Natural products have received great attention for disease prevention owing to their various health benefits, noticeable lack of toxicity and side effects, and the limitations of chemotherapeutic agents. Rice is one of the world's most important food crops and more than half of the people in the world eat rice as the main part of their diets. y-oryzanol (y-ORZ), a phytosteryl ferulate extracted from rice bran oil. y-ORZ has been using as a medicine in Japan since 1962. Each year Japan manufactures approximately 7,500 tons of y-ORZ from 150,000 tons of rice bran. Imagine a single compound that possesses the multiple health benefits properties, the compound with such wide-ranging power is y-ORZ. This is of particular importance that y-ORZ is effective in the treatment of a broad range of gastrointestinal disorders, including peptic ulcer, gastritis, the irritable bowel syndrome, and nonspecific gastrointestinal complaints. Moreover, y-ORZ is widely used as an anabolic drug by bodybuilding athletes, hypolipedimic agent for both hypercholesterolaemic and hypertriglyceridaemic patients, for the treatment of chronic schizophrenia, postmenopausal syndrome in women and lowering the risk of colon cancer as well. Considering the aforementioned background, research on rice bran phytochemicals was designed based on structure-antioxidant and anti-inflammatory potentiality both in in vivo and in vitro models.

Chapter 1: Antioxidant, free radical scavenging and NF-KB activity of phvtosteryl ferulates: structure-activity studies

There is emerging interest in the use of naturally occurring antioxidants for the management of a number of pathophysiological conditions, most of which involve free radical damage. In this study, initially antioxidant and free radical scavenging activities were measured using TBARS and DPPH assays, respectively. The results demonstrated that y-ORZ, CAF, 24-mCAF, and 13-SF and also FA had strong free radical scavenging and antioxidant potency, which was comparable to a-tocopherol. However, the sterol moiety such as CA does not have both activities. Therefore, it is suggested that the common ferulic acid-derived structure of y-ORZ, CAF, 24-mCAF and j3-SF with phenolic-hydroxyl group under the similar situation exhibited the antioxidant potency and scavenging activity.

There are numerous sources of ROS production in the cell complex defense mechanism which cannot be ruled out with the chemical reactions in solution. Therefore, cell based ROS production system was employed. CAF and eFA, a membrane permeable FA, greatly inhibited the ROS production in NIH 3T3 fibroblast cells induced by H202 in this system. It is notable that sterol moiety alone (CA) also significantly inhibited the intracellular ROS production.

NF-KB has been shown to play an important role in regulating the expression of many genes involved in cell survival, immunity, and inflammation. NF-KB activity was analyzed by measuring translocation of NF-KB p65 in LPS activated RAW 264.7 macrophages, and found that y-ORZ, CAF, 24-mCAF, B-SF as well as eFA and CA, all these chemicals significantly inhibited the NF-KB activity. CAF was also investigated on LPS activated macrophages for iNOS and COX-2 gene evaluation. The results demonstrated that CAF significantly inhibited the LPS-stimulated increased iNOS and COX-2 mRNA in a dose (10 and 30μM) dependant manner in RAW 264.7 macrophages.

Recently, great attention has been focused on the relationship between NF-KB and ROS. Inflammatory stimuli such as LPS triggers ROS production via the activation of NADPH-oxidase system in macrophages and ROS enhance the signal transduction pathways for NF-KB activation in the cytoplasm and translocation into the nucleus. In addition, there is evidence that exogenous oxidant such as H2O2 induces NF-KB activation in some cell lines and high concentration of antioxidants abolish NF-KB activation in response to LPS or hypoxia, which suggests that ROS is involved in NF-xB activation. However, the relationship between NF-KB activation and ROS is still controversial.

Chapter 2: Effects of phvtostervl ferulates in experimental colitis in mice

Colorectal cancer is an important public health problem, one of the most fearsome complications of inflammatory bowel diseases (IBDs) mainly ulcerative colitis (UC) but also Crohn's colitis which affect millions of people worldwide. The conventional medical treatment of IBD relies on the use of aminosalicylates, corticosteroids, immunosuppressive drugs (azathioprine, 6-mercaptopurin, methotrexate, cyclosporin) and antibiotics. However, aminosalicilates (5-amino salicylic acid (5-ASA) derivatives) and/or glucocorticoids remain the principals of therapy for IBD at different stage of disease. However, the clinical effects are often transitory and frequently induce side effects. Therefore, there is a need for better therapeutic agents that effectively induce remission and alter the natural course of the disease with minimum or no side effects of the treatment.

The anti-inflammatory effects of y-ORZ, CAF, FA, a possible metabolite of y-ORZ were investigated in vivo, on dextran sulfate sodium (DSS)-induced colitis in mice by monitoring disease activity index (DAI), histopathology score, tissue myeloperoxidase (MPO) activity, mRNA expressions of cytokines and COX-2, colon length, and nuclear factor-KB (NF-KB) activity in colitis tissue. Both DAI and histopathology score revealed that DSS induced a severe mucosal colitis, with a marked increase in the thickness of the muscle layer, distortion and loss of crypts, depletion of goblet cell and infiltration of macrophages, granulocytes and lymphocytes. MPO activity was correlated with the development of colonic inflammation, and administration of y-ORZ significantly ameliorated these conditions. y-ORZ significantly reduced the up-regulated IL-1B, IL-6, TNF-a and COX-2 mRNA expressions in DSS-induced colitis mice. The nuclear NF-KB p65 significantly increased and IKB-a in the cytoplasmic extract significantly decreased in the DSS-induced colitis tissue, which was significantly ameliorated by the treatment with y-ORZ. CAF had a similar ameliorative potency as y-ORZ did.

It is documented that reactive oxygen species activates NF-KB which leads to the generation of pro-inflammatory cytokines and inducible enzymes, such as COX-2 and iNOS, in leukocytes and macrophages. Conversely, the pro-inflammatory cytokines causes oxidative stress by promoting the release of reactive oxygen species by immune cells and also by non-immune cells. Thus, inflammation and oxidative stress are involved in the spiraling vicious cycle that contributes to the severity of the intestinal inflammation. Role of reactive oxygen species in initiating and controlling the phosphorylation cascades that result in NF-KB activation is still controversial. The redox-sensitive mechanism responsible for the regulation of NF-KB, however, is still not fully understood, and further studies are necessary to solve the precise mechanism.

Chapter 3: Inducible nitric oxide synthase and cyclooxygenase-2 signal transduction in LPS activated RAW 264.7 macrophages

LPS activated intracellular signaling pathways are largely unresolved. In spite of unresolved, most of the scientific reports suggest that LPS activates the IKB kinase (IKK)-NF-KB pathway, extracellular signal-regulated kinases (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38 in human monocytes and macrophages. Intracellular signaling cascade involved in pro-inflammatory response such as iNOS and COX-2 expression were investigated in LPS activated RAW 264.7 macrophages. LPS (10ng/mL) stimulation of the macrophages increased iNOS and COX-2 gene differently in a time-dependant manner (1-4 hours), where, COX-2 expression started to increase earlier than iNOS. Treatment with CAY 10404 (a COX-2 inhibitor) significantly inhibited LPS-mediated iNOS induction but not the induction of COX-2 gene. In addition, PGE2 analogs ONO-AE1259 (an EP2 agonist) and ONO-AE1329 (an EP4 agonist) also significantly upregulated iNOS gene in RAW 264.7 macrophages.

There are many intracellular signaling pathways that can not be explained precisely and logically. Finally, involvement of MAP kinase pathways for the iNOS and COX-2 gene upregulation in LPS activated macrophages were investigated. PD98059 (a p44/42 MAP kinase inhibitor) inhibited iNOS mRNA level partially but significantly (p<0.05) but not COX-2 gene in LPS activated macrophages. However, PD169316 (a p38 MAP kinase inhibitor) neither inhibited iNOS nor COX-2 gene in LPS activated macrophages. Therefore, ERK and p38 inhibitors differently affect iNOS and COX-2 expression in activated macrophages. It has been reported that activation of MAP kinase phosphorylation transduces signals to activate the transcription of NF-KB-mediated pro-inflammatory cytokines in which the activation of ERK is involved.

Conclusion

The effects of phytosteryl ferulates on antioxidation may come via ROS scavenging and inhibition of ROS production in the living cells. The anti-inflammation may come from the iNOS/NO rather COX-2 pathway via the inhibition of LPS -induced NF-kB activation. Rice bran phytosteryl ferulates having potential anti-inflammatory, antioxidant and free radicals scavenging activities could be new potential therapeutic and/or preventive agents for gastrointestinal inflammatory diseases and disease induced by oxidative stress. Further study needs for more precise conclusion.

米を主食とする我が国では年間に約100万トンもの米糠が自然界に廃棄され、その環境への影響が懸念され廃棄バイオマスとしての再利用法の開発が期待されている。米糠には古くから生理活性を持つ有効成分が多数含まれることが知られている。γ-oryzanolは米糠に含まれる脂溶性成分で植物性sterolとferulic acidが結合した成分の総称であり、抗酸化作用を持つことが知られているが、その他の生理活性作用の詳細は不明である。

本研究では、米糠由来γ-oryzanolの生理活性作用について、特に抗酸化作用と抗炎症作用についてIn vitroとIn vivoの病態モデルを用いて解明することを目的としている。

1. γ-oryzanolならびにその誘導体の抗酸化作用ならびにNFκB阻害作用

新潟産コシヒカリより単離したγ-oryzanolはcycloartenyl ferulate (CAF)を主成分とし、次いで24-Methylenecycloartenyl ferulate (24-Met CAF)、campesteryl ferulate (CSF)、β-sitosteryl ferulate (β-SSF)が含まれていた。また、ferulic acid (FA)は米糠脂溶性成分にはほとんど含まれていなかった。我々は、FA、ferulic acid ester (eFA)、cycloartenol (CA)、CAF、24-Met CAF、β-SSFを用いて、抗酸化作用とNFκB活性阻害作用について解析した。抗酸化作用はラジカル捕集機能をDPPH assay (2,2-diphenyl-1-picrylhydrazyl assay)で、抗酸化作用をTBARS assay(thiobarbituric acid reactive substances assay)で評価し、NIH3T3細胞へのH2O2刺激によるreactive oxygen species (ROS)の産生能をDCFH-DA assay (dichlorodihydrofluorescein diacetate assay)にて評価した。また、NFκB活性への影響はNFκB p65タンパク質のLPS刺激したRAW264.7マクロファージにおける核内移行を免疫染色することで評価した。γ-oryzanol、CAF、β-SSF、FAはいずれも濃度依存性にラジカル捕集作用を示し、抗酸化作用を持つことが示唆された。一方、CAはラジカル捕集作用、抗酸化作用を示さなかった。NIH3T3細胞を用いたROS産生能に対する作用において、H2O2刺激により約4倍のROS産生が認められ、CAF、eFAならびにCAはいずれも10μMで顕著にROS産生を抑制する成績を得た。LPS刺激はROSの産生を引き起こすことが知られており、ROS産生はNFκB転写因子を活性化する。そこで、LPS刺激によるNFκB p65タンパク質の核内移行について解析した。結果、CAを含む全ての化合物がLPS刺激によるNFκB p65タンパク質核内移行を顕著に抑制することがわかった。以上の成績から、CAFを主成分とするγ-oryzanolには強い抗酸化作用を示すこと、その作用はラジカル捕集作用とROS産生抑制作用によることが明らかになった。また、CAFの化学構造のCAはラジカル捕集作用を持たないが、ROS産生抑制作用を持つことを明らかにした。これらのγ-oryzanol類縁化合物はいずれも強いNFκB活性阻害作用を持つことが明らかになった。

2. Dextran sulfate sodium (DSS)誘発潰瘍性大腸炎モデルにおけるγ-oryzanolの抗炎症作用

第2章では病態モデルにおけるγ-oryzanolの有効性について、DSS誘発大腸炎モデルを用いて解析した。C57BL/6Jマウスに1% DSSを含む水を自由飲水させることでマウスに大腸炎を惹起させた。大腸炎の評価は、体重変化、糞便形状ならびに血便をスコア化したDisease activity index (DAI)、HE染色、好中球浸潤の指標となるmyeroperoxidase (MPO) activity、各種サイトカインmRNA発現などを指標に解析した。γ-oryzanolは0.01% Tween-20、0.5% CMC (carboxymethyl cellulose sodium)を含む生理食塩水に懸濁させ、50 mg/kg/dayでDSS投与開始二日前から経口投与した。DSS投与2日目からDAIの増加、HE染色による顕著な炎症性細胞の浸潤が認められ、粘膜層と筋層の肥厚、MPO活性の増加など顕著な腸炎症状を示し、この症状はDSS投与16日まで継続していた。γ-oryzanol投与群ではこれらの腸炎症の症状を顕著に軽減させた。また、DSS投与8日目までは炎症性サイトカインの発現増加が顕著に認められ、γ-oryzanol投与群ではこれらは有意に抑制された。γ-oryzanol処置群では腸炎病変部のNFκB p65タンパク質の核内移行の抑制と、IκBタンパク質の分解抑制が認められたことから、γ-oryzanolによる腸炎抑制機序はNFκB活性の抑制によると考えられた。また、γ-oryzanolの主成分であるCAF (50 mg/kg/day)ならびにCAFの代謝産物と考えられるFA (50 mg/kg/day)においても、DSS誘発腸炎症の顕著な抑制が認められた。さらに、3% DSSを5日間自由飲水させた後に同量のγ-oryzanolの経口投与を開始するγ-oryzanol後処置群においても腸炎症の有意な抑制が認められ、γ-oryzanolが腸炎の治療薬としても有効である可能性が示唆された。

以上のように本研究は、米糠由来のγ-oryzanolに抗酸化作用とは別にあらたにNFκB活性阻害作用があることをみいだした。さらに、γ-oryzanolがDSS誘発潰瘍性大腸炎モデルにおいてもNFκB活性を抑制することで腸炎症を有意に緩和させることを明らかにした。これらの知見は、今後γ-oryzanolが機能性食品や医薬品のリード化合物として期待されることを科学的に実証したものであり、学術上寄与するところは少なくない。よって審査委員一同は本論文が博士(獣医学)の学位に値するものと判断した。