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Development and Application of a Novel Mammalian Cell Culture System for the Biocompatibility and Toxicity of Polymer Films and Metal Plate Biomaterials (고분자필름과 금속막 의료소재에 대한 생체적합성 및 독성 평가를 위한 새로운 세포배양시스템의 개발 및 적용)

  • Kwak, Moon Hwa;Yun, Woo Bin;Kim, Ji Eun;Sung, Ji Eun;Lee, Hyun Ah;Seo, Eun Ji;Nam, Gug Il;Jung, Young Jin;Hwang, Dae Youn
    • Journal of Life Science
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    • v.26 no.6
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    • pp.633-639
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    • 2016
  • Biomaterials including polymer, metal, ceramic, and composite have been widely applied for medical uses as medical fibers, artificial blood vessels, artificial joints, implants, soft tissue, and plastic surgery materials owing to their physicochemical properties. However, the biocompatibility and toxicity for film- and plate-form biomaterials is difficult to measure in mammalian cells because there is no appropriate incubation system. To solve these problems, we developed a novel mammalian cell culture system consisting of a silicone ring, top panel, and bottom panel and we applied two polymer films (PF) and one metal plate (MP). This system was based on the principal of sandwiching a test sample between the top panel and the bottom panel. Following the assembly of the culture system, SK-MEL-2 cells were seeded onto Styela Clava Tunic (SCT)-PF, NaHCO3-added SCT (SCTN)-PF, and magnesium MP (MMP) and incubated at 37℃ for 24 hr and 48 hr. An MTT assay revealed that cell viability was maintained at a normal level in the SCT-PF culture group at 24 or 48 hr, although it rapidly decreased in the SCTN-PF culture group at 48 hr. Furthermore, the cell viability in the MMP culture group was very similar to that of the control group after incubation for 24 hr and 48 hr. Together, these results suggest the sandwich-type mammalian culture system developed here has the potential for the evaluation of the biocompatibility and toxicity of cells against PF- and MP-form biomaterials.

LP-M, a Novel Butanol-Extracts Isolated from Liriope platyphylla, could Induce the Neuronal Cell Survival and Neuritic Outgrowth in Hippocampus of Mice through Akt/ERK Activation on NGF Signal Pathway (맥문동(Liriope platyphylla)의 새로운 부탄올 추출물인 LP-M이 Akt/ERK NGF receptor signaling pathway를 통해 뇌조직에서 신경세포의 생존과 성장에 미치는 영향에 관한 연구)

  • Nam, So-He;Choi, Sun-Il;Goo, Jun-Seo;Kim, Ji-Eun;Lee, Yoen-Kyung;Hwang, In-Sik;Lee, Hye-Ryun;Lee, Young-Ju;Lee, Hong-Gu;Choi, Young-Whan;Hwang, Dae-Youn
    • Journal of Life Science
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    • v.21 no.9
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    • pp.1234-1243
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    • 2011
  • Liriope platyphylla has been used in oriental medicine as an effective medical plant to improve symptoms of cough, sputum production, neurodegenerative disorders, obesity and diabetes for long time. In order to investigate the effects of novel extracts on nerve growth factors (NGF)-stimulated neuritic outgrowth, the alteration of NGF expression and NGF receptor signaling pathway were detected in neuroblastoma cells and C57BL/6 mice. Of a total of 13 novel extracts, 4 extracts (LP-E, LP-M, LP-M50, LP2E17PJ) showed high viability on MTT assay. Also, all of these extracts induced NGF secretion and NGF mRNA expression in neuroblastoma cells. However, the NGF-induced neuritic outgrowth from PC12 cells was only stimulated by LP-E, LP-M and LP-M50. Furthermore, we selected LP-M as a best candidate, based on method and amounts of extraction, in order to verify its effect in mice. C57BL/6 mice were treated with 50 mg/kg of LP-M for 2 weeks and the effects on NGF regulation were analyzed with various methods. The expression of NGF mRNA was significantly increased in LP-M treated mice compared to vehicle treated mice. Also, the signaling pathway of p75NTR was inhibited in the cortex by LP-M treatment, with no change in the hippocampus of brain. However, the signaling pathway of TrkA was dramatically activated in only hippocampus via LP-M treatment. Therefore, these results suggest that the novel four extracts of L. platyphylla may contribute to the regulation of NGF expression and secretion in neuronal cells. LP-M was especially considered to be an excellent candidate for a neurodegenerative disease-therapeutic drug.

Anti-wrinkle Activity of $\beta$-carotene Extracted & Purified from Recombinant Escherichia coli (재조합 대장균으로부터 추출.정제된 베타-카로틴의 주름개선 활성)

  • Jo, Ji-Song;Ku, Bo-Mi;Kang, Sang-Soo;Lee, Jae-Ran;Kim, You-Geun;Lee, He;Kim, Sung-Bae;Kim, Seon-Won;Kim, Chang-Joon;Chung, In-Young
    • KSBB Journal
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    • v.23 no.6
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    • pp.513-518
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    • 2008
  • This paper described the extraction/purification of $\beta$-carotene from recombinant E.coli and evaluation of anti-wrinkle activity of purified $\beta$-carotene. No significant differences in extraction yields were observed when hexane or isobutyl acetate was used. However, extraction from wet-cell cake resulted in 2-fold higher amount of $\beta$-carotene than that from dry cells. Disruption of 5 g-wet cells by ultrasonic homogenizer, acetone dehydration, extraction with isobutyl acetate resulted in 36 mg of $\beta$-carotene corresponding to 61.2% of recovery. The formation and separation of $\beta$-carotene crystal improved the purity. 633 mg of $\beta$-carotene crystal with 93% purity was obtained from 223 g/L of wet-cell cake harvested from 2.5-L fed-batch culture broth. The cultures of normal human primary fibroblast were performed to investigate the effect of $\beta$-carotene on cytotoxicity as MTT assay and anti-wrinkle activity as collagen synthesis assays. $1.7{\mu}M$ of $\beta$-carotene was found to be optimal concentration at which 1.4-fold higher amount of collagen was synthesized than that in absence of $\beta$-carotene. This indicates that highly purified $\beta$-carotene can be obtained from recombinant E.coli by applying simple method with less toxic solvent and can be used in functional cosmetics as anti-wrinkle agent.

Antimicrobial Activity of Ethanol Extract from Sargassum thunbergii (지충이(Sargassum thunbergii) 에탄올 추출물의 항균활성)

  • Lee, So-Young;Song, Eu-Jin;Kim, Koth-Bong-Woo-Ri;Yoon, So-Young;Kim, Seo-Jin;Lee, So-Jeong;Hong, Yong-Ki;Lim, Sung-Mee;Ahn, Dong-Hyun
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.38 no.4
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    • pp.502-508
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    • 2009
  • Antimicrobial activity of Sargassum thunbergii was determined by paper disc assay and minimum concentration inhibitor (MIC) test. A water extract of S. thunbergii did not show the antimicrobial activity, but an ethanol extract of S. thunbergii (SHE) inhibited Serratia liquefaciens, Salmonella Typhimurium, Pseudomonas aerogenosa and all of the tested gram-positive bacteria at 4 mg/mL. Especially, Bacillus subtilis, Clostridium perfringens and Listeria monocytogenes were susceptible to SHE. As the results of MIC test, SHE inhibited the growth of B. subtilis, Staphylococcus aureus and Listeria monocytogenes at concentration of $0.1{\sim}0.3%$, and inhibited C. perfringens at 0.01%. In the thermal and pH stability test for SHE, antibacterial activities of SHE were maintained when the SHE was treated at $121^{\circ}C$ for 15 minutes or under pH $2{\sim}8$. SHE was partitioned in the order of n-hexane, chloroform, ethyl acetate and butanol. As the results of the MIC test for each obtained fraction, no fraction exhibited higher antibacterial activity than that of the crude SHE. However, a mixture of chloroform, ethylacetate and ethanol fractions showed higher antibacterial activity than SHE.

Immunohistochemical Study to Evaluate the Prognostic Significance of Four Biomolecular Markers in Radiotherapy of Nasopharyngeal Carcinoma (방사선 치료를 받은 코인두암의 생체분자적 예후 인자를 찾기 위한 면역조직화학염색 연구)

  • Kim, Yeon-Joo;Lee, Seung-Hee;Wu, Hong-Gyun;Go, Heoun-Jeong;Jeon, Yoon-Kyung
    • Radiation Oncology Journal
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    • v.28 no.2
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    • pp.57-63
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    • 2010
  • Purpose: We performed an immunohistochemical study with pre-treatment biopsy specimens to evaluate the prognostic significance of four biomolecular markers which can be used as a predictive assay for radiotherapy (RT) treatment of nasopharyngeal carcinoma (NPC). Materials and Methods: From January 1998 through December 2006, 68 patients were histologically diagnosed as non-metastatic NPC and treated by RT. Only 38 patients had the paraffin block for the immunohistochemical study. Thirty-one patients had undifferentiated carcinoma and 7 patients had squamous cell carcinoma. Thirtytwo patients (84%) had advanced stage NPC (2002 AJCC Stage III~IV). Immunohistochemical staining was performed for Met, COX-2, nm23-H1, and epidermal growth factor receptor (EGFR) expression using routine methods. Results: The median follow-up time was 30 months (range, 11 to 83 months) for all patients, and 39 months (range, 19 to 83 months) for surviving patients. The 5-year overall survival (OS) rate of the patients with high Met extent (${\geq}50%$) was significantly lower than that of the patients with low Met extent (48% vs. 84%, p=0.02). In addition, Met extent was also a significant prognostic factor in multivariate analysis (p=0.01). No correlation was observed between Met extent and T stage, N stage, stage group, gender, age, and the response to chemotherapy or RT. Met extent showed moderate correlation with COX-2 expression (Pearson coefficient 0.496, p<0.01), but COX-2 expression did not affect OS. Neither nm23-H1 or EGFR expression was a prognostic factor for OS in this study. Conclusion: High Met extent (${\geq}50%$) might be an independent prognostic factor that predicts poor OS in NPC treated with RT.

Comparison of Standard Culture Method and Real-time PCR Assay for Detection of Staphylococcus aureus in Processed and Unprocessed Foods (가공식품과 비가공식품에서의 황색포도상구균 검출을 위한 배지법과 Real-time PCR법의 비교)

  • Lee, Jae-Hoon;Song, Kwang-Young;Hyeon, Ji-Yeon;Hwang, In-Gyun;Kwak, Hyo-Sun;Han, Jeong-A;Chung, Yun-Hee;Seo, Kun-Ho
    • Food Science of Animal Resources
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    • v.30 no.3
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    • pp.410-418
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    • 2010
  • Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. In this study, we compared conventional culture methods and real-time PCR for detection of S. aureus in artificially inoculated milk, sausage, raw pork, and vegetable salad. The performance of a coagulase test for confirming S. aureus was also compared with a colony PCR test. Bulk food samples (500 g each) were artificially inoculated with S. aureus and divided into 20 samples (25 g or mL each). All samples were added to tryptic soy broth (225 mL/sample) with 10% NaCl and incubated at $37^{\circ}C$ for 24 h. After the enrichment, broth cultures were streaked onto Baird-Parker (BP) agar with egg yolk tellulite, and incubated at $37^{\circ}C$ for 24 h. In addition, 1 mL of broth cultures was collected to perform real-time PCR. Two suspicious colonies from the BP agar were picked up and plated on nutrient agar and incubated at $37^{\circ}C$ for 24 h followed, by a coagulase confirmation test and a colony PCR analysis. There were no statistical differences between culture methods and realtime PCR in food samples with low background microflora, such as milk and sausage. However, a significant statistical difference was found between the culture methods and real-time PCR for raw pork and vegetable salad. Furthermore, the colony PCR test of the presumptive colonies on BP agar for confirming S. aureus is more accurate and efficient than the coagulase test for unprocessed foods.

Induction of Apoptosis in HT-29 Human Colorectal Cancer by Aloin (인간 대장암 세포 HT-29에서 Aloin에 의한 Apoptosis 유도)

  • Yoo, Eun-Seon;Woo, Joong-Seok;Kim, Sung-Hyun;Lee, Jae-Han;Han, So-Hee;Jung, Soo-Hyun;Park, Young-Seok;Kim, Byeong-Soo;Kim, Sang-Ki;Park, Byung-Kwon;Jung, Ji-Youn
    • Journal of Food Hygiene and Safety
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    • v.34 no.5
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    • pp.495-501
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    • 2019
  • Aloin [1,8-Dihydroxy-10-(${\beta}$-D-glucopyranosyl)-3-(hydroxymethyl)-9(10H)-anthracenone], is a natural anthraquinone from aloe. It has been shown to have antioxidant and anticancer effects in various types of human cancer cells, but the anticancer effects of aloin in human colorectal cancer cells HT-29 have not been elucidated. In this study, possible mechanisms by which aloin exerts its apoptotic action in cultured human colorectal cancer HT-29 cells were investigated. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay shows that treatment with aloin (0, 100, 200, 300 and $400{\mu}M$) reduced cell viability in a concentration-dependent manner in HT-29 and showed no effects on cell proliferation in A375SM and AGS cells. In addition, it was confirmed that apoptotic body was significantly increased as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, and increased apoptosis rate by flow cytometry in HT-29 cells treated with aloin (0, 200 and $400{\mu}M$). We confirmed by western blotting that aloin activated Bax (pro-apoptotic), cleaved-poly (ADP-ribose) polymerase (PARP) and caspase-3, -8 and Bcl-2 (anti-apoptotic) were not changed compared with the control. Aloin induced up-regulation of phospho-p38 and down-regulation of phospho-extracellular signal-regulated kinase (ERK)1/2. Therefore, aloin suppressed the growth inhibitory effects by the induction of apoptosis in human colorectal cancer cells and has potential as a cancer preventive medicine.

Comparison of ginsenoside contents and antioxidant activity according to the size of ginseng sprout has produced in a plant factory (식물공장에서 생산된 새싹인삼의 크기에 따른 진세노사이드 함량 및 항산화 활성 비교)

  • Hwang, Seung Ha;Kim, Su Cheol;Seong, Jin A;Lee, Hee Yul;Cho, Du Yong;Kim, Min Ju;Jung, Jea Gack;Jeong, Eun Hye;Son, Ki-Ho;Cho, Kye Man
    • Journal of Applied Biological Chemistry
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    • v.64 no.3
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    • pp.253-261
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    • 2021
  • In this study, the ginseng sprout has produced through smart farm was classified according to its size and divided into above-ground (AG) and below-ground (BG) parts to compare ginsenoside contents and antioxidant activity. In the case of the AG part, the total phenolic contents were the highest at 5.16 mg/g in medium (M) size and the lowest at 2.23 mg/g in largest (L) size. The BG part also showed the highest content in the M size, but there was no significant difference. Also, the total flavonoid contents were also high in the M size in both the AG (5.16 mg/g) and BG (1.28 mg/g) parts. The major ginsenosides in the AG part were Re (20.33-24.15 mg/g) > Rd (11.36-27.42 mg/g) > Rg1 (4.48-5.54 mg/g) and the main ginsenosides in the BG part were Rb1 (5.09-8.61 mg/g) > Re (4.48-5.54 mg/g) > Rc (3.11-4.11 mg/g) in orders. In the case of M size, Re and Rd were approximately 4- and 19-folds higher at 24.15 mg/g and at 27.42 mg/g in the AG part and 5.20 mg/g and 1.43 mg in the BG part, respectively. In addition, F3 and Rh1 were detected in the AG part, but not in the BG part. 2,2-diphenyl-1-picrylhydrazyl (74.95%), 2,4,6-azino-bis (3-ethylbenzothiazoline-6-sulphnoic acid) diammonium salt (94.47%), and hydroxyl (70.39%) radical scavenging activities and FRAP (2.169) assay were the highest in M size than other sizes.

Melanogenesis regulatory constituents from Premna serratifolia wood collected in Myanmar

  • WOO, SO-YEUN
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2019.04a
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    • pp.21-22
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    • 2019
  • Melanin is a mixture of pigmented biopolymers synthesized by epidermal melanocytes that determine the skin, eye, and hair colors. Melanocytes produce two different kinds of melanin, eumelanin (dark brown/black insoluble pigments found in dark skin and dark hair and pheomelanin (lighter red/yellow). The biological role of melanin is to prevent skin damage by ultraviolet (UV) radiation. However, the overproduction or deficiency of melanin synthesis could lead to serious dermatological problems, which include melasma, melanoderma, lentigo, and vitiligo. Therefore, regulating melanin production is important to prevent the pigmentation disorders. Myanmar has a rich in natural resources. However, the chemical constituents of these natural resources in Myanmar have not been fully investigated. In the effort to search for compounds with anti-melanin deposition activity from Myanmar natural resources, five plants were collected in Myanmar. Extracts of these collected five plants were tested for anti-melanin deposition activity against a mouse melanoma cell line (B16-F10) induced with ${\alpha}$-melanocyte-stimulating hormone (${\alpha}$-MSH) and 3-isobutyl-1-methylxanthine (IBMX), and their anti-melanin deposition activities were compared with the positive control, arbutin. Among the tested extracts, the CHCl3 extracts of the Premna serratifolia (syn: P. integrifolia) wood showed anti-melanin deposition activities with IC50 values of $81.3{\mu}g/mL$. Hence, this study aims to identify secondary metabolites with anti-melanin deposition activity from P. serratifolia wood of Myanmar. P. serratifolia belongs to the Verbenaceae family and is widely distributed in near western sea coast from South Asia to South East Asia, which include India, Malaysia, Vietnam, Cambodia, and Sri Lanka. People in Tanintharyi region located in the southern part of Myanmar utilize the P. serratifolia, Sperethusa crenulata, Naringi crenulata, and Limonia acidissima as Thanaka, traditional cosmetics in Myanmar. Thanaka is applied in the form of paste onto skins to make it smooth and clear, as well as to prevent wrinkles, skin aging, excessive facial oil, pimples, blackheads, and whiteheads. However, the chemical constituents responsible for their cosmetic properties are yet to be identified. Moreover, the chemical constituents of P. serratifolia was almost uncharacterized. Investigation of the P. serratifolia chemical constituents is thus an attractive endeavor to discover new anti-melanin deposition active compounds. The investigation of the chemical constituents of the active CHCl3 extract of P. serratifolia led to isolation of four new lignoids, premnan A (1), premnan B (2), taungtangyiol C (3), and 7,9-dihydroxydolichanthin B (4), together with premnan C (5) (assumed to be an artifact), one natural newlignoid,(3R,4S)-4-(1,3-benzodioxol-5-ylcarbonyl)-3-[(R)-1-(1,3-benzo dioxol-5-yl)-1-hydroxy methyl]tetrahydro-2-furanone (6), and five known compounds (7-11)1,2). The structures of all isolated compounds were determined on the basis of their spectroscopic data and by comparison with the reported literatures. The absolute configurations of 1-3 and 5 were also determined by optical rotation and circular dichroism (CD) data analyses1). The anti-melanin deposition activities of all the isolated compounds were evaluated against B16-F10 cell line. 7,9-Dihydroxydolichanthin B (4) and ($2{\alpha},3{\alpha}$)-olean-12-en-28-oic acid (11) showed strong anti-melanin deposition activities with IC50 values of 18.4 and $11.2{\mu}M$, respectively, without cytotoxicity2). On the other hand, compounds 1-3, 5, and 7 showed melanogenesis enhancing activities1). To better understand their anti-melanin deposition mechanism, the effects of 4 and 11 on tyrosinase activities were investigated. The assay indicated that compounds 4 and 11 did not inhibit tyrosinase. Furthermore, we also examined the mRNA expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). Compounds 4 and 11 down-regulated the expression of Tyr and Mitf mRNAs, respectively. Although the P. serratifolia wood has been used as traditional cosmetics in Myanmar for centuries, there are no scientific evidences to support its effectiveness as cosmetics. Investigation of the anti-melanin deposition activity of the chemical constituents of P. serratifolia thus provided insight into the effectiveness of the P. serratifolia wood as a cosmetic agent.

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Physiological Activity and Physicochemical Properties of Condensed Prunus mume Juice Prepared with Pectinase (Pectinase처리를 한 매실 농축액의 이화학적 특성 및 생리활성)

  • Kim, Jeong-Ho;Cho, Hyun-Dong;Won, Yeong-Seon;Park, Wool-Lim;Lee, Kwan-Woo;Kim, Hyuk-Joo;Seo, Kwon-Il
    • Journal of Life Science
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    • v.28 no.11
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    • pp.1369-1378
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    • 2018
  • Prunus mume Siebold & Zucc., a member of the Rosaceae family (called Maesil in Korea), has been widely distributed in East Asia, e.g. Korea, Japan and China, and its fruit has been used as a traditional drug and health food. In this study, we evaluated physicochemical properties and physiological activities of condensed Prunus mume juice treated with pectinase (PJ). The values of total acidity, pH, sugar contents, turbidity moisture content of the PJ were 35.81%, 2.73, $54.36^{\circ}Brix$, 2.75 and 51.32%, respectively. The PJ had effective DPPH radical scavenging activity, reducing power effect, $H_2O_2$ scavenging activity and ${\beta}$-carotene bleaching effect. DPPH radical scavenging activities of PJ was 46.31%; their reducing power ($OD_{700}$) was 1.80; $H_2O_2$ scavenging activity of PJ was 91.62%; and ${\beta}$-carotene bleaching effect of PJ was 73.02%. Also, PJ showed effective levels of ${\alpha}$-glucosidase inhibition activity. The cell viability was measured by SRB assay. The PJ significantly decreased the cell viability of mouse melanoma cells (B16) and human melanoma cells (SK-MEL-2 and SK-MEL-28) in a dose-dependent manner, however, there was no effect on human keratinocyte HaCaT. In morphological study, PJ-treated SK-MEL-2 cells showed distorted and shrunken cell masses. Total polyphenol contents and total flavonoid contents of PJ were 588.31 mg% (gallic acid equivalent) and 860.45 mg% (rutin equivalent). The antiproliferative effect of PJ seems to be associated with the antioxidant activity of its flavonoid and polyphenol contents. In conclusion, PJ may be beneficial in development of a functional food material.