Recognition of Sesquiterpenoids and Piperidine Alkamides as Two Discerning Metabolite Classes in the Fruits of Piper nigrum 'Semongok Aman'
Keywords:
Alkamides, cultivar, FBMN, metabolomics, Piper nigrum, sesquiterpenoidsAbstract
The Malaysian Pepper Board (MPB) has recommended the plantation of three over seven Piper nigrum L. cultivars, owing to their beneficial agronomic traits. Currently, distinction between the cultivars is assessed based on morphological characters. The MPB has also proposed the concept of monovarietal farm, which is believed to have the potential of strengthening the quality of pepper in the global market. However, there remains a need for a fair assessment of the specialised metabolites’ variation among P. nigrum cultivars in search of a cultivar with distinctive metabolites profile, which may be the most suitable candidate for the application of such concept. We hereby describe revised protocols aimed at minimising the oxidation of the fruits of five P. nigrum cultivars and reducing the experimental run time that allowed utilisation of the same samples in GC-MS and 1H-NMR metabolomics. Subsequently, feature-based molecular network (FBMN) was used to verify the patterns observed in the principal component analysis (PCA) of the 1H-NMR and GC-MS data. PCA of both datasets revealed that the clustering pattern of the five cultivars paralleled the origin of their parent plants, with the genetically more similar cultivars, 'Kuching', 'Semongok Emas', 'India', and 'Yong Petai', being closer to each other compared to 'Semongok Aman'. 'Semongok Aman' was found to contain a higher abundance of the sesquiterpenoids germacrene B and γ-elemene, as well as the piperidine alkamides piperine and its isomers. FBMN further highlighted the higher abundance of the two metabolite classes in the fruits of 'Semongok Aman'. 'Semongok Aman' might be a suitable cultivar for the implementation of monovarietal pepper farm concept owing to its distinctive metabolite profile.
References
Adams, R.P. (2017). Identification of essential oil components by gas chromatography/mass spectrometry. Fourth Edition. DuPage County: Allured Business Media.
Afzan, A., Kasim, N., Ismail, N.H., Azmi, N., Ali, A.M., Mat, N. & Wolfender, J.L. (2019). Differentiation of Ficus deltoidea varieties and chemical marker determination by UHPLC-TOFMS metabolomics for establishing quality control criteria of this popular Malaysian medicinal herb. Metabolomics, 15(3): 1-11. DOI: 10.1007/s11306-019-1489-2
Ahmad, R., Ahmad, N., Amir, M., Aljishi, F., Alamer, M.H., Al‐Shaban, H.R., Alsadah, Z.A., Alsultan, B.M., Aldawood, N.A., Chathoth, S. & Almofty, S.A. (2020). Quality variation and standardization of black pepper (Piper nigrum): A comparative geographical evaluation based on instrumental and metabolomics analysis. Biomedical Chromatography, 34(3): e4772. DOI: 10.1002/bmc.4772
Andriamaharavo, N.R. (2014). Retention Data. NIST Mass Spectrometry Data Center. https://webbook.nist.gov/cgi/cbook.cgi?Source=2014AND%2319410M&Mask=2000
Ashokkumar, K., Murugan, M., Dhanya, M.K., Pandian, A. & Warkentin, T.D. (2021). Phytochemistry and therapeutic potential of black pepper [Piper nigrum (L.)] essential oil and piperine: A review. Clinical Phytoscience, 7(1): 52. DOI: 10.1186/s40816-021-00292-2
Babushok, V.I., Linstrom, P.J. & Zenkevich, I.G. (2011). Retention indices for frequently reported compounds of plant essential oils. Journal of Physical and Chemical Reference Data, 40(4): 043101. DOI: 10.1063/1.3653552
Bandyopadhyay, C., Narayan, V.S. & Variyar, P.S. (1990). Phenolics of green pepper berries (Piper nigrum L.). Journal of Agricultural and Food Chemistry, 38(8): 1696–1699. DOI: 10.1021/jf00098a015
Barata, L.M., Andrade, E.H., Ramos, A.R., De Lemos, O.F., Setzer, W.N., Byler, K.G., Maia, J.G.S. & Da Silva, J.K.R. (2021). Secondary metabolic profile as a tool for distinction and characterization of cultivars of black pepper (Piper nigrum L.) cultivated in Pará State, Brazil. International Journal of Molecular Sciences, 22(2): 890. DOI: 10.3390/ijms22020890
Beger, R.D., Dunn, W.B., Bandukwala, A., Bethan, B., Broadhurst, D., Clish, C.B., Dasari, S., Derr, L., Evans, A., Fischer, S., Flynn, T., Hartung, T., Herrington, D., Higashi, R., Hsu, P.C., Jones, C., Kachman, M., Karuso, H., Kruppa, G., Lippa, K., Maruvada, P., Mosley, J., Ntai, I., O’Donovan, C., Playdon, M., Raftery, D., Shaughnessy, D., Souza, A., Spaeder, T., Spalholz, B., Tayyari, F., Ubhi, B., Verma, M., Walk, T., Wilson, I., Witkin, K., Bearden, D.W. & Zanetti, K.A. (2019). Towards quality assurance and quality control in untargeted metabolomics studies. Metabolomics, 15(1): 4. DOI: 10.1007/s11306-018-1460-7
Chen, Y.S., Dayod, M. & Tawan, C.S. (2018). Phenetic analysis of cultivated black pepper (Piper nigrum L.) in Malaysia. International Journal of Agronomy, 2018. DOI: 10.1155/2018/3894924
Chen, Y.S. & Tawan, C.S. (2020a). Analysis of qualitative and quantitative trait variability among black pepper (Piper nigrum L.) cultivars in Malaysia. Pertanika Journal of Tropical Agricultural Science, 43(3): 257–274.
Chen, Y.S. & Tawan, C.S. (2020b). Phenotypic, taxonomy, phytochemical and physiological characterization of black pepper (Piper nigrum L.) cultivars in Malaysia (PhD thesis). Universiti Malaysia Sarawak.
Entebang, H., Wong, S.-K. & Mercer, Z.J.A. (2021). Development and performance of the pepper industry in Malaysia: A critical review. International Journal of Business and Society, 21(3): 1402–1423. DOI: 10.33736/ijbs.3361.2020
Evans, A.M., O’Donovan, C., Playdon, M., Beecher, C., Beger, R.D., Bowden, J.A., Broadhurst, D., Clish, C.B., Dasari, S., Dunn, W.B., Griffin, J.L., Hartung, T., Hsu, P.C., Huan, T., Jans, J., Jones, C.M., Kachman, M., Kleensang, A., Lewis, M.R., Monge, M.E., Mosley, J.D., Taylor, E., Tayyari, F., Theodoridis, G., Torta, F., Ubhi, B.K. & Vuckovic, D. (2020). Dissemination and analysis of the quality assurance (QA) and quality control (QC) practices of LC–MS based untargeted metabolomics practitioners. Metabolomics, 16(10): 113. DOI: 10.1007/s11306-020-01728-5
Fong, L.K. & Liang, S.S. (Eds). (2011). Pepper production technology in Malaysia. Kuching: Lembaga Lada Malaysia.
Gaweng, P. & Lai, J.C. (Eds). (2017). Manual penanaman lada. Kuching: Lembaga Lada Malaysia.
Gómez-Calvario, V. & Rios, M.Y. (2019). 1H and 13C NMR data, occurrence, biosynthesis, and biological activity of Piper amides. Magnetic Resonance in Chemistry, 57(12): 994–1070. DOI: 10.1002/mrc.4857
Gu, F., Tan, L., Wu, H., Fang, Y. & Wang, Q. (2013). Analysis of the blackening of green pepper (Piper nigrum Linnaeus) berries. Food Chemistry, 138(2–3): 797–801. DOI: 10.1016/j.foodchem.2012.11.033
Gul, I., Nasrullah, N., Nissar, U., Saifi, M. & Abdin, M.Z. (2017). Development of DNA and GC-MS fingerprints for authentication and quality control of Piper nigrum L. and its adulterant Carica papaya L. Food Analytical Methods, 11: 1209–1222. DOI: 10.1007/s12161-017-1088-7
Häckl, M., Tauber, P., Schweda, F., Zacharias, H.U., Altenbuchinger, M., Oefner, P.J. & Gronwald, W. (2021). An R-package for the deconvolution and integration of 1D NMR data: MetaboDecon1D. Metabolites, 11(7): 452. DOI: 10.3390/metabo11070452
Hashimoto, K., Yaoi, T., Koshiba, H., Yoshida, T., Maoka, T., Fujiwara, Y., Yamamoto, Y. & Mori, K. (1996). Photochemical isomerization of piperine, a pungent constituent in pepper. Food Science and Technology International, Tokyo, 2(1): 24–29. DOI: 10.3136/fsti9596t9798.2.24
Hashimoto, K., Yaoi, T., Koshiba, H., Yoshida, T., Maoka, T., Fujiwara, Y., Yamamoto, Y. & Mori, K. (2021). Feasibility of applying untargeted metabolomics with GC-Orbitrap-HRMS and chemometrics for authentication of black pepper (Piper nigrum L.) and identification of geographical and processing markers. Journal of Agricultural and Food Chemistry, 69(19): 5547–5558. DOI: 10.1021/acs.jafc.1c01515
Ho, W.S., Lau, E.T., Jafar, H.R., Sim, S.L. & Paulus, A.D. (2005). Evaluation of genetic relatedness among pepper (Piper nigrum L.) accessions using Direct Amplification of Minisatellite-Region DNA (DAMD). Proceedings of the 6th National Congress on Genetics, 12-14 May 2005, Kuala Lumpur, Malaysia. pp. 299–302.
Houriet, J., Allard, P.M., Queiroz, E.F., Marcourt, L., Gaudry, A., Vallin, L., Li, S., Lin, Y., Wang, R., Kuchta, K. & Wolfender, J.L. (2020). A mass spectrometry-based metabolite profiling workflow for selecting abundant specific markers and their structurally related multi-component signatures in traditional chinese medicine multi‐herb formulae. Frontiers in Pharmacology, 11: 578346. DOI: 10.3389/fphar.2020.578346
Jaidee, W., Maneerat, T., Rujanapun, N., Paojumroon, N., Duangyod, T., Banerjee, S., Kar, A., Mukherjee, P.K. & Charoensup, R. (2022). Metabolite fingerprinting of Piper nigrum L. from different regions of Thailand by UHPLC-QTOF-MS approach and in vitro bioactivities. Trends in Sciences, 19(22): 1520. https://doi.org/10.48048/tis.2022.1520
Jayawardena, R.S., Bhunjun, C.S., Hyde, K.D., Gentekaki, E. & Itthayakorn, P. (2021). Colletotrichum: Lifestyles, biology, morpho-species, species complexes and accepted species. Mycosphere, 12(1): 519–669. DOI: 10.5943/mycosphere/12/1/7
Johny, F., Saupi, N. & Ramaiya, S.D. (2020). Status of pepper farming and flower composition of different pepper varieties in Sarawak. Pertanika Journal of Tropical Agricultural Science, 43(4): 467–476. DOI: 10.47836/PJTAS.43.4.04
Jumhawan, U., Putri, S.P., Yusianto, Marwani, E., Bamba, T. & Fukusaki, E. (2013). Selection of discriminant markers for authentication of asian palm civet coffee (Kopi Luwak): A metabolomics approach. Journal of Agricultural and Food Chemistry, 61(33): 7994–8001. DOI: 10.1021/jf401819s
Kozukue, N., Park, M.S., Choi, S.H., Lee, S.U., Ohnishi-Kameyama, M., Levin, C.E. & Friedman, M. (2007). Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS. Journal of Agricultural and Food Chemistry, 55(17): 7131–7139. DOI: 10.1021/jf070831p
Krishnamoorthy, B. & Parthasarathy, V.A. (2010). Improvement of black pepper. CABI Reviews, 2010. DOI: 10.1079/PAVSNNR20105003
Liang, J., Sun, J., Chen, P., Frazier, J., Benefield, V. & Zhang, M. (2021). Chemical analysis and classification of black pepper (Piper nigrum L.) based on their country of origin using mass spectrometric methods and chemometrics. Food Research International, 140: 109877. DOI: 10.1016/j.foodres.2020.109877
Luca, S.V., Minceva, M., Gertsch, J. & Skalicka-Woźniak, K. (2021). LC-HRMS/MS-based phytochemical profiling of Piper spices: Global association of piperamides with endocannabinoid system modulation. Food Research International, 141: 11012. DOI: 10.1016/j.foodres.2021.110123
Matsuo, T., Tsugawa, H., Miyagawa, H. & Fukusaki, E. (2017). Integrated strategy for unknown EI-MS identification using quality control calibration curve, multivariate analysis, EI-MS spectral database, and retention index prediction. Analytical Chemistry, 89(12): 6766–6773. DOI: 10.1021/acs.analchem.7b01010
Mayr, S., Beć, K.B., Grabska, J., Schneckenreiter, E. & Huck, C.W. (2021). Near-infrared spectroscopy in quality control of Piper nigrum: A comparison of performance of benchtop and handheld spectrometers. Talanta, 223(Part 2): 121809. DOI: 10.1016/j.talanta.2020.121809
Mehr, S.H.M., Tang, A.W. & Laing, R.R. (2023). Automated qualitative and quantitative analysis of complex forensic drug samples using 1H NMR. Magnetic Resonance in Chemistry, 61(2): 95–105. DOI: 10.1002/mrc.5265
Miyazaki, T., Plotto, A., Goodner, K. & Gmitter, F.G. (2011). Distribution of aroma volatile compounds in tangerine hybrids and proposed inheritance. Journal of the Science of Food and Agriculture, 91(3): 449–460. DOI: 10.1002/jsfa.4205
Nothias, L.F., Petras, D., Schmid, R., Dührkop, K., Rainer, J., Sarvepalli, A., Protsyuk, I., Ernst, M., Tsugawa, H., Fleischauer, M., Aicheler, F., Aksenov, A.A., Alka, O., Allard, P.M., Barsch, A., Cachet, X., Caraballo-Rodriguez, A.M., Da Silva, R.R., Dang, T., … Dorrestein, P.C. (2020). Feature-based molecular networking in the GNPS analysis environment. Nature Methods, 17(9): 905–908. DOI: 10.1038/s41592-020-0933-6
Osman, M.F., Lee, S.Y., Sarbini, S.R., Mohd Faudzi, S.M., Khamis, S., Zainudin, B.H. & Shaari, K. (2021). Metabolomics-driven discovery of an introduced species and two Malaysian Piper betle L. variants. Plants, 10(11): 2510. DOI: 10.3390/plants10112510
Osman, M.F., Mohd Faudzi, S.M., Khamis, S., Sarbini, S.R. & Shaari, K. (2022). Shedding light on Piper’s identity via computational mass spectrometry. Malaysian Journal of Chemistry, 24(4): 150–160.
Phapale, P., Rai, V., Mohanty, A.K. & Srivastava, S. (2020). Untargeted metabolomics workshop report: Quality control considerations from sample preparation to data analysis. Journal of the American Society for Mass Spectrometry, 31(9): 2006–2010. DOI: 10.1021/jasms.0c00224
Rivera-Pérez, A., Romero-González, R. & Garrido Frenich, A. (2021). Application of an innovative metabolomics approach to discriminate geographical origin and processing of black pepper by untargeted UHPLC-Q-Orbitrap-HRMS analysis and mid-level data fusion. Food Research International, 150(Part A): 110722. DOI: 10.1016/j.foodres.2021.110722
Rivera-Pérez, A., Romero-González, R. & Garrido Frenich, A. (2022). A metabolomics approach based on 1H NMR fingerprinting and chemometrics for quality control and geographical discrimination of black pepper. Journal of Food Composition and Analysis, 105: 104235. DOI: 10.1016/j.jfca.2021.104235
Schmid, N., Bruderer, S., Paruzzo, F., Fischetti, G., Toscano, G., Graf, D., Fey, M., Henrici, A., Ziebart, V., Heitmann, B., Grabner, H., Wegner, J.D., Sigel, R.K.O. & Wilhelm, D. (2023). Deconvolution of 1D NMR spectra: A deep learning-based approach. Journal of Magnetic Resonance, 347: 107357. DOI: 10.1016/j.jmr.2022.107357
Singh, S., Kapoor, I.P.S., Singh, G., Schuff, C., Lampasona, M.P.De, & Catalan, C.A.N. (2013). Chemistry, antioxidant and antimicrobial potentials of white pepper (Piper nigrum L.) essential oil and oleoresins. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 83(3): 357–366. DOI: 10.1007/s40011-012-0148-4
Takooree, H., Aumeeruddy, M.Z., Rengasamy, K.R.R., Venugopala, K.N., Jeewon, R., Zengin, G. & Mahomoodally, M.F. (2019). A systematic review on black pepper (Piper nigrum L.): From folk uses to pharmacological applications. Critical Reviews in Food Science and Nutrition, 59(sup1): S210–S243. DOI: 10.1080/10408398.2019.1565489
Traxler, F., Schinnerl, J. & Brecker, L. (2020). Spectroscopic studies on the molecular interactions of curcumin and piperine. Monatshefte Fur Chemie, 151(3): 325–330. DOI: 10.1007/s00706-020-02563-z
Trindade, R., Almeida, L., Xavier, L., Andrade, E.H., Maia, J.G., Mello, A., Setzer, W.N., Ramos, A. & da Silva, J.K.R. (2021). Influence on secondary metabolism of Piper nigrum L. by co-inoculation with arbuscular mycorrhizal fungi and Fusarium solani f. sp. piperis. Microorganisms, 9(3): 484. DOI: 10.3390/microorganisms9030484
Wang, D., Zhang, L., Huang, J., Himabindu, K., Tewari, D., Horbańczuk, J.O., Xu, S., Chen, Z. & Atanasov, A.G. (2021). Cardiovascular protective effect of black pepper (Piper nigrum L.) and its major bioactive constituent piperine. Trends in Food Science and Technology, 117: 34–45. DOI: 10.1016/j.tifs.2020.11.024
Downloads
Additional Files
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 Borneo Journal of Resource Science and Technology

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Copyright Transfer Statement for Journal
1) In signing this statement, the author(s) grant UNIMAS Publisher an exclusive license to publish their original research papers. The author(s) also grant UNIMAS Publisher permission to reproduce, recreate, translate, extract or summarize, and to distribute and display in any forms, formats, and media. The author(s) can reuse their papers in their future printed work without first requiring permission from UNIMAS Publisher, provided that the author(s) acknowledge and reference publication in the Journal.
2) For open access articles, the author(s) agree that their articles published under UNIMAS Publisher are distributed under the terms of the CC-BY-NC-SA (Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License) which permits unrestricted use, distribution, and reproduction in any medium, for non-commercial purposes, provided the original work of the author(s) is properly cited.
3) For subscription articles, the author(s) agree that UNIMAS Publisher holds copyright, or an exclusive license to publish. Readers or users may view, download, print, and copy the content, for academic purposes, subject to the following conditions of use: (a) any reuse of materials is subject to permission from UNIMAS Publisher; (b) archived materials may only be used for academic research; (c) archived materials may not be used for commercial purposes, which include but not limited to monetary compensation by means of sale, resale, license, transfer of copyright, loan, etc.; and (d) archived materials may not be re-published in any part, either in print or online.
4) The author(s) is/are responsible to ensure his or her or their submitted work is original and does not infringe any existing copyright, trademark, patent, statutory right, or propriety right of others. Corresponding author(s) has (have) obtained permission from all co-authors prior to submission to the journal. Upon submission of the manuscript, the author(s) agree that no similar work has been or will be submitted or published elsewhere in any language. If submitted manuscript includes materials from others, the authors have obtained the permission from the copyright owners.
5) In signing this statement, the author(s) declare(s) that the researches in which they have conducted are in compliance with the current laws of the respective country and UNIMAS Journal Publication Ethics Policy. Any experimentation or research involving human or the use of animal samples must obtain approval from Human or Animal Ethics Committee in their respective institutions. The author(s) agree and understand that UNIMAS Publisher is not responsible for any compensational claims or failure caused by the author(s) in fulfilling the above-mentioned requirements. The author(s) must accept the responsibility for releasing their materials upon request by Chief Editor or UNIMAS Publisher.
6) The author(s) should have participated sufficiently in the work and ensured the appropriateness of the content of the article. The author(s) should also agree that he or she has no commercial attachments (e.g. patent or license arrangement, equity interest, consultancies, etc.) that might pose any conflict of interest with the submitted manuscript. The author(s) also agree to make any relevant materials and data available upon request by the editor or UNIMAS Publisher.