Gamma irradiation-engineered macrophage-derived exosomes as potential immunomodulatory therapeutic agents.

The modulation of macrophage polarization is a promising strategy for maintaining homeostasis and improving innate and adaptive immunity. Low-dose ionizing radiation has been implicated in macrophage immunomodulatory responses. However, studies on the relationship between exosomes and regulation of macrophage polarization induced by ionizing radiation are limited. Therefore, this study investigated the alterations in macrophages and exosomes induced by gamma irradiation and elucidated the underlying mechanisms. We used the mouse macrophage cell line RAW 264.7 to generate macrophages and performed western blot, quantitative reverse transcription-PCR, and gene ontology analyses to elucidate the molecular profiles of macrophage-derived exosomes under varying treatment conditions, including 10 Gy gamma irradiation. Exosomes isolated from gamma-irradiated M1 macrophages exhibited an enhanced M1 phenotype. Irradiation induced the activation of NF-κB and NLRP3 signaling in M1 macrophages, thereby promoting the expression of pro-inflammatory cytokines. Cytokine expression was also upregulated in gamma-irradiated M1 macrophage-released exosomes. Therefore, gamma irradiation has a remarkable effect on the immunomodulatory mechanisms and cytokine profiles of gamma-irradiated M1 macrophage-derived exosomes, and represents a potential immunotherapeutic modality.

Anti-cancer effects of plant-derived Micromonospora sp. M2 against A549 and MCF-7 cell lines.

The huge diversity of secondary bioactive metabolites, such as antibiotic and anticancer compounds produced by Micromonospora sp., makes it an attractive target for study. Here, we explored the anti-proliferative activities of Micromonospora sp. M2 extract (MBE) in relation to its pro-oxidative activities in A549 and MCF7 cell lines. Anti-proliferative effects were assessed by treating cells with MBE. We found that treatment with MBE decreased cell proliferation and increased intracellular reactive oxygen species, and that these observations were facilitated by the suppression of the PI3K-AKT pathway, alterations to the Bcl/Bad ratio, and increased caspase activity. These observations also demonstrated that MBE induced apoptotic cell death in cell lines. In addition, the phosphorylation of P38 and c-Jun N-terminal kinase (JNK) were upregulated following MBE treatment in both cell lines. Collectively, these results indicate that MBE acts as an anticancer agent via oxidative stress and JNK/mitogen-activated protein kinase pathway activation, enhancing apoptotic cell death in cell lines.

Ionizing radiation­induced modification of nialamide as an anti­inflammatory agent against lipopolysaccharide­induced RAW 264.7 and DH82 cells.

Nialamide is a non-selective monoamine oxidase inhibitor that was widely used as an antidepressant. However, it has been prohibited for decades in the depressive medicine market due to the adverse hepatotoxic side effects. The re-use of drugs that have been withdrawn from the market represents a promising approach for the development of novel incrementally modified drugs and, in this context, ionizing radiation can serve as a powerful tool for producing new drug candidates. The present study exposed nialamide to γ radiation at 50 kGy to obtain the novel cyclized benzylamide, nialaminosin (compound 2), along with five known compounds, 3-amino-N-benzylpropanamide (compound 3), 3-methoxy-N-benzylpropanamide (compound 4), 3-hydroxy-N-benzylpropanamide (HBPA; compound 5), N-benzylpropanamide (compound 6) and isonicotinamide (compound 7). Among the isolated compounds, HBPA was established to inhibit the lipopolysaccharide-induced overproduction of pro-inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 and cytokines including TNF-α, IL-6 and IL-10, without causing cytotoxicity to both RAW 264.7 and DH82 cells. Furthermore, HBPA was found to reduce the protein expression of inducible NO synthase and cyclooxygenase-2 in macrophages and compared with nialamide, it was established to have more potent radical scavenging activity. The present study therefore suggested the application of HBPA for the improvement of anti-inflammatory properties using ionizing radiation technology on the withdrawn drug nialamide.

Novel Dihydrocoumarins Induced by Radiolysis as Potent Tyrosinase Inhibitors.

A representative naturally occurring coumarin, 4-methylumbelliferone (5), was exposed to 50 kGy of gamma ray, resulting in four newly generated dihydrocoumarin products 1-4 induced by the gamma irradiation. The structures of these new products were elucidated by interpretation of spectroscopic data (NMR, MS, [α]D, and UV). The unusual bisdihydrocoumarin 4 exhibited improved tyrosinase inhibitory capacity toward mushroom tyrosinase with IC50 values of 19.8 ± 0.5 µM as compared to the original 4-methylumbelliferone (5). A kinetic analysis also exhibited that the potent metabolite 4 had non-competitive modes of action. Linkage of the hydroxymethyl group in the C-3 and C-4 positions on the lactone ring probably enhances the tyrosinase inhibitory effect of 4-methylumbelliferone (5). Thus, the novel coumarin analog 4 is an interesting new class of tyrosinase inhibitory candidates that requires further examination.

Production of Mono-Hydroxylated Derivatives of Terpinen-4-ol by Bacterial CYP102A1 Enzymes.

CYP102A1 from Bacillus megaterium is an important enzyme in biotechnology, because engineered CYP102A1 enzymes can react with diverse substrates and produce human cytochrome P450-like metabolites. Therefore, CYP102A1 can be applied to drug metabolite production. Terpinen-4-ol is a cyclic monoterpene and the primary component of essential tea tree oil. Terpinen-4-ol was known for therapeutic effects, including antibacterial, antifungal, antiviral, and anti-inflammatory. Because terpenes are natural compounds, examining novel terpenes and investigating the therapeutic effects of terpenes represent responses to social demands for eco-friendly compounds. In this study, we investigated the catalytic activity of engineered CYP102A1 on terpinen-4-ol. Among CYP102A1 mutants tested here, the R47L/F81I/F87V/E143G/L188Q/N213S/E267V mutant showed the highest activity to terpinen-4-ol. Two major metabolites of terpinen-4-ol were generated by engineered CYP102A1. Characterization of major metabolites was confirmed by liquid chromatography-mass spectrometry (LC-MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopy (NMR). Based on the LC-MS results, the difference in mass-to-charge ratio of an ion (m/z) between terpinen-4-ol and its major metabolites was 16. One major metabolite was defined as 1,4-dihydroxy-p-menth-2-ene by NMR. Given these results, we speculate that another major metabolite is also a mono-hydroxylated product. Taken together, we suggest that CYP102A1 can be applied to make novel terpene derivatives.

Inhibitory effect of γ-ray-modified hydroxymethylated baicalins on NO production.

Baicalin, a glucuronic flavone, is the major active component in the medicinal plant Scutellaria baicalensis. Herein, baicalin was irradiated by γ-rays to afford four unusual flavanones, baicalinols A (2), B (3), and C (4) and peroxybaicaleinol (5), and two known flavones, oroxylin A (6) and baicalein (7). The structures of the hydroxymethylated products were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry, and their absolute configuration was established using electronic circular dichroism spectroscopy. Novel hydroxymethylated flavanones 2 and 3 suppressed both nitric oxide (NO) production and the expression of inducible NO synthase and showed significantly higher anti-inflammatory activities in lipopolysaccharide-stimulated macrophages than the parent compound. These newly generated hydroxymethylated flavanones can be potentially used for treating inflammatory diseases.

Novel aminopyridazine derivative of minaprine modified by radiolysis presents potent anti-inflammatory effects in LPS-stimulated RAW 264.7 and DH82 macrophage cells.

Radiation molecularly transforms naturally occurring products by inducing the methoxylation, hydroxylation, and alkylation of parent compounds, thereby affecting the anti-inflammatory capacities of those compounds. Minaprine (1) modified by ionizing radiation generated the novel hydroxymethylation hydropyridazine (2), and its chemical structure was determined based on NMR and HRESIMS spectra. Compared to the original minaprine, the novel generated product showed a highly enhanced anti-inflammatory capacity inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 and DH82 macrophage cells. In addition, minaprinol (2) effectively inhibited cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) at the protein level and pro-inflammatory cytokine (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10) production in macrophages.

Inhibitory Effects of Thermolysis Transformation Products of Rotenone on Nitric Oxide Production.

Rotenone, isolated from Derris, Lonchocarpus, and Tephrosia from the family Fabaceae, has been shown to have a variety of biological properties and is used in various agricultural industries as a potent biopesticide. However, recent reports have demonstrated that rotenone has the potential to cause several adverse effects such as a neurodegenerative disease. This study aimed to induce thermolysis of the biopesticide rotenone and enhance the functionality of the degraded products. Rotenone (1) was degraded after autoclaving for 12 h, and the thermolytic reactants showed enhanced anti-inflammatory capacity against nitric oxide (NO) production. The structures of the newly modified products were spectroscopically determined. The thermal reaction products included various isoflavonoid derivatives 2-6, whose structures were characterized as being produced via chemical reactions in rotenone at the C-12 positions. Among the degraded products, (-)-tubaic acid (6) exhibited significantly improved anti-inflammatory effects compared to the original rotenone. Quantitative LC-MS analysis of the major thermolysis products generated in Derris extract containing rotenone was performed using isolate 2-5 purified from autoclaved rotenone. These results suggest that the thermal transformation of rotenone can improve the functionality of anti-inflammatory agents.

New Anti-Glycative Lignans from the Defatted Seeds of Sesamum indicum.

Seven known analogs, along with two previously undescribed lignan derivatives sesamlignans A (1) and B (2), were isolated from a water-soluble extract of the defatted sesame seeds (Sesamum indicum L.) by applying the chromatographic separation method. Structures of compounds 1 and 2 were elucidated based on extensive interpretation of 1D, 2D NMR, and HRFABMS spectroscopic data. The absolute configurations were established by analyzing the optical rotation and circular dichroism (CD) spectrum. Inhibitory effects against the formation of advanced glycation end products (AGEs) and peroxynitrite (ONOO-) scavenging assays were performed to evaluate the anti-glycation effects of all isolated compounds. Among the isolated compounds, (1) and (2) showed potent inhibition towards AGEs formation, with IC50 values of 7.5 ± 0.3 and 9.8 ± 0.5 µM, respectively. Furthermore, the new aryltetralin-type lignan 1 exhibited the most potent activity when tested in the in vitro ONOO- scavenging assay.

Mechanism of (-)-epigallocatechin gallate (EGCG) dimerization by low-temperature plasma.

The efficient dimerization of (-)-Epigallocatechin gallate (EGCG), which is the major bioactive constituent isolated from the leaves of Camellia sinensis, was initially reported without changes in its stereochemistry using low-temperature plasma. The contribution of plasma during the dimerization of EGCG in a methanolic solution was quantified using a major factor, with the major factor demonstrated based on the contents of newly generated products, in this case the sum of oolonghomobisflavans A and B depending on the plasma treatment method. Samples were treated in three methods: plasma direct treatment, an indirect treatment using only reactive species, and an indirect treatment using effects other than those by reactive species. Ozone was identified as a major factor during the plasma treatment, and the operating ranges of the ozone concentration for regulated dimerization were evaluated. The mechanism by which EGCG synthesizes dimers A and B during the treatment process using low-temperature plasma was investigated using the derived major factor and prior literature. The ozone generated by the plasma reacted with methanol to form formaldehyde, and dimers A and B were synthesized by oligomers through a methylene-bridge by the formaldehyde. A plausible pathway of regulated dimerization was deduced based on these results, and the mechanism of EGCG dimerization by plasma is described using this pathway.

Enzymatic transformation products of phloretin as potent antiadipogenic compounds.

Enzymatic structure modification of the representative chalcone phloretin (1) with polyphenol oxidase from Agaricus bisporus origin produced 2 new biphenyl-type phloreoxin (2) and phloreoxinone (3), and a previously undescribed (2R)-5,7,3',5'-tetrahydroxyflavanone (4). The structure of these new oxidized products 2-4 elucidated by interpreting the spectroscopic data (NMR and FABMS) containing the absolute stereochemistry is established by the analysis of the circular dichroism spectrum. Compared to the original phloretin, the new products (2) and (3) showed highly improved antiadipogenic potencies both toward pancreatic lipase and accumulation of 3T3-L1 cells. Also, phloreoxin (2) effectively inhibited the expression of C/EBPß, PPARγ, and aP2 at the mRNA level in the 3T3 adipocytes. Thus, phloreoxin (2), containing a biphenyl moiety catalyzed by A. bisporus polyphenol oxidase, have the potential to influence the antiadipogenic capacity.

Plasma-Induced Oxidation Products of (-)-Epigallocatechin Gallate with Digestive Enzymes Inhibitory Effects.

(-)-Epigallocatechin gallate (EGCG), the chief dietary constituent in green tea (Camellia sinensis), is relatively unstable under oxidative conditions. This study evaluated the use of non-thermal dielectric barrier discharge (DBD) plasma to improve the anti-digestive enzyme capacities of EGCG oxidation products. Pure EGCG was dissolved in an aqueous solution and irradiated with DBD plasma for 20, 40, and 60 min. The reactant, irradiated for 60 min, exhibited improved inhibitory properties against α-glucosidase and α-amylase compared with the parent EGCG. The chemical structures of these oxidation products 1-3 from the EGCG, irradiated with the plasma for 60 min, were characterized using spectroscopic methods. Among the oxidation products, EGCG quinone dimer A (1) showed the most potent inhibitory effects toward α-glucosidase and α-amylase with IC50 values of 15.9 ± 0.3 and 18.7 ± 0.3 µM, respectively. These values were significantly higher than that of the positive control, acarbose. Compound 1, which was the most active, was the most abundant in the plasma-irradiated reactant for 60 min according to quantitative high-performance liquid chromatography analysis. These results suggest that the increased biological capacity of EGCG can be attributed to the structural changes to EGCG in H2O, induced by cold plasma irradiation.

Molecular hybridization based on (-)-epigallocatechin gallate as a new class of antiglycation agents.

(-)-Epigallocatechin gallate (EGCG) and olivetol hybrid molecules 1-4 were conveniently synthesized using dielectric barrier discharge plasma irradiation. The structures of these unprecedented hybrid molecules were determined by interpretation of spectroscopic data. The unusual hybrid 1 showed improved antiglycation potency toward the advanced formation of glycation end products than the original EGCG and olivetol. The novel hybrid 1 is an interesting new class of antiglycation candidate that requires further investigation.

Novel Hybrid Molecules Based on (-)-Epigallocatechin Gallate as Potent Anti-adipogenic Agents.

A series of novel (-)-epigallocatechin gallate (EGCG)-phloroglucinol hybrid compounds 1-4 has been successfully synthesized by employing a simple and efficient methodology using a dielectric barrier discharge (DBD) plasma irradiation. The new hybrid structures were determined by interpretation of spectroscopic data, with the absolute configurations being established by analysis of the circular dichroism (CD) spectra. The novel hybrids 1 and 2 showed highly improved anti-adipogenic potencies toward both pancreatic lipase and preadipocytes differentiation in 3T3-L1 compared to the original EGCG and phloroglucinol. A novel hybrid 1 represent an interesting subclass of anti-adipogenic candidates that need further research.

Radiolytic Cyclization Products of Phloridzin as Potent Anti-Glycation Agents.

The current research examined for radiolytic structure modification and improved bioefficacy of phloridzin by γ-ray, subsequent to a 50 kGy irradiation dose. Structures of the unusual degraded products phlorocyclin, isophlorocyclin, and radiophlorisin were determined spectroscopically, by detailed nuclear magnetic resonance (NMR) and mass spectrometry (MS). Additionally, absolute configuration of the novel cyclized phlorocyclin and isophlorocyclin were proposed by circular dichroism (CD) spectrum analysis. Among the compounds tested, phlorocyclin and isophlorocyclin exhibit potent antidiabetic complication capacities toward advanced glycation end products (AGEs) formation inhibition assay, with IC50 values of 9.1±0.5 and 13.8±0.7 µM, respectively. Furthermore, the predominantly formed products phlorocyclin and isophlorocyclin exerted significantly enhanced DPPH radical scavenging activity compared to the parent phloridzin. These results indicate that γ-ray mediated cyclization of phloridzin exerts a positive influence on the bioactivity.

Nonthermal Plasma-Induced Degradation of Morin and Enhancement of Biological Activities.

In the present study, non-thermal dielectric barrier discharge (DBD) plasma of induced structural changes of morin resulted in the isolation of one previously undescribed benzofuranone derivative, along with two known compounds. The chemical structures of these degradation products were elucidated by UV, NMR and FAB-MS spectroscopic analyses. The isolated three compounds showed potent antioxidative activities in two different tests, with IC50 values in the range of 12.9-41.8 µm in the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+ ) radical scavenging activity, 19.0-71.9 µm for hydroxyl radical scavenging activity test. Furthermore, the new methoxylated benzofuranone exhibited enhancement of inhibitory effects against pancreatic lipase with an IC50 value of 90.7±1.6 µm, when compared to the parent morin. These results suggested that the degradation products isolated from plasma exposed morin might be beneficial for prevention of obesity and related diseases.

Identification of sesamol byproducts produced by plasma treatment with inhibition of advanced glycation endproducts formation and ONOO- scavenging activities.

Little is known of plasma-mediated relations between major food components and their biological capacities. In the present work, the effects of dielectric barrier discharge (DBD) plasma irradiation on pure sesamol and sesame oil were investigated using spectroscopic (LC-MS, NMR) and bioassay methods. Sesamol was degraded when subjected to plasma irradiation for 40 min, and the exposed products exhibited improved anti-glycation capacities against advanced glycation end products (AGEs) formation and better ONOO- scavenging ability. Structures of newly formed compounds were determined spectroscopically. Quantitative LC-MS analysis of the major products generated in sesamol and sesame oil was achieved using isolates 1-4 of purified sesamol plasma treated for 40 min. These results indicate that the predominant chemical changes induced in sesamol and sesame oil by DBD plasma treatment might enhance biological properties.

A new approach to procyanidins synthesis with potent anti-adipogenic effects.

Convenient structure modification of (+)-catechin (1) induced by nonthermal dielectric barrier discharge (DBD) plasma treatment afforded three novel methylene-linked flavan-3-ol oligomers, methylenetetracatechin (2), methylenetricatechin (3), and methylenedicatechin (4), together with two known catechin dimers, bis 8,8'-catechinylmethane (5) and bis 6,8'-catechinylmethane (6). The structures of the three new catechin oligomers 2-4 with methylene bridges were elucidated by detailed 1D- and 2D-NMR analysis, and the absolute configurations were established by the observation of circular dichroism (CD). The novel products 2 and 3 showed significantly enhanced anti-adipogenic capacities against both pancreatic lipase and differentiation of 3T3-L1 preadipocytes compared to the parent (+)-catechin.

Anti-diabetic effects of trans-resveratrol byproducts induced by plasma treatment.

Cold plasma processing has emerged a promising green technology with great potential to improve the quality and microbial safety of various minimally processed foods and materials. However, studies on non-thermal plasma-induced chemical interactions between major food ingredients that might change chemical structure and biological properties are very sparse. Thus, the objective of this study was to evaluate the influence of a dielectric barrier discharge (DBD) treatment on principal trans-resveratrol (TR) in several food stuffs by spectroscopic (HPLC, NMR, MS) and biological analyses. TR was dissolved in methanol and directly exposed to atmospheric non-thermal plasma field at 250 W for different durations (10, 20, 40, and 60 min), 40% relative humidity, and 25 °C. TR treated with plasma for 40 min showed greatly enhanced inhibitory activities for α-glucosidase and α-amylase than parent TR. Newly generated unusual compounds (1, 2) and known compounds (3-6) from plasma treated TR for 40 min were characterized using chromatographic and spectroscopic methods. The predominant reaction of TR induced by cold plasma followed by typical dimerization of products included methylene bridge formation and cyclization of TR. Among predominantly generated products, new compounds 1 and 2 showed more potent α-glucosidase and α-amylase inhibition capacities than parent TR. These results might be used to modify structures and enhance biological property of TR during food processing using DBD plasma treatment.

A green chemical oligomerization of phloroglucinol induced by plasma as novel α-glucosidase inhibitors.

A new facile method was developed for simple green synthesis of methylene-bridged phloroglucinol oligomers using nonthermal dielectric barrier discharge (DBD) plasma in methanolic solution. The chemical structures of these newly generated oligomers 2-5 were determined by interpretation of the spectroscopic data, and the inhibitory activity toward α-glucosidase of all isolates was evaluated. The unusual phloroglcuinol pentamer 5 connected by four methylene linkages showed a much higher potential inhibitory effect against α-glucosidase than the other generated oligomers 2-4 and appeared to be a promising lead for development as a potential antidiabetic agent. Abbreviations: T2DM, type2 diabetes mellitus; DBD, dielectric barrier discharge; HPLC, high-performance liquid chromatography; IC50, 50% inhibition concentration; NMR, nuclear magnetic resonance; FABMS, fastatom bombardment mass spectrometry.