Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 134, Issue 1, pp 153–162 | Cite as

Tissue culture and metabolome investigation of a wild endangered medicinal plant using high definition mass spectrometry

  • J. P. S. Oliveira
  • O. Hakimi
  • M. Murgu
  • M. G. B. Koblitz
  • M. S. L. Ferreira
  • L. C. Cameron
  • A. F. Macedo
Original Article


An increasing effort is dedicated to investigate the potential of native plants used in traditional medicine as a source of bioactive compounds for numerous industries. The bioprospection of the metabolome of medicinal and/or endangered plants has two important merits: confirming or revealing the biotechnological potential of that species, and assisting in its conservation. In addition, biotechnological techniques, such as tissue culture, are key strategies in conservation and multiplication of medicinal plants. This is the first in vitro development and non-targeted metabolome study by UPLC–QTOF–MSE of extracts from C. menthoides, an endangered medicinal plant. In vitro development investigation with a wide range of plant growth regulators resulted in maximum survival rate (81%) and the highest growth rate (1.74 cm ± 0.36) for plantlets cultured on Murashige and Skoog medium, supplemented with 1 µM gibberellic acid. Maximum rooting occurred on medium supplemented with 4.4 µM 6-benzyladenine, which also resulted in more leaves per plantlet (10.16 ± 1.7). We developed a protocol that can be used for the clonal propagation and ex situ conservation of this species. In terms of metabolome analysis, a total of 107 metabolites from several classes were detected and identified in its hydrophilic extract (HE), including organic acids and derivatives, glucosinolates, terpenes, phenolic compounds as well as other polar metabolites. The metabolites in HE with the greatest signal intensity included the isoquinoline alkaloid magnoflorine; the coumaric acid rosmarinic acid; the steroid-cardanolide convallatoxin; two anthraquinones including the poorly investigated ventinone A. Several molecules identified here carry potential pharmacological benefits such as anti-inflammatory and anticancer applications.


C. menthoides In vitro Growth regulators Metabolomics UPLC–MS Non-targeted 



Plant growth regulator


Indole-3-acetic acid






Gibberellic acid


Ultra-performance liquid chromatography


Quadrupole time of flight mass spectrometry


Electrospray ionization


Lipophilic extract


Hydrophlic extract



The authors are grateful to Prof. Dr. Agostini from Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, for his support in collecting and identifying the plant material; to Universidade Federal do Estado do Rio de Janeiro (UNIRIO) for scholarship; to Prof. Dr. Suellen Gomes Moreira from Federal Institute of Rio de Janeiro (IFRJ), Rio de Janeiro, Brazil, for helping in the development UPLC separation methodology; to Prof. Dr. André Ferreira from Oswaldo Cruz Foundation (FIOCRUZ) and Waters Corporation for logistical and technical support, respectively.

Authors contribution

JPSO—performed tissue culture, extraction, identification and acquisition experiments; analysed data; prepared all figures and wrote the manuscript. OH—advise; helped in experimental design; manuscript correction and final approval. MM—advise for compounds identification; manuscript correction and final approval. MGBK—advise; helped in experimental design; planned and performed extraction experiments funding of the project; manuscript correction and final approval. MSLF—planned and performed experiments of sample acquisition for metabolomics; helped in experimental design; project's funding; manuscript correction and final approval. LCC—mass spectrometry equipments funding and final approval. AFM—planned and performed experiments of plant material cultivation and extraction assays; helped in sample preparation for metabolomics, sample acquisition for metabolomics, compounds identification; project`s funding; manuscript correction and final approval.


This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Universidade Federal do Estado do Rio de Janeiro (Unirio).

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.

Supplementary material

11240_2018_1408_MOESM1_ESM.doc (70 kb)
Supplementary material 1 (DOC 69 KB)
11240_2018_1408_MOESM2_ESM.doc (52 kb)
Supplementary material 2 (DOC 52 KB)
11240_2018_1408_MOESM3_ESM.doc (366 kb)
Supplementary material 3 (DOC 366 KB)


  1. Agostini G, Echeverrigaray S (2006) Micropropagation of Cunila incisa Benth., a potential source of 1,8-cineole. Rev Bras Plantas Med 8:186–189Google Scholar
  2. Agostini F, dos Santos AC, Rossato M et al (2009) Essential oil yield and composition of Lamiaceae species growing in Southern Brazil. Braz Arch Biol Technol. Google Scholar
  3. Agostini G, Agostini F, Bertolazzi M et al (2010) Variation of the chemical composition of essential oils in Brazilian populations of C. menthoides Benth (Lamiaceae). Biochem Syst Ecol. Google Scholar
  4. Agostini G, Ribeiro TS, Moura S et al (2014) Cunila D. Royen Ex. L., Glechon Epl. and Hesperozygis Epl. (Lamiaceae) in South America: an ethnobotanical and phytochemical review. Agric Res Updat 7:49–66Google Scholar
  5. Bhattacharyya P, Kumar V, Van Staden J (2018) In vitro encapsulation based short term storage and assessment of genetic homogeneity in regenerated Ansellia africana (Leopard orchid) using gene targeted molecular markers. Plant Cell Tissue Organ Cult. Google Scholar
  6. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol. PubMedGoogle Scholar
  7. Bordignon SADL, Schenkel EP, Spitzer V (1997) The essential oil composition of Cunila microcephala and Cunila fasciculata. Phytochemistry. Google Scholar
  8. Bordignon SADL, Schenkel EP, Spitzer V (1998a) Essential oil of C. menthoides Bentham (Lamiaceae). J Essent Oil Res. Google Scholar
  9. Bordignon SADL, Schenkel EP, Spitzer V (1998b) The Essential oil composition of Cunila platyphylla Epling (Lamiaceae). Acta Farm Bonaer 17:17–20Google Scholar
  10. Bordignon SADL, Montanha JA, Schenkel EP (2003) Flavones and flavanones from south American Cunila species (Lamiaceae). Biochem Syst Ecol 31:785–788CrossRefGoogle Scholar
  11. Bose SK, Yadav RK, Mishra S, Sangwan RS, Singh AK, Mishra B, Srivastava AK, Sangwan NS (2013) Effect of gibberellic acid and calliterpenone on plant growth attributes, trichomes, essential oil biosynthesis and pathway gene expression in differential manner in Mentha arvensis L. Plant Physiol Biochem 66:150–158CrossRefPubMedGoogle Scholar
  12. Cramer CN, Brown JM, Tomczyk N et al (2017) Electron transfer dissociation of all ions at all times, MSETD, in a quadrupole time-of-flight (Q-ToF) mass spectrometer. J Am Soc Mass Spectrom. Google Scholar
  13. Delgado G, Hernández J, Pereda-Miranda R (1989) Triterpenoid acids from Cunila lythrifolia. Phytochemistry 28(5):1483–1485CrossRefGoogle Scholar
  14. Erland LA, Shukla MR, Glover WB, Saxena PK (2017) A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture. Plant Cell Tissue Organ Cult 131:459–470CrossRefGoogle Scholar
  15. Evans DA, Sharp WR, Flick CE (1981) Growth and behavior of cell cultures: embryogenesis and organogenesis. Plant cell culture: methods and applications in agriculture. Academic Press, New York, pp 45–113Google Scholar
  16. Fandeur T, Moretti C, Polonsky J (1985) In vitro and in vivo assessement of the antimalarial activity of sergeolide. Planta Med. Google Scholar
  17. Fracaro F, Echeverrigaray S (2001) Micropropagation of Cunila galioides, a popular medicinal plant of south Brazil. Plant Cell Tissue Organ Cult. Google Scholar
  18. Frank T, Engel K-H (2013) Metabolomic analysis of plants and crops. Metabolomics in food and nutrition. Elsevier, Amsterdam, pp 148–191CrossRefGoogle Scholar
  19. Gavarić N, Kovač J, Kretschmer N et al (2015) Natural products as antibacterial agents—antibacterial potential and safety of post-distillation and waste material from Thymus vulgaris L., Lamiaceae. In: Varaprasad B (ed) Concepts, compounds and the alternatives of antibacterials. InTech, RijekaGoogle Scholar
  20. Haider F, Bagchi GD, Singh AK (2009) Effect of calliterpenone on growth, herb yield and oil quality of Mentha arvensis. Int J Integr Biol 7:53–57Google Scholar
  21. Huang C-K, Yu T, de la Monte SM et al (2015) Restoration of Wnt/β-catenin signaling attenuates alcoholic liver disease progression in a rat model. J Hepatol. Google Scholar
  22. Isah T, Umar S, Mujib A, Sharma MP, Rajasekharan PE, Zafar N, Frukh A (2017) Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield. Plant Cell Tissue Organ Cult 132:239–265CrossRefGoogle Scholar
  23. Jung HA, Ali MY, Jung HJ et al (2016) Inhibitory activities of major anthraquinones and other constituents from Cassia obtusifolia against β-secretase and cholinesterases. J Ethnopharmacol. Google Scholar
  24. Kamatou GPP, Viljoen AM (2008) Linalool—a review of a biologically active compound of commercial importance. Nat Prod Commun 3:1183–1192Google Scholar
  25. Karam NS, Jawad FM, Arikat NA, Shibl RA (2003) Growth and rosmarinic acid accumulation in callus, cell suspension, and root cultures of wild Salvia fruticosa. Plant Cell Tissue Organ Cult 73:117–121CrossRefGoogle Scholar
  26. Kim M-S, Bang JH, Lee J et al (2015) Salvia miltiorrhiza extract protects white matter and the hippocampus from damage induced by chronic cerebral hypoperfusion in rats. BMC Complement Altern Med. Google Scholar
  27. Leng LW, Lai-Keng C (2004) Plant regeneration from stem nodal segments of Orthosiphon stamineus Benth., a medicinal plant with diuretic activity. Vitr Cell Dev Biol. Google Scholar
  28. Link A, Balaguer F, Goel A (2010) Cancer chemoprevention by dietary polyphenols: promising role for epigenetics. Biochem Pharmacol. PubMedPubMedCentralGoogle Scholar
  29. Macedo AF, Barbosa NC, Esquibel MA, Souza MM, Cechinel-Filho V (1999) Pharmacological and phytochemical studies of callus culture extracts from Alternanthera brasiliana. Pharmazie 54:776–777PubMedGoogle Scholar
  30. Mišić D, Šiler B, Gašić U et al (2015) Simultaneous UHPLC/DAD/(+/–)HESI-MS/MS analysis of phenolic acids and nepetalactones in methanol extracts of nepeta species: a possible application in chemotaxonomic studies. Phytochem Anal. PubMedGoogle Scholar
  31. Morris JS, Facchini PJ (2016) Isolation and characterization of reticuline N-methyltransferase involved in biosynthesis of the aporphine alkaloid magnoflorine in opium poppy. J Biol Chem. Google Scholar
  32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  33. Musembi NN, Hutchinson MJ, Waithaka K (2015) The effects of 6-benzylaminopurine and gibberellic acid on postharvest physiology of lisianthus (Eustoma grandiflorum) flowers: II. influence of dose on inflorescence architecture and quality. Acta Hortic 1077:65–74CrossRefGoogle Scholar
  34. Pourebad N, Motafakkerazad R, Kosari-Nasab M et al (2015) The influence of TDZ concentrations on in vitro growth and production of secondary metabolites by the shoot and callus culture of Lallemantia iberica. Plant Cell Tissue Organ Cult. Google Scholar
  35. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L (2011) Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chemie Int Ed. Google Scholar
  36. Schneider NFZ, Silva IT, Persich L et al (2017) Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation. Mol Cell Biochem. PubMedGoogle Scholar
  37. Scognamiglio M, D’Abrosca B, Esposito A (2015) Chemical composition and seasonality of aromatic mediterranean plant species by NMR-based metabolomics. J Anal Methods Chem. PubMedPubMedCentralGoogle Scholar
  38. Silva CL, Câmara JS (2013) Profiling of volatiles in the leaves of Lamiaceae species based on headspace solid phase microextraction and mass spectrometry. Food Res Int. Google Scholar
  39. Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–131PubMedGoogle Scholar
  40. Souza GHMF, Guest PC, Martins-de-Souza D (2017) LC-MSE, multiplex MS/MS, ion mobility, and label-free quantitation in clinical proteomics. In Multiplex biomarker technique. Human Press, New York, pp 57–73CrossRefGoogle Scholar
  41. Stansbury J (2014) Rosmarinic acid as a novel agent in the treatment of allergies and asthma. J Restor Med. Google Scholar
  42. Tang Y, Jacobi A, Vater C et al (2014) Salvianolic acid B protects human endothelial progenitor cells against oxidative stress-mediated dysfunction by modulating Akt/mTOR/4EBP1, p38 MAPK/ATF2, and ERK1/2 signaling pathways. Biochem Pharmacol. Google Scholar
  43. Trivellini A, Lucchesini M, Maggini R et al (2016) Lamiaceae phenols as multifaceted compounds: bioactivity, industrial prospects and role of “positive-stress”. Ind Crops Prod. Google Scholar
  44. Wang J, Xiong X, Feng B (2013) Cardiovascular effects of salvianolic Acid B. Evid Based Complement Alternat Med. Google Scholar
  45. Xu H, Zhou Y, Lu C et al (2012) Salvianolic acid B lowers portal pressure in cirrhotic rats and attenuates contraction of rat hepatic stellate cells by inhibiting RhoA signaling pathway. Lab Investig. PubMedCentralGoogle Scholar
  46. Xu M, Heidmarsson S, Olafsdottir ES et al (2016) Secondary metabolites from cetrarioid lichens: chemotaxonomy, biological activities and pharmaceutical potential. Phytomedicine. Google Scholar
  47. Zhao Y-Y, Lin R-C (2014) UPLC–MSE application in disease biomarker discovery: the discoveries in proteomics to metabolomics. Chem Biol Interact. Google Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Protein Biochemistry - Center of Innovation of Mass SpectrometryFederal University of Rio de Janeiro StateRio de JaneiroBrazil
  2. 2.Integrated Laboratory of Plant Biology, Department of Botany, Institute of BiosciencesFederal University of Rio de Janeiro StateRio de JaneiroBrazil
  3. 3.Waters Technologies do BrasilBarueriBrazil
  4. 4.Food and Nutrition Graduate Program, Nutritional Biochemistry CenterFederal University of Rio de Janeiro StateRio de JaneiroBrazil
  5. 5.Department of Biochemistry and Sportomics, Olympic LaboratoryBrazil Olympic CommitteeRio de JaneiroBrazil

Personalised recommendations