Advertisement

In Vitro Cellular & Developmental Biology - Plant

, Volume 36, Issue 6, pp 492–500 | Cite as

Isolation of oligomeric proanthocyanidins from flavonoid-producing cell cultures

  • F. E. Kandil
  • L. Song
  • J. M. Pezzuto
  • K. Marley
  • D. S. Seigler
  • M. A. L. Smith
Article

Summary

The extraction, fractionation, and chromatographic separation of a series of proanthocyanidin monomers and oligomers were facilitated using a flavonoid-rich cell culture of Vaccinium pahalae Skottsberg as the donor tissue. The cell cultures, after exposure to light, readily accumulated anthocyanin pigments and other flavonoids in relatively large amounts, with minimal concurrent production of pectins, enzymes, and complex sugars produced in field-grown Vaccinium berries. The absence of these interfering compounds greatly simplified the isolation and purification of proanthocyanidins and other phenolic compounds from cell cultures, primarily using vacuum chromatography. Subsequently, the structures and molecular weights of several individual compounds and the general composition of unresolved fractions were established with 1H- and 13C-NMR and MS. The initial extract of V. pahalae cell cultures was readily fractionated on silica gel to yield a series of fractions containing proanthocyanidin B-2, a series of increasingly polar proanthocyanidin oligomers ranging from dimers to heptamers largely based on (−)-epicatechin structures (some with A-type linkages), a mixture of E- and Z-p-coumaric acid, the corresponding 4-O-glucoside, and other compounds containing E- and Z-p-coumaric acid moieties. Cell culture extracts demonstrated broad antioxidant capacity and significant ability to inhibit tumor promotion in vitro, as indicated in an ornithine decarboxylase assay.

Key words

condensed tannins proanthocyanidins 4-O-glucosides of E- and Z-p-coumaric acid proanthocyanidin B-2 (−)-epicatechin (+)-catechin vacuum chromatography 1H-NMR 13C-NMR mass spectrometry 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aherne, S. A.; O'Brien, N. M. The flavonoids myricetin, quercetin, and rutin, protect against cholestan-3β, 5α, 6β-triol-induced toxicity in Chinese hamster ovary cells in vitro. Nutr. Res. 19:749–760; 1999.CrossRefGoogle Scholar
  2. Bomser, J.; Madhavi, D. L.; Singletary, K.; Smith, M. A. L. In vitro anticancer activity of fruit extracts from Vaccinium species. Planta Medica 62:212–216; 1996.PubMedCrossRefGoogle Scholar
  3. Bourgaud, F.; Bouque, V.; Guckert, A. Production of flavonoids by Psoralea hairy root cultures. Plant Cell Tiss. Org. Cult. 56:97–104; 1999.CrossRefGoogle Scholar
  4. Castonguay, A.; Gali, H.; Perchellet, E.; Gao, X.; Boukharta, M.; Jalbert, G.; Okuda, T.; Yoshida, T.; Hatano, T.; Perchellet, J.-P. Antitumorigenic and antipromoting activities of ellagic acid, ellagitannins, and oligomeric anthocyanin and procyanidin. Int. J. Oncol. 10:367–373; 1997.Google Scholar
  5. Chen, D.; Liu, Y.; Hartman, D. R. Examination of fat oxidation products by FT-NMR and FTIR. Amer. Lab. 31:15–20; 1999.Google Scholar
  6. Colatuoni, A.; Betuglia, S.; Magistretti, M.; Donato, L. Effects of Vaccinium myrtillus anthocyanosides on arterial vasomotion. Arzneimittel Forsch. 41:905–909; 1991.Google Scholar
  7. Das, D. K.; Sato, M.; Ray, P. S.; Maulik, G.; Engelman, R. M.; Bertelli, A. A. E.; Bertelli, A. Cardioprotection of red wine: role of polyphenolic antioxidants. Drugs Exp. Clin. Res. 25:115–120; 1999.PubMedGoogle Scholar
  8. Ellis, B. E. Natural products from plant tissue culture. Nat. Prod. Rep. 5:581–612; 1988.PubMedCrossRefGoogle Scholar
  9. Fang, Y.; Smith, M. A. L.; Pépin, M. F. Benzyladenine restores anthocyanin pigmentation in suspension cultures of wild Vaccinium pahalae. Plant Cell Tiss. Org. Cult. 54:113–122; 1998.CrossRefGoogle Scholar
  10. Fang, Y.; Smith, M. A. L.; Pépin, M. F., The effects of exogenous methyl jasmonate in elicited anthocyanin-producing cell cultures of ohelo (Vaccinium pahalae). In Vitro Cell. Dev. Biol.—Plant 35:106–113; 1999.Google Scholar
  11. Ferreira, D.; Reinier, J.; Bekker, R. Condensed tannins. In: Pinto, M., ed. Comprehensive natural products chemistry, Vol. 3. Carbohydrates and their derivatives including tannins, cellulose, and related lignans. Amsterdam: Elsevier; 1999:747–797.Google Scholar
  12. Fletcher, A. C.; Porter, L. J.; Haslam, E.; Gupta, R. K. Plant proanthocyanidins. Part 3. Conformational and configurational studies of natural procyanidins. J. Chem. Soc. Perkin Trans. 1:1628–1636; 1977.CrossRefGoogle Scholar
  13. Gerhäuser, C.; Mar, W.; Lee, S. K.; Suh, N.; Luo, Y.; Kosmeder, H.; Moriarty, R. M.; Luyengi, L.; Kinghorn, A. D.; Fong, H. H. S.; Mehta, R. G.; Constantinou, A.; Moon, R. C.; Pezzuto, J. M. Rotenoids mediate potent chemopreventive activity through transcriptional regulation of ornithine decarboxylase. Nature Med. 1:260–266; 1995.PubMedCrossRefGoogle Scholar
  14. Gottrand, F.; Beghin, L.; Duhal, N.; Lacroix, B.; Bonte, J. P.; Fruchart, J. C.; Luc, G. Moderate red wine consumption in healthy volunteers reduced plasma clearance of apolipoprotein AII. Eur. J. Clin. Invest. 29:387–394; 1999.PubMedCrossRefGoogle Scholar
  15. Hegnauer, R. Ericaceae. Chemotaxonomie der pflanzen, Vol. 4. Basel: Birkhäuser Verlag; 1966.Google Scholar
  16. Hegnauer, R. Ericaceae. Chemotaxonomie der pflanzen, Vol. 8. Basel: Birkhäuser Verlag; 1989.Google Scholar
  17. Higuchi, M.; Yoshida, F. Lowry determination of protein in the presence of sulfhydryl compounds or other reducing agents. Anal. Biochem. 77:542–547; 1977.PubMedCrossRefGoogle Scholar
  18. Jacques, D.; Haslam, E.; Bedford, G. R.; Greatbanks, D. Plant proanthocyanidins. Part II. Proanthocyanidin-A2 and its derivatives. J. Chem. Soc. Perkin Trans. 1:2663–2671; 1974.CrossRefGoogle Scholar
  19. Kalt, W.; McDonald, J. Chemical composition of lowbush blueberry cultivars. J. Amer. Soc. Hort. Sci. 121:142–146; 1996.Google Scholar
  20. Kashiwada, Y.; Iizuka, H.; Yoshioka, K.; Chen, R.; Nonaka, G.; Nishioka, I. Tannins and related compounds XCIII. Occurrence of enantiomeric proanthocyanidins in the Leguminosae plants, Cassia fistula L. and C. javanica L. Chem. Pharm. Bull. 38:888–893; 1990.Google Scholar
  21. Koga, T.; Moro, K.; Nakamori, K.; Yamakoshi, J.; Hosoyama, H.; Kataoka, S.; Ariga, T. Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seed. J. Agric. Food Chem. 47:1892–1897; 1999.PubMedCrossRefGoogle Scholar
  22. Koganov, M.; Dueva, O.; Tsorin, B. Activities of plant-derived phenols in a fibroblast cell culture model. J. Nat. Prod. 62:481–483; 1999.CrossRefGoogle Scholar
  23. Lichti, U.; Gottesman, U. Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells. J. Cell Physiol. 113:433–439; 1982.PubMedCrossRefGoogle Scholar
  24. Lloyd, G.; McCown, B. Commercially-feasible micropropagation of mountain laurel Kalmia latifolia, by use of shoot tip culture. Proc. Int. Plant Prop. Soc. 30:421–427; 1980.Google Scholar
  25. Macheix, J.; Fleuriet, A. Phenolic acids in fruits. In: Rice-Evans, C. A.; Packer, L., eds. Flavonoids in health and disease. New York: Dekker; 1998:35–59.Google Scholar
  26. Madhavi, D. L.; Bomser, J.; Smith, M. A. L.; Singletary, K. Isolation of bioactive constituents from Vaccinium myrtillus (bilberry) fruits and cell cultures. Plant Sci. 131:95–103; 1998.CrossRefGoogle Scholar
  27. Madhavi, D. L.; Smith, M. A. L.; Berber-Jiménez, M. D. Expression of anthocyanins in callus cultures of cranberry (Vaccinium macrocarpon Ait.). J. Food Sci. 60:351–355; 1995.CrossRefGoogle Scholar
  28. Morimoto, S.; Nonaka, G.; Nishioka, I. Tannins and related compounds. XXXVIII. Isolation and characterization of flavan-3-ol glucosides and procyanidin oligomers from cassia bark (Cinnamomum cassia Blume). Chem. Pharm. Bull. 34:633–642; 1986.Google Scholar
  29. Narayan, M. S.; Naidu, K. A.; Ravishankar, G. A.; Srinivas, L.; Venkataraman, L. V. Antioxidant effect of anthocyanin on enzymatic and non-enzymatic lipid peroxidation. Prostagland. Leukotr. Essen. Fatty Acids 60:1–4; 1999.CrossRefGoogle Scholar
  30. Neera, S.; Arakawa, H.; Ishimaru, K. Tannin production in Sapium sebiferum callus cultures. Phytochem. 31:4143–4149; 1992.CrossRefGoogle Scholar
  31. Nonaka, G.; Nishioka, I. Tannins and related compounds VII. Phenylpropanoid-substituted epicatechins, cinchonains from Cinchona succirubra. Chem. Pharm. Bull. 30:4268–4276; 1982.Google Scholar
  32. Pezzuto, J. M. Natural product cancer chemopreventive agents. In: Arnason, J. T.; Mata, R.; Romeo, J. T., eds. Recent advances in phytochemistry, Vol. 29, Phytochemistry of medicinal plants. New York: Plenum Press; 1995:9–45.Google Scholar
  33. Porter, L. Flavans and proanthocyanidins. In: Harborne, J., ed. The flavonoids: advances in research since 1986. Boca Raton, USA: CRC Press/New York: Chapman and Hall; 1993:23–54.Google Scholar
  34. Prior, R.; Cao, G.; Martin, A.; Sofic, E.; McEwen, J.; O'Brien, C.; Lischner, N.; Ehlenfeldt, M.; Kalt, W.; Krewer, G.; Mainland, M. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of Vaccinium species. J. Agric. Food Chem. 46:2686–2693; 1998.CrossRefGoogle Scholar
  35. Rogers, R.; Smith, M. A. L. Consequences of in vitro and ex vitro root initiation for miniature rose production. J. Hort. Sci. 67:535–540; 1992.Google Scholar
  36. Smith, M. A. L.; Madhavi, D. L.; Fang, Y.; Tomczak, M. M. Continuous cell culture and product recovery from wild Vaccinium pahalae germplasm. J. Plant Physiol. 150:462–466; 1997.Google Scholar
  37. Smith, M. A. L.; Marley, K. A.; Seigler, D.; Singletary, K. W.; Meline, B. Bioactive properties of wild blueberry fruits. J. Food Sci. 65:352–356; 2000.CrossRefGoogle Scholar
  38. Smith, R. C.; Hargis, J. H. Reactions of urates and other antioxidants with galvinoxyl. Microchem. J. 31:18–21; 1985.CrossRefGoogle Scholar
  39. Stafford, H.; Lester, H. Procyanidins (condensed tannins) in green suspension cultures of Douglas fir compared with those in strawberry and avocado leaves by means of C18-reversed-phase chromatography. Plant Physiol. 66:1085–1090; 1980.PubMedCrossRefGoogle Scholar
  40. Thompson, R. S.; Jacques, D.; Haslam, E.; Tanner, R. J. N. Plant proanthocyanidins Part 1. Introduction: the isolation structure and distribution in nature of plant procyanidins. J. Chem. Soc. Perkin Trans. 1:1387–1399; 1972.CrossRefGoogle Scholar
  41. Waterhouse, A.; Price, S.; McCord, J. Reversed-phase high-performance liquid chromatography methods for analysis of wine polyphenols. Part A: oxidants and antioxidants. Meth. Enzymol. 299:113–121; 1999.CrossRefGoogle Scholar
  42. Weinges, K.; Kaltenhäuser, W.; Marx, H.; Nader, E.; Nader, F.; Perner, J.; Seiler, D. Zur kenntnis der proanthocyanidine X. Procyanidine aus früchten. Annalen der Chemie 711:184–204; 1968.Google Scholar

Copyright information

© Society for In Vitro Biology 2000

Authors and Affiliations

  • F. E. Kandil
    • 1
  • L. Song
    • 2
  • J. M. Pezzuto
    • 2
  • K. Marley
    • 3
  • D. S. Seigler
    • 1
  • M. A. L. Smith
    • 2
  1. 1.Department of Plant BiologyUniversity of IllinoisUrbanaUSA
  2. 2.Department of Medical Chemistry and PharmacognosyUniversity of IllinoisChicagoUSA
  3. 3.Department of Natural Resources and Environmental SciencesUniversity of IllinoisUrbanaUSA

Personalised recommendations