• Journal of Applied Biological Chemistry
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• v.66
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• pp.29-38
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• 2023

Recent studies have highlighted the link between diseases and inflammation across our lifespan. Our sedentary lifestyle, high-calorie diet, chronic stress, chronic infections, and exposure to pollutants and xenobiotics, collectively intensify the course and recurrence of infections and inflammation in our bodies, promoting the prevalence of chronic diseases and aging. Given such phenomena and considering additional factors such as the frequency of prescription, and easy access to over-the-counter drugs, the need for anti-inflammatory therapeutics is ever-increasing. However, the readily available anti-inflammatory treatment option comes with a greater risk of side effects or high cost (biologics). Therefore in this growing competition of discovering and developing new potent anti-inflammatory drugs, we focused on utilizing the established knowledge of traditional medicine to find lead compounds. Since lead optimization is an indispensable step toward drug development, we applied this concept for the production of potent anti-inflammatory compounds achieved by structural modification of flavonoids. The derivative obtained through acetylation of myricetin, 3,3',4',5,5',7-hexaacetate myricetin, showed a greater inhibitory effect in the production of pro-inflammatory mediators such as nitric oxide, Prostaglandin E₂, and pro-inflammatory cytokines like interleukin-6, interleukin1β, in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells compared to myricetin. The increased potency of inhibition was in conjunction with an increased inhibitory effect on inducible nitric oxide synthase and cyclooxygenase-2 proteins. Through such measures, this study supports lead optimization for well-established lead compounds from traditional medicine using a simpler and greener chemistry approach for the purpose of designing and developing potent anti-inflammatory therapeutics with possibly fewer side effects and increased bioavailability.

• Natural Product Sciences
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• v.11 no.1
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• pp.16-21
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• 2005

Nitric Oxide (NO), derived from L-arginine, is produced by two types (constitutive and inducible) of nitric oxide synthase (NOS: cNOS and iNOS). The NO produced in large amounts by the iNOS is known to be responsible for the vasodilation and hypotension observed in septic shock, cancer metastasis and inflammation. The inhibitors of iNOS, thus, may be useful candidates for the treatment of inflammatory diseases accompanied by the overproduction of NO. We prepared alcoholic extracts of herbal drugs which have been used for the treatment of inflammation in oriental medicine. We have screened the inhibitory activity of NO production in lipopolysaccharide (LPS)-activated macrophages after the treatment of these extracts. Among 82 kinds of extracts of herbal drugs, 35 extracts showed the potent inhibitory activity of NO production above 50% at the concentration of $50\;{\mu}g/mL$. The inhibitory activities of NO production were also evaluated for several solvent fractions at two different concentrations. Especially, hexane and EtOAc fractions of Alpinia officinarum, Angelica gigas, Ostericum koreanum, Saussurea lappa, Torilis japonica, and hexane fractions of Agrimonia pilosa, Machilus thunbergii, Hydrangea serrata, Magnolia obovata, Prunella vulgaris, Tussilago farfara, and EtOAC fractions of Perilla frutescence showed a significant activity at 10 and/or $25\;{\mu}g/mL$. In Western blot analysis, the hexane fractions ($5\;{\mu}g/mL$) of Magnolia obovata and Saussurea lappa, and EtOAc fractions ($20\;{\mu}g/mL$) of Hydrangea Serrata, Perilla frutescence and Torilis japonica inhibited the expression of iNOS protein in LPS-activated macrophages. These plants may be promising candidates for the study of the activity-guided purification of active compounds and might be useful for the treatment of inflammatory diseases and endotoxemia accompanying overproduction of NO.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.6
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• pp.1549-1556
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• 2008

The objective of this study were to evaluate the antioxidant activity and the anti-inflammatory effects of Angelica tenuissima (AT) which has been used widely as a traditional medicine. The antioxidant activities of AT was tested by DPPH radical scavenging, superoxide anion scavenging and nitric oxide scavenging. AT showed strong antioxidant activity in all experiment. In macrophages nitric oxide (NO) is released as an inflammatory mediator and has been proposed to be an important modulator of many pathophysiological conditions and high concentratin of NO is produced by inducible nitric oxide synthase (iNOS). In this study we have examined the inhibition effects of NO by 85% methanol extracts of AT in mouse (C57BL/6) peritoneal macrophage. AT (100, 1000 ${\mu}g/m{\ell}$) suppressed nitric oxide production and iNOS expression without any notable cytotoxicity and it also inhibited the expression of inflammatoryenzymes like cyclooxygenase-2 (COX-2). These data suggest that 85% methanol extracts of AT may possibly be used as antioxidant and anti-inflammatory agent.

• Korean Journal of Pharmacognosy
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• v.38 no.2 s.149
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• pp.170-175
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• 2007

Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO$^-$), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. We tried to evaluate the effects of two kinds of varieties of Perilla frutescens var japnica Hara on the NO production in lipopolysaccharide (LPS)-activated microglia. The perilla cultivars of Namcheondeulkkae (NC) and Boradeulkkae (BR) were developed by pure line from the local variety and by a cross between 'deulkkae' and 'chajogi', respectively. Spirit, hexane, chloroform and butanol fractions of the leaves of NC and BR inhibited the production of NO in LPS-activated microglia. The fractions of BR showed stronger activity than NC and the spirit extracts was the most potent in both cultivars. The solvent fractions of BR suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells. Moreover, the extracts of NC and BR showed the activity of peroxynitrite scavenging in cell free bioassay system. These results imply that Namcheondeulkkae and Boradeulkkae might have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity.

• Environmental Analysis Health and Toxicology
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• v.15 no.3
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• pp.69-80
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• 2000

ADP-rubosylation may be involved in the process of macrophage activation. Nitric oxide (NO) has emerged as an important intracellular and interacellular regulatory molecule with function as diverse as vasodilation, neural communication or host defense. NO is derived from the oxidation of the terminal guanidino nitrogen atom of L-arginine by the NADPH -dependent enzyme, nitric oxide synthase (NOS) which is one of the three different isomers in mammalian tissues. Since NO can exert protective or regulatory functions in the cell at a low concentration while toxic effects at higher concentrations, its role may be tightly regulated in the cell. Therefore, this paper was focused on signal transduction pathway of NO synthesis, role of endogenous TGF-$\beta$ in NO production. effect of NO on superoxide formation. Costimulation of murine peritoneal macrophages with interferon-gamma (IFN-γ) and phorbol 12-myristate 13-acetate (PMA) increased both NO secretion and mRNA expression of inducible nitric oxide synthase (iNOS) when PMA abolished costimulation. Pretreatmnet of the cells with PMA abolished costimuation effects due to the depletion of protein kinase C (PKC) activities . The involvement of PKC in NO secretion could be further confirmed by PKC inhibitor, stauroprine, and phorbol ester derivative, phorbol 12,13-didecanoate. Addition of actinomycine D in IFN-γ plus PMA stimulated cells inhibited both NO secretion and mRNA expression of iNOS indication that PMA stabilizes mRNA of iNOS . Exogenous TGF-$\beta$ reduced NO secretion in IFN -γ stimulated murine macrophages. However addition of antisense oligodeoxynucleotide (ODN) to TGF-$\beta$ to this system recovered the ability of NO production and inhibited mRNA expression of TGF-$\beta$. ACAS interactive laser cytometry analysis showed that transportation of FITC -labeled antisense ODN complementary to TGF-$\beta$ mRNA could be observed within 5 min and reached maximal intensity in 30 min in the murine macrophage cells. NO released by activated macrophages inhibits superoxide formation in the same cells . This inhibition nay be related on NO-induced auto -adenosine diphosphate (ADP) -ribosylation . In addition, ADP-ribosylation may be involved in the process of macrophage activation .

• Nutritional Sciences
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• v.5 no.3
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• pp.123-128
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• 2002

Physical activity is a primary cancer control strategy that has received little attention to date. However, an Increasing number of epidemiological studies have proposed that physical exercise may be beneficial by enhancing anticancer immune system responses. We investigated the effects of acute exercise on changes in nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression. The amounts of NO generated by abdominal macrophages in mice were measured after exercise. Thirty-two mice, which were challenged with thioglycollate broth to activate peritoneal macrophages, were randomly assigned to control, exercise and recovery groups. The mice exercised on a motor-driven treadmill for 3 consecutive days, either moderately (18m/min, 30 min/day, 5％ grade) or severely (18-35m/min, 60 min/day, 5％ grade). The mice were killed immediately after exercise or after 6 hrs of recovery. Nitric oxide was quantified by the Griess assay. The exercised mice showed higher levels of NO generation than those of the control mice, but the intensity of exercise had no significant effect on NO generation. Mice allowed six hours of recovery after exercise showed higher levels of NO generation than that of animals sacrificed immediately after exercise, but there were no significant differences in NO generation with variations in the intensity of exercise. Increased levels of iNOS were found in the exercised groups, and this was greatest in the groups allowed six hours of recovery compared to those groups sacrificed immediately after exercise. The results of this study suggest that acute exercise may enhance an immune response by inducing macrophage-derived NO generation; these results support the epidemiological findings which support the benefits of exercise in the prevention and control of cancer. Further study is needed to determine the physiological significance of these findings, which could be applied to the use of therapeutic exercises to assist in the prevention and control of cancer.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.3
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• pp.771-776
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• 2003

The present study was conducted to evaluate the regulation mechanism of nitric oxide(NO) by water-extracted Cyperus rotundus (WCR) in RAW 264.7 macrophages. We investigated the effects of cell proliferation in mouse spleen cell and RAW 264.7 macrophages cells. WCR enhanced mitogenic activity in the dose-response manner in mouse spleen cells and RAW 264.7 macrophages cells. In nitric oxide (NO) synthesis by WCR, WCR alone had an effect on NO synthesis. It was found that the production of NO of RAW 264.7 cells stimulated with lipopolysaccharide (LPS) could be markedly inhibited by WCR. Inhibition of NO production was achieved by reducing inducible nitric oxide syntheses(iNOS) mRNA expression. The expression of IL-I gene by WCR was investigated using reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, IL-1 family(IL-1 α, IL-1β) expressions were induced by WCR. These finding suggested that regulation of NO production by WCR may be, at least in part, associated with the regulation of iNOS mRNA expression and IL-1 family gene expression.

• Nutritional Sciences
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• v.7 no.4
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• pp.214-217
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• 2004

Dietary intervention and simvastatin is beneficial in the prevention cardiovascular diseases by lowering plasma lipid levels. Endothelial dysfunction is associated with coronary artery disease and its risk factors and is reversed by dietary intervention. It has been suggested that hyperlipidemia contributes to the development of atherosclerosis by increasing inducible nitric oxide synthase (iNOS) expression via intimal thickening. Statins treatment has been found to decrease iNOS expression and atherogenensis in animal models. We hypothesized that dietary intervention and simvastatin therapy could decrease plasma nitric oxide in hypercholesterolemic patients, which would suggest the opportunity for modulation of iNOS expression through the use of statins in a clinical situation. We measured the plasma levels of nitrite and nitrate (NOx) in 19 hyperlipidemia patients. The subjects were under dietary intervention following simvastatin therapy for 12 weeks. As a result, the plasma level of NOx, stable metabolites of nitric oxide (NO), saw a two-fold elevation in hyperlipidemic patients as compared to normal levels. Although 12 weeks of dietary intervention did not lower NOx levels, subsequent 12-week simvastatin (10 mg/day) treatment, along with dietary intervention, lowered NOx levels significantly. This NOx reduction, induced by simvastatin therapy, positively correlated with lowered coronary risk factors (r=0.40, p=0.02). It indicated that simvastatin therapy decreases plasma NOx levels by, perhaps, decreasing iNOS expression or activity leading to the attenuation of the development of neointima.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.9
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• pp.1513-1517
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• 2013

This study examined the immunomodulatory activities of apple seed extracts (ASE). The immunomodulatory effects were estimated through nitric oxide production, cytokine induction, protein expression of inducible nitric oxide synthase (iNOS), and the phosphylation of mitogen-activated protein kinases (MAPKs) and inhibitory kappa $B{\alpha}$ ($I{\kappa}B-{\alpha}$) in the RAW 264.7 macrophage cell line. In the cytotoxicity asay, ASE (31 to $250{\mu}g/mL$) did not induce cytotoxicity; thus, the optimal concentration of ASE was confirmed to be less than $250{\mu}g/mL$. Nitric oxide (NO) and cytokines (tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-6) production significantly increased in a dose-dependent manner. Similarly, the protein expression of iNOS and the phosphorylation of MAPKs and $I{\kappa}B-{\alpha}$ were also increased by ASE treatment. Overall, our results suggest that extracts from apple seeds potentially have immunomodulatory activities on macrophages.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.32 no.3
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• pp.13-22
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• 2019

Objectives : Armeniacae semen is the seed of Prunus armenica L. var. ansu MAXIM, and this is classified into Rosaceae. Armeniacae semen has been used for centuries in traditional oriental medicine for the treatment of pain and inflammatory diseases. Amygdalin is the major compound of Armeniacae semen, and it is now being used for the treatment of pain and cancer. Methods : In the present study, we compared the effects of an aqueous extract of Armeniacae semen and a solution of amygdalin extracted from Armeniacae semen on lipopolysaccharide(LPS)-stimulated prostaglandin E2 synthesis and nitric oxide production in mouse BV-2 microglial cells. For this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, reverse transcription-polymerase chain reaction(RT-PCR), prostaglandin E2 immunoassay and nitric oxide detection were performed on mouse BV-2 microglial cells. Results : In the present study, an aqueous extract of Armeniacae semen and an amygdalin solution extracted from Armeniacae semen suppressed prostaglandin E2 synthesis and nitric oxide production by inhibiting the LPS-induced enhancement of cyclooxygenase-2(COX-2) mRNA and the inducible nitric oxide synthase mRNA in mouse BV-2 cells. For the cyclooxygenase-1(COX-1) expression, an aqueous extract of Armeniacae semen showed a more potent suppression effect compared to the amygdalin solution. However, the amygdalin solution more potently suppressed the LPS-induced COX-2 mRNA expression compared to the aqueous extract of Armeniacae semen. Conclusions : As a result, aqueous extract of Armeniacae semen and amygdalin exert anti-inflammatory and analgesic effects.