| Summary: | 碩士 === 東海大學 === 食品科學系 === 96 === Several reports have suggested that perilla seed oil has some functions for preventing atherosclerosis and chemically induced cancer and improving immune activity. The antioxidant, anti-inflammatory, anti-allergy, anti-viral and anti-cancer effects of phenolic compounds from perilla seed also have been reported in the past few years. Recently, phenolic compounds which existed in defatted perilla seed have been indicated with antimicrobial and antilipoxygenase activities. Although the defatted residue from perilla seed might be a kind of waste in oil and lipid industries, the existed polyphenols might be possible as one of good sources for developing a healthy food. In this study, we aimed to investigate the polyphenol contents of different solvent extracts from perilla seed and/or defatted seed, their antioxidant activity and the ability on inflammatory regulation in the lipopolysaccharide (LPS)-induced macrophage inflammatory response also would be studied. The preliminary crude extracts were prepared from perilla seed by using boiling water or methanol. After that, the defatted perilla seed from methanol extraction was re-extracted with n-hexane (HM). In addition, supercritical fluid extraction (carbon dioxide under 5000 psi pressure, at 40, 60 and 80 C and expressed as S40, S60, S80, respectively) was applied in the defat procedures in replace of the traditional used hexane solvent. The defatted meal was then extracted by using methanol (extract expressed as SM). In chemical composition analysis, phenolic components of different solvent extracts from perilla seed were isolated and identified by high performance liquid chromatography (HPLC) with photodiode array detection (PDA) and ion trap mass spectrometry with negative electron spray ionization mode. Four compounds, i.e. caffeic acid、rosmarinic acid、rosmarinic acid glucoside (Ra-G)、luteolin and apigenin were identified.
In the antioxidant activities assays in vitro, including reducing power, horseradish peroxidase (HRP)-H2O2-luminol system, scavenging activity on DPPH free radical and ABTS•+ cation free radical, were evaluated and compared. Results showed that the better effects of extracts on free radicals scavenging were from ME and HM. In addition, the antioxidant enzymes of superoxide dismutase (SOD) and catalase (CAT) were further compared in RAW 264.7 cell assays. In which, SOD activities were obviously up-regulated in the doses of 400 μg/mL from ME and HM, while the activities were also obviously elevated after adding ME (200 μg/mL) or HM (400 μg/mL) into the LPS-induced macrophages. CAT activities were obviously up-regulated in the doses of 100 and 200 μg/mL from ME and HM, respectively. The obviously elevated effects of ME and HM on the activities of antioxidant enzymes in LPS-stimulated cells were shown in ME and HM at 200, 400 μg/mL, respectively, in which the activities of SOD and CAT were elevated at 400 μg/mL concentrations significantly elevated the activities of CAT.
In anti-inflammatory experiments, murine macrophage RAW 264.7 cells were conducted for studying the inhibition abilities of extracts on LPS-induced inflammation markers, i.e. tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and inhibitor κB-α (IκB-α) proteins. Results showed that NO, PGE2, TNF-α, iNOS and COX-2 production were significantly inhibited by those of phenolic components found in perilla seed, including caffeic acid or rosmarinic acid in the doses of 250 μM and apigenin or luteolin in the doses of 25 μM. NO production was significantly inhibited at 200 μg/mL in ME, HM and SM40, respectively, and in a concentration-dependent manner. Moreover, the production of TNF-α was obviously (P < 0.05) inhibited at the concentration of 400 μg/mL in each extract as above. Contrarily, water extract showed no obviously effect on the productions of NO and TNF-α. PGE2 production was significantly inhibited at 400 μg/mL in ME, HM and WE, respectively. Western blot analysis revealed the suppression of iNOS, COX-2 expressions and IκB-α degradation were in the doses of 400 μg/mL from HM and SM, respectively. Our results suggested that extracts prepared from defatted perilla seed had antioxidant activities and the inhibition activities on the production of cytokines. Moreover, the production of iNOS and COX-2 proteins induced by LPS could be inhibited by extracts through blocking IκB-α degradation and therefore the inactivation of NF-κB.
|
|---|
