| Size | Price | Stock | Qty |
|---|---|---|---|
| 500mg |
|
||
| 1g |
|
||
| 2g |
|
||
| 5g |
|
||
| 10g |
|
||
| Other Sizes |
| Targets |
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].
|
|---|---|
| ln Vitro |
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]. |
| ln Vivo |
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].
|
| Enzyme Assay |
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]. |
| Cell Assay |
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]. |
| Animal Protocol |
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].
|
| References |
|
| Additional Infomation |
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]. |
| Molecular Formula |
C11H14N2
|
|
|---|---|---|
| Molecular Weight |
174.24
|
|
| Exact Mass |
174.115
|
|
| CAS # |
87-52-5
|
|
| Related CAS # |
|
|
| PubChem CID |
6890
|
|
| Appearance |
White to off-white solid powder
|
|
| Density |
1.1±0.1 g/cm3
|
|
| Boiling Point |
293.9±15.0 °C at 760 mmHg
|
|
| Melting Point |
132-134 °C(lit.)
|
|
| Flash Point |
131.5±20.4 °C
|
|
| Vapour Pressure |
0.0±0.6 mmHg at 25°C
|
|
| Index of Refraction |
1.631
|
|
| LogP |
1.9
|
|
| Hydrogen Bond Donor Count |
1
|
|
| Hydrogen Bond Acceptor Count |
1
|
|
| Rotatable Bond Count |
2
|
|
| Heavy Atom Count |
13
|
|
| Complexity |
168
|
|
| Defined Atom Stereocenter Count |
0
|
|
| InChi Key |
OCDGBSUVYYVKQZ-UHFFFAOYSA-N
|
|
| InChi Code |
InChI=1S/C11H14N2/c1-13(2)8-9-7-12-11-6-4-3-5-10(9)11/h3-7,12H,8H2,1-2H3
|
|
| Chemical Name |
3-(Dimethylaminomethyl)indole
|
|
| Synonyms |
|
|
| HS Tariff Code |
2934.99.9001
|
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
|
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
|
|||
|---|---|---|---|---|
| Solubility (In Vivo) |
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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.7392 mL | 28.6961 mL | 57.3921 mL | |
| 5 mM | 1.1478 mL | 5.7392 mL | 11.4784 mL | |
| 10 mM | 0.5739 mL | 2.8696 mL | 5.7392 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
