• 한국미생물·생명공학회지
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• 제49권1호
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• pp.75-87
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• 2021

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα-2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp₄₅ signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SP_Usp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC₅₀ values of 33.22 ㎍/ml and 127.2 ㎍/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 ㎍/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 ㎍/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codon-optimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

• 한국식품위생안전성학회지
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• 제37권1호
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• pp.29-37
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• 2022

본 연구에서는 발효노니를 건강기능식품 소재로 활용 시 기초자료로 제공하고자 발효노니 추출물 및 지표성분의 면역활성 증진 효과를 평가하였다. RAW 264.7 대식세포에서 발효노니 추출물 및 지표성분 6종을 처리하여 XTT 세포독성평가, Nitric Oxide 생성 측정, Cyokine 생성 측정, immune marker genes 발현분석 수행하였다. 뿐만 아니라 양성대조군으로 LPS와 기능성 원료로 사용되고 있는 발효홍삼 추출물을 사용하였다. 그 결과 모든 처리 농도 및 처리군에서 세포독성이 관찰되지 않았으며, 지표성분 6종 중 SCP 및 ASE에서 NO 생성이 증가됨을 확인하였다. 뿐만 아니라 ASE 처리군에서는 IL-6 및 IL-1β의 생성이 증가되었으며, iNOS 및 TNF-α의 immune marker genes 발현이 증가됨을 확인하였다. 발효노니 추출물 효능 평가에서는 발효시 NO 생성 및 IL-6, IL-1β의 생성, COX2의 발현이 증가되는 것으로 나타났다. 이러한 연구 결과는 발효노니 추출물 및 지표성분의 선천면역 활성증가를 타나내며 노니 및 발효노니 표준화 연구에서 지표성분으로 사용 가능성을 제시한다. 따라서 발효노니는 면역증진 활성을 갖는 제품 개발에 있어서 유용한 기능성 식품소재로써 사용될 수 있으며, 우수한 효능을 나타내는 지표성분은 유용성분으로 이용이 가능할 것으로 사료된다.

• Animal Bioscience
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• 제35권5호
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• pp.763-777
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• 2022

Objective: Excessive lipid accumulation in adipocytes results in prevalence of obesity and metabolic syndrome. Curcumin (CUR), a naturally phenolic active ingredient, has been shown to have lipid-lowering effects. However, its underlying mechanisms have remained largely unknown. Therefore, the study aims to determine the effect of CUR on cellular lipid accumulation in porcine subcutaneous preadipocytes (PSPA) and to clarify novel mechanisms. Methods: The PSPA were cultured and treated with or without CUR. Both cell counting Kit-8 and lactate dehydrogenase release assays were used to examine cytotoxicity. Intracellular lipid contents were measured by oil-red-o staining extraction and triglyceride quantification. Apoptosis was determined by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling assay. Adipogenic and apoptosis genes were analyzed by quantitative polymerase chain reaction and Western blot. Results: The CUR dose-dependently reduced the proliferation and lipid accumulation of PSPA. Noncytotoxic doses of CUR (10 to 20 μM) significantly inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and expression of adipogenic genes peroxisome proliferation-activity receptor-γ (PPAR-γ), CCAAT/enhancer binding protein-α, sterol regulatory element-binding protein-1c, adipocyte protein-2, glucose transporter-4 as well as key lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase, while ERK1/2 activation significantly reversed CUR-reduced lipid accumulation by increasing PPAR-γ. Furthermore, compared with differentiation induced media treated cells, higher dose of CUR (30 μM) significantly decreased the expression of AKT and B-cell lymphoma-2 (BCL-2), while increased the expression of BCL-2-associated X (BAX) and the BAX/BCL-2 expression ratio, suggesting triggered apoptosis by inactivating AKT and increasing BAX/BCL-2 ratio and Caspase-3 expression. Moreover, AKT activation significantly rescued CUR inhibiting lipid accumulation via repressing apoptosis. Conclusion: These results demonstrate that CUR is capable of suppressing differentiation by inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis via decreasing AKT and subsequently increasing BAX/BCL-2 ratio and Caspase-3, suggesting that CUR provides an important method for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.

• 생명과학회지
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• 제32권11호
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• pp.890-898
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• 2022

본 연구에서는 유산균 발효가 큰느타리버섯 수확후배지를 식이한 흰점박이꽃무지 유충의 항산화 활성에 미치는 영향을 평가하기 위하여 흰점박이꽃무지 유충을 유산균으로 발효시키고, 흰점박이꽃무지 유충(PLE)과 발효 흰점박이꽃무지 유충(FPLE) 추출물의 항산화 활성을 비교하였다. 추출물 PLE와 FPLE의 총 폴리페놀 함량은 각각 65.02±1.32 mg GAEs/extract g과 93.33±0.98 mg GAEs/extract g이었고, 플라보노이드 함량은 각각 18.3±1.57 mg QEs/extract g과 17.69±0.95 mg QEs/extract g이었다. 추출물 PLE와 FPLE의 DPPH에 의한 라디칼소거 활성은 2-4 mg/ml의 농도에서는 두 시료 간의 유의적 차이를 나타나지 않았지만, 8mg/ml 이상의 농도에서는 추출물 PLE에 비해 FPLE의 DPPH 라디칼 소거능이 우수하였다. 추출물 PLE와 FPLE의 환원력도 추출물 PLE에 비해 FPLE이 우수하였으며 1.6 mg/ml 이상의 농도에서는 PLE에 비해 FPLE의 환원력이 2배 이상 높게 나타났다. 추출물 PLE와 FPLE의 ORAC 지수는 각각 74.34±0.37 uM TEs/extract g과 79.22±0.72 uM TEs/extract g으로 추추물 PLE에 비해 FPLE의 ORAC 지수가 높게 나타났고, RAW 264.7 세포에 대한 추출물의 세포독성은 나타나지 않았다. 이상의 결과를 종합하면, 큰느타리버섯 수확후 배지를 식이한 흰점박이꽃무지 유충은 유산균 발효에 의해 항산화물질인 총 폴리페놀 함량 및 항산화 활성이 증가된 것을 확인할 수 있었으며, 발효법은 흰점박이꽃무지 유충을 식의약품 소재로 개발하는 과정에 필요한 전처리 과정이라고 판단된다.

• 대한본초학회지
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• 제28권2호
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• pp.67-73
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• 2013

Objective : Lithospermum Erythrorhizon (LE) has been used as an anti-bacterial and anti-inflammatory agent. However, it is unclear that LE aqueous extract could show the anti-inflammatory effects in RAW 264.7cells. The purpose of this study was to investigate the anti-inflammatory effect of aqueous extract from LE on lipopolysaccharide (LPS) - induced inflammatory response. Methods : To measure out the cytotoxicity of LE, we performed the MTT assay. To evaluate the anti-inflammatory effects of LE, we examined the inflammatory mediators such as nitric oxide (NO), prostaglandin E2 ($PGE_2$) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-${\alpha}$, interleukin, (IL)-$1{\beta}$ and (IL)-6) on RAW 264.7 cells. We also examined molecular mechanisms such as mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-${\kappa}B$) activation by western blot. Results : Aqueous Extract from LE itself did not have any cytotoxic effect in RAW 264.7 cells. Aqueous extract from LE inhibited LPS-induced productions of inflammatory mediators such as NO, $PGE_2$, and pro-inflammatory cytokines including TNF-${\alpha}$, IL-$1{\beta}$ and IL-6 in RAW 264.7cells. In addition, LE inhibited the phosphorylation of p38 kinases (p38), c-Jun $NH_2$-terminal kinase (JNK), and NF-${\kappa}B$ activation in RAW 264.7 cells. Conclusion : LE down-regulated LPS-induced production of inflammatory mediators through the inhibition of p38, JNK and NF-${\kappa}B$ activation. Taken together, these results could provide the evidence for the anti-inflammatory effects of LE. Therefore, LE may be a novel target in the management of inflammation and help to support a potential strategy for prevention and therapy of inflammatory diseases.

• 대한본초학회지
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• 제28권5호
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• pp.7-11
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• 2013

Objectives : Seungmagalgeun-tang (SMGGT) is traditional medicine widely used for inflammatory disease and flu. But SMGGT exhibits potent anti-inflammatory activity with an unknown mechanism. To elucidate the molecular mechanisms of SMGGT water extract on pharmacological and biochemical actions in inflammation, we examined the effect of SMGGT on pro-inflammatory mediators in Phorbol-12-myristate-13-acetate (PMA)+A23187-stimulated mast cells. Methods : In the present study, pro-inflammatory cytokine production was determined by performing enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and western blot analysis to measure the activation of MAPKs. Cells were treated with SMGGT 1 h prior to the addition of 50 nM of PMA and $1{\mu}M$ of A23187. Cell viability was measured by MTS assay. The investigation focused on whether SMGGT inhibited the expressions of interleukin-6 (IL-6), interleukin-8 (IL-8) and mitogen-activated protein kinases (MAPKs) in PMA+A23187-stimulated mast cells. Results : SMGGT has no cytotoxicity at examined concentration (100, 250, and $500{\mu}g/ml$). Also, gene expression of IL-6 and IL-8 in HMC-1 cells stimulated by PMA+A23187 was down regulated by SMGGT. Furthermore, SMGGT suppressed the PMA+A23187-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-jun N-terminal Kinase(JNK). But, SMGGT could not regulate phosphorylation of p38 MAPK. Conclusions : These results suggest that SMGGT has inhibitory effects on PMA+A23187-induced IL-6 and IL-8 production. These inhibitory effects occur through blockades on the phosphorylation of ERK and JNK.

• 대한본초학회지
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• 제32권2호
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• pp.25-32
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• 2017

Objectives : This study was conducted to investigate candidate materials as anti-inflammatory agent from extracts of Korean medicinal plants in Hwaak mountain. Ligustrum obtusifolium (LO) is a Korea medicinal plants that commonly used for robustness and hemostasis. It has been reported that LO has exhibited anti-ischemic, anti-oxidative, anti-hypolipidemic, anti-tumor and hypoglycemic effects. However, LO has not been previously reported to have an anti-inflammatory effect. Therefore, we have evaluated the anti-inflammatory effects of LO and its underlying molecular mechanisms in LPS-induced RAW 264.7 macrophages. Methods : Cell viability was determined by MTT assay in RAW 264.7 macrophages. Nitric Oxide (NO) was measured with Griess reagent and pro-inflammatory cytokines were detected by ELISA in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Protein expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and p65 subunit of nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) were determined by Western blot analysis. Results : Among 15 extracts of Korean medicinal plants tested, Ligustrum obtusifolium (LO) showed the inhibition of NO production without cytotoxicity. LO reduced the expression levels of iNOS and COX-2 proteins in LPS-simulated RAW 264.7 macrophages in dose-dependent manner. Consistent with these data, LO inhibited the productions of $TNF-{\alpha}$, IL-6, and $IL-1{\beta}$ in LPS-simulated RAW 264.7 macrophages. Furthermore, LO attenuated the LPS-induced nuclear translocation of p65 $NF-{\kappa}B$ in RAW 264.7 macrophages involving suppression of $NF-{\kappa}B$ activation. Conclusions : Taken together, these results suggest that the anti-inflammatory effects of LO is associated with regulation of inflammatory mediators via inhibition of $NF-{\kappa}B$ activation in LPS-treated RAW 264.7 macrophages.

• 대한본초학회지
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• 제28권4호
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• pp.17-23
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• 2013

Objectives : The objective of the paper was to study cutting method of Gardeniae frutus, that tends to show symptom of separations in a packing, in accordance with the comparison experiment of quantification and antioxidant effect proceed with the intact Gardeniae fructus(Gf), seed of Gardeniae fructus (Gs) and the pericarp of Gardeniae fructus (Gp) separately. Methods : The Gf, Gs, and Gp were extracted using 80% MeOH, followed by quantizing geniposide contained in each group. A MTT assay was conducted and ROS generation and NO production were measured for comparing its antioxidant effect. Results : As a result of quantizing geniposide contained in the Gf, Gp, and Gs, respectively, the geniposide content was shown to be the highest in the Gs. MTT assay showed that no cytotoxicity was observed in the groups treated with Gf, Gp, and Gs, respectively, at a dose of 500 ${\mu}g/ml$. The ROS generation was shown to have more significantly decreased in the group pretreated with Gf 500 ${\mu}g/ml$ than in the group treated with LPS. The NO level was shown to have more significantly decreased in the group pretreated with Gp 500 ${\mu}g/ml$ than in the group treated with LPS. Conclusion : As the geniposide content and antioxidant effect of Gf varies according to its each part, it is recommended that Gf should be distributed as an intact form other than segregation in packing.

• 대한본초학회지
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• 제32권2호
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• pp.77-85
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• 2017

Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan(HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells. Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. $PGE_2$ were measured using EIA kit. Pro-inflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. $NF-{\kappa}B$/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays. Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, $TNF-{\alpha}$, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of $NF-{\kappa}B$ in response to competitive LPS. Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of $NF-{\kappa}B$ delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced $PGE_2$, NO, IL-6 and $TNF-{\alpha}$.

• 대한본초학회지
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• 제34권1호
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• pp.23-31
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• 2019

Objectives : The aim of this study was to investigate the effect for allergic-inflammation of Fritillariae Thunbergii Bulbus (FTB) on HaCaT cells and RBL2H3 cells. Methods : To investigate the effects of FTB for anti-inflammation in HaCaT cells, the cells were pretreated with FTB for 1h and then stimulated with $TNF-{\alpha}/IFN-{\beta}$ for 24h. Then thymus and activation-regulated chemokine (TARC) and Macrophage-derived chemokine (MDC) levels were analyzed with ELISA kit. Also to investigate the effect of skin barrier protein, the cells were treated with FTB of various concentrations, and then cells were harvested, expressions of skin barrier protein were measured with RT-PCR. To investigate the effects of FTB for anti-allergy in RBL2H3 cells, the cells were pre-treated with FTB for 1h, and then stimulated with A23187 for 30 min. ${\beta}$-hexosaminidase, IL-4 and $TNF-{\alpha}$ were measured using cultured media. The cells were harvested to analyze the mechanism of the effect for FTB via Western blot. Results : FTB did not show cytotoxicity in HaCaT and RBL2H3. In HaCaT cells, FTB significantly suppressed the expression of TARC, MDC at a dose-dependent manner and markedly increased formation of the skin barrier proteins. In RBL2H3 cells, FTB decreased release of the ${\beta}$-hexosaminidase, IL-4 and $TNF-{\alpha}$ in RBL2H3 through inhibition of the phosphorylation of JNK and p38, which are include in the signaling mechanism of MAPK Conclusion : These results indicate that FTB has an anti-inflammatory effect on the allergic response through blocking MAPK pathway. This suggest that FTB could be a therapeutic agent for allergic response.