Skip to main content
SpringerLink
Log in

Ultra-high performance liquid chromatography Q-Orbitrap MS/MS–based profiling and quantification of limonoids in Meliaceae plants

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Meliaceae plants have been extensively used in agriculture, folklore, and traditional medicine. They are the major storehouses for structurally diverse limonoids (meliacins) possessing various bioactivities like antifeedant, insecticidal, antimicrobial, etc. However accurate detection of these tetranortriterpenes from the vast pool of metabolites in plant tissue extracts or biological sample is a crucial challenge. Though the mass spectrum (MS) provides the molecular mass and the corresponding elemental composition, it cannot be relied precisely. The exact identification of a specific metabolite demands the MS/MS spectrum containing the signature product ions. In the present study, we have developed the UHPLC Q-Orbitrap–based method for identification, quantification, and characterization of limonoids in different plant tissue extracts requiring minimum plant material. Using this method, we carried out the limonoid profiling in different tissue extracts of sixteen Meliaceae plants and the identification of limonoids was performed by comparing the retention time (RT), ESI-( +)-MS spectrum, and HCD-MS/MS of the purified fifteen limonoids used as reference standards. Our results revealed that early intermediates of the limonoid biosynthetic pathway such as azadiradione, epoxyazadiradione, and gedunin occurred more commonly in Meliaceae plants. The MS/MS spectrum library of the fifteen limonoids generated in this study can be utilized for identification of these limonoids in other plant tissue extracts, botanical fertilizers, agrochemical formulations, and bio pesticides.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Roy A, Saraf S. Limonoids: overview of significant bioactive triterpenes distributed in plants kingdom. Biol Pharm Bull. 2006;29:191–201. https://doi.org/10.1248/bpb.29.191.

    Article  CAS  PubMed  Google Scholar 

  2. Dreyer DL, Pickering MV, Cohan P. Distribution of limonoids in the rutaceae. Phytochemistry. 1972;11:705–13. https://doi.org/10.1016/0031-9422(72)80036-0.

    Article  CAS  Google Scholar 

  3. Chen J, Fan X, Zhu J, Song L, Li Z, Lin F, Yu R, Xu H, Zi J. Limonoids from seeds of Azadirachta indica A. Juss. and their cytotoxic activity. Acta Pharm Sin B. 2018;8:639–44. https://doi.org/10.1016/j.apsb.2017.12.009.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Gupta SC, Prasad S, Tyagi AK, Kunnumakkara AB, Aggarwal BB. Neem (Azadirachta indica): an Indian traditional panacea with modern molecular basis. Phytomedicine. 2017;34:14–20. https://doi.org/10.1016/j.phymed.2017.07.001.

    Article  CAS  PubMed  Google Scholar 

  5. Veitch GE, Beckmann E, Burke BJ, Boyer A, Maslen SL, Ley SV. Synthesis of azadirachtin: a long but successful journey. Angew Chemie - Int Ed. 2007;46:7629–32. https://doi.org/10.1002/anie.200703027.

    Article  CAS  Google Scholar 

  6. Ley SV, Abad-Somovilla A, Anderson JC, Ayats C, Bänteli R, Beckmann E, Boyer A, Brasca MG, Brice A, Broughton HB, Burke BJ, Cleator E, Craig D, Denholm AA, Denton RM, Durand-Reville T, Gobbi LB, Göbel M, Gray BL, Grossmann RB, Gutteridge CE, Hahn N, Harding SL, Jennens DC, Jennens L, Lovell PJ, Lovell HJ, De La Puente ML, Kolb HC, Koot WJ, Maslen SL, McCusker CF, Mattes A, Pape AR, Pinto A, Santafianos D, Scott JS, Smith SC, Somers AQ, Spilling CD, Stelzer F, Toogood PL, Turner RM, Veitch GE, Wood A, Zumbrunn C. The synthesis of azadirachtin: a potent insect antifeedant. Chem - A Eur J. 2008;14:10683–704. https://doi.org/10.1002/chem.200801103.

    Article  CAS  Google Scholar 

  7. Tan QG, Luo XD. Meliaceous limonoids: chemistry and biological activities. Chem Rev. 2011;111:7437–522. https://doi.org/10.1021/cr9004023.

    Article  CAS  PubMed  Google Scholar 

  8. Pohanish RP. Sittig’s handbook of pesticides and agricultural chemicals. Elsevier; 2015.

    Google Scholar 

  9. Murugan K, Jeyabalan D, Kumar NS, Babu R, Sivaramakrishnan S, Nathan SS. Antifeedant and growth-inhibitory properties of neem limonoids against the cotton bollworm, Helicoverpa armigera (Hübner). Int J Trop Insect Sci. 1998;18:157–62. https://doi.org/10.1017/S1742758400007803.

    Article  CAS  Google Scholar 

  10. Hashmat I, Azad H, Ahmed A. Neem ( Azadirachta indica A. Juss ) - a nature’s drugstore: an overview. Int Res J Biol Sci. 2012;1:76–9.

    Google Scholar 

  11. Alam A, Haldar S, Thulasiram HV, Kumar R, Goyal M, Iqbal MS, Pal C, Dey S, Bindu S, Sarkar S, Pal U, Maiti NC, Bandyopadhyay U. Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor. J Biol Chem. 2012;287:24844–61. https://doi.org/10.1074/jbc.M112.341321.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ilango K, Maharajan G, Narasimhan S. Anti-nociceptive and anti-inflammatory activities of Azadirachta indica fruit skin extract and its isolated constituent azadiradione. Nat Prod Res. 2013;27:1463–7. https://doi.org/10.1080/14786419.2012.717288.

    Article  CAS  PubMed  Google Scholar 

  13. Kumar D, Haldar S, Gorain M, Kumar S, Mulani FA, Yadav AS, Miele L, Thulasiram HV, Kundu GC. Epoxyazadiradione suppresses breast tumor growth through mitochondrial depolarization and caspase-dependent apoptosis by targeting PI3K/Akt pathway. BMC Cancer. 2018;18:1–17. https://doi.org/10.1186/s12885-017-3876-2.

    Article  CAS  Google Scholar 

  14. Maity P, Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. The use of neem for controlling gastric hyperacidity and ulcer. Phyther Res. 2009;23:747–55. https://doi.org/10.1002/ptr.2721.

    Article  CAS  Google Scholar 

  15. Paritala V, Chiruvella KK, Thammineni C, Ghanta RG, Mohammed A. Phytochemicals and antimicrobial potentials of mahogany family. Rev Bras Farmacogn. 2015;25:61–83. https://doi.org/10.1016/j.bjp.2014.11.009.

    Article  CAS  Google Scholar 

  16. Forim MR, da Silva MF das GF, Cass QB, Fernandes JB, Vieira PC. Simultaneous quantification of azadirachtin and 3-tigloylazadirachtol in Brazilian seeds and oil of Azadirachta indica: application to quality control and marketing. Anal Methods. 2010;2:860. https://doi.org/10.1039/c0ay00008f.

  17. Song L, Wang J, Gao Q, Ma X, Wang Y, Zhang Y, Xun H, Yao X, Tang F. Simultaneous determination of five azadirachtins in the seed and leaf extracts of Azadirachta indica by automated online solid-phase extraction coupled with LC–Q-TOF–MS. Chem Cent J. 2018;12:85. https://doi.org/10.1186/s13065-018-0453-y.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. de Paula JAM, Brito LF, Caetano KLFN, de Morais Rodrigues MC, Borges LL, Da Conceição EC. Ultrasound-assisted extraction of azadirachtin from dried entire fruits of Azadirachta indica A. Juss. (Meliaceae) and its determination by a validated HPLC-PDA method. Talanta. 2016;149:77–84. https://doi.org/10.1016/j.talanta.2015.11.005.

    Article  CAS  PubMed  Google Scholar 

  19. Schaaf O, Jarvis AP, van der Esch SA, Giagnacovo G, Oldham NJ. Rapid and sensitive analysis of azadirachtin and related triterpenoids from Neem (Azadirachta indica) by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. J Chromatogr A. 2000;886:89–97. https://doi.org/10.1016/S0021-9673(00)00492-1.

    Article  CAS  PubMed  Google Scholar 

  20. Barrek S, Paisse O, Grenier-Loustalot MF. Analysis of neem oils by LC-MS and degradation kinetics of azadirachtin-A in a controlled environment : characterization of degradation products by HPLC-MS-MS. Anal Bioanal Chem. 2004;378:753–63. https://doi.org/10.1007/s00216-003-2377-0.

    Article  CAS  PubMed  Google Scholar 

  21. Dai J, Yaylayan VA, Vijaya Raghavan GS, Parè JR, Liu Z. Multivariate calibration for the determination of total azadirachtin-related limonoids and simple terpenoids in neem extracts using vanillin assay. J Agric Food Chem. 2001;49:1169–74. https://doi.org/10.1021/jf001141n.

    Article  CAS  PubMed  Google Scholar 

  22. Sarais G, Caboni P, Sarritzu E, Russo M, Cabras P. A simple and selective method for the measurement of azadirachtin and related azadirachtoid levels in fruits and vegetables using liquid chromatography electrospray ionization tandem mass spectrometry. J Agric Food Chem. 2008;56:2939–43. https://doi.org/10.1021/jf7037407.

    Article  CAS  PubMed  Google Scholar 

  23. Grimalt S, Thompson DG, Coppens M, Chartrand DT, Shorney T, Meating J, Scarr T. Analytical study of azadirachtin and 3-tigloylazadirachtol residues in foliage and phloem of hardwood tree species by liquid chromatography–electrospray mass spectrometry. J Agric Food Chem. 2011;59:8070–7. https://doi.org/10.1021/jf2023947.

    Article  CAS  PubMed  Google Scholar 

  24. Drożdżyński D, Kowalska J. Rapid analysis of organic farming insecticides in soil and produce using ultra-performance liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem. 2009;394:2241–7. https://doi.org/10.1007/s00216-009-2931-5.

    Article  CAS  PubMed  Google Scholar 

  25. Rangiah K, Varalaxmi BA, Gowda M. UHPLC-MS/SRM method for quantification of neem metabolites from leaf extracts of Meliaceae family plants. Anal Methods. 2016;8:2020–31. https://doi.org/10.1039/C5AY03065J.

    Article  CAS  Google Scholar 

  26. Haldar S, Mulani FA, Aarthy T, Dandekar DS, Thulasiram HV. Expedient preparative isolation and tandem mass spectrometric characterization of C-seco triterpenoids from Neem oil. J Chromatogr A. 2014;1366:1–14. https://doi.org/10.1016/j.chroma.2014.09.006.

    Article  CAS  PubMed  Google Scholar 

  27. Haldar S, Phapale PB, Kolet SP, Thulasiram HV. Expedient preparative isolation, quantification and characterization of limonoids from Neem fruits. Anal Methods. 2013;5:5386–91. https://doi.org/10.1039/c3ay41136b.

    Article  CAS  Google Scholar 

  28. Jedrychowski MP, Huttlin EL, Haas W, Sowa ME, Rad R, Gygi SP. Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics. Mol Cell Proteomics. 2011;10(M111): 009910. https://doi.org/10.1074/mcp.M111.009910.

    Article  CAS  PubMed  Google Scholar 

  29. Aarthy T, Mulani FA, Pandreka A, Kumar A, Nandikol SS, Haldar S, Thulasiram HV. Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension. BMC Plant Biol. 2018;18:230. https://doi.org/10.1186/s12870-018-1447-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mulani FA, Nandikol SS, Haldar S, Thulasiram HV. Accurate identification of bioactive Meliaceae limonoids by UHPLC–MS/MS based structure–fragment relationships (SFRs). ACS Omega. 2021;6:26454–76. https://doi.org/10.1021/acsomega.1c03697.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang Y, Chen X, Wang J, Xun H, Sun J, Tang F. Comparative analysis of the terpenoid biosynthesis pathway in Azadirachta indica and Melia azedarach by RNA-seq. Springerplus. 2016;5:819. https://doi.org/10.1186/s40064-016-2460-6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hodgson H, De La Peña R, Stephenson MJ, Thimmappa R, Vincent JL, Sattely ES, Osbourn A. Identification of key enzymes responsible for protolimonoid biosynthesis in plants: opening the door to azadirachtin production. Proc Natl Acad Sci U S A. 2019;116:17096–104. https://doi.org/10.1073/pnas.1906083116.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Herrero P, Cortés-Francisco N, Borrull F, Caixach J, Pocurull E, Marcé RM. Comparison of triple quadrupole mass spectrometry and Orbitrap high-resolution mass spectrometry in ultrahigh performance liquid chromatography for the determination of veterinary drugs in sewage: benefits and drawbacks. J Mass Spectrom. 2014;49:585–96. https://doi.org/10.1002/jms.3377.

    Article  CAS  PubMed  Google Scholar 

  34. Govindachari TR, Narasimhan NS, Suresh G, Partho PD, Gopalakrishnan G. Insect antifeedant and growth-regulating activities of salannin and other c-seco limonoids from neem oil in relation to Azadirachtin. J Chem Ecol. 1996;22:1453–61. https://doi.org/10.1007/BF02027724.

    Article  CAS  PubMed  Google Scholar 

  35. Braga TM, Rocha L, Chung TY, Oliveira RF, Pinho C, Oliveira AI, Morgado J, Cruz A. Biological activities of gedunin—a limonoid from the Meliaceae family. Molecules. 2020;25:493. https://doi.org/10.3390/molecules25030493.

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

FAM and SSN acknowledge the Council of Scientific and Industrial Research (CSIR), New Delhi, and the Department of Science and Technology (DST), New Delhi, for the fellowship, respectively. This work is supported by the Department of Biotechnology, India, and CSIR, New Delhi, sponsored the network project (Project No. MLP01024, HCP0002).

Author information

Author notes

  1. Fayaj A. Mulani and Sharvani S. Nandikol contributed equally to this work.

Authors and Affiliations

  1. Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India

    Fayaj A. Mulani, Sharvani S. Nandikol & Hirekodathakallu V. Thulasiram

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Fayaj A. Mulani, Sharvani S. Nandikol & Hirekodathakallu V. Thulasiram

  3. Department of Bioscience, University of Mysore, Post Graduate Center, Hemagangothri, Hassan, 573226, India

    Jagadeesh S. Kajjihundi & Sharanappa Puttappa

  4. Department of P G Studies and Research in Biochemistry, Kuvempu University, Jnana Sahyadri, Shankaraghatta, Karnataka, 577451, India

    Niranjana Pathappa

  5. CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, 110007, India

    Hirekodathakallu V. Thulasiram

Authors
  1. Fayaj A. Mulani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sharvani S. Nandikol
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jagadeesh S. Kajjihundi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Niranjana Pathappa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sharanappa Puttappa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hirekodathakallu V. Thulasiram
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Fayaj A Mulani: investigation, methodology, and writing—original draft.

Sharvani S. Nandikol: investigation, methodology, and writing—original draft.

Jagadeesh S. Kajjihundi: collected the plants and prepared the herbarium.

Niranjana Pathappa: collected the plants and prepared the herbarium.

Sharanappa Puttappa: collection and identification of the plants.

Hirekodathakallu V. Thulasiram: conceptualization, funding acquisition, supervision of the overall work, checking of the data and manuscript preparation with inputs from FAM and SSN.

Corresponding authors

Correspondence to Sharanappa Puttappa or Hirekodathakallu V. Thulasiram.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary information associated with this article can be found in the online version of the manuscript doi.

Supplementary file 1 (PDF 1934 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mulani, F.A., Nandikol, S.S., Kajjihundi, J.S. et al. Ultra-high performance liquid chromatography Q-Orbitrap MS/MS–based profiling and quantification of limonoids in Meliaceae plants. Anal Bioanal Chem 414, 6093–6106 (2022). https://doi.org/10.1007/s00216-022-04169-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-022-04169-2

Keywords

Search

Navigation