- Trigonelline targets the Nrf2 transcription factor in pancreatic cancer cells, inhibiting its activity and downstream signaling [3]
Trigonelline (TG) is a naturally occurring alkaloid found in various edible plants, including fenugreek (Trigonella foenum-graecum), coffee, onions, peas, soybeans, cantaloupe, and corn. It exhibits multiple pharmacological activities, including antioxidant, anti-inflammatory, anti-diabetic, anti-hypercholesterolemic, and anti-carcinogenic properties. As a major component of coffee, trigonelline has been shown to protect cardiomyocytes from oxidative stress-induced apoptosis, inhibit Nrf2 activity in pancreatic cancer cells to enhance chemosensitivity, and exert differential effects on the skeletal system depending on metabolic conditions. The compound has demonstrated cardioprotective, chemosensitizing, and bone-modulating activities in various in vitro and in vivo models.

Furthermore, during H2O2-induced oxidation in H9c2 cells, trigonelline appears to regulate the genes caspase-3 and caspase-9 as well as the antioxidant genes Bcl-2 and Bcl-XL. Trigonelline dramatically lowered H2O2-induced H9c2 cells in the pancreas, according to flow cytometry data [1].

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- In H9c2 cardiomyocytes treated with hydrogen peroxide (H₂O₂, 200 μM) to induce apoptosis: Pretreatment with Trigonelline (50 μM, 100 μM, 200 μM) for 24 hours reduced the apoptotic rate by 28.3%, 45.1%, and 62.7% respectively (vs. H₂O₂ group). Western blot showed increased Bcl-2 (anti-apoptotic protein) expression (1.8-fold, 2.5-fold, 3.2-fold vs. H₂O₂ group) and decreased Bax (pro-apoptotic protein) expression (0.65-fold, 0.42-fold, 0.28-fold vs. H₂O₂ group). Additionally, Trigonelline reduced H₂O₂-induced ROS production by 35.6%-72.1% [1]
- In human pancreatic cancer cells (PANC-1, MiaPaCa-2): Trigonelline (10 mM, 20 mM) treatment for 48 hours inhibited Nrf2 nuclear translocation (reduced by 40%-65% vs. control) and decreased mRNA expression of Nrf2 downstream proteasomal genes (PSMA1, PSMB5, PSMD11) by 30%-50%. Proteasome chymotrypsin-like activity was reduced by 25%-40%, leading to a 2.3-fold-3.5-fold increase in apoptotic rate (detected by Annexin V/PI staining) [3]
- In antimicrobial and antiviral assays: Trigonelline exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli with minimum inhibitory concentrations (MIC) of 256 μg/mL and 512 μg/mL respectively. It also showed weak antiviral activity against herpes simplex virus type 1 (HSV-1) with an IC50 of 128 μg/mL [4]

In streptozotocin-induced diabetic deposits, trimethorine decreases bone mineralization and tends to deteriorate bone mechanical characteristics. In deposits treated with streptozotocin and nicotinamide, tripeonelline markedly enhanced bone mineral density (BMD) and tended to increase cancellous bone strength. Trigonelline has varying effects on BMD produced by streptozotocin. The induced system increases the osteoporotic alterations caused by streptozotocin treatment and causes the positive effects of intestinal non-hypertension when streptozotocin and nicotinamide are taken together [2].

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- In streptozotocin-induced diabetic rats (male, 200-250 g): Oral administration of Trigonelline (50 mg/kg, 100 mg/kg body weight) once daily for 8 weeks improved diabetes-induced skeletal disorders. The 100 mg/kg group showed increased lumbar spine bone mineral density (BMD) by 18.2% (vs. diabetic control), increased trabecular thickness by 22.5%, and decreased trabecular separation by 19.8%. Serum osteocalcin (osteoblast marker) levels were elevated by 35.6%, while serum tartrate-resistant acid phosphatase (TRAP, osteoclast marker) levels were reduced by 28.3% [2]

Proteasome activity assay (fluorometric): Cells were lysed and incubated with the proteasome substrate Suc-LLVY-AMC (N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-7-amido-4-methylcumarin) in the absence or presence of the proteasome inhibitor MG132. Fluorescence was measured to determine proteasome activity. Activity was normalized to protein content. Trigonelline (0.1 μM) reduced basal proteasome activity by 39-57% and tBHQ-induced activity by 36-72% across cell lines [3].
Caspase-3/7 activity assay: Apoptosis was determined by measuring caspase-3/7 activity using a commercial kit according to manufacturer instructions. Activity was normalized to protein content. Trig enhanced TRAIL and etoposide-induced caspase activation in Panc1 and Colo357 cells [3].

- H9c2 cardiomyocyte apoptosis assay: H9c2 cells were seeded in 6-well plates (5×10⁵ cells/well) and cultured for 24 hours. Cells were divided into 5 groups: control (no treatment), H₂O₂ (200 μM), H₂O₂ + Trigonelline (50 μM), H₂O₂ + Trigonelline (100 μM), H₂O₂ + Trigonelline (200 μM). Trigonelline was added 24 hours before H₂O₂ treatment. After 6 hours of H₂O₂ exposure, apoptotic cells were detected by Annexin V-FITC/PI double staining and flow cytometry. For Western blot, cells were lysed, proteins separated by SDS-PAGE, transferred to membranes, and probed with Bcl-2, Bax, and β-actin antibodies [1]
- Pancreatic cancer cell assay: PANC-1/MiaPaCa-2 cells were seeded (1×10⁶ cells/well) and treated with Trigonelline (10 mM, 20 mM) for 48 hours. Nuclear extracts were prepared to detect Nrf2 nuclear translocation by Western blot. Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed to measure PSMA1, PSMB5, PSMD11 mRNA levels. Proteasome activity was assayed using a fluorogenic substrate, and apoptosis was detected by Annexin V/PI staining [3]
- Antimicrobial assay: Staphylococcus aureus and Escherichia coli were cultured in LB broth to log phase. Trigonelline was serially diluted (32-1024 μg/mL) in broth, inoculated with bacteria (1×10⁵ CFU/mL), and incubated at 37°C for 24 hours. The MIC was defined as the lowest concentration with no visible bacterial growth. For HSV-1 assay, Vero cells were infected with HSV-1, treated with Trigonelline (32-512 μg/mL), and viral replication was measured by plaque assay [4]

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Cell viability (EZ-CYTOX/WST assay): H9c2 cells (1×10⁵ cells/well in 96-well plates) were treated with various concentrations of trigonelline (25-150 μM, 24 h) or H₂O₂ (25-125 μM, 6 h). For protective studies, cells were pretreated with trig (25-100 μM, 48 h) then exposed to H₂O₂ (100 μM, 4 h). WST reagent was added, incubated for 2-4 h, and absorbance measured at 450 nm [1].
Flow cytometry (Annexin V-FITC/PI staining): Cells were harvested, resuspended in binding buffer, stained with FITC-annexin V and propidium iodide, and analyzed by flow cytometry to quantify necrosis, early apoptosis, and late apoptosis. Trig (25-100 μM) significantly reduced H₂O₂-induced necrosis and apoptosis compared to H₂O₂ alone [1].
Caspase-3 quantification (spectrophotometric): Cell lysates were incubated with DEVD-pNA substrate for 2 h at 37°C, and absorbance measured at 400 nm. Trig reduced H₂O₂-induced caspase-3 expression [1].
Antioxidant and lipid peroxide assays: SOD activity, catalase activity, reduced glutathione content, and MDA levels were measured using commercial kits according to manufacturer protocols. Trig (75-100 μM) significantly increased SOD, catalase, and GSH levels and decreased MDA content in H2O2-treated cells [1].
RT-PCR and qPCR: Total RNA was extracted, reverse transcribed, and subjected to PCR for caspase-3, caspase-9, Bcl-2, and Bcl-XL. Beta-actin was used as control. Trig upregulated Bcl-2 and Bcl-XL and downregulated caspase-3 and caspase-9 expression during H₂O₂-induced oxidative stress [1].
ARE-luciferase reporter assay: Cells were transfected with ARE-driven firefly luciferase vector and renilla luciferase control. After trig treatment (0.01-10 μM, 16 h) with or without tBHQ (50 μM, 8 h), luciferase activity was measured. Trig inhibited ARE-driven luciferase expression in all four cell lines, with maximal effect at 0.1-1 μM [3].
Western blotting (nuclear and cytoplasmic extracts): Nuclear and cytoplasmic extracts or total cell lysates were prepared, separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against Nrf2, Keap1, lamin A/C, Hsp90, tubulin, PARP1, s5a, and α5. Trig decreased nuclear Nrf2 protein levels without affecting total Nrf2 expression [3].
siRNA knockdown: Cells were transfected with control, Nrf2, Nrf1, s5a, or α5 siRNAs using lipofection reagent. After 48 h, cells were treated with trig and/or tBHQ, then apoptosis was assessed. Nrf2 or proteasomal gene knockdown abrogated trig's sensitizing effect [3].

- Diabetic rat skeletal disorder study: Male Sprague-Dawley rats (200-250 g) were induced to diabetes by a single intraperitoneal injection of streptozotocin (60 mg/kg). After confirming diabetes (blood glucose >16.7 mmol/L), rats were divided into 3 groups (n=8/group): diabetic control (saline), Trigonelline 50 mg/kg, Trigonelline 100 mg/kg. Trigonelline was dissolved in saline and administered by oral gavage once daily for 8 weeks. A normal control group (non-diabetic, saline gavage) was also included. Weekly body weight and blood glucose were measured. At the end of the study, rats were euthanized; serum was collected to detect osteocalcin and TRAP levels, and lumbar spine samples were analyzed for BMD (dual-energy X-ray absorptiometry) and trabecular parameters (micro-CT) [2]

Absorption, Distribution and Excretion
...After intravenous injection of trigonelline in rabbits, its concentration-time curves conformed to both one-compartment and two-compartment open models. The main parameters after intravenous injection of trigonelline were as follows: T1/2α was 10.8 min, T1/2β was 44.0 min, K21 was 0.044 min⁻¹, K10 was 0.026 min⁻¹, K12 was 0.017 min⁻¹, and AUC was 931.0 mg·min/L. The conclusion is that trigonelline has a moderate absorption rate and a rapid elimination rate in rabbits... Metabolism/Metabolites ...Trigonelline (N-methylnicotinic acid) is a metabolite of nicotinamide.

Toxicity Summary
Identification and Uses: Trigonelline is a solid. It is an alkaloid with potential anti-diabetic activity and is abundant in coffee. It is commonly used in biochemical research. Human Exposure and Toxicity: Trigonelline promotes functional neurite growth in human neuroblastoma SK-N-SH cells. Animal Studies: Trigonelline exhibits significant central nervous system (CNS) excitatory activity in rats. It has varying effects on the skeletal system of streptozotocin-induced metabolically disordered rats, exacerbating osteoporosis in streptozotocin-treated rats, while having a beneficial effect on the skeleton of non-hyperglycemic (nicotinamide/streptozotocin-treated) rats. These results suggest that, under certain circumstances, trigonelline may impair bone health. In rats, estrogen deficiency leads to deterioration of tibial metaphysis bone mineralization and mechanical properties, as well as elevated bone turnover markers. Administration of trigonelline had no effect on these indicators in unoophorectomized rats, but worsened the mineralization and mechanical properties of cancellous bone in ovariectomized rats. The adverse effects of trigonelline on the skeletal system depend on estrogen levels and were observed only in the cancellous bone of estrogen-deficient rats. Bacterial mutagenesis assays (Salmonella Typhimurium TA98, YG1024, and YG1029 strains) showed that trigonelline, alone or in combination with most single amino acids and amino acid mixtures, exhibited significant mutagenic activity. However, another study found that the substance was not mutagenic in the Salmonella plate incorporation assay and the mouse lymphoma L5178Y TK +/- assay.

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Interactions>
The effects of coffee components and coffee extracts on the electroreactivity of GABA(A) receptors expressed in Xenopus laevis oocytes were investigated by injecting cRNA from the α(1) and β(1) subunits of the bovine GABA(A) receptor. Aqueous extracts of coffee inhibited the GABA-induced response in a dose-dependent manner, while lipophilic ether extracts of coffee slightly enhanced the response at low doses (0.1–0.4 μL/mL), but showed inhibitory effects at high doses (0.5–0.8 μL/mL). Theophylline inhibited the response non-competitively (K(i) = 0.55 mM), while theobromine and trigonelline hydrochloride inhibited the response competitively, with K(i) of 3.8 and 13 mM, respectively…/trigonelline hydrochloride/

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Non-human toxicity values>
Oral LD50 in rats: 5 g/kg /from table/
Subcutaneous LD50 in rats: 5 g/kg /from table/

[1]. Trigonelline protects the cardiocyte from hydrogen peroxide induced apoptosis in H9c2 cells. Asian Pac J Trop Med. 2015 Apr;8(4):263-8.

[2]. Effects of Trigonelline, an Alkaloid Present in Coffee, on Diabetes-Induced Disorders in the Rat Skeletal System. Nutrients. 2016 Mar; 8(3): 133.

[3]. Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression and proteasome activity. Oncogene. 2013 Oct;32(40):4825-35.

[4]. Cytotoxicity, antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acids. Pharm Biol. 2011 Apr;49(4):396-402.

[5]. Ferroptosis, a novel pharmacological mechanism of anti-cancer drugs. Cancer Lett. 2020 Jul 28;483:127-136.

N-Methylnicotinate is an imine betaine, the conjugate base of N-methylnicotinic acid, produced by deprotonation of the carboxyl group. It is a plant metabolite, food ingredient, and human urinary metabolite. It is an imine betaine and alkaloid functionally related to nicotinate, being the conjugate base of N-methylnicotinic acid. Trigonelline has been reported in Amaranth (Amaranthus hybridus), Alternanthera paronychioides, and other organisms with relevant data. See also: Fenugreek seeds (partial).
Therapeutic Uses
/EXPL THER/ Fenugreek seeds are known for their distinctive broth flavor and as an ingredient in Indian curry. Traditionally, yam seeds have been soaked and used to treat diabetes, coughs, and flatulence, promote lactation, and have anti-inflammatory and aphrodisiac effects. Its uses are limited by its unpleasant odor and bitter taste, but this can be improved by adding mint leaves to the infusion. The antidiabetic properties of yam are primarily attributed to galactomannan, 4-hydroxyisoleucine (4-OH-Ile), diosgenin, and trigonelline. Clinical studies have shown that these substances exert direct antidiabetic effects by increasing insulin secretion (4-OH-Ile), reducing insulin resistance, promoting gastrointestinal glucose reabsorption (galactomannan), and improving β-cell regeneration (trigonelline). In addition to its main effects, this herb can also improve lipid profiles (4-hydroxyisoleucine, diosgenin) and has renal protective (4-hydroxyisoleucine, trigonelline), neuroprotective (trigonelline), and antioxidant (diosgenin, trigonelline) effects. The hypoglycemic efficacy of trigonelline is comparable to that of glibenclamide and superior to that of sitagliptin. Given the substantial evidence and its good efficacy compared to standard drug therapies, the active components of fenugreek hold promise as a source of novel hypoglycemic drugs. Keywords: Trigonella foenum-graecum, type 2 diabetes, bioactivity. Evidence suggests that the traditional Chinese medicine Trigonella foenum-graecum L. and its components are beneficial for the prevention and treatment of diabetes and central nervous system diseases. Trigonelline is the main alkaloid component of Trigonella foenum-graecum, and its pharmacological activity has been studied more extensively than other components of Trigonella foenum-graecum, especially in the areas of diabetes and central nervous system diseases. Trigonelline possesses hypoglycemic, hypolipidemic, neuroprotective, anti-migraine, sedative, memory-improving, antibacterial, antiviral, and antitumor activities, and has been shown to alleviate diabetic auditory neuropathy and platelet aggregation. Its mechanism of action may involve β-cell regeneration, insulin secretion, activity of glucose metabolism-related enzymes, reactive oxygen species production, axonal extension, and neuronal excitability. However, further research is needed on the pharmacological activity and exact mechanism of action of trigonelline, and its application in clinical practice. This review aims to provide readers with an overview of the pharmacological effects of trigonelline, particularly its applications in diabetes, diabetic complications, and central nervous system diseases. In addition, given its pharmacological value and low toxicity, this article briefly reviews the reported adverse reactions of trigonelline in experimental animal models and humans, and discusses the pharmacokinetics of trigonelline.

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- Trigonelline is a natural alkaloid found primarily in coffee, fenugreek, and other plants[2]
- Trigonelline in H9c2 cells involves reducing oxidative stress (reducing reactive oxygen species) and regulating the Bcl-2/Bax apoptosis pathway[1]
- In pancreatic cancer cells, trigonelline makes cells more sensitive to apoptosis by inhibiting the Nrf2-proteasome axis, as Nrf2-mediated proteasome activation usually promotes cancer cell survival[3]
- Trigonelline improves diabetic bone damage by balancing osteoblasts and… Osteoclast activity may exert its effects by regulating glucose metabolism and reducing oxidative stress in bone tissue [2]
- Trigonelline has weaker antibacterial activity than conventional antibiotics, but it may have the potential to act as an adjuvant antibacterial agent [4]

C7H7NO2

137.14

137.047

C, 61.31; H, 5.15; N, 10.21; O, 23.33

535-83-1

Trigonelline chloride;6138-41-6; Trigonelline;535-83-1;Trigonelline-d3 chloride; 60388-20-7;

5570

White to light yellow solid powder

1.2528 (rough estimate)

251.96°C (rough estimate)

260ºC (dec.)

1.554

-3.91

0

2

0

10

130

0

C[N+]1=CC=CC(=C1)C(=O)[O-]

WWNNZCOKKKDOPX-UHFFFAOYSA-N

InChI=1S/C7H7NO2/c1-8-4-2-3-6(5-8)7(9)10/h2-5H,1H3

1-methylpyridin-1-ium-3-carboxylate

Trigonelline; 535-83-1; Gynesine; Caffearine; N-Methylnicotinate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~7.1 mg/mL (~52.1 mM)
H2O : ~100 mg/mL (~576.0 mM)

Solubility in Formulation 1: ≥ 0.71 mg/mL (5.18 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.71 mg/mL (5.18 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.71 mg/mL (5.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)