• Journal of Pharmaceutical Investigation
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• v.27 no.1
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• pp.15-22
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• 1997

The mucosal route of administration(nasal, buccal, conjunctival and vaginal) has recently been considered as an alternative to parenteral delivery for many peptide drugs because enzymatic degradation of these agents may be partly avoided. The objective of these study was to establish the optimal mucosal administration dosage form of $LHRH/[D-Ala^6]LHRH$, based on presystemic metabolism. We reported previously the peptidase inhibition effect of medium chain fatty acid salts(sodium caprylate, soadium caprate and sodium laurate), EDTA and STDHF on the proteolysis of $LHRH/[D-Ala^6]LHRH$ in rabbit mucosal homgenates. We also reported that EDTA, STDHF and sodium laurate markedly increased the potency of $LHRH/[D-Ala^6]LHRH$ solution applied vaginally. In the present study, by administration of polycarbophil hydrogel containing LHRH the ovulation inducing activity was 3.3 times greater than solution. These results indicate not only peptidase inhibitor but also polycarbophil hydrogel significantly improved the absorption of this drug. The results of this study would provide the feasibility as a rational dosage form for improving bioavailability and self administration of this hydrogel by the vaginal application.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.346-351
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• 2002

A bacterium producing the neutral pretense was isolated from soil, and was identified as Bacillus sp. DS-1 by 16S rRNA sequence comparison and biochemical determinations. The production of protease from Bacillus sp. DS-1 was increased 20% and 30% by the additions of 1% glucose and 1% yeast extract, respectively. The optimum pH and temperature for the protease activity were pH 7.0 and 55$^{\circ}C$. Bacillus sp. DS-1 produced a metalloprotease as a major protease in culture medium, since the pretense activity in culture supernatant was inhibited by the presence of 1 mM EDTA significantly.

• BMB Reports
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• v.28 no.4
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• pp.311-315
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• 1995

Exogenous $Cu^{2+}$ or $Zn^{2+}$ at micromolar concentration had a strong inhibitory effect on detergent-solubilized cytochrome c oxidase. A similar effect was observed when $Cu^{2+}$ was added to vesicular cytochrome c oxidase, although the extent of inhibition was significantly larger for the uncoupled state than for the coupled state. Interestingly, the inhibition by $Zn^{2+}$ was almost negligible for both the coupled and uncoupled states. These results suggest that the binding sites for $Cu^{2+}$ ions are exposed to the extravesicular side. whereas those for $Zn^{2+}$ are exposed to the matrix side. The EPR spectra of bound $Cu^{2+}$ ions at 77 K indicate that each of the first two $Cu^{2+}$ ions is ligated by three or four histidine residues, as evidenced by distinct $^{14}N$ superhyperfine splitting. These $Cu^{2+}$ ions can not be removed readily by EDTA and inhibit the enzyme activity by as much as 80%.

• Microbiology and Biotechnology Letters
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• v.24 no.3
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• pp.316-320
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• 1996

An alkaline proteinase produced by Yarrowia lipolytica TH65 was purified by 40-65% ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration with Sephadex G-100 and Sephadex G-75. The purified enzyme was shown as a single band on SDS-PAGE, and its molecular weight 31,500. Optimum temperature and pH were 40$\circ$C and 8.5-9.0, respectively, and the enzyme was stable below 40$\circ$C and in the pH range of 6-8. The enzyme was strongly inhibited by divalent ions, completely by PMSF, and partially by EDTA, EGTA, and phenanthroline. But the inhibitory effect in the presence of EDTA, EGTA and phenanthroline could be reversed by addition of Ca$^{2+}$. Thus, these results indicated that the purified enzyme was an alkaline serine proteinase (E.C. 3.4.21.14).

• Journal of Pharmaceutical Investigation
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• v.22 no.3
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• pp.173-183
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• 1992

To study the feasibility of transmucosal delivery of $[D-ala^2]-methionine$ enkephalinamide (YAGFM), its enzymatic degradation and stabilization in various rabbit mucosal extracts were investigated by HPLC method. The degradation of YAGFM was observed to follow the first-order kinetics and the half-lives of YAGFM in the nasal, rectal and vaginal mucosal extracts were found to be 25.7, 3.0 and 7.8 hr, respectively. However, there was no significant difference in degradation rates of YAGFM between the mucosal and serosal extracts obtained from the same mucosal membrane. This finding suggests that even a synthetic enkephalin analog, which is designed to be resistent to aminopeptidases, needs to be fully protected from the enzymatic degradation in mucosal sites for the delivery of the analog through mucosal routes. To inhibit the degradation of YAGFM in various mucosal extracts, effects of enzyme inhibitors such as bestatin (BS), amastatin (AM), thiorphan (TP), thimerosal (TM) and EDTA, alone or in combination, and modified cyclodextrins were observed by assaying YAGFM staying intact during 24 hr-incubation at $37^{\circ}C$. It was found from the results that mixed inhibitors such as TM (0.5 mM)/EDTA (5 mM) or AM $(50{\mu}M)/TM$ (0.5 mM)/EDTA (5 mM) provided very useful means for the stabilization in various mucosal extracts. The latter was found to protect YAGFM from the degradation in the nasal, rectal, and vaginal mucosal extracts by 90.9, 90.4 and 91.3%, respectively, after 24 hr-incubation, suggesting almost complete inhibition of YAGFM-degrading enzymes present in the incubation mixture. However, BS $(50{\mu}M)$, AM 50 $(50{\mu}M)$ or TP$(50{\mu}M)$ alone did not reveal sufficient inhibition except TM (0.5 mM) or EDTA (5 mM). The adddition of $2-hydroxylpropyl-{\beta}-cyclodextrin$(10%) to the nasal mucosal extract, and $dimethyl-{\beta}-cyclodextrin$(10%) to the rectal and vaginal mucosal extracts reduced the first-order rate constants for the degradation of YAGFM by 5.8, 17.3 and 8.9 times, respectively, compared to those with no additive.

• Applied Chemistry for Engineering
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• v.25 no.4
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• pp.363-367
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• 2014

In this study, the dietary fiber contents of Mozuku (Cladosiphon novae-caledoniae kylin) residue and the extraction condition (HCl, $H_2SO_4$, NaOH, $Na_2CO_3$, $Na_2EDTA$) of the dietary fiber was investigated. We examined that the contents of the total polyphenols and flavonoids in the dietary fiber from Mozuku residue, and the potent anti-cancer effect was also tested through the growth inhibition in human colon cancer cells (HT-29) in vitro. It was effective to extract soluble dietary fiber with 1.5% $Na_2EDTA$ and 0.05 N HCl in Mozuku residue. The extraction time and temperature affected the yields of soluble dietary fiber. The contents of the total polyphenols and flavonoids in the dietary fiber from Mozuku residue were the highest in 1% NaOH extract (Total polyphenols $34.4{\pm}0.055$ mg gallic acid/g dry basis, total flavonoids $34.7{\pm}0.023$ mg naringin/g extract dry basis). In DPPH radical scavenging activity, 1% NaOH extract showed the most potent antioxidant activity. In the result of viability in human colon cancer cells, growth inhibition was observed in D.W., 0.05 N HCl, and 0.5% $Na_2CO_3$ extracts in a dose-dependent manner. These results demonstrated that soluble dietary fiber from Mozuku residue significant antioxidant activity and anticancer in human colon cancer.

• BMB Reports
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• v.31 no.2
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• pp.161-169
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• 1998

TThe reactive oxygen species oxidatively modify the biological macromolecules, including proteins, lipids, and nucleic acids. Iron- and heme-mediated Fenton-like reactions produce different pro-oxidants. However, these reactive products have not been clearly characterized. We examined the nature of the oxidizing species from the different iron sources by measuring oxidative protein modification and spectroscopic study. Hemoglobin (Hb) and methemoglobin (metHb) were oxidatively modified in $O{\array-\\\dot{2}}$ and $H_{2}O_{2}$ generating systems. Globin and bovine serum albumin (BSA) were also modified by iron, iron-EDTA, hematin, and Hb in an $O{\array-\\\dot{2}}$ generating system. In a $H_{2}O_{2}$ generating system, the iron- and iron-EDTA-mediated protein modifications were markedly reduced while the Hb-and hematin-mediated modifications were slightly increased. In the $O{\array-\\\dot{2}}$ generating system, the iron- and iron-EDTA-mediated protein modifications were strongly inhibited by superoxide dismutase (SOD) or catalase, but heme- and Hb-mediated protein modifications were inhibited only by catalase and slightly increased by SOD. Mannitol, 5,5-dimethyl-l-pyrroline-N-oxide (DMPO), deoxyribose, and thiourea inhibited the iron-EDTA-mediated protein modification. Mannitol and DMPO, however, did not exhibit significant inhibition in the hematin-mediated modification. Desferrioxamine (DFO) inhibited protein modification mediated by iron, but cyanide and azide did not, while the hematin-mediated protein modification was inhibited by cyanide and azide, but not significantly by DFO. The protein-modified products by iron and heme were different. ESR and UV-visible spectroscopy detected the DMPO spin adduct of the hydroxyl radical and ferryl ion generated from iron-EDTA and metHb, respectively. These results led us to conclude that the main oxidizing species are hydroxyl radical in the iron-EDTA type and the ferry I ion in the hematin type, the latter being more effective for protein modification.

• Fisheries and Aquatic Sciences
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• v.5 no.3
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• pp.145-149
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• 2002

The effects of pH, temperature and various inhibitors on the proteolytic activity of the cell lysate of Uronema marium were investigated using colorimetric and substrate gel electro­phoretic methods. The cell lysate of U. marinum showed proteolytic activity over a wide range of pH, and pH optima ranged from pH 5 to 7. The proteolytic activity was increased according to a rise of temperature but decreased at $40^{\circ}$. The proteolytic activity of the parasite lysate was significantly inhibited by protease inhibitors including trans-epoxysuccinyl -L-leucylamido-(4-guanidino) butane (E-64), pepstatin A, phenyl-methanesulfonyl fluoride(PMSF), and ethylenediamine-tetraacetic acid (EDTA). Preincubation of the lysate with E-64 showed the maximum inhibition of the caseionolytic activity. Four protease bands (152, 97, 67 and 40 kDa) were detected by gelatin SDS-PAGE. Significant inhibition of caseinolytic activity and complete abolition of a 152 kDa band in gelatin SDS-PAGE by EDTA indicated that the cell lysate of U. marinum had a metalloprotease Another three proteolytic bands were inhibited by E64, a cysteine protease inhibitor. Preincubation of the cell lysate with pepstatin or PMSF had no effects on the protease bands.

• Journal of Microbiology
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• v.43 no.3
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• pp.237-243
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• 2005

The protease purified from Bacillus cereus JH108 has the function of leucine specific endopeptidase. When measured by hydrolysis of synthetic substrate (N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide), the enzyme activity exhibited optimal activity at pH 9.0, $60^{\circ}C$. The endopeptidase activity was stimulated by $Ca^{++},\;Co^{++},\;Mn^{++},\;Mg^{++},\;and\;Ni^{++}$, and was inhibited by metal chelating agents such as EDTA, 1,10-phenanthroline, and EGTA. Addition of serine protease inhibitor, PMSF, resulted in the elimination of the activity. The endopeptidase activity was fully recovered from the inhibition of EDTA by the addition of 1 mM $Ca^{++}$, and was partially restored by $Co^{++}\;and\;Mn^{++}$, indicating that the enzyme was stabilized and activated by divalent cations and has a serine residue at the active site. Addition of $Ca^{++}$ increased the pH and heat stability of endopeptidase activity. These results show that endopeptidase requires calcium ions for activity and/or stability. A Lineweaver-Burk plot analysis indicated that the $K_m$ value of endopeptidase is 0.315 mM and $V_{max}$ is 0.222 ) is $0.222\;{\mu}mol$ of N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide per min. Bestatin was shown to act as a competitive inhibitor to the endopeptidase activity.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.8 no.4
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• pp.213-216
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• 1975

Yellow sea bream contains comparatively larger amount of fat among white muscle fishes, so that rancidity might easily occur during drying and storage. For the purpose of the protection of rancidity, the effect of some antioxidants was studied when yellow sea bream was sun-dried after dipping in the solutions and packed in PVC film$0.3mm\times12cm\times30cm $ for storage at room temperature. The inhibitory effect of additives was in order of Tenox-II, BHA, Sustane and NDGA, while EDTA, potassium sorbate, CTC and $\alpha-naphthylamine$ were ineffective. The results suggest that the treatment of $0.1\%$ Tenox-II solution and packing in PVC film is better condition to improve the quality of product and during drying and storage.