Buddleja saligna (BS), and an isolated triterpenoid mixture (DT-BS-01) showed a fifty percent inhibitory concentration (IC50) of 33.80 ± 1.02 and 5.45 ± 0.19 µg/mL, respectively, against melanoma cells (UCT-MEL-1) with selectivity index (SI) values of 1.64 and 5.06 compared to keratinocytes (HaCat). Cyclooxygenase-2 (COX-2) inhibition was observed with IC50 values of 35.06 ± 2.96 (BS) and 26.40 ± 4.19 µg/mL (DT-BS-01). BS (30 µg/mL) significantly inhibited interleukin (IL)-6 (83.26 ± 17.60%) and IL-8 (100 ± 0.2%) production, whereas DT-BS-01 (5 µg/mL) showed 51.07 ± 2.83 (IL-6) and 0 ± 6.7% (IL-8) inhibition. Significant vascular endothelial growth factor (VEGF) inhibition, by 15.84 ± 4.54 and 12.21 ± 3.48%, respectively, was observed. In the ex ovo chick embryo yolk sac membrane assay (YSM), BS (15 µg/egg) significantly reduced new blood vessel formation, with 53.34 ± 11.64% newly formed vessels. Silver and palladium BS nanoparticles displayed noteworthy SI values. This is the first report on the significant anti-angiogenic activity of BS and DT-BS-01 and should be considered for preclinical trials as there are currently no US Food and Drug Administration (FDA) approved drugs to inhibit angiogenesis in melanoma. Melanoma cells secrete pro-angiogenic factors, which stimulates growth, proliferation and metastasis, and therefore are key therapeutic targets.  Over the past decade, the number of skin cancer cases has increased globally. In the US alone, the number of annually diagnosed invasive melanoma cases increased by 44% between 2011–2021, whereas the diagnosis and treatment of non-melanoma skin cancer increased by 77% between 1994–2014 sample is the concentration of PGE2 (pg/mL) produced when treated with the sample OR positive control and [PGE2]control is the concentration of PGE2 (pg/mL) produced when treated with the 5% DMSO vehicle control. The IC50 values were calculated using GraphPad Prism 4 Software. To determine the effect of BS and DT-BS-01 on the human recombinant cyclooxygenase-2 (COX-2) enzyme, a previously described method was used ,64. To eTM Cytometric Bead Array (CBA) Human Inflammatory Cytokine kit according to the manufacturer’s protocol (Cat. No. 551811) . Briefly, UCT-MEL-1 cells were seeded at a concentration of 1.0 × 105 cells/well in a 24−well plate with complete medium at 37 °C and 5% CO2 to allow for cell adherence. After 24 h, the medium was discarded and replaced with fresh complete medium containing 1 µg/mL phytohemagglutinin (PHA) to stimulate cytokine production. Stock concentrations of BS and DT-BS-01 were prepared at 1 mg/mL (in 0.25% DMSO). Cells were treated with final concentrations of the BS (30 µg/mL) and DT-BS-01 (5 µg/mL). Controls included a 0.25% DMSO vehicle control and cells grown in media (untreated). After 20 h incubation, the cells were centrifuged at 980 rpm for 5 min to collect the cell free supernatant and analyze the concentration (in pg/mL) of cytokines using the BDTM Accuri C6 cytometer and the FCAP Array™ Software V 3.0 . The percentage inhibition was calculated using the following equation:The quantification of cytokines expressed by UCT-MEL-1 cells was conducted in a similar manner as previously described . The lev5 cells/well in a 24−well plate with complete medium at 37 °C and 5% CO2 for 24 h to allow for cell adherence. After 24 h, the medium was replaced with fresh complete medium. Stock concentrations of BS and DT-BS-01 were prepared at 1 mg/mL (in DMSO). The cells were treated with final concentrations of BS (30 µg/mL) and DT-BS-01 (5 µg/mL). Controls included a 0.15% DMSO vehicle control, cells grown in medium (untreated) and cells exposed to the positive control, 6 µg/mL (~13 µM) ursolic acid. After 6 h of treatment, the plates were centrifuged at 980 rpm for 10 min and the cell free supernatant collected for quantification of VEGF using the VEGF human ELISA kit (Novex® Cat # KHG0111)  according to the manufacturer’s protocol, where absorbance was measured at 450 nm. Concentrations of VEGF were calculated from the standard curve using a linear equation. Percentage inhibition was calculated using the following equation:Quantification of VEGF production was done in a similar manner using an in vitro cell-based assay as previously described . Exponenv/v) relative humidity. During the incubation, eggs were gently turned twice a day to prevent adherence of the yolk sack to the shell. Thereafter, the eggs were opened into weighing boats (L89 × W89 × H25 mm) with the yolk sacs and blood vessels facing upwards. Weighing boats, with the same dimensions and with holes punctured in the sides, were used to cover the opened eggs. Once the opened eggs had stabilized for an additional 24 h at 37 °C and 90% (v/v) relative humidity, four sterilized silicone O-rings, with an internal diameter of 8 mm, were placed on the blood vessels. To each O-ring, 40 µL of sample was added, of which each egg contained at least one control. The controls included PBS and two vehicle treated controls (0.3 and 3% DMSO), where 0.3% was the vehicle for the compounds and 3% was used for BS. Samples included BS (15 µg/egg), DT-BS-01 (2.5 µg/egg), oleanolic acid (13 µmol/egg), ursolic acid (2.5 µmol/egg) and the positive control, combretastatin A4 (10 nmol/egg). After the addition of the samples to the O-rings, the eggs were incubated for a further 24 h at 37 °C and 90% (v/v) relative humidity. Images of each of the O-rings were taken at 0 and 24 h of incubation with the samples and controls using a digital USB microscope camera (Opti-Tekscope OT−V1). Images were analyzed using the Fiji ImageJ Software with the Analyze Skeleton plugin. The number of newly formed vessels was calculated by normalizing the number of newly formed vessels in the sample treated YSM to those of the 0.3 and 3% DMSO (vehicle) control.The YSM was used to determine the effect of BS and DT-BS-01 on angiogenesis ,70,71,72Buddleja saligna dried powdered material (400 mg leaves and stems) was weighed into a 100 mL flaks followed by the addition of 40 mL distilled water (dH2O). The solution was heated to 90 °C for 5 min to allow for the extraction of polyphenols and proanthocyanidins. After the solution had cooled, it was centrifuged at 2000 rpm for 5 min. Thereafter, 6 mL of the collected supernatant was added to a 30 mL vial followed by the addition of the 0.1 M metal precursor solution . The solution was continuously stirred overnight at room temperature to optimize total reduction of the metal salts to their respective nanoparticle  (2O for further use.S-PdNPs) . The nanUltraviolet-visible (UV-vis) spectroscopy was used to determine the characteristic surface plasmon resonance (SPR) of the respective nanoparticles. The samples (200 µL) were added to 800 µL in a quartz cuvette. The absorbance of each sample was read at a wavelength ranging between 200–800 nm using a Cary 60 UV-Vis spectrophotometer . The same sample preparations were used to measure the hydrodynamic size and the zeta potential of the respective nanoparticles using a Zetasizer NanoSeries (Nano-ZS90) . The average size of the respective nanoparticles was determined by obtaining transmission electron microscope (TEM) images on a JOEL 1400 TEM operating at 120 kV. The TEM samples were prepared by adding 5 µL of the respective nanoparticles on a carbon-coated copper grid and allowed to dry.The in vitro stability of the respective nanoparticles was evaluated in the presence of various biological buffer solutions to mimic in vivo conditions. The samples (1 mL) were added to glass vials containing 0.5 mL aqueous solutions of 5% NaCl, 0.5% cysteine, 0.2 M histidine, 0.5% human serum albumin (HSA), 0.5% bovine serum albumin (BSA) and PBS solutions at pH 5, 7 and 12, respectively. The stability was measured by monitoring the UV absorbance over a 24 and 48 h incubation period using a Cary 60 UV−Vis spectrophotometer. The stability of BS-PdNPs was monitored by DLS analysis since PdNPs do not exhibit an absorption peak.w/v) sodium carbonate solution (Na2CO3). The tube was incubated in a Microtube Thermal Mixer  at 30 °C for 30 min, where after the absorbance was measured at 760 nm using a microplate reader. The concentration of total phenolic content was expressed as gallic acid equivalence (GAE) in µg/mL.The total phenolic content of the respective nanoparticles was determined using the Folin—Ciocalteu method. The samples (500 µL) were added to a 2 mL microcentrifuge tube followed by the addition of 500 μL 1/10 dilution Folin–Ciocalteu phenol reagent and 1 mL of 7.5% (t-test (unpaired), or Dunnett’s or Tukey’s Multiple Comparison Test using the GraphPad Prism statistical software. * p < 0.05; ** p < 0.01 and *** p < 0.001 indicated statistical significance compared to the control (+).Results are reported as mean ± SD (or SEM). Samples were tested in duplicate or triplicate with at least 2 independent experiments see , unless Buddleja saligna (BS) and the triterpenoid mixture (DT-BS-01) should be considered for preclinical studies  due to the significant antiproliferative activity demonstrated against melanoma cells. Subsequently, both these samples have the potential to inhibit several pro-angiogenic factors, the most important of which is VEGF, which is required to stimulate angiogenesis for metastasis to occur. Additionally, utilizing the ex ovo YSM assay, BS was able to significantly inhibit the formation of new blood vessels demonstrating its anti-angiogenic efficacy in an in vivo model. Furthermore, due to the antiproliferative activity and noteworthy SI values, BS-AgNPs and BS-PdNPs should be explored for future anti-angiogenic investigations. Future studies should further include determining the antiproliferative potential of BS, DT-BS-01, BS-AgNPs and BS-PdNPs, against several other melanoma cell lines as well as primary non-tumorigenic melanocytes and to quantitatively determine apoptosis using the TUNEL assay. This is the first report on the antiproliferative and anti-angiogenic activities of BS against cancer cell lines. Moreover, the compound mixture (DT-BS-01), which consists of ursolic acid and oleanolic acid, has never been reported in combination for its antiproliferative and anti-angiogenic activities.Herein, we determined that D.T. and N.L. have filed a patent application related to the technology described in this article.


AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol  has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of  Therefore, it has gained increasing attention in recent years [−1 h−1), has been achieved by using a wild type Klebsiella pneumoniae strain with an optimized fermentation medium and conditions [Bacillus. licheniformis DSM 8785 is also an interesting 2,3-BDO producer supporting a high titer of 144.7 g/L in a fed batch fermentation [Klebsiella pneumoniae KCTC2242. Optimized expression of the genes involved in the 2,3-BDO biosynthetic pathway in K. pneumoniae increased the 2,3-BDO titer to 1.6 fold of that in the parental strain. It reached 101.53 g/L after 40 h fermentation with a productivity of 2.54 g L−1 h−1 in the strain with overexpression of budA (acetolactate decarboxylase) and budB (acetolactate synthase) [Enterobacter ludwigii strain has been developed for 2,3-BDO production by using Brewers’ spent grain hydrolysate as feedstock, which finally resulted in a titer of 118.5 g/L 2,3-BDO and a high yield of 0.48 g/g glucose [Nowadays, global warming caused by the huge emissions of CO biomass –13. Micrnt years , 14, 15.nt years . A high nditions . Bacilluentation . 2,3-BDOynthase) . Recentl glucose .ldhA gene, encoding lactate dehydrogenase, was identified by flux balance analysis (FBA) in K. oxytoca as the best candidate gene for single deletion to reduce lactate production and increase the 2,3-BDO yield [YodC) and heterologous expression of a formate dehydrogenase gene in Bacillus subtilis provided more NADH for 2,3-BDO production and led to less acetoin accumulation [pta and ldh genes eliminated acetate and lactate production, respectively. This was followed by deletion of the D-(–)− 2,3-BDO dehydrogenase gene (bdhA), and expression of the meso-2,3-BDO dehydrogenase gene (budC), from K. pneumoniae. This engineered B. subtilis strain produced 103.7 g/L pure meso-2,3-BDO in a semi-aerobic condition with a yield of 0.487 g/g [Most of the bacteria produced 2,3-BDO from different carbon sources with a mixed acid fermentation, which generates several different products, such as acetic acid, ethanol and lactic acid , 21, 22.DO yield . AcetoinDO yield . The elimulation . In addi.487 g/g .Saccharomyces cerevisiae is a promising alternative host for industrial production of 2,3-BDO since it does not suffer from these shortcomings [S. cerevisiae strain was engineered with introduction of acetolactate synthase  and acetolactate decarboxylase  from B. subtilis and overexpression of the endogenous 2,3-BDO dehydrogenase 1 (encoded by BDH1). The resulting strain produced 96.2 g/L 2,3-BDO in 244 h with a yield of 0.28 g/g glucose under oxygen-limiting conditions [PDC genes, however, creates a C2-auxotrophy because of deficiency in cytosolic acetaldehyde production. This can be overcome to some extent by provision of a C2-compound like ethanol in the medium or by evolutionary adaptation [The production of 2,3-BDO with bacteria is often cumbersome, because of pathogenicity, low robustness, phage sensitivity and difficulty to combat contamination with other bacteria . Sacchartcomings , 2 and htcomings –30. As ptcomings . This catcomings , 33. A pnditions . Deletioaptation .S. cerevisiae strains in which ethanol production has been eliminated and this is due to the excess NADH generated during the conversion process from glucose to 2,3-BDO [NoxE encoded water-forming NADH oxidase. With NoxE from Lactococcus lactis the 2,3-BDO yield increased to 0.359 g/g glucose and decreased the glycerol yield to 0.069 g/g glucose since part of the excess NADH was consumed by oxygen instead of generating glycerol [S. cerevisiae strain with elimination of alcohol dehydrogenase (adh1-5) and NAD-dependent glycerol 3-phosphate dehydrogenase , and expressing the NoxE gene to oxidize excess NADH and maintain the redox balance, resulted in a yield for 2,3-BDO production of 0.41 g/g glucose and no glycerol synthesis [Candida tropicalis PDC1 was used to produce ethanol as essential cytosolic C2 compound to maximize cell growth and 2,3-BDO production in the pdc-deficient S. cerevisiae strain with expression of the NoxE gene to lower glycerol production. The engineered strain produced 154.3 g/L 2,3-BDO in 78 h with a yield of 0.404 g/g glucose [Glycerol is the main by-product of 2,3-BDO production in  2,3-BDO . In 2,3- 2,3-BDO . As a re 2,3-BDO . This prglycerol . The eng glucose .S. cerevisiae mitochondria was shown to reduce the overflow of NADH under aerobic conditions of high glycolytic flux [Alternative oxidase is a cyanide-insensitive terminal oxidase integrated in the inner mitochondrial membrane in all plants, most fungi, algae and some protists. It catalyzes the four-electron transfer from ubiquinol (reduced form of ubiquinone) to oxygen to generate water with release of only heat and no ATP generation . The exptic flux , 41. TheHistoplasma capsulatum in S. cerevisiae [NoxE pathway have been explored in parallel in this study to oxidize excess NADH of the 2,3-BDO production in order to reduce glycerol formation. Both strategies were shown to be effective in NADH re-oxidation. Together with several other genetic modifications, the two strategies resulted in two top performance yeast strains displaying a combination of high yield, high titer and high productivity for 2,3-BDO production suitable for industrial implementation.In this study, we have created a new NADH oxidation pathway by overexpression of the homologous mitochondrial external NADH dehydrogenase   and the revisiae . ThroughS. cerevisiae strains used in this work have been listed in Table The E. coli Top10 cells and the transformants were selected on LB agar plates with ampicillin. The transformants were checked by colony PCR with Standard Taq polymerase to select positive colonies. Two positive colonies were inoculated into LB medium with ampicillin and grown overnight for plasmid extraction. Plasmids were sent for sequence analysis after purification according to the protocol provided by the manufacturer.Genomic DNA of yeast cells for PCR or sequence analysis was extracted by the phenol/chloroform/isoamylalcohol (25:24:1) method. The precipitated DNA was washed twice with ethanol and finally dissolved in MilliQ water. Plasmids were extracted with the commercial Nucleospin Plasmid EasyPure kit (Macherey Nagel) according to the protocol provided by the manufacturer. The plasmids were digested with relevant restriction enzymes to ligate DNA fragments via Gibson Assembly followed by transformation in pdc-negative strains, 5 g/L ethanol was supplied. Solid YPD plates were obtained by adding 15 g/L Bacto™ agar in the media. Transformants were selected on solid YPD plates with one antibiotic or a combination of different antibiotics  in a 30 °C incubator. For long-term storage of engineered strains at − 80 °C, 300 μL glycerol (87%) was mixed with 700 μL culture medium. Plasmid construction and multiplication in E. coli (Top10) was carried out in lysogeny broth (LB) medium supplemented with 100 mg/L ampicillin and incubated at 37 °C. To obtain solid LB nutrient plates, 15 g/L Bacto™ agar was added to LB medium.Yeast cells were grown in YPD medium  at 30 °C in a rotary shaker at 200 rpm. For culturing of The lithium-acetate method or electroporation was used for yeast transformation. Gene deletion or integration was carried using the CRISPR/Cas9 genome editing system. Yeast cells were first transformed with Cas9 enzyme expression plasmid (p51) and selected on YPD(E) solid plates with geneticin. The yeast cells harboring p51 were then precultured in YPD(E) medium with geneticin for the second transformation with the gRNA plasmid and the linear donor. The transformants were selected on YPD(E) solid nutrient plates with relevant antibiotics for the p51 and gRNA plasmids. Three positive colonies were inoculated in YPD(E) medium after confirmation with PCR using Standard Taq polymerase and transferred three times in YPD(E) medium to lose the plasmids.AlsS, Gene ID: 936,852), α-acetolactate decarboxylase , butanediol dehydrogenase  and alternative oxidase . The homologous NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1) and Sugar Transporter-Like protein (STL1) from Candida albicans were directly amplified from respective genomic DNA. The above genes were amplified with high-fidelity polymerase Q5 (New England Biolabs) and the amplicons were purified with Wizard SV Gel and PCR clean-up kit (Promega) and prepared for cloning into vectors using the Gibson assembly master mix (New England Biolabs). The donor DNA for gene deletion was constructed via two PCR amplifications. The upstream and downstream sequence (about 400 bp) of the ORF were amplified with high-fidelity polymerase Q5, with an overlap of 40 bp and purified for the second round of PCR (fusion PCR), which was then purified for use as donor DNA.The Open Reading Frame (ORF) of the following genes was codon optimized and sent for synthesis at IDT: α-acetolactate synthase  with a flow rate of 0.7 mL/min. The compounds were separated on a Biorad Aminex HPX 300 × 7.8 mm column, maintained at 60 °C, and detected with a UV detector  and RID detector .Yeast cells were inoculated in glass tubes with 3 mL YPD(E) medium from plates and grown to stationary phase. Then 2 mL of cell culture was transferred into a 300 mL erlenmeyer with 50 mL 10% YPD(E) medium and cultured for 16-20 h. A certain volume of cells was centrifuged and resuspended with fermentation medium and then inoculated in a 300 mL fermentation tube or 300 mL erlenmeyer containing 50 mL fermentation medium with starting ODAn overview of the native and engineered metabolic pathway for 2,3-butanediol production is shown in Fig. PDC1, PDC5 and PDC6 genes were deleted in strain HGS4 and each gene was replaced with a construct encoding the three 2,3-BDO pathway genes: AlsS, encoding α-acetolactate synthase, AlsD, encoding α-acetolactate decarboxylase, and BdhA, encoding acetoin reductase. The fermentation profile of the resulting HGS4 strain is shown in Fig. The BdhA gene by insertion of two copies in a single construct, using the TDH3 promoter and ADH1 promoter, into the AD7 integration site [To stimulate the conversion of the intermediate acetoin, we next overexpressed the ion site . The resNDE1 gene, encoding mitochondrial external NADH dehydrogenase, and the AOX1 gene, encoding a heterologous alternative oxidase from Histoplasma capsulatum, were expressed using the ADH1 and TDH3 promoter, respectively, in the Mk114 integration site [GPD2 ORF with the NDE1 ORF, which resulted in strain HGS31. This modification improved glucose consumption and lowered glycerol production to about half of the 2,3-BDO production -α-acetolactate to 2,3-dimethylglycerate and thus competes with AlsD-mediated (R)-acetoin production [GPD1 but from the weak CYC1 promoter [We next deleted the oduction . This stoduction . Hence, promoter  and inteNoxE gene from Lactococcus lactis, encoding a water-forming NADH oxidase, in the HGS7 strain, resulting in strain HGS8. The latter strain showed strong reduction of glycerol production from 69.80 g/L in the HGS7 strain to about 21.76 g/L  [+ symporter, CaStl1, by insertion in the Mk119 site in the HGS29 strain, resulting in strain HGS37. That strain consumed an initial glucose level of 310 g/L in 100 h, accumulating 133.91 g/L 2,3-BDO, with minimal levels of acetoin (5.88 g/L) and glycerol (7.75 g/L) being produced  , encodinced Fig. D. Utilizced Fig. E. This wced Fig. E. The hiced Fig. F.NoxE from Lactococcus lactis, encoding a water-forming NADH oxidase, has been used for the first time in 2015 to address the redox imbalance in 2,3-BDO production [NoxE does not compromise 2,3-BDO yield by increasing acetoin levels.The production of 2,3-BDO from sugar substrates by microbial cell factories poses several challenges. The first major challenge is the redox imbalance in the pathway. The conversion of a glucose molecule into a 2,3-BDO molecule by microbial fermentation produces one NADH molecule in excess. Without any additional adjustment, this causes a huge production of glycerol, which reaches even higher levels than that of the 2,3-BDO produced Fig. B, C. Theoduction . Since tNoxE encoding NADH oxidase as previously performed [NDE1, encoding mitochondrial external NADH dehydrogenase [AOX1, encoding a heterologous alternative oxidase [NoxE expression in HGS37 resulted in a similar 2,3-BDO productivity (1.58 g/L.h) compared to expression in HGS50 of the combination pathway with the alternative oxidase (1.57 g/L.h), but not when expressed per gCDW: 0.11 g/L.h per gCDW for the former versus 0.08 g/L.h per gCDW for the latter. HGS37 could use YPD medium without addition of ethanol to overcome the C2 deficiency in the pdc-minus strain. On the other hand, the supplementation of ethanol to the YPD for HGS50 facilitated the growth and resulted in higher biomass. Hence, the C2-deficiency may be a factor in causing the difference in productivity when expressed per gDCW. In both cases, overexpression of the NADH consuming enzymes should not be too high in order to avoid the onset of acetoin accumulation. The expression of two more copies of NoxE did not result in a difference in 2,3-BDO production. Also the combination of NoxE and expression of the Nde1/Aox1 alternative oxidase pathway did not enhance 2,3-BDO production. Probably, the activity of the 2,3-BDO synthesis pathway itself may be limiting. For oxygen consumption, the following equations were calculated. 1. for HGS37: 1 mol glucose + 0.47 mol O2 = 0.94 mol 2,3-BDO + 0.06 mol glycerol + 1.88 mol CO2, and 2. for HGS50: 1 mol glucose + 0.48 mol O2 = 0.96 mol 2,3-BDO + 0.04 mol glycerol + 1.92 mol CO2 [We have used 2 alternative approaches to address the redox imbalance: expression of erformed , 47, 51,rogenase , and AOX oxidase , 52, 53. mol CO2 , 48, 54.GPD1 and GPD2  mediates pyruvate uptake and comprises Mpc1 and Mpc2 during fermentative growth, or Mpc1 and Mpc3 during respiratory growth –57. DeleDO titer . NormallGPD genes, which results in higher glycerol levels compromising yield (Table CYC1, together with higher CSTL1 expression, could further decrease net glycerol production. In spite of this, the performance of HGS50 and HGS37 is probably high enough already for successful industrial implementation in commercial 2,3-BDO production. With bacterial cell factories, higher titers have been obtained but the use of bacteria in large-scale non-sterile industrial production is cumbersome amongst others because of the lack of robustness and difficulty of addressing contamination. On the other hand, it has to be mentioned also that the downstream processing of 2,3-BDO is expensive because of its high boiling point and high hydrophilicity. It can easily entail more than half of the production cost [The consecutive genetic modifications that we introduced in the two parallel strain lineages resulted in a gradual increase in 2,3-BDO yield (Fig. ion cost .Table 3TWe have been able to successfully engineer an industrial yeast strain for 2,3-BDO production displaying a 96% yield with glucose as substrate, close to the maximum theoretical yield, a very high titer of 120-130 g/L in batch fermentation, which likely can be further enhanced in fed-batch fermentation, and a high productivity of 1.58 g/L.h. This performance for the three crucial specifications combined is probably adequate for successful implementation of the strain in commercial 2,3-BDO production by yeast fermentation.


The Scanning Electron Microscope (SEM) image revealed that the activated carbon's exterior surface was irregular and full of various shapes and sizes of cavities.The Energy Dispersive X-Ray (EDX) indicated the existence of carbon, oxygen, silicon and potassium in banana peels-derived activated carbon, whereas carbon, oxygen, silicon and potassium, in addition to aluminium, were detected in the pomegranate peels-derived activated carbon. The Fourier-Transform Infrared Spectroscopy (FTIR) analysis of prepared activated carbon revealed several functional groups, including carboxylic acid, carbon dioxide, and aromatic compounds. Results also showed that the activated carbon significantly removed chlorpyrifos from water, recording 97.6%, 90.6%, 71.48%, and 52.00 % for activated carbon prepared from pomegranate peels, banana peels, date stones and orange peels, respectively. The study concluded that agricultural waste-derived activated carbon could be employed as an alternative pesticide adsorbent.Chlorpyrifos is an organophosphate insecticide linked to neurological dysfunctions, endocrine disturbance, cardiovascular illness, genotoxicity, histopathological abnormalities, immunotoxicity, and oxidative stress. Therefore, the aim of this study was to prepare activated carbon from agricultural waste to adsorb and remove chlorpyrifos from aqueous solutions, as well as to study the physicochemical characteristics of the prepared activated carbon.Activated carbon was prepared from agricultural waste . The activated carbon prepared showed an exterior surface that was irregular and full of cavities with Brunauer-Emmett-Teller(BET) surface areas of 94.26, 111.75, 183.89, and 289.86 m  •The SEM image revealed that the exterior surface of activated carbon was irregular and full of cavities.•The EDX examination revealed the existence of carbon, oxygen, and other elements in the prepared activated carbon.•The FTIR spectra showed the nature of the functional groups existing on the exterior of the activated carbon.•The pomegranate peel-activated carbon possessed maximum total pore volume and the highest surface area.•Pomegranate and banana peels-derived activated carbon adsorbed chlorpyrifos pesticide by 97.39% and 90.61%, respectively, Chlorpyrifos was purchased from Santa Cruz Biotechnology Inc. .2.2Citrus sinensis L.) and sweet orange from the Egyptian Ministry of Agriculture farms on December 30th, 2020. Dates (Phoenix dactylifera L.) were procured from a date farm in El Ayat, Giza,on January 4th, 2021. We purchased pomegranates (Punicagranatum L.)and bananas (Musa sp.) from the neighbourhood market on October 10th, 2020.We obtained fresh fruits of Washington Navel orange  and filtered through a 0.25 mm mesh.2.53PO4), according to Girgis et al Activated carbon was prepared from various agricultural wastes through a single-step chemical activation process using phosphoric acid (H2.62.6.1The surface morphology was investigated using the SEM equipped with Energy Dispersive X-ray spectra (EDX) to determine the precise elemental report 2.7−1using FTIR spectroscopy  according to the method described by Hussain et al The FTIR spectra of the prepared activated carbon were recorded between 450 and 4000 cm2.8O) within 0.05 – 0.20. The obtained results from the isotherms were used to define the BET surface area. The Horvath–Kawazoe (HK) method was applied to determine the micro-pore volume. The Barrett–Joyner–Halenda (BJH) method defined the pore size division and the mesopore volume.According to Tan et al 2.9The adsorption ability of activated carbon against chlorpyrifos was studied. Chlorpyrifos (8 mg) was dissolved in 10 mL acetonitrile and added to a litre of distilled water to reach a concentration of 8 mg/l.Then the prepared chlorpyrifos solution was divided into falcon tubes (50 mL) containing an amount of 0.25 and 0.50 g of agricultural waste activated carbon. The mixture was incubated in a shaker incubator at 100 rpm, 25 °C for 1 h. Then 50 mL of the aqueous solution was extracted twice with 50 mL of dichloromethane using a separating funnel. The mixed extracts were dehydrated by passing through a column of anhydrous sodium sulphate and evaporated using a rotary evaporator to approximately 1 mL. The extracts were then transferred to vials, and the volume was reduced to 0.1 mL using a nitrogen stream for HPLC analysis 2.10P < 0.05 was considered statistically significant.The results were expressed as mean ± SD. Statistical analysis was performed using the SPSS software version 16. A one-way analysis of variance (ANOVA) was performed, in which 33.13.1.1The SEM examined the morphological structure of activated carbon prepared from bananas, pomegranates, orange peels, and date stones , Fig. 4.3.2−1 were assigned to O-H stretching, carboxylic acids, C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="pc-E00C" format="PBM" resolution="300" x-size="12" y-size="9">000000000000000000000000000000000000111111111111000000000000111111111111000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>CC, allene, CC stretching, cyclic alkane, C-H bending, alkanes, C-O stretching, aliphatic ether, C-H bending,mono-substituted, and C-Cl stretching, halo compound respectively. On the other hand, the bands 2350.40 & 2325.00 were assigned to OCO carbon dioxide.The FTIR spectra of activated carbon prepared from banana, pomegranate, orange peels, and date stones were determined to understand the nature of the functional groups existing on the exterior of the activated carbon , Fig. 8.O stretching, primary amide, C-O stretching, aliphatic ether, and CC bending, alkene respectively. Meanwhile, the bands 2360.4, 2349.47, & 2337.64 were assigned to OCO, carbon dioxide.Meanwhile, CO, carbon dioxide, N = CN stretching, carbodiimide, CC stretching, cyclic alkane, and C-H bending, alkane, respectively, whereas both 1999.62, &1951.55 were assigned to CCC stretching, allene.On the other hand, N stretching, allene, C-H bending, alkane, C-O stretching, aliphatic ether, and C-Cl stretching, halo compounds.Results in −1, 1200–1300 cm−1, and 750–850 cm−1, 1 for orange peels activated carbon. Also, for date stones derived from activated carbon, results differed from Erhayem et al O, -COOH, C-N and CC. Results were also different from Sahmarani et al −1, from 900 to 1300 cm−1, and 1070–1080 cm−1, while two bands appeared at 743 and 875 cm−1.Results concerning the banana peels derived activated carbon were similar to those reported by Zheng and Wang 3.32 adsorption-desorption isotherms were used to measure the prepared activated carbon's specific surface area and porous structure. Different techniques, such as BET and BJH, are used to calculate the surface area of the prepared activated carbon (BET) and total pore volume (VT) of the prepared activated carbonare affected by the type of agricultural waste used. Although the activated carbon based on banana peel showed a maximum (SBETof 289.86 m2/g), the pomegranate peel activated carbon possessed maximum total pore volume (VT) (0.462 cm3/g) with the highest BJH surface area (125.11 m2/g). The orange peel-derived activated carbon showed a lower BET surface area (94.26 m2/g), while date stone-derived activated carbon possessed a lower total pore volume (0.132 cm3/g). According to the ratio SBJH/SBET and VBJH/VBET, it is clear that the mesoporosity character increased with pomegranate and orange peels derived activated carbon. The average pore radius of these prepared activated carbon results were 5.07 and 4.45 nm for pomegranate and orange peels derived activated carbon.The Nd carbon . The sur2/g) BET external surface area was 199.16, 140.99 and 71.49 (m2/g) for oil palm empty fruit bunch, bamboo stem and coconut shell, respectively. For banana peel-derived activated carbon, the SBET results were considered higher than those of Zheng and Wang Similarly, Ettish et al 3.4Data in Results showed that fruit waste-derived activated carbons at lower concentrations removed a higher amount of chlorpyrifos, except for date stones-derived activated carbon,whereas higher activated carbon concentrations removed higher amounts of chlorpyrifos concentrations. That might be due to the saturation that could have happened ata concentration of 0.25 g.Although there are various methods for removing pesticides from water, adsorption on activated carbons, on the other hand, is the most efficient and extensively used method for removing pesticides from water streams Paulownia tomentosa.According to many factors, including the source of the activated carbon, surface characterisation, particle size, and functional groups, the adsorption capabilities of various adsorbents differed. Similar findings were reported by Ettish et al 4Activated carbon from fruit waste  might be a promising adsorbent for removing chlorpyrifos from water. This study could be considered a new contribution to the production of activated carbon from fruit -waste. The outcomes of this research could be a guide for the practical use of fruit waste to remove pesticides from contaminated water. Carbon from fruit waste may be utilized in adsorption columns to produce safe water. Further investigations on the disposal of used carbon are required to ensure that it does not result in further environmental issues like the release of pesticides into the air, water, and soil.Omaima A. Hussain: Conceptualization, Methodology. Amal S. Hathout: Writing – review & editing. Yasmin E. Abdel-Mobdy: Visualization, Data curation. M.M. Rashed: Supervision. E.A. Abdel Rahim: Supervision. A.S.M. Fouzy: Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


NFκB/p65 protein expression was moderately increased by PEG-BA . However, significant (p < 0.05) overexpression of the proapoptotic genes TNF (23.72 ± 1.03) and CASPASE 3  compared with untreated cells was notable. The antioxidant potential of PEG-BA was greater (IC50 = 15.59 ± 0.64 µM) compared with ascorbic acid (25.58 ± 0.44 µM) and BA-only (>100 µM) and further confirmed with the improved reduction of hydroperoxide levels compared with BA-only (518.80 ± 25.53 µM vs. 542.43 ± 9.70 µM). In conclusion, PEG-BA activated both the intrinsic and extrinsic pathways of apoptosis and improved antioxidant activities in PC cells, suggesting enhanced anticancer activity upon conjugation.Pancreatic cancer (PC) is one of the most aggressive solid malignancies with poor treatment response and low survival rates. Herbal medicines such as betulinic acid (BA) have shown potential in treating various solid tumours, but with limitations that can be circumvented by polymer-drug conjugation. Polyethylene glycol-BA (PEG-BA) polymer-drug conjugate has previously shown selective anticancer activity against PC cells. Here, we elucidate the mechanism of cell death and the cell death pathway, anti-inflammatory and antioxidant activities of PEG-BA. PEG-BA induced apoptotic cell death by arresting MIA-PaCa-2 cells in the Sub-G Pancreatic cancer (PC) is a lethal solid malignancy with increasing incidence and mortality worldwide . Most paBetulinic acid (3β-hydroxy-lup-2029)-en-28-oic acid), the carboxylic acid derivative of betulin, is a naturally occurring, widely distributed pentacyclic triterpene sourced from the outer bark of the Betula alba tree . Betulin-en-28-oiUsing nanoparticles in chemotherapy provides an efficient and selective drug delivery system for targeting tumour cells . An examApoptosis occurs via two different pathways, namely the extrinsic (death receptor) and intrinsic  pathways . The extL and Mcl-1). These proteins have been associated with the nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), a mediator of inflammation, which is overexpressed in tumours [Cancer cells have developed mechanisms to evade apoptosis, a major hallmark of carcinogenesis . One suc tumours . Like ot tumours ,25. Furt tumours ,27,28.In the case of cellular injury, the immune system is activated in an attempt to alleviate any resulting potential damage ,30. One 1 phase of the cell cycle, activates proapoptotic genes of the extrinsic and intrinsic pathways and inhibits ROS formation. Overall, there was cell growth inhibition, apoptosis induction and ROS activity inhibition in PC cells treated with PEG-BA, suggesting that apoptosis is the primary anticancer mechanism.In this study, PEG-BA was assessed for its effect on apoptosis by measuring DNA content, proapoptotic gene expression, and NF-κB protein activity on MIA PaCa-2 treated cells. Its effect on the inflammation induced by ROS was also assessed. The findings demonstrated that PEG-BA induces apoptosis by arresting cells in the sub-GThe compounds were synthesised and characterised as previously described ,39. The The biological model for PC used in this study was the MIA PaCa-2 cells obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank . These cells were derived from a 65-year-old man with pancreatic adenocarcinoma located in the tail and body of the pancreas . Vero ce3—3.7 g/L) and 1% antibiotic antimycotic solution . In the case of the Vero cells, the standard DMEM (13.4 g/L DMEM powder + 3.7 g/L NaHCO3) was supplemented with 1.1 mM sodium pyruvate, 25 mM HEPES buffer, 250 µg/mL gentamicin, and 0.5% antibiotic antimycotic solution. Both media were further supplemented with 10% heat-inactivated foetal bovine serum (FBS). The cells were cultured in 75 cm2 vented flasks  at 37 °C (95% humidity and 5% CO2). The cells were harvested every 2–4 days or when they reached 80–90% confluency using a trypsin-EDTA  dissociation reagent. Cell counting was performed using the trypan blue exclusion method [Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  with slight modifications. The DMEM used for the MIA PaCa-2 cells was made up to a final concentration of 13.4 g/L with sodium bicarbonate (NAHCOn method .2).For experimental purposes, the cells were allowed to adhere for 24 h and then treated with controls and test compounds diluted with DMEM. DMSO (≥ 0.33%) was used as the vehicle control, 0.4 µM doxorubicin and 0.78–100 µM gemcitabine as control drugs, and 0.78–100 µM BA (free compound) and PEG-BA (polymer-drug conjugate) for 72 h in a humidified incubator  of BA and PEG-BA were analysed using light microscopy. The morphological changes were photographed after 24, 48 and 72 h using the Olympus IX51 microscope  at 10 X magnification.50 value, the half-maximal inhibitory concentration representing the concentration at which the compounds will inhibit cell viability by 50% [50 on Vero and MIA PaCa-2 cells, defined as the selectivity index (SI). Cytotoxicity assays were performed using tetrazolium dyes as previously described . The IC5y by 50% , was cal2). The cells were harvested using trypsin EDTA and washed (200× g). The cell pellet was resuspended with ice-cold 1X PBS followed by dropwise addition of ice-cold 70% ethanol for fixation at −20 °C for at least 24 h before staining. A total of 500 µL of the staining mixture, prepared by dissolving 2 mg of RNase A in 0.1% Triton-X100 reagent in PBS and containing 400 µL of 500 µg/mL propidium iodide dye, was added to each sample and incubated at room temperature for 30 min. Data acquisition commenced at low flow rates (< 300 events/sec) for improved resolution and adequate DNA quantification on the BD LSRFortessa™ Analyser and FACSDiva software . At least 30,000 events were recorded per sample. The fluorescent signal from the samples was detected using the 610/20 band filter pass filter. The generated Flow Cytometry Standard (FCS) files in FACSDiva were imported and analysed using FlowJo v10.8.1 .The single parameter-based propidium iodide staining technique was used to assess the cell cycle status of the cells, pre-and post-treatment with vehicle control (0.33% DMSO), 4 µM of BA and PEG-BA and 4.5 µM of gemcitabine following 72 h of treatment  according to the manufacturer’s guidelines. Briefly, 700 µL of guanidium thiocyanate (GTC)-lysis buffer containing 2% β-mercaptoethanol was added to the pelleted cells previously treated with 4 µM BA and PEG-BA and 4.5 µM gemcitabine for 72 h. This mixture was then transferred to an RNA homogeniser mini-column and centrifuged for 60 s at 13,000× g. This was followed by adding 700 µL of freshly prepared 70% ethanol and transferring to a HiBind®RNA mini-column. The mixture was centrifuged at 10,000× g for 60 s until the RNA in-solution was bound to the column. The bound RNA was washed  with 500 µL of RNA Wash Buffer I. The filtrate was discarded, and the RNA was rewashed  using a second buffer (RNA Wash Buffer II containing 100% ethanol). This step was repeated once to ensure proper washing of the RNA before drying the HiBind®RNA mini-column to remove any residual ethanol . The HiBind®RNA mini-column was transferred to a 1.5 mL Eppendorf tube, and 60 µL of nuclease-free water was added, followed by centrifugation  to elute the RNA. The eluted RNA was assessed for purity, and the concentration was determined using the Nanodrop 2000 .Total RNA was extracted using the E.Z.N.A.Total RNA (250 ng/µL) was used to make the cDNA template for each sample. The cDNA synthesis step was performed using the ProtoScript II First Strand cDNA Synthesis Kit  following the manufacturer’s protocol. Briefly, 20 µL of the reaction mixture was added to respective Eppendorf tubes: 2 µL of d(T)23 VN (50 µM) primers, 10 µL of the ProtoScript II reaction mix (2 X), 2 µL of the Protoscript II enzyme mix (10 X) and 6 µL of the RNA template diluted with the required amount of nuclease-free water to have a uniform final cDNA concentration for all the samples of 1000 ng/µL. The samples were then incubated in a SimpliAmp Thermal Cycler  under the following conditions: 42 °C for one hour, enzyme inactivation at 80 °C for 5 min, and an infinite hold at 4 °C.−∆∆CT method [Real-time PCR was performed in a 96-well plate format (on ice) according to the MIQE guidelines . BrieflyT method . The human nuclear factor kappa-B p65 subunit (NF-κB/p65) enzyme-linked immunosorbent assay (ELISA) kit from Elabscience Biotechnology Inc.  was used to determine the concentration of NF-kB/p65 present in cellular samples after treatment with complete DMEM, 4 µM of BA and PEG-BA, and 4.5 µM gemcitabine (72 h incubation). Cell supernatants and lysates derived from these samples were used to perform the ELISA according to the manufacturer’s protocol with slight modifications. Briefly, 100 µL of the standards (0–10 ng/mL), lysates (diluted 2X with sample diluent), and supernatants were added to respective wells in the protein-coated 96-well plate, in duplicate, sealed, and incubated at 37 °C for 1.5 h. The samples were discarded, and 100 µL of the biotinylated detection antibody working solution was added and incubated for an hour. A wash step was employed by adding 350 µL of the wash buffer, soaking for at least 60 s, and aspirating three times. Horseradish peroxidase (HRP) conjugate working solution (100 µL) was added to each well, sealed, and incubated for 30 min. The plate was washed five times, and 90 µL of the substrate reagent was added to the wells. The plates were sealed and covered in foil to protect them from light. The plate was incubated for at least 15 min, followed by adding 50 µL of the stop solution. The optical density was measured at 450 nm using a Multiscan Sky microplate reader .Although the aetiology of PC is not well-known, inflammation has been described as an important risk factor ,45. Two BA, PEG-BA, and the ascorbic acid standard were tested in the antioxidant activity assay at concentrations in the range 0.78–100 µM. This assay was modified from the 2014 protocol by Mistry and Shah . Briefly1/R2 solution (100 µg/mL DEPPD (R1) and 4.37 µM FeSO4 (R2) at a ratio of 1:25) was added to all wells except the blank. The plate was covered with foil for at least 1 min and analysed using a Victor™ X3 model 2030 multilabel plate reader  at a wavelength of 505 nm with a kinetic loop of 30 readings per well. The DEPPD assay was used to determine reactive oxygen metabolites (hydroperoxide) produced by reactive oxygen species (ROS) in PC cells. The oxidative status and ROS reduction potential of the MIA PaCa-2 cells pre- and post-treatment (72 h) with 0.33% DMSO and optimised working concentrations: 8 µM for BA and PEG-BA and 9 µM gemcitabine, were determined using the DEPPD assay. The assay was performed according to the protocol by William (2012), with modifications to accommodate using cell supernatants instead of the standard urine or blood samples . Brieflyp-value greater than α (0.05) meant that the null hypothesis (H0) was accepted, and the data were classified as following a normal distribution. Furthermore, quantile-quantile (Q-Q) plots were assessed with the assumption that plotting predicted residual vs. actual residual forms a roughly straight line for normally distributed data. For two groups , an unpaired Student t-test was performed. A two-way analysis of variance (ANOVA) was performed for more than two groups, followed by multiple comparisons using the Tukey post hoc test, if the data passed the normality tests and Q-Q plots. For data that only passed the normality tests but not the Q-Q plots, the non-parametric Kruskal–Wallis was performed followed by Dunn’s post hoc test. A p-value of less than 0.05 was considered statistically significant.The GraphPad Prism Version 8 software  was used to determine the statistical significance of the data. To assess whether the data were normally distributed, normality tests such as the Shapiro–Wilk (W), Anderson–Darling (A2*), D’Agostino–Pearson omnibus (K2), and Kolmogorov–Smirnov (distance) that are available in the GraphPad Prism (version 8) software were used. It was assumed that a p = 0.0255). Therefore, PEG-BA induced increased cell clumping and cytotoxicity in a concentration and time-dependent manner. At 72 h, 8 µM of PEG-BA resulted in clumping of the cells forming clusters, indicating high toxicity towards the PC cells. This was also observed at 24 and 48 h  compared with cells undergoing DNA synthesis (S: 17.67 ± 1.22%) and those preparing to enter mitotic division or already in the phases of mitosis (G2/M: 25.07 ± 2.44%)  and induced cell cycle arrest in the G1/G0 phase relative to the untreated MIA PaCa-2 cells (1 compared with the untreated sample and BA-only (PEG-BA: 39.50 ± 5.32% vs. untreated: 4.57 ± 0.82% and BA: 19.63 ± 4.49%) as shown in 2/M phases. The cell cycle status of the MIA PaCa-2 cells before and after treatment was performed using propidium iodide dye on 4 µM of BA and PEG-BA treated cells. This is a concentration in the ICrom this  and a pr± 2.44%) . The remreatment . At 4 µM-2 cells B,D. PEG-TNF, TNFSF10, CASPASES , BID and BAX) of the extrinsic and intrinsic pathways of apoptosis using quantitative real-time PCR. Compared with BA, PEG-BA treatment in PC cells resulted in a significant increase in the proapoptotic genes, CASPASE 2 , CASPASE 8  and BAX . Although not significant, TNF , TNFSF10 , BID , CASPASE 7  and CASPASE 3  were also elevated (p > 0.05) of apoptotic genes was observed when the PC cells were treated with gemcitabine, except for the non-significant overexpression of CASPASE 3 (p = 0.0872). Comparing PEG-BA to gemcitabine showed high expression of TNF , CASPASE 8 , BID (p = 0.0087), CASPASE 2 , CASPASE 7 , TNFSF10  and BAX . Furthermore, non-significant overexpression of CASPASE 3  was also observed.The cell cycle data reported here and the apoptosis assay performed previously  suggest elevated . A reducp = 0.1521) and BA  compared with the untreated sample (2.70 ± 0.21 ng/mL). Gemcitabine resulted in a slight but insignificant decrease . The protein expression levels of nuclear factor kappa B (p65-subunit) in both lysate and supernatant of treated  MIA PaCa-2 cells were quantified using a sandwich ELISA. A higher level of NF-κB was observed in the lysate of the untreated and treated MIA PaCa-2 cells compared with the corresponding supernatant . NF-κB lp = 0.1291). This trend was also observed at the remaining concentrations (50 µM: 39.57 ± 0.58 vs. 24.27 ± 2.91% (p = 0.0092) and 100 µM: 36.56 ± 1.86% vs. 24.26 ± 3.07%, p = 0.0279). In an analysis to determine the compound’s ability to inhibit at least 50% of the initial concentration of the DPPH radical (IC50), PEG-BA was found to have the lowest IC50 (15.59 ± 0.64 µM) compared with BA-only (>100 µM) and ascorbic acid .Antioxidant analysis using the DPPH assay showed that PEG-BA treatment induced a dose-dependent inhibition of DPPH compared with BA-only . BA treap < 0.0001) higher concentration of hydroperoxides (7.32-fold) in untreated cancer cells compared with the normal cells and treated cells (p = 0.5066). Notably, gemcitabine treatment reduced hydroperoxide levels (433.34 ± 33.53 µM) compared with untreated , PEG-BA treated  and BA-only  treated cancer cells.The level of ROS, specifically hydroperoxides, in the MIA PaCa-2 cells before and after treatment with PEG-BA, BA only and gemcitabine was assessed using the DEPPD assay. Overall, the results showed that there was a significantly  morphology of MIA PaCa-2 cells was observed in the untreated sample . Similar1 phase after treatment with PEG-BA, relative to the untreated and BA-treated cells, suggesting an increase in apoptosis. Studies have shown that the presence of cells in the sub-G1 phase indicates apoptotic cells or the induction of apoptosis [1 phase [1/G0 are undergoing apoptosis [2/M phase, indicative of diminished cell proliferation and viability. Similar findings have been reported using PANC-1 and IGROV-1 cells treated with BA, resulting in either a sub-G1 or a G1/G0 phase arrest [Cell cycle analysis studies, represented by the DNA content, showed an arrest of the cancer cells in the sub-Gpoptosis . This is[1 phase ,57. DNA poptosis ,57. BA hpoptosis , neuroblpoptosis ,60, cervpoptosis , colon cpoptosis , multiplpoptosis  and PC [poptosis . In thise arrest ,65. TakeTNF and TNFSF10 were upregulated post-PEG-BA treatment in PC cells , suggesting a higher radical scavenging potential than ascorbic acid and BA. On the other hand, treatment with BA resulted in an IC50 greater than 100 µM, suggesting a lower antioxidant potential than ascorbic acid with an IC50 of 25.58 ± 0.44 µM. A similar observation was made by Adesanwo and colleagues (2013), where they assessed the antioxidant capacity of BA isolated from Tetracera potatoria roots and reported an even higher IC50 value of 309 µM [PEG-BA treatment further improved antioxidant potential, demonstrated by a lower ICf 309 µM . Severalf 309 µM ,82. Thisf 309 µM ,83. Therf 309 µM . Our resThe DEPPD assay confirmed significantly high concentrations of hydroperoxides in the PC cells compared with the normal cells , which hThis study has demonstrated that the potential therapeutic efficacy of betulinic acid on PC cells improved following polymer conjugation. It also showed at the morphological and molecular level that this increased efficacy is due to its ability to upregulate proapoptotic genes and, consequently, induce apoptosis while exhibiting antioxidation properties. These findings suggest that PEG-BA is further investigated as a treatment for PC using models such as 3D culture or animal models.


The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. In this study, we adopted a QconCAT-based quantitative mass spectrometry approach to determine the stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native α-carboxysomes and their recombinant counterparts heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications.Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO Bacterial microcompartments (BMCs) are self-assembling proteinaceous organelles that are widespread among bacterial phyla , 2. The 3–7–2 fixation in all identified cyanobacteria and some chemoautotrophs  and carbonic anhydrase (CA), using a semipermeable shell, which allows the passage of negatively charged HCO3− and ribulose 1,5-bisphosphate (RuBP) and probably preclude O2 influx and leakage of CO2 from the carboxysome to the cytoplasm , and a full-length CsoS2B, translated via programmed ribosomal frameshifting   , 43, 44.H. neapolitanus and recombinant α-carboxysomes produced in Escherichia coli, using QconCAT-assisted quantitative mass spectrometry (MS) in combination with biochemical analysis, electron microscopy (EM), and enzymatic assays. Our results shed light on the molecular principles underlying the assembly and structural plasticity of α-carboxysomes and provide essential information required for design and engineering of carboxysomes in synthetic biology.In this study, we performed absolute quantification of protein components within native α-carboxysomes from H. neapolitanus using sucrose gradient ultracentrifugation ] at 0.20 ± 0.02 mM (n = 4) (n = 272) (The QconCAT-assisted mass spectrometry approach permitted a precise quantification of the absolute abundance of proteins –47. This–tion see . NADH-co (n = 4) . EM shown = 272) , consistn = 272) , 48, 49.10.1128/mbio.03629-21.2FIG S1H. neapolitanus and E. coli, respectively. (A) Sucrose gradient for carboxysome fractions. The milky band between 35% and 50% sucrose fraction interface consists of enriched carboxysomes. (B) SDS-PAGE of purified carboxysomes isolated from four replicate batches of culture showing the majority bands of α-carboxysome proteins. See also maxV at 2.96 ± 0.09 and 2.07 ± 0.12 μmol·mg−1·min−1 and mK(RuBP) at 0.20 ± 0.02 and 0.08 ± 0.02 mM, respectively. Data are presented as means ± SD from four independent biological replicates. (D) TEM images of purified native and recombinant carboxysomes. (E) Boxplot distribution for diameters of purified native and recombinant carboxysomes, at 124.6 ± 9.6 nm (n = 272) and 131.8 ± 18.0 nm (n = 152), respectively. Significant difference of average diameter was confirmed with Student t test (P < 0.05). (F) Analysis of the shell thickness of native and recombinant α-carboxysomes. The shell thicknesses of native and recombinant α-carboxysomes are 5.3 ± 0.6 nm and 5.5 ± 0.8 nm, respectively (n = 100), implicating the single-layer shell architecture. The profile region for measurements is marked by red lines . (G) Recombinant α-carboxysome expression cassette containing cso operon and cbbL/S, plus BMC-T protein-encoding gene csoS1D on the pBAD33 vector. Download FIG S1, JPG file, 1.7 MB.Purification and characterization of native and recombinant α-carboxysomes from Copyright © 2022 Sun et al.2022Sun et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. neapolitanus α-carboxysome  Schematic representation of the quantification concatamer, for quantification of proteins of interest. Thirty-five peptides in QconCAT are represented by blue boxes. Values for the [M + 2H2+] m/z peptide ions for the unlabeled QconCAT are aligned above each peptide (green text). The nontarget quantification peptides (GluFib and cMyc) and the hexahistidine tag for QconCAT purification are shaded in red and orange, respectively. (B) SDS-PAGE analysis of QconCAT expression and purification. The coding sequence of QconCAT peptide was subcloned into the cell-free expression vector pEU-E01-MCS (left). QconCAT was prepared by wheat germ cell-free synthesis in the presence of arginine and lysine and purified by virtue of the hexahistidine tag (right). The QconCAT is indicated by the arrow. Download FIG S2, JPG file, 1.2 MB.Structure and expression of the Copyright © 2022 Sun et al.2022Sun et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03629-21.6TABLE S1Table S1, DOCX file, 0.05 MB.Peptides derived from tryptic proteolysis of the QconCAT for carboxysome protein quantification. The flanking sequences that recapitulate the true native primary sequence context, together with additional sequences that are derived from the loop assembly synthesis of the QconCAT, are written in gray. Download Copyright © 2022 Sun et al.2022Sun et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03629-21.7TABLE S2Table S2, DOCX file, 0.04 MB.DNA and protein sequences of the carboxysome QconCAT peptide. Download Copyright © 2022 Sun et al.2022Sun et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the From the LC-MS/MS traces, the peptide precursor ions for analyte and standards were isolated and their relative areas were quantified using Skyline . This wa10.1128/mbio.03629-21.4FIG S3H. neapolitanus and E. coli with four biological replicates prepared for quantification by QconCAT mass spectrometry. The 10 μg of proteins was loaded per well. The protein bands with sizes between 15 and 40 kDa observed in recombinant carboxysomes might be cytoskeletal and membrane(-associated) proteins from E. coli as suggested by mass spectrometry . Download FIG S3, JPG file, 0.6 MB.SDS-PAGE of purified carboxysomes from trometry . SimilarCopyright © 2022 Sun et al.2022Sun et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.03629-21.5FIG S4H. neapolitanus carboxysomes. Overall good agreements were found within candidate peptides for the same protein, with the exception of peptide 3 of CsoSCA (34% ± 4% lower than the other two) and CbbO (21% ± 3%) in native carboxysome samples. Different colors represent distinct QconCAT peptides that were used to quantify the same carboxysome protein, which is listed in FIG S4, JPG file, 1.4 MB.Evaluation of QconCAT and label-free quantification. (A) Protein profile of all identified proteins in label-free quantification. In native carboxysome samples, McdAB-like proteins are both detected. The full protein list is provided in Copyright © 2022 Sun et al.2022Sun et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H. neapolitanus carboxysome structure, based on the shell surface area of a typical icosahedron (n = 272) measured in EM (H. neapolitanus α-carboxysome are CsoS1AC hexamers (863 copies), followed by Rubisco , CsoS2A (248 copies), CsoS2B (192 copies), CsoS1B hexamers (112 copies), and 58 copies of CsoSCA dimers. The H. neapolitanus α-carboxysome has a molecular weight (MW) of ∼346 MDa and the Rubisco enzymes account for ∼66% of the total MW. The hexameric shell proteins CsoS1A/C and CsoS1B make up ∼17.1% of the total MW. Additionally, about 11 copies of CsoS4A/B pentamers  are integrated within the α-carboxysome. CsoS1D pseudohexamers have a low abundance in the shell, with ∼3 copies per carboxysome. Moreover, the linker proteins, CsoS2A and CsoS2B, account for 13.5% of the total MW.We quantified the abundance of protein components within one sahedron  and the 10.1128/mbio.03629-21.8TABLE S3https://doi.org/10.1126/science.1151458; Y. Tsai, M. R. Sawaya, G. C. Cannon, F. Cai, et al., PLoS Biol 5:e144, 2007, https://doi.org/10.1371/journal.pbio.0050144). &Assumed packing densities of 74% (Kepler packing)  in the proposed carboxysome model based on the measured carboxysome diameters. Download Table S3, DOCX file, 0.04 MB.Calculation of carboxysome surface area, shell hexamer content, and carboxysome diameter. *CsoS1 and CsoS4 width/area obtained from previous publications  and its activases CbbQ2 and CbbO2  and a mK(RuBP) of 0.08 ± 0.02 mM (n = 4), though both were lower than those of native α-carboxysomes (n = 152), slightly larger than native α-carboxysomes , consisP < 0.05   of the culture was checked daily, and the cells were harvested before the culture entered the stationary phase. For expression of recombinant carboxysomes, the entire cso operon, as designed on pHnCBS1D reported previously  and subsequentially centrifuged at 300 × g for 15 min to sediment elemental sulfur. The supernatant was transferred to a new centrifugation tube, and cells were obtained by another round of centrifugation at 12,000 × g for 10 min. The resulting cell pellet was resuspended in 15 mL of TEMB buffer and incubated with egg lysosome  for 1 h at 30°C before cell breakage by prewashed glass beads  for 10 min . The cell extracts were further treated with 33% (vol/vol) B-PERII  and 0.5% (vol/vol) IGEPAL CA630 (Sigma-Aldrich) and placed on a rotary mixer for 2 h. The unbroken cells and large membrane debris were removed by centrifugation at 9,000 × g for 10 min. Crude carboxysome enrichment was pelleted at 48,000 × g for 30 min. The pellet was resuspended, briefly centrifuged at 9,000 × g, and then loaded to a step sucrose gradient  and ultracentrifuged at 105,000 × g for 35 min. The 3 mL of enriched carboxysome was harvested at 35% to 50% sucrose gradient fractions. Sucrose was removed by an additional round of ultracentrifugation after dilution with 30 mL of TEMB buffer. The pure carboxysome pellet was resuspended in 800 μL of TEMB buffer. Unless indicated otherwise, all procedures were performed at 4°C. The carboxysome purification from E. coli was performed according to the previous protocols  and treated with 0.5% (vol/vol) IGEPAL CA-630 detergent for 2 h. The following purification steps were the same as for the isolation of native carboxysomes from H. neapolitanus as described above.Purification of α-carboxysome from eviously . The 5-Lrotocols , with moSDS-PAGE analysis was performed following standard procedures. The 10 μg of purified carboxysomal samples or 100 μg of whole-cell fractions was loaded per well on 15% polyacrylamide gels and stained with Coomassie brilliant blue G-250 .−1) were stained with 3% uranyl acetate on carbon grids and then inspected with an FEI 120 kV Tecnai G2 Spirit BioTWIN transmission electron microscope (TEM) equipped with a Gatan Rio 16 camera. The diameters of carboxysomes were measured with ImageJ as described previously  using the WEPR8240H full-expression kit following default protocols . The QconCAT peptide was purified with Ni-Sepharose suspension  in centrifuge filters  following standard protocols. Finally, the QconCAT peptide was precipitated and resuspended in 30 μL of 25 mM ammonium bicarbonate with 0.1% (wt/vol) RapiGest SF surfactant  and protease inhibitors .Absolute quantification of the carboxysomal proteins was carried out by mass spectrometry analysis with stable isotope tryptic peptides as standards. The standard peptides were added in the form of a QconCAT, an artificial protein that is a concatenation of tryptic peptides in the same primary sequence context as the cognate analyte peptides , 50. Forincluded . The DNAeviously . The fineviously  was asse4HCO3. QconCAT (approximately 5 pmol) and [Glu1]-fibrinopeptide B (5 pmol) were added, and samples were denatured using 2.5 μL of 1% (wt/vol) RapiGest  in 25 mM NH4HCO3 followed by incubation at 80°C for 10 min. Samples were reduced by the addition of 2.5 μL of 12 mM dithiothreitol in 25 mM NH4HCO3 and incubation at 60°C for 10 min. Alkylation was carried out by the addition of 2.5 μL of 36 mM iodoacetamide in 25 mM NH4HCO3 and incubation at room temperature for 30 min in the dark. Trypsin at 2.5 μL  was added to each sample in a final digest volume of 50 μL. Samples were incubated at 37°C overnight. To remove residual RapiGest, digests were acidified by the addition of 0.5 μL of trifluoroacetic acid (TFA) followed by incubation at 37°C for 45 min. Samples were centrifuged at 17,200 × g for 30 min and transferred to fresh low-bind tubes.For QconCAT quantification, native and synthetic carboxysome preparations, four replicates of each, were diluted to a final protein concentration of 2.5 μg per 40 μL of 25 mM NH18, 75 μm by 2 cm, 3-μm packing material, 100 Å) using a loading buffer of 0.1% (vol/vol) TFA–2% (vol/vol) acetonitrile in water for 7 min at a flow rate of 12 μL min−1. The trapping column was then set in-line with an analytical column  and the peptides were eluted using a linear gradient of 96.2% buffer A (0.1% [vol/vol] formic acid)/3.8% buffer B (0.1% [vol/vol] formic acid in water/acetonitrile [80:20] [vol/vol]) to 50% A/50% B over 30 min at a flow rate of 0.3 μL min−1, followed by washing at 1% A/99% B for 5 min and reequilibration of the column to starting conditions. The column was maintained at 40°C, and the effluent was introduced directly into the integrated nano-electrospray ionization source operating in positive ion mode. The mass spectrometer was operated in MS-only mode, with survey scans between m/z 350 to 2,000 acquired at a mass resolution of 240,000 Full Width Half Maximum (FWHM) at m/z 200. The maximum injection time was 50 ms, and the automatic gain control was set to 3e6. The raw data files were incorporated in Skyline  nano-liquid chromatograph . Sample digest (2 μL) was loaded onto a trapping column  against the UniProt H. neapolitanus database  and E. coli database . A precursor mass tolerance of 10 ppm and a fragment ion mass tolerance of 0.01 Da were applied with dynamic modifications of 13C615N2 K, 13C615N4 R, and oxidation (M) and with the static modification of carbamidomethylation (C).Additionally, preparations of the four native and synthetic carboxysomes were analyzed by label-free quantification. Carboxysomes were digested as described above but without [Glu1]-fibrinopeptide B and analyzed by LC-MS/MS as described above but with a data-dependent acquisition method consisting of a 60,000-resolution full-scan MS scan with Automatic Gain Control (AGC) set to 3e6 ions with a maximum fill time of 100 ms. The 16 most abundant peaks per full scan were selected for Higher Energy Collisional Dissociation (HCD) MS/MS  with an ion selection window of 2 For single-carboxysome quantitative normalization, relative quantifications from QconCAT were normalized based on the 12-pentamer coverage or hexameric and pentameric protein coverage within a single-layer shell . Twelve-eviously .10.1128/mbio.03629-21.9TABLE S4n = 272) and 131.8 ± 18.0 nm (n = 152) for native and recombinant carboxysomes, respectively. Values used for standardization are displayed in bold. Calculation is described in Materials and Methods in the main text. Download Table S4, DOCX file, 0.04 MB.Absolute protein abundance per native and recombinant carboxysome based on 12-pentamer occupation and surface area coverage. *Based on surface area of ideal icosahedral with diameters measured from EM, 124.6 ± 9.6 nm  and the ProteomeXchange identifier (ID) PXD031494. Raw LC-MS/MS data for label-free quantification have been deposited to the ProteomeXchange Consortium via the PRIDE (https://www.ebi.ac.uk/pride/archive/projects/PXD031420). All other data are available from the corresponding author upon request.The entire Skyline project and raw data for QconCAT quantification have been deposited at Panorama Public  with thehe PRIDE  partner


Quercus serrata belongs to the Fagaceae family. There are 600 known species of Quercus worldwide. Q. serrata is distributed nationally in Korea, Japan, and China and grows to a height of 10–15 m. It exhibits a light grey bark with longitudinal furrows; the leaves are 6–12 cm long and 2.5–5 cm wide. The Quercus genus reportedly exhibits several types of bioactivity, including antioxidant, anti-inflammatory, antifungal, antimicrobial, and anticancer activity. Additionally, it has been reported that Quercus produces diverse phytochemicals, including tannins, flavonoids, and triterpenoids. Herein, we describe the column chromatographic isolation of five compounds from a Q. serrata extract. The compounds included caffeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5). We assessed the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity, antioxidant activity, NLR family pyrin domain-containing 3 (NLRP3) inflammasome  inhibitory effects, and collagenase inhibition activity of the Q. serrata extract and its constituent compounds. Our results indicated that the Q. serrata extract and the isolated constituent compounds showed inhibitory activity with reference to nitric oxide production, inflammasome component expression, and collagenase activity. Our findings imply that the Q. serrata extract and the isolated constituent compounds are potential candidates for the treatment of inflammatory diseases such as arthritis. Quercus genus is part of the Fagaceae family, with 600 known species worldwide. Several variants of this species exist in Korea, including Q. acutissima, Q. variabilis, Q. dentata, Q. aliena, Q. mongolica, Q. serrata, Q. acuta, Q. glauca, Q. myrsinifolia, Q. salicina, and Q. gilva. The species Q. serrata is widely distributed throughout Korea, Japan, and China and can reach up to 10–15 m in height. The bark is light grey in color with longitudinal furrows. The leaves are 6–12 cm long and 2.5–5 cm wide, with light grey elongated oval patterns on the underside [Quercus genus reportedly exhibits several types of bioactivity, including antioxidant [Quercus spp. revealed that it produces flavonoids [Q. serrata in this context are especially rare. We previously investigated the use of a Quercus spp. Extract as a therapeutic agent for inflammatory diseases by evaluating nitric oxide (NO) production and proinflammatory cytokine levels [β [β. In addition to cytokine production, NLRP3 inflammasome activation may also facilitate pyroptosis (caspase-1-mediated rapid cell death) [Q. serrata extract and its constituent compounds can downregulate the expression of the NLRP3 inflammasome proteins and show activity as antioxidants, anti-inflammatory agents, and collagenase inhibitors. Based on this bioactivity profile, we propose that this extract and its constituent compounds can potentially be applied for the treatment of inflammatory diseases such as arthritis.The ioxidant , anti-inioxidant –7, antifioxidant –10, antiioxidant , 12, antioxidant , 14, antioxidant , antibacioxidant , and antioxidant  activitiavonoids , triterpavonoids , 21, andavonoids –24. Natuavonoids –29. Seveavonoids , 31. Conavonoids –38. In te levels  determinevels [β . The NLRl death) , 41. Infl death) . Arthritl death) . In thisQ. serrata leaves were collected from Pocheon, Republic of Korea, in May 2020. The species was identified by Dr. Sung Sik Kim. A voucher specimen was stored at the Herbarium of the College of Pharmacy, Chung-Ang University.μm, Mitsubishi Chemical, Tokyo, Japan), ODS-B gel , and Sephadex LH-20 .Column chromatography was conducted using MCI gel CHP 20P (75–150 2SO4) or anisaldehyde-H2SO4 was heated and sprayed with ferric chloride (FeCl3) to determine the location of the stains on the chromatograph.A pre-coated silica gel 60 F254 plate  was used to perform thin-layer chromatography (TLC) with a mixture of chloroform, methanol, and distilled water  and a mixture of benzene, ethyl formate, and formic acid . A 10% solution of sulfuric acid (H13C-(150 MHz) NMR were performed and recorded using JEOL  at Chung-Ang University.To clarify the structure of the isolated compounds, 1D-nuclear magnetic resonance (NMR) analyses such as 1H-(600 MHz) and Q. serrata leaves using 80% acetone at room temperature. Such extracts were obtained three times over the course of three days. The extracts were concentrated by removing the acetone under vacuum, and this procedure yielded 350.12 g of concentrated extract. The Q. serrata extract (300 g) was then filtered using Celite 545, and the remaining extract was stored at -20°C. The filtered Q. serrata extract was loaded onto a Sephadex LH-20 chromatography column with a methanol: water (MeOH : H2O) gradient solvent system (ratio between 0 : 10 and 10 : 0) and yielded 12 subfractions (Q. serrata-1–12).Extracts were obtained from 2.2 kg of Q. serrata-8 (44.97 g) during repeated column chromatography using the MCI gel CHP 20P with a gradient solvent system of MeOH : H2O (from 0 : 10 to 10 : 0) yielded 24 subfractions. The Q. serrata-8-12 subfractions (494.9 mg) were loaded onto octadecyl silica (ODS) gel with a gradient solvent system of MeOH : H2O (from 0 : 10 to 10 : 0) and yielded compounds 1  and 2 .The fractionation of Q. serrata-7 (7.7 g) during repeated column chromatography using the MCI gel with a gradient solvent system of MeOH : H2O (from 0 : 10 to 10 : 0) yielded compound 3 .The fractionation of Q. serrata-9 (17.01 g) was loaded onto an MCI gel CHP 20P column with a gradient solvent system including MeOH : H2O (from 0 : 10 to 10 : 0) and produced 22 subfractions. Q. serrata-9-5 yielded four subfractions when loaded onto an ODS column with a gradient solvent system including MeOH : H2O (from 0 : 10 to 10 : 0). Column chromatography was repeated for Q. serrata-9-5-1 (1.55 g) using Sephadex LH-20 with a gradient solvent system including MeOH : H2O (from 0 : 10 to 10 : 0), yielding compounds 4 ((S)-2,3-HHDP-D-glucopyranoside, 21.3 mg) and 5 .Q. serrata extract were analyzed by HPLC. The mobile phase consisted of solvent A (0.2% acetic acid in H2O) and solvent B   . The Q. μL aliquot of each sample was dissolved in anhydrous ethanol and added to 180 μL of 0.2 mM DPPH  solution. The mixture was incubated for 37°C, 30 min, and the absorbance was measured at 517 nm using an enzyme-linked immunosorbent assay reader . The DPPH free radical scavenging activity was calculated in terms of percent inhibition (%). Half-maximal inhibitory concentration (IC50) values were defined as the concentrations that could scavenge 50% of the DPPH free radicals. L-ascorbic acid was used as the positive control. Inhibitory activity was calculated as follows:A DPPH radical scavenging assay was used to evaluate antioxidant activity. A 20 2) in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum , 100 IU/mL penicillin G, and 100 mg/mL streptomycin .RAW 264.7 cells were purchased from the Korean Cell Line Bank. The cells were grown at 37°C in a humidified atmosphere  was added to each well. After 4 h of incubation, the supernatant was aspirated. Finally, 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and the absorbance at 540 nm was measured using an ELISA reader . Relative cell viability was evaluated based on the quantity of MTT converted to the insoluble formazan salt. Distilled water served as the control sample. The optical density of the formazan generated in the control cells was considered to represent 100% viability. The data are expressed as the mean percentage of viable cells versus the control.The cytotoxicity of the test extract/compounds was determined prior to the biological assay based on the mitochondria-dependent reduction of 3--2,5-diphenyltetrazolium-bromide (MTT) (Sigma-Aldrich) to formazan. RAW 264.7 cells (2 × 105 cells/well) were seeded in a 96-well plate and incubated at 37°C for 16 h, treated with 20 2). The cells were then incubated in serum-free medium containing the test compound/extract and 1 μg/mL of lipopolysaccharide (LPS) (Sigma-Aldrich). After incubating for an additional 24 h, the NO content was evaluated using a Griess assay. For the Griess assay, 100 μL of Griess reagent (Sigma-Aldrich) was added to 100 μL of supernatant from the treated cells. The absorbance of the samples was recorded at 540 nm [RAW 264.7 murine macrophage cells (2 × 105 cells/well) were seeded in 96-well plates and pre-incubated for 16 h at 37°C in a humidified atmosphere , 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, and 1 mM ethylenediaminetetraacetic acid) . The cell lysates were centrifuged at 13,000 rpm for 15 min at 4°C, and the supernatants (protein: 60 μg) were electrophoresed using SDS-polyacrylamide gels (10% and 12%). The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane . The membrane was blocked using a blocking buffer (Thermo Fisher Scientific) for 30 min at room temperature. Then, the membrane was incubated overnight at 4°C with antibodies NLRP3 , ASC , and caspase-1 . After washing the membranes with Tris-buffered saline-Tween (TBS-T)  three times, they were incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary antibody  and m-IgGκ binding protein (BP)-HRP  for 1 h at room temperature. The bands were visualized using a LAS-500 luminescent image analyzer  after treating the membrane with ECL detection reagent .RAW 264.7 macrophage cells (1 × 106 cells/well) were pre-incubated for 16 h at 37°C and treated with 1 μg) and 4-phenylazobenzyloxycarbonyl-L-Pro-L-Les-Gly-L-Pro-D-Arg  (a substrate of collagenase) were incubated with or without samples in 0.1 M Tris buffer (pH 7.4) at 37°C for 30 min . To terminate the enzyme reaction, 1 mL of 25 mM citric acid solution was added. After mixing with 5 mL of ethyl acetate, the absorbance of the organic layer was measured at 320 nm. Epigallocatechin gallate (EGCG) was used as the positive control. The inhibitory activity was calculated as follows:∗O.D. control: O.D. of control with collagenase - O.D. of control without collagenase ∗O.D. sample: O.D. in the presence of test sample with collagenase - O.D. in the presence of test sample without collagenase.Collagenase assay was conducted using a previously described procedure . Collagep < 0.05.All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) and the Student–Newman–Keuls test were performed using the Statistical Package for Social Sciences (SPSS 24) software. The resulting values were considered significantly different at Q. serrata extract using Sephadex LH-20, MCI gel CHP–20P, and ODS-B gel yielded five compounds (1–5). These compounds were identified as caffeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5) based on instrumental analysis and comparisons with references (Q. serrata. 1H and 13C-NMR spectra of the compounds (1–5) are included in Supplementary Material as Figures Chromatographic analysis of the ferences . This is1 was a brown powder. A purple spot was detected by spraying the TLC plate with 10% H2SO4 followed by heating, and a black spot was detected using FeCl3.1 revealed ABX-type protons , 6.95 , 6.82 ) and two doublet signals with high J values, indicating trans-type protons , 6.21 ).The 1H-NMR spectrum of By comparing the spectral data with those from previous literature , the str2 was a brown powder. A black spot was detected using FeCl3, and a yellow spot was detected by spraying the TLC plate with 10% H2SO4 followed by heating.2 revealed a myricetin moiety , 6.34 , 6.16 ) and two anomeric proton signals  and 5.31 ).The 1H-NMR spectrum of 2 also showed myricetin moiety (δ 177.92 (C-4), 164.62 (C-7), 161.76 (C-5), 156.78 (C-9), 156.70 (C-2), 145.89 , 137.16 (C-4′), 134.00 (C-3), 120.56 (C-1′), 109.05 , 104.48 (C-10), 99.15 (C-6), and 93.88 (C-8)). The presence of a 3-O linkage was indicated by a 9.89 ppm downshift of C-2 (δ 156.70) and a 2.18 ppm upshift of C-3 (δ 134.00). A cellobiose moiety (δ 102.52 (C-1‴), 101.38 (C-1″), 78.13 (C-4″), 77.08 (C-5‴), 76.44 (C-3‴), 74.44 (C-2‴), 73.79 (C-3″), 71.72 (C-2″), 70.42 (C-5″), 68.52 (C-4‴), 61.59 (C-6‴), and 60.56 (C-6″)) was observed with a 9 ppm downshift of C-1”; this indicated that C-1″ has a linkage with the 3-hydroxyl group of myricetin.The 13C-NMR spectrum of By comparing the results with data from previous literature , 48, the3 was a brown powder. A grey spot was detected by spraying the TLC plate with 10% H2SO4 followed by heating, and a dark blue spot was detected using FeCl3.δ 6.87, and the 13C-NMR spectrum showed two signals  and 109.27 ).The 1H-NMR spectrum of 3 showed only one signal at By comparing our results with those from previous literature , the str2SO4 followed by heating, and a dark blue spot was detected using FeCl3.4 was a brown powder. A dark pink spot was detected by spraying the TLC plate with 10% H4 may have two isomers on an anomeric center on the sugar. In addition, the 1H-NMR spectrum of 4 revealed one hexahydroxydiphenoyl (HHDP) signal ) in the aromatic region and sugar protons existing as α and β forms in the sugar region. The 13C-NMR spectrum of 4 revealed four signals in the aromatic region indicating–COO  and included HHDP and two isomer sugar forms in the sugar region .The 1H-NMR of 4 showed duplicated signals below 5.34 ppm; thus, it is possible that By comparing these results to NMR data from previous literature , the str5 was a brown amorphous powder. A brown spot was detected by spraying the TLC plate with 10% H2SO4 followed by heating, and a dark blue spot was detected using FeCl3.δ 6.64, 6.63, 6.57, 6.57, 6.53, 6.48, 6.30, 6.29 ) in the aromatic region and a glucose core with 4C1 conformation (δ 3.74–5.45 (large coupling constants)) in the sugar region.Anomeric proton signals below 5.45 ppm were observed on the 1H-NMR spectrum of 5, and all signals were duplicated. Thus, 5 is thought to be a mixture of two isomers caused by unacylated anomeric centers on a sugar. The 1H-NMR spectrum also revealed two HHDP moieties  was evaluated using the DPPH radical scavenging activity. The DPPH free radical is deep purple in color and absorbs light at 517 nm. It loses electrons upon reacting with antioxidants, resulting in a reduced form, which is yellow in color. The reaction is thus monitored by measuring the absorbance at 517 nm.The antioxidant ability of the 50 of the test compounds was calculated by plotting the inhibitory activity (%) in the DPPH free radical scavenging assay against the concentration. A low IC50 value indicates a high antioxidant activity. The Q. serrata extract (IC50 = 65.84 ± 5.79 μg/mL) demonstrated superior radical scavenging activity compared with ascorbic acid (IC50 = 11.20 ± 5.97 μg/mL) (50 = 51.07 ± 1.47 μM). In particular, compound 5 (IC50 = 24.18 ± 1.06 μM) exhibited the strongest activity compared with ascorbic acid (The IC7 μg/mL) , Table 3bic acid , Table 4Q. serrata extract on cell viability and its cytotoxic activity were evaluated by the mitochondria-dependent reduction in MTT. When MTT is processed in living cells, it is reduced by the reductase in the mitochondria, forming formazan crystals. The formation of formazan indicates a lack of cytotoxicity.The effect of the Q. serrata extract at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL in RAW 264.7 cells. The cell viability was maintained at >80% at all concentrations of the extract. These results demonstrate that the inhibition of NO production and the production of inflammatory molecular products due to treatment with Q. serrata do not cause cytotoxicity  enzyme isoforms . NO regulates the cellular and toxic responses. However, excessive NO production is considered to enhance tumor development and DNA methylation. Thus, the inhibition of inflammatory response-related NO production might be a useful therapeutic and prophylactic method in arthritis.Q. serrata extract showed a potent inhibitory effect on NO production (IC50 = 6.32 ± 0.19 μg/mL); the activity was comparable to that of the positive control L-NMMA (IC50 = 5.10 ± 0.14 μg/mL) (4 (117.66 ± 2.63 μM) and compound 5 (111.79 ± 2.52 μM) showed good inhibition of NO production compared with that of the other compounds (The 4 μg/mL) , Table 5ompounds , Table 6Q. serrata extract and ellagitannin  on the expression of the components of the NLRP3 inflammasome  in RAW 264.7 cells were evaluated using Western blot analysis. The NLRP3 complex is considered important as a treatment target for inflammatory diseases because it controls inflammatory cytokine levels.The inhibitory effects of the Q. serrata extract was lower than that in the control group, indicating that the extract inhibited NLRP3 protein expression. Similarly, compound 5 also inhibited the expression of NLRP3, caspase-1, and ASC (level compared with that in the control group)  on collagenase activity were evaluated. The cartilage tissue associated with arthritis includes collagen and various proteins. Collagen plays an important role in joints, and collagenase is the key enzyme that cleaves and breaks down collagen.The inhibitory effects of the Q. serrata extract (99.68%) and compound 5 (57.52%) showed potent inhibitory activity against collagenase compared with the positive control, EGCG (85.30%), at 25 μM  is induced, and various chronic inflammatory diseases such as diabetes, inflammatory bowel disease, arthritis, and prostate hypertrophy  occur [Quercus spp. As a therapeutic agent for inflammatory diseases by evaluating the production of NO and proinflammatory cytokines [Q. serrata extract can potentially be applied for the treatment of inflammatory diseases such as arthritis treatment.We aim to evaluate an extract made from s) occur –42. Reces) occur –56. TherQ. serrata extract evaluated here, as well as compounds 1, 4, and 5 exhibited good antioxidant ability; the major component, compound 5, was most potent in this regard. In addition, the Q. serrata extract and compounds 4 and 5 exerted anti-inflammatory effects. Based on these results, we evaluated whether the Q. serrata extract and compound 5 were able to inhibit the expression of the components of the NLRP3 inflammasome. Both the extract and compound 5 suppressed the expression of the NLRP3 inflammasome components. Western blot analysis of the NLRP3 components revealed that the suppression of the protein expression of all the three components  by the extract and compound 5 was clearly concentration-dependent. The extract and compound 5 also showed potent antioxidant and anti-inflammatory abilities. Inhibition of collagenase activity is assessed to evaluate whether the test compound can potentially be applied for arthritis treatment. In our study, both the Q. serrata extract and compound 5 showed a potent collagenase inhibitory activity. There has been increased interest regarding the role of the inflammasome in arthritis. Increased secretion of NLRP3, ASC, and caspase-1 has been reported in arthritis patients, and suppression of NLRP3 activity was reported to suppress arthritis in rat models [Q. serrata extract and its component compounds. Our results showed that the QS extract and its component compounds may have therapeutic benefits towards inflammatory diseases such as arthritis.Our results in this study demonstrated that the t models . In addit models . On the t models . Based oQ. serrata, namely caffeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5). Content analysis showed that pedunculagin (5) was the main constituent of this extract. The antioxidant activity of the extract and the component compounds was evaluated by measuring DPPH radical scavenging activity. The Q. serrata extract and the isolated compounds (1–5) exhibited increased DPPH radical scavenging activity, and compound 5 performed the best in this assay. The anti-inflammatory activities were evaluated based on the inhibition of NO production. The Q. serrata extract and compounds 4 and 5 showed potential NO inhibitory activity. The Q. serrata extract and compound 5 potently inhibited the protein expression of the components of the NLRP3 inflammasome, including NLRP3, ASC, and caspase-1, further confirming their anti-inflammatory nature. The Q. serrata extract and compound 5 also showed potent collagenase inhibitory activity. The present results suggest that the Q. serrata extract and compound 5 are promising candidates for treating inflammatory diseases such as arthritis. However, further in vivo studies in arthritis animal models are required to confirm these findings.In this study, five compounds (1–5) were isolated from