University, 060-0810 Sapporo JAPAN, China

Recently, some studies have shown that diet and nutritional status of the mother during pregnancy affects not only the development of the fetus in the uterus, but also the epigenetic changes after birth, and thus prone to lead to a variety of diseases such as adult hypertensive heart disease and diabetes. Epigenetic changes mainly originate from DNA methylation, histone modifications and non-coding RNAs in the regulation of gene expression patterns, which reflects interactions between genes and their products. These heritable changes in gene expression do not involve changes in DNA sequence. However, these tiny changes can result in a series of severe harmful consequences in future. Catechin from green tea extract is an active ingredient and its polyphenol structure. Green tea-derived catechin is like polyhydric phenol extracted polyphenols from grapes, red wine, Bai Li pure. Adult moderate drinking green tea can improve the resistance to disease. It is reported that active polyphenols from Azuki bean red coat fed to lactation rats suffered from malnourished pregnancy can improve the health status of the male offspring. Since different genders have different anatomical structures and physiological characteristics, and female bears the major burden of the fetus, the health status in female plays a greater role in health status of future generations. In addition, catechin in female infants offspring under alternation of nutritional status in the mother before and after childbirth may be expected to play an important role. An imbalanced diet by expectant mother can result in a higher incidence of disease after birth such as hypertensive heart disease, diabetes, obesity, and cardiovascular disease. The aim of this study is to investigate whether pregnant females under malnutrition who continuously take in small amounts of green tea- derived catechin during lactation can improve the future health of their unborn offspring. Female rats are 1:1 mated to male rats, and recorded as 0 days of pregnancy by observation of vaginal suppository. Female rats during pregnancy (3 weeks) were divided into four groups, first group always fed of standard diet as a normal control group, the second group fed of low nutrition diet in pregnancy and standard diet in lactation as malnutrition control group, and the third group and the fourth group fed of low nutrition diet in pregnancy then standard diet with different dose catechin in lactation as the experimental groups. As results, body weight and visceral weight, plasma biochemical parameters of each group did not significantly change. However, phosphorylated AMPK and epigenetic factors (DNMTs, SIRTs) in malnutrition group administrated with catechin were significantly increased as compared with those in malnutrition group administrated without catechin. From these result, it was suggested that catechin recovered renal status of malnutrition induced in infants. In conclusion, offspring from mothers of starvation and administration with green tea-derived catechin obtained more benefits than that of without green tea treatment, which provided a scientific basis for improving diet of expectant mothers. Further investigation will be needed to clarify the relationship between the expression of DNMTs and offspring health.

－136－ P-19 Comparison of the radical scavenging activity of the polyphenol fractions of Japanese and imported black teas

Chigusa Tateyama1 and Kiharu Igarashi2

1University of Niigata Prefecture, 2Faculty of Agriculture, Yamagata University, Japan

While black teas represent around three-fourths of global tea production, Japan is dependent on imported black tea to meet demand as domestic production is low. However, both the recent westernization eating habits and the increased desire for self-sufficiency in producing a wider variety of teas have elevated demand and resulted in a trend towards the revival of domestic black tea production across Japan. Japanese black teas have mellower flavors than overseas varieties, but more specific flavor differences and functional components remain unclear. Therefore, this study aims to gather data sufficient for a better understanding of the characteristics of Japanese black teas. The present study measured the chemical components and radical scavenging activity of commercial black tea polyphenol fractions based on leaching conditions listed in the Standard Tables of Food Composition in JAPAN Fifth Revised and Enlarged Edition. Tannin content was also measured using the Folin-Denis method. Fractions were obtained from commercial domestic black tea varieties, specifically tadasnishiki, benihomare, yabukita, and beniogata, as well as imported varieties, namely Darjeeling, Assam, Uba and Qimen. Measurements were conducted primarily on available standard preparations of the following polyphenol fraction chemical components: five catechins ((+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin, (-)-epicatechin gallate, and (-)-epigallocatechin); gallic acid; and four theaflavins. As flavonol formation is closely related to the formation of catechins in the tea leaf, high performance liquid chromatography of flavonols was also performed. Polyphenol components for which standard preparations could not be obtained were analyzed using liquid chromatography-mass spectrometry (LC-MS) and high performance liquid chromatography (HPLC) with photodiode array detection. Substances such as catechins that possess strong antioxidant activity are abundant in tea. Spectrophotometry was used to measure the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the polyphenol fractions. The results showed a strong correlation was observed between tannin content and DPPH radical scavenger activity. Furthermore, catechin content tended to be higher with increased tannin content. A greater proportion of tannin content represented by theaflavins did correlate with weak DPPH radical scavenging activity. One explanation for this is the high proportion of theaflavins produced during fermentation and the unoxidized catechins in black tea tannins. Unoxidized catechins and theaflavins are generated due to differences in both tea cultivars and manufacturing methods.

－137－ P-20 Trans-fatty acids in blood and eating habits of teenagers

Eri Oomi, Nanako Doro, Takako Baba and Mari Mori

Mukogawa Women’s University Institute for World Health Development, Nishinomiya, Japan

【Aim】In Japan, there is neither regulation nor labeling about trans-fatty acids in the food because the intake of trans-fatty acids is considered to be generally low in Japanese population. The intake of trans-fatty acids per person in Japan is 0.92-0.96g, a day. This numerical value was utilized as a standard that was recommended by Ministry of Agriculture, Forestry, and Fisheries: Less than 1% of total energy should be derived from trans-fatty acids. However, we are concerned about trans-fatty acids intakes because food containing much oil and fat such as fast food, snacks are popular among teenagers. We investigated the association of the intakes of confectionery, meat, oil and fat with trans-fatty acids in the blood of teenagers. 【Method】We implemented food education and health examination from 2007-2011 for 583 students aged 12-18 (238 boys in 1 boys’ school and 345 girls in 2 girls’ schools) and we collected blood in fasting condition from 236 boy students and 264 girl students. We examined height, weight, body fat ratio, blood analysis and questionnaires (Food Frequency Questionnaire). 【Result】Trans-fatty acids were detected in the blood of 200 girl students（75.8%） and 16 boy students (6.8%) . Elaidic acid were detected in the blood of 83 girl students (31.4%) and 2 boy students (0.85%). 34 girl students (12.8%) and 25 boy students (10.6%) exceeded the standard of the arteriosclerosis index (LDLC/HDLC>2.0). The girl students’ average values of trans- pajlmiteladic acid, trans-elaidic acid, total of trans-fatty acids, docosa-hexaenoic acid, LDLC, total cholesterol were significantly higher than those of the boy students.221 girl students (83.7%) and 191 boy students (80.9%) were taking confectionery that exceeded their standard. Moreover, 89 girl students (33.7%) and 38 boy students (16.1%) were taking oil and fats that exceeded their standard intakes 【Conclusion】Boys and girls were taking a lot of lipid. However, more girl students had trans- fatty acids in their blood than boy students. In conclusion girl students were taking foods containing more trans-fatty acids than boy students. Moreover, many boys and girls were taking lipid over their standards. By this examination, we could not identify food from which trans-fatty acids were derived, but the study indicated the excess intake of lipid by teenagers. Therefore, dietary education about how to take proper lipid is needed for boys and girls.

－138－ P-21 Oxidative stability of rice-based diets may affect growth performance of broiler chickens

Chiaki Ito, Fumika Nanto, Tomomi Kamizono, Tomoko Matsueda, Motoi Kikusato and Masaaki Toyomizu

Graduate School of Agricultural Science, Tohoku University, Japan

【Aim of the study】A previous study by our group highlighted that chicks fed a diet containing 43% whole-grain paddy rice and 10% soybean oil exhibited growth retardation compared with a control group fed a corn-based diet containing 6% soybean oil. As the feeding of chicks with 40.7% dehulled rice or 43% whole-grain paddy rice, with 6% soybean oil resulted in normal chick growth comparable to that of the control group, it is conceivable that the growth retardation was caused by the combination of whole-grain paddy rice and the high level of soybean oil which was added to diets to maintain the overall energy content. The present study was carried out to identify elements in the diet that are important determinants of the growth retardation. 【Material and Methods】Thirty-six chicks (1-day-old) were divided into 6 equal-sized groups that were fed one of the following six experimental diets ad libitum for 28 d: two kinds of dehulled rice-based diets containing 5% or 10% soybean oil (DS5%:control or DS10%), another three kinds of whole-grain paddy rice-based diets containing 10% soybean oil, corn oil, or rendering oil* (WS10%, WC10%, WR10%), and a WS10% diet supplemented with vitamin B12 (+90µg/kg), methionine (+3.5g/kg) and ethoxyquin(+150ppm) (WS10%+B12+Met+EQ). Lipid peroxidation levels in tissues were assayed colorimetrically in terms of the production of 2-thiobarbituric acid reactive substances (TBARS). The oxidative stability of diets was assessed in terms of the peroxide value (POV) of fats extracted from the experimental diets; with POV determined using a method that measures the oxidation of ferrous to ferric ions by hydroperoxides. The vitamin B12 content of the plasma was analyzed by a chemiluminescent immunoassay method. *Rendering oil is extracted from inedible parts of birds and domestic animals, such as the feathers, head and some internal organs. This oil is widely employed in feedstock used in the production of broiler feed. 【Results】 The average weight gain of birds fed the DS10% diet was lower than that of birds fed the DS5% diet (control), but there were no significant differences in feed efficiency between the two groups. The average weight gain and feed efficiency of groups fed the WS10% and WC10% diets were significantly lower than those of the control group. In addition, the liver tissue in birds from the WS10% and WC10% diet groups exhibited significantly higher concentrations of TBARS, and fats extracted from the feed given to these groups showed a slightly higher POV compared with that for the control group. In comparison, supplementation of the WS10% diet with vitamin B12, methionine and ethoxyquin dramatically improved growth and feed efficiency. No significant differences in the plasma vitamin B12 content were observed between groups for the different treatments. 【Conclusions】These results clearly indicate that combinations of untreated whole-grain paddy rice and a high level of vegetable oil (10% soybean oil or corn oil) in diets have adverse effects on performance, suggesting that lipid peroxidation may be a contributing factor to the growth retardation seen in birds fed such diets.

－139－ Section 3 P-22 Effects of water-soluble undenatured type II collagen in collagen-induced arthritis mice

Orie Yoshinari1, Yoshiaki Shiojima1, Hiroyoshi Moriyama1 and Manashi Bagchi2

1Ryusendo Co., Ltd., 2NutriToday LLC., Japan

Rheumatoid arthritis (RA) is a severe inflammatory autoimmune disorder with a prevalence of 0.5-1% in the global population. It is characterized by persistent inflammation of the synovium and progressive erosion of cartilage and bone. The most common model of RA in rats and mice is collagen-induced arthritis (CIA) that elicits both antibody and T cell responses to type II collagen (C II). Oral administration of C II has been proven to improve signs and symptoms in CIA animal and clinical study for RA. However, there is no report which described quality of the collagen and detailed mechanism. The present study is designed to examine the oral administration of water-soluble undenatured type II collagen (NATUC- II®) in CIA mice. NATUC- II® was administrated after arthritis development enough, not the prevention. To investigate the mechanism, we assayed arthritis index, serum interleukin (IL)-2, -6 levels, histological assessment and proportion of CD4+CD25+ cells in spleen. After the acclimatization period, DBA1/J mice (8-week-old) were divided in 2 groups, normal group (n=7) and collagen-induced group (n=25). Bovine collagen type II was dissolved in 0.05 M acetic acid at 4 mg/ml and emulsified in an equal volume of Freund’s complete adjuvant. One hundred microlitres of this emulsion was injected intradermally into the dorsal root of the tail of the collagen-induced group mice. This day was defined as day 0. On day 21, the booster injection with same amount of above mention was given to the collagen- induced group. On day 39, the collagen-induced group mice were assigned in 3 groups (CIA (n=8), glucosamine hydrochloride (Glu) (n=8), and NATUC (n=9) group), and administered with glucosamine hydrochloride (300 mg/kg of body weight), NATUC-II® (1 mg/kg, as undenatured C II level) or saline only (for CIA group) until the day 48. Oral dose of NATUC- II® was decided by measuring undenatured C II. Glucosamine hydrochloride, as an index of the improvement of CIA. Development of arthritis was assessed once in three days. On day 48, after operating rat’s abdomen with anesthesia, blood was collected by cardiac puncture from mice. The spleen and the hind limbs were removed from each mouse. As a result, arthritis index in the NATUC group significantly decreased in comparison with that of the CIA group. In the histological analysis of hind limb, the NATUC group showed limited synovial infiltration, well-restored articular cartilage and discernible joint space. Serum IL-6 level in the NATUC group was significantly decreased compared to the CIA group, conversely serum IL-2 level was increased. Furthermore, oral administration of NATUC-II® enhanced the proportion of CD4+CD25+ T cells, and gene expressions of forkhead box p3 (Foxp3), transforming growth factor (TGF)-β1 and CD25. These results suggest that orally NATUC-II® is highly efficacious in the suppression of CIA through induction CD4+CD25+ Treg cells.

－140－ P-23 Effects of liver hydrolysate on the blood glucose in metabolic syndrome model rats (SHR/NDmcr-cp)

Naonori Inoue1, Shuji Hidaka2, Naoyoshi Miura1, Masahiro Fukahori1, Masugi Maruyama2, Satoshi Kawahara2, Kazuyoshi Ohta2 and Michio Muguruma2

1Zeria Pharmaceutical Co., Ltd., 2University of Miyazaki, Japan

Lifestyle-related diseases such as hypertension, diabetes, and dyslipidemia have become major social problems in Japan. Metabolic syndrome is a pathology in which hypertension, hyperglycemia, and dyslipidemia occur simultaneously in the same patient with the common basal condition of insulin resistance accompanying visceral fat obesity. The relationship between the renin-angiotensin system and insulin resistance has gradually been elucidated, and antihypertensive agents such as angiotensin converting enzyme (ACE) inhibitors and angiotensin II (Ang II) receptor antagonists have been reported to prevent the onset of diabetes. Moreover, oxidative stress has been reported to be involved in the onset and progression of diabetes and its complications. Recent years there are numerous researches into functional foods, and peptides possessing ACE-inhibitory and antioxidant activities have been found in the hydrolysates of soybeans, whey, meats, and other foods. These functional foods are expected to contribute to the prevention and improvement of lifestyle-related diseases. Liver hydrolysate is obtained via hydrolysis of mammalian liver. Many peptides, various amino acids, nucleotides, vitamins, and minerals are included in liver hydrolysate. In this study, the effect of liver hydrolysate administration on the blood glucose was examined in SHR/NDmcr-cp (SHR- cp) rats that show spontaneously occurring metabolic syndrome-like abnormalities. In addition, we investigated the ACE-inhibitory and antioxidant activities of liver hydrolysate in vitro. The SHR-cp rats were fed diets containing 5% liver hydrolysate for 12 weeks, and the fasting blood glucose levels and HbA1c were determined every 3 weeks. After administration of the liver hydrolysate-containing feed for 12 weeks, an oral glucose tolerance test was conducted and the plasma AngII concentrations were determined. The results of the oral glucose tolerance test showed that although administration of liver hydrolysate did not affect the blood insulin level, it did significantly inhibit the rise of blood glucose after administration of d-glucose. Furthermore, the liver hydrolysate had almost no effect on the fasting blood glucose level, but tended to inhibit the increase of HbA1c. The plasma AngII concentration after the 12-week administration of liver hydrolysate remained significantly lower than that in the control group. This study confirmed that liver hydrolysate possesses ACE-inhibitory activity (IC50 value: 0.18 mg/mL). It also confirmed that liver hydrolysate possesses DPPH radical scavenging ability (55.6 μM Trolox equivalent/g). These results indicate that a component of liver hydrolysate inhibits d-glucose -induced increase of the blood glucose level, and may improve insulin resistance. The ACE-inhibiting effect and antioxidant effect of liver hydrolysate may be involved in this effect.

－141－ P-24 The enzyme-treated Asparagus offcinalis extract shows anti-stress effects in neural cells and prevents cognitive impairment in senescence-accelerated mice

Takuya Sakurai1, Kentaro Kitadate2, Hiroshi Nishioka2, Koji Wakame2, Hajime Fujii2, Junetsu Ogasawara1, Takako Kizaki1, Shogo Sato1, Yoshinaga Ishibashi1, Tomonori Fujiwara1, Kimio Akagawa1, Kazuhiko Imaizumi3, Daizoh Saitoh4, Tetsuya Izawa5 and Hideki Ohno1

1Kyorin University, School of Medicine, 2Amino Up Chemical Co., Ltd., 3Waseda University, 4National Defense Medical College Research Institute, 5Doshisha University, Japan

Undue psychological stresses in the modern society bring about various health impairments, such as sleep disorders and depressions. Moreover, sharp increases in the number of patients with dementia, involving Alzheimer’s disease (AD), are considered a grave public health problem. In neurodegenerative disorders involving AD, biological stresses induce neural cell damage. Therefore, strategies for the prevention of stress-induced neural cell damage are thought to be an extremely important public health problem. Asparagus offcinalis is a popular vegetable in the world, and the extract from this vegetable has been reported to possess various biological activities, such as antitumor and immunoprotective functions. In the present study, we investigated effects of the enzyme-treated Asparagus offcinalis extract (ETAS: Amino Up Chemical Co., Ltd., Sapporo) on the cell stresses in neural cells and cognitive impairment in senescence-accelerated mouse prone-8 (SAMP8) mice, which exhibit impaired learning and memory in their early stages and are thought to be animal models of AD. Neuroblastoma ×glioma hybrid NG108-15 cells were treated with ETAS (2 mg/ml), and the expression of several mRNAs was examined by DNA array and RT-PCR analysis. Expression of the gene for stress protein heat shock protein (HSP) 70 was upregulated in NG108-15 cells by treatment with ETAS, whereas the expression level of genes for other HSPs (HSP89 and 50) was not significantly changed by such treatments. Moreover, the expression of mRNAs for several anti-apoptotic factors, such as heme oxygenase 1 (Hmox) and growth differentiation factor 15 (GDF15), was also enhanced with ETAS. In addition, lactate dehydrogenase release from damaged NG108-15 cells increased definitely when the cells were cultured under culture medium containing nitric oxide donor sodium nitroprusside or hypoxia mimic reagent cobalt chloride. Expectedly, ETAS significantly attenuated such cell damages. Four-month-old male SAMP8 mice were given ETAS (1,000 mg/kg/day) for 15 weeks, and then conditioned fear memory test was conducted. Contextual fear memory, which is considered hippocampus- dependent memory, in SAMP8 mice is significantly impaired as compared with that in senescence-accelerated-resistant 1 (SAMR1) mice. ETAS definitely attenuated such cognitive impairment in SAMP8 mice. On the other hand, no significant differences in cued fear memory, which is considered amygdala-dependent memory, were observed between SAMR1 and SAMP8 mice. ETAS did not affect the cued fear memory of SAMP8 mice. These results suggest that ETAS enhances the expression of mRNAs for HSP70 and some anti-apoptotic factors, such as Hmox1. Moreover, ETAS exhibits anti-stress effects in neural cells, and has preventive effects on cognitive impairment in SAMP8 mice.

－142－ P-25 Effect of brown rice extracts on immune response in mice

Kayoko Kawakami1, Hitomi Mori2, Misugi Uraji1, Masayo Kimura1, Tadashi Hatanaka1 and Hideyuki Ito2

1Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, 2Okayama University, JAPAN

【Background 】It has come to our attention that concerns have been raised regarding the increasing patient of allergic disease such as pollenosis, asthma and atopic dermatitis in Japan. The prevention or modulation of immune response through the regulation of Th2 type helper T cell differentiation is recently demonstrated to attenuate symptoms which related to allergy. In fact, target the immune system by functional foods has a potential especially in the intestinal immune responses. This immune responses against pathogens by a secretion of immunoglobulin A (IgA) which is produced by Peyer’s patch (PP) in intestinal mucosa. On the other hand, several reports have revealed that brown rice had various biological activities. However, there are few reports about immune response. Here, we investigated the effect of brown rice extracts (BRE) on immune response in mice. 【Methods】Commercial brown rice protein (NutriBiotic Vegan Rice Protein) was extracted with distilled water at 40℃ for 15 hr and then lyophilized. The BRE were resuspended in distilled water and fractionated using an AmiconUltra-centrifugal filter. The molecular weight distributions of each preparation were analyzed by gelfiltrationchromatography (GFC). We evaluated the ability of BRE to enhance the secretion of IgA by culturing PP cells obtained from BALB/c mice. PP cells were cultured with or without BRE at 37℃ at a 5% CO2. Supernatants were collected on Day 7 to determine IgA production. We also evaluated the ability of BRE to induce the production of interferon (IFN)-γ and interleukin (IL)-4 by culturing murine splenocytes. Splenocytes were cultured in the presence or absence of BRE. Supernatants were collected on Day 5 to measure induced cytokines (IFN-γ and IL-4). The concentration of IgA and cytokines in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). In an in vivo experiment, female BALB/c mice were administered orally BRE (0, 20 or 200 mg/kg body weight) using a stomach tube for 21 days. Feces were collected, and IgA was determined by ELISA. 【Results】When a protein from brown rice was extracted for 15 hr, GFC peak area of molecular weight approximately 9,800 decreased as compared to a extraction for 3 hr. The treatment of 5%-15 hr extraction dose-dependently increased the production of IgA in PP cells and splenocytes. In order to determine the active component, we separated BRE by ultrafiltration. High molecular weight (>30,000) fraction enhanced IgA production. Moreover, BRE increased Th1 type cytokine IFN-γ production in a dose-dependent manner, but did not affect to the Th2 type cytokine IL-4 production in splenocytes. We further examined whether oral administration of BRE could have a beneficial effect on immune system. Fecal IgA was slightly higher in the group given 20 mg/kg BRE than that in the control group. The oral administration of BRE induced IFN-γ production by splenocytes in a dose-dependent manner. In contrast, oral administration of BRE had no effect on IL-4 production. 【Conclusion】These results suggest that BRE might improve the Th1/Th2 balance toward Th1 dominance.

－143－ P-26 Therapeutic effect of petit vert on mice with acute DSS colitis

Kohsuke Matsumoto1, Kenji Suzuki1, Yuki Nishizawa1, Masaki Yonezawa1, Yutaka Honda1, Junji Yokoyama1, Masaki Nagata2, Somasundaram Arumugam3, Rajarajan A. Thandavarayan3, Kenichi Watanabe3, Tetsuya Konishi3 and Yutaka Aoyagi1

1Niigata University School of Medicine, 2Niigata University School of Dentistry, 3Niigata University of Pharmacy and Applied Life Sciences, Japan

Petit vert is a hybrid of Brussels sprout and kale, and was newly developed in Japan. Compared with other vegetables, Petit vert contains high vitamine and minerals, and its health benefits has been recently attracting much attention. Inflammatory bowel disease (IBD) comprises ulcerative colitis and Crohn’s disease, and its prevalence has been increasing steadily in Japan as well as in European and North American countries. The pathogenesis of IBD is not fully understood, but is considered as an multifactorial process resulting from genetic factors, environmental factors, intestinal flora, which together result in a dysregulated innate and adaptive mucosal immune response. Western diets with rich contents in certain fats, polyunsaturated fatty acids, and meat, are considered to be risk factors for IBD, especially Crohn’s disease. In contrast, high vegetable intake is thought to protect against ulcerative colitis. In this study, we examined the therapeutic effect of Petit vert on mice with dextran sulphate sodium (DSS)-induced acute coltis, which is considered as an animal model for ulcerative colitis. Acute DSS colitis was induced in C57/BL/6 mice by administration of 3% DSS in drinking water for 6 days. Petit vert enema was performed on day 0, 2, and 4. The mice were divided into 3 groups: Group A (given normal water, and vehicle enema), Group B (given DSS, and vehicle enema), Group C (given DSS, and Petit vert enema). Clinical condition of each mouse was evaluated daily with Disease Activity Index (DAI) score. The mice were sacrificed on day 6, and the colon was taken and used for further analysis. Petit vert enema significantly ameliorated the clinical condition of acute DSS colitis such as body weight loss, diarrhea and bloody stool, shown by significant decrease of DAI score. The shortening of the colon is a typical feature of acute DSS colitis, and Petit vert also prevented the shortening of the colon significantly. Western blotting analysis for cytochrome c and Caspase 7 revealed that Petit vert ameliorated the elevation of these apoptosis-related molecules in the colon of DSS colitis. In contrast, Petit vert increased the expression of bcl-2, anti-apoptois molecule in the colon of DSS colitis. Western blotting for gp91, p22phox, and p57phox revealed that expression of these NADPH-related molecules were increased in DSS colitis, and Petit vert significantly decreased them. Comet assay showed that increased apoptosis was observed in the colon of acute DSS coltis, and that Petit vert significantly inhibited the apoptosis. These data indicate that Petit vert has the therapeutic effect on mice with acute DSS colitis through anti-oxidative stress and anti-apoptosis mechanism, suggesting Petit vert as an potential food relating complementary medicine for IBD.

－144－ P-27 The protective effect of squalene on inflammatory responses in BALB/c mice

Naoto Tatewaki, Hiroshi Nishida, Yurie Kuwabara, Yuuki Fuse, Takahiro Eitsuka and Tetsuya Konishi

Niigata University of Pharmacy and Applied Life Sciences, Japan

【Objective】Squalene (SQ) is a naturally occurring polyprenyl compound primarily known as the precursor of cholesterol biosynthesis. SQ has been used as a health beneficial dietary supplement with chemopreventive function and also as a biological response modulator to suppress the adverse effects associated with cancer chemo- and radio therapy. We previously demonstrated SQ is able to enhance tumor growth suppression by DOX in allograft mouse model. In the present study, we further examined the potential anti-inflammatory activity of SQ in different experimental mouse models. 【Methods】Experimental mice were used 8 week old male BALB/c. The anti-inflammatory activity of SQ was evaluated by acetic acid-induced writhing test and formalin-induced pain test, respectively. We also analyzed pro-inflammatory mediator prostaglandin E2 (PGE2) by ELISA. Writhing test: Different groups of BALB/c mice were treated oral administration of SQ (2 mol/kg, 200 μl as squalene oil) and Aspirin (500 mg/kg, Mineral oil of 200 μl as solvent). Muscular contraction was induced by intraperitoneal injection of 0.7 % acetic acid at a volume of 200 μl for 1 h after the treatment. The number of muscular contractions (writhes) was counted starting at 5 min after injection for a period of 10 min. Data represent the average of the total numbers of writhes observed. Formalin test: Mice were treated with oral administration of mineral oil (CT), SQ (2 mol/kg) or Aspirin (250 mg/kg) 30 min before injection of 5 % formalin at a volume of 10 μl. Formalin was injected in the paw of left leg of mice. Reaction to pain was quantified by counting the number of bites, licks, flinches, and shakes of the injected paw. Then, mice were observed for 60 min. ELISA: Colon26, mouse colon cancer cell line, was transplanted in the paw of left leg of male BALB/c mice. On day 18 of transplantation, blood was collected for the analysis. Blood samples were collected 1 h after SQ or Aspirin administration. 【Results and Conclusion】Oral administration of SQ and Aspirin significantly inhibited as compared to the CT group (about 30 % and 50%, respectively) in writhing test. The formalin test also tended to decrease count by SQ and Aspirin as compared to the CT. ELISA analysis showed that SQ decreased PGE2 level, a marker of inflammation, in the blood plasma. These data suggest that the complementary use of SQ will be promised in cancer therapies because of anti-inflammatory functions.

－145－ P-28 Anti-allergic immune effects of Aspergillus oryzae and Lactobacillus sakei on an ovalbumin induced allergy mice

Hideaki Suzuki1, Koji Kobayashi1, Koji Kobayashi1, Yuuko Kosuge1, Masami Igarashi1 and Tsuyoshi Sugiyama2

1Kitasato Junior College of Health and Hygienic Sciences, 2OTO corporation, Japan

Probiotics may be considered for treatment on the basis of their immunomodulating properties. Preclinical and clinical evidences for a role of oral probiotics in the management of allergic diseases are emerging. Allergy is an inflammation associated with an elevated T helper (Th) 2 lymphocyte responses to allergens and elevated serum IgE levels and cytokines. The filamentous fungus Aspergillus oryzae has been used in the Japanese food fermentation industry for more than 1,000 years. Although the Lactobacillus sakei as the number of lactic acid bacteria is often relatively low on the surface of vegetables, the anaerobic and low salt conditions engender the rapid growth of lactic acid bacteria at the end of the fermenting stages. We aimed at testing the immunomodulatory effects of intranasal versus intragastric administration of Aspergillus oryzae and Lactobacillus sakei in a model of ovalbumin (OVA) allergic mouse. Two probiotic bacterial preparations were obtained from OTO corporation (odawara, Japan). Six-week-old male BALB/c mice were divided into six groups: normal control, allergy control and four allergy groups each treated with Aspergillus oryzae 1mg/kg weight/day intake, Aspergillus oryzae 10mg/kg weight/day intake, Lactobacillus sakei 1mg/kg weight/day intake or Lactobacillus sakei 10mg/kg weight/day intake. To determine serum antibody responses, blood was collected after the first OVA stimulating 26th day. Sera were stored at -80℃ until measurement of OVA-specific IgE and total IgE levels using an enzyme-linked immunosorbent assay (ELISA). Spleen cells were collected the spleen of the OVA allergy induced mice, Splenpcyte ware cultured in vitro with OVA for three days, and amount of cell multiplication activity by WST-1 kit. In order to inspect the immunity activity of the macrophage with Aspergillus oryzae or Lactobacillus sakei, the mouse macrophage cell line, J774.1 (5×105 cells/mL) was stimulated with those probiotic bacterial preparations, and the levels of tumor necrosis factor α (TNF-α) and interleuin-12 (IL-12) production were determined using ELISA 24h after incubation. The statistical significance of mean differences were evaluated by one-way ANOVA with the Dunnett’s multiple comparison test using JMP (version 8.0.1). A P-value of less than 0.05 was considered statistically significant. Both Aspergillus oryzae and Lactobacillus sakei were significantly reduced the production of anti-OVA IgE antibodies, also decreased the splenocyte multiplication activity on OVA adding culture. On mouse macrophage cell line study, when it cultured with Aspergillus oryzae or Lactobacillus sakei, secretion of TNF-α was increased. These results show that it is possibility of Aspergillus oryzae or Lactobacillus sakei can reduce an allergic reaction by balancing Th1/Th2 immune responses and that it might be used in the OVA-induced allergy mice.

－146－ Section 4 P-29 Antioxidant activity of ramie and perilla leaves grown in Niigata

Tomoko Yamaguchi, Azusa Kawasaki and Jun-ichi Sakai

Niigata University, Japan

Ramie (Boehmiria nivea var. nipononivea) is a perennial plant in the nettle family, and grown in Japan, China, Korea, and as well as in Brazil. The stem of Ramie, Aoso, provides raw material for Japanese textile, especially Kimono for summer. The leaves contain large amount of potassium, calcium, and β-carotene. Perilla (Perilla frutescens (L.) Britton var. japonica Hara) is a yearly plant in the labiates family, and grown in China, Korea, and Southeast Asia. Perilla oil made by the seeds contain α-linolenic acid as main component of fatty acid. The leaves contain flavonoids such as luteolin, apigenin and rosmarinic acid, which possess antioxidant, anti-inflammatory, .and anti-allergic effects. In this study, we measured the total phenol content and antioxidant activity in the leaves of Ramie and Perilla grown at Odiya and Agano in Niigata. The total phenol content was measured by Folin-Ciocalteu method. The antioxidant activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) were also measured. In addition, we investigated the method of pretreatment of application to food of Ramie and Perilla, drying and boiling Five different types of Ramie and Pellira powder, A:freeze-dried powder, B: air-dried powder (dried at room temperature), C:air-dried powder (dried at 55℃), D:air-dried powder (dried at 80℃), E: freeze-dried powder after boiling, were prepared for investigation. The total phenol content and ORAC value of freeze-dried powder of Ramie were 1.5 times higher than that of Perilla. The DPPH radical-scavenging activity of freeze-dried powder of Ramie was the same as that of Perilla. Among the powder made by different dried methods, total phenol content, DPPH radical-scavenging activity, and ORAC value in freeze-dried powder were the highest, followed by air-dried powder (dried at room temperature), air-dried powder (dried at 80℃), and air-dried powder (dried at 55℃). After boiling of Ramie leaves, total phenol content, DPPH radical-scavenging activity, and ORAC value were decreased to 19, 6, and 14 %, respectively. In the case of Perilla, however, only a small decrease of total phenol content, DPPH radical-scavenging activity, and ORAC value, and these remain rate were 60, 47, and 66 %, respectively. It seems that the Ramie contain a lot of water soluble components, therefore, the large amount of phenolic components were released in boiling water after boiling. Antioxidants, which can neutralize free radicals, are known to have important function for prevention of cancer, aging, and several diseases induced by free radicals. Therefore, antioxidants in Ramie and Perilla leaves have received considerable attention for their role in human health.

－147－ Section 5 P-30 Effect of quercetin intake during lactation on the AMPK activation in the hypothalamus of adult male rat offspring programmed by maternal protein restriction

Masato Tanaka1, Mai Turuga1, Norihito Takahashi1, Aya Sasaki1, Miki Miyajima1, Ryo Nishimura1, Naoko Hishioka1, Yuuka Mukai2, Shin Sato2, Masaaki Kurasaki1, and Takeshi Saito1

1Hokkaido University, 2Aomori University of Health and Welfare, Japan

Fetal and neonatal environments are important determinants of disease risk in adult life. Epidemiological and experimental studies indicate a relationship between the periconceptional, fetal and early infant phases of life and the subsequent development of diseases as an adult. For instance, maternal low-protein diets are early-life inducers of glucose intolerance, hypertension, renal disease and obesity. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities including activation of adenosine-5’-monophosphate-activated protein kinase (AMPK). AMPK is a serine/threonine protein kinase that plays a central role in regulating cellular metabolism and energy balance. Hypothalamic AMPK regulates feeding behavior via the metabolic changes in nerve cells. We investigated the effects of quercetin intake during lactation on the AMPK activation in the hypothalamus of adult offspring programmed by maternal protein restriction during gestation. Pregnant Wistar rats were fed control and low-protein diets during gestation. Following delivery, each dam received a control or 0.2% quercetin-containing control diet during lactation as follows: control on control (CC), control on restricted (MC) and 0.2% quercetin-containing control on restricted (MCQ). At weaning (week 3), some of the pups from each dam were killed, and the remaining pups continued to receive a standard laboratory diet and were killed at week 23. The animals were weighed, and blood samples were collected under anesthesia, and the brains were dissected into 7 discrete regions (cerebellum, medulla oblongata and pons, hypothalamus, mid-brain and thalamus, striatum, hippocampus, and cerebral cortex) by the method of Glowinski and Iversen (1996) on an ice-cold plastic plate. The samples were then transferred to a desalted vessel and kept at -80℃ prior to experimentation. Blood chemistry (insulin and triglyceride) and phosphorylation levels of AMPKα in the hypothalamus of male offspring were examined. The body weights of the MC group were lower than those of CC group, on the other hand, the body weights of MCQ group were same levels of CC group. The levels of insulin in MCQ group were significantly higher than those in MC group at week 23. The levels of plasma triglyceride in MCQ group were significantly lower than those in CC group at week 3. On the other hand, the levels of plasma triglyceride in MCQ group significantly higher than those in CC group at week 23. The abundance of phosphorylated AMPK protein that in MCQ group in the hypothalamus increased compared with that in MC group at week 23. In conclusion, quercetin intake during lactation up-regulates phosphorylated AMPK in the hypothalamus of adult offspring of protein-restricted dams and may up-regulate the feeding behavior. －148－ P-31 Isomaltulose used as a sugar substitute for rice cooking controls changes in blood glucose and insulin response

Kiyoaki Miyasaka1, Yumiko Tezuka1, Kaori Araki2, Kazuki Mochizuki3, Masae Sakuma2, Hidekazu Arai2 and Yoko Ichikawa2

1Mitsui sugar Co., Ltd., 2University of Shizuoka, 3University of Yamanashi, Japan

【Background】Carbohydrate intake-derive postprandial hyperglycemia is considered to have relation on the increase of the risk of diabetes and cardiac diseases. For Japanese people, rice diet has a major impact on postprandial glycemic response. We have been studying the physiological effect of carbohydrate intake, with a particular focus on digestion and absorption speed in the small intestine. Isomaltulose (6-O-D-glucopyranosyl-D-fructose, Palatinose®) is a natural disaccharide contained in honey, which has a caloric value of 4 kcal/g, the same as that of sucrose. Isomaltulose is digested slowly, resulting in a slow availability for absorption, and is reported that it delays the digestion and absorption of other carbohydrates. In this study, we investigated isomaltulose impact on blood glucose and insulin as substitute sweetener in rice cooking. 【Methods】Experiments was performed on 9 normal healthy volunteers, who belong to University of Shizuoka (aged 19.3±0.5 yrs, BMI 21.4±2.4). After overnight fast, blood glucose and insulin were measured. Japanese dishes including rice as main components (like rice bowl dishes and Sushi etc.) were prepared by using equal amounts of isomaltulose or sucrose as a sweetener and both were given to the subjects on different days. Blood glucose was measured by Self-Monitoring of Blood Glucose every 15min thereafter for a period of 2h. Insulin response was measured at 30min and 60min after taking dishes. Differences between sucrose and isomaltulose group were compared based on the variation from the fasting state. Statistical analysis of all date in this study was performed with Student’s paired t-test (p<0.05). The study design was approved by the ethical committee of University of Shizuoka. 【Results】Sanshoku rice bowl (minched chicken, sakura denbu and egg bowl) (15, 30min), Teriyaki-chicken rice bowl (15, 60min) and Inari-zushi (fried bean-curd stuffed with sushi rice) (120min) prepared with isomaltulose showed significantly lower blood glucose rise than those of sucrose (P<0.05). There is no significant difference in insulin secretion between the groups. 【Overview】In this study, it was found that isomaltulose used rice cooking have lower postprandial glucose reaction than those of sucrose, without the increase of insulin secretion. Slow digestion and absorption of carbohydrates intake has many physiological benefits, such as prevention of visceral fat accumulation, improvement of insulin sensitivity and sustaining a sense of satiety. We focused on the absorption speed of carbohydrate and lunched “Slow Calorie Project”. With approved companies and researchers, we continue to promote and to research for the importance of intake slowly-absorbable carbohydrate.

－149－ P-32 Effect of quercetin intake during lactation on the AMP-activated protein kinase activity in the livers of adult male rat offspring programmed by maternal protein restriction

Shin Sato and Yuuka Mukai

Aomori University of Health and Welfare, Japan

Fetal and neonatal environments are important determinants of disease risk in adult life. For instance, maternal low-protein diets are early-life inducers of glucose intolerance, hypertension, renal disease, and obesity. Epidemiological and experimental studies have indicated a relationship between the periconceptional, fetal, and early infant phases of life and the subsequent development of diseases as an adult. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities including activation of AMP-activated protein kinase (AMPK). We investigated the effects of quercetin intake during lactation on the AMPK activation in the livers of adult offspring programmed by maternal protein restriction during gestation. Pregnant Wistar rats were fed control and low-protein diets during gestation. Following delivery, each dam received a control or 0.2% quercetin-containing control diet during lactation as follows: control on control (CC), control on restricted (LPC), and 0.2% quercetin-containing control on restricted (LPQ). At weaning (week 3), some of the pups from each dam were killed, and the remaining pups continued to receive a standard laboratory diet and were killed at week 23. Blood chemistry and phosphorylation levels of AMPKα, acetyl-CoA carboxylase (ACC), endothelial nitric oxide synthase (eNOS), and mammalian target of rapamycin (mTOR) in the livers of male offspring were examined. Body weights among CC, LPC, and LPQ at week 3 were not significantly different. However, at week 23, the body weights of the LPQ group were higher than those of the LPC group and comparable to those of the CC group. The level of total AMPK protein did not change at postnatal week 3 in any of the groups. However, the level of phosphorylated AMPK protein in LPQ increased about 1.5- and 2.1-fold compared with LPC and CC, respectively. Likewise, the protein abundance of phosphorylated AMPK in LPQ offspring at week 23 was higher than in CC and LPC offspring, indicating that quercetin intake during lactation activated AMPK in the livers of adult offspring of dams fed a protein-restricted diet. A significant increase in phosphorylated ACC and eNOS levels was found in LPQ. There was no significant difference among the 3 groups in the level of phosphorylated mTOR protein. In conclusion, the feeding of quercetin, an AMPK activator, to protein-restricted dams during lactation is likely to upregulate AMPK activation and may, at least in part, lead to long- term alterations of AMPK pathway in the livers of adult offspring of protein-restricted dams.

－150－ Section 6 P-33 Antihypertensive effect of boysenberry seed polyphenols on spontaneously hypertensive rats and identification of orally absorbable proanthocyanidins with vasorelaxant activity

Ryo Furuuchi, Akito Matsushima, Yuta Nagakura, Tadayuki Yokoyama and Masao Hirayama

Bourbon Corporation, Japan

【Background and Aim】Hypertension is one of the major risk factors for cardiovascular diseases. Recently, we reported that boysenberry seeds contained five polyphenoilc classes, rich of short-oligomeric proanthocyanidins1). The aim of this study was to assess the antihypertensive effect of boysenberry seed polyphenols (BSP) after orally administrating to spontaneously hypertensive rats (SHRs) and to investigate candidates for the active components by determining their oral absorbability in vivo and vasorelaxant activity in vitro. 【Methods and materials】Defatted boysenberry seeds were extracted with 60% aqueous ethanol, and the polyphenols were fractionated through an Amberlite XAD-7HP column to afford a boysenberry seed polyphenols XAD fraction (PPh fr). Further fractionation of PPh fr was carried out as follows. After a solution of PPh fr in water was extracted with hexane:ethyl acetate (3:1 v:v) three times to remove highly lipid-soluble substances, the water fraction was then partitioned with ethyl acetate seven times. The resulting ethyl acetate and water fractions were evaporated in vacuo, leaving proanthocyanidin (PA fr; PA content, 8.88%) and ellagitanin fractions (ET fr; PA content, none detectable), respectively. In vitro vasodilatory activity was measured using rat aorta rings and the mechanism was deduced in comparison with and without specific inhibitors on vasorelaxation. Effects of BSP on blood pressure were measured for 24 hr after a single oral administration to SHR. In a separate set of experiments, the PA fr were similarly administered to SHRs. After 4 h, the rats from both groups were injected intraperitoneally with L-NAME and SBPs were measured after 2 h. Orally absorbable compounds were detected in plasma by LC/MS/MS. 【Results and conclusions】A single oral administration of PPh fr was found to decrease the systolic blood pressure significantly, reaching its minimum at 6 hr, -17.3 mmHg of baseline. When the PA or ET fraction was orally administered to SHRs, a significant decrease in SBP was observed only in the PA fr group. This SBP decrease was abolished by an NG-nitro-L- arginine methyl ester (L-NAME) injection. An analysis of the orally absorbable components showed that intact dimeric and trimeric procyanidins and propelargonidins were detectable in the plasma with a maximal concentration 2 h post administration. The vasorelaxant activity of PA fr was also confirmed by in vitro assay using rat aorta rings. These results suggest that proanthocyanidins in boysenberry seeds may have played an important role in the observed antihypertensive effect. 1) Furuuchi R, et al, J Agr Food Chem, 2011, 59, 3738-46.

－151－ P-34 Novel functions of Xylose to maintain slow proliferation and undifferentiated state of ES cells

Sakiko Takizawa-Shirasawa1, Tadayuki Yokoyama1, Susumu Yoshie2, Ken Matsumoto3, Mika Nagai1 and Katsunori Sasaki2

1Bourbon Corporation, 2Shinshu University of Medicine, 3Nissui Pharmaceutical Co.LTD., Japan

In order to define the effects of saccharides on mouse embryonic stem cells (ES Cells), embryoid body (EB) formation and EB outgrowth, D-glucose of DMEM was replaced respectively with D-cellobiose, D-fructose, D-galactose, maltose monohydrate, D-raffinose, sucrose, trehalose or D-xylose, and containing with glucose-deprived knockout-serum replacement (KSR) as ES medium and with glucose-deprived fetal bovine serum (FBS) as EB medium. Proliferation and undifferentiated state of ES cells were tested in the control medium containing glucose and were also observed moderately in Xylose, Galactose, Fructose or Maltose-contained medium. EB’s formation was poor in these saccharides, but their outgrowth in xylose and galactose was unique in their morphology and in the view of undifferentiated marker expression. Moreover, in addition to gene expression, the protein expression of undifferentiated markers such as Oct3/4 and Nanog were also observed only in Xylose-cultured EB with immunocytochemistry. It was speculated that xylose could have a novel function. Therefore, further experiments were performed focusing on Glucose and Xylose. ES colonies cultured in Xylose for a long term did not change so much in morphology, and changed drastically in Glucose. The expression of undifferentiated markers such as Oct- 3/4, Nanog and Rex-1 was kept in both monosaccharides for 8 days. High level expression of Oct 3/4 either with real-time PCR or immunocytochemistry, though LIF and feeder cells which are necessary to keep undifferentiation status of ES cells were used in glucose, but not in xylose culture medium. EB formed in Xylose showed undifferentiated state for longer time than in Glucose. In addition, Co-culturing with PA6, though ES colonies differentiated into Tuj1-positive cells in Glucose, they remained undifferentiated in Xylose. These results showed xylose has novel functions to maintain slow proliferation, long-time survival and undifferentiated status of ES cells without LIF.

－152－ P-35 Effect of Isomaltulose on liver and skeletal muscle glycogen accumulation in rats

Katsumi Sasagawa1, Shigeru Mineo1, Hiroshi Nishida2, Shinji Sato2 and Masao Hirayama1

1Bourbon Corporation, 2Niigata Univ. of Pharm. and Appl. Life Sci.,Japan

【Background/Aim】Recent studies showed that liver and skeletal muscle glycogen played an important role in enhancing the performance in the endurance-related exercise. It is known that glycogen accumulation is closely associated with the type of carbohydrate and the intake timing. Isomaltulose (Palatinose®, 6-O-D-glucopyranosyl-D -fructose) is a sucrose isomer naturally found in honey. It is commercially produced by enzymatic transformation from sucrose. Physiological studies demonstrated that isomaltulose was digested slowly compared to sucrose (5-fold) though it had equivalent energy (4kcal/g) with sucrose. Thus, isomaltulose has a potential to attenuate a rapid increase of blood glucose level after intake. However, a detail of metabolic pathways after isomaltulose absorption remains unclear. This study aims to investigate how isomaltulose administration contributes to glycogen accumulation in rats. 【Methods】Male Wistar rats were divided into three groups (saline, sucrose and isomaltulose administration group), and experiments were performed as follows. Glycogen content: Saline and 5% aqueous saccharide solutions were given freely, and 24 hr later liver and skeletal muscle were collected surgically. Glycogen content was measured by enzymatic method. Intravenous glucose tolerance test (IVGTT): Saline and two test saccharides were administered orally, and then glucose was administered intravenously 90 min post administration. Blood was collected by the jugular cannula, and the plasma glucose and insulin levels were monitored for 120 min. The plasma glucose area under the curve (AUC) was calculated from the plasma glucose level until 30 min after intravenous administration of glucose. Western blot analysis: Skeletal muscle was collected 90 minutes after the oral administration of test saccharides. Phosphorylation levels of Akt and AMPK in skeletal muscle were detected by using phosphor- specific antibodies. 【Results】The liver glycogen content in the isomaltulose group was significantly higher than that of sucrose group (p < 0.05). The skeletal muscle glycogen contents in the sucrose and the isomaltulose treated groups were slightly increased compare to saline group though there were no significant differences. The decrease of plasma glucose level in the isomaltulose group was earlier than that of other groups. The plasma glucose AUC in the isomaltulose group was significantly lower than in the saline group (p < 0.05). The insulin levels were almost same among three groups. Akt phosphorylation was increased in the isomaltulose group compared with the saline group. AMPK phosphorylation level had no significant difference among three groups. 【Conclusion】In this study, liver glycogen content after isomaltulose administration was found to increase than that after sucrose. The stimulation of the glucose uptake through the Akt signal might be associated with the increase of glycogenesis. These results suggest a possibility that isomaltulose had the potential to enhance an endurance performance through glycogen accumulation.

－153－

Poster Session Abstract

MEXT*-Supported Program for the Strategic Research Foundation at Private Universities, 2010-2012

Special INSDH poster session on “Establishing the base for developing next generation functional food research by collaboration of pharmaceutical and food sciences and for its practical application for health promotion and complementary medicine”

MEXT*-Supported Program for the Strategic Research Foundation at Private Universities, 2010-2012

Food as medicine is well known tale since ancient period in human history both in oriental and western society. Current development of the sciences on functional food revived this idea from the molecules called food factor, many active ingredients are identified in the food resources and their mechanism of function is being unveiled. Thus the role of food in the health preservation and promotion attracted much attention and functional or health foods are getting established its social status and a new category of food is legally established in certain countries such as the food for special health use (HOSHU) in Japan. As understanding the molecular bases of how the food ingredient functions, the dose issue becomes more apparent for effective and safely application of food functions in either disease prevention or disease control. In the present project, the research teams from Pharmaceutical and Life Science Faculty of Niigata University of Pharmacy and Applied Life Sciences formed a core to establish the scientific background for developing the new category of application of food functions in human health and medicine, that is, the next generation functional food. We simply categorized the food functions into two in terms of the function, one is the preventive and nourishing function (Shokuyou) and the other is the complimentary function in medical treatments (Shokuryou). In the former category, the food functions are materialized in the forms of supplement or health food for promoting quality of life (QOL) and disease prevention such as by modulating immune system, mental activity, motor activity and antiaging. In the latter category, the food called such as medicinal food can be applied clinically together with western medical treatment to enhance the treating efficacy and decrease the adverse effect, or accelerate recovery. In this case, the mechanism of function has to be fully clarified and the effective and toxic doses also have been determined. Here in the 6th INS, we are happy to have a special session on this project and shire the time to have discussion with the participant of 6th INS. Critical and suggestive comments are highly welcomed.

Tetsuya Konishi Project leader and coordinator

－157－ Section 7 P-36 Amadori-glycated phosphatidylethanolamine up-regulates telomerase activity -A link between diabetes and cancer-

Takahiro Eitsuka1, Kiyotaka Nakagawa2, Hiroshi Nishida1, Tadao Kurata1 and Teruo Miyazawa2

1Niigata University of Pharmacy and Applied Life Sciences, 2Tohoku University, Japan

【Objectives】 Membrane phospholipids such as phosphatidylethanolamine (PE) are abnormally glycated under hyperglycemic conditions, and lipid glycation may contribute to the pathogenesis of diabetic complications (e.g., retinopathy, nephropathy, neuropathy, and atherosclerotic macrovascular disease). We previously found that Amadori-PE (an early glycation product of PE) caused lipid peroxidation and angiogenesis, and demonstrated its accumulation in blood plasma of diabetic patients. Numerous epidemiologic studies suggest that type 2 diabetes significantly increases overall cancer risk and mortality. Recent meta- analysis estimated that individuals with diabetes have a two-fold greater relative incidence of pancreatic cancer compared with individuals without diabetes. Although there are many epidemiologic researches suggesting the involvement of diabetes in tumor development, little is known regarding the molecular mechanism underlying this phenomenon to date. Hence, we hypothesized that Amadori-PE may participate not only in the pathogenesis of diabetic complications but also in tumor progression. In the present study, this hypothesis was investigated in cell-culture study, with particular emphasis on the effect of Amadori-PE on telomerase activity which contributes to the infinite replicative potential of cancer cells. 【Methods and results】PANC-1 human pancreatic carcinoma cells were cultured in medium containing Amadori-PE. Telomerase activity was examined by stretch PCR assay. Amadori- PE induced cellular telomerase activity of PANC-1 in time- and dose-dependent manner. Treatment of the cells with 5 μM Amadori-PE resulted in a 2-fold increase in telomerase activity compared with control. On the other hand, intact PE had virtually no effect on telomerase activity. To evaluate the mechanism for telomerase induction by Amadori-PE, real- time RT-PCR and Western blotting were performed. Amadori-PE elicited cellular telomerase activity by up-regulating hTERT (human telomerase reverse transcriptase) mRNA expression through induction of c-myc expression. A 2',7'-dichlorofluorescin (DCFH) assay revealed that Amadori-PE significantly augmented intracellular ROS levels in PANC-1. We also found that telomerase activity was significantly decreased by a combination treatment of Amadori-PE and α-tocopherol, an antioxidant vitamin, suggesting that Amadori-PE up-regulates telomerase activity through intracellular ROS generation. 【Conclusion】These results indicate for the first time that Amadori-PE may be an important compound that promotes tumor progression as a result of telomerase activation and provide experimental evidence for a novel role of Amadori-PE in linking diabetes and cancer. Our observation also raises the possibility that α-tocopherol might contribute to a reduction in cancer risk in diabetic patients.

－158－ P-37 Involvement of AMPK-PKC-ζ-NF-κB signaling pathway and nitrosative stress on development of diabetic cardiomyopathy in streptozotocin- induced diabetic rats and its prevention by curcumin

Vivian Soetikno, Arun Prasath Lakshmanan, Somasundaram Arumugam, Vigneshwaran Pitchaimani, Meilei Harima, Kenji Suzuki and Kenichi Watanabe

Niigata University of Pharmacy and Applied Life Sciences, Indonesia

【Objectives】Macro- and microvascular disease are the most common cause of morbidity and mortality in patients with diabetes mellitus. Hyperglycemic episodes, which complicate even well-controlled cases of diabetes, are closely associated with increased oxidative and nitrosative stress, which can trigger the development of diabetic complications. Cardiomyopathy in diabetes is associated with a cluster of features including decreased diastolic compliance, interstitial fibrosis, and myocyte hypertrophy. It is also characterized by excessive lipid accumulation, with increased triacylglycerol stores, and inflammation in left ventricle. We hypothesized that chronic treatment with curcumin, a powerful antioxidant, would normalized diabetes-induced cardiomyopathy through modulation of AMP-activated protein kinase (AMPK)-protein kinase C (PKC)-ζ-nuclear factor (NF)-κB pathway and nitrosative stress. 【Methods】Diabetes was induced in male Sprague Dawley rats by single intraperitoneal injection of streptozotocin (STZ) (55 mg/kg). After three weeks of STZ injection, diabetic rats were divided into two groups and treated with either curcumin (100 mg/kg/day) or vehicle by gavage for 8 weeks. Age-matched normal rats without STZ injection were also included in this study. At termination, rats were surgically prepared for hemodynamic measurement, subsequent to which their hearts were removed to evaluate cardiac performance, histological and biochemical changes. We performed Western blot analysis for the measurement of myocardial AMPK, PKC-ζ, and NF-κB activities, endothelial nitric oxide synthase (eNOS), and transforming growth factor (TGF)-β1. In addition, we employed hematoxylin eosin, Azan Mallory, and Oil Red O staining methods to demonstrate the hypertrophy, fibrosis, and lipid accumulation, respectively. Further, the mRNA expression of fatty acid synthase (FAS) and acetyl CoA-carboxylase (ACC) were assessed by reverse transcription-polymerase chain reaction analysis. 【Results】Myocardial functional parameters were significantly improved by treatment with curcumin compared with vehicle-treated rats. Diabetes-induced decreased phosphorylation of AMPK was associated with translocation of PKC-ζ and NF-κB to membranous and nuclear fractions, respectively. Curcumin could significantly prevent these changes. Diabetic rats also showed relative cardiac hypertrophy and a higher protein expression of eNOS and TGF-β1 as well as a higher mRNA expression of FAS and ACC; treatment with curcumin could ameliorate these changes in diabetic cardiomyopathy. Furthermore, curcumin significantly lowered plasma glucose, plasma triglyceride, lipid peroxidation, and increased anti-oxidant enzyme activity. Finally, lipid accumulation as determined by Oil-Red O staining was decreased by curcumin. 【Conclusion】Our results suggest that curcumin treatment, by increasing phosphorylation of AMPK and inhibiting PKC-ζ and NF-κB activities, may be useful as an adjuvant therapy for the prevention of diabetic cardiomyopathy.

－159－ P-38 Mulberry leaf diet protects against progression of experimental autoimmune myocarditis to dilated cardiomyopathy via modulation of oxidative stress and adverse cardiac remodeling

Somasundaram Arumugam, Rajarajan Amirthalingam Thandavarayan, Vivian Soetikno, Vigneshwaran Pitcahimani, Meilei Harima, Kenji Suzuki, Makoto Kodama and Kenichi Watanabe

Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan

Mulberry is commonly used as silkworm diet and an alternative medicine in Japan and China, has recently reported to contain many antioxidative flavanoid compounds and having the free radical scavenging effects. Antioxidants reduce cardiac oxidative stress and attenuate cardiac dysfunction in animals with pacing-induced congestive heart failure. It is of interest to study the effect of dietary antioxidants against oxidative stress mediated ailments. Hence we investigated the cardioprotective effect of mulberry leaf powder in rats with experimental autoimmune myocarditis. In this study, we used a rat model of cardiac myosin-induced experimental autoimmune myocarditis to test the effects of ML diet (MLD) (5%) on various markers of cardiac remodeling and function. After 4 weeks of immunization, the rats were fed with 5% MLD for 4 weeks. By the end of the study, echocardiography was performed to assess the myocardial dimensions. The heart tissue was used for histopathology and western blotting analyses. Our study showed that the post-myocarditis rats exhibited increased oxidative stress and MLD supplementation suppressed this change, compared to vehicle treatment. In addition, postmyocarditis rats showed significant elevation of the endoplasmic reticulum stress markers, which was prevented by the MLD supplementation. Similarly the vehicle treated rats suffered with the adverse myocardial remodeling in the form of fibrosis as evidenced by the Azan- Mallory staining and immunohistochemistry for collagen-III levels, compared with the control rats. However, MLD treatment not only markedly attenuated cardiac fibrosis, but also improved the left ventricular ejection fraction and fractional shortening. Interestingly, the myocardial levels of hypertrophy markers were also significantly attenuated by MLD, indicating its antihypertrophic effects. Collectively, these results suggest that supplementation of rats with 5% MLD has the ability to regulate cardiac remodeling and improves cardiac function and hence contributes to prevent the development of postmyocarditis dilated cardiomyopathy.

－160－ P-39 Dominant negative 14-3-3η promotes the myocardial apoptosis through the angiotensin-II type 1 receptor dependent-down-regulation of AMPK in the early stage of type 1 diabetes mellitus in mice

Arun Prasath Lakshmanan, Flori Ratna Sari, Vivian Soetikno, Harima Meilei, Sayaka Mito, Somasundaram Arumugam, Vigneshwaran Pitchaimani and Kenichi Watanabe

Niigata University of Pharmacy and Applied Life Sciences, Japan

Previously, we have reported that the transgenic (TG) dominant negative (DN) 14-3-3η promotes the myocardial apoptosis through the activation of JNK in the early stage of type 1 diabetes mellitus (DM) induced by streptozotocin (STZ-150 mg/kg, bw) in mice. The AMP- activated protein kinase (AMPK) is considered as the ‘master sensor’ of cellular energy balance and its up-regulation or down-regulation for the attenuation of myocardial apoptosis is remains to be controversial. Moreover, recently unearthed angiotensin-II (AT)/AMPK cascade could also implicate in the mechanism of apoptosis signaling. So, therefore, in this study we tried to elucidate the principle mechanism behind the exacerbated myocardial apoptosis in the DN 14-3-3η, using metformin at a dose of 250mg/kg, bw - an AMPK activator and olmesartan at a dose of 5 mg/kg, bw – a specific AT-1R blocker) treatment during the early stage (on day 7) of type 1 DM in the wild type (WT) and TG (DN-14-3-3η) mice . We employed western blot analysis for the measurement of myocardial protein expressions, such as TGF-beta activated kinase 1 (Tak1), p-AMPKα, angiotensin-II type 1 receptor (AT-1R), angiotensin-II type 2 receptor (AT-2R), phospho-c-Jun N-terminal kinase (p-JNK) and caspase-12 and TUNEL assay for the measurement of myocardial apoptosis in the WT and TG mice. The phosphorylation of AMPKα was not activated in the diabetic DN 14-3-3η mice when comparison with the WT mice, and the metformin and olmesartan treatment, were involved for the activation of p-AMPKα in the diabetic DN-14-3-3η mice and down-regulation of p-AMPKα in the diabetic WT mice. There were no significant differences in the protein expressions of AT-1R and AT- 2R between the diabetic TG and WT mice. In addition, the myocardial protein expressions of p-JNK and caspase-12 were significantly up-regulated in the diabetic TG mice, in comparison to the diabetic WT mice. Interestingly, metformin and olmesartan treatment significantly elevated the phosphorylation of JNK, but not the caspase-12 protein expression in the diabetic WT mice. These results indicate for the first time that the 14-3-3η protein could play an unprecedented role for the attenuation of myocardial apoptosis through the up-regulation of p-AMPKα in the presence of AT-1R-dependent manner in the early stage of type 1 DM condition and any chemical entity which modulates or stimulates the 14-3-3η protein could provide a new strategic treatment for the treatment of diabetes-associated cell death.

－161－ P-40 The differential regulation of brain mitogen-activated protein kinase cascade in rats after the induction of diabetes mellitus and heart failure

Vigneshwaran Pitchaimani, Arun Prasath Lakshmanan, Vivian Soetikno, Vijayakumar Sukumaran, Harima Meilei, Sayaka Mito, Somasundaram Arumugam and Kenichi Watanabe

Niigata University of Pharmacy and Applied Life Sciences, Japan

It has been strongly believed that diabetes mellitus (DM) is closely associated with the emergence of higher rates of neurodegenerative disorders, especially Alzheimer`s disease. In addition, inflammatory response has also been proposed for the neurodegenerative disorders. But the exact mechanism for the pathogenesis of brain disorders is not fully known. Furthermore, mitogen-activated protein kinase (MAPK) is also known as `stress response cascade` has been demonstrated to be activated in presence of hyperglycemia and inflammation, and has been implicated in brain disorders. So, therefore, in this study we attempted to find out the regulation of brain MAPK and its downstream pathway after the induction of DM by streptozotocin, and heart failure (HF) by cardiac myosin injection in rats. The protein expressions of oxidative stress markers as well as the malonaldehyde (MDA) level, angiotensin-II type 1 receptor (AT-1R), p-p38 MAPK and p-Tau, TGF-beta activated kinase 1 (Tak1), cytochrome C, and caspase-12, were significantly increased, whereas the protein expression of angiotensin converting enzyme-2 (ACE-2) was significantly down- regulated, in brain from the DM- and HF-induced rats, in comparison to their respective normal brain. In addition, increased phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular regulated kinase 1 and 2 (ERK1/2), and slight increase in phosphorylation of phosphoionositide-3 kinase (PI3K) and protein kinase B (Akt), Bcl-2 protein expression were observed only in brain from HF-induced rats, and significant attenuation in the phosphorylation of JNK, ERK1/2, PI3K and Akt were observed in brain from DM-induced rats, in comparison to their respective normal brain. Furthermore, interestingly the phosphorylation of p38 MAPK, Tak1 and Tau, oxidative stress markers, caspase-12, decrease in ACE-2 expressions were found to be higher in brain from DM-induced rats than brain from HF-induced rats. Taken together, it is suggested that both DM and HF conditions are prone to cause neurological disorders through differential activation of MAPK cascade and DM condition due to increased oxidative stress might play more dominant role for the pathogenesis of brain damage rather than HF condition.

－162－ P-41 New inhibitory effect of flavanones and isoflavones on cholesterol synthesis

Saori Nakagawa, Mayuri Watanabe, Tomoko Tanaka and Susumu Yamato

Niigata University of Pharmacy and Applied Life Sciences, Japan

【Backgrounds】Many studies show that an increased cholesterol level in blood is one of the major risk factors for atherosclerosis or coronary heart diseases. Lowering cholesterol level in blood is, therefore, one of the approaches for the prevention of these diseases. Several studies using animal models, cell cultures or biochemical tools suggest that polyphenols have a hypocholesterolaemic effect. The studies of mechanisms of lowering plasma cholesterol have focused on the inhibition of lower cholesterol biosynthesis since it reported only a few substance inhibited the lower cholesterol biosynthesis. This lower cholesterol biosynthesis, there are two alternative pathways, i.e., via lathosterol and via desmosterol, from lanosterol to cholesterol. In this study, we have examined that effects of polyphenols on cholesterol biosynthesis pathways were evaluated by measuring the level of cholesterol precursors (squalene, desmosterol, 7-dehydrocholesterol and lathosterol) and cholesterol in human hepatoma cells by gas chromatography-mass spectrometry (GC-MS). 【Materials & Methods】HepG2 cells, a human liver carcinoma cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). After 10 hours cultivation, medium were changed to DMEM containing 1% penicillin/streptomycin and 10% lipoprotein deficient bovine serum (LPDS) to promote cholesterol biosynthesis, and then added polyphenols(hesperetin, naringenin and eriodictyol of citrus flavonoid, daidzein and genisitein of soybean isoflavones to the cell culture medium. The concentration ranges of polyphenols were examined from 0.1 to 100 μmol/L. After 36 or 72 hours cultivation, the cells were collected, and then pretreated by saponification with potassium hydroxide. After neutralization with phosphoric acid, cholesterol precursors and cholesterol were extracted with n-hexane and then they were derivatized with trimethylsilyl (TMS) reagent. The TMS-derivatives of cholesterol precursors and cholesterol were determined by GC-MS with selective ion monitoring (SIM). 【Results】Hesperetin, naringenin and eriodictyol of citrus flavonoids decreased the level of cholesterol, however, increased that of lathosterol and 7-dehydrocholesterol with a dose dependent manner, suggesting they inhibit sterol-delta7-reductase (DHCR7). Daidzein and genisitein of soybean isoflavones slightly decreased the level of cholesterol, but increased that of desmosterol suggesting that it inhibits sterol-delta24-reductase (DHCR24). It was recognized that polyphenols (hesperetin, naringenin, eriodictyol, daidzein and genistein) have a new inhibition site on cholesterol biosynthesis pathway (DHCR7 or DHCR24).

－163－ P-42 Herbal extracts against fatty liver

Shinichi Ichikawa, Tomohiro Takahashi, Yuya Takiguchi, Kento Takizawa, Wataru Sugawara and Michiyo Nagano-Ito

Niigata Univ. of Pharm. & Appl. Life Sci., Japan

【Introduction】Fatty liver is a condition where lipid droplets accumulate in liver cells. Lipid droplets are intracellular storage sites of triglyceride. The main cause of fatty liver is excess calorie intake. Fatty liver induces inflammation and may finally cause cancer. Thus, herbs which inhibit the formation of lipid droplets are of great importance. In the present study, we report inhibitory effect of herbal extract(s) on lipid droplet formation in liver cells. 【Methods】Cell based assay-A mouse hepatoma cell line Hepa1-6 was cultured over night in DMEM supplemented with 10% FBS, oleate conjugated with bovine serum albumin, and herbal extracts to be tested. The next day, the cells were fixed and lipid droplets formed by excess oleate were visualized by Oil Red O staining. For the extracts with the inhibitory effect, the lipid pattern of the extract-treated cells was further analyzed. One of the extracts (hereafter designated as Herb A extract) which showed strong inhibitory effect on oleate-induced lipid droplet formation was further examined by mouse model of fatty liver. Animal experiments-Male C57/BL6J mice were used throughout the experiments. Mice were divided into three groups (n=6 per group), ND, HFD and HFD + herb A groups. ND group was fed normal diet. HFD and HFD + herb A groups were fed with a high fat diet HFD32 (CLEA Japan, Inc., Tokyo Japan). Herb A extract was orally administered in mice in HFD + herb A group. Four weeks after, the mice were sacrificed and liver samples were collected. Histological studies were carried out on liver samples. The use of animals complied with the guidelines established by Animal Care Comitee of Niigata University of Pharmacy and Applied Life Sciences. 【Results and Conclusions】Extracts from 199 herbs have been tested. Among them, 4 herbal extracts had inhibitory effect. The decrease of triglyceride content was observed in the cells treated with all the extracts. The mechanisms of the triglyceride decrease are now under examination. In the mouse experiments, intake of herb A extract did not affect body weight gain in mice fed by HFD32, although it protected fatty liver effectively. The result suggests direct effect of herb extract on liver cells. Oil Red O staining of cryosectioned liver tissue showed smaller number and size of lipid droplets in herb A extract-treated mice. In addition, dramatic decrease of triglyceride content in liver was observed in herb A extract-treated mice. Our studies suggest herb A is promising herb for preventing or treating fatty liver. MEXT*-Supported Program for the Strategic Research Foundation at Private Universities, 2010-2012

－164－ P-43 A vinegar production process using simultaneous fermentation by Saccharomyces cerevisiae and Acetobacter pasteurianus

Toru Shigematsu1, Sakae Ikarashi1, Mayumi Hayashi1, Akinori Iguchi1 and Masao Hirayama2

1Niigata Univ. Pharm. Appl. Life Sci. (NUPALS), 2Bourbon Corporation, Japan

Using an organic-acid resistant strain of Acetobacter pasteurianus NBRC3283, we constructed an acetic acid fermentation process from boysenberry juice1). The vinegar made from boysenberry juice showed an increased in vitro vasodilation using rat aorta, compared with the boysenberry juice. This result suggested acetic acid fermentation process enabled increase in the vasodilatory function of the boysenberry juice. In this process, ethanol was added into the culture broth at the start of the acetic acid fermentation as the substrate for acetic acid production. During the acetic acid fermentation, a significant part of the ethanol would vaporize without being utilized by A. pasteurianus, resulting low acetic acid production efficiency. So, it is important to construct a simultaneous fermentation by Saccharomyces cerevisiae strain KA31a and A. pasteurianus NBRC3283, which could allow a low ethanol concentration in the culture broth through the fermentation process. The process constructed in this study was a simultaneous fermentation by S. cerevisiae and A. pasteurianus using glucose as the sole carbon source. S. cerevisiae converts glucose to ethanol, which A. pasteurianus converts to acetic acid. The affinity of S. cerevisiae to glucose, which evaluated as the saturation constant Ks of Monod equation was higher than that of A. pasteurianus. This result suggested that S. cerevisiae can preferentially utilize glucose in coculture with A. pasteurianus. The affinity of A. pasteurianus to glucose was lower than that of A. pasteurianus to ethanol. This result suggested that A. pasteurianus can preferentially utilize ethanol whereas glucose exists in the cultivation medium. From these results, cocultivation of the two strains with glucose as the sole carbon source could allow the preferential utilization of glucose by S. cerevisiae to ethanol, as well as the preferential utilization of ethanol by A. pasteurianus. A batchwise cocultivation of S. cerevisiae and A. pasteurianus was carried out at 30℃ using a jar fermentator with a working volume of 1 L. Air was introduced at 1 vvm. The cultivation medium was YPD medium containing 150 g/L glucose, at which concentration S. cerevisiae converts glucose to ethanol even under aerobic conditions. After start of the cultivation, the dissolved oxygen (DO) concentration decreased, reaching approximately 0% (based on atmosphere concentration) at 5 h. The DO concentration continued approximately 0% until 45 h, and then it rapidly increased to 100% at 46 h. The pH of the culture broth decreased with cultivation period to 3.2 at 46.5 h. The final acetic acid concentration was 35.5 g/L. These results suggested that glucose was successfully converted to acetic acid in simultaneous fermentation by S. cerevisiae and A. pasteurianus. However, the conversion efficiency of glucose to acetic acid was only 25.6%. Although the optimization of the cultivation conditions is still needed, this study provided the prospect of a vinegar production by the simultaneous fermentation process. Further study should provide an efficient simultaneous fermentation by S .cerevisiae and A. pasteurianus from boysenberry juice, which could produce vinegar with an increased vasodilatory function. This study was financially supported by MEXT-Supported Program for the Strategic Research Foundation at Private Univerisies.

1) Toru Shigematsu et al. Abstracts and Synopsis of The 5th International Niigata Symposium on Diet and Health p. 108, Toki Messe, Niigata, Japan (30, 31 Oct. 2010)

－165－ P-44 Enzymatic synthesis of a novel sialyl mimic molecules by use of marine bacterial sialyltransferase

Tatsuo Miyazaki1, Toshiki Mine2, Takayuki Watanabe1, Hitomi Kajiwara2, Katsumi Ajisaka1 and Takeshi Yamamoto2

1Niigata University of Pharmacy and Applied Life Sciences, 2Japan Tobacco Inc., Japan

Sialic acids are essential constituents of carbohydrate chains and are usually found at the non-reducing end of oligosaccharides in glycoconjugates, which include the glycoprotein and glycolipids. Many studies have been reported that the sialosides of glycoconjugates play significant roles in a large number of cellular biological recognition events, such as inflammatory, cellular adhesiveness, immunological responses, cancer metastasis and viral infection. To investigate these biological events, the natural and modified sialosides as drug targets and functional molecules for the inhibition of carbohydrate binding of cells have been designed and synthesized. Sialyltransferases generally transfer N-acetylneuraminic acid (NeuAc) from the common donor substrate of these enzymes, cytidine 5’-monophospho-N-acetylneuraminic acid (CMP- NeuAc), to either the 3- or 6-position of terminal galactose (Gal) or N-acetylgalactosamine (GalNAc) residues, which are typical acceptor substrates. Some sialyltransferases have been shown to accept CMP-NeuAc derivatized at the 4-, 5- or 9-position of the sialic acid moiety. Analogues of the acceptors Galβ1-4GlcNAc and Galβ1-3GalNAc, in which the NHAc group is replaced by the N3, phthalimide, carbamate, or pivaloyl group, are also recognized as the substrates for these enzymes. However the adaptation range of the typical sialyltransferase in synthetic chemistry was restrictive because the enzymes are very specific for their natural substrates. To date, we have demonstrated that marine bacterial sialyltransferases show broad acceptor substrate specificities. For instance, the α2,3-sialyltransferase from Photobacterium sp. JT-ISH-224 can transfer NeuAc residue to both the 3’-OH and the 2-OH groups of lactose simultaneously to give 2,3’-disialyllactose as the reaction product [1]. Furthermore, the sialyltransferase reaction towards the mannose as the substrate gave the sialylmannoside in which the α-Neu5Ac residue is coupled to the anomeric position of mannose via β-form [2]. These two novel substrates, the α-glucose and β-mannose have a 1,2-cis-diol structure corresponding to the 3- and 4-positon of the Gal, which is typical acceptor toward the sialyltransferases. To elucidate the limitation of unique acceptor substrate specificity of the marine bacterial α2,3-sialyltransferase from Photobacterium sp. JT-ISH-224, we investigated α2,3-sialyltransferase reaction toward inositols with 1,2-diol and 6-membered ring structure. The myo-, 1D-chiro- and muco-inositol with a 1,2-cis-diol were recognized by the α2,3-sialyltransferase [3]. Additionally, the sialyltransferase reactions toward the neo- and cis-inositols having two or three 1,2-cis-diol gave both monosialoside and disialoside simultaneously. These results clearly show that the α2,3-sialyltransferase from Photobacterium sp. JT-ISH-224 recognizes acceptor substrates through the cis-diol structure corresponding to the 3- and 4-positon of the Gal. This novel class of sialyl mimic molecules might be utilized as the functional food additives. 1. Mine, T.; Kajiwara, H.; Murase, T.; Kajihara, Y.; Yamamoto, T. J. Carbohydr. Chem. 2010, 29, 51 2. Mine, T.; Miyazaki, T.; Kajiwara, H.; Naito, K.; Ajisaka, K.; Yamamoto, T. Carbohydr. Res. 2010, 345, 1417 3. Mine, T.; Miyazaki, T.; Kajiwara, H.; Tateda, N.; Ajisaka, K.; Yamamoto, T. Carbohydr. Res. 2010, 345, 2485

－166－ P-45 pH-sensing mechanism of sweet taste-modifying proteins

Takayuki Okubo, Minoru Tamiya and Masaji Ishiguro

Niigata University of Pharmacy and Applied Life Sciences, Japan

Neoculin and Miraculin are known as taste-modifying proteins as they are not sweet at natural conditions but sweet at acidic conditions. Through several mutational experiments, His11 of neoculin was found to be responsible for their pH sensing property, whereas His30 of miraculin play an important role in the receptor activation ( i.e. sweetness ). Molecular Dynamics (MD) calculation on the dimeric structure of the wild type and the His11 mutants constructed from the crystal structure at pH7 and pH3 suggested that neoculin forms a dimeric structure at pH7 through hydrogen bonds at the interface while it forms a monomeric structure which would be responsible for the receptor activation. Thus, His11 acts as a pH sensing residue for receptor activation. On the other hand, structural models of miraculin were constructed through homology modeling and docking of two monomeric miraculin molecule to form dimeric structures linked by disulfide bond. Among the generated dimeric structures, a “ closed “ structure was a plausible candidate for inactive miraculin at pH7. This closed structure forms a hydrogen bond network at the interface of the monomeric protein and His30 are covered by hydrophilic residues at the interface, so the closed structure may inhibit an interaction between the sweet receptor and the His30 of miraculin. On the other hand, molecular dynamics calculations on miraculin at pH3 gave an “ open “ structure in which His30 is exposed at the aqueous phase and the residue can interact with the sweet receptor. Thus, the open structure may represent an active form of miraculin at pH3. As shown above, the active forms of neoculin and miraculin have a common characteristic as they form dimeric structure forming closed structures with hydrogen bonds at their interface. These hydrogen bonds are sensitive to the pH change, particularly at acidic conditions which break their hydrogen bonds to lead to monomeric structures. The formation of tight dimeric structures may cover the receptor interacting residues and then block the interactions with sweet receptor. On the other hand, disturbance of these tight interactions at lower pH may expose the receptor interacting residue at the aqueous surface and then may enable the residues to interact with the receptor for activation. The details of the molecular dynamic calculations and the comparison between the data observed by NMR measurement will be discussed to confirm the proposed mechanism of the activation of the taste-modifying proteins.

－167－ P-46 Biosynthesis of triterpene in higher plants

Masaaki Shibuya

Niigata University of Pharmacy and Applied Life Sciences, Japan

Triterpene saponin is a major constituent of crude drugs such as Glycyrrhiza uralensis, Panax ginseng, Bupleurum falcatum, etc. Distribution of triterpene saponin is not limited in medicinal plants. Many plants used as food also contain triterpene saponins. Soybean (Glycine max), one of the most famous crops, contains triterpene saponin, which is called soyasaponin. Soyasapogenol B, which is an aglycone of soyasaponin I, is a major source of promising anti- hepatitis drug. In order to obtain substantial amount of soyasapogenol B using transgenic yeast (Saccharomyces cerevisiae), cloning of beta-amyrin synthase from G. max and squalene epoxidase from S. cerevisiae were carried out. ⑴ cDNA cloning of beta-amyrin synthase from G. max A sequence annotated as a beta-amyrin synthase from G. max is registered in GenBank DNA datab ase with accession number AY095999.1, which lacks 69 base of nucleotides at the position around 254th amino acid from N terminal compared with that of Pisum sativum beta-amyrin synthase (GenBank accession number AB034802.1). We obtained 2.2 kbp DNA fragment by PCR using primers designated from the sequence registered in database and cDNA pool which obtained from soybean seedlings as a template. The obtained DNA fragment was ligated pYES2, an yeast expression vector (Invitrogen). Resultant plasmid was used to transform a lanosterol synthase deficient yeast strain GIL77. After cultivation of transformant and induction of introduced gene, yeast cell was extracted with hexane. Product was purified by silica gel column chromatography, which structure was identified as beta-amyrin by 1H- and 13C-NMR spectroscopy. Based on these results, we concluded that the obtained gene encodes beta-amyrin synthase. The obtained gene consists of 2289 bp nucleotides and encodes a peptide chain consisting of 763 amino acids which shows 90% amino acid identities. And also, missing sequences of 69 bp nucleotides corresponding to 23 amino acids was uncovered. ⑵ cDNA cloning of squalene epoxidase from S. cerevisiae Key points for increasing the production of soyasapogenol B in transgenic yeast are supposed to be sufficient supply of substrate and prompt excretion of product into media. In this period, we tried the former one, improvement of substrate supply. Substrate of beta-amyrin synthase is oxidosqualene which is produced from squalene by squalene epoxidase in yeast cells. We supposed that overexpression of squalene epoxidase may conquer insufficient supply of substrate, and lead to high production of beta-amyrin. Squalene epoxidase from S. cerevisiae is registered in GenBank DNA database with accession number M64994. We obtained 1.5 kbp DNA fragment by PCR using primers designated from the sequence registered in database and genomic DNA which obtained from S. cerevisiae as a template. The obtained DNA fragment was ligated pYES2. Sequencing of obtained DNA and over-expression were now underway.

－168－ P-47 First total synthesis and revision of absolute configuration of (-)-1,3,4,5-tetragalloylapiitol

Yutaka Nakamura, Masaru Kojima and Seiji Takeuchi

Niigata Univ. Pharmacy and Applied Life Sciences (NUPALS), Japan

(-)-1,3,4,5-tetragalloylapiitol (1) was isolated from the aqueous extract of the African plant Hylodendron gabunensis by screening for substances that possess HIV ribonuclease H (RNase H) inhibitory activity. The structure of 1 consists of four gallic acids and apiitol, and the acids are connected to the hydroxyl groups at C-1, C-3, C-4, and C-5 of the sugar via ester linkages. The absolute configuration of 1 was originally assigned as S by comparing the optical rotation of the compound, which was obtained through the hydrolysis of the natural product 1 under basic conditions, with that of authentic D-apiitol. Herein, we report the total synthesis of the proposed structure of (S)-1 and the enantiomer (R)-1 and the structure revision and determination of the absolute configuration of the natural 1,3,4,5- tetragalloylapiitol. The total synthesis of the previously proposed structure of (S)-1,3,4,5-tetragalloylapiitol was accomplished from L-ribose in seven steps. The optical rotations of the synthesized product (S)-1 and the natural product exhibited the opposite signs. These results suggested that the absolute configuration at C-3 in the natural product should be revised to R. The correct absolute stereochemistry was further confirmed through the first total synthesis of the enantiomer of (S)-1 from inexpensive D-ribose and the analysis of the CD spectra of both compounds. A comparison of the optical rotations and the CD spectra of (S)- and (R)-apiitol tetra-O-benzoates with those published in the literature confirmed that our determination of their absolute configurations at C-3 was correct.

－169－ P-48 Future prospects of plant defensin Rs-AFP1 as a food preservative agent

Hiroaki Takaku, Yoshifumi Oguro, Harutake Yamazaki and Masamichi Takagi

Niigata University of Pharmacy and Applied Life Sciences, Japan

Food spoilage by yeasts and filamentous fungi is a significant problem in the food industry. Chemical and biological (nature-derived) preservative agents are required to ensure that manufactured foods remain safe and unspoiled. Synthetic chemical antifungals are becoming less attractive as food preservative agents in view of safety and environmental hygiene. Plant defensins, one of biologically active natural products, are small cysteine-rich proteins that have the potential to replace synthetic chemical antifungals. Many plant defensins can inhibit the growth of a broad range of fungi at micromolar concentrations but are nontoxic to both mammalian and plant cells. Defensin isolated from Brassica juncea, Rs-AFP1, showed broad range of antifungal activity against various yeasts and filamentous fungi. To investigate the biological properties of Rs-AFP1, we produced the functional recombinant Rs-AFP1 (rRs-AFP1) using the yeast Pichia pastoris heterogous expression system and purified. The biological function of rRs- AFP1 was found highly stable in extremes pH (range from 3 to 11) and temperature (upto 100 ℃) conditions. Understanding how rRs-AFP1 works against target microorganisms could lead to insights and new treatments for food preservation. The exact mechanism of action of most plant defensins is not completely understood, but there is evidence that plant defensins interact with specific targets on the fungal cell membrane. Therefore, we have also characterized a functional role of the fungal sphingolipid glucosylceramide (GlcCer) in regulating sensitivity of the yeast species to rRs-AFP1. A GlcCer deletion mutant of human pathogenic yeast Candida albicans becomes resistant to rRs-AFP1. The rRs-AFP1 induces plasma membrane permeabilization in C. albicans. In contrast, no such rRs-AFP1-induced plasma membrane permeabilization was detected in C. albicans GlcCer deletion mutant, which is resistant to rRs-AFP1. We also show that rRs-AFP1 induces reactive oxygen species (ROS) in C. albicans in a dose-dependent manner, but not all in GlcCer deletion mutant of C. albicans. These findings indicate that upstream binding rRs-AFP1 to GlcCer is needed for plasma membrane permeabilization and ROS production leading to yeast cell death. Based on our findings, we conclude that GlcCer is essential for rRs-AFP1-mediated growth inhibition of C. albicans. Finally, the rRs-AFP1 has the potential of being applied in multiple situations, such as crop protection, food preservation or human health.

－170－ P-49 Prevention of metabolic syndrome and second meal effect

Shinji Sato

Niigata Univ. of Pharm. & Appl. Life Sci., Japan

【Background/Aim】Reduction of postprandial hyperglycemia is important in the prevention of the metabolic syndrome and treatment of type 2 diabetes and impaired glucose tolerance. Glycemic index (GI) is a classification of carbohydrate foods based on acute plasma glucose response. On the fundamental assumption of the GI concept, it has been evaluated that low GI foods produce a low glycemic response as a result of a slower rate of digestion of carbohydrate in the intestinal lumen and subsequent suppression of absorption of glucose into the plasma circulation. Absorption of glucose from the small intestine into the plasma circulation is influenced by the intrinsic properties of the carbohydrate, fiber and fat content of respective foods. Variability in the GI is dependent on numerous factors such as amount and type of ingested carbohydrates, composition of preceding meal, and gastric emptying rate. Consumption of low glycemic index (GI) breakfast diminished the glucose response after lunch, and the ability of this breakfast is known as the second meal effect (SME). The aim of this study was to examine the SME of white rice, rice cracker and chocolate. 【Methods】Nineteen healthy subjects (ten men and nine women) with a mean age of 23.1 ± 3.6 years and with a normal body mass index of 21.5 ± 2.4 kg/m2 participated in this study. None of the subjects had impaired glucose tolerance, nor were they receiving medication. The subjects ate the reference (glucose 0, 25, 50, 75 g) and the test (white rice, rice cracker and chocolate; 50 g available carbohydrate) breakfast. Three hours later, the subjects were fed the same standard (white rice containing 65.8 g available carbohydrate) lunch. Finger prick capillary blood samples were taken for measurement of glucose, insulin, non-esterified fatty acids (NEFA), and triglyceride (TG). 【Results and Discussion】The values of the post-lunch incremental area under the curve of plasma glucose levels (AUCG) in glucose 75 g, white rice and rice cracker ingestion groups were significantly lower than that in glucose 50 g breakfast group. The value of AUCG decreased in accordance with the decrease of the post-lunch area under the curve of plasma NEFA levels (AUCN) in all breakfast groups except the chocolate breakfast group. There was a significantly positive correlation between AUCN and AUCG (γ2 = 0.663, P<0.05). It was clarified that white rice and rice cracker have the potential to regulate postprandial responses to a second meal by reducing NEFA competition for glucose disposal. These results suggested that white rice for breakfast may play the beneficial effect in the prevention of metabolic syndrome.

－171－ P-50 Developing a self-pollinate buckwheat based on reverse genetics approach

Jotaro Aii, Ayana Nakano, Shingo Sato, Taketo Funaki, Yuya Sato and Hiroshi Tanaka

Niigata University of Pharmacy and Applied Life Sciences, Japan

Buckwheat is used either as health foods or in the form of various processed products such as noodles, tea and various forms of juices and soups. Its consumption has increased gradually over the years, because of its high nutritional values. But it has never attained the status of major cereal crop from the production and yield point of view. Major causes of low productivity come from the heteromorphic self-incompatibility (SI). Two different forms of flowers are present in buckwheat, where an individual plant possesses either long style type flowers or short style type flowers. Buckwheat exhibits self-incompatibility and the breeding between individuals with the same type of flowers fails. This heterostylous self-incompatible trait is governed by a single genetic locus or gene complex (S-locus) and the genotypes for the plants with short and long style flowers are S/s and s/s, respectively. Despite recent progress in our understanding of the molecular basis of flower development and plant SI systems, the molecular mechanisms underlying heteromorphic SI remain unresolved. To reveal the genetic basis of this trait, we have been performed transcriptomic analyses of genes expressed in styles, ion-beam mutagenesis, and various genetic and evolutionary analyses. By examining differentially expressed genes from the styles of the two floral morphs, we successfully identified four short-style-specific genes (SSG1-SSG4) that are expressed only in short-styled plants. To confirm the physical linkage between S locus and the four SSGs, we conducted mutagenesis procedure using heavy-ion-beam irradiation. Flower morphology was observed for 1,400 M1 plants grown in a closed experimental room. During the mutagenesis screening of buckwheat, a single chimeric plant possessing a branch that sets long-styled flowers on a short- styled plant was obtained. PCR analysis of the genomic DNA of this chimeric plant yielded intriguing results. PCR successfully amplified SSG2 and SSG3 from the total DNA of the short- styled part of the plant but not from the total DNA of the long-styled part of the plant. On the other hand, PCR successfully amplified SSG1 and SSG4 from DNA isolated from both short- and long-styled parts of the plant. Considering that SSG2 and SSG3 are tightly linked and separated by about 100 kb, this result suggests that somatic deletion of the region that includes SSG2 and SSG3 occurred in the chimeric plant, and raises the possibility that these genes are located at the S-locus, which determines the floral phenotype. BLAST search identified no homologs with SSG2. On the other hand, homology between SSG3 and Arabidopsis thaliana ELF3 was detected. Phylogenetic analysis based on the deduced amino acid sequences showed that SSG3 is not an ortholog but a paralog of ELF3. We named this new gene S-LOCUS EARLY FLOWERING 3 (S-ELF3). The S-ELF3 gene is present only among the genomes of plants with short style flowers in the heterostylous self-incompatible buckwheat but becomes pseudogene in a self-compatible species such as tartary buckwheat. This result collectively indicates that the S-ELF3 gene play some role in the heterosylous self-incompatibility in buckwheat.

－172－ P-51 Structural stability studies on various functional peptides

Hiromu Yoshihara, Jun Saito, Hiroshi Hoshino, Yuuki Ishiguro, Kanako Iiyoshi and Toshinari Asakura

Niigata University of Pharmacy and Applied Life Sciences, Japan

【Purpose】The peptidic/protein drugs or food-derived functional peptides such as insulin formulations, glucagon-like peptides or soybean-derived peptides form higher-order structures. Since the protein/peptide-structure is usually sensitive to the severe conditions, they must be stored under appropriate environment. These drugs/peptides are handled by patients/users after deliver/purchase, the storage conditions may vary. The effects on the structural stability of these peptides under the users’ conditions are not well studied. In this study, we examined the structural stability of these peptides under various storage conditions which may easily occurred by the patients/purchasers. 【Methods】Insulin formulations and incretin formulations were stored under high temperature, longtime vibration, or re-freezing. The secondary structure was determined by measuring its circular dichroism (CD) spectrum, and the quaternary structure was determined by size- exclusion chromatography (SEC). The stability of these peptides was evaluated by their secondary and quaternary structures. 【Results and Discussions】Both secondary and quaternary structure had been shown to be changed among the many types of insulin formulations under hifh-temperature (90℃), long- time vibration (24h) or re-freezing condition. Although these insulin analogs are highly homolog each other, the structural stabilities vary. On other hand, no significant structural change is found among the incretin formulations under these conditions. These results suggested that the structural stability of peptidic/protein drugs and functional peptide foods may vary among those products, and these detailed analysis under various storage condition is necessarily proceeded to evaluate the safety of medicinal/food produts.

－173－ P-52 Survey on the use of supplements by questionnaire ～The difference of consciousness between some group of professional and citizen～

Noriaki Yohkoh, Manabu Abe and Mikio Saito

Niigata University of Pharmacy and Applied Life Sciences, Japan

We will focus on the supplements as a functional food. We are advancing the study mainly of a research by questionnaire. We have conducted a survey to target the general public, household medicine distributor and dentist about the actual use of supplements. We discussed the difference between consciousness of these profession and that of the general public 【Materials and methods】We conducted a survey by questionnaire to 155 people of general public, 141 people of household medicine distributors and 90 people of dentists. Questionnaire items are about the presence or absence of the use of supplements of the past year, the frequency of dosing, dosing reason, consciousness about the effects of supplements, discomfort caused by supplement , and presence or absence of a combination of medicine and supplements. 【Results and discussion】Fifty percent people of surveyed general public, 72% people of household medicine distributors and 49% people of dentists were taking supplements in the past year. Utilization of supplements of the household medicine distributor was higher than the dentist and general public. It was found that 42% of the general public, 46% of household medicine distributors and 41% of dentists are taking in an almost daily dosing in supplement use. In the people of household medicine distributors, "because I think that it is good for health" occupied 54%, and the reason for using supplements turned into most common reasons. In the people of the general public and dentist, otherwise, "because I think that it was good for health", reason "in order to compensate for the lack of nutrition", "because I think it is good for health," had become almost about 25%. Thirty three percent of surveyed general public, 47% of household medicine distributors, and 25% of dentists think that a supplement has an effect. Some differences were observed in the three parties. Discomfort symptom caused by the supplement was 5% of general public, 8% of household medicine distributors, and 0% of dentists. As described above, a little different result was obtained in the questionnaire survey between the general public, the household medicine distributor and the dentist. We are planning to continue the investigation, such as those added for the background of these considerations.

－174－ Corporate Sponsors

Amino Up Chemical Co., Ltd.

A. O. A. Japan Co., Ltd.

Bourbon Corporation

DAISYO CORPORATION

Echigoseika Co., Ltd.

Niigata Bio-Research Park, Inc

Ryusendo Co., Ltd.

(As of Sept. 26, 2012)