오미자 (Schizandra chinensis Baillon.)를 포함한 생약 소재들의 Helicobacter pylori에 대한 항균 활성 연구

Abstract

Approximately 46% of the Korean adults and 50% of over 50-year-old adults in the world are reported of infection by Helicobacter pylori. In order to remove this bacteria some antibiotics (i.e., amoxicillin), proton pump inhibitors (i.e., omeprazole), urease inhibitors (i.e., acetohydroxamic acid), and vaccines are presently treated singularly or complexly. But these medical treatments causes medicine-resistant H. pylori and and the patients treated with this medicine show some side effets of constipation, eruption, or reflux esophagitis. What we call Schizandra chinensis in general indicates fruit of S. chinensis Baillon. In oriental medicine and folk remedies, fruit of S. chinensis Baillon is used for nutrition. As food, it, exactly, its pigment, is used to make a kind of pattern-pressed candy (made from starch) or rice cake after being soaked in water. It is also used to make tea, wine or juice. Most of the existing researches of S. chinensis Baillon were focused on its ingredients, and in relation to its functions, there were some papers reporting that it lowers hyperlipidemia and that it is effective for protection of liver cells, for antioxidation, and for antibiosis. Recently, some researches of examining antibiotic activities of natural materials are being reported because H. pylori antibiotics have high side effects. This paper examined antibiotic activities against H. pylori, targeting the extracts from 188 kinds of natural edible biological resources. As a result of it, S. chinensis Baillon was finally selected as a final test sample. As a part of a basic study for development of functional food or of an intermediary material of a medicine by use of S. chinensis Baillon, this paper examined antibiotic activities of the ethanol extracts of S. chinensis Baillon against H. pylori and separated and identified the active substances. In addition, through urease inhibitory activities and hemagglutination inhibition assay of 188 natural herb medicines, it had an antiadhesive activities assay and tried to construct database of H. pylori in vitro from various aspects. In order to control H. pylori with 188 kinds of natural herb medicines, this paper examined them from three aspects sof growth inhibition, urease inhibitory activity and adhestion inhibition assay. The results obtained are as following. 1. Using paper disc method, 95% ethnaol(EtOH) extracts of 54 medical plants, 70% EtOH extracts of 70 medical plants and water extracts of 60 medical plants were tested an antimicrobial activities against the Helicobacter pylori. Valeriana fauriei (95% EtOH extract), Schizandra nigra M. (70% EtOH extract) and Panax notoginsengs (water extract) showed the inhibition zone each 10.0, 9.0 and 9.0mm. And Paeonia suffiructicosa A., Artemisia capillaris T. and Torilis japonica 70% EtOH extracts showed a weak anti-H. pylori activities. Their inhibition zone were a 7.5mm each. 2. The EtOH extracts cencentrations of 1mg/disc, 3mg/disc and 5mg/disc of Schizandra chinensis Baillon showed the inhibition zone 8.0, 15.0, 20.0mm at each concentration. And ethylacetate(EtOAc) fractions of S. chinensis showed the inhibition zone 9.0, 19.0, 25.0mm at same concentration. 3. The yield of EtOH extract of S. chinensis B. had the highest percentage (39.5%); MC fraction(8.66%), EtOAc fractions (17.03%) and water fraction (4.98%). 4. MICs of H. pylori from S. chinensis B. found that EtOH extract and EtOAc fraction had concentrations of 1mg/mL. 5. After heat treatment of EtOAc fraction of S. chinensis B. at 100℃ for each 15, 30, 60 min growth rate of H. pylori were checked. According to the result of growth rate, there were not a differences of antimicrobial activity between all of test and control. In result of growth of H. pylori in the midium adding EtOAc fraction treated various pH conditions (2, 4, 6, 8) there were no differences of antimicrobial activity. So its stability on pH was high quality. 6. Using MPLC, the EtOAc fraction obtained from EtOHl extracts of S. chinensis was separated 11 subfractions. Then, in the result of identifying antimicrobial effect on the 11 subfractions by paper disc method, two subfractions had the inhibitory effect only. 1) Compound 1: S-EA-3 Bright yellow oil (form EtOAc fraction) C_(8)H_(16)O₃, ethyl 3-hydroxyhexanoate IR spectrum: 3218.61cm^(-1) hydroxy group(-OH), 1718.26cm^(-1) carbonyl group(C=O), 1179.26cm^(-1) carboxyl group(C-O) ¹H-NMR spectrum(CDCl3, 400 MHz, δ): 1.24ppm(s, 1H), 1,26ppm(s, 1H), 1.27ppm(s, 1H), 1.28ppm(s, 1H), 1.29ppm(s, 1H), 1.30ppm(s, 1H), 2.85ppm(q, 2H, J=6.5), 2.89ppm(q, 2H, J=4.1), 4.17ppm(q, 2H, J=7.1), 4.24ppm(q, 2H, J=2.01), 4.54ppm(d, 1H, J=1.38), 4.59ppm(s, 1H) ^(13)C-NMR spectrum(CDCl₃, 100 MHz, δ): 173.404(C-1), 38.505(C-2), 67.172(C-3), 62.238(C-4), 61.385(C-5), 14.016(C-6), 38.458(C-1´), 14.016(C-2´) 2) Compound 2: S-EA-5 Bright yellow oil (from EtOAc fraction) C_(8)H_(16)O₃ ,ethyl 5-hydroxyhexanoat IR spectrum: 3197.4.cm^(-1) hydroxy group(-OH), 1712.48cm^(-1)`carbonyl group, 1190.83cm^(-1) carboxyl group ¹H-NMR spectrum(CDCl3, 400 MHz, δ): 1.74ppm(d, 3H, J=6.61), 1.20ppm(d, 3H, J=7.02), 2.69ppm(d, 2H, J=8.34), 2.75ppm (d, 2H, J=8.28), 2.80ppm(d, 2H, J=5.98), 4.04ppm(d, 1H, J=7.1), 4.11ppm(d, 2H, J=7.11), 4.13ppm(s, 1H) ^(13)C-NMR spectrum(CDCl3, 100 MHz, δ): 172.034(C-1), 61.457(C-2), 43.704(C-3), 79.696(C-4), 73.675(C-5), 40.383 (C-6), 60.732(C-1'), 40.383(C-2´) 7. Urease inhibitory activities from the extracts of 188 medical plants lead as follow. M. vulgare(95% EtOH ex.) had the highest percentage, 231.25%; 95% EtOH extract of S. buergeriana (200.56%), 70% EtOH extract of L. japonica(164.54%), water extract of I. japonica (137.5%) and water extract of C. versicolor (97.72%). 8. In the result of hemagglutination inhibition assay to the extracts of 188 medical plants, 6 kinds (M. vulgare, S. commixta, C. trilobus, P. amurense, O. ficus-indica var. saboten and R, coreanus M.) among the 95% EtOH extracts of 55 medical plants were showed effect of hemagglutination inhibition. And, C. unshin and R. satirus among the 70% EtOH extracts of 73 medical plnats were showed effect it. 11 kinds (P. thunbergiana, A. sessiliforun S., L. chinense M., P. cocas W., C. japonica C., A. gramineus, C. officinale, P. oleracea, R. glutinosa L., H. macrophylla for. otaksa, and P. notoginsengs) among the water extracts of 60 medical plants were showed effect it. Adhesion inhibitory assay from the 95% EtOH extract of R. coreanus in its evolution (immature, well-timed and ripen) by hemagglutination inhibition assay lead as follow. The EtOH extract of ripen R. coreanus was showed effect of hemagglutination inhibition at 0.1mg/mL concentration. It was very excellent. On one side, adhesion inhibitory assay from the EtOH extract and EtOAc fraction of S. chinensis B. by hemagglutination inhibition assay lead as follow. Both of EtOH extract and EtOAC fraction were showed effect of hemagglutination inhibition at 1mg/mL concetration. But, at 0.5mg/mL concetration EtOAC fraction were showed weak effet it only. For growth inhibition, S. chinensis Baillon, with which Koreans are familiar, was selected as a sample. A growth inhibitory substance for H. pylori was separated and identified. For urease inhibitory activity and hemagglutination inhibition assay, some extracts with excellent activity were selected out of 188 kinds of natural herb medicine extracts. This paper constructed database of the in vitro test for control of H. pylori, which will become useful for future in vitro researches.