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Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes NAD(P)+-dependent glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

Background/Aims: Excessive melanogenesis often causes unaesthetic hyperpigmentation. In a previous report, our group introduced a newly synthesized depigmentary agent, Melasolv™ (3,4,5-trimethoxycinnamate thymol ester). In this study, we demonstrated the significant whitening efficacy of Melasolv using various melanocytes and human skin equivalents as in vitro experimental systems. Methods: The depigmentary effect of Melasolv was tested in melan-a cells (immortalized normal murine melanocytes), α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 murine melanoma cells, primary normal human melanocytes (NHMs), and human skin equivalent (MelanoDerm). The whitening efficacy of Melasolv was further demonstrated by photography, time-lapse microscopy, Fontana-Masson (F&M) staining, and 2-photon microscopy. Results: Melasolv significantly inhibited melanogenesis in the melan-a and α-MSH-stimulated B16 cells. In human systems, Melasolv also clearly showed a whitening effect in NHMs and human skin equivalent, reflecting a decrease in melanin content. F&M staining and 2-photon microscopy revealed that Melasolv suppressed melanin transfer into multiple epidermal layers from melanocytes as well as melanin synthesis in human skin equivalent. Conclusion: Our study showed that Melasolv clearly exerts a whitening effect on various melanocytes and human skin equivalent. These results suggest the possibility that Melasolv can be used as a depigmentary agent to treat pigmentary disorders as well as an active ingredient in cosmetics to increase whitening efficacy.

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized

Abutilon crispum L. Medik, better known as bladdermallow, is used as a traditional remedy in India, for its anti-inflammatory effect due to its high content of flavonoids. However, research about its anti-inflammatory effect at the molecular level has not been performed. In this study, we aimed to investigate the mechanism of Abutilon crispum methanol extract (Ac-ME) in inhibiting the inflammatory response by conducting several experiments including cellular and molecular assays. Ac-ME inhibited the production of nitric oxide (NO) in RAW264.7 cells during treatment of LPS and Pam3CSK4 without exhibiting cytotoxicity. Ac-ME also suppressed the mRNA expression of inducible nitric oxide (iNOS) and proinflammatory cytokines such as interleukin (IL)-1β and IL-6. Moreover, Ac-ME was shown to inhibit the NF-κB pathway, according to the luciferase reporter gene assay performed with a NF-κB-Luc construct containing NF-κB-binding promoter regions under MyD88 and TRIF overexpression conditions, and immunoblotting analysis by determining the phospho-form levels of IκBα, IKKα/β, and p85, a regulatory domain of phosphatidylinositide 3-kinase (PI3K). Finally, we observed that the level of phospho-p85 induced by the overexpression of spleen tyrosine kinase (Syk) and Src was decreased by Ac-ME at 200 μg/ml. Therefore, these results suggest that Ac-ME has an anti-inflammatory effect by targeting PI3K in the NF-κB signaling pathway.

Tyrosinase 1 is a copper-containing enzyme which is widely distributed in nature. This enzyme is associated with the production of melanin for the protection of the skin from solar irradiation. However, overproduction of melanin in the skin is of particular concern to woman, for example, in the conditions melasma and lentigo. 2 Tyrosinase catalyzes the first two steps of melanin biosynthesis, namely the hydroxylation of L-tyrosine to L-dopa and the subsequent oxidation of L-dopa to dopaquinone. Tyrosinase is contained in vegetables and fruits and is responsible for the undesirable enzymatic browning that occurs upon long term storage of these. 3 Therefore, tyrosinase inhibitors have attracted attention as important depigmenting agents for the treatment of hyperpigmentation and as anti-browning agents in vegetables and fruits. Kojic acid 4 (1) is a well known tyrosinase inhibitor. However, its inhibitory activity is not potent enough for the above purposes. To overcome this drawback, many semisynthetic kojic acid derivatives have been synthesized, usually by modification of the C-2 hydroxyl group. 5 Recently, we synthesized kojic acid derivatives containing thioether 6 and ester 7 linkers and evaluated their tyrosinase inhibitory activities. Among them, 2-((cyclohexylthio)methyl)-5-hydroxy-4H-pyran-4-one (2b), showed the most potent inhibitory activity. Compound 2b is composed of three parts, including the kojic acid moiety, a thioether linker, and a hydrophobic cyclohexane group. We synthesized and evaluated additional compounds to get more structural insight into the tyrosinase inhibitory activity of the kojic acid derivatives. The crystallographic structure of tyrosinase has recently been established, enabling closer examination of its three-dimensional structure, which revealed the presence of a hydrophobic protein pocket adjoining the binuclear copper active site. 8 We also conducted a molecular docking study to elucidate the structural importance of the kojic acid derivatives in binding with the active site of tyrosinase. The synthetic pathways of the kojic acid derivatives are shown in Scheme 1. Thioether derivatives (2a-2e) were synthesized by the condensation of kojyl chloride 4 with the potassium salts of thiols. Ester derivatives (3a-3e) were synthesized by the condensation of kojyl chloride 4 with the potassium salts of acids. The inhibitory activities of the kojic acid derivatives on mushroom tyrosinase were investigated using kojic acid as a positive control. The results are summarized in Table 1.

E-mail: snpark@seoultech.ac.krReceived April 22, 2014, Accepted June 5, 2014Key Words : Dimeric cinnamoylamide, Melanin, Depigmentation, TyrosinaseHuman skin color is primarily determined by melaninproduction. Melanin is a biopigment synthesized by melano-cytes in a specialized cellular organelle called the melano-some.

Seed oil triacylglycerol (TAG) composition of 32 soybean varieties were determined and compared using ¹H-NMR. The contents of linolenic (Ln), linoleic (L), and oleic (O) ranged from 10.7% to 19.3%, 37.4%-50.1%, and 15.7%-34.1%, respectively. As is evident, linoleic acid was the major fatty acid of soybean oil. Compositional differences among the varieties were observed. Natural oils containing unsaturated groups have been regarded as important nutrient and cosmetic ingredients because of their various biological activities. The TAG profiles of the soy bean oils could be useful for distinguishing the origin of seeds and controlling the quality of soybean oils. To the best of our knowledge, this is the first study in which the TAG composition of various soybean oils has been analyzed using the ¹H-NMR method.

E-mail: snpark@seoultech.ac.krReceived June 13, 2011, Accepted December 26, 2011Key Words : Depigmenting activity, Hydroxyl carboxamide, Hydrophobic moietySkin pigmentation results from an increase in either thenumber of melanocytes or the activity of tyrosinase, wherethe latter is responsible for melanin synthesis.

Tyrosinase 1 is widely distributed in microorganisms, animals, and plants and is responsible for melanization in animals and browning in plants. Melanin plays a crucial protective role against the photodamage of skin. However, the production of abnormal melanin pigmentation is a serious esthetic problem for human beings. In the food industry, tyrosinase is responsible for enzymatic browning reactions in damaged fruit during post-harvest handling and processing. Therefore, the development of tyrosinase inhibitors that can be used as skin whitening agents 2 or preservatives 3 for fresh food has been pursued. Kojic acid 4 (1) is produced by various fungi and bacteria and is widely used as a skin whitening agent because of its tyrosinase inhibitory activity. Kojic acid has also been shown to prevent photodamage due to its free radical scavenging activity. 5 However, its inhibitory activity and storage properties are insufficient for use in cosmetics and as an anti-browning agent for food. To increase its activity, many semi-synthetic kojic acid derivatives have been synthesized. Many of these compounds were formed by modifying the C-2 hydroxyl group to form esters, 6 ethers, 7 sulfides 8 and peptide 9 derivatives because C-5 enolic hydroxyl group is considered as a pharmacophore for tyrosinase inhibition. Recently, we synthesized a 2,4-dihydroxy benzoate ester of kojic acid containing an adamantane moiety (2) and evaluated its tyrosinase activity. 10 Although compound 2 has a kojic acid moiety for chelation to copper in the active site of tyrosinase, tyrosinase inhibitory activity is not found. We also synthesized a benzoate ester of kojic acid without an adamantane moiety and investigated its tyrosinase inhibitory and radical scavenging activities. Scheme 1 shows the synthetic pathways for the benzoate ester of kojic acid. The reaction of kojic acid with thionyl chloride produces compound 6, which is conveniently converted to compound 7 using dimethylsulfate and potassium carbonate in acetone under reflux conditions. Compounds 6 and 7, which are both chlorides, react with potassium salts of benzoic acids in dimethylformamide at 110 o C - 120 o C to give the corresponding ester derivatives 4a-4m and 5a-5c. First, we evaluated the tyrosinase inhibitory activities of the kojic acid and benzoate ester derivatives. Tyrosinase is known to be the enzyme responsible for the oxidation of tyrosine. The inhibitory activities were evaluated by measuring the transformation rate of L-tyrosine to L-dopaquinone. Compounds 4a-4m exhibited potent inhibitory activities against tyrosinase. The hydrophobic benzoate increased the inhibitory activity of kojic acid. However, the numbers and

Ginsenoside (G) Rp₁ is a ginseng saponin derivative with anti-cancer and anti-inflammatory activities. In this study, we examined the mechanism by which G-Rp₁ inhibits inflammatory responses of cells. We did this using a strategy in which DNA constructs containing cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) promoters were transfected into HEK293 cells. G-Rp₁ strongly inhibited the promoter activities of COX-2 and iNOS; it also inhibited lipopolysaccharide induced upregulation of COX-2 and iNOS mRNA levels in RAW264.7 cells. In HEK293 cells G-Rp₁ did not suppress TANK binding kinase 1-, Toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-b (TRIF)-, TRIF-related adaptor molecule (TRAM)-, or activation of interferon regulatory factor (IRF)-3 and nuclear factor (NF)-κB by the myeloid differentiation primary response gene (MyD88)-induced. However, G-Rp₁ strongly suppressed NF-κB activation induced by Iκβ kinase (IKK)β in HEK293 cells. Consistent with these results, G-Rp₁ substantially inhibited IKKβ-induced phosphorylation of Iκβα and p65. These results suggest that G-Rp₁ is a novel anti-inflammatory ginsenoside analog that can be used to treat IKKβ/NF-κB-mediated inflammatory diseases.

Ginsenoside (G)-F1 is an enzymatic metabolite generated from G-Rg1. Although this metabolite has been reported to suppress platelet aggregation and to reduce gap junction-mediated intercellular communication, the modulatory activity of G-F1 on the functional role of skin-derived cells has not yet been elucidated. In this study, we evaluated the regulatory role of G-F1 on the cellular responses of B16 melanoma cells. G-F1 strongly suppressed the proliferation of B16 cells up to 60% at 200 μg/mL, while only diminishing the viability of HEK293 cells up to 30%. Furthermore, G-F1 remarkably induced morphological change and clustering of B16 melanoma cells. The melanin production of B16 cells was also significantly blocked by G-F1 up to 70%. Interestingly, intracellular signaling events involved in cell proliferation, migration, and morphological change were up-regulated at 1 h incubation but down-regulated at 12 h. Therefore, our results suggest that G-F1 can be applied as a novel anti?skin cancer drug with anti-proliferative and anti-migration features.

is involved in the pathogenesis of numerous diseases such as chronic inflammation, cancer, rheumatoid arth-ritis, and autoimmune diabetes. Macrophages play a pivotal role in inflammation by producing various mediators. One such primary inflammatory mediator, nitric oxide (NO), is produced enzymatically by inducible nitric oxide synthase (iNOS).

) is well known depigmenting agent that is used to reduce melanin content by inhibition of tyrosinase. Although hydroquinone exhibits highly potent depigmenting activity, its application in cosmetics isrestricted because of its severe side effects such as melanocyte destruction, irritative dermatitis, and contact dermatitis. To over-come this disadvantage, its glycoside derivative, arbutin

is bifunctional enzyme, which catalyzes the hydroxylation of L-tyrosine to L-dopa and enhances the oxidation of L-dopa to dopaquinone. There-fore, inhibitors of tyrosinase should be useful as therapeutic agents for the treatment of melanin hyperpigmentation. Many metal ion chelators, for example, kojic acid

Data were obtained as meanvalue of 1/[V], inverse of the increase of optical density at 450 nm per min. (OD450/min), of three independent tests with differentconcentrations of L-tyrosine as a substrate. (A) with 100 μM (rectangle), 50 μM (triangle), 25 μM (circle), or no kojic acid (B) with 40 μM(rectangle), 20 μM (triangle), 10 μM (circle), or no compound

Lipid peroxidation has been suggested to play a key role in damaging biological processes resulting from excessive exposure to UV light. Aging of skin has been associated with increased peroxidation of skin lipid. In particular, squalene, which is a main component of skin surface polyunsaturated lipids, is easily peroxidized. Squalene-monohydroperoxide (Sq-OOH), the initial product of peroxidized squalene, is produced at the human skin by natural exposure to sunlight during day time activities. Recently, a few researchers reported on the damages of hairless mice skin by the topical application of Sq-OOH. Repeated application of Sq-OOH to the hairless mice induced its the skin wrinkles with clear and fine appearance. Histologically were seen, more keratinocyte hypertrophy with Sq-OOH treated skin than with chronic UVB irradiated group in epidermis. In dermis, characteristics, found in UVB irradiated skin, such as the loss of polarity, fractured collagen bundle and elastosis, were not observed severely. The alteration of epidermal layer might be the main factor for the formation of wrinkle caused by Sq-OOH. From the previous works, Sq-OOH is considered as an important source of damage in the early stage of aging process to result in epidermal wrinkle. Thus, it is very important to minimize such damage that would be induced mainly by Sq-OOH against healthy skin condition. Therefore, we tried to develop new anti-aging materials for protecting skin damage by Sq-OOH. Hydroxamic acids, the naturally occurring products exhibit low toxicities in general, and are of interest for many therapeutic applications. Recently, hydroxamic acids and derivatives were reported largely as effective antibacterial and antifungal agents. Their activities originate from their ability to coordinate on metal ions. That is, hydroxamic acid group is regarded as an one electron donor. Hydroxamic acid moiety has also drawn much attention as a reactive oxygen species (ROS) scavenger. In this study, a new skin protecting compound, (4-(Nhydroxycarbamoyl)phenyl)-N-adamantanylcarboxamide (1) is synthesized to show inhibitory activities of lipid peroxidation both in vitro and in vivo. The targeted compound 1 is a hydroxamic acid derivative which possesses an amide linker between hydroxamic acid and adamantane moiety. Adamantane group was adopted as a hydrophobic moiety to optimize antioxidant activity of hydroxamic acid in animal test. Compound 1 is believed to be adequate in skin permeation because of its balance in hydrophilic and hydrophobic character.

Two kaempferol glycosides were isolated from green tea seed extract (GTSE). After conducting a structure analysis, these two compounds were identified as kaempferol-3-O-[2-O-beta-D-galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-beta-D-xylopyranosyl-6-O-alpha-L-rhanmopyranosyl]-beta-D-glucopyranoside (compound 2). These two compounds were hydrolysed by o-glycolytic enzymes for the production of kaempferol. After performing several reactions, we found the optimum enzyme combination, a reaction with beta-galactosidase and hesperidinase. Finally, we produced kaempferol of above 95% purity. The 5alpha-reductase inhibition activities of GTSE hydrolysate (GTSE-H) containing kaempferol were evaluated by the contact cell-based metabolic method using a stable HEK 293 cell line. GTSE-H showed a good inhibition effect on HEK 293 cell lines both type 1 and type 2 on 5alpha-reductase. Especially, GTSE-H inhibited type 2 with kaempferol content dependency. The results indicate that the inhibition activity of hydrolysate on 5alpha-reductase type 2 increases in accordance with kaempferol content.

Π-A isotherms and phase behaviors of ‘tocomide’, a newly synthesized 1,3-bis(N-(2-hydroxyethyl)-tocopherol succinylamino)-2-hydroxypropane, mixed with cholesterol and linoleic acid, was studied for its monolayer miscibility and a stable delivery formulation for antioxidant applications. The monolayer of tocomide and cholesterol was formed in a homogeneously mixed state at air–water interface. The ternary mixtures with linoleic acid showed various bulk structures, including a stable and transparent solution of thermodynamically stable lamellar phase. The lamellar structure was confirmed by the X-ray diffraction (XRD) patterns and polarized microscopy such that pure tocomide formed a liquid crystal at room temperature with a lamellar periodicity of 36.7 A (2θ=2.41°)

21-O-Angeloyltheasapogenol E3 (ATS-E3) is a triterpenoid saponin recently isolated from the seeds of the tea treeCamellia sinensis(L.) O. Kuntze. ATS-E3 has several beneficial properties including anti-inflammatory, antidiabetic, antiatherosclerotic, and anticancer effects. Unlike other phenolic compounds isolated from tea plants, there are no studies reporting the pharmacological action of ATS-E3. In this study, we therefore aimed to explore the cellular and molecular inhibitory activities of ATS-E3 in macrophage-mediated inflammatory responses. ATS-E3 remarkably diminished cellular responses of macrophages such as FITC-dextran-induced phagocytic uptake, sodium nitroprusside- (SNP-) induced radical generation, and LPS-induced nitric oxide (NO) production. Analysis of its molecular activity showed that this compound significantly suppressed the expression of inducible NO synthase (iNOS), nuclear translocation of nuclear factor- (NF-)κB subunits (p50 and p65), phosphorylation of inhibitor ofκB kinase (IKK), and the enzyme activity of AKT1. Taken together, the novel triterpenoid saponin compound ATS-E3 contributes to the beneficial effects of tea plants by exerting anti-inflammatory and antioxidative activities in an AKT/IKK/NF-κB-dependent manner.

In this study, we aimed to examine the cellular and molecular mechanisms of lancemaside A fromCodonopsis lanceolata(Campanulaceae) in the inflammatory responses of monocytes (U937 cells) and macrophages (RAW264.7 cells). Lancemaside A significantly suppressed the inflammatory functions of lipopolysaccharide- (LPS-) treated RAW264.7 cells by suppressing the production of nitric oxide (NO), the expression of the NO-producing enzyme inducible NO synthase (iNOS), the upregulation of the costimulatory molecule CD80, and the morphological changes induced by LPS exposure. In addition, lancemaside A diminished the phagocytic activity of RAW264.7 cells and boosted the neutralizing capacity of these cells when treated with the radical generator sodium nitroprusside (SNP). Interestingly, lancemaside A strongly blocked the adhesion activity of RAW264.7 cells to plastic culture plates, inhibited the cell-cell and cell-fibronectin (FN) adhesion of U937 cells that was triggered by treatment with an anti-β1-integrin (CD29) antibody and immobilized FN, respectively. By evaluating the activation of various intracellular signaling pathways and the levels of related nuclear transcription factors, lancemaside A was found to block the activation of inhibitor ofκB kinase (IKK) and p65/nuclear factor- (NF-)κB. Taken together, our findings strongly suggest that the anti-inflammatory function of lancemaside A is the result of its strong antioxidative and IKK/NF-κB inhibitory activities.

Tyrosinase is a copper-containing enzyme, which catalyzes two distinct reactions of melanin synthesis, the hydroxylation of L-tyrosine to L-dopa and the oxidation of L-dopa to dopaquinone. These processes are involved in local hyperpigmentation such as melasma and lentigo. Therefore, the inhibition of tyrosinase to treat the pigmentation disorders has been a recent subject of many studies. Kojic acid (1) is well known as one of the most popular tyrosinase inhibitors and it has been widely used as a skin-whitening agent. However, the inhibitory activity of kojic acid is not potent enough. To overcome this drawback, many semi-synthetic kojic acid derivatives were synthesized usually by the modification of C-2 hydroxyl group into ester, hydroxyphenyl ether, and glycoside. Recently, two molecules of kojic acid were connected by ester and ethylene. Their inhibitory activities were dramatically enhanced. Thus we became interested in new kojic acid derivatives containing two molecules of kojic acid. In this study, we connected two molecules of kojic acid by various chemical bonds such as ester, amide, and thioether. Anti-melanogenic activities of compounds (4a-4f) were compared with that of kojic acid (Figure 1).

genous supplementation of antioxidants may be an effective strategy to counteract the deleterious effects of the ROS generated from the excessive exposure to UV irradiation. Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxyl stilbene, is considered an essential antioxidative constituent of red wine. 4 It showed broad antioxidative activities such as scavenging of the free radicals, 5 inhibition of lipid peroxidation, 6 inhibition of the platelet aggregation 7 and anti-cancer activity. 8 However, because of its unfavorable photo stability, the application of transresveratrol has been limited. Various UV irradiation conditions showed that trans-resveratrol was easily transformed into cis-resveratrol. 9 Therefore, an attempt to search for photo stable alternative material of trans-resveratrol is desirable. In this study, a mimic compound of trans-resveratrol, 3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide (2), containing an amide linker between two hydroxyl phenyl groups, is synthesized. We evaluated various biological activities of compound 2 compared with trans-resveratrol (Fig. 1).

Long chain alkanolamides have attracted considerable interest because of their intrinsic amphiphilic properties. They are frequently used as a surfactant for cosmetics and pharmaceutical use. In the course of the synthesis of alkanolamides, control of chemoselective amide formation over competing O-acylation of neighboring hydroxyl groups plays a key step. Several methods have been developed which include a few enzymatic synthesis and the reaction of carboxylic acid with amino alcohols by the aid of N-ethoxy1-ethoxy-dihydroquinone (EEDQ) in ethanol reflux condition. However, these known methods have drawbacks for example narrow application scope in enzymatic reaction, longer reaction time and harsh reaction condition. Recently, we have reported that chemoselective N-acylation of amino alcohols promoted by magnesium oxide in aqueous organic solution. Although the use of this method was successful in many cases, preparation of acid chloride is not always easy so there is still a strong need for more convenient amidation method of amino alcohols. Now we report a useful procedure for the efficient and chemoselective amidation of hydrophilic amino alcohols with mixed anhydride generated from carboxylic acid and ethylchloroformate (Scheme 1). Treatment of benzoic acid 1a with ethylchloroformate (1.1 equiv) in the presence of N-methylmorpholine (1.1 equiv) in THF gave mixed anhydride, which subsequently reacted with tris(hydroxymethyl)aminomethane 3a in DMF and TEA (1.1 equiv) to give alkanolamide 4a in 85% yield (entry 1 in Table 1). The formation of by-products such as O-acylated and N, O-diacylated derivatives have not been observed in HPLC analysis. After the disappearance of mixed anhydride checked by TLC analysis, DMF was evaporated in vacuo. The residue of reaction mixture was extracted with ethyl acetate, washed with water. The organic layer was analyzed by HPLC. As shown in Figure 1, small amount of benzoic acid were detected without by-products. Benzoic acid was easily separated by flash chromatography. As an additive for the synthesis of mixed anhydride, TEA as well as N-methylmorpholine was effective. Of solvents tested, THF and ether were suitable. Using aliphatic carboxylic acid 2b, alkanolamide 4b was also obtained in high yield (entry 2). Further, this method was successfully extended to other acids containing acid or base-sensitive groups such as cyclic disulfide, ester and acetate (entry 3, 4 and 5). In all cases, byproducts that are usually formed in the reaction of acid chlorides with amino alcohols were not detected. Preferential amine selectivity can be explained by assuming low electrophilicity of mixed anhydride comparing with acid chloride. Because of its low electrophilicity, more nucleophilic amino groups may only react with mixed anhydride. The reaction also worked well for the other hydrophilic amine such as ethanolamine, DL-serine methyl ester and diethanolamine. The desired alkanolamides 4f, 4g and 4h

The sprouts, microgreens and leafy greens of common and tartary buckwheat of Nepalese strain were compared for the phenolic contents and biological activity. The tartary buckwheat samples expressed higher total phenolic and flavonoid contents compared to the common buckwheat. The sprouts had the highest total phenolic contents (9333.48 ± 150.23 and 6976.21 ± 213.65 mgGAE/100g dw in tartary and common buckwheat, respectively) whereas, the highest total flavonoid content was present in the leafy greens (7635.39 ± 141.40 and 4414.61 ± 70.85 mgRE/100g dw in tartary and common buckwheat respectively). The high performance liquid chromatography (HPLC) results revealed that the tartary buckwheat vegetables had higher rutin, (3800.28 ± 434.41 mg/100g in leafy greens), quercetin (159.75 ± 9.04 mg/100g in sprouts) and chlorogenic acid (293.47 ± 65.06 mg/100g in microgreens) contents than those of common buckwheat. However, other phenolics like vitexin, isovitexin, orientin and isoorientin contents were more abundant in common buckwheat. In biochemical assay, all three types of vegetable of common and tartary buckwheat showed higher antioxidant and α-glucosidase inhibition effect in dose dependent manner. Based on these results, it can be conformed that all the vegetables (microgreens, sprouts and leafy greens) of both varieties of buckwheat of Nepalese strains can be regarded as a potent sour ce of functional food.Key words: Antioxidant, α-glucosidase, buckwheat vegetables, Nepalese strain buckwheat, phenolics.

The objective of this study was to examine the biological activity of kaempferol and its rhamnosides. We isolated kaempferol (1), a-rhamnoisorobin (2), afzelin (3), and kaempferitrin (4) as pure compounds by far-infrared (FIR) irradiation of kenaf (Hibiscus cannabinus L.) leaves. The depigmenting and anti-inflammatory activity of the compounds was evaluated by analyzing their structure-activity relationships. The order of the inhibitory activity with regard to depigmentation and nitric oxide (NO) production was kaempferol (1) > a-rhamnoisorobin (2) > afzelin (3) > kaempferitrin (4). However, a-rhamnoisorobin (2) was more potent than kaempferol (1) in NF-kB-mediated luciferase assays. From these results, we conclude that the 3-hydroxyl group of kaempferol is an important pharmacophore and that additional rhamnose moieties affect the biological activity negatively.

Three kinds of prenylated flavonols, icariside I, icariside II, and icaritin, were isolated from an icariin hydrolysate and their effects on melanogenesis evaluated based on mushroom tyrosinase inhibition and quantifying the melanin contents in melanocytes. Although none of the compounds had an effect on tyrosinase activity, icariside II and icaritin both effectively inhibited the melanin contents with an IC50 of 10.53 and 11.13 MM, respectively. Whereas icariside II was obtained from a reaction with beta-glucosidase and cellulase, the icariin was not completely converted into icariside II. Thus, for the high-purity production of icariside II, the reaction was optimized using the response surface methodology, where an enzyme concentration of 5.0 mg/ml, pH 7, 37.5 degrees C;, and 8 h reaction time were selected as the central conditions for the central composite design (CCD) for the enzymatic hydrolysis of icariin into icariside II using cellulase. Empirical models were developed to describe the relationships between the operating factors and the response (icariside II yield). A statistical analysis indicated that all four factors had a significant effect (p0.01) on the icariside II production. The coefficient of determination (R2) was good for the model (0.9853), and the optimum production conditions for icariside II was an enzyme concentration of 7.5 mg/ml, pH 5, 50 degrees C, and 12 h reaction time. A good agreement between the predicted and experimental data under the designed optimal conditions confirmed the usefulness of the model. A laboratory pilot scale was also successful.

Kojic acid, 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4one, is produced from carbohydrate sources in an aerobic process by a variety of microorganisms. It showed broad biological activities such as inhibition of tyrosinase, scavenging of the free radicals, chelating activity of metal ions and prevention of photodamage. Its various activities are due to γ-pyranone structure having enolic hydroxyl group. Recently, enolic hydroxyl group of kojic acid has been focused as an alternative of carboxylic acid in retinoid structure. We synthesized 3,4-methylenedioxy cinnamic acid ester of kojic acid as a new retinoidal compound. In this study, we evaluated biological activities of new kojic acid derivative 1, 2-((3E)-4(2H,3H-benzo[3,4-d]1,3-dioxolan-5yl)-2-oxo-but-3-enyloxy)-5-hydroxy-4H-pyran-4-one.

The binding of sphingoid bases to peroxisome proliferator-activated receptor (PPAR) has been detected in a solid-phase binding assay. However, sphingoid base-induced changes in PPAR transactivation activity have not been examined. In this report, we show by reporter gene analyses that phytosphingosine (PS), a natural sphingoid base, activates the transcriptional activity of PPARs in the immortalized human keratinocyte, HaCaT. Real-time PCR analyses showed that the mRNA level of PPARgamma was increased after PS treatment in HaCaT cells in a dose- and time-dependent manner. Because PPARs play important roles in skin barrier homeostasis by regulating epidermal cell growth, terminal differentiation, and inflammatory response, we examined the effect of PS on normal human epidermal keratinocytes (NHEKs) and mouse skin. PS increased the production of cornified envelope in NHEKs by approximately 1.8-fold compared with controls. Epidermal differentiation marker proteins such as involucrin, loricrin, and keratin1 were also increased in PS-treated NHEKs, by ELISA or Western blotting analysis. A [(3)H]thymidine incorporation assay showed that PS inhibited DNA synthesis in NHEKs to 20% compared with controls. The antiproliferative and anti-inflammatory effects of PS were examined in a mouse model of irritant contact dermatitis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). PS blocked epidermal thickening and edema and the infiltration of inflammatory cells into the dermis in the skin of TPA-treated hairless mice. The anti-inflammatory effects of PS were confirmed by the observation that PS blocked the TPA-induced generation of prostaglandin E(2) in peripheral mononuclear leukocytes. Taken together, our results provide an insight into the multiple regulatory roles of PS in epidermal homeostasis, and furthermore point to the potential use of PS as a therapeutic agent in the treatment of inflammatory and proliferative cutaneous diseases.

To obtain effective and safe topical depigmenting agents, we synthesized hydroxybenzoates, alkoxybenzoates, and 3,4,5-trimethoxycinnamate containing a thymol moiety and screened then for high-level inhibitory activity against melanin synthesis in cultured melanocytes. Eight compounds were tested for their depigmenting effect and cytotoxicity using a murine melanocyte cell line. We found that 3,4,5-trialkoxybenzoates and 3,4,5-trimethoxycinnamate, synthesized by conjugating 3,4,5-trialkoxybenzoic acids and 3,4,5-trimethoxycinnmic acid with thymol, showed a potent depigmenting effect and low cytotoxicity. Compound 4h, 5-methyl-2-(methylethyl)phenyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate, showed the most potent depigmenting effect (IC50=10 microM) with low cytotoxicity (IC50=200 microM).

Skin is a highly complicated tissue which possesses the largest surface area in the body and serves as the protective layer for internal organs. It is designed to give both physical and biochemical protection, and is equipped with a large number of defense mechanisms. 1 However, skin is very susceptible to oxidative reaction, because there are rich contents of lipids, proteins and DNA in the skin cell, all of which are very sensitive to the oxidation process. 2 Lipid peroxidation could be induced from excessive exposure to UV light, and has been considered as a major factor for skin aging processes. 3 In particular, squalene, main component of skin surface polyunsaturated lipids, is easily peroxidized. 4 The initial product of peroxidized squalene, squalene-monohydroperoxide (Sq-OOH), is produced at the human skin by natural exposure to sun light. Recently, the damages of hairless mice skin by the topical application of Sq-OOH 5 have been reported. Repeated application of Sq-OOH to the hairless mice induced clear skin wrinkles. Thus, in order to prevent or retard skin aging process, prevention of skin lipid peroxidation by natural or chemical antioxidants is desirable. To identify efficient antioxidants in cosmetic applications, a convenient assay method suitable for the estimation of skin lipid peroxidation is necessary. Rancimat 6 is a convenient method which can evaluate the lipid peroxidation by observing the change of electrical conductivity due to the formation of volatile dicarboxylic acids. It has been used in food industry to determine the peroxidation of unsaturated fatty acid components. However, the rancimat method is not suited for skin research, because the composition of oils used as a lipid source in this method is very different from that of skin lipid. 6c In this report, we demonstrate that modified rancimat method is a powerful tool for screening natural antioxidants for skin lipid peroxidation. We changed composition of lipid source to what is very close to skin lipid. At first, we tested antioxidative activities of several natural extracts by using general rancimat method. As shown in Table 1, extracts from Korean medicinal plants presented different induction time on retarding the peroxidation of corn oil. Their activities were compared with BHT as a positive control. Among the three natural extracts, Scutellaria baicalensis Georgi (SBG) extract was found to have the most powerful antioxidative activity. Portulaca oleracea L. (POL) extracts appeared to be the compound with the next activity and Rheum coreanum Nakai (RCN) extract was less active than other extracts. The order of activity is BHT > SBG > POL > RCN. However, ranges of Pf values of tested natural product are too small (from 1.2 to 1.6). Thus, sensitivity is not enough for primary screening of antioxidative activity. To establish the modified rancimat system suitable for skin lipid peroxidation studies, we prepared the artificial skin lipid and used it instead of corn oil. The prepared artificial skin lipid 7 was consisted of pork lard (33%), stearic acid (24%), lanolin (22%), squalene (12%) and cholesterol (4%). This composition is similar to that of human skin. According to the Table 2, the results of new rancimat method were different from previous general rancimat data. Surprisingly, SBG extract shows the strongest antioxidative activity among the tested samples. Induction period of SBG was 11.4 times longer than that of control with this new method. The sensitivity for evaluation of antioxidative activity was dramatically increased due to the wide ranges of Pf value (from 2.2 to 11.4). It is interesting to observe that the order of activity between RCN and ROL was reversed. In contrast to previous rancimat data, RCN is more active than POL. These interesting and meaningful differences between general rancimat and modified method may be due to changing of lipid composition which is suited to skin research. In search of new antioxidant applicable to prevent skin lipid peroxidation, we selected meso-dihydroguaiaretic acid (MDGA) 8 as a candidate. MDGA, a naturally occurring polyphenol

Histone acetylation is linked to the control of chromatin remodeling, which is involved in cell growth, proliferation, and differentiation. It is not fully understood whether cyclic adenosine monophosphate (cAMP), a representative differentiation-inducing molecule, is able to modulate histone acetylation as part of its anticancer activity. In the present study, we aimed to address this issue using cell-permeable cAMP, i.e. dibutyryl cAMP (dbcAMP) and C6 glioma cells. As reported previously, under the conditions of our studies, treatment with dbcAMP clearly arrested C6 cell proliferation and altered their morphology. Its antiproliferative and differentiation-inducing activity in C6 glioma cells involved upregulation of p21WAF/CIP, p27kip1, glial fibrillary acidic protein (GFAP), and Cx43, as well as downregulation of vimentin. Furthermore, dbcAMP modulated the phosphorylation of ERK and Akt in a time-dependent manner and altered the colocalization pattern of phospho-Src and the actin cytoskeleton. Interestingly, dbcAMP upregulated the enzyme activity of histone acetyltransferase (HAT) and, in parallel, enhanced cellular acetyllysine levels. Finally, the hyperacetylation- inducing compound, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, displayed similar anticancer activity to dbcAMP. Therefore, our data suggest that antiproliferative and differentiation-inducing activities of dbcAMP may be generated by its enhanced hyperacetylation function

Background: Korean ginseng is an ethnopharmacologically valuable herbal plant with various biological properties including anticancer, antiatherosclerosis, antidiabetic, and anti-inflammatory activities. Since there is currently no drug or therapeutic remedy derived from Korean ginseng, we developed a ginsenoside-enriched fraction (AP-SF) for prevention of various inflammatory symptoms. Methods: The anti-inflammatory efficacy of AP-SF was tested under in vitro inflammatory conditions including nitric oxide (NO) production and inflammatory gene expression. The molecular events of inflammatory responses were explored by immunoblot analysis. Results: AP-SF led to a significant suppression of NO production compared with a conventional Korean ginseng saponin fraction, induced by both lipopolysaccharide and zymosan A. Interestingly, AP-SF strongly downregulated the mRNA levels of genes for inducible NO synthase, tumor necrosis factor-α, and cyclooxygenase) without affecting cell viability. In agreement with these observations, AP-SF blocked the nuclear translocation of c-Jun at 2 h and also reduced phosphorylation of p38, c-Jun N-terminal kinase, and TAK-1, all of which are important for c-Jun translocation. Conclusion: Our results suggest that AP-SF inhibits activation of c-Jun-dependent inflammatory events. Thus, AP-SF may be useful as a novel anti-inflammatory remedy.