• The Korea Journal of Herbology
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• v.34 no.6
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• pp.117-124
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• 2019

Objective : Broccoli is edible green plant that has a wide variety of health benefits including cancer prevention and cholesterol reduction. However, leaves of broccoli are not eaten and are mostly left as waste. This study was conducted to evaluate the effects of the broccoli leaf extract (BLE) on prostaglandin E₂ (PGE₂) production related to nuclear factor kappa B (NF-κB) signaling in lipopolysaccharide (LPS)-activated macrophages. Methods : BLE was prepared by extracting dried leaf with ethanol. Cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PGE₂ and inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Expression level of each protein was monitored by Western blot analysis. Results : In LPS-activated Raw264.7 cells, PGE₂ release into culture medium was dramatically enhanced compared to control cells. However, increased PGE₂ was attenuated dose-dependently by treatment with BLE. Inhibition of PGE₂ production by BLE was due to the suppression of cyclooxygenase-2 (COX-2) expression determined by Western blot analysis. BLE also inhibited the production of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Inhibition at PGE₂ and cytokine was mediated from inhibition of nuclear translocation of NF-κB due to the repression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation. Conclusion : This study showed that BLE exerted inhibitory activities against PGE₂, which is critical for the initiation and resolution of inflammatory responses, and that inhibition of PGE₂ was mediated by suppression of NF-κB signaling. These results suggest that the waste broccoli leaves could be used for controlling inflammation.

• Natural Product Sciences
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• v.23 no.3
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• pp.175-182
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• 2017

This study evaluated the anti-Helicobacter and anti-inflammatory effects of Sohamhyungtang (SHHT). The minimum inhibitory concentration (MIC) of SHHT against Helicobacter pylori (H. pylori) was determined by the agar dilution method. Expression of the H. pylori cagA gene in the presence of SHHT was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Inhibition of H. pylori urease by SHHT was determined by the phenol-hypochlorite assay. Antiadhesion activity of SHHT was measured by urea-phenol red reagent. Inhibition of nitric oxide (NO) production in AGS cells was measured with Griess reagent. Inducible nitric oxide synthase (iNOS) and IL-8 mRNA expression in AGS cells which were infected with H. pylori was determined by qRT-PCR. IL-8 level was measured by enzyme-linked immunosorbent assay (ELISA). The MIC of SHHT was $100{\mu}g/mL$ and the expression of cagA gene was decreased about 25 folds in the presence of SHHT. H. pylori urease was inhibited 90% by SHHT. SHHT inhibited H. pylori adhesion on AGS cell in a concentration dependent manner. mRNA expression of iNOS and IL-8 and the production of NO and IL-8 were significantly decreased in the presence of SHHT. In conclusion, SHHT showed anti-Helicobacter activity and has potent anti-inflammatory effect on H. pylori-induced inflammation in human gastric epithelial AGS cells.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.20 no.2 s.33
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• pp.68-76
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• 2007

Objectives : This experimental study was performed to investigate the effects of Sulfur extract on anti-inflammation and anti-Propionibacterium acnes. Methods : The cytotoxicity of Sulfur extract about viability of Raw 264.7 cell were tested using a colorimetric tetrazolium assay(MTT assay). To investigate the anti-inflammation effects of Sulfur extract on LPS-induced macrophage Raw 264.7 cell, we used ELISA kit and Western blots. We evaluated anti-oxidation effects of Sulfur extract on HaCaT cell by Enzyme recycling method. And we investigated the inhibitory effects of Sulfur extract on Propionibactrium acnes using paper disk diffusion method. Results: 1. Sulfur extract has a little cytotoxicity in Raw 264.7 cell. 2. Concentration of $100\;{\mu}g/m{\ell}$ Sulfur extract inhibited the production of NO in the Raw 264.7 cell stimulated with LPS. 3. Sulfur extract showed a oxidation inhibition effect by decreasing the DPPH radicals. 4. Sulfur extract has not the significant inhibition effect of Propionibactrium acnes. Conclusions: These results indicate that Sulfur extract has anti-inflammation and anti-oxidation effects. If further study is performed, the use of Sulfur extract will be valuable and benificial in the therapy of Propionibactrium acnes.

• KSBB Journal
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• v.16 no.6
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.255-255
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• 1994

MIEF는 소의 초유중에 함유되어 있는 분자량 22,000 dalton의 peptide로서 말초혈액 림파구의 배양액에 첨가할 경우 자연살해세포를 활성화 시켜 암세포에 대한 자연살해능을 증가시키는 것으로 이미 보고된 바 있다. 본 연구에서는 수유기간별 MIEF의 함량 및 MIEF가 B세포의 증식 및 분화에 미치는 영향을 조사하였다. 수유기간별 MlEF의 함량을 competitive ELISA inhibition assay를 이용하여 조사한 결과 MIEF의 함량은 수유 첫째날의 초유에 109$\mu\textrm{g}$/ml로 가장 높았으며, 그 이후로 급격히 감소하여 3일째 이후에서는 3-4$\mu\textrm{g}$/ml이하로 존재하였다. MIEF가 B세포의 분화에 미치는 영향은 사람의 편도선에서 분리한 림파구의 배양액에 MIEF를 가하고 5-9일간 배양한후 항체를 생산하는 세포의 수를 ELISPOT assay로 측정하여 조사하였다. MIEF는 10-30$\mu\textrm{g}$/ml의 농도에서 B세포의 분화를 촉진시켰으며, 그 정도는 양성대조군으로 사용한 LPS와 유사하여 MIEF가 B세포의 분화를 유도하는 작용이 아주 강력함을 알수 있었다.

• Journal of Korean Society on Water Environment
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• v.33 no.5
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• pp.538-545
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• 2017

The purpose of this study is to investigate growth response and toxigenicity under various temperature and nutritional conditions, in order to understand the physioecological characteristics of Aphanizomenon flos-aquae, which is a bloom-forming cyanobacterium in the Nakdong River. The strain was inoculated into media under combinations of four temperatures (4, 12, 21, $30^{\circ}C$) and three nutrients (modified CB medium, P-depleted CB medium, N-depleted CB medium) for 28 days. The algae-inhibition tests were performed to assess the potential allelopathic effects of the strains' filtrates on the growth of four algae strains (Microcystis aeruginosa, Aulacoseria ambigua f. spiralis, Aphanizomenon flos-aquae, Scenedesmus obliquus). Toxin production of a strain was measured by Enzyme-Linked ImmunoSolbent Assay (ELISA). The optimal growth temperature (Topt) of strains was $19.9^{\circ}C$ ($18.3-21.2^{\circ}C$), and the temperature range for growth was from $-0.3^{\circ}C$ to $34.3^{\circ}C$. Specific growth rate (${\mu}$) in modified CB medium varied from 0.10 to $0.16day^{-1}$, and the maximum growth rate (${\mu}_{max}$) was $0.17day^{-1}$. Although growth curves under N-existed and N-depleted conditions were almost the same, growth under N-depleted condition was relatively slowed (${\mu}=0.09$ to $0.14day^{-1}$), with a decreased maximum cell density. However, growth under the P-depleted condition was restricted for all temperatures, Two stains of Aphanizomenon flos-aquae were confirmed as not producing toxins, because saxitoxin and cylindrospermopsin were not detected by ELISA. The exudates or filtrates from the Aphanizomenon flos-aquae (DGUC003) resulted in significant inhibition of algal growth on the Aulacoseira ambigua f. spiralis (DGUD001) and Aphanizomenon flos-aquae (DGUC001) (p < 0.01).

• The Journal of Korean Medicine
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• v.40 no.2
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• pp.35-50
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• 2019

Objectives: Orostachys japonicas (O. japonicus) has been known for its anti-tumor effect. In the present study, it was investigated whether O. japonicus EtOH extracts could induce apoptosis and autophagy which are part of the main mechanism related to anti-tumor effect in THP-1 cells. Methods: Cells were treated with various concentrations of O. japonicus EtOH extracts ($0-300{\mu}g/ml$) for 24, 48, and 72h. Cell viability was evaluated by MTS/PMS assay and apoptosis rate was examined by flow cytometry and ELISA assay. The mRNA expression of apoptosis-related genes (Bcl-2, Mcl-1, Survivin, Bax) and autophagy-related gene (mTOR) was evaluated using real-time PCR. The protein expression of Caspase-3, Akt, LC3 II, Beclin-1, Atg5, $NF-{\kappa}B$, p38, ERK was evaluated using western blot analysis. Results: O. japonicus EtOH extracts inhibited cell proliferation and apoptosis rate was increased in both flow cytometry and ELISA assay. Bcl-2, Mcl-1, Survivin (anti-apoptosis factors) mRNA expressions were decreased and Bax (pro-apoptosis factor) mRNA level was increased. mTOR mRNA expressions was decreased and LC3 II protein expressions was increased. Activation of $NF-{\kappa}B$ was decreased and phosphorylation of p38 was increased. Conclusion: O. japonicus is regarded to inhibit cell proliferation, to induce apoptosis and to regulate autophagy-related genes in THP-1 cells via $NF-{\kappa}B$ and p38 MAPK signaling pathway. This suggests O. japonicus could be an effective herb in treating acute myeloid leukemia.

• Ocean and Polar Research
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• v.40 no.4
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• pp.249-257
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• 2018

Matrix metalloproteinases (MMPs) are associated with the invasion and metastasis of malignant tumors composed of cancer cells in an increased state of expression. This study evaluates the inhibitory effect of Carex pumila on MMP-2 and MMP-9 activity in phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 human fibrosarcoma cells using gelatin zymography, MMPs enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. C. pumila was extracted twice with dichloromethane ($CH_2Cl_2$) and methanol (MeOH). Treatment with $CH_2Cl_2$ extract and MeOH extract in PMA-stimulated HT-1080 cells effectively reduced the production of MMP-2 and 9. Also, the combined crude extracts ($CH_2Cl_2$ and MeOH) significantly inhibited the enzymatic activities and the expression of MMP-2 and MMP-9 in mRNA and protein levels. The combined crude extracts were partitioned between $CH_2Cl_2$ and water. The organic layer was further fractionated with n-hexane, 85% aqueous methanol (85% aq.MeOH) and the aqueous layer was separated into n-butanol and water, successively. Of the fractions, 85% aq.MeOH fraction showed the highest inhibitory activity of MMP-2 and MMP-9 in gelatin zymography and MMP ELISA kit. Furthermore, 85% aq.MeOH fraction most significantly suppressed cell migration. In RT-PCR and Western blot assay, n-butanol and 85% aq.MeOH fractions exerted the greatest inhibition on mRNA and protein expression of MMP-2 and MMP-9, respectively. As a result, C. pumila can be used as a good anti-invasive agent source.

• Korean Journal of Veterinary Research
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• v.63 no.4
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• pp.37.1-37.7
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• 2023

Canine respiratory coronavirus (CRCoV) is a significant pathogen that causes respiratory diseases in dogs, collectively known as a canine infectious respiratory disease. The virus is highly contagious and exhibits high seroprevalence worldwide. Currently, bovine coronavirus (BCoV) enzyme-linked immunosorbent assay (ELISA) kits are used to detect CRCoV antibodies. However, BCoV-ELISA kits cannot differentiate between infections caused by BCoV and those caused by CRCoV. In this study, we evaluated the hemagglutination inhibition (HI) test for CRCoV by comparing it with the virus neutralization (VN) test. Subsequently, we evaluated the seroprevalence of CRCoV in 383 dog serum samples collected from South Korea utilizing the HI test. The HI test for CRCoV showed a strong correlation with the VN test (R = 0.83, p < 0.001). The analysis of seroprevalence revealed that 52.2% (95% confidence interval [CI], 47.2%-57.1%) of the Korean dog serum samples were positive. The seroprevalence exhibited varied with age, with a positivity rate of 43.9% in dogs under 1 year of age and 66.7% in dogs aged 3 to 5 years (odds ratio, 2.54; 95% CI, 1.43-4.59). In conclusion, the HI test to monitor CRCoV antibody proved to be closely related to the VN test. Furthermore, over half of the dogs in Korea tested positive for CRCoV antibodies. These findings contribute to a better understanding of the sero-epidemiology of CRCoV.

• Proceedings of the SCSK Conference
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• 2003.09b
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• pp.12-25
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• 2003

L-camitine ($\beta$ -hydroxy-${\gamma}$ -trimethyl-ammoniumbutyric acid) is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-camitine, we investigated the effects of in vitro MMP inhibition and activity and expression of UVA-induced MMP 1 in human skin fibroblasts. Fluorometric assays of the proteolytic activities of MMP-l were performed using fluorescent collagen substrates. ELISA (enzyme linked immuno sorbent assay), gelatin-substrate zymography, and RT-PCR ELISA techniques were used for the effects of L-camitine on MMP expression and activity, MMP mRNA expression in UVA irradiated fibroblast. L-camitine inhibited the activities of MMP-l in a dose-dependent manner and the $IC_{50}$/ values calculated from semi-log plots were 2.45mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP expression was reduced 40% by treated with L-carnitine, and MMP-l mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibition the MMP activity, regulation of MMP expression in protein and mRNA level. All these results suggest that L-carnitine may be useful as new anti-aging cofactor for protection against UVA induced MMP expression and activity.