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Genomic analysis of Mycobacterium fortuitum by pulsed-field gel electrophoresis (Pulsed-field Gel Electrophoresis를 이용한 Mycobacterium fortuitum의 유전형 분석)

  • Lee, Tae-Yoon;Do, In-A;Kim, Sung-Kwang
    • Journal of Yeungnam Medical Science
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    • v.12 no.2
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    • pp.366-385
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    • 1995
  • Epidemiological studies are important in both the prevention and treatment of mycobacterial infections. This study was initiated to establish the pulsed-field gel electrophoresis (PFGE) method, which are not yet extensively studied. The most apprpriate restriction endonucleases included DraI, AsnI, and XbaI. The optimal PFGE condition was different according to the enzymes used. Two stage PFGE was performed, in case of DraI first stage was performed with 10 seconds of initial pulse and 15 seconds of final pulse, while the second stage was performed with 60 seconds of initial pulse and 70 seconds of final pulse. The electrophoresis time for DraI-PFGE was 14 hours for each stage. Electrophoresis was performed for 22 hours, in case of XbaI, with 3 seconds of initial pulse and 12 seconds of final pulse. Electrophoresis was performed for 22 hours, in case of AsnI, with 5 seconds of initial pulse and 25 seconds of final pulse. In all cases the voltage of the electrophoresis was maintained constantly at 200 voltage. Standard mycobacterial strains, which included Mycobacterium bovis BCG, M. tuberculosis, and M. fortuitum, could not be differentiated by PFGE analysis. PFGE analysis was performed to differentiate 9 clinically isolated M. fortuitum strains using AsnI. All M. fortuitum strains showed different genotypes except 2 strains. Cluster analysis divided M. fortuitum strains into 2 large groups. PFGE analysis was performed to further differentiate M. fortuitum isolates using XbaI. The undifferentiated 2 M. fortuitum strains showed different PFGE patterns with Xba I. Cluster analysis of the XbaI-PFGE patterns showed more complex grouping than AsnI-PFGE patterns, which showed that XbaI-PFGE analysis was better than AsnI-PFGE in M. fortuitum genotyping. The top dissimilarity values of AsnI-PFGE and XbaI-PFGE were 0.74 and 0.75, respectively. This value was higher than that of arbitrarily primed polymerase chain reaction (AP-PCR) analysis and lower than that of restriction fragment length polymorphism (RFLP) analysis. This suggested that PFGE can be used as a supportive or alternative genotyping method to RFLP analysis.

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Inhibitory Effect of Metal Surface on the Antimicrobial Resistance Microorganism (금속표면이 항생제 내성균주의 생육억제에 미치는 영향)

  • Kim, Jung-Beom;Kim, Jae-Kwang;Kim, Hyunjung;Cho, Eun Jung;Park, Yeon-Joon;Lee, Hae Kyung
    • Annals of Clinical Microbiology
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    • v.21 no.4
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    • pp.80-85
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    • 2018
  • Background: The aim of this study was to comparatively evaluate the bactericidal effects of copper, brass (copper 78%, tin 22%), and stainless steel against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VREFM), and multidrug-resistant Pseudomonas aeruginosa (MRPA). Methods: The isolates (MRSA, VREFM, MRPA) used in this study were mixed wild type 3 strains isolated from patients treated at Uijeongbu St. Mary's Hospital in 2017. These strains showed patterns of multidrug resistance. The lyophilized strains were inoculated into and incubated for 24 hr in tryptic soy broth at $35^{\circ}C$. The initial bacterial inoculum concentration was adjusted to $10^5CFU/mL$. A 100-mL bacterial suspension was incubated in containers made of brass (copper 78%, tin 22%), copper (above 99% purity), and stainless steel at $35^{\circ}C$. Viable counts of bacteria strains were measured for 9 days. Results: In this study, the bactericidal effects of copper and brass on MRSA, VREFM, and MRPA were verified. The bactericidal effect of stainless steel was much weaker than those of copper and brass. The bactericidal effect was stronger on MRPA than on MRSA or VREFM. Conclusion: To prevent cross infection of multidrug resistant bacteria in hospitals, further studies of longer duration are needed for testing of copper materials on objects such as door knobs, faucets, and bed rails.

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens (임상 객담검체에서 Peptide Nucleic Acid Probe를 이용한 결핵과 비결핵 항산균의 구분)

  • Lee, Seung Hee;Kim, Shine Young;Kim, Hyung Hoi;Lee, Eun Yup;Chang, Chulhun L.
    • Annals of Clinical Microbiology
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    • v.18 no.2
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    • pp.37-43
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    • 2015
  • Background: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. Methods: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. Results: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. Conclusion: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears.

Single Crossing Condition of Miscanthus sacchariflorus and Miscanthus sinensis to Breed Miscanthus x giganteus Cultivar (이질3배체 억새(Miscanthus x giganteus) 품종육성을 위한 물억새(M. sacchariflorus)와 참억새(M. sinensis) 단교배 조건구명)

  • Moon, Youn-Ho;Kim, Kwang-Soo;Lee, Ji-Eun;Kwon, Da-Eun;Kang, Yong-Ku;Cha, Young-Lok
    • Korean Journal of Plant Resources
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    • v.32 no.5
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    • pp.509-518
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    • 2019
  • This study was conducted to investigate single crossing condition of M. sacchariflrous and M. sinensis for breeding of M. ${\times}$ giganteus cultivar. Compared with natural day length condition, cultivation in short day length condition shorten days to heading to 18~27 days in both species. Pollen germination ratio of were 75.8% at 6 o'clock in M. sacchariflorus and 51.9% at 7 o'clock in M. sinensis but decreased to below 10% at 8 o'clock in both species. When cut ears immerged in 150 mL of cut-flowers conservation solution and isolated with covering of white non-woven fabric, flowering and pollen dispersal were persisted for 7 days, and the ratio of pollen germination were above 40% for 4 days. The ratio of self-fertilization of both species were below 2.5%, but open pollenation ratio were above 50%. We obtained 437 seeds with experimental single cross of 14 combinations between tetraploid M. sacchariflorus and diploid M. siensis by application of developed single crossing methods. In the single cross, numbers of seed set were different by mother plants. Thus, the newly investigated single crossing condition will be used to breed M. ${\times}$ giganteous cultivar which is sterile and has superior characteristics of biomass yield.

Comparison of Gene Expression Profile in Eutopic Endometria with or without Endometriosis: A Microarray Study (자궁내막증 환자와 대조군에서의 자궁내막 유전자 발현의 차이: Microarray를 이용한 연구)

  • Chung, Min-Ji;Chung, Eun-Jung;Lee, Shin-Je;Kim, Moon-Kyu;Chun, Sang-Sik;Lee, Taek-Hoo
    • Clinical and Experimental Reproductive Medicine
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    • v.34 no.1
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    • pp.19-31
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    • 2007
  • Objective: Pathogenesis of the endometriosis is very complex and the etiology is still unclear. Our hypothesis is that there may be some difference in gene expression patterns between eutopic endometriums with or without endometriosis. In this study, we analyzed the difference of gene expression profile with cDNA microarray. Methods: Endometrial tissues were gathered from patients with endometriosis or other benign gynecologic diseases. cDNA microarray technique was applied to screen the different gene expression profiles from early and late secretory phase endometria of those two groups. Each three mRNA samples isolated from early and late secretory phase of endometrial tissues of control were pooled and used as master controls and labeled with Cy3-dUTP. Then the differences of gene expression pattern were screened by comparing eutopic endometria with endometriosis, which were labeled with Cy5-dUTP. Fluorescent labeled probes were hybridized on a microarray of 4,800 human genes. Results: Twelve genes were consistently over-expressed in the endometrium of endometriosis such as ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ transporting (ATP5C1) and TNF alpha factor. Eleven genes were consistently down-regulated in the endometriosis samples. Many extracellular matrix protein genes (decorin, lumican, EGF-containing fibulin-like extracellular matrix protein 1, fibulin 5, and matrix Gla protein) and protease/protease inhibitors (serine proteinase inhibitor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1), and insulin like growth factor II associated protein were included. Expression patterns of selected eight genes from the cDNA microarray were confirmed by quantitative RT-PCR or real time RT-PCR. Conclusion: The result of this analysis supports the hypothesis that the endometrium from patients with endometriosis has distinct gene expression profile from control endometrium without endometriosis.

Cancer Chemopreventive Effects of Ginsenoside $Rg_3,\;Rg_5,\;Rh_2$ and BST from Enzymatically Fermented Korean Ginseng Extract

  • Yun Taik-Koo
    • Proceedings of the Ginseng society Conference
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    • 2002.10a
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    • pp.35-46
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    • 2002
  • Panax ginseng C. A. Meyer has been one of the most highly recognized medicinal herbs in the Orient. Previous experiments have demonstrated that $Rg_3,\;and\;Rg_5$ statistically significantly decreased the incidence of benzo(a)pyrene-induced mouse lung tumor, $Rh_2$ showed tendency of decrease and $Rh_1$ showed no effect. It was, therefore, concluded that $Rg_3,\;Rg_5\;and\;Rh_2$ are active cancer chemopreventive components in red ginseng and they either singularly or synergistically act in the prevention of cancer. This study was undertaken to compare the cancer chemopreventive effects of $Rg_3,\;Rg_5\;and\;Rh_2$(purity: more than $60\%$) isolated from fermented ginseng extract and BST fermented ginseng with fortified ginsenoside $Rg_3\;and\;Rh_2$. The cancer chemopreventive effects were investigated in experimental groups treated with benzo(a)pyrene(BP) with ginsenoside $Rg_3,\;Rg_5\;Rh_2\;or\;BST$ at three doses of $50^{\circ}C/ml,\;100^{\circ}C/ml\;and\;200^{\circ}C/ml$ When mice given with $50^{\circ}C/ml$ concentration of ginsenoside $Rg_3$ combined with BP for 6 weeks after BP administration, $Rg_3\;showed\;60\%$ of lung tumor incidence, where as $100^{\circ}C/ml\;and\;200^{\circ}C/ml\;of\;Rg_3$ combined with BP groups had significant decrease of incidence $(40.0\%)$ respectively, with the inhibition rate being $35.5\%.$ While the tumor incidence was not decreased in the group treated with BP and 50 of $Rg_5,$ the incidence was $34.0\%\;and\;32.0\%$ in the group treated with BP and 100 and 200 of $Rg_5$, respectively. These incidences were significantly less than the group treated with BP alone, with the inhibition rate being $45.2\%\;and\;48.4\%,$ respectively. On the other hand, in the group treated with BP and 50 of ginsenoside $Rh_2,$ the tumor incidence was not decreased. However, the incidence was $40.0\%\;and\;38.8\%$ in the experimental treated with BP and 100 and 200 of $Rh_2,$ respectively, with the inhibition rate being $45.2\%\;and\;48.4\%,$ respectively. In addition, the incidence showed the tendency to decrease in the experimental group treated with BP and 50 of BST which contained $16.2\%\;of\;Rh_2,\;15.4\%\;of\;Rg_3\;and\;2.5%\;of\;Rg_5.$ The tumor incidence was $54.0\%$ in this group. In the group treated with 100 and 200 of EST, the incidence was $34.0\%\;and\;30.0\%,$ respectively, the incidences significantly being lower than the group treated with BP alone, with the inhibiting rate being $45.2\%\;and\;51.6\%,$ respectively. The results of this study strongly suggested that ginsenoside $Rg_3,\;Rg_5\;and\;Rh_2$ are the active components of red ginseng having a cancer chemopreventive activity and $Rg_5$ is the strongest cancer chempopreventive among them. On the other hand, the results demonstrating that the incidence of lung tumor was more markedly reduced by BST fermented ginseng with fortified ginsenoside $Rh_2\;or\;Rg_3$ compared to the single component alone, suggest that the combination of these components may remarkablely improve the cancer preventive effect

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Selection of Preventers of Rusty Ginseng Roots from Natural Resources (천연자원으로부터 인삼 적변방제물질의 선발)

  • Ban, Sung-Hee;Shin, Sun-Hee;Woo, Hyun-Jung;Yang, Deok-Cho
    • Journal of Ginseng Research
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    • v.26 no.2
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    • pp.89-95
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    • 2002
  • We screened biotic and abiotic preventative,i(preventers) from natural resources to prevent the rusty phenomenon in ginseng roots. To select preventatives(preventers), soil microbes such as Agrobacterium and certain microbes isolated from the rusty ginsengs and the soil in which the rusty ginsengs were planted and used. It is also performed with germination tests of the seeds of Latuca Sativa L. We identified that how selected preventatives(preventers) effect the germination of ginseng seeds. Furthermore, how these influence on the rusty phenomenon and the growth of 1 -year-old ginsengs treated in the pavement. The final preventatives; ICPE-C1$\sub$05/, ICPE-P$\^$107/ were effective in not only the growth of ginseng, but also inhibition of the rusty phenomenon. Moreover, we selected abiotic soil improvers; called P, R, and W, respectively; to promote the effects of preventatives. R and W was excellented among choring improvers. The germination rate of 2-year-old ginsengs treated with ICPE-C$\sub$105/P, and ICPE-P$\sub$107/P was the highest under the effects of naturally selected preventatives mixing with abiotic soil improvers. All treat which was compounding preventers & improvers were so excellented of growth ginseng. Especially treats of ICPE-C$\sub$105/R and ICPE-P$\sub$107/R showed growth increased of each 67.3% and 52.7% As well, the growth of ginseng was the highest in the treatment of ICPE-C$\sub$105/R, and ICPE-P$\sub$105/R. Though rusty of rate was emerged 35% in control, preventers ICPE-C$\sub$105/R and ICPE-P$\sub$107/R were emerged 5.3%. It was affirmed effective of preventer. On the other hands, amounts of ginsenoside treated with preventatives showed to be changed. The ginsenoside was increased to 14.2% with treatment with ICPE-P$\sub$107/R which is highest among groups compared to control, and ICPE-C$\sub$105/P was increased to 5.0%. To sum up with total results, it is judged that biotic preventatives (ICPE-C$\sub$105/R, and ICPE-P$\sub$107/R) which we created improve both a high yield of ginseng and the inhibition of the rusty phenomenon. phenomenon.

New Whitening Agent : Selina-4(14), 7(11)-dien-8-one (신규 미백제 : Selina-4(14), 7(11)-dien-8-one)

  • Kim, Cheong-Taek;Chang, Yun-Hee;Lee, Sang-Hwa;Kang, Sang-Jin;Cho, Wan-Goo
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.31 no.1 s.49
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    • pp.17-23
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    • 2005
  • We had previously reported that Selina (selina-4(14), 7(11)-dien-8-one) was isolated from methanol extract of Afractylodes rhizome and has strong whitening activity in B16 melanoma cells. In this report, we demonstrated its action mechanism in melan-a cells, non-tumorigenic melanocytes. We also investigated the clinical efficacy of cosmetic preparation containing Selina. Selina reduced the melanin synthesis of Melan-a cells by $50\%$ at a concentration of $10 {\mu}g/mL$ without any apparent cytotoxicity. We also found that the treatment of cells with Selina decreased tyrosinase activity by $60\%$ at a concentration of $10 {\mu}g/mL$ but Selina was not a direct inhibitor of tyrosinase activities. To elucidate the action mechanism of Selina, we investigated the changes in mRNA and protein level of tyrosinase, TRP-1 and TRP-2 using RT-PCR and western blotting, respectively. As a result, the mRNA and protein level of tyrosinase were markedly reduced at $10 {\mu}g/mL$ of Selina without any effect on TRP-1 and TRP-2. These results suggest that Selina exerts its whitening effect mainly through regulating expression of tyrosinase. A 7 week-clinical trial using formulation containing $0.2\%$ selina-4(14), 7(11)-dien-8-one with 20 volunteers resulted in statistically significant whitening effect (p < 0.05), without any adverse effect. Based on these results, Selina (selina-4(14), 7(11)-dien-8-one) can be s useful and safe ingredient for the cleanness and brightness of skin.

Effect of Halophilic Bacterium, Haloarcula vallismortis, Extract on UV-induced Skin Change (호염 미생물(Haloarcula vallismortis) 용해물의 자외선유발 피부변화에 대한 효과)

  • Kim, Ji Hyung;Shin, Jae Young;Hwang, Seung Jin;Kim, Yun Sun;Kim, Yoo Mi;Gil, So Yeon;Jin, Mu Hyun;Lee, Sang Hwa
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.41 no.4
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    • pp.341-350
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    • 2015
  • Skin carrys out protective role against harmful outer environment assaults including ultraviolet radiation, heavy metals and oxides. Especially, ultraviolet-B (UVB) light causes inflammatory reactions in skin such as sun burn and erythma and stimulates melanin pigmentation. Furthermore, the influx of UVB into skin cells causes DNA damage in keratinocytes and dermal fibroblasts, inhibition of extracellular matrix (ECM) synthesis which leads to a decrease in elasticity of skin and wrinkle formation. It also damages dermal connective tissue and disrupts the skin barrier function. Prolonged exposure of human skin to UVB light is well known to trigger severe skin lesions such as cell death and carcinogenesis. Haloarcula vallismortis is a halophilic microorganism isolated from the Dead Sea, Its growth characteristics have not been studied in detail yet. It generally grows at salinity more than 10%, but the actual growth salinity usually ranges between 20 to 25%. Because H. vallismortis is found mainly in saltern or salt lakes, there could exist defense mechanisms against strong sunlight. One of them is generation of additional ATP using halorhodopsin which absorbs photons and produces energy by potential difference formed by opening the chloride ion channel. It often shows a color of pink or red because of their high content of carotenoid pigments and it is considered to act as a defense mechanism against intense UV irradiation. In this study, the anti-inflammatory effect of the halophilic microorganism, H. vallismortis, extract was investigated. It was found that H. vallismortis extract had protective effect on DNA damage induced by UV irradiation. These results suggest that the extract of halophilic bacterium, H. vallismortis could be used as a bio-sunscreen or natural sunscreen which ameliorate the harmful effects of UV light with its anti-inflammatory and DNA protective properties.

Healthy Functional Food Properties of Phenolic Compounds Isolated from Ulmus pumila (유근피(Ulmus pumila)로부터 분리한 phenol성 물질의 건강기능식품 활성)

  • Kim, Kyung-Bum;Jo, Bun-Sung;Park, Hye-Jin;Park, Ki-Tae;An, Bong-Jeun;Ahn, Dong-Hyun;Kim, Myung-Uk;Chae, Jung-Woo;Cho, Young-Je
    • Food Science and Preservation
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    • v.19 no.6
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    • pp.909-918
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    • 2012
  • The phenolic compounds which were extracted with 70% ethanol from Ulmus pumila for 12 hr were the highest as $17.9{\pm}1.0\;mg/g$. DPPH scavenging activity of 70% ethanol extracts was also the highest as $89.5{\pm}1.9%$ and it was confirmed to be high as 80% over in both of water and 70% ethanol extracts containing $50{\mu}g/mL$ over phenolic concentration. ABTS radical cation decolorization activities of water and 70% ethanol extracts were higher as $96.8{\pm}2.9%$, antioxidant protection factor (PF) was 2.0 PF in 70% ethanol and showed higher activities in both of water and 70% ethanol extracts containing $200{\mu}g/mL$ phenolic concentration as 2.5 PF than BHA. TBARs of 70% ethanol extracts was $86.5{\pm}4.6%$, it showed high anti-oxidative activity in $50{\sim}200{\mu}g/mL$ phenolic concentrations of water and 70% ethanol extracts as 80% over. The angiotensin converting enzyme (ACE) inhibitory activity of Ulmus pumila extracts against hypertension was 77.4% and 90.6% in water and 70% ethanol extracts of $200{\mu}g/mL$ phenolic concentration. Xanthine oxidase inhibitory activity of Ulmus pumila extracts for anti-gout effect was not observed in water extracts, but it showed 30% inhibitory activity in 70% ethanol extracts, and 48.1% at $200{\mu}g/mL$ phenolics concentration.