Physicochemical Properties
| Molecular Formula | C23H48NO7P |
| Molecular Weight | 481.603488922119 |
| CAS # | 97281-40-8 |
| Appearance | White to off-white solid powder |
| SMILES | P(=O)(O)(OCCN)OC[C@@H](COC(CCCCCCCCCCCCCCCCC)=O)O |
| Synonyms | Lyso-PE (egg); LPE (egg); 97281-40-8; 1-stearoyl-2-linoleoyl-GPE; L-alpha-Lysophosphatidylethanolamine (Egg, Chicken); GPE(18:0/18:2); GPE(18:0/18:2(9Z,12Z)); CHEBI:133599; YDTWOEYVDRKKCR-KNERPIHHSA-N; 1-18:0-2-18:2-phosphatidylethanolamine; L-α-lysophosphatidylethanolamine |
| 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) |
Biological Activity
| Targets | Naturally-occurring lysophospholipid |
| ln Vitro | We found that Grifola frondosa extracts induced the activation of mitogen-activated protein kinase (MAPK) in cultured PC12 cells, a line of rat pheochromocytoma cells. The active substance was isolated by a few chromatographic steps, including high-performance liquid chromatography, and was identified to be Lysophosphatidylethanolamine (LPE) from various structural analyses. LPE from G. frondosa (GLPE) was confirmed to induce the activation of MAPK of cultured PC12 cells and was found to suppress cell condensation and DNA ladder generation evoked by serum deprivation, suggesting that the GLPE had antiapoptotic effects. Moreover, GLPE caused morphological changes in and upregulation of neurofilament M expression of PC12 cells, demonstrating that the GLPE could induce neuronal differentiation of these cells. The activation of MAPK by GLPE was suppressed by AG1478, an antagonist of epidermal growth factor receptor (EGFR), and by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, an inhibitor of TrkA, or by pertussis toxin. These results demonstrate that GLPE induced the MAPK cascade [EGFR-MEK1/2-extracellular signal-regulated protein kinases (ERK1/2)] of PC12 cells, the activation of which induced neuronal differentiation and suppressed serum deprivation-induced apoptosis. This study has clarified for the first time the involvement of the MAPK signal cascade in LPE actions [1]. |
| Enzyme Assay |
Analysis of the chemical structure of the active substance for MAPK activation [1] HPLC-time of flight MS was performed using a micro-time of flight focus equipped with a Develosil ODS 60-5 column (4.5 × 250 mm, 5 μm particle size), and the active components were eluted by methanol. Infrared spectra were recorded with a Nicolet Magna-500 infrared spectrometer. 1H-NMR spectra were recorded with a JEOL AL-300 spectrometer, with chemical shifts being reported on the δ scale in ppm relative to Me4Si. 13C-NMR, 1H-1H total correlation spectroscopy, 1H-13C heteronuclear single quantum correlation, and 1H-13C 1H-detected heteronuclear multiple bond connectivity spectra were recorded with a Bruker AV-400 spectrometer. Detection of phosphorylated proteins [1] Proteins (20 μg) in each supernatant were mixed with SDS sample buffer and incubated for 5 min at 80°C. Protein samples were separated on SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride filters (Fluorotrans membrane W, 0.2 μm). Immunoblotting analysis was performed using monoclonal antibodies against p44/42 ERK, phospho p44/42 ERK, and neurofilament M (NF-M) as primary antibodies, followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG as the secondary antibody. The blots were developed by the enhanced chemiluminescence method. Treatment with specific inhibitors of signal transduction [1] Each inhibitor was added to serum-containing medium to result in a final concentration of 1 μM (K252a, PTX, and AG1478), 50 μM (U0126), or 200 μM (PD98059). Cells were preincubated with each inhibitor for 20 h (PTX) or 4 h (all others) and cultured in the serum-containing medium supplemented with each extract or reagent for the appropriate times. The cells were then collected and subjected to analysis of phosphorylation or expression of proteins as described above. Cytotoxicity of U0126 or PD98059 was measured by MTT assay. Analysis of apoptotic DNA fragmentation [1] PC12 cells maintained for 2 days on six-well plates (2 × 106 cells/well) at 37°C in an atmosphere containing 5% CO2 were washed with PBS and incubated in serum-free medium for 0, 1, 2, or 8 days. Then, the cells were collected and centrifuged at 500 g for 5 min. DNA was extracted from the cells using a QIAamp DNA mini kit. Fragmentation of DNA was confirmed on 3% agarose gels after electrophoresis and staining with ethidium bromide for visualization under ultraviolet transillumination. |
| Cell Assay |
Cell culture and bioassay [1] PC12 cells were cultured as described previously. In brief, the cells were maintained in DMEM supplemented with 10% heat-inactivated horse serum and 5% heat-inactivated FBS or in serum-free medium (DMEM supplemented with 1% BSA), unless stated otherwise. All G. frondosa samples, such as the chloroform-methanol extract, fractions obtained by HPLC, and reagents, were prepared in serum-free DMEM and sonicated until fully emulsified. PC12 cells were seeded at a cell density of 2 × 106 cells/well into collagen-coated six-well plates containing medium with serum and precultured for 2 days at 37°C in an atmosphere of 95% air and 5% CO2. The cells were then washed with PBS and incubated with the above-mentioned culture medium containing a given sample from G. frondosa or various agents for 10 min at 37°C. Then, the culture plates were placed on ice, and each well was washed with 3 ml of 2 mM TBS containing 0.33 M NaF and 6.25 M Na3VO4 and subsequently lysed with 150 μl of 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (w/v), 1% sodium deoxycholate (w/v), 0.1% SDS (w/v), 50 mM NaF, 0.1% aprotinin (w/v), 0.1% leupeptin (w/v), 1 mM Na3VO4, and 1 mM PMSF. Cell lysates were collected using a cell scraper, transferred to 1.5 ml microcentrifuge tubes, and centrifuged at 15,000 g for 30 min at 4°C. The supernatant was collected and transferred to another tube, and the overall protein concentration was determined using the BCA Protein Assay Reagent Kit with BSA as the standard. NF-M expression in PC12 cells [1] PC12 cells plated on poly-d-lysine-coated cover glasses (13 × 13 mm) were precultured in serum-containing medium for 24 h, washed with PBS, and incubated with the same medium containing mushroom extract or agent for the appropriate period. Cells were fixed with paraformaldehyde solution (4%, w/v) and incubated for 20 min in PBS containing Triton X-100 (0.3%, v/v). Nonspecific binding was blocked with Block Ace solution (2%, v/v). Cells were treated with anti-NF-M monoclonal antibody and then reacted with Alexa Fluor 488-conjugated anti-mouse IgG antibody. The stained samples were mounted on slide glasses by use of Tissue-Tek and observed with a confocal laser scanning microscope. |
| References |
[1]. Lysophosphatidylethanolamine in Grifola frondosa as a neurotrophic activator via activation of MAPK. J. Lipid Res. 47(7), 1434-1443 (2006). |
| Additional Infomation |
A well-known neurotrophic factor, NGF, acts on cultured PC12 cells and induces many processes, including neurite outgrowth for their neuronal differentiation into sympathetic neuron-like cells. These NGF actions require both phosphorylation of the NGF receptor, TrkA, expressed on the cell surface, as a trigger and subsequent activation of the MAPK cascade. Based on these facts, extracts of G. frondosa were tested and found to induce ERK1/2 phosphorylation of PC12 cells as efficiently as NGF. An active component was isolated from the extracts and identified as Lysophosphatidylethanolamine/LPE. To date, the pharmacological effects of LPE are unclear; although phosphatidylethanolamine is a common cell membrane component, and LPE is easily derived from phosphatidylethanolamine by deacylation by phospholipase A2.
The LPE/Lysophosphatidylethanolamine concentration necessary to induce the activation of ERK1/2 of PC12 cells was almost 200 μM in our assay system. On the other hand, Howe and Marshall demonstrated that the concentration of lysophosphatidic acid (LPA), an analog of LPE, necessary to induce the activation of ERK1/2 of PC12 cells was <200 nM. In this study, the effect of 1-oleoyl-LPE on ERK1/2 activation was greater than that of an equal concentration of 1-oleoyl-LPA (Fig. 3). As phospholipids such as LPA and LPE have polar and nonpolar groups in their structures, they can be precipitated or emulsified by addition to aqueous medium. In recent studies, various solvents or emulsifiers such as DMSO and methanol were used to dissolve phospholipids into aqueous solution, which may cause serious problems for estimation of the activities of these phospholipids. Therefore, in this study, LPE was added to the medium by supersonic dispersion, using neither solvents nor emulsifier. We confirmed by the following methods that LPE was the single active component contained in GLPE. First, NMR analysis of GLPE gave no signals other than those of LPE. Two kinds of TLC conditions using silica-gel plates or ODS plates developed with chloroform-methanol-water (120:70:2) or chloroform-acetic acid-water (46:25:2.5) were used to check for impurities with the same relative mobility value as LPE. From the results mentioned above, even if a compound other than LPE was contained in the GLPE sample, it would seem to be less important. It is necessary to compare the activity of the phospholipids with consideration of their solubility in the near future. Phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine are major phospholipids of the plasma membrane of mammalian cells; phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol are major phospholipids in the plant kingdom. Pharmacological studies on phosphatidylethanolamine demonstrated suppression of MAPK activation, Raf-1 kinase inhibition, induction of human keratinocyte differentiation, and activation of heterotrimeric G protein. LPE/Lysophosphatidylethanolamine is a phosphatidylethanolamine molecule lacking a fatty acid. Although the induction of transformin growth factor-β release from the extracellular matrix of chondrocytes by LPE was reported recently, the function of LPE has not yet been fully clarified. To our knowledge, this study is the first to show the antiapoptotic activity and neuronal differentiation-stimulating property of LPE. Among the lysophospholipids, platelet-agglutinating factor, lysophosphatidylcholine (LPC), and LPA have been investigated for their pharmacological activities. It is noteworthy that LPA activates MAPK via GPCR or induces neurite retraction in PC12 cells. This study has shown that LPE, the functional group of which is different from that of LPA, activated MAPK of PC12 cells via the EGFR and facilitated neurite outgrowth. Of interest, LPE induced the activation of the MEK-ERK pathway via the EGFR, not through the NGF receptor (Fig. 5). However, the effects of LPE on PC12 cells were neuroprotective and/or neurotrophic, like those of NGF and different from those of EGF (Figs. 7, 8). G2A and S1P1–3 have been identified as receptors of LPC and sphingosine-1-phosphate (S1P), respectively. G2A is activated not only by LPC but also by LPA. Neurite outgrowth is enhanced by the activation of S1P1 as part of a chain reaction that is triggered by the activation of TrkA by NGF and suppressed by the activation of S1P2 that accompanies cell differentiation. As the mechanism of MAPK phosphorylation induced by LPE, direct stimulation by LPE through its unknown receptor(s) or indirect stimulation by LPA or LPC through its receptor G2A generated from hydrolysis or base-exchange reaction of LPE may be possible. A relationship between the degree of MAPK phosphorylation and the concentration of LPE, S1P, or LPS was preliminarily examined. The minimal concentrations of LPE necessary for the induction of MAPK phosphorylation were equal or rather lower than those of S1P or LPC (data not shown). Therefore, it is difficult to assume that LPE is converted into S1P, LPC, or LPA and induces the phosphorylation of MAPK. Although the possibility that LPE induces the phosphorylation of MAPK through EGFR was suggested by this study, it could not be excluded that LPE directly stimulates G2A and/or S1P1, the receptors of S1P, LPC, or LPA. Kinetic analysis of LPE binding to these receptors is an urgent matter to clarify the mechanisms of LPE signal transduction. As shown in Fig. 2, the major fatty acids bound to GLPE were oleic and linoleic acids. The contribution of a fatty acid base to pharmacological activity might be small, because the activities of synthetic 1-myristoyl-LPE, 1-palmitoyl-LPE, and 1-oleoyl-LPE were almost equal to those of GLPE. Moreover, it is clear that LPE itself induces the activation of ERK, because the GLPE activity was equal to the synthetic LPE activity. In this study, the effects of LPE on the MAPK cascade have been partly clarified. The MAPK signal cascade is known to regulate cell growth and differentiation. For instance, tyrosine kinase receptors on cell membranes are activated by growth factors, and their signals are transduced to Raf, MEK1/2, ERK1/2, Elk1, and/or p90RSK. Because activation of ERK1/2 by GLPE was suppressed by a MEK1/2 inhibitor, we may assume that GLPE activated ERK1/2 via MEK1/2. We also tested the effect of a Raf-1 kinase inhibitor (5-lodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]2-indolinone) on the activation of ERK1/2 by GLPE. After PC12 cells were treated with Raf-1 kinase inhibitor, NGF, an activator of Raf-1/MEK/ERK, was added to the medium. ERK1/2 of PC12 cells was phosphorylated by the addition of NGF as well as in the Raf-1 kinase inhibitor-free medium. Thus, it has been confirmed that the Raf-1 kinase inhibitor mentioned above does not inhibit the activity of Raf in PC12 cells. To clarify the effect of GLPE on Raf, it will be necessary to use another Raf-1 kinase inhibitor that has better cell membrane permeability or to try another method, such as Raf-1 kinase assay. The results shown in Fig. 5 suggest that EGFR, not the NGF receptor or pertussis toxin-sensitive GPCR, was necessary for the activation of ERK1/2 by GLPE. It is known from a recent study that LPA, an analog of LPE/Lysophosphatidylethanolamine, activates both EGFRs and pertussis toxin-sensitive GPCRs of PC12 cells, resulting in the induction of activation of the MAPK pathway. On the other hand, because PTX was irrelevant to the activation of ERK1/2 by GLPE, LPE would appear to differ from LPA in terms of pathway, as it induces the activation of ERK1/2 chiefly via the EGFR. It is known that U0126 binds to MEK1/2 of all types and inhibits the phosphorylation of ERK1/2, whereas PD98059 only binds to MEK of inactive type and inhibits the phosphorylation of MEK by Raf. The possibility that the phosphorylation of MEK by Raf is unnecessary to the phosphorylation of ERK by GLPE is likely, because the phosphorylation of ERK1/2 induced by GLPE was inhibited weakly by PD98059 but strongly by U0126. Elucidation of the signal pathway from the EGFR to MEK1/2 is an important issue to be resolved in the future. From earlier experimental results showing a relationship between the fatty acid species bound to LPA and biological activity, 1-oleoyl-LPA had the strongest activity. However, the fatty acid species associated with LPE in our study did not appear to be related to the activity of LPE. We assume that the difference of the interactive functional group between LPE and LPA was responsible for the difference in the effects of the bound fatty acid on activity. In conclusion, we found that GLPE induced the activation of the MAPK cascade via EGFR-MEK1/2-ERK1/2 in PC12 cells, which resulted in neuronal differentiation and suppression of serum deprivation-induced apoptosis. Our results clarified for the first time the involvement of the MAPK signal cascade in LPE actions, such as the induction of neuronal differentiation and the suppression of apoptosis. Thus, the clinical application of LPE, a low-molecular-weight compound having neuroprotective and neurotrophic activities, is hopeful. The potential use of LPE to prevent and treat neurological disorders such as Alzheimer's disease and the neuronal apoptosis that occurs after brain ischemia should be evaluated in the future.[1] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0764 mL | 10.3821 mL | 20.7641 mL | |
| 5 mM | 0.4153 mL | 2.0764 mL | 4.1528 mL | |
| 10 mM | 0.2076 mL | 1.0382 mL | 2.0764 mL |