Pinocembrin and its linolenoyl ester derivative induce wound healing activity in HaCaT cell line potentially involving a GPR120/FFA4 mediated pathway.

Wound healing represents an urgent need from the clinical point of view. Several diseases result in wound conditions which are difficult to treat, such as in the case of diabetic foot ulcer. Starting from there, the medicinal research has focused on various targets over the years, including GPCRs as new wound healing drug targets. In line with this, GPR120, known to be an attractive target in type 2 diabetes drug discovery, was studied to finalize the development of new wound healing agents. Pinocembrin (HW0) was evaluated as a suitable compound for interacting with GPR120, and was hybridized with fatty acids, which are known endogenous GPR120 ligands, to enhance the wound healing potential and GPR120 interactions. HW0 and its 7-linolenoyl derivative (HW3) were found to be innovative wound healing agents. Immunofluorescence and functional assays suggested that their activity was mediated by GPR120, and docking simulations showed that the compounds could share the same pocket occupied by the known GPR120 agonist, TUG-891.

Folate-Targeted Polyacrylamide/Punicic Acid Nanomicelles for Flutamide Delivery in Prostate Cancer: Characterization, In Vitro Biological Evaluation, and its DFT Study.

BACKGROUND: Targeted nanocarriers can be used for reducing the unwanted side effects of drugs in non-target organs. Punicic acid, the polyunsaturated fatty acid of pomegranate seed oil, has been shown to possess anti-cancer effects on prostate cancer and the study also covers recent patents related to prostate cancer. The objective of the current study was to synthesize a co-polymeric micelle for delivery of Flutamide (FL) in prostate cancer using Polyacrylamide (PAM) and Punicic Acid (PA). METHODS: The co-polymer of PAM and PA was synthesized and conjugated to folic acid. The successful conjugation was studied computationally by the density functional theory method and was confirmed by the FT- IR and 1HNMR. The folate-PAMPA micelles produced by the film casting method were characterized physically. FL was loaded in the nanomicelles and its release test was done at different pH. The Critical Micelle Concentration (CMC) was measured by pyrene as a fluorescent probe. Their cellular uptake and cytotoxicity were evaluated on PC3 prostate cancer cells. The molecular geometry and vibrational frequencies of two different possibilities for conjugation were calculated using the B3LYP/6-31G basis set. RESULTS: The CMC of the micelles and their particle size were 79.05 µg/ml and 88 nm, respectively. The resulting nanocarriers of FL showed significantly more cytotoxic effects than the free drug at a concentration of 25 µM. The calculated results showed that the optimized geometries could well reproduce the structural parameters, and the theoretical vibrational frequencies were in good agreement with the experimental values. CONCLUSION: Folate-PAMPA nanomicelles may be promising for the enhancement of FL cytotoxicity and seem to potentiate the effect of chemotherapeutic agents used in prostate cancer treatment.

Punicic Acid Inhibits Glioblastoma Migration and Proliferation via the PI3K/AKT1/mTOR Signaling Pathway.

BACKGROUND: Punicic Acid (PA) is a polyunsaturated fatty acid that accounts for approximately 70%- 80% of Pomegranate Seed Oil (PSO). PA possesses strong antioxidant, anti-inflammatory, anti-atherogenic effects, and anti-tumorigenic properties. Pomegranate extracts have been shown to have anticancer activity in many studies. However, there is no evidence for the effect of PSO on T98 glioblastoma cells. Therefore, the present study was the first to investigate the mechanisms induced by PA on T98 cells, which is one of the major compounds extracted from PSO. METHODS: The effects of PA on cell viability; oxidative stress; and migration, proliferation, and apoptosis at the IC50 dose were studied. RESULTS: The proliferation and migration were inhibited in the treated group compared to the non-treated group by 9.85µl/ml PA. The difference was statistically significant (***p<0.001). Furthermore, PA-induced apoptosis in the T98 glioblastoma cells compared to non-treated group and the difference was statistically significant (***p<0.001). Apoptosis was determined via immunocytochemistry staining of caspase-3, caspase-9 and TUNEL methods. Apoptosis was checked by flow cytometry (using caspase 3 methods) and Scanning Electron Microscopy Analysis. We also investigated the potential signaling pathway underlying this apoptotic effect. The immunocytochemical stainings of PI3K/ Akt-1/ mTOR-1 demonstrated that Akt-1 staining was increased with PA treatment similar to mTOR-1 and PI3K staining (***p<0.001). These increases were statistically significant compared to the non-treated group. CONCLUSION: PA exhibited exceptional abilities as an anticancer agent against GBM cells. The use of punicic acid in combination with other drugs used in the treatment of glioblastoma may increase the efficacy of the treatment. This study provided a basis for future investigation of its use in preclinical and clinical studies.

Linolenic acid-modified methoxy poly (ethylene glycol)-oligochitosan conjugate micelles for encapsulation of amphotericin B.

Introduction of linolenic acid (LNA) and methoxy poly (ethylene glycol) (MPEG) to the backbone of oligochitosan (CS) afforded LNA-modified MPEG-CS conjugate (MPEG-CS-LNA). Amphotericin B-loaded MPEG-CS-LNA micelles (AmB-M) were prepared via dialysis method with 82.27 ± 1.96% of drug encapsulation efficiency and 10.52 ± 0.22% of drug loading capacity. The AmB-M enhanced AmB's water-solubility to 1.64 mg/mL, being 1640-folds higher than native AmB. The AmB-M obviously reduced hemolytic effect and renal toxicity of AmB when compared to marketed AmB injection (AmB-I). Its antifungal activity against Candida albicans was equivalent to AmB-I although AmB's release from AmB-M was significantly retarded. According to fluorescence microscopy test, the unchanged activity should be attributed to enhanced fungal cellular uptake of AmB-M caused by combined inducement of LNA and CS. The pharmacokinetic studies demonstrated that AmB-M also improved the pharmacokinetic parameters of AmB with AmB-I as control. Conclusively, developed LNA-modified MPEG-CS micellar system could be a viable alternative to the current toxic commercial AmB-I as a highly efficacious drug delivery system.

Synthesis of 3-oxalinolenic acid and beta-oxidation-resistant 3-oxa-oxylipins.

3-Oxalinolenic acid (3-oxa-9(Z),12(Z),15(Z)-octadecatrienoic acid or (6(Z),9(Z),12(Z)-pentadecatrienyloxy)acetic acid) was synthesized from 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid by a sequence involving the C15 aldehyde 3(Z),6(Z),9(Z),12(Z)-pentadecatetraenal as a key intermediate. Conversion of the aldehyde by isomerization and two steps of reduction afforded 6(Z),9(Z),12(Z)-pentadecatrienol, which was coupled to bromoacetate to afford after purification by HPLC >99%-pure 3-oxalinolenic acid in 10-15% overall yield. 3-Oxalinolenic acid was efficiently oxygenated by soybean lipoxygenase-1 into 3-oxa-13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid, and this hydroperoxide could be further converted chemically into 3-oxa-13(S)-hydroxy-9(Z),11 (E),15(Z)-octadecatrienoic acid and 3-oxa-13-oxo-9(Z),11 (E),15(Z)-octadecatrienoic acid. The 3-oxa-hydroperoxide also served as the substrate for the plant enzymes allene oxide synthase, divinyl ether synthase, and hydroperoxide lyase to produce 3-oxa-12-oxo-10,15(Z)-phytodienoic acid and other 3-oxa-oxylipins that were characterized by MS. 3-Oxalinolenic acid was not oxygenated by 9-lipoxygenase from tomato but was converted at a slow rate into 3-oxa-9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid by recombinant maize 9-lipoxygenase. Recombinant alpha-dioxygenase-1 from Arabidopsis thaliana catalyzed the conversion of 3-oxalinolenic acid into a 2-hydroperoxide, which underwent spontaneous degradation into a mixture of 6,9,12-pentadecatrienol and 6,9,12-pentadecatrienyl formate. A novel alpha-dioxygenase from the moss Physcomitrella patens was cloned and expressed and was found to display the same activity with 3-oxalinolenic acid as Arabidopsis thaliana alpha-dioxygenase-1. Lipoxygenase-generated 3-oxa-oxylipins are resistant toward beta-oxidation and have the potential for displaying enhanced biological activity in situations where activity is limited by metabolic degradation.

Possible anti-Parkinson properties of N-(alpha-linolenoyl) tyrosine: a new molecule.

Tyrosine is unable to cross the blood-brain barrier and is therefore unable to improve the status of brain dopamine (DA) and to provide relief for patients with Parkinson's disease (PD) or other DA-insufficient disorders. We report the creation of an amide bond molecule [N-(alpha-linolenoyl)tyrosine (NLT)] that combines tyrosine with a fatty acid mixture. NLT significantly improves the rotational behavior of rats [following unilateral striatal lesions (as a model for Parkinson's)] and overcomes the exaggerated eye-blinking induced by a potent DA-depleting agent (as a model for essential blepharospasm). These results are supported by the finding that NLT's mode of action, in striatum, is the same as the mode of action of D-amphetamine. They both induce an increase in the DA level, DA turnover and release.

Analysis of oxylipins by high-performance liquid chromatography with evaporative light-scattering detection and particle beam-mass spectrometry.

The metabolism of 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phaseolus radiatus L.). Hydroperoxide-metabolizing activity was mainly due to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis and cyclization of the allene oxide synthase product 12,13-epoxy-9Z,11,15Z-octadecatrienoic acid and from peroxygenase catalysis were identified by high-performance liquid chromatography (HPLC) particle beam-mass spectrometry (PB-MS) and quantified by normal-phase HPLC with an evaporative light-scattering detector (ELSD). An advantage of this methodology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owing to a comparable sample inlet system, the ELSD served an important analytical pilot function for the PB-MS: Qualitatively identical chromatographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12-oxo-phytodienoate, methyl 11-hydroxy-12-oxo-9Z,15Z-octadecadienoate, methyl 12-oxo-13-hydroxy-9Z,15Z-octadecadienoate, methyl 9-hydroxy-12-oxo-10E,15Z-octadecadienoate, methyl 13-hydroxy-9Z,11E,15Z-octadecatrienoate, methyl 15,16-epoxy-13-hydroxy-9Z,11E-octadecadienoate, and methyl 13-hydroperoxy-9Z,11E,15Z-octadecatrienoate on a Lichrospher DIOL column within 33 min. Compared with a diode array detector, the ELSD proved to be more sensitive, in the case of methyl 12-oxo-13-hydroxy-9Z, 15Z-octadecadienoate by a factor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase product 2E-hexenal was 49%, whereas the sum of oxylipins reached about 15%.

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase.

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase-catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40 °C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20 °C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase-catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to -30 °C.

Synthesis of mayolene-16 and mayolene-18: larval defensive lipids from the European cabbage butterfly.

A tandem Wittig approach has been employed for the synthesis of both (11S,9Z,12Z,15Z)- and (11R,9Z,12Z,15Z)-hydroxyoctadeca-9,12,15-trienoic acid (11-hydroxylinolenic acid, 11-HLA) from (R)-glyceraldehyde acetonide. From (11R)-HLA we have prepared the corresponding palmitic acid and stearic acid esters, mayolene-16 (1) and mayolene-18 (2), insect defensive compounds recently identified from Pieris rapae larvae. In addition, we describe the synthesis of three macrocyclic oligomers (24-26) derived from (11R)-HLA.

Analysis of 1,2-diols of linoleic, alpha-linolenic and arachidonic acid by gas chromatography--mass spectrometry using cyclic alkyl boronic esters.

Arachidonic acid is metabolized by hepatic and renal cortical microsomes in the presence of NADPH to vicinal dihydroxyeicosatrienoic acids as some of the major metabolites. Other polyunsaturated, long-chain fatty acids might be metabolized to vicinal dihydroxy acids (1,2-diols) in the same way. To facilitate identification of 1,2-diols in biological samples, a series of unsaturated 1,2-diols were synthesized from linoleic, alpha-linolenic and arachidonic acid and the electron-ionization mass spectra of cyclic methane- and n-butaneboronic ester derivatives and of trimethylsilyl (TMS) ether derivatives were compared. The TMS ether derivatives gave rise to weak molecular ions but prominent informative fragmentation ions were formed by alpha-cleavage as well as cleavage between the carbons with the TMS ethers. The TMS ether derivative of methyl 15,16-dihydroxy-9,12-octadecadienoate had a considerably larger carbon value than the other C18 diols, while the cyclic boronates were poorly separated on gas chromatography. The methane- and n-butaneboronic acid derivatives showed strong molecular ions and a characteristic but not very informative fragmentation, although the position of the hydroxyls could be deduced from one or two fragments formed by alpha-cleavage. Linoleic and alpha-linolenic acid are metabolized in the rabbit to many polar products by hepatic and renal cortical microsomes and NADPH. 12,13-Dihydroxy-9-octadecenoic acid and other metabolites of linoleic acid were identified by gas chromatography--mass spectrometry.