Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
권호 표지
DOI QR코드

DOI QR Code

Convenient Method for Selective Isolation of Immuno-Stimulating Polysaccharides from Persimmon Leaves

감잎으로부터 면역 활성 다당의 선택 분리를 위한 간편 방법

  • Received : 2015.10.15
  • Accepted : 2015.11.27
  • Published : 2016.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

The biological activity of polysaccharide is greatly influenced by polysaccharide structure and molecular distribution. Here, we developed a rapid and convenient isolation method for fractionating polysaccharides with different characteristics and optimized it using a polysaccharide mixture from Korean persimmon leaves. A crude polysaccharide mixture, persimmon leaves-enzyme (PLE) fraction, was isolated from persimmon leaves digested with pectinase and ethanol precipitation. The PLE fraction was further fractionated with a serially diluted ethanol solution (ethanol : deionized water=4:1, 2:1, 1.5:1, 1:1, and 0.5:1) to produce 10 subfractions (five precipitate fractions labeled from PLE-4 to PLE-0.5 and five supernatant fractions labeled from PLE-4S to PLE-0.5S). HPLC analysis indicated that PLE-4 and -2 consisted of diverse polysaccharides, whereas PLE-1.5, -1, and -0.5 contained high molecular weight (MW) polysaccharides. The fractions from PLE-4 to PLE-1 were mostly composed of 13 different characteristic sugars in rhamnogalacturonan (RG) I and II, and the sugars contained an arabino-${\beta}$-3,6-galactan moiety. However, PLE-0.5 did not contain RG-II or ${\beta}$-arabino-3,6-galactan. Treatment of macrophages with fractions PLE-1.5S and PLE-1S led to a $10{\mu}g/mL$ increase in interleukin (IL)-6 production, whereas treatment with PLE-4S and PLE-2S fractions composed of low MW polysaccharides resulted in reduced levels of IL-6. These results indicate that this isolation method may be useful for the rapid and convenient fractionation of bioactive RGs from polysaccharide mixtures with various properties.

다당이 갖는 각종 생물 활성은 그 구조의 특성 및 분자량 분포에 의해 달라지므로 특정 다당을 분리하기 위한 정제과정은 다당 연구에 있어 필수적으로 요청된다. 본 연구에서는 서로 다른 특성을 소유한 다당을 분획하기 위한 간편하고 신속한 분리법을 개발하기 위해 한국산 감잎으로부터 조제한 다당 혼합물을 이용, 본 분리법을 최적화하였다. 감잎은 pectinase 처리 후 에탄올 침전법을 통해 조다당 획분인 PLE로 조제되었으며, PLE는 재차 농도별로 연속 희석된 에탄올 용액(EtOH : DIW=4:1, 2:1, 1.5:1, 1:1 및 0.5:1)을 이용하여 총 10개 획분(5개 침전물 획분: PLE-4~PLE-0.5, 5개 상등액 획분: PLE-4S~PLE-0.5S)으로 분획하였다. HPLC 분석 결과 PLE-4, PLE-2 획분은 저분자와 고분자 획분이 혼합된 다당이, PLE-1.5~0.5 획분에는 고분자 다당이 주로 검출되었다. 또한 PLE-4~PLE-1 획분은 구성당 분석 결과 RG(rhamnogalacturonan)-I과 RG-II 다당의 지표 구성당인 총 13종의 서로 다른 당으로 구성되어 있음이 확인되었으며, ${\beta}$-arabino-3,6-galactan 잔기를 함유하고 있는 것으로 확인되었다. 하지만 PLE-0.5 획분에서는 RG-II 및 ${\beta}$-arabino-3,6-galactan 잔기를 함유하고 있지 않았다. 한편 PLE-1.5S~PLE-1S 획분을 처리한 마우스 복강 대식세포에서는 농도 의존적인 IL-6의 생산 증가가 관찰된 반면, 저분자 다당으로 구성된 PLE-4S 및 PLE-2S 획분에서는 활성이 매우 낮음이 확인되었다. 이상의 결과로부터 본 분리 방법이 다양한 특성을 갖는 다당의 혼합물로부터 생물 활성을 갖는 RG류를 신속하고 간편하게 분리하는 데 있어 유용한 방법임을 확인할 수 있었다.

Keywords

References

  1. Ridley BL, O'Neill MA, Mohnen D. 2001. Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry 57: 929-967. https://doi.org/10.1016/S0031-9422(01)00113-3
  2. O'Neill M, Albersheim P, Darvill A. 1990. The pectic polysaccharides of primary cell walls. In Methods Plant Biochem. Dey PM, ed. Academic Press, London, UK. Vol 2, p 415-441.
  3. Engelsen SB, Cros S, Mackie W, Perez S. 1996. A molecular builder for carbohydrates: application to polysaccharides and complex carbohydrates. Biopolymers 39: 417-433. https://doi.org/10.1002/(SICI)1097-0282(199609)39:3<417::AID-BIP13>3.3.CO;2-R
  4. Ishii T, Matsunaga T. 2001. Pectic polysaccharide rhamnogalacturonan II is covalently linked to homogalacturonan. Phytochemistry 57: 969-974. https://doi.org/10.1016/S0031-9422(01)00047-4
  5. Perez S, Rodriguez-Carvajal MA, Doco T. 2003. A complex plant cell wall polysaccharide: rhamnogalacturonan II. A structure in quest of a function. Biochimie 85: 109-121. https://doi.org/10.1016/S0300-9084(03)00053-1
  6. Srivastava R, Kulshreshtha DK. 1989. Bioactive polysaccharides from plants. Phytochemistry 28: 2877-2883. https://doi.org/10.1016/0031-9422(89)80245-6
  7. Shin KS, Kiyohara H, Matsumoto T. 1998. Rhamnogalacturonan II dimers cross-linked by borate diesters from the leaves of Panax ginseng C.A. Meyer are responsible for expression of their IL-6 production enhancing activities. Carbohydr Res 307: 97-106. https://doi.org/10.1016/S0008-6215(98)00016-0
  8. Mueller EA, Anderer FA. 1990. Chemical specificity of effector cell/tumor cell bridging by a Viscum album rhamnogalacturonan enhancing cytotoxicity of human NK cells. Immunopharmacology 19: 69-77. https://doi.org/10.1016/0162-3109(90)90028-D
  9. Shin KS, Lee H. 1997. Structural analysis of the unusual sugar-containing oligosaccharides formed by the selective cleavage of weakly acidic polysaccharide. Korean J Food Sci Technol 29: 1105-1112.
  10. Mueller EA, Anderer FA. 1990. Synergistic action of a plant rhamnogalacturonan enhancing antitumor cytotoxicity of human natural killer and lymphokine-activated killer cells: chemical specificity of target cell recognition. Cancer Res 50: 3646-3651.
  11. McNeil M, Darvill AG, Albersheim P. 1980. Structure of plant cell walls: X. Rhamnogalacturonan I , a structurally complex pectic polysaccharide in the walls of suspensioncultured sycamore cells. Plant Physiol 66: 1128-1134. https://doi.org/10.1104/pp.66.6.1128
  12. Jung WY, Jeong JM. 2012. Change of antioxidative activity at different harvest time and improvement of atopic dermatitis effects for persimmon leaf extract. Kor J Herbology 27: 41-49. https://doi.org/10.6116/kjh.2012.27.1.41
  13. Jung UJ, Lee JS, Bok SH, Choi MS. 2011. Effects of extracts of persimmon leaf, buckwheat leaf, and Chinese matrimony vine leaf on body fat and lipid metabolism in rats. J Korean Soc Food Sci Nutr 40: 1215-1226. https://doi.org/10.3746/jkfn.2011.40.9.1215
  14. Kawakami K, Aketa S, Sakai H, Watanabe Y, Nishida H, Hirayama M. 2011. Antihypertensive and vasorelaxant effects of water-soluble proanthocyanidins from persimmon leaf tea in spontaneously hypertensive rats. Biosci Biotechnol Biochem 75: 1435-1439. https://doi.org/10.1271/bbb.100926
  15. Kim HJ, Kim MK. 2003. Anticancer effect of persimmon leaf extracts on Korean gastric cancer cell. Korean J Nutr 36: 133-146.
  16. Moon SH. 2002. Inhibitory effect of persimmon leaves on the mutagenicity in spore rec assay and on the growth of human cancer cells. Korean J Food & Nutr 15: 23-28.
  17. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356. https://doi.org/10.1021/ac60111a017
  18. Blumenkrantz N, Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Anal Biochem 54: 484-489. https://doi.org/10.1016/0003-2697(73)90377-1
  19. Bradford MM. 1976. 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. https://doi.org/10.1016/0003-2697(76)90527-3
  20. Karkhanis YD, Zeltner JY, Jackson JJ, Carlo DJ. 1978. A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of gram-negative bacteria. Anal Biochem 85: 595-601. https://doi.org/10.1016/0003-2697(78)90260-9
  21. Jones TM, Albersheim P. 1972. A gas chromatographic method for the determination of aldose and uronic acid constituents of plant cell wall polysaccharides. Plant Physiol 49: 926-936. https://doi.org/10.1104/pp.49.6.926
  22. van Holst GJ, Clarke AE. 1985. Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion. Anal Biochem 148: 446-450. https://doi.org/10.1016/0003-2697(85)90251-9
  23. McNeil M, Darvill AG, Aman P, Franzen LE, Albersheim P. 1982. Structural analysis of complex carbohydrates using high-performance liquid chromatography, gas chromatography, and mass spectrometry. In Methods in Enzymology. Ginsburg V, ed. Academic Press, New York, NY, USA. Vol 83, p 3-45.
  24. Yu KW, Kim YS, Shin KS, Kim JM, Suh HJ. 2005. Macrophage-stimulating activity of exo-biopolymer from cultured rice bran with Monascus pilosus. Appl Biochem Biotechnol 126: 35-48. https://doi.org/10.1007/s12010-005-0004-6
  25. Yu KW, Hwang JH. 2001. Characterization of bone marrow cell proliferating arabinogalactan through Peyer's patch cells from rhizomes of Atractylodes lancea DC. J Food Sci Nutr 6: 180-186.
  26. van Holst GJ, Clarke AE. 1985. Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion. Anal Biochem 148: 446-450. https://doi.org/10.1016/0003-2697(85)90251-9
  27. Keller R, Keist R, Wechsler A, Leist TP, van der Meide PH. 1990. Mechanisms 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. https://doi.org/10.1002/ijc.2910460422
  28. Nathan CF, Murray HW, Cohn ZA. 1980. The macrophage as an effector cell. N Engl J Med 303: 622-626. https://doi.org/10.1056/NEJM198009113031106
  29. Ohta Y, Lee JB, Hayashi K, Fujita A, Park DK, Hayashi T. 2007. In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans. J Agric Food Chem 55: 10194-10199. https://doi.org/10.1021/jf0721287
  30. Meyer RA. 2007. Immunology: from cell biology to disease. 1st ed. Wiley Publisher, Hoboken, NJ, USA. p 102-107.
  31. Shin YA, Park HR, Hong HD, Shin KS. 2012. Immuno-stimulating activities of polysaccharide fractions isolated from persimmon leaves. Korean J Food & Nutr 25: 941-950. https://doi.org/10.9799/ksfan.2012.25.4.941

Cited by

  1. 쥐눈이콩-노루궁뎅이버섯 균사체 발효물의 생리활성 vol.30, pp.6, 2016, https://doi.org/10.9799/ksfan.2017.30.6.1348