• Title/Summary/Keyword: growth inhibitory effects

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Processing and Quality Analysis of Seasoned Low-salt Fermented Styela clava Supplemented with Fermentation Alcohol for Extended Shelf-life (발효주정 첨가 저염 미더덕(Styela clava) 양념젓갈의 제조 및 품질)

  • Hwang, Young-Sook;Lee, Hyun-Jin;Hwang, Seok-Min;Oh, Kwang-Soo
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.54 no.1
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    • pp.1-8
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    • 2021
  • In order to develop value-added low-salt fermented seafood with a long shelf-life, we prepared seasoned low-salt fermented Mideoduck (Styela clava) supplemented with fermentation alcohol (SME). The SME was produced by washing and dewatering shelled Mideoduck, followed by cutting and salting for 24 h at 0℃. The salted Mideoduck was seasoned and fermented with ingredients, including garlic, ginger, monosodium glutamate, red pepper, sesame, sorbitol and sugar, for 7-8 days at 0℃. After adding 3-5% fermentation alcohol, the Mideoduck was packed in a polyester container. The salinity, volatile basic nitrogen, and amino nitrogen content of the SME was 4.5%, 20.9 mg/100 g and 92.0 mg/100 g, respectively. In comparison with the control, the addition of 3-5% fermentation alcohol showed inhibitory effects of decreased freshness, texture degradation, and growth of residual bacteria. Additionally, the SME had good storage stability and organoleptic qualities when stored at 4±1℃ for 40 days. Therefore, it is suitable for commercialization as a seasoned low-salt fermented product with a long shelf-life. The total amino acid content of the SME was 11,774.5 mg/100 g, majorly comprising glutamic acid, aspartic acid, lysine, arginine, and leucine, and the free amino acid content was 506.4 mg/100 g, majorly comprising hydroxyproline, taurine, and glutamic acid.

Antioxidant and Antifungal Activities of Essential Oils against Contaminating Fungi Isolated from Fermented Sausages (발효소시지 유래 오염 곰팡이에 대한 에센셜 오일의 항곰팡이능과 항산화능 분석)

  • Lee, Eun-Seon;Kim, Jong-Hui;Kim, Bu-Min;Oh, Mi-Hwa
    • Journal of the Korean Society of Food Culture
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    • v.37 no.5
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    • pp.446-453
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    • 2022
  • In this study, we analyzed the antifungal activities of five essential oils (clove, rosemary, thyme, basil, and oregano) against three fungi (Penicillium oxalicum, Penicillium commune, and Cladosporium cladosporioides) isolated from farm-type fermented meat products Though their antifungal activities differed for each fungus, thyme had the greatest effect. Notably, C. cladosporioides showed the highest sensitivity to essential oils, and growth inhibitory effects were greater than for the other two strains. Additionally, ABTS, DPPH, and FRAP analysis confirmed that the five essential oils studied had antioxidant activity. ABTS analysis showed that clove (75%) and oregano (75%) oils had the highest antioxidant activities (both 93.7%). DPPH analysis showed that clove (75%) and rosemary (75%) oils had significantly greater antioxidant activities (both 93.8%) than thyme, basil, or oregano oils. FRAP results indicated that clove and basil oils were the strongest reductants. Comprehensive comparative analysis indicated that clove oil had more antioxidant activity than the other four essential oils. Overall, the study shows that the excellent antifungal properties of clove oil could be harnessed to produce safe fermented meat products by preventing rancidity and mold contamination.

Involvement of adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 in diallyl trisulfide-induced cytotoxicity in hepatocellular carcinoma cells

  • Guan, Feng;Ding, Youming;He, Yikang;Li, Lu;Yang, Xinyu;Wang, Changhua;Hu, Mingbai
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.6
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    • pp.457-468
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    • 2022
  • It has been demonstrated that APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) is involved in the regulation of several growth-related signaling pathways and thus closely associated with the development and progression of some cancers. Diallyl trisulfide (DAT), a garlic-derived bioactive compound, exerts selective cytotoxicity to various human cancer cells through interfering with pro-survival signaling pathways. However, whether and how DAT affects survival of human hepatocellular carcinoma (HCC) cells remain unclear. Herein, we tested the hypothesis of the involvement of APPL1 in DAT-induced cytotoxicity in HCC HepG2 cells. We found that Lys 63 (K63)-linked polyubiquitination of APPL1 was significantly decreased whereas phosphorylation of APPL1 at serine residues remained unchanged in DAT-treated HepG2 cells. Compared with wild-type APPL1, overexpression of APPL1 K63R mutant dramatically increased cell apoptosis and mitigated cell survival, along with a reduction of phosphorylation of STAT3, Akt, and Erk1/2. In addition, DAT administration markedly reduced protein levels of intracellular TNF receptor-associated factor 6 (TRAF6). Genetic inhibition of TRAF6 decreased K63-linked polyubiquitination of APPL1. Moreover, the cytotoxicity impacts of DAT on HepG2 cells were greatly attenuated by overexpression of wild-type APPL1. Taken together, these results suggest that APPL1 polyubiquitination probably mediates the inhibitory effects of DAT on survival of HepG2 cells by modulating STAT3, Akt, and Erk1/2 pathways.

Porphyra tenera induces apoptosis of oral cancer cells (구강암 세포주에서 김 추출물에 의한 세포자멸사 유도)

  • Kim, Sang Chan;Lee, Jong Rok;Park, Sook Jahr
    • The Korea Journal of Herbology
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    • v.30 no.2
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    • pp.25-30
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    • 2015
  • Objectives : Laver (Porphyra tenera), a red algae species, is one of the most widely consumed edible seaweed in Korea. Laver contains various substances such as essential amino acid, fiber, minerals and polyphenols that benefit human health. In the present study, we prepared ethanol extracts from commercially processed product of Porphyra tenera, and evaluated the growth inhibitory effect against human oral squamous carcinoma YD-10B cells. Methods : Cell viability was measured by MTT assay. Apoptosis was confirmed by TUNEL assay and flow cytometry with the green fluorescent dye FITC annexin V entering apoptotic cells and the red fluorescent dye PI not entering. The expression of the relevant proteins was detected using Western blot. Results : Ethanol extracts of Porphyra tenera (PTE, $50-200{\mu}g/m{\ell}$) caused a significant decrease of cell viability in a dose dependant manner. The cell death occurred as a result of apoptotic process as determined by TUNEL assay and flow cytometric analysis. In line with this observation, decrease in procaspase proteins and increase in cytosolic cytochrome c were observed in cells treated with PTE. In addition, exposure to PTE decreased the expression levels of Bcl-2, and induced PARP cleavage and AIF translocation from mitochondria to nucleus. Conclusions : In conclusion, PTE exerts anti-cancer effects by inducing apoptosis via caspase activation and AIF nuclear translocation in YD-10B cells. These results provide evidence for the possible therapeutic effect of Porphyra tenera in oral cancer cells.

Anti-inflammatory Effect of Angelicae Gigantis Radix Water Extract on LPS-stimulated Mouse Macrophages (Lipopolysaccharide로 유발된 마우스 대식세포의 염증매개성 Cytokine 생성증가에 대한 참당귀 물추출물의 효능 연구)

  • Han, Hyo-Sang
    • The Korea Journal of Herbology
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    • v.28 no.5
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    • pp.113-119
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    • 2013
  • Objectives : The purpose of this study was to investigate the effects of Angelicae Gigantis Radix Water Extract(AG) on the production of proinflammatory mediators in RAW 264.7 cells stimulated with lipopolysaccharide(LPS). Method : RAW 264.7 cells were cotreated with AG(50 and 100 ug/mL) and lipopolysaccharide(LPS; 1 ug/mL) for 24 hours. After 24 hour treatment, using Bead-based multiplex cytokine assay, concentrations of various cytokines such as interleukin(IL)-6, IL-$1{\beta}$, IL-10, tumor necrosis factor-alpha(TNF-${\alpha}$), granulocyte colony-stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF), interferon inducible protein-10(IP-10), leukemia inhibitory factor(LIF), lipopolysaccharide-induced chemokine(LIX), monocyte chemoattractant protein-1(MCP-1), macrophage colony-stimulating factor(M-CSF), macrophage inflammatory protein(MIP)-$1{\alpha}$, MIP-$1{\beta}$, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted(RANTES) and vascular endothelial growth factor(VEGF) were measured. Result : AG significantly inhibited LPS-induced production of TNF-${\alpha}$, MIP-$1{\alpha}$, G-CSF, RANTES, IL-10, and M-CSF from LPS-stimulated RAW 264.7 cells at the concentrations of 50 and 100 ug/mL. AG significantly inhibited LPS-induced production of MIP-$1{\beta}$, MIP-2, GM-CSF, and IL-6 from LPS-stimulated RAW 264.7 cells at the concentrations of 50 ug/mL. AG significantly inhibited LPS-induced production of VEGF from LPS-stimulated RAW 264.7 cells at the concentrations of 100 ug/mL. But AG did not show any significant effect on the production of MCP-1, LIF, LIX, IP-10 and IL-$1{\beta}$ from LPS-induced RAW 264.7 cells. Conclusion : These results suggest that AG has anti-inflammatory effect related with its inhibition of proinflammatory mediators such as TNF-${\alpha}$, MIP-$1{\alpha}$, G-CSF, RANTES, IL-10, MIP-$1{\beta}$, MIP-2, GM-CSF, IL-6, VEGF and M-CSF in LPS-induced macrophages.

Anticancer effects of Ulva compressa extracts on FaDu human hypopharangeal squamous carcinoma cells in vitro

  • Jang, Ji Yun;Jung, Seo Yun;Park, Bo-Ram;Lee, Seul Ah;Kim, Chun Sung
    • International Journal of Oral Biology
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    • v.47 no.3
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    • pp.41-48
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    • 2022
  • Ulva compressa Linnaeus (UCL) is a green algae seaweed that performs photosynthesis and is used as a food material in some Asian regions including Korea. It is known to be the dominant species in copper ion-contaminated seas, and many studies on copper ion resistant mechanisms have been reported. UCL is known to have an excellent antioxidant effect, but limited information is available regarding its other physiological activities. In this study, we investigated the anticancer activity of 30% prethanol extracts of Ulva compressa Linnaeus (30% PeUCL) and the underlying mechanisms of its activity on human FaDu hypopharyngeal squamous carcinoma cells. The 30% PeUCL extracts suppressed FaDu cell viability without affecting normal cells (L929), as determined by MTT and viability assays. Furthermore, the 30% PeUCL extracts induced apoptosis, as determined by DAPI staining. The 30% PeUCL extracts inhibited colony formation effectively as well as wound-healing of FaDu cells, even at noncytotoxic concentrations. In addition, 30% PeUCL extracts induced apoptosis significantly through proteolytic cleavage of caspase-3, -7, and -9, and poly (ADP-ribose) polymerase, and by downregulation of Bcl-2 and upregulation of Bax in FaDu cells, as determined by Western blot analysis. Collectively, these results suggest that the inhibitory effect of 30% PeUCL extracts on the growth of oral cancer cells, colony formation and wound-healing may be mediated by caspase- and mitochondrial-dependent apoptotic pathways in human FaDu hypopharyngeal squamous carcinoma cells. Therefore, 30% PeUCL extracts can be administered as a natural chemotherapeutic drug for the treatment of human oral cancers.

Studies on Sclerotium rolfsii Sacc. isolated from Magnolia kobus DC. in Korea (목련(Magnolia kobus DC.)에서 분리한 흰비단병균(Sclerotium rolfsii Sacc.)에 관한 연구)

  • Kim Kichung
    • Korean journal of applied entomology
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    • v.13 no.3 s.20
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    • pp.105-133
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    • 1974
  • The present study is an attempt to solve the basic problems involved in the control of the Sclerotium disease. The biologic stranis of Sclerotium rolfsii Sacc., pathogen of Sclerotium disease of Magnolia kobus, were differentiated, and the effects of vitamins, various nitrogen and carbon sources on its mycelial growth and sclerotial production have been investigated. In addition the relationship between the cultural filtrate of Penicillium sp. and the growth of Sclerotium rolfsii, the tolerance of its mycelia or sclerotia to moist heat or drought and to Benlate (methyl-(butylcarbamoy 1)-2-benzimidazole carbamate), Tachigaren (3-hydroxy-5-methylisoxazole) and other chemicals were also clarified. The results are summarizee as follows: 1. There were two biologic strains, Type-l and Type-2 among isolates. They differed from each other in the mode of growth and colonial appearance on the media, aversion phenomenon and in their pathogenicity. These two types had similar pathogenicity to the Magnolia kobus and Robinia pseudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-l being more virulent. 2. Except potassium nitrite, sodium nitrite and glycine, all of the 12 nitrogen sources tested were utilized for the mycelial growth and sclerotial production of this fungus when 10r/l of thiamine hydrochloride was added in the culture solution. Considering the forms of nitrogen, ammonium nitrogen was more available than nitrate nitrogen for the growth of mycelia, but nitrate nitrogen was better for sclerotia formation. Organic nitrogen showed different availabilities according to compounds used. While nitrite nitrogen was unavailable for both mycelial growth and sclerotial formation whether thiamine hydrochlioride was added or not. 3. Seven kinds of carbon sources examined were not effective in general, as long as thiamine hydrochloride was not added. When thiamine hydrochloride was added, glucose and saccharose exhibited mycelial growth, while rnaltose and soluble starch gave lesser, and xylose, lactose, and glycine showed no effect at all,. In the sclerotial production, all the tested carbon sources, except lactose, were effective, and glucose, maltose, saccharose, and soluble starch gave better results. 4. At the same level of nitrogen, the amount of mycelial growth increased as more carbon Sources were applied but decreased with the increase of nitrogen above 0.5g/1. The amount of sclerotial production decreased wi th the increase of carbon sources. 5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l for maximun growth of mycelia. At a higher concentration of more than 20r/l, however, mycelial growth decreased as the concentration increased, and was inhibited at l50r/l to such a degree of thiamine-free. 6. The effect of the nitrogen sources on the mycelial growth under the presence of thiamine were recognized in the decreasing order of $NH_4NO_3,\;(NH_4)_2SO_4,\;asparagine,\;KNO_3$, and their effects on the sclerotial production in the order of $KNO_3,\;NH_4NO_3,\;asparagine,\;(NH_4)_2SO_4$. The optimum concentration of thiamine was about 12r/l in $KNO_3$ and about 16r/l in asparagine for the growth of mycelia; about 8r/l in $KNO_3$ and $NH_4NO_3$, and 16r/l in asparagine for the production of sclerotia. 7. After the fungus started to grow, the pH value of cultural filtrate rapidly dropped to about 3.5. Hereafter, its rate slowed down as the growth amount increased and did not depreciated below pH2.2. 8. The role of thiamine in the growth of the organism was vital. If thiamine was not added, the combination of biotin, pyridoxine, and inositol did not show any effects on the growth of the organism at all. Equivalent or better mycelial growth was recognized in the combination of thiamine+pyridoxine, thiamine+inositol, thiamine+biotin+pyridoxine, and thiamine+biotin+pyridoxine+inositol, as compared with thiamine alone. In the combinations of thiamine+biotin and thiamine+biotin+inositol, mycelial growth was inhibited. Sclerotial production in dry weight increased more in these combinations than in the medium of thiamine alone. 9. The stimulating effects of the Penicillium cultural filtrate on the mycelial growth was noticed. It increased linearly with the increase of filtrate concentration up to 6-15 ml/50ml basal medium solution. 10. $NH_4NO_3$. as a nitrogen source for mycelial growth was more effective than asparasine regardless of the concentration of cultural filtrate. 11. In the series of fractionations of the cultural filtrate, mycelial growth occured in unvolatile, ether insoluble cation-adsorbed or anion-unadsorbed substance fractions among the fractions of volatile, unvolatile acids, ether soluble organic acids, ether insoluble, cation-adsorbed, cation-unadsorbed, anion-adsorbed and anion-unadsorbed. and anion-un-adsorbed substance tested. Sclerotia were produced only in cation-adsorbed fraction. 12. According to the above results, it was assumed that substances for the mycelial growth and sclerotial formation and inhibitor of sclerotial formation were include::! in cultural filtrate and they were quite different from each other. I was further assumed that the former two substances are un volatile, ether insotuble, and adsorbed to cation-exchange resin, but not adsorbed to anion, whereas the latter is unvolatile, ether insoluble, and not adsorbed to cation or anion-exchange resin. 13. Seven amino acids-aspartic acid, cystine, glysine, histidine, Iycine, tyrosine and dinitroaniline-were detected in the fractions adsorbed to cation-exchange resin by applying the paper chromatography improved with DNP-amino acids. 14. Mycelial growth or sclerotial production was not stimulated significantly by separate or combined application of glutamic acid, aspartic acid, cystine, histidine, and glysine. Tyrosine gave the stimulating effect when applied .alone and when combined with other amino acids in some cases. 15. The tolerance of sclerotia to moist heat varied according to their water content, that was, the dried sclerotia are more tolerant than wet ones. The sclerotia harvested directly from the media, both Type-1 and Type-2, lost viability within 5 minutes at $52^{\circ}C$. Sclerotia dried for 155 days at$26^{\circ}C$ had more tolerance: sclerotia of Type-l were killed in 15 mins. at $52^{\circ}C$ and in 5 mins. at $57^{\circ}C$, and sclerotia of Type-2 were killed in 10 mins. both at $52^{\circ}C$ or $57^{\circ}C$. 16. Cultural sclerotia of both strains maintained good germinability for 132 days at$26^{\circ}C$. Natural sclerotia of them stored for 283 days under air dry condition still had good germinability, even for 443 days: type-l and type-2 maintained $20\%$ and $26.9\%$ germinability, respectively. 17. The tolerance to low temperature increased in the order of mycelia, felts and sclerotia. Mycelia completely lost the ability to grow within 1 week at $7-8^{\circ}C$> below zero, while mycelial felts still maintained the viability after .3 weeks at $7-20^{\circ}C$ below zero, and sclerotia were even more tolerant. 18. Sclerotia of type-l and type-2 were killed when dipped into the $0.05\%$ solution of mercury chloride for 180 mins. and 240 mins. respectively: and in the $0.1\%$ solution, Type-l for 60 mins. and Type-2 for 30 mins. In the $0.125\%$ uspulun solution, Type-l sclerotia were killed in 180 mins., and those of Type-2 were killed for 90 mins. in the$0.125\%$solution. Dipping into the $5\%$ copper sulphate solution or $0.2\%$ solution of Ceresan lime or Mercron for 240 mins. failed to kill sclerotia of either Type-l or Type-2. 19. Inhibitory effect on mycelial growth of Benlate or Tachi-garen in the liquid culture increased as the concentration increased. 6 days after application, obvious inhibitory effects were found in all treatments except Benlate 0.5ppm; but after 12 days, distingushed diflerences were shown among the different concentrations. As compared with the control, mycelial growth was inhibited by $66\%$ at 0.5ppm and by $92\%$ at 2.0ppm of Benlate, and by$54\%$ at 1ppm and about $77\%$ at 1.5ppm or 2.0ppm of Tachigaren. The mycelial growth was inhibited completely at 500ppm of both fungicides, and the formation of sclerotia was checked at 1,000ppm of Benlate ant at 500ppm or 1,000ppm of Tachigaren. 20. Consumptions of glucose or ammonium nitrogen in the culture solution usually increased with the increment of mycelial growth, but when Benlate or Tachigaren were applied, consumptions of glucose or ammonium nitrogen were inhibited with the increment of concentration of the fungicides. At the low concentrations of Benlate (0.5ppm or 1ppm), however, ammonium nitrogen consumption was higher than that of the ontrol. 21. The amount of mycelia produced by consuming 1mg of glucose or ammonium nitrogen in the culture solution was lowered markedly by Benlate or Tachigaren. Such effects were the severest on the third day after their treatment in all concentrations, and then gradually recovered with the progress of time. 22. In the sand culture, mycelial growth was not inhibited. It was indirectly estimated by the amount of $CO_2$ evolved at any concentrations, except in the Tachigaren 100mg/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10mg/g sand of Benlate or Tachigaren. 23. There was no visible inhibitory effect on the germination of sclerotia when the sclerotia were dipped in the solution 0.1, 1.0, 100, 1.000ppm of Benlate or Tachigaren for 10 minutes or even 20 minutes.

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Biological Activities of Extracts from Gamma-irradiated Aralia elata Cortex (감마선 조사한 총목피(Aralia elata Cortex) 추출물의 생리활성)

  • Park, Hye-Jin;Lee, Eun-Ho;Kim, Myung-Uk;Lee, Seon-Ho;An, Dong-Hyun;An, Bong-Jeun;Kwon, Joong-Ho;Cho, Young-Je
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.43 no.8
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    • pp.1236-1247
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    • 2014
  • Gamma irradiated-treatment of natural medicinal plants can be used to improve extraction transference number and for qualitative improvement of color when applied to functional material exploration. This study investigated the biological activities of Aralia elata cortex extracts upon gamma irradiation. In addition, different physical techniques [photostimulated luminescence (PSL) and thermoluminescence (TL)] were used for irradiation identification of Aralia elata cortex. In PSL analysis, non-irradiated (0 kGy) sample showed a negative result of 400 photon counts (PCs), whereas irradiated (5, 10, and 30 kGy) samples showed positive results of 90,100.00, 312,614.33, and 321,661.67 PCs, respectively. In the TL method, growth curve showed very unusual behaviors around $200^{\circ}C$ upon natural-irradiation of the non-irradiated (0 kGy) sample and around $150{\sim}250^{\circ}C$ for the irradiated (5, 10, and 30 kGy) samples. The TL ratio was 0.1 in non-irradiated samples at 0.011, whereas the values of irradiated samples (5, 10, and 30 kGy) were 0.1 at 1.105, 1.009, and 2.206, respectively. For phenolics of gamma-irradiated Aralia elata cortex, water and 50% ethanol extracts had the highest amounts, $17.30{\pm}0.40mg/g$ and $18.87{\pm}0.46mg/g$ at 10 kGy irradiation, respectively. The inhibitory activities of angiotensin-converting enzyme and xanthin oxidase were higher in both irradiated water and 50% ethanol extracts than in non-irradiated ones. For pancreatin ${\alpha}$-amylase and ${\alpha}$-glucosidase inhibitory activities, water and 50% ethanol extracts containing $200{\mu}g/mL$ of phenolics showed high inhibitory activities of 60~100% at all irradiation doses (0~30 kGy). This result confirmed that Aralia elata cortex extracts have greater anti-diabetic effects than acabose as a diabetic remedy. Gamma-irradiated Aralia elata cortex extracts are useful as a functional material with anti-diabetic effects. Thus, Aralia elata cortex extracts can be used as a functional material with various biological activities, and gamma-irradiation can be used to amplify biological activities in plants.

Effects of Major Phenolic Acids Identified from Barley Residues on the Germination of Paddy Weeds (보리 잔여물(殘餘物)속에 함유(含有)된 주요(主要) Phenolic Acids가 논 잡초(雜草) 발아(發芽)에 미치는 영향(影響))

  • Kwak, S.S.;Kim, K.U.
    • Korean Journal of Weed Science
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    • v.4 no.1
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    • pp.39-51
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    • 1984
  • Effects of major phenolic acids identified from barley residues (straw, root) on the germination of rice and 3 paddy weeds such as Echinochloa crusgalli, Cyperus serotinus, and Potamogeton distintus were evaluated to obtain the basic informations on the development of naturally occurring herbicides. The predominant phenolic compounds extracted from barley residues in both straw and root were identified as p-coumaric, p-hydroxybenzoic, ferulic, vanillic, and salicylic acids by means of paper chromatography. Total phenol content of barley straw and root at the harvesting stage was 0.169% and 0.127% per dry weight, respectively. During the decomposing process, total phenol content slightly increased and then decreased. The germination of test plants was inhibited by treatments of 4 major authentic phenolic acids identified, most significantly on rice, and less on E. crusgalli, and C. serotinus. P. distintus, however, was markedly stimulated by them as the concentration increased, and then sprouted buds of pondweeds were changed to dark brownish color, resulting in the death as the treatment prolonged. The greater inhibitory effect appeared on shoot growth rather than germination. The aqueous extracts of barley residues showed the similar inhibitory effect on the germination and shoot growth of rice and three paddy weeds as the treatments of 4 authentic phenolic acids.

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BCR/ABL mRNA Targeting Small Interfering RNA Effects on Proliferation and Apoptosis in Chronic Myeloid Leukemia

  • Zhu, Xi-Shan;Lin, Zi-Ying;Du, Jing;Cao, Guang-Xin;Liu, Gang
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.12
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    • pp.4773-4780
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    • 2014
  • Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.