Trigonelline: A Versatile Bioactive Alkaloid of Medicinal Plants. A Mini-Review

Trigonelline (TRG, N-methylnicotinic acid, C7H7NO2) is a pyridine alkaloid first isolated from fenugreek seeds in 1885 [1]. It is widely distributed in medicinal and dietary plants, occurring at 1-3% of dry weight in green coffee beans and at substantial levels in pumpkin and wax gourd [2,3]. Beyond its classical roles in planta as a hormone modulating the cell cycle and nodulation and as an osmoprotectant [4], TRG has attracted growing biomedical interest. A substantial body of evidence supports its multi-targeted antidiabetic, neuroprotective, and antioxidant activities with a remarkable safety profile, positioning TRG as a promising lead for nutraceuticals and pharmaceuticals against metabolic and agerelated disorders [2,3]. Nevertheless, TRG content varies markedly among genotypes, tissues, and developmental stages, and the genetic determinants of its accumulation remain largely unknown in most medicinal crops, constraining the breeding of stable, high- TRG varieties. Recent advances in metabolomics and genomics, particularly metabolite-based genome-wide association studies (mGWAS), now provide a powerful framework to bridge this gap [5-7]. This mini-review summarizes the pharmacological potential of TRG and highlights how mGWAS-driven strategies can accelerate the genetic dissection and molecular breeding of TRG-enriched medicinal plants.

TRG is synthesized from nicotinic acid (vitamin B3) through N-methylation catalyzed by S-adenosyl-L-methionine-dependent nicotinate N-methyltransferases, an extension of the conserved pyridine nucleotide cycle in plants [4,8]. In coffee, TRG accumulates during fruit development and can be demethylated back to nicotinic acid during roasting, thereby contributing to dietary vitamin B3 intake [8]. The conservation of this pathway offers a tractable entry point for the metabolic engineering of TRG content. Pharmacologically, TRG exerts antidiabetic effects through multiple mechanisms, including inhibition of alpha-glucosidase and DPP- 4, modulation of PPARgamma signaling, protection of pancreatic beta-cells, and improvement of insulin sensitivity [2,3]. Its neuroprotective activities involve inhibition of Abeta aggregation, upregulation of neurotrophic factors such as NGF and BDNF, and suppression of neuroinflammation via TLR4/NF-kappaB pathways [3]. TRG also enhances antioxidant defenses (SOD, CAT, GSH) while reducing oxidative damage markers such as MDA and NO [2,3]. Importantly, no acute oral toxicity was observed up to 5000 mg/ kg in mice, and epidemiological evidence links higher serum TRG levels with lower HbA1c over a five-year follow-up, supporting its translational potential [2]. These findings underpin the growing demand for standardized, high-TRG plant materials for functional food and pharmaceutical applications.

Bioactive metabolite accumulation is a complex quantitative trait controlled by multiple loci and strongly modulated by the environment. mGWAS integrates high-throughput metabolomics with genome-wide polymorphism data, enabling the genetic dissection of metabolite accumulation at population scale [6,9]. Widely targeted metabolomics based on LC-MS/MS now allows simultaneous profiling of hundreds to thousands of metabolites across large germplasm collections [10], while mixed linear models accounting for population structure and kinship ensure statistical robustness [9]. Landmark mGWAS studies in medicinal plants illustrate the power of this approach. In wolfberry (Lycium), metabolomic profiling of 307 re-sequenced accessions identified 563 annotated metabolites, and mGWAS mapped significant SNP-metabolite associations for 340 metabolites, leading to the functional validation of genes controlling flavonoids (LbUGT, LbCHS, LbMYB), betaine (LbSSADH), and spermidine (LbBEAT) [5]. Importantly, the derived KASP markers enable markerassisted selection (MAS) for quality traits, demonstrating the direct breeding utility of mGWAS outputs [5]. More recently, an mGWAS in Andrographis paniculata dissected the genetic basis of andrographolide accumulation and pinpointed a bifunctional regulatory gene (ApNB-ARC25) whose role was validated by virusinduced gene silencing [7]. These studies confirm that mGWAS can translate natural metabolic variation into actionable gene targets and molecular markers across diverse medicinal species.

Applying the mGWAS framework to TRG holds great promise. In our laboratory, we are conducting an mGWAS in wax gourd, a major cucurbit vegetable for which a chromosome-level genome is available [11], to dissect the genetic architecture of TRG accumulation. The strategy comprises:

a. Widely targeted metabolomic profiling of TRG and related pyridine alkaloids across a diverse natural population;

b. Population-scale re-sequencing and genome-wide association analysis;

c. Prioritization and functional validation of candidate genes, particularly nicotinate N-methyltransferase family members and their transcriptional regulators; and

d. Translation of elite alleles into KASP markers for MAS, together with exploration of CRISPR-based enhancement of key biosynthetic genes [5,7,12].

Integrating transcriptomic and genomic data will further resolve the regulatory network controlling TRG content. Meanwhile, clinical validation of efficacy, safety, and bioavailability remains essential to translate high-TRG germplasm into evidence-based nutraceutical products [2,3].

Trigonelline is a safe, multi-targeted bioactive alkaloid with substantial potential for functional food and pharmaceutical applications. The combination of metabolomics, genomics, and genetics—exemplified by mGWAS—now offers a realistic roadmap from natural metabolite variation to causal genes and molecular markers. Continued efforts, including our ongoing mGWAS in wax gourd, are expected to accelerate the development of trigonellineenriched medicinal crops, meeting the growing demand for highquality natural bioactive products.

1. Zhou J, Chan L, Zhou S (2012) Trigonelline: a plant alkaloid with therapeutic potential for diabetes and central nervous system disease. Curr Med Chem 19(21): 3528-3534.
2. Nguyen V, Taine EG, Meng D, Cui T, Tan W (2024) Pharmacological activities, therapeutic effects, and mechanistic actions of trigonelline. Int J Mol Sci 25(6): 3385.
3. Liang Y, Dai X, Cao Y, Xian Wang, Jing Lu, et al. (2023) The neuroprotective and antidiabetic effects of trigonelline: A review of signaling pathways and molecular mechanisms. Biochimie 206: 93-104.
4. Ashihara H, Ludwig IA, Crozier A (2015) Trigonelline and related nicotinic acid metabolites: occurrence, biosynthesis, taxonomic considerations, and their roles in planta and in human health. Phytochem Rev 14(5): 765-798.
5. Zhao J, Yuhui Xu, Haoxia Li, Wei An, Yue Yin, et al. (2024) Metabolite-based genome-wide association studies enable the dissection of the genetic bases of flavonoids, betaine and spermidine in wolfberry (Lycium). Plant Biotechnol J 22(6): 1435-1452.
6. Zhu G, Shouchuang Wang, Zejun Huang, Shuaibin Zhang, Qinggang Liao, et al. (2018) Rewiring of the fruit metabolome in tomato breeding. Cell 172(1-2): 249-261.
7. Shen Q, Yuxia Wang, Xu Li, Bin Jin, Jiawen Chen, et al. (2026) Metabolome-based genome-wide association study provides genetic insights into the andrographolide accumulation in Andrographis paniculata. J Genet Genomics S1673-8527(26)00101-3.
8. Zheng XQ, Nagai C, Ashihara H (2004) Pyridine nucleotide cycle and trigonelline (N-methylnicotinic acid) synthesis in developing leaves and fruits of Coffea arabica. Physiol Plant 122(1): 104-111.
9. Korte A, Farlow A (2013) The advantages and limitations of trait analysis with GWAS: a review. Plant Methods 9: 29.
10. Chen W, Liang Gong, Zilong Guo, Wensheng Wang, Hongyan Zhang, et al. (2013) A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: application in the study of rice metabolomics. Mol Plant 6(6): 1769-1780.
11. Xie D, Yuanchao Xu, Jinpeng Wang, Wenrui Liu, Qian Zhou, et al. (2019) The wax gourd genomes offer insights into the genetic diversity and ancestral cucurbit karyotype. Nat Commun 10(1): 5158.
12. Gao C (2021) Genome engineering for crop improvement and future agriculture. Cell 184(6): 1621-1635.