Multiple emulsions, also called complex emulsions or multiphase emulsions, include water-in-oil-in-water (W/O/W)-type and oil-in-water-in-oil (O/W/O)-type emulsions. W/O/W-type multiple emulsions, obtained by utilizing lamellar liquid crystal with a layer structure showing optical anisotropy at the periphery of emulsion droplets, are superior in stability to O/W/O-type emulsions. In this study, we investigated a two-step emulsification process for a W/O/W-type multiple emulsion utilizing liquid crystal emulsification. We found that a W/O/W-type multiple emulsion containing lamellar liquid crystal can be prepared by mixing a W/O-type emulsion (prepared by primary emulsification) with a lamellar liquid crystal obtained from poly(oxyethylene) stearyl ether, cetyl alcohol, and water, and by dispersing and emulsifying the mixture in an outer aqueous phase. When poly(oxyethylene) stearyl ether and cetyl alcohol are each used in a given amount and the amount of water added is varied from 0 to 15 g (total amount of emulsion, 100 g), a W/O/W-type multiple emulsion is efficiently prepared. When the W/O/W-type multiple emulsion was held in a thermostatic bath at 25°C, the droplet size distribution showed no change 0, 30, or 60 days after preparation. Moreover, the W/O/W-type multiple emulsion strongly encapsulated Uranine in the inner aqueous phase as compared with emulsions prepared by one-step emulsification.

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Gas chromatography (GC) of docosenoic acid (22:1) has been performed for the separation of positional isomers on the novel SLB-IL100 column with a highly polar ionic liquid stationary phase. A test mixture of 22:1 methyl esters prepared from total lipids of flounder was subjected to GC on a 60 m×0.32 mm i.d. column at an isothermal temperature of 150°C–180°C. On this column, all five positional isomers separated in the elution order of 22:1n-15, 22:1n-13, 22:1n-11, 22:1n-9, and 22:1n-7. The positional isomers, 22:1n-15, 22:1n-13, and 22:1n-11, unresolvable on conventional polar polymer phase columns, were almost completely separated from each other within 24 min at 170°C. The equivalent chain length values of 22:1n-11 to 22:1n-7 were parallel to those on polyethylene glycol and cyanopropyl polysiloxane columns, whereas 22:1n-15 and 22:1n-13 were relatively lower and closer to saturated 22:0 acid. Similar findings were also obtained for co-injected 20:1n-15 to 20:1n-11 isomers. Analysis of fish 22:1 revealed that 22:1n-13 is not always a minor isomer, as previously reported for several samples. The results of this study confirm the view that SLB-IL100 is a powerful tool for GC analysis of monounsaturated fatty acids.

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Suppression of leukemia, colon cancer, myeloma, and fibrosarcoma to some extent by omega 3 fatty acid bound phospholipids has been reported in the last two decade. However, the anti-angiogenic activity of those phospholipids is still not known. Four kinds of marine phospholipid molecular species i.e. starfish EPA bound diacyl phospholipid (EPA-PC), EPA bound monoacyl phospholipid (EPA-LPC) which was prepare via Lipozyme RMIM mediated partial hydrolysis of EPA-PC, squid DHA bound diacyl phospholipid (DHA-PC), and DHA bound monoacyl phospholipid (DHA-LPC) which was also prepare via Lipozyme RMIM mediated partial hydrolysis of DHA-PC, were subjected to antiangiogenic activity assay by using a piece of rat main artery and a human umbilical cord vein endothelial cell. The lengths of micro vein generated from those tissues after incubation with the above four kinds of phospholipid molecular species were measured and compared. EPA-LPC and DHA-LPC showed strong antiangiogenic activity on the rat main artery tissue, while on the human umbilical cord vein endothelial cells, 100 μM of EPA-LPC in the culture medium, exhibited the most effective suppression on angiogenesis, followed by 100 μM of DHA-LPC. It was concluded that EPA-LPC obtained via Lipozyme RMIM mediated partial hydrolysis of EPA-PC is the most effective omega 3 phospholipid on anti-angiogenesis.

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Conjugated fatty acid is a collective term used for fatty acids with conjugated double bond systems. Seed oils from certain plants include conjugated linolenic acids, which have a conjugated triene system and are geometrical and positional isomers of α-linolenic acid. One of these isomers, jacaric acid (JA, 8c, 10t, 12c-18:3), has not been examined widely. Therefore, we investigated the absorption and metabolism of JA in normal animals (ICR mice). An oral dose of JA of 5 mg/day for 1 week had no effects on body weight, food intake and tissue weight of mice. JA was detected in the serum, kidney, liver, lung and epididymal white adipose tissue. Analysis of the fatty acid composition in liver and white adipose tissue showed a tendency to increase levels of saturated fatty acids (SFAs) such as palmitic acid (16:0) and stearic acid (18:0) and to decrease levels of monounsaturated fatty acids (MUFAs) such as palmitoleic acid (16:1) and oleic acid (18:1). Thus, JA treatment decreased the desaturation index (16:1/16:0, 18:1/18:0) in liver and white adipose tissue. This index is used as an indicator of the activity of stearoyl coenzyme A desaturase (SCD), an endoplasmic reticulum enzyme that catalyzes the biosynthesis of MUFAs from SFAs. The change in this index indicates that JA inhibited SCD activity in ICR mice, and further experiments showed that JA also decreased the expression level of SCD-1 mRNA. Inhibition of SCD activity may have anti-obesity and anti-diabetes effects, and therefore the findings in this study suggest that JA may be effective for preventing obesity and diabetes.

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In order to clarify the physiological significance of stereospecificities of peroxisomal multifunctional enzyme (MFE) type 1 (MFE1) and MFE2, we developed a chiral separation analysis for 3-hydroxyacyl-CoA using high performance liquid chromatography (HPLC) equipped with a chiral separation column. To demonstrate the utility of this technique, we cloned the hydratase domain from wild-type human MFE2 hydratase (MFE2Hwt) and expressed it as a GFP-tagged protein (GFP-MFE2Hwt) in Escherichia coli (E. coli). GFP-MFE2H was purified by diethylaminoethyl (DEAE) Sephacel from an E. coli sonication solution. As anticipated, we observed the formation of 3R-hydroxyhexadecanoyl-CoA (3R-OH-16-CoA) on the HPLC chromatogram after incubating trans-2-enoyl-CoA (16eno-CoA) with GFP-MFE2Hwt. GFP-MFE2Hwt was readily purifiable and could be assayed because of its traceability. We used site-directed mutagenesis to construct GFP-MFE2H variants corresponding to 17 reported MFE2H missense mutations and measured their hydratase activities using our HPLC method. Hydratase activity was completely lost or markedly decreased in the same variants corresponding to MFE2H mutations in patients with D-bifunctional protein (DBP) deficiency type II. On the other hand, the nonpathological variants did not markedly affect hydratase activity.

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A 9 GHz EPR imager that is capable of the imaging surface area of thin materials has been built. The EPR imager resolved samples spaced 1 mm apart. The developed TE₁₁₁ cavity was able to detect easily ~1.0 mM aqueous TEMPOL solution in ~1.0 mm (i.d.) glass capillary placed just above the cavity. The sensitivity measured using the TEMPOL solution showed ~0.3 of that for the modified JEOL cavity, which was in a qualitative agreement with the calculations considering the difference in the filling factors of the cavities. The relatively low measured sensitivity of the TE₁₁₁ cavity is due to utilization of the microwave field from ~3 mm aperture (hole) in the cavity wall. More importantly, the TE₁₁₁ cavity does not require inserting the sample into the cavity and placing samples into EPR tubes.

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Mannosylerythritol lipids (MELs) are biosurfactants known for their versatile interfacial and biochemical properties. To broaden their application in cosmetics, we investigated the antioxidant properties of different MEL derivatives (MEL-A, -B, and -C) by using a 1,1-diphenyl-2-picryl hydrazine (DPPH) free-radical- and superoxide anion-scavenging assay. All MEL derivatives tested showed antioxidant activity in vitro, but at lower levels than those of arbutin. Of the MELs, MEL-C, which is produced from soybean oil by Pseudozyma hubeiensis, showed the highest rates of DPPH radical scavenging (50.3% at 10 mg/mL) and superoxide anion scavenging (>50% at 1 mg/mL). The antioxidant property of MEL-C was further examined using cultured human skin fibroblasts (NB1RGB cells) under H₂O₂-induced oxidative stress. Surprisingly, MEL-C had a higher protective activity against oxidative stress than arbutin did: 10 μg/mL of MEL-C and arbutin had protective activities of 30.3% and 13%, respectively. Expression of an oxidative stress marker, cyclooxygenase-2, in these cells was repressed by treatment with MEL-C as well as by arbutin. MEL-C was thus confirmed to have antioxidant and protective effects in cells, and we suggest that MELs have potential as anti-aging skin care ingredients.

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