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1 작품번호 1316 생리활성기능이강화된화합물유도체합성과 유도체의효능분석 출품분야학생부출품부문동물 구분성명 출품학생장영오 박완 지도교사한만위 - 1 -

2 . Acteoside. Acteoside,. acteoside caffeic acid phenethyl phenolic acid acteoside,. acteoside caffeic acid phenethyl phenolic acid phenethyl urea(papu). a-tocopherol acteoside DPPH, hydroxyl. PAPU lipopolysaccharide nitric oxide.,

3 ) DPPH ) Glucose oxidase

4 .,..,,.,. (reactive oxygen species, ROS) (1-3)., superoxide dismutase, glutathione peroxidase, catalase thioredoxin reductase,, glutathione NADPH. -,. -,, (4,5). -. a-tocopherol, ascorbic acid( C) b-carotene (6).,,, 4,000,. (7-10). (11,12). DNA,...,,

5 ., (prooxidant) (13,14).,.,,,,.. Acteoside(Fig. 1),,, (15).,,. Fig. 1 acteoside phenolic acid. Acteoside, phenolic acid. acteoside Fig. 1. Chemical structure of acteoside, phenolic acid. acteoside.,... acteoside, phenolic acid (caffeic acid, ferulic acid, coumaric acid ) (ascorbic acid, a-tocopherol ) Sigma-Aldrich. (Jurkat), (NIH/3T3), (MCF-7), (B16F10), (RAW264.7)

6 Fig. 2. Scheme of organic synthesis for acteoside derivatives (phenolic acid phenethyl ureas; PAPU). Six PAPU compounds were finally synthesized using phenolic acid and phenethyl in this study and their molecular weights were from to Acteoside,,., acteoside phenolic acid, Curtius rearrangement 6 (PAPU, phenolic acid phenethyl urea) (Fig. 2). 2 5, caffeic acid monomethoxy- dimethoxy-caffeic acid DPPA azide (1 ), isocyanate (2 ), dihydroxyphenylethylamine dihydroxyphenylethanol (3 ), caffeic acid urea (4 ), demethylation (5 ).,.. 10% (100 IU/ml G mg/ml ) Dulbecco s modified Eagle s medium(dmem), 6-well 96-well

7 (0, 10, mm), ) DPPH DPPH Blios (16),. DPPH 0.2 mm, PAPU 0, 10, 50, mm ELISA reader 517 nm. 2) Glucose oxidase Glucose oxidase(go) hydroxyl, MTT assay. 96-well Jurkat, PAPU phenolic acid 0, 10, 50, 100 mm GO(10 mu/ml). 24~48 5 mg/ml MTT(3-(4,5-Dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide) 10 ml well 4, well 70 ml acidic isopropanol 1 ELISA reader 560 nm.. MTT assay. 70~80% (0~100 mm) 48 MTT assay.., lipopolysaccharide(lps) nitric oxide(no) - 6 -

8 . RAW well PAPU (0, 10, 50, 100 mm) LPS(1 mg/ml) NO Griess reaction (17). NO 540 nm nitrite.. (standard deviation, SD). One-way ANOVA Scheffe's test, p PAPU Table 1 Table I. Chemical name of the synthesized phenolic acid phenethyl ureas (PAPUs) Compound No PAPU1 PAPU2 PAPU3 PAPU4 PAPU5 PAPU6 Chemical name (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea (E)-1-(3,4-dihydroxystyryl)-3-phenethylurea (E)-1-(3,4-dihydroxystyryl)-3-(4-hydroxyphenethyl)urea (E)-1-(3,4-dihydroxystyryl)-3-(4-dihydroxyphenethyl)urea (E)-1-(4-methoxyphenethyl)-3-(4-methodystyryl)urea (E)-1-(3,4-dihydroxyphenethyl)-3-(4-hydroxystyryl)urea.. DPPH PAPU5 (Fig. 3)., PAPU a-tocopherol ascorbic acid, acteoside. PAPU2 PAPU4, PAPU6 20 mm - 7 -

9 Fig. 3. DPPH free radical scavenging activity of natural antioxidants and synthesized acteoside derivatives. The natural antioxidants or PAPU compounds were mixed with DPPH radical and then their scavenging activities were measured by MTT assay as described. T, a-tocopherol; AA, ascorbic acid; AC, acteoside. DPPH 80%, a-tocopherol acteoside 50%. hydroxyl ( OH) PAPU. Jurkat 5 mm glucose 10 mu/ml glucose oxidase(go) hydroxyl,, 24 MTT assay (Fig. 4). Hydroxyl 17%, PAPU5 5 (Fig. 4B)., 100 mm PAPU1, PAPU2, PAPU3, PAPU4 PAPU ± 3.1%, 56.0 ± 5.3%, 32.6 ± 4.8%, 32.3 ± 1.1% 31.9 ± 4.2% GO 17%., phenolic acid(e-cinnamic acid, p-coumaric acid, 4-methoxycinnamic acid, caffeic acid, 3,4-dimethoxycinnamic acid ferulic acid) ( 4A). PAPU phenolic acid.. (Fig. 5). B16F10 PAPU phenolic acid 0, 10, mm 48 MTT assay, 6 phenolic acid 100 mm - 8 -

10 Fig. 4. Protective effects of PAPUs on cell death induced by hydroxyl radicals produced in G/GO system. Jurkat cells were treated with various concentrations (0-100 mm) of phenolic acids (A) or PAPU samples (B) and then exposed to hydroxyl radicals generated by 5 mm glucose and 10 mu glucose oxidase (G/GO system) for 24 h. Each bar represents the mean ± SD values of triplicates. *p < 0.05 and **p < 0.01 vs. the untreated control values. CIA, e-cinnamic acid; COA, p-coumaric acid; MEA, 4-methoxycinnamic acid; CAA, caffeic acid; DIA, 3,4-dimethoxycinnamic acid; FEA, ferulic acid. (Fig. 5A)., PAPU 100 mm (Fig. 5B). PAPU1, PAPU2 PAPU5 B16F10, 50 mm 23.1 ± 4.5%, 19.5 ± 2.4% 16.1 ± 2.3%. 10 mm PAPU3. PAPU B16F10 2 (NIH/3T3 MCF-7) (Fig. 6). PAPU., PAPU B16F10, NIH/3T3 PAPU1, PAPU2 PAPU ± 1.9%, 57.4 ± 5.2% 47.1 ± 1.1%. MCF-7 PAPU1, PAPU2 PAPU3,. PAPU,

11 Fig. 5. Inhibitory effects of PAPUs on the viability of B16F10 melanoma cells. B16F10 cells were treated with the indicated concentrations of phenolic acids (A) or synthetic PAPUs (B) for 48 h and then processed for the MTT assay. Each bar represents the mean ± SD values of triplicates. *p < 0.05, **p < 0.01, and ***p < vs. the untreated control values. CIA, e-cinnamic acid; COA, p-coumaric acid; MEA, 4-methoxycinnamic acid; CAA, caffeic acid; DIA, 3,4-dimethoxycinnamic acid; FEA, ferulic acid... (RAW 264.7) 1 mg/ml LPS nitric oxide(no) PAPU 48 NO., PAPU LPS NO, PAPU1, PAPU2 PAPU3., PAPU1 PAPU2 PAPU3 (Fig. 7). LPS NO 5, PAPU3 (Fig. 7A). NO PAPU3., 10 mm PAPU3 NO, 50 mm NO. LPS(E.LPS) NO PAPU3 LPS(P.LPS) (Fig. 7B)

assay. Each bar represents the mean ± SD values of triplicates. *p < 0.05, **p < 0.01, and ***p < 0.001 vs.

12 Fig. 6. Inhibitory effects of PAPUs on cell viability are different according to the type of cells examined. B16F10, NIH/3T3, and MCF-7 cells were treated with the indicated concentrations of PAPUs for 48 h and then were processed for MTT assay. Each bar represents the mean ± SD values of triplicates. *p < 0.05, **p < 0.01, and ***p < vs. the untreated control values.. Phenolic acid. caffeic acid, p-coumaric acid ferulic acid (18-21). phenolic acid (22). acteoside caffeic acid phenethyl (15).,, acteoside.. PAPU. PAPU

13 Fig. 7. Effect of PAPU3 on NO production in LPS-stimulated RAW cells. (A) Cells were exposed to the indicated concentrations (0-100 mm) of PAPU3 for 48 h in the presence and absence of 1 mg/ml LPS. Produced NO was analyzed by measuring nitrite concentrations in the culture media. *p < 0.05 and ***p < vs. the LPS treatment alone. (B) In addition, cells were stimulated with 1 mg/ml of E.LPS or P.LPS in the presence and absence of 100 mm PAPU3, and after 48 h of incubation, the levels of NO in the culture media were determined. **p < 0.01 and ***p < mean significant differences between the experiments.. 1. Mello-Filho, A. C., Meneghini, R Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals. Mutat. Res. 251: Rollet-Labelle, E., Grange, M. J., Elbim, C., Marquetty, C., Gougerot-Pocidalo, M. A., Pasquier, C Hydroxyl radical as a potential intracellular mediator of polymorphonuclear neutrophil apoptosis. Free Radic. Biol. Med. 24:

14 3. Mates, J. M., Sanchez-Jimenez, F. M Role of reactive oxygen species in apoptosis: Implications for cancer therapy. IJBCB 32: Ames, B. N., Shinenaga, M. K Oxidants are a major contributor to cancer and aging. In DNA and Free Radicals, Halliwell, B., Aruoma, O. I. Eds. pp. 1-15, Fllis Horwood Limited, Chichester, West Sussex, England. 5. Slater, A. F. G., Orrenius, S Oxidative stress and apoptosis. In Oxidative Stress and Aging, Cutler, R. G., Packer, L., Bertram, J., Mori, A. Eds. pp , Birkhauser Verlag Basel, Switzerland. 6. van Poppel, G., van den Berg, H Vitamins and cancer. Cancer Lett. 114: Morel, I., Lescoat, G., Cogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P., Cillard, J Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 45: Rice-Evans, C. A., Miller, N. J., Bolwell, P. G., Bramley, P. M., Pridham, J. B The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic. Res. 22: Hollman, P. C., Katan, M. B Dietary flavonoids: intake, health effects and bioavailability. Food Chem. Toxicol. 37: Caltagirone, S., Rossi, C., Poggi, A., Ranelletti, F. O., Natali, P. G., Brunetti, M., Aiello, F. B., Piantelli, M Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential. Int. J. Cancer. 87: Yang, E. B., Zhang, K., Cheng, L. Y., Mack, P Butein, a specific protein tyrosine kinase inhibitor. Biochem. Biophys. Res. Commun. 245: Gamet-Payrastre, L., Manenti, S., Gratacap, M. P., Tulliez, J., Chap, H., Payrastre, B Flavonoids and the inhibition of PKC and PI 3-kinase. Gen. Pharmacol. 32:

15 13. Yoshino, M., Haneda, M., Naruse, M., Murakami, K Prooxidant activity of flavonoids: copper-dependent strand breaks and the formation of 8-hydroxy-2'-deoxy-guanosine in DNA. Mol. Genet. Metab. 68: Metodiewa, D., Jaiswal, A. K., Cenas, N., Dickancaite, E., Segura-Aguilar, J Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic. Biol. Med. 26: Kim, S. S., Son, Y. O., Chun, J. C., Kim, S. E., Chung, G. H., Hwang, K. J., Lee, J. C Antioxidant property of an active component purified from the leaves of paraquat-tolerant Rehmannia glutinosa. 10: Blios, M. S Antioxidant determination by the use of a stable free radical. Nature. 181: Weissman, B. A., Gross, S. S Measurement of NO and NO synthase. Curr. Prot. Neurosci. Chapter 7: Hudson, E. A., Dinh, P. A., Kokubun, T., Simmonds, M. S., Gescher, A Characterization of potentially chemopreventive phenols in extracts of brown rice that inhibit the growth of human breast and colon cancer cells. Cancer Epidemiol. Biomarkers Prev. 9: Mathew, S., Abraham, T. E Bioconversions of ferulic acid, an hydroxycinnamic acid. Crit. Rev. Microbiol. 32: Shahidi, F., Alasalvar, C., Liyana-Pathirana, C. M Antioxidant phytochemicals in hazelnut kernel (Corylus avellana L.) and hazelnut byproducts. J. Agric. Food. Chem. 55: Srinivasan, M., Sudheer, A. R., Menon, V. P Ferulic Acid: therapeutic potential through its antioxidant property. J. Clin. Biochem. Nutr. 40: Konishi, Y., Hitomi, Y., Yoshioka, E Intestinal absorption of p-coumaric and gallic acids in rats after oral administration. J. Agric. Food Chem. 52:

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical