LC–MS–MS and GC–MS analyses of biologically active extracts of Tunisian Fenugreek (Trigonella foenum-graecum L.) Seeds

  • Henda Keskes
  • Sahla Belhadj
  • Lobna Jlail
  • Abdelfattah El Feki
  • Sami Sayadi
  • Noureddine Allouche
Original Paper


The objective of this study is to investigate the chemical composition of biologically active extracts from Fenugreek (Trigonella foenum-graecum L.) Seeds. A variety of in vitro models such as β-carotene-linoleic acid and 1,1-diphenyl-2-picryl hydrazyl (DPPH) were used to measure the antioxidant activity. Alpha-amylase inhibitory activity was evaluated by enzyme inhibition using in vitro assay. The hexane extract with a potent inhibition ability against linoleic acid (95.4%) as well as a powerful α-amylase inhibitory activity (IC50 = 26.9 µg/mL) was analyzed by gas chromatography coupled with mass spectrometry (GC–MS). A total of 20 compounds were identified, some of them being reported for the first time in fenugreek seeds [e.g. β-thujone (10.8%), (2E, 4E)-decadienal (7.8%), heptanal (6.7%), and (2E, 4E)-heptadienal (5.3%)]. On the other hand, methanol extract exhibited a strong DPPH radical-scavenging activity (IC50 = 19.0 µg/mL) compared to that of BHT as well as the highest alpha-amylase inhibitory activity (IC50 = 52.1 μg/mL). HPLC technique coupled with negative ion electrospray ionization mass spectrometry and diode array detection (HPLC-DAD–ESI/MS) was employed to identify polyphenols in the methanol extract. The obtained results allowed the detection of 3 flavonoids glycosides and one amino acid identified on the basis of their UV and MS spectra, and comparisons with standards when available, as well as with literature data. A systematic study of the obtained MS spectra and the observed fragmentation allowed identifying for known compounds: vicenin-2, 4-hydroxyisoleucine, isoschaftoside and isoorientin. These two last compounds were identified for the first time in Fenugreek Seeds.


Fenugreek Seeds GC/MS HPLC-DAD–ESI/MS Flavonoid C-glycosides Biological activities 



The authors are grateful for the financial support of the Ministry of Higher Education and Scientific Research, Tunisia, granted to the Laboratory of Organic Chemistry, Natural Substances Team, Faculty of Sciences of Sfax, [Grant Number LR17ES08].

Compliance with ethical standards

Conflict of interest

The authors report that they have no conflicts of interest.


  1. 1.
    S. Barnes, M. Kirk, L. Coward, Isoflavones and their conjugates in soy foods: extraction conditions and analysis by HPLC-mass spectrometry. J. Agric. Food Chem. 11, 2466–2474 (1994)CrossRefGoogle Scholar
  2. 2.
    L.Z. Lin, X.G. He, M. Lindenmaier, J. Yang, M. Cleary, S.X. Qiu, G.A. Cordell, LC-ESI-MS study of the flavonoid glycoside malonates of red clover (Trifolium pratense). J. Agric. Food Chem. 48, 354–365 (2000)CrossRefGoogle Scholar
  3. 3.
    W. Andlauer, M.J. Martena, P. Fürst, Determination of selected phytochemicals by reversed-phase high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection. J. Chromatogr. A 849, 341–348 (1999)CrossRefGoogle Scholar
  4. 4.
    P. Waridel, J.L. Wolfender, K. Ndjoko, K.R. Hobby, H.J. Major, K. Hostettmann, Evaluation of quadrupole time-of-flight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic flavonoid isomers. J. Chromatogr. A 926, 29–41 (2001)CrossRefGoogle Scholar
  5. 5.
    J.B. Harborne, T.J. Mabry, H. Mabry, The Flavonoids. (Chapman and Hall, London, 1975)CrossRefGoogle Scholar
  6. 6.
    W. Wagner, S. Bladt, E.M. Zgainski, in Plant Drug Analysis. (Springer, Heidelberg, 1984)CrossRefGoogle Scholar
  7. 7.
    V. Cody, E. Middleton, J.B. Harborne, Plant flavonoids in biology and medicine: biochemical, pharmacological, and structure-activity relationships: proceedings of a symposium held in Buffalo, New York. Prog. Clin. Biol. Res. 213, 22–26, (1986)Google Scholar
  8. 8.
    M. Becchi, D. Fraisse, Fast atom bombardment and fast atom bombardment collision-activated dissociation/mass-analysed Ion kinetic energy analysis of C-glycosidic flavonoids. Biomed. Environ. Mass. Spectrom. 18, 122–130 (1989)CrossRefGoogle Scholar
  9. 9.
    E. Basch, C. Ulbricht, G. Kuo, P. Szapary, M. Smith, Therapeutic applications of fenugreek. Altern. Med. Rev. 8, 20–27 (2003)Google Scholar
  10. 10.
    A. Djeridane, M. Yousfi, B. Nadjemi, D. Boutassouna, P. Stocker, N. Vidal, Antioxidant activity of some algerian medicinal plants extracts containing phenolic compounds. Food Chem. 97, 654–660 (2006)CrossRefGoogle Scholar
  11. 11.
    Y. Massada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry. (Wiley, New York, 1996)Google Scholar
  12. 12.
    N.W. Davies, Gas chromatographic retention indices of monoterpenes on methyl silicone and Carbowax 20M phases. J. Chromatogr. A 503, 1–24 (1990)CrossRefGoogle Scholar
  13. 13.
    N. Fki, S. Allouche, Sayadi, The use of polyphenolic extract, purified hydroxytyrosol and 3, 4-dihydroxypheny l acetic acid from olive mill wastewater for the stabilization of refined oils: a potential alternative to synthetic antioxidants. Food Chem. 93, 197–204 (2005)CrossRefGoogle Scholar
  14. 14.
    N. Allouche, I. Fki, S. Sayadi, Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters. J. Agric. Food Chem. 52, 267–273 (2004)CrossRefGoogle Scholar
  15. 15.
    F.J. Gella, G. Gubern, R. Vidal, C. Francesca, Determination of total and pancreatic α amylase in human serum with 2-chloro-4-nitrophenyl-α-D-maltotrioside as substrate. Clin. Chim. Acta 259, 147–160 (1997)CrossRefGoogle Scholar
  16. 16.
    G. Mazza, D. Di Tommaso, S. Foti, Volatile constituents of sicilian fenugreek (Trigonella foenum-graecum L.) seeds. Sci. Aliments 22, 249–264 (2002)CrossRefGoogle Scholar
  17. 17.
    R. Mebazaa, A. Mahmoudi, M. Fouchet, M. Dos Santos, F. Kamissoko, A. Nafti, R. Ben Cheikh, B. Rega, V. Camel, Characterisation of volatile compounds in Tunisian fenugreek seeds. Food Chem 115, 1326–1336 (2009)CrossRefGoogle Scholar
  18. 18.
    J. Blank, S. Lin, R. Devaud, L.B. Fumeaux, Fay, The principal flavor component of fenugreek (Trigonella foenum-graecum L.)., in S.J. Risch, C.T. Ho eds., Spices, flavor chemistry and antioxidant properties, ACS symposium series no. 660, 12–28 (1997)Google Scholar
  19. 19.
    P. Girardon, J.M. Bessiere, J.C. Baccou, Y. Sauvaire, Volatile constituents of fenugreek seeds. Planta Med. 6, 533–544 (1985)CrossRefGoogle Scholar
  20. 20.
    Y. Sauvaire, P. Girardon, J.C. Baccou, A.M. Ristérucci, Changes in growth, proteins and free amino acids of developing seed and pod of fenugreek. Phytochemistry 23, 479–486 (1984)CrossRefGoogle Scholar
  21. 21.
    O. Belguith-Hadriche, M. Bouaziz, K. Jamoussi, S.J.S. Monique, A. El Feki, F. Makni-Ayedi, Comparative study on hypocholesterolemic and antioxidant activities of various extracts of fenugreek seeds. Food Chem. 138, 1448–1453 (2013)CrossRefGoogle Scholar
  22. 22.
    F. Cuyckens, M. Claeys, Mass spectrometry in the structural analysis of flavonoids. J. Mass. Spectrom. 39, 1–15 (2004)CrossRefGoogle Scholar
  23. 23.
    Q.M. Li, H. Van den Heuvel, L. Dillen, M. Claeys, Differentiation of 6-C and 8-C-glycosidic flavonoids by positive ion fast atom bombardment and tandem mass spectrometry. Biol. Mass. Spectrom. 21, 213–321 (1992)CrossRefGoogle Scholar
  24. 24.
    T. Iwashina, The structure and distribution of the flavonoids in plants. J. Plant. Res. 113, 287–299 (2000)CrossRefGoogle Scholar
  25. 25.
    B. Domon, C.E. Costello, A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 5, 397–409 (1988)CrossRefGoogle Scholar
  26. 26.
    Z. Benayad, C. Gómez-Cordovés, N. Es-Safi, Identification and quantification of flavonoid glycosides from fenugreek (Trigonella foenum-graecum) germinated seeds by LC–DAD–ESI/MS analysis. J. Food. Compost. Anal. 35, 21–29 (2014)CrossRefGoogle Scholar
  27. 27.
    D. Barreca, E. Bellocco, C. Caristi, U. Leuzzi, G. Gattuso, Distribution of C- and O-glycosyl flavonoids, (3-hydroxy-3-methylglutaryl) glycosyl flavanones and furocoumarins in Citrus aurantium L. juice. Food Chem. 124, 576–582 (2011)CrossRefGoogle Scholar
  28. 28.
    F. Sánchez-Rabaneda, O. Jáuregui, I. Casals, C. Andrés-Lacueva, M. Izquierdo-Pulido, R.M. Lamuela-Raventós, Liquid chromatography/electrospray ionization tandemmass spectrometric study of the phenolic composition of cocoa (Theobroma cacao). J. Mass Spectrom. 38, 35–42 (2003)CrossRefGoogle Scholar
  29. 29.
    P.G. Pietta, Flavonoids as antioxidants. J. Nat. Prod. 63, 1035–1042 (2000)CrossRefGoogle Scholar
  30. 30.
    W. Bors, W. Heller, C. Michel, M. Saran, Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods Enzymol 186, 343–355 (1990)CrossRefGoogle Scholar
  31. 31.
    K. Henda, L. Marc, C. Atef, B. Sahla, H. Besma, E.F. Abdelfattah, D. Vincent, B.S. Abdelhamid, D. Mohamed, A. Noureddine, Antioxidant and α-amylase inhibitory activities of extract and isolates from Zygogynum pancheri subsp. arrhantum. J. Asian Nat. Prod. Res. 16, 1132–1138 (2014)CrossRefGoogle Scholar
  32. 32.
    E.N. Frankel, S.W. Huang, J. Kanner, J.B. German, Interfacial phenomena in the evaluation of antioxidants: bulk oils vs. emulsions. J. Agric. Food Chem. 42, 1054–1059 (1994)CrossRefGoogle Scholar
  33. 33.
    E.N. Frankel, A.S. Meyer, The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric. 80, 1925–1940 (2000)CrossRefGoogle Scholar
  34. 34.
    R.P. Singh, K.N.C. Murthy, G.K. Jayprakasha, Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J. Agric. Food Chem. 50, 81–86 (2002)CrossRefGoogle Scholar
  35. 35.
    C.M. Liyana Pathirana, F. Shahidi, Antioxydant propreties of commercial soft and hard winter wheats (Triticum aestivium L.) and their milling fractions. J. Sci. Food Agric. 86, 477–485 (2006)CrossRefGoogle Scholar
  36. 36.
    G. Ruberto, M. Baratta, Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem. 69, 167–174 (2002)CrossRefGoogle Scholar
  37. 37.
    L.H. Cazarolli, L. Zanatta, E.H. Alberton, M.S. Figueiredo, P. Folador, R.G. Damazio, M.G. Pizzolatti, F.R. Mena Barreto Silva, Flavonoids: prospective drug candidates. Mini. Rev. Med. Chem. 8, 1429–1440 (2008)CrossRefGoogle Scholar
  38. 38.
    K. Hamden, H. Keskes, S. Belhaj, K. Mnafgui, A. Feki, N. Allouche, Inhibitory potential of omega-3 fatty and fenugreek essential oil key enzymes of carbohydrate-digestion and hypertension in diabetes rats. Lipids Health Dis. 10, 226 (2011)CrossRefGoogle Scholar
  39. 39.
    B. Christophe, M. Michèle, G. René, B. Yves, J. Marc, P. Pierre, S. Yves, R. Gérard, 4-Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion. Eur. J. Pharmacol. 3, 339–345 (2000)Google Scholar
  40. 40.
    Y. Sauvaire, P. Petit, C. Broca, M. Manteghetti, Y. Baissac, J. Fernandez-Alvarez, R. Gross, M. Roye, A. Leconte, R. Gomis, G. Ribes, 4-hydroxy isoleucine: a novel amino acid potentiator of insulin secretion. Diabetes 47, 206–210 (1998)CrossRefGoogle Scholar
  41. 41.
    J.S. Kim, C.S. Kwon, K.H. Son, Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid. Biosci. Biotech. Biochem. 64, 2458–2461 (2000)CrossRefGoogle Scholar
  42. 42.
    R.K. Gupta, K. Dharmendra, K.C. Amrendra, M. Mukesh, S. Ranjit, Antidiabetic activity of Passiflora incarnata Linn. in streptozotocin-induced diabetes in mice. J. Ethnopharmacol. 139, 801–806 (2012)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Henda Keskes
    • 1
  • Sahla Belhadj
    • 2
  • Lobna Jlail
    • 3
  • Abdelfattah El Feki
    • 2
  • Sami Sayadi
    • 3
  • Noureddine Allouche
    • 1
  1. 1.Laboratory of Organic Chemistry, Natural Substances Team (LR17ES08), Faculty of Sciences of SfaxUniversity of SfaxSfaxTunisia
  2. 2.Laboratory of Animal Ecophysiology, Faculty of Sciences of SfaxUniversity of SfaxSfaxTunisia
  3. 3.Laboratory of Environmental BioprocessesCenter of Biotechnology of SfaxSfaxTunisia

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