Biotechnology Letters

, Volume 31, Issue 4, pp 607–614 | Cite as

Production of imidazole alkaloids in cell cultures of jaborandi as affected by the medium pH

  • N. L. Andreazza
  • I. N. Abreu
  • A. C. H. F. Sawaya
  • M. N. Eberlin
  • P. Mazzafera
Original Research Paper


The effect of pH (from 4.8 to 9.8) on the production of pilosine and pilocarpine and on their partition between cell and medium was studied in two lineages (P and PP) of Pilocarpus microphyllus cell suspension cultures. Highest mass accumulation was observed at high pHs and both lineages produced pilocarpine while only lineage PP produced pilosine. Both alkaloids were released in the medium but higher accumulation occurred in the cells. The highest production of pilocarpine was at pH 8.8–9.8 in both cell lineages. Other imidazole alkaloids were also identified in both lineages. At all pHs tested, the pH in the media cultures tended to stabilize around 6 after 10–15 days of cultivation. NO3 and NH4 + variation in the media might partially explain the pH stabilization.


Medium pH Membrane transporters Pilocarpine Pilocarpus microphyllus Pilosine 



N.L.A., I.N.A. and A.C.H.F.S. thank the Conselho Nacional de Desenvolvimento Científico Tecnológico do Brasil (CNPq-Brasil) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for student fellowships. M.N.E. and P.M. thank CNPq-Brasil for research fellowships. This work was partially supported by FAPESP.


  1. Abreu IN, Sawaya ACHF, Eberlin MN, Mazzafera P et al (2005) Production of pilocarpine in callus of Jaborandi (Pilocarpus microphyllus Stapf). In Vitro Cell Dev Biol Plant 41:806–811CrossRefGoogle Scholar
  2. Abreu IN, Andreazza NL, Sawaya ACHF, Eberlin MN, Mazzafera P (2007a) Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi. Plant Biol 9:793–799PubMedCrossRefGoogle Scholar
  3. Abreu IN, Mazzafera P, Eberlin MN, Zullo MAT, Sawaya ACHF (2007b) Characterization of the variation of the imidazole alkaloid profile of Pilocarpus microphyllus in different seasons and parts of the plant by electrospray ionization mass spectrometry fingerprinting and identification of novel alkaloids by tandem mass spectrometry. Rapid Commun Mass Spectrom 21:1205–1213PubMedCrossRefGoogle Scholar
  4. Andrade-Neto M, Mendes PH, Silveira ER (1996) An imidazole alkaloid and other constituents from Pilocarpus trachyllophus. Phytochemistry 42:885–887CrossRefGoogle Scholar
  5. Avancini G, Abreu IN, Saldana MDA, Mohamed RS, Mazzafera P (2003) Induction of pilocarpine formation in jaborandi leaves by salicylic acid and methylasmonate. Phytochemistry 63:171–175PubMedCrossRefGoogle Scholar
  6. Balestri E, Cinelli F (2001) Isolation and cell wall regeneration of protoplasts from Posidonia oceanica and Cymodocea nodosa. Aquat Bot 70:237–242CrossRefGoogle Scholar
  7. Baumann T, Rohrig L (1989) Formation and intracellular accumulation of caffeine and chlorogenic acid in suspension cultures of Coffea arabica. Phytochemistry 28:2667–2669CrossRefGoogle Scholar
  8. Biondi S, Antognoni F, Perellino NC, Sacchetti G, Minghetti A, Poli F (2004) Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl. Plant Biosyst 138:117–124CrossRefGoogle Scholar
  9. Brodelius P, Deus B, Mosbach K, Zenk MH (1979) Immobilized plant cells for the production and transformation of natural products. FEBS Lett 103:93–97PubMedCrossRefGoogle Scholar
  10. Buckland E, Townsley PM (1975) Coffee cell suspension cultures: caffeine and chlorogenic acid content. J Inst Can Sci Technol 8:164–165Google Scholar
  11. Cataldo DA, Haroon M, Schrader LE, Youngs VL (1975) Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun Soil Sci Plant Anal 6:71–80CrossRefGoogle Scholar
  12. Charlet S, Gillet F, Villarreal ML, Barbotind JN, Fliniauxa MA, Nava-Saucedod JE (2000) Immobilisation of Solanum chrysotrichum plant cells within Ca-alginate gel beads to produce an antimycotic spirostanol saponin. Plant Physiol Biochem 38:875–880CrossRefGoogle Scholar
  13. Davies AN, Broadley K, Beighton D (2001) Xerostomia in patients with advanced cancer. J Pain Symp Manag 22:820–825CrossRefGoogle Scholar
  14. Dong H, Zhong J (2001) Significant improvement of taxane cell production in suspension cultures of Taxus chinensis by combining elicitation with sucrose feed. Biotechnol Eng J 8:145–150Google Scholar
  15. El-Sayed M, Choi YH, Frédérich M, Roytrakul S, Verpoorte R (2004) Alkaloid accumulation in Catharanthus roseus cell suspension fed with stemmadenine. Biotechnol Lett 26:793–798PubMedCrossRefGoogle Scholar
  16. Goodman CD, Casati P, Walbot V (2004) A multidrug resistance associated protein involved in anthocyanin transport in Zea mays. Plant Cell 16:1812–1826PubMedCrossRefGoogle Scholar
  17. Haldimann D, Brodelius P (1987) Redirecting cellular metabolism by immobilization of cultured plant cells: a model study with Coffea arabica. Phytochemistry 26:1431–1434CrossRefGoogle Scholar
  18. Kintzios S, Makri O, Panagiotopoulos E, Scapeti M (2003) In vitro rosmarinic acid accumulation in sweet basil (Ocimum basilicum L.). Biotechnol Lett 25:405–408PubMedCrossRefGoogle Scholar
  19. Martinoia E, Klein M, Geisler M, Bovet L, Forestier C, Kolukisaoglu Ü, Müller-Röber B, Schulz B (2002) Multifunctionality of plant ABC transporters—more than just detoxifiers. Planta 214:345–355PubMedCrossRefGoogle Scholar
  20. McCullough H (1967) The determination of ammonia in whole blood by a direct colorimetric method. Clin Chim Acta 17:297–304PubMedCrossRefGoogle Scholar
  21. Migdal C (2000) Glaucoma medical treatment: philosophy, principles and practice. Eye 14:515–518PubMedGoogle Scholar
  22. Minocha SC (1987) pH of the medium and the growth and metabolism of cells in culture. In: Bonga JM, Durzan DJ (eds) Cell and tissue culture in forestry vol. 1: general principles and biotechnology. Martinus Nijhoff Publishers, Dordrecht, pp 125–141Google Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. Pinheiro CUB (1997) Jaborandi (Pilocarpus sp., Rutaceae): a wild species and its transformation into a crop. Econ Bot 51:49–58Google Scholar
  25. Sakai K, Shitan N, Sato F, Ueda K, Yazaki K (2002) Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein. J Exp Bot 53:1879–1886PubMedCrossRefGoogle Scholar
  26. Salisbury FB, Ross CW (1992) Plant physiology. Wadsworth Publishing Company, BelmontGoogle Scholar
  27. Sandhu SS, Abreu IN, Colombo C, Mazzafera P (2006) Pilocarpine content and molecular diversity in Jaborandi. Sci Agric 63:478–482CrossRefGoogle Scholar
  28. Sawaya ACHF, Abreu IN, Andreazza NL, Eberlin M, Mazzafera P (2008) HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus. Molecules 13:1518–1529PubMedCrossRefGoogle Scholar
  29. Takayama S, Misawa M, Ko K, Misato T (1977) Effect of cultural conditions on the growth of Agrostemma githago cell in suspension culture and concomitant production of an anti-plant virus substance. Physiol Plant 41:313–320CrossRefGoogle Scholar
  30. Terasaka K, Sakai K, Sato F, Yamamoto H, Yasaki K (2003) Thalictrum minus cell cultures and ABC-like transporter. Phytochemistry 62:483–489PubMedCrossRefGoogle Scholar
  31. Van Hengel AJ, Harkes MP, Wichers HJ, Hesselink PGM, Buitelaar RM (1992) Characterization of callus and camptothecin production by cell lines of Camptotheca acuminate. Plant Cell Tisss Org Cult 28:11–18CrossRefGoogle Scholar
  32. Verpoorte R, Memelink J (2002) Engineering secondary metabolite in plant. Curr Opin Biotechnol 13:181–187PubMedCrossRefGoogle Scholar
  33. Verpoorte R, van der Heijden R, ten Hoopen HJG, Memelink J (1999) Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals. Biotechnol Lett 21:467–479CrossRefGoogle Scholar
  34. Wynn RL (1996) Oral pilocarpine (Salagen): a recently approved salivary stimulant. Gen Dent 44:29–30Google Scholar
  35. Yazaki K (2006) ABC transporters involved in the transport of plant secondary metabolites. FEBS Lett 580:1183–1191PubMedCrossRefGoogle Scholar
  36. Yu F, Zhang D, Bai F, An L (2005) The accumulation of isocamptothecin A and B in suspension cell culture of Camptotheca acuminate. Plant Cell Tissue Org Cult 81:159–163CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • N. L. Andreazza
    • 1
  • I. N. Abreu
    • 1
    • 2
  • A. C. H. F. Sawaya
    • 3
  • M. N. Eberlin
    • 3
  • P. Mazzafera
    • 1
  1. 1.Departamento de Fisiologia Vegetal, Instituto de BiologiaUniversidade Estadual de CampinasCampinasBrazil
  2. 2.Plant Physiology DepartmentUmeå UniversityUmeåSweden
  3. 3.Instituto de QuímicaUniversidade Estadual de CampinasCampinasBrazil

Personalised recommendations