Anti-inflammatory effects of a traditional Korean medicine: Ojayeonjonghwan.

OBJECTIVE: To study the anti-inflammatory properties of OJ. CONTEXT: Ojayeonjonghwan (OJ) is a traditional Korean prescription, which has been widely used for the treatment of prostatitis. However, no scientific study has been performed of the anti-inflammatory effects of OJ. MATERIALS AND METHODS: Peritoneal macrophages were isolated 3-4 days after injecting a C57BL/6J mouse with thioglycollate. They were then treated with OJ water extract (0.01, 0.1, and 1 mg/mL) for 1 h and stimulated with lipopolysaccharide (LPS) for different times. Nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and proinflammatory cytokine levels were determined by NO assay, Western blotting, RT-PCR and ELISA. RESULTS: NO generation and iNOS induction were increased in the LPS-activated mouse peritoneal macrophages. However, NO generation and iNOS induction by LPS were suppressed by treatment with OJ for the first time. The IC50 value of OJ with respect to NO production was 0.09 mg/mL. OJ did not influence LPS-stimulated COX-2 induction, but did significantly decrease LPS-stimulated secretions and mRNA expressions of tumour necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß. Inhibition rates of TNF-α, IL-6, and IL-1ß at an OJ concentration of 1 mg/mL were 77%, 88%, and 50%, respectively. OJ also suppressed the LPS-induced nuclear translocation of NF-κB. High-performance liquid chromatography showed schizandrin and gomisin A are major components of OJ. CONCLUSIONS: OJ reduces inflammatory response, and this probably explains its positive impact on the prostatitis associated inflammation.

cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4.

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthiaaurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5'-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Cionaintestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (-) bacteria, while evidencing no hemolytic activity toward human erythrocytes.

Process development for heme-enriched peptide by enzymatic hydrolysis of hemoglobin.

An efficient production method of heme-iron-enriched peptide was developed based on enzymatic hydrolysis. Hemoglobin hydrolysis, carried out stepwise with commercially available exopeptidase and endopeptidase, resulted in an increased degree of hydrolysis (DH). Exopeptidase-catalyzed protein hydrolysis formed low molecular weight peptides and amino acids. Different process parameters including dialysis and ultra- and diafiltration were evaluated. Heme/peptide ratio increased as molecular weight cut-off (MWCO) of the dialysis membrane increased. When the hydrolysate was dialyzed against sodium phosphate buffer, a higher heme/ peptide ratio was obtained. The heme/peptide ratio of the hydrolysate reached up to 25.4% when the dialysis was carried out with a membrane of 12-14 kDa MWCO. Also, the ratio was improved by the use of ultrafiltration and diafiltration on the pilot-scale.

Effects of degree of hydrolysis and pH on the solubility of heme-iron enriched peptide in hemoglobin hydrolysate.

Hemoglobin was hydrolyzed with Esperase and Flavourzyme as the endopeptidase and exopeptidase, respectively. The solubility of the heme-iron enriched peptide fraction decreased as the degree of hydrolysis of the hydrolysate increased. When the pH of a hydrolysate was adjusted to 5.0 after simultaneous hydrolysis with the two enzymes, the solubility of heme-iron enriched peptide was nearly zero, and 98% of the heme-iron enriched peptide fraction was recovered as a precipitate. These results indicated that an effective separation method for the production of heme-iron enriched peptide could be established by pH adjustment of the hemoglobin hydrolysate with high degree of hydrolysis.