Earticle

A novel purification method was developed for producing (+)-dihydromyricetin, an active component in the fruit of Hovenia dulcis, giving high purity and yield. The developed method, which consisted of fractional precipitation and chromatography, was found to be a simple and efficient procedure for the isolation and purification of (+)-dihydromyricetin from the crude extracts. The use of fractional precipitation in the pre-purification process allowed for the efficient separation of (+)-dihydromyricetin from interfering compounds and dramatically increased the purity of (+)-dihydromyricetin compared to alternative processes. This is a fractional precipitation of (+)-dihydromyricetin by difference of solubility in acetone solution. The crude extracts were efficiently pre-purified, increasing in purity from 55.7% to over 84.9% with a yield of 97% by fractional precipitation. Fractional precipitation was performed to obtain (+)-dihydromyricetin with a high purity, which is adjustable to apply to the column employed in a subsequent HPLC purification step.

In this study, the correlation between structural characterization and immuno-stimulating activity was investigated. This work was based on the structural characterization affecting molecular immuno-modulating activity of polysaccharide. The correlation was established through onverting the structure of polysaccharide by acetylation and determining NO production indicating the immune activity. The degree of acetylation of polysaccharide was measured by FT-IR, In results, it was found that NO production was increased with the increase of acetylation, resulting in showing the high immune activity.

Molecular imprinting is a versatile and facile technique for the preparation of synthetic polymers with predetermined molecular recognition sites for a desired template. It enables the formation of tailor-made materials, which can be used as chiral stationary phases in various chromatographic techniques. In our previous investigations, MIP microbeads were prepared by a modified suspension polymerization method using D-Phe as template. In the current study, these microbeads, packed into a stainless steel column, were evaluated as HPLC stationary phase for the enantioseparation of phenylalanine isomers. The selection of a suitable mobile phase, mobile phase composition, pH and flow rate were investigated for the best resolution. Baseline separation was achieved with EtOH-acetate buffer solution as a mobile phase. Maximum resolution was obtained for a mobile phase of pH 4.75. The separation factor and resolution decreased with an increase in the mobile phase flow rate. These microbeads were found superior to the majority of the reported molecularly imprinted polymers with respect to chiral separation ability.

A HPLC method is described for the analysis of ribose, arabinose and mixtures obtained from chemical isomerization processes. These processes gain importance since the molecules can be used for the synthesis of antiviral therapeutics. The method uses Ca+ resin additive to enhance the separation on FPLC system. Dow99Ca350resin is ion- exchange resin, and primarily resin. This resin has styrene-divinylbenzene matrix and formed Ca+ form. The aim of this study was analysis ribose and arabinose. FPLC of low pressure use on glass column that paked Ca+ resin. This resin use separation of ribose and arabinose. Area of weak resolution has fractionation. Fractional sample analysis by NH2 column in HPLC.

The principal cause of red tide and eutrophication is nitrate in waste water. High concentrations of nitrate in water can cause public health problems. The objective of this study is to remove the nitrate from nitrogenous compound by using adsorbents (Bio magmaⓇ, zeolite, chitosan resin) Batch adsorption experiments were carried out to evaluate adsorption efficiency as time changes. Nitrate determined directly using UV detection with HPLC system, which consisted anion exchange column. Experiment with adsorbents confirmed efficiency of nitrate adsorption among the three adsorbents. Using the MATLAB, we predicted aspect of adsorption and desorption according to retention time.

Transmissible spongeform encephalopathies including bovine spongeform encepalopathies and human Creutzfeldt-Jakob diseas are fatal neurodegenerative diseases. The protein-only hypothesis holds that structurally converted transmembrane prion proteins play a major role in the procedure of these diseases. According to this hypothesis, the wild type of prion protein (PrPC) is converted into the infectious form (PrPSC) under unknown conditions and this PrPSc is believed to act as a seed for forming infectious fibril like oligomers of prion proteins. Between PrPC and PrPSc, there are no differences in sequences of deoxyribonucleic acids and amino acids but, only the structure is changed which leads PrPSc to obtain features of resistance to proteaseK and the increase of -β sheet contents than PrPC. In the present study, we focused on the mechanism how normal prion proteins are converted into infectious forms and assumed that the breakage of intra-disulfide bond of prion proteins by a reducing agent would make normal PrPC to tend to be like the infectious one. It is shown that the in vitro converted PrPSc-like structure exhibits a binding affinity to membranes. Circular dichroism spectroscopy shows that the disulfide bond-broken PrPC has more β-sheet contents than normal PrPC in the presence of membrane. Membrane-encapsulated calcein, a self-quenching fluorophore, is released upon the treatment of both prion protein and reducing agent. The color of polydiacetylene biosenseor composed of PDA and phospholipid (25% negative charged) is shifted from blue to red when disulfide bond-defective PrPC is present. Results indicate that the prion protein gains membrane binding ability when the intramolecular disulfide bond is reduced, leating to membrane disruption.

The enzyme-catalyzed resolution of racemic compounds is one of methods for obtaining optically pure substances. Lipase is an enzyme that catalyzes the hydrolysis of (S)-Naproxen 2,2,2-trifluoroethyl thioester to (S)-Naproxen. Hydrolytic reaction of (R,S)-naproxen 2,2,2-trifluoroethyl thioester by lipase was carried out in water-saturated isooctane for 72 h. Rreaction conditions such as temperature, concentrations of substrate and enzyme, and agitation speed was optimized by using response surface methodology (RSM). Based on statistical analysis, optimal conditions were determined to be 42.5, 3.28 mM, 32.95 m℃g/mL and 164 rpm, respectively. Under optimal reaction conditions, the concentration of (S)-naproxen produced by the hydrolytic reaction of (R,S)-naproxen 2,2,2-trifluoroethyl thioester was 0.260 mM, similar to that of (S)-naproxen (0.266 mM) predicted by the model.

Tetragonia tetragonioides has long been used as a traditional remedy for stomach cancer and furuncle. We investigated antioxidant activity, tyrosinase activity and acetylcholinesterase activity of the solvent extracts of Tetragonia tetragonioides. The solvent fractions were extracted by 100% methanol (MeOH) and successively extracted by n-hexane, methyl chloride(CH2Cl2), ethyl acetate(EtOAc) and butanol(BuOH). Antioxidant activites of solvent fractions from Tetragonia tetragonioides were examined by free radical scavenging assay, superoxide radical (O2 -) scavenging activity, total phenolic compound content, vitamin E content and total antioxidnat capacity. The CH2Cl2 and EtOAc extracts of Tetragonia tetragonioides were found to exhibit a distinctive antioxidant activity. The tyrosinase inhibitory activity of MeOH, n-hexane, CH2Cl2, EtOAc and BuOH extracts of Tetragonia tetragonioides was investigated. In addition, the MeOH extract of Tetragonia tetragonioides was tested for acetylcholinesterase inhibitory activity using the Ellman colorimetric method. Extracts of Tetragonia tetragonioides exhibited moderate inhibition of the enzyme, defined as more than 50% at 0.1 mg/ml.

Staurosporine and its derivatives have aroused considerable interest as they were shown to have strong inhibitory activity against protein kinase C.1 Staurosporine also inhibits several other kinases such as protein kinases A and G, myosin light-chain kinase, and tyrosine kinases.2 The search for new staurosporine derivatives was further intensified after the discovery that staurosporine inhibits platelet aggregation and smooth muscle contraction and blocks certain phases of the cell-growth cycle.3 The most promising activity is the reversal of multidrug resistance by some derivatives.4 The potential of staurosporines as anticancer agents is clearly supported by the example of 7-hydroxystaurosporine (UNC-01), which is in clinical phase 1 trials at the NCI.5 As part of our continuing program to develop the biomedical potential of marine microorganisms, we have focused considerable attention on the marine actinomycetes. From a marine-derived actinomycete, we isolated two indolocarbazole alkaloids, staurosporines. We present here the discovery of staurosporines from marine Streptomyces sp. The isolation, structure determination, and cytotoxicity of staurosponies from marine Streptomyces will be presented. The isolation of staurosporines from a marine actinomycete supports the hypothesis that associated marine microorganisms are the real producers of staurosporines in the ascidian Eudistoma toealensis.

Most of Basidiomycetes mushrooms contain biologically active polysaccharides from fruit bodies, mycelium and culture broth. Grifola frondosa is commonly used in the treatment of various diseases, due to its considerable biological activities. In this study, we evaluated the suitability of various carbon, nitrogen sources and mineral sources for the effective production of biomass and exo-polysaccharide from G. frondosa. All the basic liquid culture conditions were carried out in flasks(250mL) containing 100 ml working volume based on the basal medium(YMK) which the compositions were (in g/L) glucose 20, yeast extract 5, KH2PO4 2 and MgSO4․7H2O 1. Under the basic conditions (25℃, Working vol. 100mL, 150 rpm)without pH control, the optimal temperature, initial pH and working volume were identified respectively. The objective of this work was to optimize the medium composition of various factors to increase the yields of mycelial growth and exo-polysaccharide.

Grapefruit Seed Extract (GSE), thyme and lemongrass Oil have been well known as natural materials with its distinguished antimicrobial activities. In this study, antifungal and antimicrobial susceptibility of methyl paraben and propyl paraben, which are both synthetic and common cosmetic food grade preservatives, and also that of natural preservatives were tested. From the results of test with liquid culture medium revealed that GSE had the highest antimicrobial effect with Minimum Inhibitory Concentration (MIC) value and Minimum Bactericidal Concentration value, and that thyme and lemongrass had similar range of inhibitory concentration. It was confirmed that all natural materials had high antimicrobial activity, and based on this test result, relevant cosmetics were made and the possibility of a preservative was tested accordingly. Also, we tested preservative activity of the mixture of essential oil, it was unable to confirm the synergic effects caused by mixture use. And it showed similar results to the antimicrobial activity of essential oil alone. In consequence, it was concluded that GSE, thyme and lemongrass oil could replace existing synthetic preservative effectively.

Spirulina extract is a possible candiate for cosmetic ingredients because it contains vitamin A, E and Calong with other strong antioxidatives. But its skin efficacy has not been studied well. Previously spirulina extracts fermented by Lactobacillus plantarumP23 and Bacillus subtilis TP6 were shown to have strong antioxidant activites analysed by DPPH methods. In addition, both extracts reduced significantly UV-induced TNF-aand IL-6 in cultured keratinocytes. In this study, anti-aging efficacy of the fermented spirulina extracts was investigated. The mechanism of these skin aging are closely assosiated with MMPs(matrix metalloproteinases) activities. UV-induced MMPs cause connective tissue damage, as a result, skin become wrinkled and aged. Zymography was used to determine the expression of UV-induced MMPs. Also collagen synthesis was measured in cultured human dermal fibroblasts by a sircol assay. Treatment with fermented spirulina extract resulted in the increase of collagen synthesis and reduced the expression of UV-induced MMPs in human dermal fibroblasts in vitro. The results suggest that fermented spirulina extracts have a potential as anti-aging cosmetic ingredients.

In the cascade of neurotransmission, neuronal SNARE proteins play a central role. Neuronal SNARE proteins mediate membrane fusion between presynaptic membrane and synaptic vesicle. By the result of synaptic membrane fusion, a fusion pore is made through which neurotransmitters are released. SNARE complex is thought to generate fundamental force that is required for the membrane fusion. As is well-known, a few types of botulinum neurotoxin have been developed and commercialized for the purpose of cosmetic and therapeutic uses. For example, Botox, a popular cosmetic agent, is to cleave SNARE proteins disabling neurotransmission. When neurotransmission is disabled, muscle cannot contract or relax. That is the way Botox works. So, it is that SNARE folding modulator may act like Botox by inhibiting the folding of SNARE complex not by cleaving the SNARE proteins. In the present study, we could draw some candidate SNARE folding modulators from phenolic compounds. A few compounds inhibited SNARE complex formation. As expected, the phenolic compounds inhibited membrane fusion and neurotransmitter release from PC12 cells, too. Probably, the phenolic compounds may play a role as an effective anti-wrinkle agent.

In the present work, we report the results of a study aimed at evaluating the antioxidant activity, tyrosinase activity and the acetylcholinesterase inhibitory capacity of methanol, n-hexane, CH2Cl2, EtOAc and n-buOH extracts from Alnus sp. The EtOAc extract of Alnus sp. showed the highest radical scavenging activity (DPPH test) and superoxide anion radical scavenging activity values IC50 65.83 and 14.53 /ml, respectively. The inhibition of tyrosinase activity ㎍ was higher in the EtOAc extract. The highest activity was found for EtOAc with an IC50 130.05 ㎍/ml. The plant of methanol, n-hexane, CH2Cl2, EtOAc and n-buOH extracts were tested for acetylcholinesterase inhibitory activity using the Ellman colorimetric method. The results showed that the MeOH extract, n-hexane extract and CH2Cl2 extracts from Alnus sp. at concentraction of 0.1 mg/ml inhibitied more than 80% of AChE activity. these finging may contribute to biological significance in that acetylcholinesterase (AChE) inhibitor has been used as a drug for the symptomatic treatment of Alzheimer's desease.

The comparison between surfactants, SLS showed a significant difference in all age groups when compared with other surfactants (p<0.001). The skin erythema response of SLS was seen in decreasing order of age group, aged over 50’, 20’s~40’s and teens. Although decyl-glucoside is a semi-natural surfactant, it showed high skin erythema after SLS in individuals over 50 years in age. In the comparison of formulations, the toner type formula of the four surfactants showed significantly higher skin erythema than the cream types in all age group except the visual evaluation of teens (p<0.05). Also, the mexameter and spectrophotometer presented a higher correlation with the visual assessment than LDPI. This study indicated that decyl-glucoside, one of two semi-surfactants, was thought to have higher irritant potential in the age group over 50’ than tween-80, synthetic surfactant. The skin irritant response of SLS seem to show the increasing skin erythema in the age group over 50’ than those in the age group teens and in the age group 20’s~40’s by the weakened skin barrier function in elderly. Moreover, the toner type formula was associated with an increase in damage to the skin barrier than the cram type formula and caused an increase in skin erythema. Also, the mexameter and spectrophotometer seem to be more sensitive for measuring skin irritant evaluation than the LDPI.

It is known that Spirulina extracts have strong antioxidant activities since it contain diverse antioxidants such as phytocyanin, β-carotene, vitamin E and other carotenoids. In order to obtain an enhanced antioxidant activity of Spirulina, in this study, Spirulina extracts were fermented by Lactobacillus plantarum P23 and Bacillus subtilis TP6. The resulting fermented supernatants were analyzed for their antioxidant activities by DPPH(2,2-diphenyl-1-picrylhydrazyl) method. The results indicated that fermentation process significantly enhanced total antioxidant activities. Increased levels of UV-induced TNF-α and IL-6, two of known proinflammatory cytokines, were reduced back to normal level even by treatment of all three of the spirulina extracts. However the spirulina extracts fermented by Lactobacillus plantarum P23 showed the same antiinflammatory effect at 0.01% while the unfermented extract was needed 0.1%. The result suggested that the fermentation process enhanced the antiinflammatory activities at least 10 X higher than the simple extract. In conclusion, the fermented Spirulina extracts are expected to be used as cosmeceuticals.

Neuronal SNARE proteins are crucial protein component in membrane fusion between presynaptic membrane and synaptic vesicle. The formation of SNARE assembly drives membrane fusion, leading to the formation of fusion pore through which neurotransmitters are released. SNARE complex is composed of 3 proteins(Snap25, VAMP, Syntaxin 1a) and generates fundamental force that is required for the membrane fusion. It is thought that the regulation of this SNARE assembly may inhibit membrane fusion leading to the inhibition of neurotransmitter release. In the present study, we have designed small peptide that regulates SNARE complex formation. High-throughput screening technology was also developed by mimicking SNARE-driven membrane fusion in vitro. Then, the developed fusion assay was applied to measure the efficacy of synthetic peptides on membrane fusion. Among various 17mer and 6mer peptides tested, we could draw 6mer synthetic peptide that harbors much stronger membrane fusion inhibition efficacy than the well-known "Argirelline." The found peptide might function as anti-wrinkle agent by blocking SNARE complex formation while Botox cleaves SNARE proteins. Further, the found peptide might be developed to potential pain-killer.

Silk Fibroin is made up of multiple repeats of the sequence of Ala–Gly–Ala–Gly–Ser-Gly-1),2). Thus, the compositions of the hydrolyzed peptides was found to be mixtures of short peptides varying by two residues of amino acid3). The hydrolyzed peptides of silk fibroin were hardly separated by conventional chromatography, because of structural similarity. This report describes the determination of the major components of fractionated silk peptides using isopropanol by Electrospray Ionization-Time Of Flight Mass Spectrometer(ESI-TOF MS). Silk peptide was solubilized in varying alcohol/water mixtures and precipitates were isolated. The yield of isolated peptide was determinated by dry weight and by UV-Vis Spectrometer. The peptide compositions of the supernatant and the precipitate were identical in ethanol. However, the major composition of silk peptide of Gly, Ala, Ser, Tyr and Val residues was concentrated in the supernatant more than in the precipitate when using isopropanol. Thus, isopropanol solubilization can be used to enrich the composition of major peptide residues.

Bisethylhexyloxyphenolmethoxyphenyltrizine(BEMT) is one of the most widely used ydrophobic UVA absorber in sunscreen cosmetic product. But topical dermal application of BEMT is restricted due to its poor solubility in the cosmetic product. In this study, organic UV absorber loaded Solid lipid nano-particles(SLN) were performed in a way that release was prolonged and improving the solubility.(1,2) The maximum loading capacity of BEMT-SLN was obtained formulation using polyol 50%.(HPLC analysis) The average particle size for all formulation were in the range of 140 ∼ 500nm. To compare with the IR pattern of BEMT free and BEMT loading SLN, we used cetyl palmitate and obtained satisfactory result. Through the DSC analysis obtained crystallinity index(C.I) was small, CI value of lipid 4% formulation obtained 9.14%, meaning that lipid matrix might be in a super-cooled melting state. Ultimately it appeared that Solid lipid nano-particle was the most effective to adopt BEMT and may be used for a useful sunscreen carrier system.

Mungbean(Phaseolus radiatus) has been used for the relief of heat and blood pressure & cholesterol, promotion of urination, treatment of pesticide and lead poisoning and treatment of burn. Especially, it is known to have distinguished strong activity for various skin diseases. GABA is known as a neurotransmitter. It was recently reported that topical application of GABA accelerated barrier recovery on damaged skin and played an important part on skin barrier homeostasis. In this study, mungbean extract was fermented by Lactobacillus sakei B2-16 a GABA producing Lactobacillus strain. The resulting fermentation product contained up to 4 % GABA, organic acids such as lactic acid and mungbean peptides naturally hydrolyzed by a unique fermentation process. Antiinflammation efficacy of the fermented mungbean was analyzed by in vitro and in vivo using animal model and found that it had a strong anti-inflammatory activity. In addition. it markedly enhanced the collagen synthesis in cultured fibroblasts. Results showed that the mungbean fermentation extract could be used for cosmetics as a skin soothing active ingredient.

Background/purpose: A recently developed method to estimate skin smoothness is the replica method which may have the limitation of the roughness difference of actual skin due to the skin-replicating process. Therefore, observation of dermal layer change is very important. For this purpose, ultrasonic display equipment is generally used. The purpose of the present study was to investigate the correlation between skin roughness and dermal density in wrinkle evaluation. Methods: We evaluated the crow’s feet of 95 Korean females using mechanical assessments; Skin-Visiometer SV 600 and Dermascan C. Transparency profilometry (Skin Visiometer) use a very thin skin print, which allows parallel light to pass through and is analyzed immediately after production. High-frequency (20MHz) ultrasonography (Dermascan C) enables non-invasive evaluation of skin thickness and echodensity. Results: We found a correlation between skin roughness and dermal density. Particularly, we found a significant correlation between skin roughness (R2) and dermal thickness. Also, we found a significant negative correlation between dermal density and dermal thickness (p<0.05). Conclusion: Therefore, the ultrasonography system may be considered a very useful method in wrinkle evaluation with the transparency profilometry. However, further study will be required.

Active compounds of coffee were prepared by supercritical fluid extraction. Various active compounds of coffee, such as fatty acids, α-tocopherol, and β -sitosterol were extracted by supercritical fluid at temperature of 50℃∼70℃ and pressure of 250 bar∼400 bar. The mechanism of melanogenesis by extracts were investigated. Inhibitory effects on melanogeneis were observed with in vitro cultures of B16 melanoma cells. Proteome analysis were made to explain the inhibitory melanogenesis by supercritical fluid extracts. B16 melanoma cells treated with and without extracts were analyzed using 2-D PAGE. Isoelectric focusing was carried out using commercial Immobiline Dry Strips 3-10NL 13cm and SDS-PAGE was made using 18⨯24 cm gels1). Gels were stained with silver nitrate and analyzed by Image Master program2).

Skin aging appears to be principally related to a decrease in the levels of Type I collagen, the primary component of the skin dermis. Asiaticoside, a saponin component which has been isolated from Centella asiatica, has been shown to induce Type I collagen synthesis in human dermal fibroblast cells. However, the mechanism underlying asiaticoside-induced Type I collagen synthesis, especially at a molecular level, remains only partially understood. In this study, we have attempted to characterize the action mechanism of asiaticoside in Type I collagen synthesis. Asiaticoside was determined to induce the phosphorylation of both Smad 2 and Smad 3. In addition, we detected the asiaticoside-induced binding of Smad 3 and Smad 4. In a consistent result, the nuclear translocation of the Smad 3 and Smad 4 complex was induced via treatment with asiaticoside, pointing to the involvement of asiaticoside in Smad signaling. In addition, SB431542, an inhibitor of the TGFβ receptor I (TβRI) kinase, which is known to be an activator of the Smad pathway, was not found to inhibit both Smad 2 phosphorylation and type 1 collagen synthesis induced by asiaticoside. Therefore, our results show that asiaticoside can induce Type I collagen synthesis via the activation of the TβRI kinase-independent Smad pathway.

Inhibitory effects and mechanism of aging by natural products were studied using proteome analysis. The fibroblast were cultured in Dulbecco's Modified Eagle's Medium(DMEM) with or without natural products. The proteome of cultured human fibroblast were profiled using 2D-PAGE. Isoelectric focusing was carried out using commercial immobiline dry strips 3-10NL 13cm. SDS-PAGE was performed using 18 x 24cm, 10.5 % acrylamide gel1). Gels were stained with silver nitrate, analyzed by image master 2D software program. In conclusion, we could identify that many factors such as collagen, MMPs, which affect the skin aging, were up or down-regulated by natural products2).

Betula platyphylla var. japonica has been used as a Chinese medicine for diuresis and alleviation of pain and fever. Recently antioxidant and anticancer activity were found in the extract from Betula platyphylla var. japonica.1) In order to seek other functions of it, its bark extacted by hot water and methanol, and then we performed some experiments with it. At a non-cytotoxic dose it was found that the extract is effective on inhibition of tyrosinase activity which plays a key role in melanin synthesis. Moreover the extract attenuated the amount of melanin sythesis in B16 melanoma cells. By western blotting we investigated the expression level changes of tyrosinase, TRP1, TRP2 and PAR2, which is related to translocation of melanosome.2) However, there were no differences between the extract-treated cells and control cells in the changes of those expression levels. Taken together, the Betula platyphylla extract has depigmentation activity of skin and the depigmentation mechanism is not to block the translocation of melanosome mediated by PAR2 nor to change the expression level of tyrosinase but to inhibit the activity of tyrosinase.

We investigated the effect of a novel phytosphingosine derivative, N-acetyl phytosphingosine (NAPS), on inhibition of α-MSH (melanocyte-stimulating hormone) induced-melanogenesis in B16F1 melanoma. At non-cytotoxic concentrations, NAPS decreased the contents of melanin overproduced by α-MSH induction in both intracellular and the secreted form of it. Furthermore, we observed that NAPS reduced the activity of intracellular tyrosinase increased by α-MSH. Accordingly, we examined whether NAPS could have influence on the expression of melanogenic proteins. Western blot analysis demonstrated that NAPS decreased the expression of tyrosinase and its key transcription factor, microphthalmia-associated transcription factor (MITF), in a concentration-dependent manner. Taken all together, NAPS showed the inhibitory effect on α-MSH-induced melanogenesis through the down-regulation of MITF-tyrosinase expression. Thus, it suggests that NAPS may show skin whitening effect, exerting anti-melanogenic activity as a potent hypopigmenting agent.

Exposure to UV appears to be the most important environmental factor involved in the development of skin photoaging. It has been known that oxidative stress induced by UV lead to the skin cell death, the formation of reactive oxygen species(ROS), and the increase of matrix metalloproteinases (MMPs). In our previous study, it has been shown that silkworm hemolymph extract(SHEX) has anti-oxidative activities as determined by in vitro assay. In this study, we investigated the effect of SHEX on oxidative stress induced by UV in immortalized human keratinocytes, HaCaT. Treatment of HaCaT cells with SHEX inhibited UV-induced cell death and also inhibited the formation of ROS, which was determined using DCFH-DA. Moreover, there was a reduction in the expression of MMP-1(interstitial collagenase) induced by UV. To identify the antioxidant compounds, SHEX was separated by gel permeable chromatography. Seven fractions were obtained, and the anti-oxidative activities of each fraction were assayed. These results suggest that SHEX contains some components protecting human skin cells against oxidative stress induced by UV and such components will be used as cosmetic supplements.

Type I collagen is the primary component of the skin dermis. Both the quantity and quality of extracellular collagen are primarily related to skin ageing. In this study, we investigated the possibility that Camellia japonica oil (CJ oil) may be introduced as an anit-wrinkle agent. As a first step to this end, human COL1A2 promoter luciferase assay was performed in human dermal fibroblast cells. CJ oil was determined to activate human COL1A2 promoter in a concentration-dependent manner. In a consistent with this result, while MMP (matrix metalloproteinase)-1 activity was inhibited by CJ oil, human type I procollagen synthesis was also induced by CJ oil. These results suggest the possibility that CJ oil may be involved in the skin ageing. For the evaluation of CJ oil’s safety and efficiency on human skin, human skin primary irritation test and trans-epidermal water loss (TEWL) were performed. Transepidermal water loss (TEWL) was measured before treatment then, 1 h and 2 h after treatment; the forearm site was selected to measure TEWL. Also, a human skin primary irritation test was performed on the normal skin (upper back) in 30 volunteers to see if a certain material included in CJ oil has irritation or sensitization potential. In these assays, CJ oil reduced trans-epidermal water loss (TEWL) and did not induce any adverse reactions. Therefore, based on these results, we suggest the possibility that CJ oil may be considered as possible wrinkle-reducing candidates for topical application.

Background: In a recent study, human skin tests to evaluate the wrinkles’ improvement or whitening effect were performed by previous study guideline. Concerning the data of the visual assessment and machinery evaluation of the result for the human skin test, unpaired t-test have been used in order to compare between the test and the control groups and paired t-test for the comparison of effects for before and after treatment. The methodology and statistical analysis of American and European test centers were similar to ours. The data obtained by repeated application from identical individual has high relation. For this reason it is desirable to apply repeated measures ANOVA (RM ANOVA) to visual assessment and machinery evaluation. Objective: The purpose of this study was to compare the usability between the RM ANOVA and the t-test. Methods: In the present investigation we researched the clinical trial methodology, represents the clinical trial of phase III study and for the propriety investigation of statistical analysis methods for human test data of cosmetics about Repeated measures ANOVA. Results: Our results, which were confirmed by the machinery evaluation data, showed significant improvement by repeated measures ANOVA. But this data was not statistically significant in the unpaired t-test and paired t-test. Conclusion: We suggested that RM ANOVA is the new approach to statistical analysis of human test data of cosmetics.

A cell-based assay system for monitoring COL1A2 promoter activity was developed to determine the influence of activated COL1A2 promoter in human dermal fibroblast cells. A pLuc-COL1A2 promoter plasmid that expresses the luciferase reporter gene in response to COL1A2 promoter activity was constructed. The pLuc-COL1A2 promoter plasmid and pCI-neo plasmid containing the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells were cotransfected into human dermal fibroblast cells. COL1A2 promoter activities were measured by luciferase reporter gene assay using a luminescence detection method. Fibroblast cell transfectants treated with TNF-α, known to be an inhibitor of COL1A2 promoter expression, showed a reduction of COL1A2 promoter activity in a concentration-dependent manner, whereas TGF-β, known to be a stimulator of COL1A2 promoter expression, increased COL1A2 activity in a concentration-dependent manner. This assay system could be used to quantitatively measure COL1A2 promoter activity in human dermal fibroblast cells and allow the screening of anti-wrinkle agents from various synthetic chemicals and natural products.