Drug-Free Liposomes Containing Mannosylated Ligand for Liver-Targeting: Synthetic Optimization, Liposomal Preparation, and Bioactivity Evaluation

This research was performed to optimize the enzymatic synthesis of mannosylated ligand with which to prepare mannosy-lated liposomes and investigate their bioactivity. Based on single-factor studies, lipase dose, substrate molar ratio (diester lauric diacid-cholesterol to mannose) and temperature were identified as significant parameters, and optimal reaction conditions were determined through response surface methodology (RSM) with central composite design. The optimum operating parameters, 61.23 mg of lipase, a substrate molar ratio of 5.36, and 56.64 °C temperature offered a predicted yield (71.11%) which was consistent with the actual yield (69.08%). Drug-free mannosylated liposomes were prepared film-dispersion. The characterizations of these liposomes showed that mannosylated liposomes were well-dispersible spherical particles with an average particle size of 142.3 nm, the polydispersity index of 0.16, and a zeta potential of −19.8 mV. Pyrogen examination, hemolytic studies and cytotoxicity assays revealed no substantial safety concern for drug-free mannosylated liposomes. Cellular uptake efficiency of mannosylated liposomes by HepG2 cells was significantly higher than that of unmodified liposomes, demonstrating that mannosylated ligands have a positive effect on liver targeting. Overall, mannosylated liposomes could be active drug delivery system for combatting the therapy of hepatic diseases.

Interesterification of Egg-Yolk Phosphatidylcholine with p-Methoxycinnamic Acid Catalyzed by Immobilized Lipase B from Candida Antarctica

The p-methoxycinnamic acid (p-MCA) is one of the most popular phenylpropanoids, the beneficial impact of which on the human health is well documented in the literature. This compound has shown many valuable activities including anticancer, antidiabetic, and neuro- and hepatoprotective. However, its practical application is limited by its low bioavailability resulting from rapid metabolism in the human body. The latest strategy, aimed at overcoming these limitations, is based on the production of more stability in systemic circulation bioconjugates with phospholipids. Therefore, the aim of this research was to develop the biotechnological method for the synthesis of phospholipid derivatives of p-methoxycinnamic acid, which can play a role of new nutraceuticals. We developed and optimized enzymatic interesterification of phosphatidylcholine (PC) with ethyl p-methoxycinnamate (Ep-MCA). Novozym 435 and a binary solvent system of toluene/chloroform 9:1 (v/v) were found to be the effective biocatalyst and reaction medium for the synthesis of structured p-MCA phospholipids, respectively. The effects of the other reaction parameters, such as substrate molar ratio, enzyme dosage, and reaction time, on the degree of incorporation of p-MCA into PC were evaluated by use of an experimental factorial design method. The results showed that substrate molar ratio and biocatalyst load have significant effects on the synthesis of p-methoxycinnamoylated phospholipids. The optimum conditions were: Reaction time of three days, 30% (w/w) of Novozym 435, and 1/10 substrate molar ratio PC/Ep-MCA. Under these parameters, p-methoxycinnamoylated lysophosphatidylcholine (p-MCA-LPC) and p-methoxycinnamoylated phosphatidylcholine (p-MCA-PC) were obtained in isolated yields of 32% and 3% (w/w), respectively.

Development and Optimization of Lipase-Catalyzed Synthesis of Phospholipids Containing 3,4-Dimethoxycinnamic Acid by Response Surface Methodology

The interesterification reaction of egg-yolk phosphatidylcholine (PC) with ethyl ester of 3,4-dimethoxycinnamic acid (E3,4DMCA) catalyzed by Novozym 435 in hexane as a reaction medium was shown to be an effective method for the synthesis of corresponding structured O-methylated phenophospholipids. The effects of substrate molar ratios, time of the reaction and enzyme load on the process of incorporation of 3,4DMCA into PC were evaluated by using the experimental factorial design of three factors and three levels. The results showed that a substrate molar ratio is a crucial variable for the maximization of the synthesis of 3,4-dimethoxycinnamoylated phospholipids. Under optimized parameters of 1/10 substrate molar ratio PC/E3,4DMCA, enzyme load 30% (w/w), hexane as a medium and incubation time of 3 days, the incorporation of aromatic acid into phospholipid fraction reached 21 mol%. The modified phosphatidylcholine (3,4DMCA-PC) and modified lysophosphatidylcholine (3,4DMCA-LPC) were obtained in isolated yields of 3.5% and 27.5% (w/w), respectively. The developed method of phosphatidylcholine interesterification is the first described in the literature dealing with 3,4DMCA and allows us to obtain new O-methylated phenophospholipids with potential applications as food additives or nutraceuticals with pro-health activity.

Optimization of Enzymatic Synthesis of Betulinic Acid Amide in Organic Solvent by Response Surface Methodology (RSM)

Optimization of the lipase catalyzed enzymatic synthesis of betulinic acid amide in the presence of immobilized lipase, Novozym 435 from Candida antartica as a biocatalyst was studied. Response surface methodology (RSM) and 5-level-4-factor central-composite rotatable design (CCRD) were employed to evaluate the effects of the synthesis parameters, such as reaction time (20–36 h), reaction temperature (37–45 °C), substrate molar ratio of betulinic acid to butylamine (1:1–1:3), and enzyme amounts (80–120 mg) on the percentage yield of betulinic acid amide by direct amidation reaction. The optimum conditions for synthesis were: reaction time of 28 h 33 min, reaction temperature of 42.92 °C, substrate molar ratio of 1:2.21, and enzyme amount of 97.77 mg. The percentage yield of actual experimental values obtained 65.09% which compared well with the maximum predicted value of 67.23%. The obtained amide was characterized by GC, GCMS and 13C NMR. Betulinic acid amide (BAA) showed a better cytotoxicity compared to betulinic acid as the concentration inhibited 50% of the cell growth (IC50) against MDA-MB-231 cell line (IC50 < 30 µg/mL).

Effect of parameters on butyl butyrate synthesis using novel Aspergillus niger lipase as biocatalyst

A novel “green” Aspergillus niger lipase, obtained from the fermentation of pumpkin seeds, was used in a free form and encapsulated in sol-gel matri x in butyl butyrate (pineapple flavor) synthesis. Esterification reactions were performed with varying substrate molar ratio (butanol: butyric acid) ranging between 1:1 and 5:1; temperature between 30 and 60°C and biocatalyst mass between 0 and 1g, respectively, according to experimental design 23 with 6 axial and 3 central points. Maximum butyl butyrate production was obtained when substrate molar ratio (butanol:butyric acid) 3:1, temperature at 60°C and 0.5 g free or encapsulated lipase as biocatalyst, were used. Temperature was the most significant parameter for production with the two biocatalysts, indicating that higher rates mean greater compound synthesis. Response surface plots showed that higher butyl butyrate production may be obtained with higher temperature and molar ratio rates (butanol:butyric acid) and with lower rates of biocatalyst mass in reactions catalyzed by free or encapsulated lipase. Aspergillus niger lipase obtained from agro-industrial waste could be employed as biocatalyst in esterification reactions in the production of natural aroma as butyl butyrate.

Optimization of A Two-Step Method to Synthesize Azelaic Acid from Oleic Acid

The present study deals with the oxidative cleavage of oleic acid (OA) using hydrogen peroxide and tungstic acid as a catalyst to produce azelaic acid. A two-step method has been expanded for the optimization of a new route of azelaic acid synthesis with the addition of sodium hypochlorite as the co-oxidation. The Central Composite Design (CCD) and Response Surface Methodology (RSM) were performed to optimize the production of azelaic acid. The interaction effect among catalyst concentration, substrate molar ratio and temperature were done for optimization the conversion of oleic acid. Maximum oleic acid conversion of 99.11% was reached at substrate molar ratio of 4/1 (H2O2/OA), a catalyst concentration of 1.5% (w/wOA) and temperature of 70oC. The GC analysis shows that the yield of azelaic acid is 44.54% and pelargonic acid is 34.12%. These results indicate that the proposed process show a good strategy for the synthesis of azelaic acid from oxidative cleavage of oleic acid.

SINTESIS STEARAMIDA DARI ASAM STEARAT DAN UREA MENGGUNAKAN PELARUT CAMPURAN : PENGARUH TEMPERATUR DAN WAKTU REAKSI

Surfactant is a molecule which has hydrophilic group and a lipophilic group which can unify a mixture consisting of water and oil. Stearamide is one kind of surfactant non-ionic which has used on any aplication. This study aims to know about optimum of temperature and time on synthesis of stearamide from stearic acid and urea with zirconium (IV) chloride in mixed solvent. This study will be observed the effect of temperature and reaction time on the synthesis of stearamide surfactant from stearic acid and urea with zirconium (IV) chloride as catalyst in mixed solvent. This research was conducted at temperature of 45°C, 55°C, 65°C, 75°C, 85°C and 95°C, reaction time of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr and 8 hr, substrate molar ratio at 1:6 (w/w), solvent ratio at 2:1 (v/v), weight of catalyst 3% (%w), and stirring speed at 250 rpm. Based on this research, the optimum condition to synthesis stearamide surfactant was obtained at 85 oC and reaction time at 3 hours.

Use of Echium oil fatty acids and tricaprylin as substrates of enzymatic interesterification for the production of structured lipids

Structured lipids (SLs) were produced from the free fatty acids of Echium oil and tricaprylin by enzymatic acidolysis reactions. Lipozyme® RM IM, immobilized sn-1,3 specific lipase was used in the enzymatic reactions. In order to optimize the incorporation of stearidonic acid (SDA), three factors were chosen [Reaction temperature (50-60 ºC), reaction time (6-12 hour) and substrate molar ratio (3-6 mol/mol (total free fatty acids/tricaprylin)] for the application of response surface methodology (RSM) using a central composite circumscribed design (CCC) with five levels. The optimum temperature, time and substrate molar ratio obtained from the models were 60 ºC; 6 h, 6 mol/mol, respectively. Furthermore, SLs with 6.2% SDA content at sn-2 position were produced by scaling up the process. SL was obtained with nearly 78-79% of long-chain fatty acids at the sn-2 position. According to the melting profile analysis, the melting peaks of tricaprylin and Echium oil were sharper and narrower while the SL had more broadened peaks.

Identification and Optimisation of Lipase-Catalysed Synthesis of Betulinic Acid Amide in a Solvent System

Betulinic acid amide was synthesized from the enzymatic reaction of betulinic acid and butylamine catalysed by Novozym 435. The effects of different reaction parameters, such as effect of reaction time, reaction temperature, amount of enzyme, and substrate molar ratio (betulinic acid : butylamine), were studied and conventionally optimised. Based on this study, the enzymatic synthesis of betulinic acid amide was found to be 64.6% at the optimum conditions of 24 h, 40°C, 100 mg enzyme, and 1 : 1 substrate molar ratio in 9 : 1 mixture of chloroform and hexane as solvent. The identification of final product was carried out using TLC, melting point, and FTIR and NMR showed the presence of betulinic acid amide.

Improvement on the Catalytic Performance Using Dual Lipases System in the Synthesis of Ferulate Esters

A novel approach of dual lipases system was successfully carried out in improving the synthesis of ferulate esters between ethyl ferulate and olive oil. Combination of Novozym 435 and Lipozyme RMIM were used as biocatalyst to improve the reaction performance. Different reaction parameters (ratio of lipases, reaction time, lipase dosage, substrate molar ratio and reaction temperature) were analyzed systematically. A high conversion of ferulate esters (85%) was obtained after 12 hrs of reaction time at optimal conditions of 1:9 w/w (Novozym 435/Lipozyme RMIM), 80 mg of lipase and 1:4 ethyl ferulate:olive oil at 60 oC.