Gramine targets adiponectin receptor 1 (AdipoR1) with IC50 of 4.2±0.53 μM (validated) [1]; adiponectin receptor 2 (AdipoR2) with IC50 of 3.2±0.2 μM (validated) [1]; mouse β2-adrenergic receptor (β2-AR) (agonist activity confirmed, no numerical EC50 provided) [2]; human β2-adrenergic receptor (β2-AR) (agonist activity confirmed, no numerical EC50 provided) [2].
Gramine primarily targets the adiponectin receptor (AdipoR), acting as an active agonist. It exhibits IC50 values of 3.2 µM for AdipoR2 and 4.2 µM for AdipoR1. Additionally, Gramine is a human and mouse β2-Adrenergic receptor (β2-AR) agonist. Beyond these primary receptors, Gramine has been shown to target CDK2 in cervical cancer cells through a ncRNA-mediated suppression mechanism. Its diverse receptor targeting profile underlies its broad pharmacological activities, including anti-tumor, anti-viral, and anti-inflammatory properties. The compound's ability to modulate multiple receptors makes it a valuable tool for studying adiponectin signaling, adrenergic pathways, and their roles in cancer and metabolic diseases.

Gramine is an active agonist of the adiponectin receptor (AdipoR), having IC50 values of 3.2 μM for AdipoR2 and 4.2 μM for AdipoR1, respectively [1]. Potential β2-AR agonistic agent is gramine [2]. With IC50 values of 9.6±0.9 and 0.1±0.1 μM, respectively, gramme (20 μM to 1.2 nM) dose-dependently suppresses the development of AdipoR1/adipoR2-activated liver cancer lines (MCF-7 cells) [1].

---

In vitro assays: Gramine was identified as a hit compound from a natural product library screening using a fluorescence polarization-based competitive binding assay, with primary screen IC50 of 4.5 μM against AdipoR1 and 3.6 μM against AdipoR2; validated IC50 values were 4.2±0.53 μM (AdipoR1) and 3.2±0.2 μM (AdipoR2) [1].
In MDA-MB-231 human breast cancer cells, treatment with Gramine at IC50 concentrations for 6 hours elevated AMPK phosphorylation as determined by western blot [1].
In MCF-7 human breast cancer cells, treatment with Gramine at IC50 concentrations for 6 hours inhibited p-PPARα level as determined by western blot [1].
Gramine inhibited proliferation of MDA-MB-231 cells with an IC50 of 9.6±0.9 μM after 72-hour treatment measured by CellTiter-Blue assay [1].
Gramine inhibited proliferation of MCF-7 cells with an IC50 of 0.1±0.1 μM after 72-hour treatment measured by CellTiter-Blue assay [1].
In HEK293 cells expressing mouse β2-AR, Gramine (100 μM) increased CRE-luciferase activity after 3-hour incubation; the effect was blocked by pre-incubation with the selective β2-AR antagonist ICI-118551 (1 μM). Gramine increased luciferase activity in a concentration-dependent manner (concentrations tested from 0 to 100 μM) in β2-AR-expressing HEK293 cells [2].
In HEK293 cells expressing human β2-AR, Gramine (100 μM) increased CRE-luciferase activity after 3-hour incubation, and this increase was cancelled by ICI-118551 pre-treatment [2].
In vitro studies have demonstrated that Gramine significantly inhibits proliferation and migration, induces apoptosis, and triggers G0/G1 phase cell cycle arrest in HeLa cervical cancer cells. It has also been shown to suppress triple-negative breast cancer by inducing ferroptosis via the CUL3–MTDH axis. Gramine exhibits cytotoxic properties and holds research potential in antitumor, anti-inflammatory, and antiviral applications. Studies using Gramine-loaded nanoparticles have demonstrated outstanding anticancer activity against the HCT-116 cell line, with increased oxidative stress indicators such as NO, LPO, and ROS levels compared to controls. These findings position Gramine as a promising lead compound for cancer therapy, particularly in drug-resistant cancers.

In vivo assays: Intramuscular injection of Gramine at 20 mg/kg body weight into mouse quadriceps (100 μL per leg, diluted in 10% DMSO in sterile water) resulted in a significant increase in Nr4a1 mRNA expression (p<0.01) in quadriceps muscle 6 hours after injection, as determined by real-time PCR. No significant changes were observed in Nr4a3 or PGC-1α4 mRNA levels under the same conditions [2].
In vivo experiments using 4T1 syngeneic and MDA-MB-231 xenograft mouse models confirmed that Gramine significantly suppressed tumor growth at doses of 10 and 20 mg/kg without observable systemic toxicity or body weight loss. In both 4T1 and MDA-MB-231 mouse models, Gramine significantly inhibited tumor growth without causing systemic toxicity. These findings position Gramine as a promising lead compound for TNBC therapy and open new avenues for targeting ferroptosis in drug-resistant cancers. Gramine also demonstrates anti-inflammatory and antiviral properties in vivo, though specific detailed in vivo studies for these activities are less extensively documented. The compound's favorable safety profile at therapeutic doses supports its continued investigation as a potential therapeutic agent.

The fluorescence polarization (FP) binding assay was developed to identify adiponectin receptor ligands. The assay used fluorescein-labeled probe 1 (FITC-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2) at 100 nM and adiponectin receptor (AdipoR1 or AdipoR2) at 2 μM in a reaction volume of 19 μL. Test compounds (including Gramine) were prepared at half-dilution concentrations from 10 μM to 0.078 μM in 5% DMSO and transferred to assay plates. The mixture was incubated for 30 minutes at 25°C. Fluorescence polarization was measured at excitation 485 nm and emission 538 nm. Competitive binding was assessed by decrease in FP values, and IC50 values were determined by non-linear least square fitting [1].
Luciferase reporter assay for β2-AR agonist activity: HEK293 cells were plated in 12-well plates at 1.0×10^5 cells/well, cultured for 20 hours, and transfected by calcium phosphate method with 100 ng of pCRE-luc reporter plasmid (containing four copies of cAMP response element), 10 ng of p3×FLAG-β2-AR (mouse or human expression plasmid) or empty vector, and 100 ng of pCMV-β-Gal (β-galactosidase expression plasmid). Four hours after transfection, medium was replaced with DMEM supplemented with 5% dextran-charcoal-stripped FBS. After 24 hours, test compounds (including Gramine) or DMSO control were added (final DMSO concentration 0.1%). After 3-hour (or 6-hour for dose-response) incubation, cells were lysed and luciferase and β-galactosidase activities were measured. Normalized luciferase values were calculated by dividing luciferase activity by β-galactosidase activity [2].
Non-cellular binding assays for Gramine typically involve measuring its affinity for the adiponectin receptor (AdipoR) and β2-adrenergic receptor (β2-AR). These assays often use radioligand binding techniques or surface plasmon resonance to determine IC50 or Ki values. For AdipoR, Gramine demonstrates IC50 values of 3.2 µM for AdipoR2 and 4.2 µM for AdipoR1. For β2-AR, Gramine acts as an agonist, and its binding affinity can be assessed using membrane preparations from cells expressing the receptor. Competitive binding assays with labeled reference ligands are commonly employed to determine the potency and selectivity of Gramine at these receptors. These assays are typically performed in buffer systems at physiological pH and temperature, with incubation times optimized for equilibrium binding.

Cell culture: MCF-7 and MDA-MB-231 cells were maintained in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2 atmosphere [1].
Western blotting: Cells were grown to 80% confluence, harvested, and treated with different doses of Gramine (IC50 concentrations) in serum-free media for 6 hours. Cells were washed with ice-cold PBS and lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined by Bio-Rad protein assay. Equal amounts of total protein (50 μg) were subjected to 10% Tris-HCl gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked, incubated with primary antibodies (anti-p-AMPKα, anti-AMPKα, anti-p-PPARα, anti-β-actin), then with secondary antibodies, and detected by ECL plus western blotting detection system [1].
Cell proliferation assay: Cells were seeded into 384-well plates (45 μL per well), allowed to attach overnight, then treated with serial dilutions of Gramine (20 μM to 1.2 nM) for 72 hours. Cell viability was evaluated using CellTiter-Blue reagent; fluorescence was measured at excitation 530 nm and emission 590 nm. Viability values were normalized to vehicle controls [1].
Luciferase assay for β2-AR activity: HEK293 cells were cultured in DMEM with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO2. Cells were plated in 12-well plates at 1.0×10^5 cells/well, cultured for 20 hours, then transfected by calcium phosphate method with pCRE-luc, p3×FLAG-β2-AR or empty vector, and pCMV-β-Gal. After 4 hours, medium was replaced with DMEM containing 5% dextran-charcoal-stripped FBS. After 24 hours, cells were treated with Gramine (100 μM, or indicated concentrations for dose-response) or DMSO control for 3–6 hours. For antagonist studies, cells were pre-incubated with ICI-118551 (1 μM) for 30 minutes before compound addition. Luciferase and β-galactosidase activities were then measured [2].
In vitro cell-based assays for Gramine typically involve cancer cell lines such as HeLa (cervical cancer), HCT-116 (colorectal cancer), and MDA-MB-231 or 4T1 (breast cancer). Cells are cultured in appropriate media and treated with Gramine at various concentrations for defined periods (e.g., 24-72 hours). Cell viability is assessed using MTT, CCK-8, or similar assays. Proliferation and migration are evaluated using colony formation, wound healing, or Transwell assays. Apoptosis is measured using flow cytometry with Annexin V/PI staining, and cell cycle analysis is performed using propidium iodide staining. Oxidative stress markers such as NO, LPO, and ROS levels are measured to assess mechanisms of action. Gramine is typically dissolved in DMSO and diluted in culture medium, with DMSO controls included to account for solvent effects.

Animal protocol for intramuscular injection: Male 7-week-old C57BL/6J mice were anesthetized with isoflurane. The quadriceps in both legs were injected directly with Gramine at 20 mg/kg body weight, diluted in 10% DMSO in sterile water (100 μL per leg). Control mice received 10% DMSO in sterile water (100 μL per leg). Six hours after administration, mice were killed under isoflurane anesthesia, and quadriceps muscles were rapidly excised for RNA extraction [2].
In vivo animal studies for Gramine commonly use 4T1 syngeneic and MDA-MB-231 xenograft mouse models to evaluate its anti-tumor efficacy. Tumor-bearing mice are treated with Gramine at doses of 10 and 20 mg/kg, typically administered orally or intraperitoneally. Tumor volume and body weight are monitored regularly throughout the study. At the end of the experiment, tumors are excised and weighed, and tissues are collected for histological analysis and biomarker evaluation. Systemic toxicity is assessed by monitoring body weight loss, organ weights, and histopathological examination of major organs. Gramine has been shown to significantly inhibit tumor growth without causing observable systemic toxicity. These protocols are standard for evaluating the anti-tumor activity of candidate compounds in preclinical settings.

Pharmacokinetic data for Gramine are limited in publicly available sources. As a small molecule alkaloid with a molecular weight of approximately 174.24 g/mol, Gramine is expected to be absorbed from the gastrointestinal tract and distributed to tissues. Its solubility in various solvents is a key factor for formulation in both in vitro and in vivo studies. Gramine is metabolized by hepatic enzymes, though specific metabolic pathways are not well characterized. The compound's anti-tumor, anti-viral, and anti-inflammatory properties suggest it may have favorable tissue distribution and bioavailability. However, detailed PK parameters such as half-life, Cmax, AUC, and bioavailability have not been extensively reported. Gramine is typically administered orally or intraperitoneally in animal studies, indicating reasonable oral bioavailability.

Toxicological data for Gramine indicate a favorable safety profile at therapeutic doses. In vivo experiments using 4T1 syngeneic and MDA-MB-231 xenograft mouse models confirmed that Gramine significantly suppressed tumor growth at doses of 10 and 20 mg/kg without observable systemic toxicity or body weight loss. In both 4T1 and MDA-MB-231 mouse models, Gramine significantly inhibited tumor growth without causing systemic toxicity. These findings suggest that Gramine has a wide therapeutic window. However, comprehensive toxicological studies, including acute and chronic toxicity, genotoxicity, and reproductive toxicity assessments, are not extensively documented. As a natural alkaloid, Gramine's toxicity profile may vary depending on the source and purity of the compound. Research-grade Gramine is intended for laboratory use only.

[1]. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay. PLoS One. 2013 May 14;8(5):e63354.

[2]. Identification of Functional Food Factors as β2-Adrenergic Receptor Agonists and Their Potential Roles in Skeletal Muscle. J Nutr Sci Vitaminol (Tokyo). 2018;64(1):68-74.

Gramine is an aminoalkylindole with a dimethylaminomethyl substituent at the 3-position. It is a plant metabolite with serotonin antagonist, antiviral, and antibacterial activities. It is an aminoalkylindole, an indole alkaloid, and a tertiary amine compound. It is the conjugate base of granulamine (1+). Granamine has been reported in barley (Hordeum vulgare), hartwegii (Pinus hartwegii), and other organisms with relevant data.

---

Gramine is a natural indole alkaloid that exists in higher plants such as giant reed (Arundo donax Linn.) [2] and silver maple [1]. It has been demonstrated that gramine derivatives exhibit anti-viral activity against enterovirus 71 and neuroprotective activity by inhibiting Ca2+ entry into neuroblastoma cells [2]. In the context of adiponectin receptor agonism, Gramine showed activity in both AdipoR1 and AdipoR2 systems, and was derived from silver maple according to the natural product library [1]. In the context of β2-AR agonism, Gramine may increase cAMP levels through its β2-AR agonistic activities, and a high concentration of a gramine analog was previously reported to increase cAMP levels in rat aorta [2]. Gramine is considered a potential drug candidate for hypoadiponectin-related diseases and for maintaining skeletal muscle function [1][2].
Gramine (Donaxine) is a natural indole alkaloid isolated from giant reed (Arundo donax L.). It acts as an active adiponectin receptor (AdipoR) agonist with IC50 values of 3.2 µM for AdipoR2 and 4.2 µM for AdipoR1, and also as a human and mouse β2-adrenergic receptor (β2-AR) agonist. Gramine exhibits anti-tumor, anti-viral, and anti-inflammatory properties. In vitro, it significantly inhibits proliferation and migration, induces apoptosis, and triggers G0/G1 phase cell cycle arrest in cancer cells. In vivo, it suppresses tumor growth in mouse models without systemic toxicity. Gramine is not approved for clinical use and is strictly a research compound. Its diverse receptor targeting profile makes it a valuable tool for studying adiponectin signaling, adrenergic pathways, and cancer biology.

C11H14N2

174.24

174.115

87-52-5

6890

White to off-white solid powder

1.1±0.1 g/cm3

293.9±15.0 °C at 760 mmHg

132-134 °C(lit.)

131.5±20.4 °C

0.0±0.6 mmHg at 25°C

1.631

1.9

1

1

2

13

168

0

OCDGBSUVYYVKQZ-UHFFFAOYSA-N

InChI=1S/C11H14N2/c1-13(2)8-9-7-12-11-6-4-3-5-10(9)11/h3-7,12H,8H2,1-2H3

3-(Dimethylaminomethyl)indole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (11.94 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 (11.94 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 (11.94 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.

---

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