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| Other Sizes |
| Targets |
The primary molecular target of gamma-Linolenic Acid methyl ester is the leukotriene B4 (LTB4) receptor. LTB4 is a potent pro-inflammatory lipid mediator derived from arachidonic acid via the 5-lipoxygenase pathway. By binding to and antagonizing the LTB4 receptor (BLT1 and BLT2), gamma-Linolenic Acid methyl ester can reduce the chemotaxis of neutrophils and the subsequent inflammatory cascade. This inhibition is relatively weak, with a Ki of 1 microM against [3H]-LTB4 binding to porcine neutrophil membranes. The compound may also be metabolized in vivo to GLA, which can be elongated to dihomo-gamma-linolenic acid (DGLA) and further metabolized to anti-inflammatory prostaglandins of the 1-series. Thus, its overall mechanism is likely a combination of direct receptor antagonism and metabolic conversion to other bioactive lipids.
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| ln Vitro |
γ-Linolenic Acid methyl ester (1-4 μg/mL; 72 h) causes apoptosis in A-549 lung cancer cell lines grown in vitro, with the use of the SRB assay. Nevertheless, GLA is caused by changes in BCl-2 expression and actions at the gene/oncogene level[1].
In vitro, gamma-Linolenic Acid methyl ester is a weak antagonist of the leukotriene B4 (LTB4) receptor. It was identified as an omega-6 fatty acid that could be elongated to arachidonic acid for endogenous eicosanoid synthesis. In a competitive binding assay, GLA was found to inhibit [3H]-LTB4 binding to porcine neutrophil membranes with a Ki (inhibition constant) of 1 microM. This indicates that the compound can bind to the LTB4 receptor, albeit with moderate affinity. The methyl ester form is used in research to formulate GLA-containing diets and dietary supplements. The free acid GLA, which is the bioactive form, has been studied for various other in vitro activities, including its effects on cancer cell proliferation, particularly as a melanoma cell proliferation inhibitor. The methyl ester itself is a prodrug that is likely hydrolyzed in vivo to the active free acid. |
| ln Vivo |
γ-Linolenic Acid Methyl Ester reduces the histological signs of fatty liver caused by EtOH as well as the hepatic triglycerides[2]. In rats that are normal or hyperlipidemic, γ-Linolenic Acid methyl ester has the potential to dramatically lower levels of plasma total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), MDA, atherosclerosis index (AI), and liver TC and MDA while raising levels of high density lipoprotein cholesterol (HDL-C).
In vivo, gamma-Linolenic Acid methyl ester (Methyl GLA) has demonstrated anti-inflammatory activity in a model of LTB4-induced bronchoconstriction. In this in vivo model, intravenous (i.v.) administration of gamma-Linolenic acid methyl ester at a dose of 1 mg/kg resulted in 53% inhibition of LTB4-induced bronchoconstriction. This effect was comparable to that of other fatty acids like ricinelaidic acid (46% inhibition) and supports the hypothesis that essential fatty acids act as LTB4 receptor antagonists, which may account, in part, for their reported anti-inflammatory activities. This suggests that the compound is systemically available and can exert its effects in vivo. It has also been investigated in nutritional studies for weight regain and as a potential tumor suppression agent. The methyl ester form is often used in these studies because it is more stable and easier to handle. |
| Enzyme Assay |
A typical non-cellular binding assay for gamma-Linolenic Acid methyl ester or its free acid GLA is a radioligand binding assay using porcine neutrophil membranes. Porcine blood is collected, and neutrophils are isolated by dextran sedimentation and density gradient centrifugation. The isolated neutrophils are lysed by sonication in a hypotonic buffer, and the crude membrane fraction is collected by ultracentrifugation (100,000 × g for 60 minutes). The membrane pellet is resuspended in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2). For the competition assay, 100 ug of membrane protein is incubated with 1 nM [3H]-LTB4 and varying concentrations of GLA (0.1 - 100 uM) in a total volume of 200 uL. Non-specific binding is determined by the addition of 1 uM unlabeled LTB4. The reaction mixture is incubated at 4degC for 60 minutes. Bound and free radioligand are separated by rapid filtration through Whatman GF/C glass fiber filters. The filters are washed three times with 3 mL of ice-cold assay buffer. The retained radioactivity on the dried filters is measured by liquid scintillation counting. The Ki value is calculated from the IC₅0 using the Cheng-Prusoff equation. The reported Ki for GLA is 1 uM.
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| Cell Assay |
Apoptosis Analysis[1]
Cell Types: A-549 lung cancer cell line Tested Concentrations: 1, 2, 3, and 4 μg/mL Incubation Duration: 72 hrs (hours); observed at 24 h, 48 h, and 72 h Experimental Results: demonstrated cytotoxicity potentially due to the induction of apoptosis of tumor cells by augmenting free radical generation only in the tumor cells but not normal cells. A typical in vitro cell-based assay for the bioactivity of GLA (the free acid of methyl GLA) is a proliferation assay using melanoma cell lines. Human melanoma cells, such as A375 or SK-MEL-28, are cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. Cells are seeded in 96-well plates at 5 × 103 cells/well and allowed to adhere overnight. The next day, the medium is replaced with fresh medium containing various concentrations of GLA (0-200 uM), prepared by complexing the fatty acid with fatty acid-free bovine serum albumin (BSA) to improve solubility. After 48-72 hours of incubation, cell viability is measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The IC₅0 (half-maximal inhibitory concentration) is calculated using non-linear regression. For a neutrophil chemotaxis assay, human neutrophils are isolated and placed in the upper chamber of a transwell plate. The test compound (GLA methyl ester or free acid) is added to the lower chamber along with a chemoattractant, such as LTB4 (10 nM). After incubation, the number of cells that have migrated to the lower chamber is quantified by flow cytometry or by measuring the activity of a cytoplasmic marker like myeloperoxidase (MPO). Inhibition of LTB4-induced chemotaxis is calculated. |
| Animal Protocol |
Animal/Disease Models: Hepatic pathol rats model induced by EtOH (male SD (Sprague-Dawley) rats, 250-300 g)[2]
Doses: 90 mL, 50-60 mL Route of Administration: intraperitoneal (ip) injection; one time/day; administered 90 mL during day 1-5 and day 9, 50-60 mL during day 6-8 Experimental Results: diminished the hepatic triglycerides of 25.6 mg/g compared with saline (40.2 mg/g) or olive oil (42.8 mg/g) treatment. diminished liver index (the ratio of liver weight and body weight) in hyperlipidemic rats, but had no significant effect in normal rats. The in vivo protocol for testing gamma-Linolenic Acid methyl ester in the LTB4-induced bronchoconstriction model is performed in male Dunkin-Hartley guinea pigs (250-350 g). The animals are anesthetized with an intraperitoneal (IP) injection of pentobarbital sodium (60 mg/kg). A tracheal cannula is inserted, and the animals are mechanically ventilated. The carotid artery is cannulated for blood pressure monitoring, and the jugular vein is cannulated for compound administration. The animals are paralyzed with pancuronium bromide (0.1 mg/kg, IV). Bronchoconstriction is measured as an increase in insufflation pressure via a pressure transducer connected to the tracheal cannula. gamma-Linolenic Acid methyl ester is formulated in a vehicle such as 0.9% saline containing 0.2% BSA. The compound is administered intravenously (IV) at doses of 0.3, 1, and 3 mg/kg, 5 minutes before LTB4 challenge. LTB4 (0.3 ug/kg, IV) is injected to induce bronchoconstriction. The peak increase in insufflation pressure is recorded. The percentage inhibition of bronchoconstriction is calculated by comparing the treated group's pressure increase to that of a vehicle-treated control group. All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC). |
| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of gamma-Linolenic Acid methyl ester itself are not fully characterized. As a methyl ester, it is a prodrug that is likely hydrolyzed rapidly in vivo by esterases to the free acid, gamma-linolenic acid (GLA). GLA is a naturally occurring fatty acid, and its metabolism is well-studied. Following oral administration, the methyl ester is likely absorbed and then hydrolyzed. GLA is incorporated into cell membrane phospholipids and is metabolized by desaturation and elongation enzymes to produce other long-chain polyunsaturated fatty acids (LCPUFA), including dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (AA). DGLA is the precursor for the anti-inflammatory prostaglandin E1 (PGE1). GLA is beta-oxidized in the mitochondria for energy production. The elimination half-life of GLA in plasma is relatively short. The methyl ester is used in nutritional research to improve the stability and formulation of GLA in animal diets. Detailed human PK data is limited.
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| Toxicity/Toxicokinetics |
No detailed toxicity data is available for gamma-Linolenic Acid methyl ester. As a common dietary fatty acid, GLA is generally recognized as safe (GRAS) and has a low order of toxicity. It is a natural component of various vegetable oils, such as evening primrose oil, black currant seed oil, and borage oil. No significant adverse effects are associated with GLA consumption at typical nutritional levels. In research settings, the compound is well-tolerated at the doses tested. Standard safety precautions for handling fatty acids should be observed, including avoiding contact with skin and eyes and working in a well-ventilated area. It is for research use only and not for human consumption as a pharmaceutical.
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| References |
[1]. Jubie S, et al. Isolation of methyl gamma linolenate from Spirulina platensis using flash chromatography and its apoptosis inducing effect. BMC Complement Altern Med. 2015 Aug 4;15:263.
[2]. Segarnick DJ, et al. Gamma-linolenic acid inhibits the development of the ethanol-induced fatty liver. Prostaglandins Leukot Med. 1985 Mar;17(3):277-82. [3]. Xiuqin K, et al. Studies on the hypolipidemic effects of gamma-linolenic acid methyl ester derived from Spirulina maxima[J]. Zhongguo hai Yang yao wu= Chinese Journal of Marine Drugs, 2003, 22(6): 30-34. [4]. Williams, et al. Antithrombosis agent containing γ-linolenic acid or a functional derivative of it: Federal Republic of Germany, DE2749492[P]. 1978-05-11. [5]. Hiyamuta, et al. Melanoma cell proliferation inhibitors containing γ-linolenic acid or its derivatives: Japan, JP2014141427[P]. 2014-08-07. |
| Additional Infomation |
Methyl gamma-linolenic acid (GLA) is a fatty acid methyl ester formed by the condensation of methanol and GLA. It is a bacterial metabolite with apoptosis-inducing, antitumor, and antibacterial activities. Its functions are related to those of GLA.
gamma-Linolenic Acid methyl ester is not an approved drug. It is a research-grade chemical used primarily in nutritional and pharmacological studies. Its main mechanism of action is as a weak LTB4 receptor antagonist, which contributes to its anti-inflammatory properties. It is also a source of GLA in dietary supplementation research, where it is used to study the effects of omega-6 fatty acids on various health conditions, including atopic eczema, rheumatoid arthritis, and diabetic neuropathy. No clinical trials have been registered for the methyl ester form. For research use only; not for human therapeutic or diagnostic use. |
| Molecular Formula |
C19H32O2
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|---|---|
| Molecular Weight |
292.46
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| Exact Mass |
292.24
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| CAS # |
16326-32-2
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| PubChem CID |
6439889
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| Appearance |
Typically exists as solid at room temperature
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| Density |
0.9±0.1 g/cm3
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| Boiling Point |
385.4±0.0 °C at 760 mmHg
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| Flash Point |
101.5±23.2 °C
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| Vapour Pressure |
0.0±0.8 mmHg at 25°C
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| Index of Refraction |
1.476
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| LogP |
7.03
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
14
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| Heavy Atom Count |
21
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| Complexity |
314
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCCCC/C=C\C/C=C\C/C=C\CCCCC(=O)OC
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| InChi Key |
JFRWATCOFCPIBM-JPFHKJGASA-N
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| InChi Code |
InChI=1S/C19H32O2/c1-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19(20)21-2/h7-8,10-11,13-14H,3-6,9,12,15-18H2,1-2H3/b8-7-,11-10-,14-13-
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| Chemical Name |
methyl (6Z,9Z,12Z)-octadeca-6,9,12-trienoate
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: 100 mg/mL (341.93 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.55 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), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4193 mL | 17.0964 mL | 34.1927 mL | |
| 5 mM | 0.6839 mL | 3.4193 mL | 6.8385 mL | |
| 10 mM | 0.3419 mL | 1.7096 mL | 3.4193 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.
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