Characterization of Immuno-stimulating Polysaccharides Isolated from Korean Persimmon Vinegar

감식초에서 분리한 면역활성다당의 특성

  • Hwang, Yong-Chul (Department of Food Science & Biotechnology, Kyonggi University) ;
  • Shin, Kwang-Soon (Department of Food Science & Biotechnology, Kyonggi University)
  • 황용철 (경기대학교 식품생물공학과) ;
  • 신광순 (경기대학교 식품생물공학과)
  • Published : 2008.04.30

Abstract

In this study, polysaccharides were isolated from Korean persimmon vinegar to characterize the polysaccharides existing as soluble forms within traditional Korean fermented beverages, and their immuno-stimulating activities were examined. Three successive chromatographies were used to purify the main polysaccharide in the persimmon vinegar, PV-1b-I, to homogeneity from the crude polysaccharide (PV-0). The molecular mass of PV-1b-I was estimated as 110 kDa and it contained significant proportions of mannose (46.8%), galactose (28.5%) and arabinose (19.1%). PV-1b-I strongly reacted with ${\beta}$-glucosyl Yariv reagent, suggesting the presence of an arabino-3,6-galactan moiety. PV-1b-I also induced high levels of macrophage activation and mitogenicity on murine splenocytes in vitro. The intravenous administration of PV-1b-I significantly augmented NK cytotoxicity against YAC-1 tumor cells. PV-1b-I also showed potent anticomplementary activity in a dose-dependent manner. Finally, C3 activation products were identified by crossed immunoelectrophoresis using anti-human C3 and the anti-complementary activity of PV-1b-I under $Ca^{2+}$-free conditions, suggesting that this PV-1b-I causes complementary activations via both alternative and classical pathways. From these results, one can conclude that Korean persimmon vinegar contains select polysaccharides in addition to healthy components, and these polysaccharides appear to provide immuno-stimulating activities beneficial to human health.

한국 전통발효음료 중에 존재하는 특이다당류의 화학적 특성 및 생물활성을 규명할 목적으로, 농가에서 직접 발효한 감식초로부터 다당류를 분리하고 이들의 각종 면역증강활성에 대해 검토하였다. 감식초에 80% ethanol 침전을 행하여 얻어진 조다당 획분 PV-0을 이용, 연속적인 3회의 column chromatography를 행하여, 감식초의 주요 다당인 PV-1b-I으로 정제할 수 있었다. PV-1b-I획분은 HPLC상에서 대칭을 유지하는 단일 peak로 검출되었으며, 분자량은 약 110 kDa으로 평가되었다. 정제다당인 PV-1b-I의구성당 조성을 확인한 결과, mannose(46.8%), galactose(28.5%) 및 arabinose(19.1%)를 높은 비율로 함유하고 있었다. 또한 본 정제다당은 ${\beta}$-glucosyl Yariv reagent와 강하게 반응하는 특성을 보여주었으며 이러한 사실로부터 PV-1b-I이 arabino-3,6-galactan 부위를 소유함을 확인할 수 있었다. 한편 감식초에서 정제한 다당 PV-1b-I은 in vitro상에서 macrophage의 활성화를 높은 비율로 유도하였으며, 비장세포에 대해 높은 증식능을 보였다. 또한 PV-1b-I을 정맥 투여한 경우, NK cell을 활성화하여 YAC-1 종양세포에 대한 세포독성을 증가시킴이 관찰되었다. 한편 고분자 정제 다당인 PV-1b-I는 비특이적 면역계에 있어 중요 역할을 담당하고 있는 보체계에 대하여 농도 의존적인 활성화 경향을 보였다. 이들은 $Ca^{2+}$ 이온이 제거된 상태에서의 항보체 활성과 anti-human C3를 이용한 2차원 면역전기영동에 의하여 C3 산물을 동정한 결과로부터 보체계의 classical pathway와 alternative pathway 양 경로를 모두 경유하여 활성을 나타냄을 확인할 수 있었다.

Keywords

References

  1. Wollin SD, Jones PJH. Alcohol, red wine and cardiovascular disease. J. Nutr. 131: 1401-1404 (2001)
  2. Nakanc S. Food useful for preventing alcohol intoxication containing persimmon-vinegar and optimum fruits, with blood alcohol concentration reducing action. Jpn. Patent 63: 562-566 (1988)
  3. Kerry NL, Abbey M. Red wine and fractionated phenolic compounds prepared from red wine inhibit low density lipoprotein oxidation in vitro. Atherosclerosis 135: 93-102 (1997) https://doi.org/10.1016/S0021-9150(97)00156-1
  4. Tedesco I, Russo M, Russo P, Iacomino G, Russo GL, Carraturo A, Faruolo C, Moio L, Palumbo R. Antioxidant effect of red wine polyphenols on red blood cells. J. Nutr. Biochem. 11: 114-119 (2000) https://doi.org/10.1016/S0955-2863(99)00080-7
  5. Whitehead TP, Robinson D, Allaway S, Syms J, Hale A. Effect of red wine ingestion on the antioxidant capacity of serum. Clin. Chem. 41: 32-35 (1995)
  6. Fuhrman B, Lavy A, Aviram M. Consumption of red wine with meals reduces the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation. Am. J. Clin. Nutr. 61: 549-554 (1995) https://doi.org/10.1093/ajcn/61.3.549
  7. Gorinstein S, Zensler M, Wietz M, Halvey S, Bartnikowska E. Fluorometric analysis of phenolics in persimmon. Biosci. Biotech. Bioch. 58: 1078-1092 (1994)
  8. Gorinstein S, Zachwjeja Z, Folta M, Barton H, Plotrowicz J, Zemser M, Welsz M, Trakhtenberg S, Martin-Belloso O. Comparative contents of dietary fiber, total phenolics, and minerals in persimmons and apples. J. Agr. Food Chem. 49: 952-957 (2001) https://doi.org/10.1021/jf000947k
  9. National Rural Living Science Institute. Food Composition Table . 5th ed. pp. 142, Suwon, Korea. p.142 (1996)
  10. Matsuo T, Ito S. The chemical structure of kaki-tannin from immature fruit of the persimmon (Diospyros kaki). Agr. Biol. Chem. 42: 1637-1643 (1978) https://doi.org/10.1271/bbb1961.42.1637
  11. Choi C. Studies on Investigation into Biologically Activated Substances from Korean Persimmon Leaves and Developing High Function Beverages. Report of Ministry of Agriculture and Forestry. Gwacheon, Korea. pp. 149-150 (2000)
  12. Kotani M, Matsumoto M, Fujita A, Higa S, Wang W, Suemura M, Kishimoto T, Tanaka T. Persimmon leaf extract and astragalin inhibit development of dermatitis and IgE elevation in NC/Nga mice. J. Allergy Clin. Immun. 106: 159-166 (2000) https://doi.org/10.1067/mai.2000.107194
  13. Uchida S, Edamatsu R, Hiramatsu M, Mori A, Nonaka Gy, Nishioka I, Niwa M, Ozaki M. Condensed tannins scavenge active oxygen free radicals. Med. Sci. Res. 15: 831-834 (1987)
  14. Funayama S, Hikino H. Hypotensive principles of Diospyros kaki leaves. Chem. Pharm. Bull. 27: 2865-2868 (1979) https://doi.org/10.1248/cpb.27.2865
  15. Jeong YJ, Lee GD, Kim KS. Optimization for the fermentation condition of persimmon vinegar using response surface methodology. Korean J. Food Sci. Technol. 30: 1203-1208 (1998)
  16. Jeong YJ, Seo JH, Park NY, Shin SR, Kim KS. Changes in the components of persimmon vinegars by two stages fermentation (II). Korean J. Postharvest Sci. Technol. 6: 233-238 (1999)
  17. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356 (1956) https://doi.org/10.1021/ac60111a017
  18. Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acid. Anal. Biochem. 54: 484-489 (1973) https://doi.org/10.1016/0003-2697(73)90377-1
  19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Anal. Biochem. 72: 248-254 (1976) https://doi.org/10.1016/0003-2697(76)90527-3
  20. Karkhanis YD, Zeltner JY, Jackson JJ, Carlo DJ. A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative. Anal. Biochem. 85: 595-601 (1978) https://doi.org/10.1016/0003-2697(78)90260-9
  21. Jones TM, Albersheim P. A gas chromatography method for the determination of aldose and uronic acid constituents of plant cell wall polysaccharide. Plant Physiol. 49: 926-936 (1972) https://doi.org/10.1104/pp.49.6.926
  22. Holst GJ, Clarke AE. Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion. Anal. Biochem. 148: 446-450 (1985)
  23. Suzuki I, Tanaka H, Kinoshita A, Oikawa S, Osawa M, Yadomae T. Effects of orally administered ${\beta}$ -glucan on macrophage function in mice. Int. J. Immunopharmaco. 12: 675-684 (1990) https://doi.org/10.1016/0192-0561(90)90105-V
  24. Mayer MM. Complement and complement fixation. pp. 133-240. In: Experimental Immunochemistry. 2nd ed. Kabat EA, Mayer MM (eds). Charles C. Thomas Publisher, Springfield, IL, USA (1971)
  25. Shimura K, Ito H, Hibasami H. Screening of host-mediated antitumor polysaccharides by crossed immunoelectrophoresis using fresh human serum. Jpn. J. Pharmacol. 33: 403-408 (1983) https://doi.org/10.1254/jjp.33.403
  26. Ridley BL, O'Neill MA, Mohnen D. Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57: 929-967 (2001) https://doi.org/10.1016/S0031-9422(01)00113-3
  27. O'Neill M, Albersheim P, Darvill A. The pectic polysaccharides of primary cell walls. pp. 415441. in: Methods In Plant Biochemistry. Vol. 2, Carbohydrates. Dey PM (ed). Academic, London, England (1990)
  28. Ishii T. O-Acetylated oligosaccharides from pectins of potato tuber cell walls. Plant Physiol. 113: 1265-1272 (1997) https://doi.org/10.1104/pp.113.4.1265
  29. Engelsen SB, Cros S, Mackie W, Perez S. A molecular builder for carbohydrates: Application to polysaccharides and complex carbohydrates. Biopolymers 39: 417-433 (1996) https://doi.org/10.1002/(SICI)1097-0282(199609)39:3<417::AID-BIP13>3.0.CO;2-8
  30. Klis FM, Mol P, Hellingwerf K, Brul S. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 26: 239-256 (2002) https://doi.org/10.1111/j.1574-6976.2002.tb00613.x
  31. Aspinall GO. Carbohydrate polymers of plant cell wall. pp. 95- 115. In: Biogenesis of Plant Cell Wall Polysaccharides. Loewus F (ed). Academic Press, New York, NY, USA (1973)
  32. Clarke AE, Anderson RL, Stone BA. Form and function of arabinogalactans and arabinogalactan-proteins. Phytochemistry 18: 521-540 (1979) https://doi.org/10.1016/S0031-9422(00)84255-7
  33. Abbas AK, Lichtman AH, Pober JS. In: Cellular and Molecular Immunology. 4th ed. WB Sounders, London. UK (2000)
  34. Keller R, Keist R, Wechsler A, Leist TP, van der Meide PH. Mechanism of macrophage-mediated tumor cell killing: A comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor. Int. J. Cancer. 46: 682-686 (1990) https://doi.org/10.1002/ijc.2910460422
  35. Nathan CF, Murray HW, Cohen ZA. Current concepts: The macrophage as an effector cell. N. Engl. J. Med. 303: 662-665 (1980) https://doi.org/10.1056/NEJM198009183031202
  36. Shida K, Suzuki T, Kiyoshima-Shibata J, Shimada S, Nanno M. Essential roles of monocytes in stimulating human peripheral blood mononuclear cells with Lactobacillus casei to produce cytokines and augment natural killer cell activity. Clin. Vaccine Immunol. 13: 997-1003 (2006) https://doi.org/10.1128/CVI.00076-06
  37. Hunter CA, Chizzonite R, Remington JS. IL-1 beta is required for IL-12 to induce production of IFN-gamma by NK cells. A role for IL-1 beta in the T cell-independent mechanism of resistance against intracellular pathogens. J. Immunol. 155: 4347-4354 (1995)
  38. Saito H, Tomioka H, Sato K. PSK, a polysaccharide from Coriolus versicolor, enhances oxygen metabolism of murine peritoneal macrophages and the host resistance to listerial infection. J. Gen. Microbiol. 134: 1029-1035 (1988)
  39. Kwon MH, Sung HJ. Characteristics of immune response by polysaccharides with complement system activity. Food Sci. Indus. 30: 30-43 (1997)
  40. Whaley K. The complement system. pp. 1-35 In: Complement in Health and Disease. Whaley K (ed). MTP Press, Lancaster, UK (1986)
  41. Kim JH, Shin KS, Lee H. Chracterization and action mode of anti-complementary substance prepared from Lactobacillus plantarum. Korean J. Food Sci. Technol. 34: 290-295 (2002)
  42. Hudson L, Hay FC. Two dimensional or crossed immunoelectrophoresis. pp. 244-246. In: Practical Immunology. Blackwell Scientific Publications, Oxford, UK (1989)