China has richly and inexpensive fat and oils from animal and plants, but these resources could not get effectively utilization. In order to make the best of these resources, lipase-catalyzed acidolysis of lard with caprylic acid to produce functional lipid in solvent free system was investigated. Of the five lipases that were tested in the initial screening, immobilized lipase TL IM fromca T. languginosa resulted in the highest incorporation of capry lic acid into lard. This enzyme was further studied for the effect of enzyme load, substrate ratib, reaction time, reaction temperature and added water content on the incorporation of caprylic acid into lard. HPLC analyzed the products from the acidolysis reaction. The highest incorporation was attained at 20% enzyme load. Time course studied suggest that the incorporation of caprylic acid into lard was increased up to 38.77 mol% after 24h. Desirable mole ratio of substrates was 1:2 (lard: caprylic acid), caprylic acid incorporation up to 30.95 mol%. In the range of 45 - 60 degrees C , temperature had no significant effect on enzyme activity and caprylic acid incorporation changed little. When temperature was above 60 degrees C, incorporation of caprylic acid into lard was decreased. The highest incorporation of caprylic acid into lard 35.76 mol% was attained when added water content was 2.5%.
Caprylates ; chemistry ; Catalysis ; Chromatography, High Pressure Liquid ; Dietary Fats ; metabolism ; Enzymes, Immobilized ; metabolism ; Lipase ; metabolism ; Lipids ; chemical synthesis ; Solvents

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.
Cricetinae ; Animals ; Antibodies, Monoclonal/metabolism* ; Caprylates/chemistry* ; Cell Culture Techniques ; Chromatography, Affinity ; CHO Cells ; Cricetulus ; Chemical Precipitation

The skin of Bufo bufo gargarizans, originated from Bufo bufo gargarizans Cantor (Bufonidae), is widely used in traditional Chinese medicine for the treatment of hepatoma, lung cancer and etc. The preparation of the aqueous components has significant therapeutic effect against the digestive tract cancer. The water-soluble chemical constituents in the skin of Bufo bufo gargarizans were then investigated to make clear the active compounds. Six compounds were isolated and purified by recrystallization and column chromatography on silica gel and ODS, their structures were elucidated as 4-amido-3-hydroxymethyl-cyclooctylamidezotetra-alpha-furanone (I), bufogargarizanine C (II), bufothionine (III), dehydrobufotenine hydrobromide (IV), suberic acid (V) and succinic acid (VI) on the basis of physicochemical properties and spectral data (UV, IR, 1H NMR, 13C NMR and MS). Of the above compounds, compounds I and II are new compounds and named bufogargarizanine B and C, respectively.
Animals ; Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; isolation & purification ; Bufo bufo ; Caprylates ; chemistry ; isolation & purification ; Dicarboxylic Acids ; chemistry ; isolation & purification ; Medicine, Chinese Traditional ; Molecular Conformation ; Molecular Structure ; Skin ; chemistry

A new dynamic in vitro intestinal absorption model for screening and evaluating lipid formulations was established by means of the characteristics of the intestinal digestion and absorption of the lipid formulations. This model was composed of two systems, including intestinal digestion and the intestinal tissue culture, which drew the evaluation method of intestinal absorption into the in vitro lipolysis model. The influence of several important model parameters such as Ca2+, D-glucose, K+ on the two systems of this model has been investigated. The results showed that increasing of Ca2+ concentration could be significantly conductive to intestinal digestion. The increasing of D-glucose concentration could stepped significantly down the decay of the intestinal activity. K+ was able to maintain intestinal activity, but the influence of different concentration levels on the decay of the intestinal activity was of no significant difference. Thus the model parameters were set up as follows: Ca2+ for 10 mmol x L(-1), D-glucose for 15 mmol x L(-1) and K+ for 5.5 mmol x L(-1). Type I lipid formulation was evaluated with this model, and there was a significant correlation between the absorption curve in vitro and absorption curve in vivo of rats (r = 0.995 6, P < 0.01). These results demonstrated that this model can be an attractive and great potential method for the screening, evaluating and predicting of the lipid formulations.
Animals ; Calcium ; metabolism ; Caprylates ; chemistry ; Digestion ; Glucose ; metabolism ; Glycerides ; chemistry ; Indomethacin ; chemistry ; Intestinal Absorption ; Lipids ; chemistry ; Lipolysis ; Models, Biological ; Potassium ; metabolism ; Rats ; Rats, Sprague-Dawley

The distribution fate and solubilization behavior of indomethacin through the intestinal tract were investigated with in vitro lipolysis model, by comparing the Capmul MCM and Labrafil M 1944 CS type I lipid formulations. The results showed that the more favorable solubilization was in the aqueous digestion phase from each lipid formulations for indomethacin. The lipolysis rate and extent were decided with chemical constitution of the lipid excipients, which meant that less indomethacin was transferred from the long chain polar oil lipid solution into the aqueous digestion phase. Increasing the concentration of indomethacin in the lipid formualitons from a solution to a suspension led to a linear increase in the concentration of indomethacin attained in the aqueous digestion phase from lipid formulations. This study also implied that adverse effects of the lipolysis rate and extent on drug absorption were could be taken into consideration when screening lipid formulations. Lipid suspensions likely had better enhancement of drug absorption. Last, this study demonstrated that a potential basis for optimizing and assessing type I lipid formulations and also researching in vivo-in vitro correlations of lipid formulations were provided by an in vitro lipolysis model.
Caprylates ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Digestion ; Excipients ; Glycerides ; chemistry ; Indomethacin ; administration & dosage ; pharmacokinetics ; Lipolysis ; Models, Biological ; Polyethylene Glycols ; chemistry ; Solubility ; Suspensions

Hydroxypropyl methylcellulose (HPMC) propels self-emulsifying drug delivery systems (SEDDS) to achieve the supersaturated state in gastrointestinal tract, which possesses important significance to enhance oral absorption for poorly water-soluble drugs. This study investigated capacities and mechanisms of HPMC with different viscosities (K4M, K15M and K100M) to inhibit drug precipitation of SEDDS in the simulated gastrointestinal tract environment in vitro. The results showed that HPMC inhibited drug precipitation during the dispersion of SEDDS under gastric conditions by inhibiting the formation of crystal nucleus and the growth of crystals. HPMC had evident effects on the rate of SEDDS lipolysis and benefited the distribution of drug molecules across into the aqueous phase and the decrease of drug sediment. The mechanisms were related to the formed network of HPMC and its viscosities and molecular weight. These results offered a reference for selecting appropriate type of HPMC as the precipitation inhibitor of supersaturatable SEDDS.
Caprylates ; chemistry ; Chemical Precipitation ; drug effects ; Crystallization ; Drug Delivery Systems ; methods ; Emulsifying Agents ; chemistry ; Emulsions ; Ethylene Glycols ; chemistry ; Glycerides ; chemistry ; Hypromellose Derivatives ; administration & dosage ; chemistry ; pharmacology ; Indomethacin ; administration & dosage ; chemistry ; Lipolysis ; drug effects ; Molecular Weight ; Polyethylene Glycols ; chemistry ; Viscosity

Novel solid lipid nanoparticle (SLN) system is prepared with Compritol ATO 888 and tricaprylic glyceride. DSC, XRD, SAXS and NMR are employed to study the novel carrier property and microstructure. When the peak melting point decreased from 70.8 degrees C to 61.4 degrees C, the enthalpy sharply decreased. It could be concluded that the regular crystal lattices in the novel carriers are broken out for the oil joined in them. Melting behavior is occurred at -17.7 degrees C while novel SLN is composed of oil and solid lipid mixture from the DSC measurement. Most alpha phase and least beta' phase are in the nano carrier system whether drug loading or not from the XRD investigation. There is only 0.1 nm change of long space among the novel SLN made of mixture and the lipid matrix and traditional SLN; therefore, it is impossible of the oil molecular insert into the solid glyceride structure. Since the different melting behavior (DSC measurements) and molecular move state (NMR investigations), two lipid matrix are still in two state of liquid and solid lipid in the novel SLN carrier. Presume the microstructure of the novel SLN prepared by our experiment would be that liquid oil has formed superfine nano accommodation encapsulated with solid lipid, but the whole particle is still in nano size range.
Calorimetry, Differential Scanning ; Caprylates ; chemistry ; Diterpenes ; administration & dosage ; chemistry ; Drug Carriers ; chemistry ; Drug Delivery Systems ; Epoxy Compounds ; administration & dosage ; chemistry ; Fatty Acids ; chemistry ; Magnetic Resonance Spectroscopy ; Nanoparticles ; Particle Size ; Phenanthrenes ; administration & dosage ; chemistry ; Triglycerides ; chemistry ; X-Ray Diffraction

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.
Aeropyrum ; chemistry ; enzymology ; Archaeal Proteins ; genetics ; metabolism ; Binding Sites ; Biocatalysis ; Caprylates ; metabolism ; Cloning, Molecular ; Dimethyl Sulfoxide ; chemistry ; Escherichia coli ; Hot Temperature ; Industrial Microbiology ; methods ; Kinetics ; Laurates ; metabolism ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; methods ; Peptide Hydrolases ; genetics ; metabolism ; Protein Binding ; Protein Conformation ; Recombinant Proteins ; genetics ; metabolism ; Solvents ; chemistry ; Substrate Specificity

OBJECTIVE: This study aimed to investigate the effects of caprylic acid (C8:0) on lipid metabolism and inflammation, and examine the mechanisms underlying these effects in mice and cells. METHODS: Fifty-six 6-week-old male C57BL/6J mice were randomly allocated to four groups fed a high-fat diet (HFD) without or with 2% C8:0, palmitic acid (C16:0) or eicosapentaenoic acid (EPA). RAW246.7 cells were randomly divided into five groups: normal, lipopolysaccharide (LPS), LPS+C8:0, LPS+EPA and LPS+cAMP. The serum lipid profiles, inflammatory biomolecules, and ABCA1 and JAK2/STAT3 mRNA and protein expression were measured. RESULTS: C8:0 decreased TC and LDL-C, and increased the HDL-C/LDL-C ratio after injection of LPS. Without LPS, it decreased TC in mice ( P < 0.05). Moreover, C8:0 decreased the inflammatory response after LPS treatment in both mice and cells ( P < 0.05). Mechanistic investigations in C57BL/6J mouse aortas after injection of LPS indicated that C8:0 resulted in higher ABCA1 and JAK2/STAT3 expression than that with HFD, C16:0 and EPA, and resulted in lower TNF-α, NF-κB mRNA expression than that with HFD ( P < 0.05). In RAW 264.7 cells, C8:0 resulted in lower expression of pNF-κBP65 than that in the LPS group, and higher protein expression of ABCA1, p-JAK2 and p-STAT3 than that in the LPS and LPS+cAMP groups ( P < 0.05). CONCLUSION Our studies demonstrated that C8:0 may play an important role in lipid metabolism and the inflammatory response, and the mechanism may be associated with ABCA1 and the p-JAK2/p-STAT3 signaling pathway.
ATP Binding Cassette Transporter 1/immunology* ; Animals ; Caprylates/chemistry* ; Cholesterol/metabolism* ; Diet, High-Fat/adverse effects* ; Humans ; Inflammation/metabolism* ; Janus Kinase 2/immunology* ; Lipid Metabolism/drug effects* ; Macrophages/immunology* ; Male ; Mice ; Mice, Inbred C57BL ; RAW 264.7 Cells ; STAT3 Transcription Factor/immunology* ; Signal Transduction