• The Korea Journal of Herbology
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• v.27 no.3
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• pp.67-74
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• 2012

Objectives : The present study was designed to investigate effects of Ssanghwa-tang (Shu$\bar{a}$nghu$\bar{a}$-g$\bar{e}$ng) on oxidation/reduction reaction-related and aging-related enzymes $in$ $vitro$. Methods : We performed MTT assay, collagenase inhibition assay, elastase inhibition assay, tyrosinase inhibition assay, DPPH free radical scavenging assay, SOD-like activity and xanthine oxidase (XO) inhibition assay. Results : The 50% ethanol (EtOH) extract of Ssanghwa-tang (SHT) showed 55% inhibition of collagenase activity, and 42% inhibition of elastase activity at 1 mg/ml concentration. Also it's treatment showed 18% inhibition of tyrosinase activity, to relate whitening effect, at the same dose of 50% ethanol extract of SHT. Antioxidant activities were determined by DPPH radical scavenging, XO inhibiting activity and SOD-like activity. These scavenging, XO-inhibiting and SOD-like activities were measured in 80%, 75%, and 28% inhibitions, respectively, at a 1 mg/ml treated dose, compared to those of control. The inhibitory effects of 50% EtOH extract on aging and oxidation-related enzyme activities were higher than those of water extract and 95% EtOH extract. Conclusions : Taken together, our findings suggest that the SHT has potential and applicable benefits for development of cosmetics to have anti-aging (anti-wrinkle and whitening) and anti-oxidation functions.

• Advances in Traditional Medicine
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• v.6 no.2
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• pp.134-143
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• 2006

The capacities of bacterial DNA, extracted from Salmonella typhimurium, and lipopolysaccharide (LPS), extracted from Salmonella minnesota, to activate mouse peritoneal macrophages in vitro were compared. Activation was assessed by estimating e levels of 3 cytokines, IL-10, IL-12, and $IL-1{\beta}$, at time intervals of 3, 6, 9, and 24 h after addition of LPS and/or DNA to macrophage cultures. Cytokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA levels were estimated based on band intensity in cultured cells by reverse transcriptase-polymerase chain reaction (RT-PCR). Results obtained demonstrated the ability of DNA and LPS to elicit increased production of all 3 cytokines as compared to controls. In the amount tested, LPS appeared to be a more potent inducer of IL-12, and $IL-1{\beta}$, whereas DNA induced higher levels of IL-10. DNA and LPS, used in combination, exhibited neither an additive nor a synergistic effect. Rather, an antagonist effect appeared to occur. RT-PCR results correlated well with ELISA.

• The Plant Pathology Journal
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• v.19 no.3
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.372-379
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• 2017

The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or $100{\mu}M$ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with $10{\mu}M$ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.210-215
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• 2011

Objective: This study was performed to identify whether growth and differentiation factor-9 (GDF-9) and transforming growth factor-${\beta}1$ (TGF-${\beta}1$) expressions would be lower in the follicular fluid (FF) of those over age 35 who underwent IVF than under age 35. Methods: A total of 24 IVF cycles (20 patients) were included in this study. All of patients were stimulated for IVF by the GnRH short protocol and divided into two groups for analysis, according to their age: <35 group (14 cycles, 11 patients) vs. ${\geq}35$ group (10 cycles, 9 patients). The expression levels of GDF-9 and TGF-${\beta}1$ were determined by western blotting and quantitative enzyme-linked immunosorbent assay. Results: The numbers of retrieved oocytes and metaphase II oocytes were significantly lower in the ${\geq}35$ group. Lower expression of GDF-9 and TGF-${\beta}1$ by western blotting in the ${\geq}35$ group were observed as well. The mean GDF-9 and TGF-${\beta}1$ levels by enzyme-linked immunosorbent assay were lower in the ${\geq}35$ group. The values were $6,850.5{\pm}928.4$ ng/L vs. $3,333.3{\pm}1,089.2$ ng/L of GDF-9 ($p$ <0.05) and $3,844.1{\pm}571.1$ ng/L vs. $2,187.7{\pm}754.0$ ng/L of TGF-${\beta}1$ ($p$ <0.05). A negative correlation between GDF-9 and age was observed (r=-0.546, $p$=0.006). Conclusion: GDF-9 and TGF-${\beta}1$ production from stimulated ovaries during IVF appears to decrease with age.

• Herbal Formula Science
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• v.25 no.4
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• pp.537-551
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• 2017

Objectives : Jinnoe-san (JNS) is a novel herbal formula consisting of five oriental medicinal herbs including Polygalae Radix, Prunellae Spica, Perillae Herba, Betulae Cortex, and Lonicerae Flos. In this study, we investigated the effects and molecular mechanism of JNS on Parkinson's disease in vitro model. Methods : The effects of JNS on 1-methyl-4-phenylpyridinium ($MPP^+$)-induced cell death in SH-SY5Y cells were evaluated with a cell viability assay, flow cytometry, and western blots analysis. The effects of JNS on lipopolysaccharide (LPS)-stimulated BV2 microglia were determined with a nitric oxide (NO) assay, enzyme linked immunosorbent assays, and western blots analysis. Result : $MPP^+$-induced cell death in SH-SY5Y cells was significantly reduced by JNS pre-treatment in a dose-dependent manner. JNS inhibited the production of reactive oxygen species, mitochondria dysfunction, and apoptosis induced by $MPP^+$ in SH-SY5Y cells. Furthermore, JNS significantly activated Akt and ERK in SH-SY5Y cells and the ability of JNS to prevent mitochondria dysfunction by $MPP^+$ was antagonized by pre-treatment of LY294002 and PD98059, an Akt and ERK inhibitor, respectively. In addition, JNS inhibited LPS-induced NO and $PGE_2$ production as well as iNOS expression and secretion of TNF-${\alpha}$, pro-inflammatory cytokines without affecting the cell viability. JNS also suppressed LPS-induced ERK activation. Conclusions : These results demonstrate that JNS has a protective effect on the dopaminergic neurons against $MPP^+$-induced neurotoxicity and anti-inflammatory effect on the LPS-stimulated microglia. These findings provide evidences for JNS to be considered as a new prescription for treating Parkinson's disease.

• BMB Reports
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• v.35 no.3
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• pp.348-351
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• 2002

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed $\alpha$ and $\beta$) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the $\alpha$ and $\beta$ subunits of M.AquI into expression vectors. The overexpressed His-fusion $\alpha$ and $\beta$ subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The $\alpha$ and $\beta$ subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-L-methionine to DNA is reconstituted. We also showed that the $\beta$ subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the $\alpha$ subunit.

• The Korean Journal of Food And Nutrition
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• v.25 no.2
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• pp.246-252
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• 2012

After a mixed carbohydrate diet, inhibition of ${\alpha}$-amylase and ${\alpha}$-glucosidase involved in the digestion and absorption of carbohydrates can significantly decrease the postprandial increase of blood glucose level. In the course of screening these useful enzyme inhibitors, we selected five kinds of bean, using an in-vitro enzyme inhibition assay method. To evaluate the effect of germination process on the functionality of the bean, we investigated the inhibitory activities of the water extracts of non-germinated bean and germinated bean against ${\alpha}$-amylase and ${\alpha}$-glucosidase, relevant to postprandial hyperglycemia. We also investigated the oxygen radical absorbance capacity(ORAC), total phenolics content, and postprandial blood glucose lowering effect in rats(Sprague-Dawley rat model). Most germinated beans showed significantly higher ${\alpha}$-glucosidase inhibitory activity, compared with non-germinated beans. Among germinated beans, Glycine max had the highest ${\alpha}$-glucosidase inhibitory activity(53.3%). The water extract of germinated Phaseolus vulgaris L. had the highest ${\alpha}$-amylase inhibitory activity(95.1%), followed by Glycine max(58.7%), and Glycine max L. Merr(54.1%). Furthermore, the five germinated beans also showed high antioxidant activities in ORAC assay. Results suggested that the germination process may improve and enhance the anti-hyperglycemia potential and antioxidant activity of the bean.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.28 no.1
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• pp.135-149
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• 2002

To date, research on the regulation of melanogenesis has focused on factors which affect tyrosinase, the rate-limiting enzyme in the melanogenic pathway, by searching for chemicals which competitively inhibit tyrosinase function. Many types of tyrosinase inhibitors have been developed, but no satisfactory results have been made clinically until now, To find a new whitening agent, which effectively inhibits melanogenesis, we synthesized several compounds and selected compounds by cell-based assay system. Finally, 3, 4, 5-trimethoxy cinnamaie thymol ester(TCTE, Melasolv) was selected and the effects of TCTE on melanogenesis were investigated. Treatment of mouse-derived melanocyte melan-a cells with TCTE results in a marked down-regulation of tyrosinase activity. 80% decrease of tyrosinase activity occurs with 30uM TCTE treatment for 72 hours without affecting cell growth. The inhibition of tyrosinase activity is dose-dependent and melanin content was also decreased to 40%. From the in vitro tyrosinase assay using cell extract, TCTE does not act as a direct inhibitor of the enzyme. Treatment of melan-a cultures with TCTE blocks the increase in tyrosinase activity by either forskolin, 3-isobutyl-1-methtyl-xanthine. TCTE decreased the expression of tyrosinase, TRP-1 without effects on TRP-2 protein expression through the down regulation of tyrosinase and TRP-1 mRNA. From the results of cAMP immunoassays, intracellular levels of the cyclin nucleotide are unaffected in cells treated with TCTE. The inhibitory effects of melanin synthesis were also shown in reconstitute human epidermis model by topical application. These findings suggest that TCTE can be used for studying the regulation of melanogenesis and depigmenting agent.

• Korean Journal of Pharmacognosy
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• v.37 no.3
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• pp.200-205
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• 2006

Ecklonia cava is brown alga which ow in the sea all over the world. It has been shown that Ecklonia cava has antioxidative, anticoagulant, and antiviral effect, lately. In this study, we examined the antihypertensiye effect of Ecklonia cava extract (Eca) on 2-kidney 1-clip Goldblatt Hypertensive Rats (GB-HT). In vitro assay, the angiotensin converting enzyme (ACE) activity was significantly inhibited by Eca (27-95% inhibition at $10-50\;{\mu}g/ml$). The systolic blood pressure of Eca-treated GH-HT significantly was decreased compared to that of control CB-HT (15% decrease at 50 mg/kg). The serum ACE activity was also inhibited 19% with 50 mg/kg Eca treatment. These results suggest that Eca can be used as antihypertensive functional foods and nutraceuticals.