• Journal of Life Science
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• v.19 no.6
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• pp.728-734
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• 2009

In this work, a simple, inexpensive and reproducible technique of flotation density gradient centrifugation was developed to isolate monocytes with high purity and yield from peripheral blood mononuclear cells (PBMC) using Histopaque solution, density and osmolarity of which were modified to 1.072 g/ml and 335 mOsm with phosphate-buffered saline (PBS) and sodium chloride (NaCl) solution, respectively. The average purity of monocytes was 74.75${\pm}$3.84%, with the individual purity ranging from 71.44% to 82.38%. The average yield of monocytes was 32.62${\pm}$11.16%, with the individual yields ranging from 21.02 to 53.63%. The monocytes isolated by floatation density gradient centrifugation could be successfully cultured into morphologically, phenotypically and functionally dendritic cells in vitro. In conclusion, the entire procedure seemed to be faster and more convenient, simple and cost-effective than other monocyte isolation methods, including plastic adherence and density gradient methods, and has the potential to be developed as a closed system for clinical scale generation of dendritic cells.

• Food Science and Preservation
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• v.16 no.4
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• pp.554-561
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• 2009

We investigated the antioxidative effects of solvent extracts of doenjang fermented using Bacillus subtilis DJI (DJI doenjang) in vitro. The solvents used for extraction were ethanol, n-hexane, and water. The antioxidative activities of DJI doenjang solvent extracts were measured by estimation of peroxide value, the presence of linoleic acid level, and nitrite scavenging activity, the Rancimat test, and 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical generation, in comparison with the commercial antioxidant butylated hydroxytoluene (BHT). The peroxide value of an ethanol extract was lower than those obtained using n-hexane and water extracts. Furthermore, the peroxide value of the ethanol extract was similar to that obtained after BHT treatment. The nitrite scavenging activity was 23.36% after addition of 600 ppm DJI doenjang ethanol extract, and the DPPH free-radical scavenging activity was 19.06% under same condition, which shows that DJI doenjang ethanol extract exhibited lower antioxidative capacities than did BHT. In the Rancimat test, the ethanol extract (11.20 min induction time), n-hexane extract (7.58 min induction time), and water extract (8.26 min induction time) after treatment with 600 ppm DJI doenjang extracts demonstrated longer induction periods than did BHT (6.94 min). These results indicate that DJI doenjang has potential anti-oxidative activity.

• Journal of Nutrition and Health
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• v.49 no.3
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• pp.135-143
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• 2016

Purpose: Ultraviolet (UV)-induced oxidative stress contributes to several adverse biological effects on skin. Many phenolic phytochemicals have been shown to have antioxidant properties and protect skin cells from UV-induced oxidative damage. In this study, we investigated whether or not Aralia elata (AE) has a protective effect against UVB-induced reactive oxygen species (ROS), ultimately leading to photoaging. Methods: Phenolic content of dried AE and antioxidant properties of AE extract in 70% ethanol weredetermined by measuring DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power (FRAP). The effect of AE extract on cellular ROS generation and expression levels of oxidative stress-response proteins such as superoxide dismutase (SOD)-1, catalase, nuclear factor-erythroid 2-related factor (Nrf)-2, and heme oxygenase (HO)-1 in UVB-irradiated ($75mJ/cm^2$) human keratinocytes (HaCaT) were further determined by 2'-7'-dichlorofluoresceine diacetate assay and Western blotting, respectively. Results: The total phenolic and flavonoid contents of dried AE were 20.15 mg tannic acid/g and 18.75 mg rutin/g, respectively. The $IC_{50}$ of AE extract against DPPH radical was $98.5{\mu}g/mL$, and ABTS radical scavenging activity and FRAP upon treatment with $1,000{\mu}g/mL$ of AE extract were $41.8{\mu}g\;ascorbic\;acid\;(AA)\;eq./mL$ and $29.7{\mu}g\;AA\;eq./mL$,m respectively. Pretreatment with AE extract significantly reduced (p < 0.05) ROS generation compared to that in UVB-irradiated control HaCaT cells. Pretreatment with AE extract reversed reduction of Nrf-2 and SOD-1 protein expression and induction of HO-1 protein expression caused by UVB exposure in HaCaT cells, whereas it did not affect catalase expression. Conclusion: AE extract in 70% ethanol demonstrated a protective effect against UVB-induced oxidative stress and decreased expression of Nrf-2 and SOD-1 in human keratinocytes. These findings suggest that AE ethanol extract might have potential as a natural resource for a skin anti-photoaging product in the food and cosmetic industry.

• Proceedings of the Ginseng society Conference
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• 1988.08a
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• pp.115-121
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• 1988

Although Saponin A from Panax ginseng has previously been shown to inhibit carageenin induced edema. a paucity of information exists on the effects of components from ginseng on the cellular inflammatory response. specifically polymorphonuclear leukocyte (PMNL) function. The purpose of this study was 10 determine the effects of isolated neutral dammarane saponins from ginseng (i.e..glycosidic derivatives of 20(S)-protopanaxadiol [ginsenoside $Rb_1,\;Rb_2$ and Rc] and 20(S)-protopanaxatriol [ginsenosides Re and $Rg_1$]) on in vivo PMNL function and to compare their effects with those produced by a steroidal anti-inflammatory agent (dexamethasone) and commercially available saponin. Dexamethasone. the ginsenosides and saponin were all shown to he potent inhibitors of PMNL chemotaxis using the $^{51}Cr$ assay with $5{\times}10^{-8}M$ f-met-leu-phe [FMLP] as the chemoattractant. Inhibition or PMNL chemotaxis by dexamethasone. the ginsenosides and saponin were all shown to be both time-and dose-dependent and these agents did not affect cellular viability at the concentrations tested Saponin and the ginsenosides were more potent inhibitors of chemotaxis than was dexamethasone. while oxidant generation (as measured by the luminol-enhaneed chemil-uminescence of PMNL using FMNL $[10^{-6}]$ as the stimulus) was inhibited by dexamethasone. the ginsenosides $(Rb_1\;Rb_2\;Rc\;Re\;and\;Rg_1)$ and saponin at a concentration of 1 ${\mu}M$ had no significant effect on PMNL chemiluminescence. Thus. the neutral dammarane saponins are potentially important modulators or PMNL function and their inhibitory effects may he differentiated from those of the Steroidal anti-inflammatory agents.

• Journal of Chest Surgery
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• v.35 no.7
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• pp.501-510
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• 2002

Background: The various pathogeneses of acute respiratory distress syndrome have been suggested but not established yet. In the present study, the role of group II phospholipase $A_2$($PLA_2$) in the pathogenesis of gut ischemia-reperfusion(I/R) induced acute lung injury (ALI), especially in the pulmonary oxidative stress with infiltration of neutrophils was investigated. Material and Method: To induce ALI, reperfusion of mesentery was done for 120 min after clamping of superior mesenteric artery for 60 min in Sprague-Dawley rats that weighed about 300g. To exmaine the role of group II $PLA_2$ in ALI, especially endothelial injury associated with the action of neutrophils, lung myeloperoxidase activity, lung leak index, bronchoalveolar lavage fluid protein were measured, and pulmonary $PLA_2$ activity changes in gut I/R were also measured. The role of group II $PLA_2$in the neutrophilic generation of free radicals was assessed by inhibiting group II $PLA_2$ with rutin, manoalide and scalaradial. Furthermore, to verify the oxidative stress in the lung, histologic and free radical detecting cytochemical electron microscopy were done. Result: After reperfusion, ALI was developed with accumulation of neutrophils in the lung, which was confirmed by the increase of myeloperoxidase activity, lung leak index and bronchoalveolar lavage protein (p<0.001). The pulmonary and intestinal group II $PLA_2$ activities significantly increased after gut I/R which were reversed by rutin(p<0.001). In vitro, cytochrome-c reduction assay denoted the inhibitory effects of rutin, scalaradial and manoalide on the production of free radicals from isolated human neutrophils. Histologically, neutrophilic accumulation and pericapillary edema in the lung after gut I/R was detected by light microscopy which was suppressed by rutin. In $CeCl_3$ cytochemical electron microscopy, the increased production of hydrogen peroxide in the lung after gut I/R was confirmed and also the production of hydrogen peroxide was decreased by rutin. Conclusion: On the basis of these experimental results, the inhibition of group II $PLA_2$ seemed to mitigate gut I/R-induced ALI by suppressing the production of free radicals from the infiltrated neutrophils. Collectively, group II $PLA_2$ seems to play a crucial role in gut I/R-induced ALI by neutrophilic oxidative stress.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.2
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• pp.529-541
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• 2003

Yukmijihwang-tang has been widely used as an and-aging herbal medicine for hundred years in Asian countries. Numerous studies show that Yukmijihwangtang has anti-oxidative effect both in vivo and in vitro. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective herb in Yukmijihwang-tang on undifferentiated PC12 cells upon oxidative-stressed with hydrogen peroxide. The purpose of this study is to; 1) evaluate the recovery of neuronal damage by assessing the anti-oxidant effect of MCR on PC12 cells differentiated with nerve growth factor (NGF), 2) identify candidate genes responsible for anti-oxidative effect on differentiated PC12 cells by oligonucleotide chip microarray. PC12 cells, which were differentiated by treating with NGF, were treated without or with hydrogen peroxide in the presence or absence of various concentration of MCR. Cell survival was determined by using MTS assay. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2DCFDA assay The viability of cells treated with MCR was significantly recovered from stressed PC12 cell. In addition, wide rage of concentrations of MCR shows dose-dependent inhibitory effect on ROS production in oxidative-stressed cells. Total RNAs of cells without treatment(Control group), only treated with H₂O₂ (stressed group) and treated with both H₂O₂ and of MCR (MCR group) were isolated, and cDNAs was synthesized using oligoT7(dT) primer. The fragmented cRNAs, synthesized from cDNAs, were applied to Affymetrix GeneChip Rat Neurobiology U34 Array. mRNA of Calcium/calmodulin-dependent protein kinase II delta subunit(CaMKII), neuron glucose transporter (GLUT3) and myelin/oligodendrocyte glycoprotein(MOG) were downregulated in Stressed group comparing to Control group. P2X2-5 receptor (P2X2R-5), P2X2-4 receptor (P2X2R-4), c-fos, 25 kDa synaptosomal attachment protein(SNAP-25a) and GLUT3 were downregulated, whereas A2 adenosine receptor (A2AR), cathechol-O-methyltransferase(COMT), glucose transporter 1 (GLUT1), EST223333, heme oxygenase (HO), VGF, UI-R-CO-ja-a-07-0-Ul.s1 and macrophage migration inhibitory factor (MIF) were upregulated in MCA group comparing to Control group. Expression of Putative potassium channel subunit protein (ACK4), P2X2A-5, P2X2A-4, Interferon-gamma inducing factor isoform alpha precursor (IL-18α), EST199031, P2XR, P2X2 purinoceptor isoform e (P2X2R-e), Precursor interleukin 18 (IL-18) were downregulated, whereas MOO, EST223333, GLUT-1, MIF, Neuronatin alpha, UI-R-C0-ja-a-07-0-Ul.s1, A2. adenosine receptor, COMT, neuron-specific enolase (NSE), HO, VGF, A rat novel protein which is expressed with nerve injury (E12625) were upregulated in MCR group comparing to Stressed group. The results suggest that decreased viability and AOS production of PC12 cell by H₂O₂ may be, at lease, mediated by impaired glucose transporter expression. It is implicated that the MCR treatment protect PC12 cell from oxidative stress via following mechanisms; improving glucose transport into the cell, enhancing expression of anti-oxidative genes and protecting from dopamine cytotoxicity by increment of COMT and MIF expression. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the anti-oxidative effects of herbal extract Moutan Cortex Radicis.

• Tuberculosis and Respiratory Diseases
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• v.63 no.6
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• pp.497-506
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• 2007

Background: The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase $A_2$ ($cPLA_2$) activation by plateletactivating factor (PAF). Methods: The possible formation of the positive feedback loop of the $cPLA_2$ activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung. Results: Five hours after intratracheal instillation of PAF ($5{\mu}g/rat$), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic $cPLA_2$ activity were increased following PAF exposure and exposure to hydrogen peroxide increased $cPLA_2$ activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by $CeCl_3$ cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment. Conclusion: PAF mediates acute oxidative lung injury by the activation of $cPLA_2$, which may provoke the generation of free radicals in neutrophils.

• Journal of Plant Biotechnology
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• v.33 no.3
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• pp.195-207
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• 2006

Stem cells are the primordial, initial cells which usually divide asymmetrically giving rise to on the one hand self-renewals and on the other hand progenitor cells with potential for differentiation. Zygote (fertilized egg), with totipotency, deserves the top-ranking stem cell - he totipotent stem cell (TSC). Both the ICM (inner cell mass) taken from the 6 days-old human blastocyst and ESC (embryonic stem cell) derived from the in vitro cultured ICM have slightly less potency for differentiation than the zygote, and are termed pluripotent stem cells. Stem cells in the tissues and organs of fetus, infant, and adult have highly reduced potency and committed to produce only progenitor cells for particular tissues. These tissue-specific stem cells are called multipotent stem cells. These tissue-specific/committed multipotent stem cells, when placed in altered environment other than their original niche, can yield cells characteristic of the altered environment. These findings are certainly of potential interest from the clinical, therapeutic perspective. The controversial terminology 'somatic stem cell plasticity' coined by the stem cell community seems to have been proved true. Followings are some of the recent knowledges related to the stem cell. Just as the tissues of our body have their own multipotent stem cells, cancerous tumor has undifferentiated cells known as cancer stem cell (CSC). Each time CSC cleaves, it makes two daughter cells with different fate. One is endowed with immortality, the remarkable ability to divide indefinitely, while the other progeny cell divides occasionally but lives forever. In the cancer tumor, CSC is minority being as few as 3-5% of the tumor mass but it is the culprit behind the tumor-malignancy, metastasis, and recurrence of cancer. CSC is like a master print. As long as the original exists, copies can be made and the disease can persist. If the CSC is destroyed, cancer tumor can't grow. In the decades-long cancer therapy, efforts were focused on the reducing of the bulk of cancerous growth. How cancer therapy is changing to destroy the origin of tumor, the CSC. The next generation of treatments should be to recognize and target the root cause of cancerous growth, the CSC, rather than the reducing of the bulk of tumor, Now the strategy is to find a way to identify and isolate the stem cells. The surfaces of normal as well as the cancer stem cells are studded with proteins. In leukaemia stem cell, for example, protein CD 34 is identified. In the new treatment of cancer disease it is needed to look for protein unique to the CSC. Blocking the stem cell's source of nutrients might be another effective strategy. The mystery of sternness of stem cells has begun to be deciphered. ESC can replicate indefinitely and yet retains the potential to turn into any kind of differentiated cells. Polycomb group protein such as Suz 12 repress most of the regulatory genes which, activated, are turned to be developmental genes. These protein molecules keep the ESC in an undifferentiated state. Many of the regulator genes silenced by polycomb proteins are also occupied by such ESC transcription factors as Oct 4, Sox 2, and Nanog. Both polycomb and transcription factor proteins seem to cooperate to keep the ESC in an undifferentiated state, pluripotent, and self-renewable. A normal prion protein (PrP) is found throughout the body from blood to the brain. Prion diseases such as mad cow disease (bovine spongiform encephalopathy) are caused when a normal prion protein misfolds to give rise to PrP$^{SC}$ and assault brain tissue. Why has human body kept such a deadly and enigmatic protein? Although our body has preserved the prion protein, prion diseases are of rare occurrence. Deadly prion diseases have been intensively studied, but normal prion problems are not. Very few facts on the benefit of prion proteins have been known so far. It was found that PrP was hugely expressed on the stem cell surface of bone marrow and on the cells of neural progenitor, PrP seems to have some function in cell maturation and facilitate the division of stem cells and their self-renewal. PrP also might help guide the decision of neural progenitor cell to become a neuron.

• Korean Journal of Food Science and Technology
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• v.31 no.1
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• pp.238-245
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• 1999

Antioxidative effects of the water or ethanol extracts from Rhus verniciflua Stokes (RVS) were measured by protection against hydroxyl radicals in mouse brain tissue culture. In the water extracts from RVS, cell viabilities were estimated 60.0, 66.0, 72.0, 84.0 and 90.0% at addition of 1, 2, 4, 7 and $10{\mu}L$, respectively, compared with GO (20 mU/mL) alone. The cell viability in the ethanol extracts was similarly with water extracts. In the antitumor effects, the results showed that percentages of the HeLa cell death were approximately 24% for 12 hrs, 57% for 48 hrs at addition of 10%/well ethanol extracts respectively. To know inhibition of tumor growth, in vivo, mice (BALB/c) were inoculated with 0.25 mL CT-26 $(1{\times}10^6\;cells/mL)$ subcutaneously. After the generation of tumor, the results of RVS extracts (ethanol, water) injection showed generally that the tumor size in BALB/c was reduced. For physicochemical characterization of the RVS extracts, purified substances of water or ethanol extracts were analized with SDS-PAGE and ICP spectrometer. In electrophoresis, gel showed 2 bands (210, 230 KDa). The results of ICP verified that RVS extracts contain $Cu^{2+}$ in both samples. Conclusively, this substance might be a laccase which has a biological effective function, as a natural bioactive substance.

• Tuberculosis and Respiratory Diseases
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• v.43 no.2
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• pp.210-220
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• 1996

Background : Neutrophils or monocytes separated in vitro by the adherence to plastic surface are known to be activated by surface adherence itself and subsequent experimental data might be altered by surface adherence. In the process of surface adherence, adhesion molecules have a clear role in intracellular signal pathway of cellular activation. Human alveolar macrophages(HAM) are frequently purified by the adherence procedure after bronchoalveolar lavage. But the experimental data of many reports about alveolar macrophages have ignored the possibility of adhesion-induced cellular activation. Method : Bronchoalveolar lavage was performed in the person whose lung of either side was confirmed to be normal by chest CT. With the measurement of hydrogen peroxide release from adherent HAM to plastic surface and non-adherent HAM with or without additional stimulation of phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), we observed the effect of the adherence to plastic surface. We also evaluated the effect of various biological surfaces on adhesion-induced activation of HAM. Then, to define the intracellular pathway of signal transduction, pretreatment with cycloheximide, pertussis toxin and anti-CD11/CD18 monoclonal antibody was done and we measured hydrogen peroxide in the culture supernatant of HAM. Results : 1) The adherence itself to plastic surface directly stimulated hydrogen peroxide release from human alveolar macrophages and chemical stimuli such as phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine(fMLP) colud not increase hydrogen peroxide release in these adherent macrophages which is already activated. 2) PMA activated human alveolar macrophages irrespective of the state of adhesion. However, fMLP stimulated the release of hydrogen peroxide from the adherent macrophages, but not from the non-adherent macrophages. 3) HAM adherent to A549 cell(type II alveolar epithelium-like human cell line) monolayer released more hydrogen peroxide in response to both PMA and fMLP. This adherence-dependent effect of fMLP was blocked by pretreatment of macrophages with cycloheximide, pertussis toxin and anti-CD18 monoclonal antibody, Conclusion : These results suggest that the stimulatory effect of PMA and fMLP can not be found in adherent macrophage because of the activation of human alveolar macrophage by the adherence to plastic surface and the cells adhered to biologic surface such as alveolar epithelial cells are appropriately responsive to these stimuli. It is also likely that the effect of fMLP on the adherent macrophage requires new protein synthesis via G protein pathway and is dependent on the adhesion between alveolar macrophages and alveolar epithelial cells by virtue of CD11/CD18 adhesion molecules.