Micro-methods to determine the composition of selected secondary metabolites in Colombian forages

Year 2025, Volume: 12 Issue: 3, 593 - 603, 04.09.2025

Ana Maria Calvo Salamanca , Erika Duran , Andrea Sierra Ronnal Ortiz Olga Mayorga Claudia Ariza-nieto

https://doi.org/10.21448/ijsm.1532400

Abstract

Plants used as forage in livestock production are a source of secondary metabolites (SM) that are involved in important interaction in life, health, nutrition and animal performance. This study aimed to adapt and validate six spectrophotometric micro-methods for quantifying SM such as total phenols (TP), total tannins (TT), condensed tannins (CT), total saponins (TS), total alkaloids (TA), and total sterols (TE), in more than 700 Colombian tropical forage samples. The sample and reagent quantities used in the assays were reduced, preparation conditions were modified in some steps, and the colorimetric reactions were performed in 96-well microplates. The micro methods were validated for linearity, limits of detection and quantification, accuracy, precision, and percent recovery in materials such as grasses, legumes, and tree forages from six Colombian ecoregions. The coefficients of variation (CV), repeatability and reproducibility for all methods were less than 0.4 and 5.6%, respectively. The recoveries ranged from 84.8 to 88.4% for TP, 65.7 to 92.3 for TT, 59.8 to 80.2 for CT, 49.9 to 69.5 for TA, and 61.9 to 78.8% for TS. Micro assays promote minimal waste generation in the environment and allow the processing of a larger number of samples, thereby reducing reagent consumption. This is particularly useful in the analysis of forage materials when making decisions about the effects and functionality of these components in animal diets and their performance.

Keywords

Secondary metabolites , Grass forages , Legume forages , Tree forages , Micro assays

Supporting Institution

Agrosavia

Thanks

The authors would like to thank the Colombian Ministry of Agriculture and Rural Development (MADR) and the Corporación Colombiana de Investigación Agropecuaria AGROSAVIA for supporting this study. The forage samples were collected according to the Colombian Resolution No. 1466 of 3rd December 2014, by which Agrosavia has permission to collect biological diversity samples for non-commercial and scientific research purposes

References

• Adu, J.K., Amengor, C.D.K., Kabiri, N., Orman, E., Patamia, S.A.G., & Okrah, B.K. (2019). Validation of a simple and robust liebermann–burchard colorimetric method for the assay of cholesterol in selected milk products in Ghana. International Journal of Food Science, 2019, 1–7. https://doi.org/10.1155/2019/9045938
• Al-Khayri, J.M., Rashmi, R., Toppo, V., Chole, P.B., Banadka, A., Sudheer, W.N., Nagella, P., Shehata, W.F., Al-Mssallem, M.Q., Alessa, F.M., Almaghasla, M.I., & Rezk, A.A.-S. (2023). Plant secondary metabolites: The weapons for biotic stress management. Metabolites, 13(6), 716. https://doi.org/10.3390/metabo13060716
• AOAC. Official Methods of Analysis, (2005). Method 931-01 phosphorus in plants. In Official methods of analysis. Arlington: Association of official analitycal chemists.
• AOAC. Official Methods of Analysis (2024). AOAC International. https://www.aoac.org/official-methods-of-analysis/
• Araújo, L.B.D.C., Silva, S.L., Galvão, M.A.M., Ferreira, M.R.A., Araújo, E.L., Randau, K.P., & Soares, L.A.L. (2013). Total phytosterol content in drug materials and extracts from roots of Acanthospermum hispidum by UV-VIS spectrophotometry. Revista Brasileira de Farmacognosia, 23(5), 736–742. https://doi.org/10.1590/s0102-695x2013000500004
• Ariza-Nieto, C., Mayorga, O., Mojica, B., Parra, D., & Afanador-Tellez, G. (2018). Use of LOCAL algorithm with near infrared spectroscopy in forage resources for grazing systems in Colombia. Journal of Near Infrared Spectroscopy, 26(1), 44 52. https://doi.org/10.1177/0967033517746900.
• Avendaño R.J., Carlos, O.M., & Ramírez S.M. (2003). Caracterización de los componentes vegetales consumidos por ovinos y bovinos en plantas de tagasaste (Chamaecytisus proliferus ssp. palmensis). Agricultura Técnica, 63(1). https://doi.org/10.4067/s0365-28072003000100006
• Arowolo, M.A., & He, J. (2018). Use of probiotics and botanical extracts to improve ruminant production in the tropics: A review. Animal Nutrition 4(3), 241 249. https://doi.org/10.1016/j.aninu.2018.04.010
• Awad, A.B., Chan, K.C., Downie, A.C., & Fink, C.S. (2009). Peanuts as a Source of β-Sitosterol, a Sterol with Anticance Properties. Nutrition and Cancer, 36(2), 238-241. https://doi.org/10.1207/S15327914NC3602_14.
• Busquet, M., Calsamiglia, S., Ferret, A., & Kamel, C. (2006). Plant extracts affect in vitro rumen microbial fermentation. Journal of Dairy Science, 89(2), 761 771. https://doi.org/10.3168/jds.s0022-0302(06)72137-3
• Bush, L., & Burton, H. (2015). Intrinsic Chemical Factors in Forage Quality. ASA, CSSA, and SSSA Books, 367–405. https://doi.org/10.2134/1994.foragequality.c9
• Ellis, B. (1993). Phenolic Metabolism in Plants.Helen A. Stafford , Ragai K. Ibrahim. The Quarterly Review of Biology, 68(4), 596–597. https://doi.org/10.1086/418355
• Guerriero, G., Berni, R., J. Armando Muñoz-Sanchez, Apone, F., Abdel-Salam, E. M., Qahtan, A.A., Alatar, A.A., Cantini, C., Cai, G., Hausman, J.-F., Siddiqui, K.S., Hernández-Sotomayor, T., & Faisal, M. (2018). Production of plant secondary metabolites: Examples, tips and suggestions for biotechnologists. Genes, 9(6), 309 309. https://doi.org/10.3390/genes9060309
• Hallé, F. (2010). Arquitectura de los árboles. Bol. Soc. Argent. Bot. 45(3-4), 405-418. https://www.uv.mx/personal/tcarmona/files/2016/08/Halle-2010.pdf
• Harborne, J.B (1993). Introduction to Ecological Biochemistry. Academic Press England.
• Harborne, J.B. (1992). Chapter 20 Phenolic compounds. In E. Heftmann, Chromatography Fundamentals and Applications of Chromatographic and Electmphoretic Methods. Elsevier.
• Hiai, S., Oura, H., & Nakajima, T. (1976). Color reaction of some sapogenins and saponins with vanillin and sulfur1c acid. Planta Medica, 29(02), 116–122. https://doi.org/10.1055/s-0028-1097639
• Holman, B.W.B., & Malau‐Aduli, A.E.O. (2012). Spirulina as a livestock supplement and animal feed. Journal of Animal Physiology and Animal Nutrition, 97(4), 615–623. https://doi.org/10.1111/j.1439-0396.2012.01328.x
• IUPAC. (1997). Compendium in chemical terminology. Oxford: Blackwell Scientific publications.
• Jayanegara, A., Togtokhbayar, N., Makkar, H., & Becker, K. (2009). Tannins determined by various methods as predictors of methane production reduction potential of plants by an in vitro rumen fermentation system. Animal Feed Science and Technology, 150(3) 230-237. https://doi.org/10.1016/j.anifeedsci.2008.10.011
• Jenko, C., Bonato, P., Fabre, R., Perlo, F., Tisocco, O., & Teira, G. (2018). Adición de taninos a dietas de rumiantes y su efecto sobre la calidad y rendimiento de la carne. Ciencia, docencia y tecnología, 224-241. http://www.scielo.org.ar/scielo.php?script=sci_arttext&pid=S1851-17162018000100010&lng=es&tlng=es
• Kenny, A. (1952). The determination of cholesterol by the Liebermann-Buchard reaction. Biochemistry Journal, 52(4), 611-619. https://doi.org/10.1042/bj0520611
• Koura, B.I., Shipandeni, M., & Cutrignelli, M.I. (2023). Sustainable Feeds for Animal Nutrition in Tropical Areas. Animals, 13(8), 1379. https://doi.org/10.3390/ani13081379
• Kuppusamy, P., Dong, K., Song, C.E., Ilavenil, S., Srigopalram, S., Valan, M., & Choon, K. (2018). Quantification of major phenolic and flavonoid markers in forage crop Lolium multiflorum using HPLC DAD. Revista Brasileira de Farmacognosia 28(3), 282 288. https://doi.org/10.1016/j.bjp.2018.03.006
• Makkar, H.P.S., Siddhuraju, P., & Becker, K. (2007). Trypsin inhibitor. Methods in Molecular BiologyTM, 1–6. https://doi.org/10.1007/978-1-59745-425-4_1
• Makkar, H., Blummel, M., Borowy, N., & Becker, K. (1993). Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. Journal of the Science of Food and Agriculture, 61(2), 161 165. https://doi.org/10.1002/jsfa.2740610205
• Marques G.S, Leão, W.F., Lyra, M.A.M., Peixoto, M.S., Monteiro, R.P.M., Rolim, L.A., Xavier, H.S., Neto, P.J.R., Soares, L.A.L. (2013). Comparative evaluation of UV/VIS and HPLC analytical methodologies applied for quantifi cation of fl avonoids from leaves of Bauhinia forfi cata. Brazilian Journal of Pharmacognosy, 23(1), 51 57. https://doi.org/10.1590/S0102-695X2012005000143
• Martin, C., Morgavi, D.P., & Doreau, M. (2010). Methane mitigation in ruminants: from microbe to the farm scale. Animal, 4(3), 351 365. https://doi.org/10.1017/S1751731109990620
• Munidasa, S., Eckard, R., Sun, X., Cullen, B., McGill, D., Chen, D., & Cheng, L. (2021). Challenges and opportunities for quantifying greenhouse gas emissions through dairy cattle research in developing countries. Journal of Dairy Research, 88(1), 3 7. https://doi.org/10.1017/S0022029921000182
• Patra, A., & Saxena, J. (2011). A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen. Phytochemistry Journal, 71(11 12), 1198 1222. https://doi.org/10.1016/j.phytochem.2010.05.010
• Patra, A., & Saxena, K. (2009). The effect and mode of action of saponins on the microbial populations and fermentation in the rumen and ruminant production. Nutrition Research Reviews, 22(2) 204-219. https://doi.org/10.1017/S0954422409990163
• Pensiero, J.F., Zabala, J.M., Lorena, & Richard, G.A. (2020). Native and Naturalized Forage Plant Genetic Resources for Saline Environments of the Southernmost Portion of the American Chaco. Springer EBooks, 339–380. https://doi.org/10.1007/978-3-030-52592-7_18
• Ramírez-Restrepo, C., & Barry, A. (2005). Alternative temperate forages containing secondary compounds for improving sustainable productivity in grazing ruminants. Animal Feed Science and Technology, 120(3), 179-201. https://doi.org/10.1016/j.anifeedsci.2005.01.015
• Ramos, G., Frutos, P., Giráldez, F.J., & Mantecón, A.R. (1998). Ramos, G.; Frutos, P.; Giráldez, F. J. & Mantecón, A. R. Secondary plant compounds in herbivore nutrition. Arch. Zootecn. 47, 597-620.
• Santo, M., & Lecumberry, G. (2005). El proceso de medición: Análisis y comunicación de datos experimentales [The measurement process: Analysis and communication of experimental data]. Rio cuarto: Universidad Nacional de Rio Cuarto.Argentina.
• Santocoloma-Varón, L.E. (2010). Evaluación del contenido de metabolitos secundarios en dos especies de plantas forrajeras encontradas en dos pisos térmicos de Colombia. Revista de Investigación Agraria y Ambiental, 31-35. https://doi.org/10.22490/21456453.890
• Sepúlveda-Jiménez, G., Porta-Ducoing, H., & Rocha-Sosa, M. (2004). La participación de los metabolitos secundarios en la defensa de las plantas [The participation of secondary metabolites in plant defense]. Revista Mexicana de Fitopatología, 355-363. https://www.redalyc.org/pdf/612/61221317.pdf
• Sreevidya, N., & Mehrotra, S. (2003). Spectrophotometric method for estimation of alkaloids precipitable with dragendorff reagent in plant materials. Journal of AOAC International, 86(6), 1124-1127.
• Stafford, H.A. (1967). Biosynthesis of phenolic compounds in first internodes of Sorghum: Lignin and related products. Plant Physiology, 450 455. https://doi.org/10.1104/pp.42.3.450
• Vaou, N., Stavropoulou, E., Voidarou, C.C., Tsakris, Z., Rozos, G., Tsigalou, C., Bezirtzoglou, E. (2022). Interactions between medical plant-derived bioactive compounds: Focus on antimicrobial combination effects. Antibiotics (Basel), 11(8), 1014. https://doi.org/10.3390/antibiotics11081014
• Verdecía, D.H.-H.-M. (2021). Metabolitos primarios y secundario de seis especies de árboles, arbustos y leguminosas herbáceas. Cuban Journal of Agricultural Science, 77-93. http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S207934802021000100008&lng=es&tlng=es
• Wallace, R.J. (2004). Antimicrobial properties of plant secondary metabolites. Proceedings of the Nutrition Society, 63, 621-629. https://doi.org/10.1079/PNS2004393
• White, R.G., & Lawler, J.P. (2002). Can methane suppression during digestion of woody and leafy browse compensate for energy costs of detoxification of plant secondary compounds? A test with muskoxen fed willows and birch. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology, 133, 849-859. https://doi.org/10.1016/S1095-6433(02)00152-6