Superoxide dismutase (SOD) is an antioxidant protein. When administered orally, it has low bioavailability due to its low permeation. In a previous study we fused gliadin peptide P51 (LGQQQPFPPQQPYPQPQPF) and gliadin peptide P61 (QQPYPQPQPF) with SOD Citrus limon (SOD_Cl), namely GliSOD_P51 and GliSOD_P61 to increase permeation of SOD_Cl through intestine. In this work, the permeation of fluorescein isothiocyanate (FITC)-Dextran 10 kDa, FD10 and 40 kDa, FD40 as paracellular transport markers across excised rat intestinal wall was investigated with the presence of GliSOD_P51 and GliSOD_P61. A permeability study was performed using non-everted rat intestine by incubating FD10 or FD40 with SOD_Cl, and GliSOD_P61. The presence of SOD_Cl, GliSOD_P51 or GliSOD_P61 inside intestine (apical) and outside intestine (basolateral) was analyzed by protein electrophoresis. The concentration of FD that penetrated to the basolateral solution was analyzed by spectrofluorometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the presence of GliSOD_P51 and GliSOD_P61 but not SOD_Cl in basolateral compartment. The percentage of FD10 but not FD40 and SOD_Cl that penetrated to the basolateral solution significantly increased with the presence of gliadin in GliSOD_P51 and GliSOD_P61. GliSOD_P51 and GliSOD_P61 are able to penetrate the rat intestinal epithelial membrane and the gliadin peptides facilitate FD10 to penetrate the epithelial.

Matrix hydrophobicity hinders cell attachment in tissue engineered scaffolds. We proposed a facile seeding method to optimize cell presentation in nanofibrous mats formulated in a mixture of hydrophobic polycaprolactone (PCL), hydroxyapatite (HAp) mimicking bone surface roughness, and a ligand-supporting biopolymer. High-density osteoblast suspensions in serum-deficient media were seeded into composites arranged on a glass carrier sandwiched between cylindrical supports (seeding constructs) for 4 hours initial seeding and subsequently statically cultured in a complete medium for 7 days. Cell behavior and growth were analyzed by viability assays, LIVE/DEAD fluorescence labeled imaging, and electron microscopy. The zeta potential and contact angle of PCL-HAp nanofibers were altered by the addition of biopolymers, which directed cell attachment and proliferation. Modified seeding proved the benefit of collagen reinforcement to mediate cell-matrix interactions, which was demonstrated by enhancing cell spread with nearly twofold substantial growth during culture. The addition of gelatin showed a lower level of increased cell adhesion than collagen. Interestingly, clusters of spheroid cells were found in the chitosan composite with high cell adhesion on the first day, but the cells did not grow further until the end of the culture. In contrast, poor cell adhesion and inconsistent growth were found after conventional seeding and thus emphasized the potential role of modified seeding in supporting matrix performance as a cell carrier.

Superoxide dismutase (SOD) is an Mn-containing enzyme that catalyzes the conversion of superoxide (O2•−) into O2 and H2O2. To develop a small, non-protein Mn-containing SOD mimic, an [Mn(salen)Cl] complex was synthesized from MnCl2.4H2O and H2salen. The product yielded 75-90% reddish dark brown crystals indicative of the presence of manganese(III) metal ions. An elemental analysis showed that the compound contained: C = 48.36%; H = 4.34%; and N = 7.13%. Infrared spectra of this compound showed peaks at: υ(OH)=3426 (medium), υ(C=N)=1634 (very high), υ(CO)=1292 (high). An ESI-MS analysis (m/z) showed the presence of ionic species from the complex including (salen)(H+) = 269.0 and [Mn(salen)]+ = 320.9. Conductivity measurements revealed that the compound is neutral with a molar conductivity of 0.17 mS. Its magnetic moment of 5.2 BM was obtained from a magnetic susceptibility balance measurement, showing that the compound is paramagnetic. The SOD activity of this compound was given by IC50 = 2.5 ± 0.3 µM in a non-enzymatic riboflavin photoreduction in vitro test.

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Abstrak: Pada penelitian ini dilakukan pengujian aktivitas penangkal radikal bebas superoksida secara in vitro terhadap senyawa kompleks polimerik [Mn(salen)N(CN) 2 ] n , baik dengan metode non-enzimatik maupun enzimatik. Aktivitas penangkal radikal bebas superoksida yang didapatkan dari kedua metode ini dinyatakan dengan IC 50 . Melalui metode non-enzimatik riboflavin-nitrobluetetrazolium (Rb-NBT), radikal superoksida yang dihasilkan dari penyinaran riboflavin, bereaksi dengan nitrobluetetrazolium sebagai indikator dan senyawa kompleks, lalu serapan NBT tereduksi diukur pada panjang gelombang 560 nm. Dengan metode non-enzimatik ini, dihasilkan nilai IC 50 yang tinggi, konsisten, dan standar deviasinya sangat kecil, yaitu 3,4 ± 0,4 µM. Sedangkan secara enzimatik, radikal superoksida yang dihasilkan dari reaksi antara xantin dan xantin oksidase ditangkap oleh sitokrom C (sebagai indikator) dan senyawa kompleks, kemudian sitokrom C tereduksi diukur serapannya pada panjang gelombang 450 nm. Melalui metode enzimatik McCord-Fridovich (McCF) ini, dihasilkan beberapa nilai IC 50 kompleks [Mn(salen)N(CN) 2 ] n yang tidak konsisten, yang disebabkan oleh tidak berfungsinya enzim. Dengan demikian, metode non-enzimatik Rb-NBT lebih mudah dan lebih akurat untuk digunakan sebagai teknik penentuan aktivitas penangkal radikal anion superoksida dibandingkan dengan metode enzimatik.Kata kunci: penangkal radikal bebas, superoksida, non-enzimatik, [Mn(III)(salen)N(CN) 2 ], riboflavin-NBT Abstract: In this research, the in vitro activity test of superoxide free-radical scavenger has been done to the polymeric complex compound of [Mn(salen)N(CN) 2 ], either by non-enzymatic or enzymatic method. The activity of superoxide free radical scavenger obtained from these two methods expressed by IC 50 . Through the nonenzymatic method of riboflavin-nitrobluetetrazolium (Rb-NBT), superoxide radicals generated by riboflavin irradiation, was reacted with nitrobluetetrazolium as an indicator and complex compound, then the reduced NBT absorbances were measured at a wavelength of 560 nm. With the non-enzymatic method, resulted the IC 50 value that are high, consistent, and its standard deviation was very small, i.e. 3.4 ± 0.4 µM. While enzymatically, superoxide radicals generated from the reaction between xanthine and xanthine oxidase were captured by the cytochrome C (as an indicator) and complex compound, then the reduced cytochrome C absorbances were measured at a wavelength of 450 nm. Through the McCord-Fridovich (McCF) enzymatic method, produced some of IC 50 values of [Mn(salen)N(CN) 2 ] n complex which inconsistent, caseud by unfunctional of enzyme. Thus, the non-enzymatic method Rb-NBT is more simple and accurate to be used as a technique of determining the activity of anion superoxide radical scavenger than the enzymatic method.