Trigonelline targets multiple pathways. It inhibits nuclear factor E2-related factor 2 (Nrf2) activity, with maximal inhibition at 0.1-1 μM, reducing Nrf2 nuclear accumulation without affecting total Nrf2 protein expression [3]. It suppresses Nrf2-dependent proteasome activity and proteasomal gene expression (s5a/psmd4, s5/psma5) [3]. It also increases antioxidant enzyme activities (SOD, catalase) and reduces MDA levels in H9c2 cells [1]. In pancreatic cancer cells, trig reduces proteasome activity by 36-72% depending on cell line [3].

Cardiocyte protection (H9c2 cells): Trigonelline (25-100 μM, 48 h pretreatment) significantly rescued H9c2 cell morphology from H₂O₂ (100 μM, 4 h)-induced oxidative damage, increased cell viability (from ~50% to ~80-90%), reduced necrosis and apoptosis (flow cytometry), decreased caspase-3 expression, and increased Bcl-2 and Bcl-XL expression. It also enhanced SOD, catalase, and GSH levels while reducing MDA content. Higher concentrations (125-150 μM) induced cell death. Quercetin (100 μM) was used as positive control [1].
Nrf2 inhibition in pancreatic cancer cells: Trigonelline (0.1-1 μM, 1-16 h) dose-dependently inhibited ARE-driven luciferase expression in Panc1, Colo357, MiaPaca2, and H6c7 cells, with maximal inhibition at 0.1-1 μM (higher doses less effective). It decreased nuclear Nrf2 protein levels without affecting total Nrf2 expression, reduced proteasome activity (by 39-57% for basal; 36-72% for tBHQ-induced), and decreased proteasomal gene expression (s5a and α5). Trig treatment (0.1 μM) reduced GCLC expression in Panc1 and Colo357 cells [3].
Apoptosis sensitization: In Panc1 and Colo357 cells, trigonelline (0.1 μM, 16 h pretreatment) enhanced TRAIL and etoposide-induced caspase 3/7 activity (2.8-4.3-fold for TRAIL; 2.3-3.1-fold for etoposide) and PARP1 cleavage. In MiaPaca2 and H6c7 cells, trig (0.1 μM) blocked tBHQ-induced protection from apoptosis, restoring sensitivity to TRAIL and etoposide [3].
Nrf2 and proteasome dependency: siRNA knockdown of Nrf2 or proteasomal genes (s5a, α5) abrogated trig's sensitizing effect, confirming Nrf2-dependent mechanism [3].

---

It was discovered that trigonelline considerably improved the morphology of H9c2 cells. Trigonelline treatment decreases H2O2-induced cell death and improves antioxidant activity in cells. Furthermore, research has demonstrated that trigonelline controls the expression of the antioxidant genes Bcl-2 and Bcl-XL as well as the oxidant genes caspase-3 and caspase-9 in H9c2 cells during the H2O2-induced oxidation process. Trigonelline chloride appears to also greatly diminish H2O2-induced H9c2 cell connections and anodes, as evidenced by flow cytometry studies [1].

Cardioprotective activity (not described in full animal model): The study did not include in vivo cardioprotective experiments; only in vitro H9c2 cell studies were conducted [1].
Skeletal effects in diabetic rats: Trigonelline (50 mg/kg p.o. daily for 4 weeks) in streptozotocin-induced diabetic rats worsened bone mineralization (decreased bone mineral mass/bone mass ratio, increased bone water mass/bone mass ratio) and tended to worsen cancellous bone mechanical properties. In nicotinamide/streptozotocin-treated (non-hyperglycemic) rats, trig significantly increased bone mineral density (tibia and vertebra) and tended to improve cancellous bone strength. No effect on body mass, blood glucose, or serum bone turnover markers (osteocalcin, RatLaps) was observed [2].
Chemosensitization in xenograft model: In SCID-beige mice bearing Colo357 or Panc1 pancreatic tumor xenografts, combination treatment with trigonelline (1 mg/kg body weight, i.p., daily for 4 days with a 3-day interval) plus etoposide (10 mg/kg) significantly reduced tumor size and weight compared to etoposide alone or vehicle control (Colo357: 391±155 mm³ vs 661±461 mm³; Panc1: 113±45 mm³ vs 216±126 mm³). Immunohistochemistry showed reduced nuclear phospho-Nrf2 levels in combination-treated tumors [3].

---

In streptozotocin-induced diabetes condition, trimetholine chloride decreases bone mineralization and tends to deteriorate bone mechanical characteristics. Trigonelline significantly increased bone mineral density (BMD) and tended to increase cancellus in nicotinamide/streptozotocin treated material. Trigonelline has varying effects on the induction produced by streptozotocin. has positive effects on the induction of psychiatric non-hypertension (nicotinamide/streptozotocin treatment) and strengthens the osteoporotic alterations of streptozotocin treatment [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].

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].

Rat model (streptozotocin-induced diabetes): Three-month-old female Wistar rats received streptozotocin (60 mg/kg i.p. in citrate buffer) or nicotinamide (230 mg/kg i.p.) 15 min before streptozotocin. Two weeks later, trigonelline (50 mg/kg p.o. daily) or vehicle (tap water, 2 mL/kg) was administered by gastric tube for 4 weeks. Non-fasting blood glucose was measured weekly via tail tip. At termination, serum was collected, and femurs, tibias, and L-4 vertebrae were isolated for bone mechanical testing (three-point bending, compression test), DXA (BMD, BMC), and bone composition analysis (mineral, water, organic content). Some rats died during the experiment; final n=7-10/group [2].
SCID-beige mouse xenograft model: Female SCID-beige mice (8-week-old, ~20 g) were inoculated subcutaneously with 2×10⁶ Colo357 or Panc1 cells in 200 μL saline. When tumors reached 5×5 mm², mice were randomized into 3 groups (n=6): Group I (0.9% NaCl), Group II (etoposide 10 mg/kg i.p.), Group III (trigonelline 0.02 mg/kg + etoposide 0.2 mg/kg in 0.9% NaCl). Treatment was given i.p. daily for 4 days, with a 3-day break, then repeated. Tumor sizes were measured weekly. After scarification (day 21), tumors were excised, weighed, and snap-frozen. Immunohistochemistry was performed on cryosections using anti-phospho-Nrf2 (Ser40) antibody to detect activated nuclear Nrf2. Expression score was calculated as intensity × distribution [3].

Trigonelline pharmacokinetics have been reported previously: in rats, trigonelline has a half-life (t₁/₂) of 215.9 minutes. It is well absorbed after oral administration. The trigonelline dose used in rat studies (50 mg/kg p.o. daily) corresponds to approximately 5 mg/kg in humans (350 mg/70 kg person) using a conversion factor of 10 due to faster rat metabolism. Moderate to heavy coffee drinkers may be exposed to similar trigonelline doses, as trigonelline content in roasted coffee is comparable to that of caffeine [2].

Trigonelline at concentrations up to 100 μM had no toxic effect on H9c2 cells; higher concentrations (125-150 μM) induced cell death and cytotoxicity [1]. In rat studies, trigonelline (50 mg/kg p.o. for 4 weeks) caused no significant changes in body mass or gross toxicity. However, in streptozotocin-induced diabetic rats, trig worsened bone mineralization (decreased bone mineral mass/bone mass ratio, increased bone water mass/bone mass ratio) and tended to worsen cancellous bone mechanical properties, indicating a potential adverse skeletal effect in severe hyperglycemic conditions [2]. In contrast, in non-hyperglycemic (nicotinamide/streptozotocin-treated) rats, trig improved bone mineral density. No LD50, hepatotoxicity, or nephrotoxicity data are reported in these studies [1][2][3].

[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.

Trigonelline is a naturally occurring alkaloid (pyridine derivative) found in fenugreek, coffee, onions, peas, soybeans, cantaloupe, and corn. As a major component of coffee brew, it is present in roasted coffee at concentrations of 2.8-9.6 mg/g in Arabica samples (∼5.3 mg/g in strongly roasted espresso-type coffee). Trigonelline is thermolabile and degrades during roasting. It is readily absorbed after coffee consumption and is excreted in urine as N-methylpyridinium and trigonelline metabolites [2].
The compound exhibits a biphasic dose dependency for Nrf2 inhibition, with maximal efficiency at submicromolar doses (0.1-1 μM) and less efficiency at higher doses. It is currently used as a tool compound in studies on Nrf2, as an Nrf2 inhibitor, and has been shown to have potential in combination therapy for highly resistant tumors such as pancreatic cancer [3]. No adverse effects of this natural coffee constituent have been reported so far, and its suitability for human application has been demonstrated in diabetes and metabolic syndrome patients [3]. Trigonelline acts as an Nrf2 inhibitor by reducing nuclear import of Nrf2, independent of nuclear export (crm1 pathway) and intranuclear degradation (proteasome pathway), as shown by experiments with leptomycin-B (nuclear export inhibitor) and MG132 (proteasome inhibitor) [3].

---

Trigonelline hydrochloride is citrate-based.

C7H8CLNO2

173.5969

173.024

C, 48.43; H, 4.65; Cl, 20.42; N, 8.07; O, 18.43

6138-41-6

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

134606

White to off-white solid powder

~260 °C (dec.)

1

3

1

11

136

0

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

TZSYLWAXZMNUJB-UHFFFAOYSA-N

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

1-methylpyridin-1-ium-3-carboxylic acid;chloride

Trigonelline hydrochloride; trigonelline HCl; 6138-41-6; 3-Carboxy-1-methylpyridinium chloride; Trigonelline, chloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~125 mg/mL (~720.05 mM)
H2O : ~100 mg/mL (~576.04 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (11.98 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 20.8 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (11.98 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (11.98 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 100 mg/mL (576.04 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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