• Ocean and Polar Research
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• 제41권2호
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• pp.79-88
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• 2019

In this study, the inhibitory effect of Atriplex gmelinii C. A. Mey. against the activity of MMP-2 and MMP-9 secreted from phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 cells was evaluated by gelatin zymography and enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase-chain reaction (RT-PCR), and Western blot assay. Specimens of the halophyte A. gmelinii were extracted twice for 24 hr with methylene chloride ($CH_2Cl_2$), and then twice with methanol (MeOH), in turn. Each extract significantly inhibited the enzymatic activities in gelatin zymography and MMP ELISA kit, and expression of MMP-2 and 9 in mRNA and protein levels. Two crude extracts were combined and then the combined crude extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water ($H_2O$) fractions, according to solvent polarity. Among solvent-partitioned fractions, the 85% aq.MeOH fraction showed the strongest inhibitory effect against MMP-2 and -9 in gelatin zymography and MMP ELISA kit. In RT-PCR, all solvent-partitioned fractions significantly suppressed mRNA expression of MMP-2 and -9. On the other hand, in Western blot assay, all solvent-partitioned fractions except $H_2O$ significantly reduced expression levels of protein. HT 1080 cell migration was most significantly inhibited by the n-BuOH fraction followed by the 85% aq.MeOH and $H_2O$ fractions. These results suggest that A. gmelinii could be used as a potential source to inhibit tumor cell metastasis.

• 대한본초학회지
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• 제24권3호
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• pp.29-37
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• 2009

Objectives : The present study was carried out to investigate the effect of Trichosanthis Radix extract (TRE) on the proliferation and activation of eosinophils which were prepared from lung cells of asthma-induced mice by ovalbumin (OVA) treatment. Methods : C57BL/6 mouse was exposed to OVA three times a week for 6 weeks. The mouse lung tissues were dissected out, chopped and dossiciated with collagenase (1 $\mu$g/ml). Eosinophils were activated by rmIL-3/rmIL-5 co-treatments. The lung cells were treated with TRE, incubated for 48 hr at 37$^{\circ}C$, and analyzed by flow cytometer, ELISA and RT-PCR methods Results : To measure cytotoxicity, mouse lung fibroblast cells (mLFCs) were pretreated with various concentrations of TRE. TRE at 100 $\mu$g/ml, the highest concentration, examined did not have any cytotoxic effects on mLFCs. In FACS analysis, number of granulocyte/lymphocyte, CD3e-/CCR3+, CD3e+/CD69+, CD4+/CD8+ T cells in asthma-induced lung cells were significantly decreased by TRE treatment compared to the control group. But CD4+/CD25+ T cells were not examined significant change in lung cells treated with TRE. In ELISA analysis, production levels of IL-3, IL-5, IL-13 and histamine in asthma-induced lung cells, which were induced by rIL-3 plus rmIL-5 co-treatment, were significantly decreased by TRE treatment. Conclusions : The present data suggested that Trichosanthis Radix on the inhibition of parameters associated with asthma responses in eosinpophils, and thus implicate the possibility for the clinical application of Trichosanthis Radix.

• BMB Reports
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• 제29권2호
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• pp.105-110
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• 1996

Polyclonal antiserum R101 against aflatoxin $B_1$ ($AFB_1$) was raised in New Zealand white rabbits after injection of bovine serum albumin-$AFB_1$ conjugate. Competitive ELISA (enzyme linked immuno-sorbent assay) demonstrated that antiserum R101 has the highest binding for $AFB_1$ (50% inhibition at 170 fmol) and aflatoxicol II (50% inhibition at 112 fmol). It also reacts with other aflatoxins such as $AFB_2$, $AFG_1$, $AFG_2$, and aflatoxin metabolites ($AFM_1$, $AFM_2$, $AFP_1$, and $AFQ_1$), but it does not cross-react with $AFG_2a$. Using this antiserum, aflatoxins were quantitated in 100 urine samples of undergraduate students at the College of Pharmacy, Sung Kyun Kwan University, Republic of Korea. By ELISA, $AFB_1$ and its metabolites were detected in human urine samples (N=100, male=89, female=11, ages=20~31 yrs) with a range of 1.4~200.6 ng/kg/day (mean$\pm$SD=$18.11{\pm}33.01\;ng\;AFB_1/kg/day$ in males, $3.82{\pm}2.65\;ng/kg/day$ in females). Assuming that urinary excretion is about 7.6% of $AFB_1$ intake (Groopman et al., 1992), we estimated that Koreans were daily exposed to a total dietary $AFB_1$ of $240.20{\pm}438.67\;ng/kg/day$ in males and $50.35{\pm}29.88\;ng/kg/day$ in females, respectively. When the human monitoring data was applied to a linear regression model of Y=21.67X-10.04 {Y=liver cancer incidence per 100,000, X=Log $AFB_1$ intake (ng/kg/day), r=0.99} developed from previously reported epidemiological data, calculated liver cancer incidences attributed to $AFB_1$ exposure were 41.56/100,000 in males and 26.84/100,000 in females. The incidences were similarly correlated with liver cancer mortality rates of 43.43/100,000 in males and 11.23/100,000 in females in Korea. These results suggest that aflatoxin exposure may be an important risk factor for the high incidence of liver cancer in Korea.

• Journal of Dairy Science and Biotechnology
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• 제21권2호
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• pp.97-104
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• 2003

본 연구에서는 우유 앨러지의 원인물질중의 하나인 casein단백질을 생체가 알르겐 물질로 인식할 수 없는 수준의 분자로까지 분해하여 항원성을 저감시키고 또한 풍미가 좋은casein분해물을 제조하기 위하여 분해특성이 다른 효소를 사용하여 만들어진 분해물의 특성과 항원성을 조사하였다. 박테리아 효소인 Neutrase, Alcalase, Protamex, Pescalase을 처리한 casein가수분해물의 평균분자량은 1,100~2,300 dalton이었고 곰팡이 효소인 MP, Flavourzyme, LP, Promod경우에는 거의 770 ~ 1,140 dalton범위였다. 항원성 저감정도를 측정하기 위하여 토끼항casein항혈청을 이용하여 ELISA억제시험을 실시한 결과 항체결합을 50%저해하는 미분해casein단백질의 농도는 $10^{3.6}$ng/ml였다. 박테리아 효소인 Neutrase, Alcalase, Protamex, Pescalase에 의한 Casein가수분해물의 값은 각각 $10^{4.8},\;10^{5.5},\;10^{5.9},\;10^{6.1}\;ng/ml$이었고, 곰팡이 효소인 MP, Flavourzyme, LP, Promod경우에는 각각 $10^{6.3},\;10^{6.3},\;10^{6.5},\;10^{6.8}\;ng/ml$로 나타났다. 박테리아와 곰팡이효소의 조합인 Fla+prota, Pro+prota처리구에서는 $10^{7.4},\;10^{7.3}ng/ml$로 나타났다. 따라서 미분해 casein의 항원성을 1로 하면, casein분해물의 항원성은 최고 1/8,000 이하로 저하되었다. 수신피부아나피랙시스반응(PCA)을 이용한 항원성시험에서 casein으로 피하 감작할 경우 푸른색 반점이 나타났으나 효소처리구에서는 이러한 반점이 나타나지 않아 충분히 항원성이 저감화 되었음을 확인하였다.

• 생약학회지
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• 제34권3호통권134호
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• pp.210-217
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• 2003

Korean mistletoe (Viscum album) extract has been found to posses immunostimulatory activity. In this study, Korean mistletoe extract, M11C (non-lectin components), was used to know whether this extract might activate mouse splenic macrophages to produce tumor necrosis $factor-{\alpha}\;(TNF-{\alpha}$) and might play a role in anticancer. To know the effect of M11C on the production of $TNF-{\alpha}$, the splenic macrophages were treated by the M11C, and then collected the supernatant (M11C stimulated splenic macrophage-conditioned media; MSCM). MSCM was analyzed for the $TNF-{\alpha}$ secretion by means of ELISA and immunoblotting, and mRNA expression was analyzed by RT-PCR. The S-180 murine sarcoma model was established to know the effect of M11C on the inhibition of tumor growth. M11C had the effect of $TNF-{\alpha}$ production from splenic macrophages performed by ELISA technique. This ELISA data was reconfirmed by immunoblotting assay. The effects of M11C on the expression of $TNF-{\alpha}$ mRNA from the macrophages was also shown. M11C also had the inhibitory effect of S-180 tumor growth. These data suggest that Korean mistletoe extract M11C may be used for an immunomodulator.

• 한국식품과학회지
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• 제26권5호
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• pp.532-538
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• 1994

Chymotrypsin, trypsin, pancreatin, 그리고 Aspergillus oryzae 유래 단백질분해효소의 in vitro 처리에 의하여 유청단백질(WPI)의 가수분해물(WPH)중 ${\beta}-LG$유래의 항원성변화를 조사하기 위하여 토끼 항${\beta}-LG$항혈청을 이용한 competitive inhibition ELISA(cELISA)와 heterologous PCA를 실시하였다. cELISA에 의하여 WPH의 monovalent항원성을 분석한 결과, 전체적으로 ${\beta}-LG$유래의 monovalent항원성은 효소처리에 의하여 $10^{-1.7}{\sim}10^{-4.1}$배 또는 그 이하로 저하되었으며, 특히 pepsin전처리후 Asp. oryzae유래의 효소로 가수분해한 경우(OUP)의 항원성은 거의 상실되었다. Guinea pig를 이용한 PCA test에 의하여 ${\beta}-LG$유래의 polyvalent항원성을 분석한 결과, WPH의 항원성은 $1/2{\sim}1/128$ 또는 그 이하로 저하되었다. 특히, WPH중에서 열변성이나 pepsin의 전처리없이 Asp. oryzae유래의 효소로 가수분해한 경우(OUN), 가수분해도(DH)가 그다지 높지 않고 monovalent항원성도 여전히 잔존하였음에도 불구하고($10^{-3.2}$배로 저하) 알레르기의 발증과 밀접한 관련이 있는 polyvalent항원성은 거의 상실되었다. 이는 OUN의 분해효율이 아주 높지는 않으나 ${\beta}-LG$상의 항원결정기가 효과적으로 파괴되어, polyvalent항원성이 제거되었기 때문으로 추측된다. 이 결과는, Asp. oryzae유래의 효소를 WPI에 처리하면 우유 알레르기의 주요 원인물질인 ${\beta}-LG$의 polyvalent항원성이 제거됨으로써 저알레르기성 infant formula용 WPH가 제조될 수 있음을 시사하고 있다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권11호
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• pp.4609-4615
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• 2014

Background: To investigate tumor inhibition effects and mechanisms of Angelica sinensis and Sophorae flavescentis ait decoction (ASSF) combined with diamine-dichloroplatinum (DDP). Materials and Methods: Bodyweight, tumor inhibition rate and q value were calculated for single ASSF or ASSF combined with DDP on H22 carcinoma xenograft KM mice. Biochemical methods for serum LDH, AST, ALT, and AKP, ELISA method for serum HIF-$1{\alpha}$, pathological assessemnt of thymus, immunohistochemistry detection of tumor tissue caspase3 and mutant p53 protein, and qRT-PCR detection of bax/ bcl-2 mRNA were applied. Results: Compared with DDP control group, the bodyweight increased in ASSF-DDP group (p<0.01). Tumor inhibition rates for DDP, ASSF, ASSF-DDP were 62.7%. 43.7% and 71.0% respectively, with a q value of 0.90. Compared with other groups, thymus of DDP control group had obvious pathological injury (p<0.01), serum LDH, AST, ALT, AKP increased significantly in DDP control group (p<0.01), while serum HIF-$1{\alpha}$ was increased in the model control group. Compared with this latter, the expression of mutant p53 protein and bcl-2 mRNA were decreased in all treatment groups (p<0.01), but there were no statistical difference between DDP control p and ASSF-DDP groups. The expression of caspase3 protein and bax mRNA was increased in all treatment groups, with statistical differences between the DDP and ASSF-DDP groups (p<0.01). Conclusions: ASSF can inhibit bodyweight decrease caused by DDP, can inhibit tumor growth synergistically with DDP mainly through increasing serum HIF-$1{\alpha}$ and pro-apoptotic molecules such as caspase 3 and bax, rather than through decreasing anti-apoptotic mutant p53 and bcl-2. ASSF can reduce DDP toxicity due to decreasing the release of LDH, AST, ALT, AKP into blood and enhancing thymus protection.

• 한국가금학회지
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• 제18권3호
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• pp.183-196
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• 1991

세계적으로 가장 출현빈도가 높은 혈청형인 마사추셋형 IBV를 발육란에서 증식시킨 다음 요막강액을 채취, 바이러스를 농축 정제하여 항원으로 사용하고 야외 IBV감염계군에서 채취한 혈청중 HI반응에 의해 양성 및 음성혈청을 선발, 표준혈청으로 사용하여 ELISA를 시도한 결과 다음과 같은 성적을 얻었다. 1. 정제항원은 ELISA plate의 well당 40ng 단백량으로 coating하였을 때 높은 P/N치를 나타냈고 혈구응집항원은 well당 1.2~2.5 HA unit로 coating하였을 때 정제항원과 유사한 결과를 보였다. 2. 항원의 coating시 온도와 시간은 37$^{\circ}C$, 1시간이나 4$^{\circ}C$에 12~16시간 처리하였을 때 P/N에서 유의성 있는 차이를 보이지 않았으며 항원을 건조시켜 4$^{\circ}C$에 1개월 보관하여도 항원성의 변화를 인정할 수 없었다. 3 제품이 다른 3종류의 plate에서 항원 coating의 차이를 비교한 결과 제품간에 항원 coating의 균일도와 농도에 있어서 뚜렷한 차이가 인정되었다. 4. 음성혈청희석배수 1：50에서도 비특이 반응은 인정되지 않았으며 가경혈청은 1：100희석했을 때 높은 P/N치를 보여서 screen용 희석배수로 적당하였다. 5. Substrate처리한 후 발색 정도는 15분 이후에는 일정하여 30분까지 변화가 없었으며 이때 발색정지제를 처리하였을 때 치리직후부터 4시간까지도 흡광도에 있어서 유의성 있는 차이가 인정되지 않았다. 6. 74개의 혈청에 대해 ELISA에 의한 P/N치와 HI항체가와의 상관관계는 r＝0.42였으며 HI가 2$^{6}$이상, P/N치 1.4이상을 기준으로 하였을 때 양성 case의 일치율은 98.7%였다. 1. 백신접종 시험계군에서 ELISA와 HI test에 의한 항체 소장 비교에서는 10주째를 제외하고는 양 test에서 유사한 추이를 보였다.

• 대한한방내과학회지
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• 제29권3호
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• pp.730-741
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• 2008

Objective : To examine the efficacy of CY, the improvement of atopy dermatitis(AD)-like lesions on the rostral back of the NC/Nga mice, which took CY orally and were treated with a chemical substance called DNFB, the inhibition of ear swelling, and the inhibition of inflammatory response of the ear tissue of the mice were observed and compared, and the inhibition of the serum IgE count and the IL-4 and IFN-${\gamma}$ count produced by CD4+ T cells of the lymph node were observed and compared. Materials and Methods : For measurement of ear thicknesses, 0.15% DNFB $25{\mu}l$ mixed with acetone/olive oil(3:1) was applied to the right ear and dorsal skin of the NC/Nga mice 5 times at an interval of 7 days. Ear thickness was measured every day over a five-week period after the first application of DNFB. The total serum IgE count was measured with ELISA by taking blood samples 24 hours after the fifth application of DNFB. To measure the IL-4 and IFN-${\gamma}$ count, the lymph node was cut off, and the CD4+ T cells were purified and stimulated to activate the T cells, and then the IL-4 and IFN-${\gamma}$ count was measured with ELISA. Results : Continuous oral administration of CY improved the AD-like skin lesions on the rostral back and inhibited the ear swelling in NC/Nga mice treated with DNFB and inhibited the inflammatory response in the NC/Nga mice treated with DNFB NC/Nga mice. Continuous oral administration of CY did not significantly inhibit the serum IgE count and failed to significantly reduce the IL-4 count generated after TCR stimulation in the CD4+ T cells of the NC/Nga mice treated with DNFB. Continuous oral administration of CY significantly reduced the IFN-${\gamma}$ count generated after TCR stimulation in the CD4+ T cells of the NC/Nga mice treated with DNFB.

• 한국식품과학회지
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• 제26권1호
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• pp.74-80
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• 1994

효소에 의한 단백질분해가 유청단백질의 항원성의 저하에 미치는 영향을 조사하기 위한 기본연구로서, 유청단백질의 가수분해특성을 조사하고 competitive inhibition enzyme-linked immunosorbent assay(cELISA)에 의한 항원성의 변화를 검토하였다. 유청단백질의 가수분해는 chymotrypsin, trypsin, pancreatin, 그리고 Aspergillus oryzae유래의 protease를 각기 4시간 동안 행하였다. TNBS(trinitrobenzensulfonic acid)법에 의하여 측정한 유청단백질의 가수분해도(DH)는 chymotrypsin이나 trypsin을 처리한 경우$(5.05{\sim}11.47)$보다 Aspergillus oryzae유래의 protease 및 pancreatin을 처리한 경우$(15.67{\sim}20.20)$가 훨씬 높게 나타났으며, 각 효소의 처리전에 열처리($75^{\circ}C$, 20분)나 pepsin의 처리를 한 경우에 대체로 약간 높게 나타났다. High performance size exclusion chromatography(HPSEC)에 의하여 분자량분포를 조사한 결과, 가수분해물에 따라 10kDa 이상의 polypeptide가 $12{\sim}36%$ 정도 존재하였고, 평균분자량은 $4,252{\sim}9,132$ dalton, 평균길이는 아미노산 $38{\sim}83$개로 나타났다. 또한 쓴맛은 형성되지 않았다. SDS-PAGE의 결과 처리구에 따라 분자량 14.2kDa 이상의 polypeptide가 일부 존재하였으나 native 유청단백질은 대부분 가수분해에 의하여 제거되었음을 확인하였다. 토끼 항WPI항혈청에 의한 cELISA로 검토한 유청단백질 가수분해물의 monovalent 항원성은 효소처리에 의하여 약 $10^{-1.7}{\sim}10^{-4.9}$배 또는 그 이하로 저하되었으며 대체로 가수분해가 많이 일어난 분해물은 그 항원성이 낮아지는 것으로 나타났다. 또한 각 처리구내에서는 열 및 pepsin의 전처리후 다음 효소 분해한 유청단백질 가수분해물(CDP, TDP, PDP, ODP)의 경우 그 항원성이 가장 낮았다. 그중에서도 pancreatin 가수분해물(PDP)의 경우 항원성이 거의 상실된 것으로 나타났다.