Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin. In this study, we aimed to use a preprocessed aquaculture sidestream for production of carotenoids, including the fish feed ingredient astaxanthin by C. glutamicum. The addition of a preprocessed aquaculture sidestream to the culture medium did not inhibit growth, obviated the need for addition of several components of the mineral salt’s medium, and notably enhanced production of astaxanthin by an engineered C. glutamicum producer strain. Improved astaxanthin production was scaled to 2 L bioreactor fermentations. This strategy to improve astaxanthin production was shown to be transferable to production of several native and non-native carotenoids. Thus, this study provides a proof-of-principle for improving carotenoid production by C. glutamicum upon supplementation of a preprocessed aquaculture sidestream. Moreover, in the case of astaxanthin production it may be a potential component of a circular economy in aquaculture.Circular economy holds great potential to minimize the use of finite resources, and reduce waste formation by the creation of closed-loop systems. This also pertains to the utilization of sidestreams in large-scale biotechnological processes. A flexible feedstock concept has been established for the industrially relevant  The consumer demand for seafood is rising as the world population is growing and healthier diets are becoming more important. The worldwide fish production was 178.5 million tons (mt) in 2018, and is anticipated to reach 204.4 mt in 2030 [Recirculating aquaculture systems (RAS) present a promising alternative to the traditional cultivation systems as they have greatly reduced land and water requirements, and can be built without exploitation of farmland ,6. Moreode novo carotenoids are mixed into the feed of farmed fish and crustaceans [Haematococcus pluvialis are stronger than those of synthetic astaxanthin [Yarrowia lipolytica [Paracoccus carotinifaciens [Escherichia coli [Saccharomyces cerevisiae [Corynebacterium glutamicum [One of the most important quality criteria of aquaculture bred fish, such as salmon (2.4 mt in 2018) and rainbow trout (0.8 mt in 2018), and crustaceans (9.4 mt in 2018) ,23. As fstaceans ,25. Astastaceans ,27 and fstaceans ,29, algastaceans ,31,32, mstaceans , and bacstaceans ,34, havestaceans . Currentstaceans ,36. Howeaxanthin . Howeveraxanthin ,39. Takepolytica ,41, Paraifaciens , Escherihia coli ,44, Saccrevisiae , and Corutamicum  are natu+ soil bacterium C. glutamicum that is a natural producer of the yellow C50 carotenoid decaprenoxanthin and its glucosides [C. glutamicum, which efficiently produces the endogenous decaprenoxanthin [eYfEbcrtY [Fulvimarina pelagi [Astaxanthin can be synthesized by the Gramucosides   of dry matter fraction). The nutrient analysis of the aquaculture sidestream (−1) is the major macronutrient present in the aquaculture sidestream, followed by potassium  and sulphate (2 g L−1). Furthermore, elemental sulfur, phosphorus, calcium, zinc, and boron were detected. The analysis also covered ammonium derived nitrogen (NH4-N) (<1 g L−1), P2O5 (<3 g L−1), CaO (<2 g L−1), Mg (<1 g L−1), MgO (<2 g L−1), Mn, Cu, and Mo , but these nutrients could not be detected.The liquid phase of an aquaculture sidestream from a Norwegian RAS salmon farm was analyzed regarding its physical parameters and nutrient composition in order to get insights about which components of the standard minimal growth medium CGXII of destream  showed tω-amino acid 5-aminovaleric acid (5-AVA), and the diamines putrescine and cadaverine are present in AQ. The concentrations of the proteinogenic amino acids range from 0.5 to 10.7 mg L−1, while 5-AVA (76 mg L−1), putrescine (62 mg L−1), and cadaverine (53 mg L−1) are present in higher concentrations.Furthermore, the amino acid and amine composition of preprocessed aquaculture sidestream (AQ) was analyzed via HPLC. The results  display Moreover, an ion exchange chromatography (detection with a RID at λ = 210 nm) analysis of AQ was performed via HPLC . Four PeC. glutamicum WT was used to verify if AQ as a new complex media component is compatible with growth. Therefore, cultivations in different media compositions were performed in a Biolector® microcultivation system. Addition of AQ to the standard minimal growth medium CGXII to 20% (v/v) led to significantly increased biomass formation (from OD600 nm 52.0 ± 0.9 to 62.0 ± 0.8) and decaprenoxanthin content (from 1.1 ± 0.2 to 1.6 ± 0.0 mg L−1), while the growth rate was only slightly reduced (v/v) AQ , or trace elements by AQ led to comparable biomass formation and growth rates compared to CGXII with addition of 20% (v/v) AQ. This indicated that addition of 20% (v/v) AQ is sufficient to replace these components of CGXII. However, replacing PCA or trace elements with 20% (v/v) AQ significantly increased decaprenoxanthin production . The addition of AQ instead of phosphorous reduced biomass formation , but decaprenoxanthin production was comparable. The replacement of nitrogen had the strongest negative effect: biomass formation was reduced to one-fifth (∆OD600 nm of 10 ± 0.3), the maximal growth rate by one-third (0.3 ± 0.0 h−1), and the decaprenoxanthin production by more than half (0.5 ± 0.0 mg L−1) compared to CGXII. To test whether AQ could function as a carbon source for C. glutamicum, the standard carbon source glucose was replaced by 5%, 10%, 20%, or 40% (v/v) AQ (v/v) AQ only one-sixth of biomass formation (∆OD600 nm of 8.0 ± 0.0) was observed  AQ A. Replac(v/v) AQ B. In allobserved B. These C. glutamicum WT could grow with 20% (v/v) AQ (adjusted to pH 7) as sole medium component. However, the biomass and decaprenoxanthin formation were negligible  and glucose were added, some growth (∆OD600 nm of 10 ± 0.2) and decaprenoxanthin production (0.4 ± 0.0 mg L−1) were detected , or combined with ammonium sulfate (3.5 ± 0.1 mg L−1). The decaprenoxanthin titer was 3-fold higher than from the standard CGXII medium.Second, we tested if gligible C. When Mdetected C. The adC. glutamicum, in which all components that could be replaced by AQ without reducing biomass formation or decaprenoxanthin production  were omitted. The new medium was named CGAQ, and it contained 20% (v/v) AQ, 42 g L−1 MOPS buffer, 40 g L−1 glucose, 20 g L−1 (NH4)2SO4, 5 g L−1 urea, 1 g L−1 K2HPO4, and 1 g L−1 KH2PO4). Growth of C. glutamicum was supported by the medium CGAQ  AQ.Taken together, we have developed two growth media using AQ that are suitable for ® microcultivation system using strains overproducing various carotenoids. Strain ASTA* produces astaxanthin while ASTALYS*  AQ or CGAQ , but reduced by supplementation of CGXII with 20% (v/v) AQ (−1) was comparable to the one in CGXII, but was reduced by 24% when AQ was supplemented.Decaprenoxanthin and biomass formation by MB001∆(v/v) AQ . Lycopenv/v) AQ , and further improved in CGAQ , while the biomass formation was decreased in CGAQ. Production of two other non-native C50 carotenoids C.p.450 and sarcinaxanthin by strains CP1 and SAX1, respectively, was reduced in CGAQ as was biomass formation. However, upon addition of AQ to CGXII, carotenoid production increased considerably: plus 280% C.p.450 (47.5 ± 1.2 mg L−1), and plus 360% sarcinaxanthin (72.3 ± 4.3 mg L−1).Production of BABR by strain BARBR1 was increased by supplementation of CGXII with 20%  and canthaxanthin  were not significant compared to CGXII, considerable and statistically significant increases were observed for zeaxanthin  and astaxanthin .A similar pattern was observed for production of astaxanthin and its precursors by BETA4, ZEA5, CAN5, and ASTA*, respectively. In CGAQ, these strains exhibited reduced biomass and carotenoid formation. By contrast, the addition of AQ to CGXII had a positive impact on biomass formation and carotenoid production. While the slight increases in production of β-carotene (127.4 ± 2.7 mg L−1) upon addition of AQ to CGXII. Lysine production was increased 1.8-fold in CGAQ (1.40 ± 0.03 g L−1), and more than 6-fold (5.52 ± 0.14 g L−1) upon supplementation of CGXII with AQ.The strain that co-produces astaxanthin with lysine, ASTALYS*, showed very low biomass formation compared to the other carotenoid production strains, which increased significantly upon addition of AQ to the medium  or in the CGAQ medium . Strain ASTALYS* showed 1.5-fold increased astaxanthin production  AQ to CGXII medium in microcultivation, lab-scale bioreactors (2 L) were used to test if this improvement was stable at larger scale. Two parallel 2 L batch fermentations using C. glutamicum ASTA* were performed. They contained either CGXII medium (v/v) AQ (600 nm of 58 as compared to 46), as was the growth rate (0.31 h−1 compared 0.24 h−1). In CGXII, astaxanthin accumulated to a cellular content of 0.38 mg g−1 cell dry weight (CDW)  during 77 h, with a maximal volumetric productivity of 0.05 mg L−1 h−1 (−1 (cellular content of 0.44 mg g−1 CDW) was reached already after 61 h, increasing the maximal volumetric productivity to 0.09 mg L−1 h−1 (After having shown that astaxanthin production by I medium A or CGXI(v/v) AQ B. After  L−1 h−1 A. Upon A L−1 h−1 B. Thus, v/v) AQ medium was used as a batch medium, and either 600 mL CGXII concentrate or CGXII concentrate supplemented with 20% (v/v) AQ were used as feed medium  AQ as feed). With CGXII concentrate as feed, a maximal ∆OD600 nm of 206, and an astaxanthin concentration of 6.1 mg L−1 (cellular content of 0.15 mg g−1) accumulated during 64 h with a maximum productivity of 0.10 mg L−1 h−1 (600 nm was 208 and an astaxanthin concentration of 3.8 mg L−1 (cellular content of 0.08 mg g−1 CDW) accumulated during 64 h with a maximum volumetric productivity of 0.08 mg L−1 h−1  AQ, respectively) compared to the batch fermentations (max. 24.8 mg L-1 in CGXII and max. 30.3 mg L-1 in CGXII plus 20% (v/v) AQ) (C. glutamicum strain ASTA* required supplementation of 20% (v/v) AQ in the batch phase, but not during the feeding phase.With the aim to improve astaxanthin production in bioreactor cultivation, fed-batch fermentations were performed. In a comparison, 1 L CGXII with 20% (d medium . Both cu L−1 h−1 . When AQ L−1 h−1 . Therefov/v) AQ) . Thus, tC. glutamicum. Fermentative production of several carotenoids, including the aquaculture feed ingredient astaxanthin was increased by usage of AQ.In this study, a promising example with potential for the circular economy was presented. The liquid phase of a sidestream from a recirculating aquaculture system for salmon served as a sustainable feedstock for C. glutamicum was supported by AQ as the sole medium component. However, it was a poor source of carbon and nitrogen. The addition of glucose, urea, and/or ammonium sulfate restored the growth to levels comparable with the standard medium. While L-glutamine, L-glutamate and a number of other amino acids present in AQ, e.g., L-alanine, L-asparagine, L-serine, and L-threonine, can be used as nitrogen sources by C. glutamicum [C. glutamicum [C. glutamicum is able to metabolize nitrate to nitrite as part of a respiratory chain [C. glutamicum, it was reported that the supplementation of an aquaculture sidestream with aquaculture sludge improved growth and omega-3 fatty acid production of an algal co-cultivation [Growth of utamicum , their cutamicum ,97,98. Lry chain ,100; howry chain ,102. Pritivation .C. glutamicum was improved by supplementation with AQ. However, since AQ is a complex source of macro- and micro-elements, it is very difficult to speculate which component(s) cause the improvement. It is tempting to hypothesize that trace elements may be involved as several enzymes of carotenogenesis use metal ions as cofactors. For example, prenyltransferases use divalent metal ions, such as Mg2+ and Mn2+ as cofactors [C. glutamicum, the geranylgeranyl pyrophosphate synthases IdsA and CrtE require Mg2+ for their activity [−1). It is not known whether the substrate specificities of the prenyltransferases used here  are affected when Mg2+ is replaced by Mn2+, as was recently observed for a flavonoid prenyltransferase from Artocarpus heterophyllus [C. glutamicum. For example, 28 reduced ferredoxins are required for the biosynthesis of the isoprenoid diphosphate precursors DMAPP, IPP and HMBPP, while their conversion to decaprenoxanthin yields only three reduced ferredoxins [−1 S and 2 g L−1 SO4) increased sulfur availability. Taken together the diverging effects of AQ supplemented CGXII and CGAQ on the production of the tested native and heterologous carotenoids prompt that the production of each carotenoid may be improved by optimization of the (trace) element composition of the growth medium. Earlier studies in carotenoid producing yeast [Carotenoid production by ofactors ,104,105.activity ; howeverophyllus . Ferredoredoxins . For theredoxins . These ing yeast ,110 and ng yeast ,112 suggXanthophyllomyces dendrorhous. Cultures of X. dendrorhous produced up to 9.69 µg L−1 astaxanthin when grown for five days on pre-treated whey, a sidestream of the dairy industry (volumetric productivity of 0.08 µg L−1 h−1), and up to 1.88 mg L−1 astaxanthin when cultivated in a fruit and vegetable waste derived medium for 7 days (0.01 mg L−1 h−1) [−1 astaxanthin were produced by X. dendrorhous after about 200 h of cultivation (0.13 mg L−1 h−1) in a partially-saccharified mussel wastewater [Bacillus subtilis and the alga H. pluvialis for astaxanthin production from a starch-containing sidestream of a potato processing plant a titer of 8 mg L−1 astaxanthin was achieved after 350 h of cultivation (0.02 mg L−1 h−1) [−1 in 48 h; 0.15 mg L−1 h−1) upon supplementation with AQ and partially also higher titers than those achieved by X. dendrorhous. The yeast P. rhodozyma produced about 130 mg L−1 astaxanthin after 120 h by direct fermentation of food wastes (1.08 mg L−1 h−1) [S. cerevisiae, about 18 mg L−1 astaxanthin accumulated within one week of fermentation (0.10 mg L−1 h−1) [−1 observed in this study may indicate that more astaxanthin can be produced if all precursor carotenoids are converted to astaxanthin. This may be approached by adjusting the copy number and/or expression levels of crtZ and crtW as previously demonstrated for E. coli and S. cerevisiae [Bacterial astaxanthin production from AQ was demonstrated here for the first time. Previously, sidestream derived astaxanthin production was described for the yeast L−1 h−1) . Moreovestewater . In a coL−1 h−1) . Our stuL−1 h−1) . On stilL−1 h−1) . Both sirevisiae ,119.C. glutamicum to fish or crustaceans may be an option to provide astaxanthin and additional components relevant as feed additives. For this application, the strain ASTALYS* may be beneficial as it secretes L-lysine to the culture medium and accumulates astaxanthin in the cells [L-lysine producing C. glutamicum were already commercialized for poultry feeds [C. glutamicum ASTALYS* may support growth of poultry and coloration of egg yolks. In this regard, it has to be noted that several amino acid producing C. glutamicum strains have been approved by the European Food Safety Authority (EFSA) as a feed additive for all animal species [In aquatic feeds, bacterial biomass proved to be a sustainable, protein rich substrate to partially or completely replace traditional protein sources, such as fish meal . In aquahe cells . Lysine he cells ,128 and he cells ,127. Whory feeds . Thus, s species . Today, ®  with 0.22 µm pore diameter. The resulting AQ was used in the growth experiments.The aquaculture sidestream was collected from the sump of a post-smolt RAS for salmon operated by Lumarine AS  outside of Trondheim (Norway). A plastic canister was filled with aquaculture sidestream, and was left to settle by gravitation for 0.5 h. The supernatant (liquid aquaculture sidestream) was thereafter gently poured off, frozen (−20 °C), and transported to our research facility in Bielefeld (Germany). The liquid aquaculture sidestream was defrosted, stirred, and frozen (−20 °C) in smaller containers until further use. Preprocessing of the liquid aquaculture sidestream in order to use it as a growth medium component was implemented by a 90 min centrifugation step at 4000 rpm and subsequent sterile filtration of the supernatant. The filtration was performed with a vacuum driven SteritopC. glutamicum or E. coli respectively. Precultures of E. coli DH5α, C. glutamicum ATCC 13032, and derived strains were grown in LB medium [C. glutamicum ASTALYS* precultures were grown in BHIS  supplemented with 10 g L−1 glucose. For main cultures CGXII minimal medium (20 g L−1 (NH4)2SO4, 1 g L−1 K2HPO4, 1 g L−1 KH2PO4, 5 g L−1 urea, 42 g L−1 MOPS buffer, 0.2 mg L−1 biotin, 30 mg L−1 PCA, 10 mg L−1 CaCl2, 250 mg L−1 MgSO4·7 H2O, trace elements: 10 mg L−1 FeSO4·7 H2O, 10 mg L−1 MnSO4·H2O, 0.02 mg L−1 NiCl2·6 H2O, 0.313 mg L−1 CuSO4·5 H2O, and 1 mg L−1 ZnSO4·7 H2O) [−1 glucose was used as a control. Comparisons of AQ-based or -supplemented media were carried out using 20% (v/v) AQ. When appropriate, 25 µg mL−1 kanamycin, 100 µg mL−1 spectinomycin, and 1 mM IPTG were added to the medium. Cultures were inoculated to an initial OD600 nm of 1 after washing in TN buffer . For OD measurements a Shimadzu UV-1202 spectrophotometer  was used. Growth experiments were conducted in 1 mL scale with gas permeable sealing foil in a flowerplate of the Biolector® microcultivation system  at 30 °C and 1100 rpm in triplicates.Strains and plasmids used in this study and their characteristics and references are listed in 4·7 H2O)  suppleme−1 was maintained from the bottom through a ring-sparger. The fermentations were performed with a head space overpressure of 0.3 bar. An automatic control of the stirrer speed between 400 rpm and 1500 rpm kept the relative dissolved oxygen saturation (rDOS) at 30%. The temperature was kept at 30 °C, and a pH of 7.0 was automatically maintained by the addition of 10% (v/v) H3PO4 and 4 M KOH. For inoculation a first pre-culture was grown in 10 mL LB medium with addition of 10 g L−1 glucose and 25 µg mL−1 kanamycin in a 100 mL shake flask. A second pre-culture in 200 mL CGXII medium with addition of 40 g L−1 glucose and 25 µg mL−1 kanamycin in a 2 L shake flask was inoculated to an OD600 nm of 1. For the batch fermentation an initial working volume of 2 L was inoculated to an OD600 nm of 2 from the second pre-culture. The fed-batch fermentations were performed with an initial working volume of 1 L, inoculated to an OD600 nm of 2 with the second pre-culture. An amount of 600 mL feed medium (CGXII concentrate) was used with the following formulation: 433.7 g L−1 glucose, 34.4 g L−1 (NH4)2SO4, 8.7 g L−1 K2HPO4, 8.7 g L−1 KH2PO4, 8.6 g L−1 Urea, 5.2 g L−1 MgSO4·7 H2O, 100 mg L−1 FeSO4·7 H2O, 100 mg L−1 MnSO4·H2O, 0.2 mg L−1 NiCl2·6 H2O, 3.13 mg L−1 CuSO4·5 H2O, 10 mg L−1 ZnSO4·7 H2O, 1 mg L−1 Biotin, and 6 mL L−1 Antifoam 204. If indicated, 20% (v/v) AQ was added to this feed formulation. A steady airflow of 0.5 NL min−1 was maintained from the bottom through a ring sparger for the first 24 h of cultivation, afterwards the airflow was increased to 1 NL min−1. The feed was primed when the rDOS fell below 30% for the first time. The feed pump activated every time the rDOS exceeded 60% and stopped when it subsequently fell below 60%, to prevent oversaturation with glucose. Foam formation during the fermentation was reduced by addition of antifoam 204 controlled via an antifoam probe. Samples during the fermentations were collected with an autosampler and cooled down to 4 °C until further use.A glass bioreactor with a total volume of 3.7 L  was used for the fermentations. Two six-bladed Rushton turbines were placed on the stirrer axis with a distance of 6 cm and 12 cm from the bottom of the reactor. A mechanical foam breaker was installed on the stirrer axis with a distance of 22 cm to the bottom of the reactor. The stirrer to reactor diameter ratio was 0.39, and the aspect ratio of the reactor was 2.6:1.0. A steady airflow of 1 NL minrepA gene in the pSH1 vector. This mutation (exchange of Gly at position 429 of RepA protein by Glu) resulted in an increased plasmid copy number. Site-directed mutagenesis was performed via plasmid backbone amplification . The plasmids pSH2_FpcrtZ and pSH2_FpcrtW were constructed in E. coli DH5α on the basis of pSH2. Polymerase chain reaction (PCR) fragments of FpcrtZ and FpcrtW were generated from Fulvimarina pelagi using the primers HA34 + HA35, and FpW1 + FpW4, respectively. The PCR products were cloned into the BamHI  digested pSH2 vector by Gibson assembly [2-competent E. coli DH5α were prepared and transformed via heat shock [C. glutamicum strains were transformed by electroporation [crtZ~W [The pSH2 expression vector was constructed by site-directed mutagenesis of the assembly . The CaCat shock . Transfoporation . For theporation  was tran [crtZ~W .−2 dilution of the untreated liquid aquaculture sidestream in water was sent to Eurofins Agraranalytik Deutschland GmbH  to analyze the pH, organic substance content, and total nitrogen, ammoniacal nitrogen, phosphorous, phosphorous as P2O5, potassium, potassium as K2O, magnesia, magnesia as MgO, calcium, calcium as CaO, sulfur, sulfur as SO4, boron, manganese, molybdenum, copper, and zinc content of the untreated liquid aquaculture sidestream.A 1·10For all HPLC analysis an Agilent 1200 series system  was used. Culture samples (200 or 500 µL) were centrifuged at 14,000 rpm for 10 min, the supernatants and pellets were stored separately at −20 °C until analysis.ortho-phthaldialdehyde (OPA) were used with a reversed-phase main column . l-asparagine was used as an internal standard. The separation was achieved at 40 °C with 0.25% (v/v) sodium acetate (pH 6.0) (A) and methanol (B) as mobile phases, with the following gradient and flow profile: 0 min 20% B 0.7 mL min−1, 3 min 38% B 0.7 mL min−1, 6 min 42% B 0.1 mL min−1, 7 min 46% B 0.7 mL min−1, 14.5 min 70% B 1.2 mL min−1, 14.8 min 75% B 1.2 mL min−1, 16.8 min 85% B 1.2 mL min−1, 17.8 min 20% B 1.2 mL min−1, and 19.5 min 20% B 1.2 mL min−1 adapted from [For the analysis of extracellular amino acids and their derivatives 5 µL sample and an automatic pre-column derivatization with ted from . The flu−1 as described previously [The carbohydrates in the cultivation medium were quantified with an organic acid resin column  under isocratic conditions with a flow of 0.8 mL mineviously . The anaThe analysis of carotenoids was performed as previously described . SamplesC. glutamicum. The beneficial effect of adding this sidestream to growth media was observed for strains overproducing either native or non-native carotenoids. In particular, astaxanthin production more than doubled upon AQ supplementation in small-scale cultivation, and in 2 L batch and fed-batch bioreactor fermentations. Thus, our proof-of-principle example for production of the fish feed supplement astaxanthin from AQ holds the potential to contribute to the establishment of the circular economy in aquaculture.The preprocessed liquid phase of a sidestream from a recirculating aquaculture system for salmon was shown to be suitable to support growth and carotenoid production by


The present study uses simple, innovative methods to isolate, characterize and fractionate LDL in its main components for the study of specific oxidations on them that characterize oxidized low-density lipoprotein (oxLDL) status, as it causatively relates to atherosclerosis-associated cardiovascular disease (CVD) risk assessment. These methods are: (a) A simple, relatively time-short, low cost protocol for LDL isolation, to avoid shortcomings of the currently employed ultracentrifugation and affinity chromatography methodologies. (b) LDL purity verification by apoB100 SDS-PAGE analysis and by LDL particle size determination; the latter and its serum concentration are determined in the present study by a simple method more clinically feasible as marker of CVD risk assessment than nuclear magnetic resonance. (c) A protocol for LDL fractionation, for the first time, into its main protein/lipid components , as well as into LDL carotenoid/tocopherol content. (d) Protocols for the measurement, for the first time, of indicative specific LDL component oxidative modifications  out of the many (known/unknown/under development) that collectively define oxLDL status, which contrasts with the current non-specific oxLDL status evaluation methods. The indicative oxLDL status markers, selected in the present study on the basis of expressing early oxidative stress-induced oxidative effects on LDL, are studied for the first time on patients with end stage kidney disease on maintenance hemodialysis, selected as an indicative model for atherosclerosis associated diseases. Isolating LDL and fractionating its protein and main lipid components, as well as its antioxidant arsenal comprised of carotenoids and tocopherols, paves the way for future studies to investigate all possible oxidative modifications responsible for turning LDL to oxLDL in association to their possible escaping from LDL’s internal antioxidant defense. This can lead to studies to identify those oxidative modifications of oxLDL , which are recognized by macrophages and convert them to foam cells, known to be responsible for the formation of atherosclerotic plaques that lead to the various CVDs. C1016)Calcium chloride granular Chloroform Coomassie Brilliant Blue G-250 Diethyl ether Ethanol ; caution, highly flammableEthyl acetate Glycerol Glycine Heparin 5000 i.u./ml – 5 ml  Hexane Hydrochloric acid 7-Keto-cholesterol LDL-cholesterol kit 26 H2O; Merck, cat. no. 102367)Magnesium chloride hexahydrate Methanol (100%) for HPLC 8H18N2O4S; Serva, cat. no. 25245)N-(2-hydroxyethyl)piperazine-N′-2-ethane sulfonic acid Octane (-iso) Polyethylene glycol 6000 Rhodamine 6G Silica gel high-purity grade, pore size 60 Å, 70–230 mesh, 63–200 μm, for column chromatography Sodium chloride 6H5Na3O72H2O; Sigma, cat. no. W302600)Sodium citrate Sodium hydroxide 3PO4⋅12H2O; Merck, cat. no. 106578)Sodium (tri-)phosphate dodecahydrate TEMED 2-Thiobarbituric acid Trichloroacetic acid Tris-base Tris hydrochloride Xylenol orange tetrasodium salt .•64 mM Citrate buffer, pH 5.12: For 200 ml, dissolve (in ddH2O) 3.764 g anhydrous Na-citrate, and adjust pH with HCl. Before use adjust pH to exactly 5.12 at RT.•Heparin stock: 5,000 U ml–1.•2-CaCl2, pH 7.2 (Hepes solution):Hepes-NaCl-MgCl For 20 ml, dissolve (in ddH2O) 0.026 g Hepes , 0.023 g NaCl , 0.0080 g MgCl2⋅6H2O , and 0.0088 g anhydrous CaCl2 , and adjust pH with HCl.•4% NaCl: For 5 ml, dissolve (in ddH2O) 0.2 g NaCl.•20 mM Tris buffer, pH 7.7: For 100 ml, dissolve (in ddH2O) 0.242 g Tris-base and adjust pH with HCl.•2:5 M MgCl For 7.5 ml, dissolve 7.625 g MgCl2⋅6H2O in ∼2.25 ml ddH2O at 37°C, and adjust volume up to 7.5 ml.•400/400 mM BHA/BHT: For 4 ml, dissolve in 3.39 ml EtOH, 0.288 g BHA and 0.353 g BHT.•50/50 mM BHA/BHT: For 2 ml, dissolve in 2 ml EtOH, 0.018 g BHA and 0.022 g BHT.•50 mM Phosphate (Pi) buffer, pH 7.4: For 20 ml, dissolve (in ddH2O) 0.38 g Na3PO4⋅12H2O and adjust pH with HCl.•3:MetOH 3:1 (v/v):CHCl For 16 ml, mix 12 ml CHCl3 with 4 ml MetOH.•80:1 Isooctane:EA (v/v) mixture: For 81 ml, mix 80 ml isooctane with 1 ml ethyl acetate.•20:1 Isooctane:EA (v/v) mixture: For 21 ml, mix 20 ml isooctane with 1 ml ethyl acetate.•75:25 Isooctane:EA (v/v) mixture: For 10 ml, mix 7.5 ml isooctane with 2.5 ml ethyl acetate.•50:50:1 Hexane:diethyl ether:acetic acid (v/v/v): For 11 ml, mix 5 ml hexane, 5 ml diethyl ether and 1 ml acetic acid.•0.005% Rhodamine 6G: For 100 ml, dissolve (in ddH2O) 0.005 g Rhodamine 6G.•2.5% Diethyl ether: For 6 ml, mix 5.85 ml benzene with 0.15 ml diethyl ether.•30% Acrylamide: For 100 ml, dissolve (in ddH2O) 29.2 g acrylamide and 0.8 g bis-acrylamide.•10% Ammonium persulfate: For 10 ml, dissolve (in ddH2O) 1 g of ammonium persulfate.•10% SDS: For 10 ml, dissolve (in ddH2O) 1 g SDS.•1.5 M Tris–HCl buffer, pH 8.8: For 250 ml, dissolve (in ddH2O) 45.4 g Tris–HCl and adjust pH with HCl.•0.5 M Tris–HCl buffer, pH 6.8: For 100 ml, dissolve (in ddH2O) 6 g Tris–HCl and adjust pH with HCl.•5% Separation electrophoresis gel: For 8 ml, mix 4.5 ml ddH2O, 1.33 ml 30% acrylamide, 2 ml 1.5 M Tris pH 8.8, 80 μl 10% SDS, 80 μl 10% ammonium persulfate, and 8 μl TEMED.•3% Stacking electrophoresis gel: For 5 ml, mix 3.17 ml ddH2O, 0.5 ml 30% acrylamide, 1.25 ml 0.5 M Tris pH 6.8, 50 μl 10% SDS, 50 μl 10% ammonium persulfate, and 5 μl TEMED.•CBB G-250 stain solution: For 2 L, first dissolve 1 g CBB G-250 in 860 ml MetOH. Then add 140 ml of acetic acid and 1 L ddH2O.•Gel destain solution: For 2 L, mix 200 ml MetOH and 140 ml acetic acid with 1.66 L ddH2O.•Electrophoresis sample buffer (4×): For 9 ml, dissolve 0.02 g bromophenol blue and 0.8 g sodium dodecyl sulfate in 4 ml glycerol and 5 ml 0.5 M Tris–HCl buffer, pH 6.8.•Running buffer (10×): For 1 L, dissolve (in ddH2O) 30.3 g Tris-base , 144.4 g glycine  and 10 g SDS .•Running buffer (1×): For 1 L, mix 100 ml of 10× running buffer with 900 ml ddH2O.•FOX (−Fe) reagent: For 5 ml, dissolve (by stirring for 30 min) in ddH2O 0.0076 g XO, and add 0.07 ml sulfuric acid. Centrifuge for 5 min, at RT and at 13,000 g and collect the supernatant. The reagent can be stored at −20°C.•FOX (+Fe) reagent: For 1 ml, dissolve (by vortexing) in 1 ml FOX (−Fe2+) reagent 0.0015 g ammonium ferrous sulfate. Prepare fresh.•20 mM SDS + 0.18% PEG-6000 + 0.1 M NaOH solution: For 50 ml, dissolve (in ddH2O) 0.288 g SDS, 0.09 g PEG-6000 and 0.5 ml 10 M NaOH.•20 mM SDS + 0.18% PEG-6000: For 50 ml, dissolve (in ddH2O) 0.288 g SDS and 0.09 g PEG-6000.•20 mM SDS: For 50 ml, dissolve (in ddH2O) 0.288 g SDS.•Solution A: For 0.75 ml, mix 625 μl TCA 100% with 125 μl 12 M HCl.•Solution B: For 1 ml, dissolve (by vortexing) in 1 ml 0.2 M NaOH 0.025 g TBA.•TBA reagent: For 1 ml, mix 0.5 ml solution A with 0.5 ml solution B.•TBA solvent: For 1 ml, mix 0.5 ml solution A with 0.5 ml 0.2 M NaOH.•10 M NaOH: For 100 ml, dissolve (in ddH2O) 40 g NaOH.•0.2 M NaOH: For 10 ml, mix 9.8 ml ddH2O with 0.2 ml 10 M NaOH.•0.1 M BHA: For 2 ml, dissolve in 1.966 ml EtOH, 0.036 g BHA.•TCA 100%: For 5 ml, dissolve (in ddH2O) 5 g TCA.•3.3% PEG-6000: For 10 ml, dissolve (in ddH2O) 0.33 g PEG-6000.•ApoB100 1 μg/μl: For 0.5 ml, dissolve 500 μg pure apoB100 in 500 μl 20 mM SDS + 0.18% PEG-6000.from blood serum of LDL particles (LDL-P) and their diameter size determination, (b) protocols for LDL fractionation into its main components: apoB100, cholesteryl esters, triglycerides, free cholesterol, phospholipids, carotenoids, and tocopherols, and (c) assays for the quantification of the following LDL components’ certain specific oxidative modifications: apoB100 malondialdehyde (apoB100-MDA) and dityrosines (apoB100-DiTyr), and the lipid hydroperoxides (-OOH) cholesteryl ester-OOH, triglyceride-OOH, free cholesterol-OOH, and phospholipid-OOH. These methods are diagrammatically outlined in The methods employed in the present study involve: (a) Protocols for isolation 2-precipitation  by heparin, which removes most contaminating serum proteins , and can be either used immediately to isolate LDL, or stored frozen at −80°C. LDL is isolated from serum by combined modifications of the heparin-citrate precipitation at pH 5.12 , and thepitation  methods.proteins ; (2) by proteins , 33. Theproteins , 35. The1.g at 25°C (using a refrigerated centrifuge).One milliliter serum sample is mixed (in a 15-ml glass Corex centrifuge tube) with 10 ml 64 mM citrate buffer, pH 5.12, to which are added 150 μl 5 KU/ml heparin stock  and 25 μl 400/400 mM BHA/BHT solution , and incubated at 25°C for 10 min. The crude LDL precipitate is collected by centrifugation for 10 min at 10,000 Notes: (1) Higher centrifugation speeds can be used as long as 25°C is kept constant to ensure citrate buffer pH stability. (2) At pH 5.12, VLDL and HDL particles remain in solution, whereas LDL particles are selectively precipitated  is washed twice with 1 ml Hepes, pH 7.2, containing 1/1 mM BHA/BHT (by adding 2.5 μl 400/400 mM BHA/BHT), each time by scraping, with a metallic spatula, the inner wall of the tube toward its bottom, followed by centrifugation at 10,000 3.2 , followed by incubation for 10 min in an ice-water bath, and precipitation of the pure LDL pellet by centrifugation at 10,000 g for 15 min at 4°C. It is important to withdraw (and discard) the supernatant immediately  to avoid resuspension of the LDL pellet.Small quantities of serum proteins that may have contaminated the 2×-washed LDL precipitate are removed as follows: The 2×-washed LDL pellet is dissolved in 0.2 ml 4% NaCl, containing 1/1 mM BHA/BHT (by adding 4 μl 50/50 mM BHA/BHT), and to the resulting solution are sequentially added 10 ml 20 mM Tris, pH 7.7 , and 0.3 ml 5 M MgClNote: MgCl2 concentration set at exactly 143 mM is crucial for LDL precipitation to take place; small increases (≥188 mM) result in ineffective precipitation, possibly related to a salting-out effect. That is, at MgCl2 concentrations ≥188 mM LDL particles are not precipitated (MgCl2 is not chelated with the phosphate groups of the LDL phospholipids), since Cl– compete with LDL phospholipid PO4– group, and “steal” the phospholipid-bound Mg+2, thus preventing LDL precipitation.4.step 3 is washed with 1 ml Hepes, pH 7.2, containing 1/1 mM BHA/BHT (by adding 2.5 μl 400/400 mM BHA/BHT), and centrifuged at 10,000 g for 5 min at 4°C, followed by supernatant discarding. The resulting pellet is resuspended in 1 ml Hepes + 1/1 mM BHA/BHT (by mild suction using a 1 ml pipette tip), transferred in a 1.5-ml microcentrifuge tube, and centrifuged at 10,000 g for 5 min at 4°C, followed by supernatant discarding. At this point, the resulting pure LDL pellet can be either stored frozen at −80°C or suspended in 0.15 ml 4% NaCl (by vortexing) and subjected to the following method (in section 3.2).The resulting LDL pellet from Low-density lipoprotein particles in the NaCl-suspension are initially fractionated into their protein and total lipid + antioxidant components: the protein fraction consists of a single protein (apoB100), the total LDL lipid + antioxidant fraction consists of cholesteryl esters, triglycerides, free cholesterol, and phospholipids together with the antioxidant components carotenoids and tocopherols. Fractionation of the two aforementioned components is achieved by a modification of a previously developed lipid extraction method  as follo1.step 4, section 3.1), are added 0.1 ml 50 mM Pi buffer, pH 7.4, adjusted to contain 1/1 mM BHA/BHT (by adding 5 μl 50/50 mM BHA/BHT).To the 0.15 ml purified LDL suspension (2.3/MetOH 3:l (v/v), containing 1/1 mM BHA/BHT (by adding 2.5 μl 400/400 mM BHA/BHT), by 1 min-vortexing, followed by centrifugation at 13,000 g for 5 min at 4°C, which forms an upper (aqueous) phase, a middle phase (containing the apoB100 protein disc) and a lower yellowish chloroform phase . The lower chloroform phase is collected (into a 2-ml microcentrifuge tube). To the remaining aqueous and apoB100 disc layer phases, 0.75 ml CHCl3 are added (containing 1/1 mM BHA/BHT), followed by 1 min-vortexing and centrifugation at 13,000 g for 5 min at 4°C. The upper aqueous phase is discarded and the lower CHCl3 phase is combined with the initial CHCl3 phase and vacuum-dried as dry extract of total LDL lipids, which are either stored frozen at −80°C, or immediately fractionated in their main classes , and also the co-extracted carotenoids and tocopherols.The resulting 0.25 ml LDL suspension is delipidated with 1 ml CHCl3.step 2 is washed 3× in 0.55 ml ice-cold acetone as follows: the apoB100 pellet is initially mixed with 50 μl ice-cold acetone and frittered into small pieces . Then, another 0.5 ml of ice-cold acetone are added to the loose apoB100 acetone suspension, followed by 30-s mild vortexing and centrifugation at 13,000 g for 5 min at 4°C. After each wash the acetone supernatant is discarded and the 3×-acetone-washed apoB100 is vacuum-dried, collected in the (previously detached/pre-weighted) cap of a 1.5-ml microtube by inversion, and accurately weighted using a semi-micro analytical balance (0.1 mg sensitivity). At this point, the resulting apoB100 pellet can be stored at −80°C, or dissolved in ∼200 μl 20 mM SDS + 0.18% PEG-6000 (using a glass rod). For apoB100 solubilization, the protein pellet must be incubated for several minutes in 20 mM SDS + 0.18% PEG-6000 in order to be thoroughly impregnated. By doing so, the majority of the protein pellet is solubilized. Following centrifugation at 13,000 g for 1 min at 4°C, the soluble supernatant is collected, while a small insoluble, gel-like, remnant (possibly made up of serum protein impurities) is discarded.The chloroform-washed apoB100 pellet from step 3, section 3.2) is assessed via SDS-PAGE electrophoresis . The gel is loaded by 30 μl LDL-isolated apoB100 and pure apoB100 (control), each added as duplicates of 10 and 20 μg, made by diluting their 1 μg/μl standard solutions with sample buffer containing 3% 2-mercaptoethanol. The 30 μl apoB100 loaded quantities are previously incubated for 3 min at 100°C and cleared by centrifugation at 13,000 g, at RT, for 4 min. The gel is stained using CBB G-250 stain solution, and apoB100 band purity is determined using Image Lab software by Bio-Rad.The purity of the delipidated apoB100 (from d), and comparing it against that reported in literature. LDL-P average diameter is indirectly calculated from the correspondence of 1 molecule apoB100 per one LDL-P, and its phospholipid circular surface (A), which is determined by its phospholipid numbers , multiplied by the average phospholipid surface area 75 Å2  per phospholipid, which is used to calculate the d [by the formula A = π(d/2)2] corresponding to the circular LDL-P. LDL-P serum concentration (nmoles/L) is determined from apoB100% purity, weight and MW 550 kDa.The aim of this experiment is to provide evidence for the purity of LDL by determining its diameter  by 2×-wash with 1 ml 80:1 isooctane:EA as follows: the 1 ml 80:1 isooctane:EA/silica gel suspension is mildly mixed by hand inversion, followed by centrifugation at 13,000 2.step 2, section 3.2) is extracted 2× with 0.25 ml 80:1 v/v isooctane:EA, each time followed by vigorous vortexing and centrifugation at 15,000 g for 5 min at 4°C, resulting in a 2×-washed small clear pellet (seen by contrast difference), which contains the free cholesterol and phospholipid fractions. The combined (0.5 ml) yellowish supernatant is mixed with the wet 0.27 g activated silica gel from step 1 and the tube is stirred by hand inversion, followed by centrifugation at 15,000 g for 5 min at 4°C, and the supernatant is collected. The silica gel pellet is washed 3× with 1 ml of 80:1 isooctane:EA as follows: 1 ml 80:1 isooctane:EA is added in the tube, stirred by hand inversion, and centrifuged at 15,000 g for 5 min at 4°C, the supernatant is collected, and the resulting silica gel pellet undergoes the same process two more times. The 3×-washed silica gel pellet (of yellowish color) is saved as it contains the LDL-triglyceride fraction (see step 3). Then, the combined supernatants (∼3.5 ml) are vacuum-dried, with the dry pellet being the LDL cholesteryl ester fraction containing also tocopherols  to assexμl), and of control sample blank (SBxμl) and reagent blank (RBxμl), composed of mixtures of reagents (placed in 1.5-ml Eppendorf tubes) as shown in A − SBA) − [RB (+Fe2+)A − RB (−Fe2+)A]. Finally, net A is converted to cumene hydroperoxide (cum-OOH) nmole equivalents using a cum-OOH standard curve, using solvents same as those used in the assay, and at their maximum volumes. Lipid sub-fraction hydroperoxide cum-OOH equivalents are expressed as cum-OOH nmole/mg apoB100 .Measurement of LOOH in the solubilized LDL lipid sub-fractions of cholesteryl esters (cholesteryl ester-OOH), triglycerides (triglyceride-OOH), free cholesterol (free cholesterol-OOH), and phospholipids (phospholipid-OOH), sub-fractionated from LDL isolated from 2 ml blood serum (following sections 3.1 to 3.3), is performed by minor modification of a previously reported assay, developed by our group . For eacstep 3 of section 3.2) is performed by modification of a previously reported assay, developed by our group  are measured at ex/em 535/550 nm (setting at low sensitivity the spectrofluorometer in use). Net FU for each sample is calculated from the formula net FU = SFU − SBFU − RBFU, which is converted to MDA pmoles, using a corresponding standard curve of pure MDA (in concentrations ranging from 0.08 μM to 0.66 μM in 20 mM SDS + 0.18% PEG-6000 + 0.1 M NaOH), and expressed as MDA pmoles/mg apoB100 .Measurement of MDA bound to apoB100 (apoB100-MDA) in the LDL apoB100 fraction  are further diluted in 20 mM SDS to final 0.3 ml, and their FU are measured at ex/em 320/405 nm (setting at low sensitivity the spectrofluorometer in use) against a triplicate RB [containing 0.3 μl 20 mM SDS and 2 μl 3.3% PEG-6000 ]. Sample DiTyr FU are converted to pmoles  via a standard curve of DiTyr . Similarly, there is no statistically significant correlation of years of hemodialysis with the levels of free cholesterol-OOH , since years of hemodialysis explain only 1% of the variance . Same statistical analysis shows that age does not correlate with triglyceride-OOH levels , since age explains only 2% of the variance . Similarly, there is no statistically significant correlation of age with the levels of free cholesterol-OOH , since age explains only 2% of the variance .We further investigate whether years of hemodialysis and age of CKD-5d patients correlate with the increase in the levels of oxLDL triglyceride-OOH and free cholesterol-OOH markers, using Simple Linear Regression analysis. It is found that total cholesterol, triglycerides, vitamin D, CRP, iPTH, Ca, P, and CaxP do not significantly correlate with the levels of the aforementioned oxLDL markers. Focusing on the LDL-C clinical marker, since its increased levels are used to assess high risk for CVDs, we investigate  its possible association with the increased triglyceride-OOH and free cholesterol-OOH levels in CKD-5d patients , since LDL-C levels explain only 3% of the variance  , since LDL-C levels explain 10% of the variance  , since HDL-C levels explain 5% of the variance  , since HDL-C levels explain 12% of the variance  . Regarding LDL-P serum concentration as predictor for CVDs risk development, the ROC analysis shows that higher concentration of LDL-P is statistically insignificant (p = 0.11), whereas smaller LDL-P concentrations do not predict higher CVDs risk (p = 0.11).We also investigated, using The present study uses clinically applicable, simple, innovative methods  to isola•Development of a simple, relatively time-short, low-cost protocol for LDL isolation, to avoid shortcomings of the ultracentrifugation •Development of a method for LDL-P size and serum concentration determination in contrast to the, specialized equipment requiring, NMR –55 and g•Development, for the first time, of simple protocols for the fractionation of LDL into its main protein/lipid/antioxidant components. Their application can be extended to other biological sources such as blood serum, organ tissues, plants, microorganisms, and cells.•Measurement, for the first time, of specific LDL protein/lipid component oxidative modifications, selected on the basis of representing early OS-prone oxidative effects. These are applied on CKD-5d patients as indicative markers of oxLDL and for possible association with CKD-5d CVDs risk assessment. The selected markers are cholesteryl ester-OOH, triglyceride-OOH, free cholesterol-OOH, phospholipid-OOH, apoB100-MDA, and apoB100-DiTyr, and they partially express oxLDL status as being part of the many known  and unknThe choice of the study group and the selected oxLDL status markers, representing early OS appearing oxidative modifications of LDL components, are justified as such because chronic kidney disease (CKD) patients exhibit high OS  and deveThe selected in the present study oxLDL protein/lipid component markers are measured in CKD-5d patients  and compared to the control group. Only the lipid peroxidation related markers triglyceride-OOH and free cholesterol-OOH are elevated in CKD-5d patients compared to the control group, which signifies a possible causative association of oxidized cholesterol with CKD-5d. It is very interesting to note also that the increased levels of the aforementioned markers are not associated with patients’ underlying medical conditions, suggesting that CKD can be related mostly to high OS. This is also corroborated by the fact that not even statin medication correlates with the two OS markers in CKD-5d patients, which is in accordance with the observation of no beneficial effect by statins in any underlying CVD of these patients –81. ThisBy isolating LDL and fractionating LDL’s protein and main lipid components, as well as its antioxidant arsenal comprised of carotenoids and tocopherols, the present study paves the way for future studies to investigate additional oxidative modifications that complement the markers that define oxLDL status, thus its more reliable association with CVDs risk assessment. Moreover, the separate fractions of carotenoids and tocopherols isolated by the present study, facilitate the identification of their individual species , 58, andThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Scientific Committee of the General University Hospital of Patras. The patients/participants provided their written informed consent to participate in this study.PP: methodology, investigation, formal analysis, data visualization, statistical analysis, and writing—original draft, and review and editing. MSk, EK, AV, and MSp: investigation, formal analysis, and writing—review and editing. MP, EP, and DG: clinical study design on chronic kidney disease patients and review and editing. AO: investigation. ER and IM: writing—review and editing. CG: conceptualization, methodology, validation, formal analysis, graphical data/graphical visualization, supervision, writing—original draft and review and editing, and project administration. All authors contributed to the article and approved the submitted version.


Entada africana Guill. & Perr. from the family of Fabaceae is used to treat inflammatory conditions in the management of fractures, wounds, and sprains in the northern region of the Republic of Benin. The aim of the present study was to isolate and elucidate phenolic compounds from a hydroalcoholic leaf extract from E. africana and to identify compounds with anti-inflammatory activity in vitro. Eleven compounds were purified from three fractions, which have shown strong to medium anti-inflammatory activity. The isolated compounds were characterized by HRESI-MS and NMR methods as gallic acid (1), ethyl gallate (2), 5,7-dihydroxychromen-4-one (3), 3′,4′,7-trihydroxyflavone (4), dihydrokaempferol-7-O-glucoside (5), catechin (6), quercetin-3-O-[β-apiosyl-(1‴→2″)-β-glucoside] (7), quercetin-3-O-glucoside (8), naringenin-7-O-glucoside (9), aromadendrin (10), and myricetin-3-O-glucoside (11). Nine of the major phenolic compounds were tested using TNF-α stimulated human keratinocytes (HaCaT) as skin inflammation model to identify molecules, which may explain the use of the plant leaves as an anti-inflammatory remedy by assessing the release of proinflammatory cytokines IL-8 and IL-6. The hydroacoholic leaf extract of E. africana exerted a medium inhibitory effect on the release of IL-8. 3′,4′,7-trihydroxyflavone, aromadendrin, dihydrokaempferol-7-O-glucoside and ethyl gallate demonstrated a strong to medium effect on the release of IL-6. For the release of IL-8, 3′,4′,7-trihydroxyflavone demonstrated a medium activity. This study provides for the first time a detailed screening of phenolic compounds occurring in the hydroethanolic leaf extract of E. africana. Additionally, it is shown that E. africana contains active compounds which may justify its traditional medicinal use as an anti-inflammatory remedy to treat inflammatory and pain-related skin conditions in the Republic of Benin.In West African medicine,  Entada africana is described in several African cultures for its numerous ethnomedicinal uses  were used as a skin inflammation model to identify molecules, which may explain the use of the plant’s leaves as an anti-inflammatory remedy.In African medicine, plants have been frequently used for the management of inflammatory and pain-related conditions such as wounds, joint inflammation, inflammatory skin disorders, and other inflammatory diseases . While anal uses . It is anal uses . The planal uses . In Burknal uses . In the nal uses . Apart fnal uses . Additio extract . Active ifoliola . Betulinafricana . Concernrophages . Maceratrophages . HoweverIf not stated otherwise, all solvents for extraction and fractionation were purchased from VWR International GmbH, Darmstadt, Germany. All solvents were of analytical grade. DMSO was obtained from Sigma-Aldrich, Steinheim, Germany, 3--2,5-diphenyltetrazolium (MTT) from Sigma-Aldrich, Taufkirchen, Germany, sodium dodecyl sulfate (SDS) from ThermoFisher Scientific, Rockford, United States, and 2-aminoethyl diphenyl borate from Carl Roth, Germany.E. africana were harvested in December 2015 in Bassila in the department of Donga (9°41′59.99″ N/1°39′59.99″ E as coordinates) in the Republic of Benin. Samples were authenticated and collected with the assistance of a botanist, and a voucher specimen was deposited at the National Herbarium of the University of Abomey-Calavi (No.: YH 455/HNB). Exportation allowance for pharmacological testing and chemical characterization was obtained from the Ministry of Agriculture, Livestock and Fisheries, Republic of Benin.The leaves of v/v/v) constituted the mobile phase. The developed plates were allowed to air dry, and chromatograms were detected at 254 nm using CAMAG TLC Visualizer 2 operated under the software vision CATS 2.5. In addition, derivatization was performed with natural product reagent (2-aminoethyl diphenyl borate) as a detecting reagent for flavonoids and chromatograms were evaluated at 366 nm.Thin layer chromatography was performed on TLC Silica Gel 60 F254 plates . Samples were prepared in the range of 1–2 mg per mL, and 4 µL of each sample were applied as 10 mm bands by means of the CAMAG Automatic TLC Sampler 4 (AST4). The chromatograms were developed in a CAMAG chamber over 8 cm running distance with prior chamber saturation. The system water: formic acid: ethyl acetate . Separation was performed on an RP-18 stationary phase  with the following binary gradient  at a flow rate of 0.5 mL/min: t0min 2% B, t0.5min 2% B, t5.5min 5% B, t6.5min 12% B, t30min 25% B, t41.5min 70% B, t46min 95% B to t50min 100% B. Samples were generally prepared at a concentration of 1–2 mg/mL.Analytical UPLC for fractionation and purity control was performed using Acquity™ Ultra Performance LC, PDA λe Detector and QDa™ Detector, autosampler, in-line degasser and Waters Empower 3t0min 0% B, t0.4min 5% B, t9.9min 100% B, t15min 100% B. The injection volume was 2 µL. Eluted compounds were detected using a Dionex Ultimate DAD-3000 RS over a wavelength range of 200–400 nm and a Bruker Daltonics micrOTOF-QII time-of-flight mass spectrometer equipped with an Apollo electrospray ionization source in positive mode at 4 Hz over a mass range of m/z 50–1,500 using the following instrument settings: nebulizer gas nitrogen, 4 bar; dry gas nitrogen, 9 L/min, 220°C; capillary voltage 4500 V; end plate offset -500 V; transfer time 100 µs; collision gas nitrogen; collision energy and collision RF settings were combined to each single spectrum of 1,250 summations as follows: 624 summations with 80 eV collision energy and 130 Vpp + 313 summations with 16 eV collision energy and 130 Vpp + 313 summations with 16 eV collision energy and 130 Vpp. Internal dataset calibration (HPC mode) was performed for each analysis using the mass spectrum of a 10 mM solution of sodium formiate in 50% isopropanol that was infused during LC reequilibration using a divert valve equipped with a 20 µL sample loop.Chromatographic separations for LC-HRESI-MS were performed on a Dionex Ultimate 3000 RS Liquid Chromatography System on a Dionex Acclaim RSLC 120, C18 column  with a binary gradient  at 0.4 mL/min: 1H) or 150 MHz (13C). Samples were dissolved in methanol-d4  and chemical shifts were referenced to the respective residual solvent signals.NMR spectra were recorded at 299 K on an Agilent DD2 spectrometer  at 600 MHz  was defatted with petroleum ether solvent using a Soxhlet apparatus. The defatted plant material (183.3 g) was then extracted with ethanol:water  three times, resp. by Ultra Turrax®  at 13,000 rpm for 10 min. The extract was centrifuged for 5 min at 3,000 × g, supernatants were collected and lyophilized after evaporation of the organic solvent. The dried extract  was stored at −20°C for further analysis.The leaves of E. africana (20.02 g) was dissolved in ethanol-water (1:1 v/v) and loaded onto a chromatography column packed with Sephadex® LH-20 as stationary phase. The fractionation of the extract was carried out using a step gradient from ethanol, methanol/water (1:1 v/v), methanol to acetone/water (7:3 v/v), and the fractions collected were monitored by TLC. Fractions were combined according to their fingerprints to obtain 25 fractions that were analyzed by UPLC-PDA.The hydroalcoholic extract of v/v) and loaded at the top of the column (36 × 500 mm) filled with RP18 material . A step gradient of methanol 10, 20, 50, 70, and 100% (v/v) was used at a flow rate of 4 mL/min. The fractions collected were analyzed by TLC and combined to yield 6 sub-fractions (FC4-I to FC4-VI). FC4-I was entirely composed of gallic acid .MPLC technique used for preparative separation of organic compounds was applied for subfractionation and isolation of compounds from FC4. 1 g of the fraction was diluted in 8 mL methanol 30%  and acetonitrile (B). Ethyl gallate , 5,7-dihydroxychromone  and 3′,4′,7-trihydroxyflavone  were isolated from fraction FC3. Subfraction FC4-II yielded dihydrokaempferol-7-O-β-glucopyranoside  and catechin . Quercetin-3-O-[β-apiosyl-(1‴→2″)-β-glucopyranoside] , isoquercitrin , prunin  and aromadendrin  were obtained from fraction FC4-III. Finally, FC8 yielded myricetin-3-O-β-glucopyranoside , with further amounts of 8 (0.9 mg) and 9 (3 mg).For the isolation of pure compounds from FC3, FC8 and sub-fractions FC4-II and FC4-III, the fractions were dissolved to 10 mg/mL in a mixture of acetonitrile and water resp. and 1,500 µL were injected for each run. Preparative HPLC was carried out using Quaternary Gradient Module 2545, Photodiode Array Detect or 2998, Autosampler 2707, Waters Prep Degasser and Waters Fraction Collector III. Software: Waters ChromScope v1.40 Beta . Stationary phase: Nucleodur2. Experiments were conducted at a cell confluency of 80–90%. Cells were seeded at 5.0 × 104/500 µL after detachment using trypsin-EDTA (0.05%) solution (Biowest), in 24 well plates and incubated for 24 h.Immortalized human keratinocyte cells (HaCaT)  were purThe experiments were carried out according to ® M 200 plate reader .Cell viability of the TNF-α stimulated HaCaT cells was tested with a subsequent MTT assay. Thus, after collecting the supernatant, cells were treated with an MTT solution (0.3 mg/mL in DPBS) and incubated for 2 h. The resulting formazan salt was dissolved with 500 µL SDS lysis buffer . The control well is treated with the culture medium, and in another well, cells are lysed with triton. After an incubation overnight, the amount of formazan was quantified spectrophotometrically at 570 nm using an Infinitep ≤ 0.0001, ***p ≤ 0.001 **p ≤ 0.01, and *p ≤ 0.05 compared to TNF-α control were considered statistically significant. Half-maximal inhibitory concentrations (IC50) were computed based on the non-linear regression of the concentration–response plots. Maximum inhibition (MI) was calculated from normalized values and represents inhibition at 100 µg/mL for plant extract and fractions or at 100 µM for pure compounds.Spectrophotometric data from cytokine release experiments and cytotoxicity testing were processed with Microsoft Excel version 2016. Statistical analysis was performed with GraphPad Prism (version 9.3.1). Interleukin amount and metabolic activity were normalized to the TNF-α stimulated untreated control (set as 100%). One-way analysis of variance (ANOVA) was used to evaluate the differences between the control and treatment groups. Values ****E. africana were defatted and extracted with ethanol-water (1:1 v/v) for further fractionation steps. Then, the extract was subjected to a fractionation which led to a total of 642 single fractions collected. Similar fractions were combined, resulting in 25 fractions (FC1—FC25) and screened for their phenolic profile by TLC and UPLC-PDA-MS. The fractionation pattern is presented in E. africana (Ea) were tested for their anti-inflammatory properties in vitro using an immortalized keratinocyte cell line (HaCaT). The stimulation of HaCaT cells with TNF-α led to the secretion of interleukin IL-6  and IL-8 . In addition, budesonide 10 mM used as a positive control reduced IL-6 and IL-8 release to 57.5 ± 4.7% and 44.4 ± 5.5% respectively (p < 0.05). The hydroalcoholic extract of E. africana inhibited the release of IL-8 to 48.1 ± 3.5% with an IC50 = 59.2 µg/mL, 95% CI (36.1–97.0) while there was no effect on the release of IL-6. Fractions FC3, FC4 and FC22 demonstrated a strong to medium activity on IL-6 release while IL-8 is strongly and moderately reduced respectively by FC22 and FC8. The remaining fractions  did not affect the release of IL-6 and IL-8. All results are summarized in p < 0.0001 by the hydroalcoholic leaves extract compared to the negative control, while none of the fractions showed any reduction in cell viability up to 100 µg/mL.The leaves of m/z value of 171.0298 ([M + H] +) which corresponds to gallic acid (1). The structure was further confirmed by NMR in comparison to published data (2) (3) (4) (O-glucoside (5) (6) (O-[β-apiosyl-(1‴→2″)-β-glucopyranoside] (7) (8) (O-glucoside (9) (10) (O-glucoside (11) . Subfraction FC4-I was revealed to be a pure compound showing an hed data . The isodata (2) , 5,7-dih (2) (3) , and 3′, (3) (4)  were isoside (5)  and cate (5) (6)  were obtide] (7) , isoquer (7) (8) , prunin side (9) , and aro(9) (10) . Finallyide (11)  togetherE. africana leaves hydroalcoholic extract. Compounds 2 to 5 and 7 to 11 were assessed for their in vitro anti-inflammatory effect. Since there is sufficient literature on compounds 1 and 6 as compounds with anti-inflammatory activity, no analysis was performed. The results reported in 50 = 17.8 µg/mL, 95% CI (4.5–69.6)   see  followedE. africana is a traditional medicinal plant which has been used in different cultures. Different parts of the medicinal plant have been investigated but until now only two flavonoids (apigenin and robonetin) were reported in the roots of the plant (v/v) since this resembles the traditional preparation (® LH-20 was performed to collect groups of compounds which could then be further partitioned. This method is one of the most common methods applied for isolation and purification of various flavonoid derivatives (in vitro using an immortalized keratinocyte cell line in order to assess their influence on proinflammatory cytokine IL-8 and IL-6 release. IL-6 is induced in acute inflammation and triggers the transition from acute to chronic inflammation by enhancing monocyte recruitment (E. africana inhibited IL-6 or IL-8 release in a concentration dependent manner. The purification led to 11 compounds out of which 9 were tested. Catechin and gallic were not investigated, since they are frequently occurring molecules with reported anti-inflammatory activity (for reviews see E. africana are flavonoids which are known to possess different biological properties such as anticancer, antidiabetic, anti-inflammatory, etc. The identified flavonoids are differing in the number of hydroxyl groups, their position and their attachment to a sugar moiety (O-rhamnoside and its aglycone. Besides that, aromadendrin, dihydrokaempferol-7-O-glucopyranoside and ethyl gallate exerted a strong to moderate effect on IL-6 release. Thus, an anti-inflammatory effect of E. africana and its secondary metabolites may be mediated by a downregulation of proinflammatory cytokine production e.g., in chronic wound healing.he plant . Moreoveparation . The fraivatives . The hydruitment  whereas ruitment . The relruitment . While nruitment . In thisruitment . Althougruitment . Furtherr moiety . 3′,4′,7r moiety . Accordir moiety . Thus, tE. africana was reported for the first time and 11 compounds were isolated. 3′,4′,7-trihydroxyflavone exerted strong to moderate inhibitory activity on the release of IL-6 and IL-8 from TNF-α stimulated HaCaT cells. Aromadendrin, dihydrokaempferol-7-O-glucoside, and ethyl gallate demonstrated a moderate influence on the release of interleukin IL-6. This finding supports the use of E. africana leaves in the traditional medicine for the treatment of inflammatory and pain-related skin conditions in the Republic of Benin.In the present study, the chemical characterization of phenolic compounds in


In contrast, linear HO–PEG–OH and MeO–PEG–OMe do not confer stability to AgNPs, and HS–PEG–OMe, which is often used for gold nanoparticles, sulfidates the surface to considerably degrade the properties. TEM shows an essentially intact nanostructure of c-PEG-physisorbed AgNPs even after heating at 95 °C, while complete disturbance is observed for other AgNPs. Molecular weight- and concentration-dependent stabilization by c-PEG is investigated, and DLS and ζ-potential measurements prove the formation of a c-PEG layer on the surface of AgNPs. Furthermore, c-PEG-physisorbed AgNPs exhibit persistent antimicrobial activity and cytotoxicity.Silver nanoparticles (AgNPs) are practically valuable in biological applications. However, no steady PEGylation has been established, which is essential for internal use in humans or animals. In this study, cyclic PEG ( Cyclic PEG without any chemical inhomogeneity was physiosorbed onto AgNPs to PEGylate and drastically enhance the dispersion stability against a physiological condition, white light, and high temperature, while its linear counterparts could not. However, unlike gold nanoparticles (AuNPs), AgNPs are not a stable material and susceptible to light, dissolved electrolytes, and various chemical species, and transformations occur leading to aggregation, dissolution, change in structure, activity loss, etc.9 In diverse fields of nanoscience, the instability of AgNPs often hinders their applications and commercialization. Although several capping agents have been explored for AgNPs, transformations, dissolution and agglomerations in various environments still remain a significant issue.The emerging importance of nanoscience cannot be overemphasized, and the field of nanotechnology has drawn special worldwide attention especially in the cases of noble metals such as gold and silver.10,11 sodium dodecyl sulfate (SDS),10,12 and other surfactants has been well reviewed with limitations of non-biocompatibility and instability. The utmost crucial factors of stability and biocompatibility have been desirable and sought after in the application of nanoparticles. In this regard, poly(ethylene glycol) (PEG), a non-ionic polymer with a flexible structure, is the most commonly used biocompatible polymer accepted by the United States Food and Drug Association and has wide applications including food, commodities, and drugs as well as uses in agriculture and manufacturing industries.13–16 In order to use PEG as a stabilizer for metal nanoparticles, especially AuNPs, thiol-functionalized PEG (HS–PEG–OMe) is often employed through the chemisorption between the sulfur atom and metal surface.17 However, the use of HS–PEG–OMe for AgNPs results in the formation of a silver sulfide (Ag2S) layer on the surface drastically disturbing the nanoparticles' properties18 and leading to dissolution,19,20 inhibiting PEGylation of AgNPs by the thiol chemisorption. A few reports show that AgNO3 is reduced by hydroxy-terminated PEG (HO–PEG–OH), and the resulting nanoparticles are capped by the same polymer in the process.21–23 However, the number of reports using this method is quite limited, and thus the capping structure and properties of the formed nanoparticles are not well studied. For these reasons, polyvinylpyrrolidone (PVP) is an alternative polymer often used for AgNPs but has limited biocompatibility compared to PEG.24–26 Therefore, stable PEGylation of AgNPs to curb the various restrictions is utmost needed.The use of stabilizers such as cetyltrimethylammonium bromide (CTAB),27,28 Of the various polymer topologies, cyclic polymers have shown unique physical and chemical properties such as increased glass transition temperature, higher refractive index, less entanglement, slow hydrolytic degradation, and self-assembling behaviors, different from their linear counterparts with the same molecular weight.29–35 In biomedical applications, cyclic polymers have promising potential due to their superior properties such as higher efficacy of gene delivery,36 higher cancer cell take up,37 longer circulation time in vivo,38,39 and controlled release of drugs.40 We reported that cyclic amphiphilic block copolymers form micelles with strong salt and thermal stabilities.41,42 Recently, cyclized PEG (c-PEG) without any chemical inhomogeneity was found to endow AuNPs with high dispersion stability by physisorption.43 Moreover, AuNPs PEGylated by c-PEG can be used for the colorimetric detection of bovine serum albumin through a unique complexation.44 The strong physisorption of c-PEG likely arises from the less entropic penalty upon adsorption to the surface compared to the linear counterparts,45,46 which is also suggested by theoretical and computational studies.47–51 Bearing this in mind, the physisorption of c-PEG is considered suitable for PEGylation and dispersion stabilization of AgNPs because AgNPs are much less chemically stable than AuNPs, and the thiol chemisorption is essentially inapplicable. The present work provides a new approach for the stabilization of AgNPs in the presence of c-PEG against physiological conditions, white light, and high temperature, as well as to examine the antimicrobial activity and cytotoxicity of AgNPs. We found that, in contrast to HS–PEG–OMe and other linear PEG, c-PEG readily stabilized AgNPs against the various harsh conditions and retained the biological activities  dispersed in a 2 mM sodium citrate solution with a silver mass concentration of 0.02 mg mL−1 were purchased from nanoComposix, USA and used as received. AgNPs with a size of 80 nm functionalized with methoxy polyethylene glycol sulfhydryl with a molecular weight of 5 kDa (HS–PEG5k–OMe) in Milli-Q water with a silver mass concentration of 0.02 mg mL−1 were also purchased from nanoComposix, USA and used as received. Poly(ethylene glycol) 2000 (HO–PEG2k–OH) , poly(ethylene glycol) 4000 (HO–PEG3k–OH) , and poly(ethylene glycol) 6000 (HO–PEG9k–OH) were purified by passing them through a silica gel column using chloroform/methanol  as an eluent. mPEG-SH, 10k (HS–PEG9k–OMe)  was purified by recycling preparative SEC. The AgNPs' and PEGs' names in the parentheses above are the ones used in the paper. The molecular weights of PEG in the catalogs deviated from our measurement to some extent, and the measurement values are used. Tetrahydrofuran (THF), dehydrated and stabilizer free , anthralin , silver trifluoroacetate , n-heptane , potassium hydroxide , iodomethane , chlorobenzene , chloroform , tosyl chloride , acetone , methanol , dichloromethane , magnesium sulfate , sodium dihydrogen phosphate , disodium hydrogen phosphate , sodium chloride , chloroform-d , Wakosil C-300 , E. coli JM 109 , Muller Hinton Broth , ampicillin , HeLa cells , fetal bovine serum (FBS) , penicillin–streptomycin, and trypsin (2.5%) , Dulbecco's modified Eagle's medium (DMEM) with low glucose , and D-PBS (−)  were used as received.Citrate-capped AgNPs with a size of 10, 20, and 30 nm  and tosyl chloride (190 mg) in 100 mL of dry THF was added over 144 h to a dispersion of powdered potassium hydroxide (3.3 g) in 100 mL of a mixture of dry THF and n-heptane (75/25 v/v) at 40 °C using a syringe pump in a dry nitrogen atmosphere. Additional 24 h of stirring was allowed for complete cyclization. The reaction mixture was filtered, and the solvent was removed under reduced pressure. Chloroform was added to the filtrate, washed with brine followed by deionized water. The organic phase was dried with magnesium sulfate, and the solvent was removed under reduced pressure. Silica gel column chromatography was carried out with a mixture of chloroform/acetone (9/1 v/v) to elute polymeric products that underwent intermolecular reactions, followed by a mixture of chloroform/methanol (9/1 v/v) to elute the crude containing c-PEG. The crude was dissolved in dichloromethane, and n-heptane was slowly added until the solution turned cloudy. The cloudy solution was heated to 40 °C and cooled to 25 °C with two resultant layers. The upper clear layer containing a relatively large proportion of c-PEG was collected, and this procedure was repeated several times to obtain pure c-PEG as a white solid. The yields of c-PEG2k, c-PEG3k, and c-PEG9k were 303 (6.1%), 247 (4.9%), and 145 mg (2.9%), respectively. Concerning the very low isolated yields, the purity was prioritized over the yield, resulting in a major loss during the isolation.Cyclization of HO–PEG–OH was carried out by the previously reported method.2.352 Typically, in a dry nitrogen atmosphere, chlorobenzene (20 mL) was added to finely powdered potassium hydroxide (2.3 g), and the mixture was stirred at 25 °C. Iodomethane (0.35 g) was added to the mixture, followed by slow addition of HO–PEG2k–OH (5.0 g) dissolved in chlorobenzene (50 mL) over 25 min. The mixture was stirred for 24 h. Filtration was carried out, and the filtrate was reduced to a small volume under reduced pressure, followed by washing with distilled water and deionized water. The organic phase was dried with magnesium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column with a mixture of chloroform/acetone (9/1 v/v), and the product was eluted with a mixture of chloroform/methanol (9/1 v/v). The solvent was removed and vacuum dried to obtain dimethylated poly(ethylene glycol) (MeO–PEG2k–OMe)  as a white solid.Methylation of HO–PEG–OH was carried out according to the previous method.2.41H NMR (400 MHz) and 13C NMR (100 MHz) were recorded on a JEOL JNM-ESC400 instrument at room temperature at a polymer concentration of 20 mg mL−1. Deuterated chloroform was used as a solvent.2.5−1. Calibration was performed with PEG standard samples.Size exclusion chromatography measurements were performed on a Shodex GPC-101 gel permeation chromatography system  equipped with a Shodex KF-G guard column  and two Shodex KF-804L columns (8 mm × 300 mm) in series. THF was used as an eluent at a flow rate of 1.0 mL min2.6−1, 25 μL) and an ionization agent . The mixture (0.4 μL) was dropcast on an opti-TOF 384-Well Insert (123 × 81 mm) plate for the measurement.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed at the Open Facility, Hokkaido University using an ABSCIEX TOF/TOF 5800 mass spectrometer. PEG (1.5 mg) dissolved in THF (10 μL) was mixed with a matrix  was used. JAIGEL-2H and 3H columns and a pre-column were connected in series. Chloroform was used as a solvent, and the flow rate was set at 3.5 mL min2.8600) in the antimicrobial activity experiment was determined by the intensity of an incubated specimen at 600 nm with that of the medium subtracted.UV-Vis absorption spectra were recorded using a JASCO Ubset V-670 spectrophotometer at 25 °C in a micro quartz cuvette  with a path length of 10 mm. Deionized water was used as a blank. Spectra were acquired at a wavelength range of 300–800 nm. Optical density at 600 nm . A micro quartz cuvette  and Zetasizer nano cell  were used. Measurements were carried out at 25 °C with a 120 s equilibration time. A cumulant analysis performed using the inbuilt software of the instrument was used to determine the z-average size.DLS and 2.1010 (0.54 mL) was added to HO–PEG9k–OH, MeO–PEG9k–OMe, HS–PEG9k–OMe, or c-PEG9k (1.5 mg) in a 1.5 mL Eppendorf tube, and the mixture was vortexed for 1 min to form AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k, respectively. The PEG concentration was varied by changing the amount of PEG. AgNPs10/No PEG was prepared by vortex mixing an aqueous dispersion of AgNPs10 (0.54 mL).Typically, an aqueous dispersion of AgNPs2.112HPO4 81 mM, NaH2PO4 14.7 mM) was prepared in advance. AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k (0.54 mL) prepared above was placed in a micro quartz cuvette. Subsequently, the tenfold-concentrated PBS solution (0.06 mL) was added to the cuvette, and the resulting mixture was 0.6 mL with pH 7.4 and a NaCl concentration of 150 mM with a PEG concentration of 0.25 wt%. A time-course UV-Vis measurement was performed for 1000 min.A tenfold-concentrated phosphate-buffered saline (PBS) solution  was added to HO–PEG9k–OH, MeO–PEG9k–OMe, HS–PEG9k–OMe, or c-PEG9k (1.5 mg) in a glass vial and mixed by pipetting to dissolve PEG to form AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k, respectively. AgNPs10/No PEG was prepared by pipetting an aqueous dispersion of AgNPs10 (0.54 mL). Subsequently, the tenfold-concentrated PBS solution (0.06 mL) was added to the glass vial and mixed by pipetting. The resulting mixture was 0.6 mL with pH 7.4 and a NaCl concentration of 150 mM with a PEG concentration of 0.25 wt%. A color change was observed.For ESI Movie 1,2.1210 (0.54 mL) was added to c-PEG2k, c-PEG3k, or c-PEG9k (0.15 mg) in a 1.5 mL Eppendorf tube, and the mixture was vortexed for 1 min to form AgNPs10/c-PEG2k, AgNPs10/c-PEG3k, or AgNPs10/c-PEG9k, respectively. Subsequently, the tenfold-concentrated PBS solution (0.06 mL) was added to the cuvette, and the resulting mixture was 0.6 mL with pH 7.4 and a NaCl concentration of 150 mM with a PEG concentration of 0.25 wt%. A time-course UV-Vis measurement was performed for 1000 min.An aqueous dispersion of AgNPs2.1310 (0.54 mL) was added to c-PEG9k  in a 1.5 mL Eppendorf tube, the mixture was vortexed for 1 min to form AgNPs10/c-PEG9k. Subsequently, the tenfold-concentrated PBS solution (0.06 mL) was added to the cuvette, and the resulting mixture was 0.6 mL with pH 7.4 and a NaCl concentration of 150 mM with a PEG concentration of 0.05, 0.25, 0.40, or 1.25 wt%, respectively. A time-course UV-Vis measurement was performed for 1000 min.An aqueous dispersion of AgNPs2.14c-PEG9k (1.5 mg) was added to an aqueous dispersion of AgNPs10, AgNPs20, or AgNPs30 (0.54 mL), and the mixture was vortexed for 1 min. Subsequently, the tenfold-concentrated PBS solution (0.06 mL) was added to the cuvette, and the resulting mixture was 0.6 mL with pH 7.4 and a NaCl concentration of 150 mM with a PEG concentration of 0.25 wt%. A time-course UV-Vis measurement was performed for 1000 min.2.152 100 mM, pH unadjusted) was prepared in advance. AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k (0.54 mL) prepared above was placed in a micro quartz cuvette. Subsequently, the tenfold-concentrated CaCl2 solution (0.06 mL) was added to the cuvette, and the resulting mixture was 0.6 mL with 10 mM of CaCl2 with a PEG concentration of 0.25 wt%. A time-course UV-Vis measurement was performed for 1000 min.A tenfold-concentrated calcium chloride solution (CaCl2.1610 (2.1 mL) was two times diluted with deionized water (2.1 mL) and added to HO–PEG9k–OH, MeO–PEG9k–OMe, HS–PEG9k–OMe, or c-PEG9k (10.5 mg) in a 50 mL Falcon tube. The mixture was vortexed for 1 min to form AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k, respectively, where the PEG concentration was 0.25 wt%. AgNPs10/No PEG was prepared by diluting AgNPs (2.1 mL) with deionized water (2.1 mL) and vortexed for 1 min. The mixtures were kept under 860–990 lux light intensity using a white light emitting tube at 25 °C for 35 d. 0.60 mL of the mixtures was withdrawn from the Falcon tubes immediately after mixing (day 0) and subsequently at a 7 day interval for an absorption measurement. The measured samples were not returned to the Falcon tubes.An aqueous dispersion of AgNPs2.1710 (0.30 mL) was two times diluted with deionized water (0.30 mL) and added to HO–PEG9k–OH, MeO–PEG9k–OMe, HS–PEG9k–OMe, or c-PEG9k (1.5 mg) in a 1.5 mL Eppendorf tube. The mixture was vortexed for 1 min to form AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k, respectively, where the PEG concentration was 0.25 wt%. AgNPs10/No PEG was prepared by diluting AgNPs (0.30 mL) with deionized water (0.30 mL) and vortexed for 1 min. The mixtures were incubated for 4 h at 4, 37, or 95 °C, and UV-Vis absorption spectra were recorded.An aqueous dispersion of AgNPs2.1810/No PEG, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k heated at 95 °C for 4 h were placed on a carbon coated Formvar TEM grid and air blown with a blower. Measurements were performed with a Japan Electron Optics Laboratory JEM-2010 operated at 200 kV.A few drops from the above AgNPs2.19E. coli was grown in a Muller Hinton Broth (MHB) medium containing ampicillin (100 μg mL−1) at 37 °C for 24 h and standardized using 0.5 McFarland standard (108 CFU mL−1). A tenfold-concentrated PBS solution  was added to AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k (2.7 mL) with a PEG concentration of 0.25 wt% and incubated for 24 h. The resulting mixture was centrifuged at 3000 rpm for 20 min, and the supernatant or dispersion was ultrafiltered to reduce the volume to 100 μL and mixed with 10 μL of 105 CFU mL−1E. coli in a 1.5 mL Eppendorf tube. The mixture was added to a test tube containing an MHB medium (2.9 mL) and incubated at 37 °C and 200 rpm for 24 h. UV-Vis absorption spectra were recorded.2.209k–OH, MeO–PEG9k–OMe, HS–PEG9k–OMe, or c-PEG9k was dissolved into DMEM (FBS (−)) at a concentration of 5 mg mL−1. The PEG solution was mixed with an aqueous dispersion of AgNPs (20 μg mL−1) at a 1 : 1 volume ratio. The mixture was diluted with DMEM (FBS (−)) by 10-fold for the final concentrations of AgNPs (1.0 μg mL−1) and of PEG (0.25 mg mL−1). For AgNPs/No PEG, AgNPs (20 μg mL−1) were diluted with DMEM (FBS (−)) to form AgNPs (1.0 μg mL−1). HeLa cells were cultured in cell culture dishes (Eppendorf) containing DMEM with 10% FBS, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin at 37 °C in 5% CO2. For the cell viability assay, the cells were seeded at a concentration of 6 × 103 per well in a 96 well microplate (Thermo Scientific) and grown for 24 h. The cells were treated with 100 μL of AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k followed by incubation for 2 h at 37 °C in 5% CO2. After the incubation, the cells were washed with D-PBS (−), and the medium was replaced with 100 μL of fresh DMEM (FBS (+)). The cells were additionally incubated for 24 h at 37 °C in 5% CO2. The cell viability was measured using a CellTiter-Glo 2.0 cell viability assay kit (Promega) according to the manufacturer's protocol.HO–PEG2.2110/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, and AgNPs10/c-PEG9k were prepared in the same manner as the cell viability assay with the final concentrations of AgNPs (1.0 μg mL−1) and of PEG (0.25 mg mL−1) in DMEM (FBS (−)). HeLa cells were seeded at a concentration of 8 × 104 per well in a 24 well microplate (Corning) and grown for 24 h. The cells were treated with 250 μL of AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, or AgNPs10/c-PEG9k followed by incubation for 2 h at 37 °C in 5% CO2. After incubation, the cells were washed with D-PBS (−) and scratched using a pipette tip followed by washing with D-PBS (−). The medium was replaced with 100 μL of fresh DMEM (FBS (+)). The scratched regions were observed using a microscope . Subsequently, the cells were incubated for 22 h at 37 °C in 5% CO2, and the scratched regions were measured again.AgNPs3.3.12k–OH, HO–PEG3k–OH, and HO–PEG9k–OH, respectively) was successfully cyclized by etherification. Thus, the chain ends of HO–PEG–OH were intramolecularly connected in the presence of tosyl chloride and potassium hydroxide in dilution. Highly pure c-PEG2k, c-PEG3k, and c-PEG9k were obtained after column chromatography and repeated separation using dichloromethane and n-heptane. SEC of c-PEG showed a unimodal trace with a peak shift to the lower molecular weight region compared to the prepolymer HO–PEG–OH 44H + Ag]+, where Ag+ was from silver trifluoroacetate, an ionization agent, whereas c-PEG2k had a peak at m/z = 2044.26 for [(C2H4O)44 + Ag]+ with the difference arising from the elimination of a water molecule. However, a MALDI-TOF mass spectrum for PEG9k was not obtainable due to its large molecular weight. The expected diameter of c-PEG2k, c-PEG3k, and c-PEG9k was 4.5, 6.8, and 22 nm, respectively, when they form an ideal right circular conformation. Furthermore, methylation of HO–PEG–OH resulted in the successful synthesis of MeO–PEG–OMe with features similar to its precursor HO–PEG–OH in terms of the appearance of the carbon atoms adjacent to the methoxy end group , was simply mixed with an aqueous dispersion of AgNPs with a size of 10, 20, or 30 nm . The addition of HO–PEG–OH, MeO–PEG–OMe, or c-PEG to AgNPs had no effect on the surface plasmon resonance (SPR) shown in λmax remained at 398 nm. On the other hand, the addition of HS–PEG9k–OMe abruptly reduced the absorption, broadened the peak, and deepened the yellow color of the AgNPs dispersion to yellowish brown. This change in SPR is explained as being a result of increase in the local dielectric environment upon thiol coordination to the Ag surface.19According to the procedure we previously established,ζ-potential measurements proved the distinct formation of a c-PEG layer on the surface of AgNPs as in the case of AuNPs.43 Thus, by DLS, AgNPs10/No PEG had a size of 18 nm, which on addition of HO–PEG9k–OH and MeO–PEG9k–OMe slightly enlarged to 24 and 20 nm, respectively  on silica.45,46 When AgNPs20 and AgNPs30 were used with c-PEG9k , the increase in size was less intense compared to AgNPs10 (109 nm). Thus, c-PEG9k with a diameter of 22 nm in the ideal right circular conformation exhibited the strongest interaction with AgNPs10 with a size of 18 nm.DLS and ectively . On the ζ-potential of AgNPs10/No PEG showed a negative value of −31 mV, which on addition of non-ionic PEG reduced to −25 mV for AgNPs10/HO–PEG9k–OH and −16 mV for AgNPs10/MeO–PEG9k–OMe  to each AgNPs dispersion (0.54 mL) to form the intended physiological conditions (pH 7.4 and a NaCl concentration of 150 mM), there was an immediate color change from yellow to light brown in the case of AgNPs10/No PEG and AgNPs10/HO–PEG9k–OH or to dark brown for AgNPs10/MeO–PEG9k–OMe , which was likely caused by dissolution of AgNPs in the presence of thiol.18Subsequently, the dispersion stability of AgNPsEG9k–OMe , which wpitation . The relc-PEG was investigated. versus time for AgNPs10/c-PEG2k, AgNPs10/c-PEG3k, and AgNPs10/c-PEG9k in a PBS buffer solution with pH 7.4 and a NaCl concentration of 150 mM. A significant increase in the dispersion stability was observed with an increase in the molecular weight. AgNPs10/c-PEG9k retained 74% of Rel. Abs after 1000 min, while AgNPs10/c-PEG3k retained only 39%, and AgNPs10/c-PEG2k also showed a weak stabilization with Rel. Abs of 37%. Moreover, the concentration of c-PEG9k was varied from 0.05 to 1.25 wt%  and AgNPs30/c-PEG9k (Rel. Abs = 57%). We showed above that the DLS size and ζ-potential were dependent on the molecular weight and AgNPs' size . This further proved the strong dispersion stability endowed by c-PEG.Moreover, divalent ionic salts have been reported to exert stronger dissolution and agglomeration effects on nanoparticles than the monovalent counterparts.% Fig. S6. On the 58 Thus, in production, storage, and applications, light is a well-known limiting factor which causes transformational changes of AgNPs.59,60 Also, emphasis is always made in the safety data sheets with respect to light. Thus, we tested the stability endowed by c-PEG against photoirradiation. AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, and AgNPs10/c-PEG9k with a PEG concentration of 0.25 wt% were exposed to white light at 860–990 lux , AgNPs10/HO–PEG9k–OH (blue-marked tube), and AgNPs10/MeO–PEG9k–OMe (green-marked tube) changed from light yellow to deep yellow, which intensified as the days progressed via the aggregation of AgNPs  along with stain-like objects with a few hundred nanometer in size were observed. The coordination of thiol to the AgNPs surface leads to an Ag2S layer.18,19 At high temperature, dissociation of Ag2S from AgNPs was stimulated to reduce the median particle size shown in 2S aggregated upon drying. What needs to be emphasized here is that AgNPs10/c-PEG9k was intact after heating with no significant change in size, and the particles were still well dispersed  in a Muller Hinton Broth (MHB) medium was evaluated. Thus, AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, and AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, and AgNPs10/c-PEG in PBS  were added to an E. coli-containing medium. Upon addition of AgNPs10/c-PEG to E. coli, immediate change in the color to brownish was observed, while the other mixtures did not cause the change. After 24 h of incubation, the E. coli-containing medium with AgNPs10/No PEG, AgNPs10/HO–PEG9k–OH, and AgNPs10/MeO–PEG9k–OMe became cloudy, suggesting the growth of E. coli : AgNPs10/No PEG, 0.79; AgNPs10/HO–PEG9k–OH, 0.63; AgNPs10/MeO–PEG9k–OMe, 0.82; AgNPs10/HS–PEG9k–OMe, 0.86; AgNPs10/c-PEG9k, 0.08. This revealed that the antimicrobial efficacy was preserved in AgNPs10/c-PEG9k but lost in all other specimens. Because HO–PEG9k–OH and MeO–PEG9k–OMe could not maintain the dispersibility of AgNPs in PBS, the antimicrobial potency was quenched before the addition to E. coli. In the case of AgNPs10/HS–PEG9k–OMe, no precipitate formed, but the sulfidation of AgNPs nullified the antimicrobial efficacy. In contrast, physisorption of c-PEG exhibited an improved dispersion stability of AgNPs and evidently retained the antimicrobial efficacy.Antimicrobial activity is inherent and one of the most important properties of AgNPs, and that against Gram-negative  E. coli . For AgN E. coli . The groc-PEG via cytotoxicity and scratch assay experiments using HeLa cells were performed. Fig. S810/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, and AgNPs10/c-PEG9k after incubation with HeLa cells in a DMEM medium for 24 h. AgNPs10/c-PEG9k had the lowest cell viability of 79% which was statistically significant (p < 0.05) compared to AgNPs10/No PEG with a viability of 85% with sextuplicate experiments. This suggests that c-PEG helps in the dispersion of AgNPs in the medium and preserves the cytotoxicity. On the other hand, no cytotoxicity was seen in AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, and AgNPs10/HS–PEG9k–OMe likely due to precipitation under the conditions.Following the various dispersion stability and antimicrobial activity experiments, further evaluation of the biological properties of AgNPs/10/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, AgNPs10/HS–PEG9k–OMe, and AgNPs10/c-PEG9k after 2 h of incubation (Fig. S910/No PEG, AgNPs10/HO–PEG9k–OH, AgNPs10/MeO–PEG9k–OMe, and AgNPs10/HS–PEG9k–OMe specimens were recovered to some extent after 22 h. However, most of the HeLa cells in AgNPs10/c-PEG9k were stripped off from the plates upon scratching, and basically no recovery was observed. This could be explained by AgNPs10/c-PEG9k leading to cell death in the large area, thereby inhibiting adhesion of the cells. These results further confirmed the cytotoxicity through the enhanced dispersion stability of AgNPs by c-PEG.In addition, migration and recovery of HeLa cells were evaluated by a cell scratch assay. A confluent monolayer was scratched on AgNPsn Fig. S9. The scr4.c-PEG, which cannot be attained with HS–PEG–OMe due to the formation of silver sulfide. Physisorption of c-PEG provided outstanding dispersion stability to AgNPs, against physiological conditions, white light, and high temperature, whereas HO–PEG–OH or MeO–PEG–OMe did not provide such dispersion stability. This method further exhibited persistent antimicrobial activity and cytotoxicity, which are two of the most important properties of AgNPs. Coupled with the excellent biocompatibility of PEG and the simple physisorption method, c-PEG would pave the way and broaden the uses of AgNPs especially in the biological and medicinal fields. Moreover, as we previously proved that the physisorption of c-PEG can also enhance the dispersion stability of AuNPs,43 the present method has great potential for application in a wide variety of metal nanoparticles.Our research has shown the first steady PEGylation method for AgNPs conferred by physisorption of The authors declare no competing interest.NA-004-D1NA00720C-s001NA-004-D1NA00720C-s002