A proteomic analysis of Curcuma comosa Roxb. rhizomes

<p>Abstract</p> <p>Background</p> <p>The similarly in plant physiology and the difficulty of plant classification, in some medicinal plant species, especially plants of the Zingiberaceae family, are a major problem for pharmacologists, leading to mistaken use. To overcome this problem, the proteomic base method was used to study protein profiles of the plant model, Curcuma comosa Roxb., which is a member of the Zingiberaceae and has been used in traditional Thai medicine as an anti-inflammatory agent for the treatment of postpartum uterine bleeding.</p> <p>Results</p> <p>Due to the complexity of protein extraction from this plant, microscale solution-phase isoelectric focusing (MicroSol-IEF) was used to enrich and improve the separation of Curcuma comosa rhizomes phenol-soluble proteins, prior to resolving and analyzing by two-dimensional polyacrylamide gel electrophoresis and identification by tandem mass spectrometry. The protein patterns showed a high abundance of protein spots in the acidic range, including three lectin proteins. The metabolic and defense enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase, that are associated with antioxidant activity, were mainly found in the basic region. Furthermore, cysteine protease was found in this plant, as had been previously reported in other Zingiberaceae plants.</p> <p>Conclusion</p> <p>This report presents the protein profiles of the ginger plant, Curcuma comosa. Several interesting proteins were identified in this plant that may be used as a protein marker and aid in identifying plants of the Zingiberaceae family.</p

Free Radical Scavenging Properties and Induction of Apoptotic Effects of Fa Fraction Obtained after Proteolysis of Bioactive Peptides from Microalgae Synechococcus sp. VDW

Svrha ovog istraživanja bila je odrediti antioksidacijsku aktivnost bioaktivnih peptida izoliranih iz stanica mikroalge Synechococcus sp. VDW nakon 21 dana uzgoja. Hidrolizati proteina iz mikroalge Synechococcus sp. VDW dobiveni su pomoću tripsina, te su zatim pročišćeni ultrafiltracijom pomoću membrana veličine od 10, 5 i 3 kDa. Frakcija proteina FA molekularne mase od 3 kDa imala je najveću sposobnost uklanjanja slobodnih radikala 2,2\u27-azino-bis(3-etilbenzotiazolin-6-sulfonske kiseline) (ABTS), IC50 vrijednosti od (11,5±0,3) μg/mL, i 2,2\u27-difenil-1-pikrilhidrazila (DPPH), IC50 vrijednosti od (13,6±0,2) μg/mL. Frakcija proteina FA razdvojena je pomoću HPLC kromatografije obrnutih (reverznih) faza u četiri podfrakcije (F1–4). Subfrakcija F4 imala je maksimalni učinak uklanjanja ABTS radikala od (3,55±0,61) %, pa je odabrana za daljnju analizu pomoću metode ESI-Q-TOF-MS/MS, temeljene na sekvenciranju peptida de novo. Identificirano je pet peptida s antioksidacijskim učinkom, od kojih je najveću sposobnost uklanjanja ABTS radikala imao peptid AILESYSAGKTK. Nadalje, frakcija FA imala je snažan citotoksični učinak na stanične linije humanih karcinoma, pogotovo staničnu liniju raka debelog crijeva (SW620), pri čemu je IC50 vrijednost bila (106,6±21,5) μg/mL, no nije imala citotoksični učinak na netransformiranu staničnu liniju Wi38. Uzrokovala je početak apoptoze u staničnoj liniji SW620 nakon 24, 48 i 72 sata, s najvećom aktivnosti kaspaza 3, 8 i 9 nakon 72 sata. Dobiveni rezultati pokazuju da se mikroalga Synechococcus sp. VDW može upotrijebiti za proizvodnju prirodnih antitumorskih lijekova.This study aims to determine the antioxidant activity of bioactive peptides derived from Synechococcus sp. VDW cells cultured for 21 days. Synechococcus sp. VDW protein hydrolysates were prepared with trypsin and purified by ultrafiltration with molecular mass cut-off membranes of 10, 5 and 3 kDa. The M<3 kDa (FA) fraction had the highest 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, with IC50 values of (11.5±0.3) and (13.6±0.2) µg/mL, respectively. The FA fraction was separated by reversed phase HPLC to yield four subfractions (F1–4). The F4 subfraction showed the highest maximum ABTS radical scavenging activity (3.55±0.61) % and it was selected for further analysis by electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) based on de novo peptide sequencing. Five antioxidant peptides were identified, of which AILESYSAGKTK had the highest ABTS radical scavenging activity. Furthermore, the FA fraction showed high cytotoxic activities against human cancer-derived cell lines, especially the colon cancer cell line (SW620) with an IC50 value of (106.6±21.5) µg/mL, but not the untransformed Wi38 cell line. The FA fraction activated the apoptotic pathway in SW620 cells after treatment for 24, 48 and 72 h, with the highest activities of caspases-3, -8 and -9 being observed after treatment for 72 h. These findings suggested that microalgae Synechococcus sp. VDW may be used to develop natural anticancer drugs

Nitric Oxide Synthesis Inhibition and Anti-Inflammatory Effect of Polypeptide Isolated from Chicken Feather Meal in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Dušikov oksid ima ključnu ulogu u mehanizmu nastanka upalnog procesa te uzrokuje ozljedu tkiva. Brašno od perja kokoši bogato je aminokiselinama te može poslužiti za inhibiciju sinteze dušikovog oksida kao hidrolizat peptida. Pripremljeni su hidrolizati brašna od perja s protuupalnim svojstvima, koji su razdvojeni u pet skupina prema molekulskoj masi. Najmanja je frakcija (<0,65 kDa) najbolje inhibirala sintezu dušikovog oksida, a da nije djelovala citotoksično na stanice mikrofaga RAW 264.7. Daljnjim razdvajanjem dobivene su subfrakcije u količinama dovoljnim za provedbu analize aminokiselinskog slijeda metodom Q-TOF LC-MS/MS ESI. Peptid SNPSVAGVR (885,97 Da) i odgovarajući čisti sintetizirani peptid inhibirali sintezu dušikovog oksida (IC50=(55,2±0,2)) u makrofagu RAW 264.7, a da nisu imale citotoksični učinak. Rezultati dobiveni pomoću metoda RT-PCR i kvantitativne RT-PCR pokazuju da dobiveni peptid SNPSVAGVR smanjuje ekspresiju upalnih citokina iNOS, TNF-α, COX-2 i IL-6 u stanicama RAW 264.7 stimuliranim pomoću lipopolisaharida. Time je potvrđeno da se peptidi iz proteina brašna od perja mogu primijeniti kao dodatak funkcionalnoj hrani ili nutraceuticima, a za smanjenje upalnih procesa.Nitric oxide (NO) plays a key role in the pathogenesis of inflammation and has been implicated in endotoxin-induced tissue injury. Chicken feather meal is a rich source of amino acids that may serve as a peptide hydrolysate to inhibit NO activity. Anti-inflammatory hydrolysates of chicken feather meal were prepared and fractionated into five samples based on molecular mass. The smallest fraction (<0.65 kDa) exhibited the highest NO inhibitory activity without cytotoxicity towards macrophage RAW 264.7 cells. Further subfractions were sufficient to obtain amino acid sequences by Q-TOF LC-MS/MS ESI analysis. Of these, the SNPSVAGVR (885.97 Da) peptide and its corresponding pure synthetic peptide have inhibitory activity against NO production by RAW 264.7 cells (IC50=(55.2±0.2) mM) without cytotoxicity. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR results revealed that the peptide of the obtained fraction reduced transcript expression levels of the pro-inflammatory cytokines iNOS, TNF-α, COX-2 and IL-6 in lipopolysaccharide-stimulated RAW 264.7 cells. These results suggest that the peptides derived from the chicken feather meal protein could potentially be used as a promising ingredient in functional foods or nutraceuticals against inflammatory diseases

Publication License of Fig.11

Publication Licensing Rights of Fig.11 (©BioRender – biorender.com)</p

<b>S3 Table.</b> Lipase inhibitory results of DLSH ultrafiltered fractions.

S3 Table. Lipase inhibitory results of DLSH ultrafiltered fractions.</p

<b>S8 Table. </b>The influence of the GRSPDTHSG peptide on lipid accumulation in 3T3-L1 cells.

S8 Table. The influence of the GRSPDTHSG peptide on lipid accumulation in 3T3-L1 cells.</p

<b>S7 Table.</b> 3T3-L1 cell viability after treatment with different concentrations of GRSPDTHSG peptide and simvastatin.

S7 Table. 3T3-L1 cell viability after treatment with different concentrations of GRSPDTHSG peptide and simvastatin.</p

Tyrosinase inhibition.png

"""..</p

<b>S6 Table.</b> Kinetic parameters.

S6 Table. Kinetic parameters. The reciprocals of the velocity and substrate concentration of lipase activity at varying DLSH peptide concentrations and the inhibitor constant (Ki) determination of the non-competitive inhibition by GRSPDTHSG.</p