Grifola frondosa (G. frondosa) have anti-obesity and anti-inflammatory activities. In this study, the major type, molecular weight, homogeneity and structure of a polysaccharide purified from G. frondosa (denoted GFPA) were determined. In high-fat diet (HFD)-treated mice, 8 weeks of GFPA administration efficiently decreased body weight and blood glucose concentration and counteracted hyperlipidemia. GFPA efficiently decreased adipocyte size and ameliorated inflammatory infiltration in the three types of white adipose tissue and alleviated steatosis, fat accumulation and inflammatory infiltration in the livers of HFD-fed mice. GFPA also decreased the concentrations of aspartate aminotransferase, alanine aminotransferase and pro-inflammatory factors in the sera and livers of HFD-treated mice. Furthermore, GFPA was found to regulate lipid metabolism via the inhibition of ceramide levels in HFD-treated mice. GFPA exhibited strong anti-obesity effects via the modulation of chronic inflammation through Toll-like receptor 4/nuclear factor kappa-B signaling, which supports the use of GFPA for the treatment of obesity.Polysaccharides from  Obesity is characterized by excessive weight gain and fat deposition. It has become a major threat to human health globally, affecting 51% of the world’s population .The link between chronic inflammation and obesity has been widely researched , 4. ChroMost patients with obesity show poor compliance with lifelong behavioral therapies, such as a balanced diet and physical exercise . The surGanoderma lucidum, which is a β-D-glucan containing (1→3)-β-D-Glcp, -β-D-Glcp, (1→6)-β-D-Glcp and terminal-β-D-Glcp moieties, suppresses obesity and inflammation by inhibiting Toll-like receptor 4 (TLR4)/myeloid differential protein-88 (Myd88)/NF-κB signaling in mice fed a high-fat diet (HFD) , have anti-obesogenic and anti-inflammatory effects through the regulation of the intestinal microbiome and inflammatory cascades in HFD-fed mice (Polysaccharides isolated from mushrooms mainly contain a β-linked glucose (Glc) backbone, with different patterns and degrees of branching in various species. These polysaccharides have anti-obesity activity . The polet (HFD) . Dictyopfed mice .Grifola frondosa (G. frondosa), which belongs to the Polyporaceae family, regulates lipid metabolism by inhibiting Cer, which further contributes to its anti-obesity effects in mice with diet-induced obesity (DIO)  . As one fed rats . One of r injury . HoweverG. frondosa (termed GFPA). The anti-obesity effects of GFPA were found to be related to the inhibition of Cer, via the downregulation of TLR4-mediated pro-inflammatory signaling cascades in mice with DIO. These results support the use of GFPA as an anti-obesity agent that targets chronic inflammation.In this study, we performed the structural characterization of a polysaccharide purified from G. frondosa fruiting bodies  were identified by Prof. Zhang Bo, Jilin Agricultural University, Changchun, China, and extracted twice with hot water at 80°C for 2 h. After concentrating the extracts, they were deproteinized with Sevag reagent  and precipitated with 80% alcohol. The resulting polysaccharides were further purified using a diethylaminoethyl cellulose-52 (DEAE-52) anion exchange column  and HiPrep™ 26/60 Sephacryl™ S-400 high-resolution  and EzLoad 26/60 Chromdex 200  prep grade columns. The collected polysaccharides (GFPA) were used in subsequent experiments  and ultraviolet (UV) spectra  of GFPA were determined as previously described .A high-performance anion-exchange chromatography system  equipped with an electrochemical detector (Dionex ICS 5000 system) and a Dionex™ CarboPac™ PA20 column  was used to determine the monosaccharide component of hydrolyzed GFPA. The following conditions were used: column temperature, 30°C; mobile phase A, 0.1 M NaOH; mobile phase B, 0.1 M NaOH and 0.2M NaAc; flow rate, 0.5 mL/min; injection volume, 5 μL and gradient elution , 95:5 at 0 min, 80:20 at 30 min, 60:40 at 30.1 min, 60:40 at 45 min, 95:5 at 45.1 min and 95:5 at 60 min .3; flow rate, 0.4 mL/min; injection volume, 100 μL and isocratic elution time, 100 min ; a laser light scattering detector  and a differential detector . The detection conditions were as follows: column temperature, 45°C; mobile phase, 0.1 M NaNO 100 min .Methylation analysis was performed by gas chromatography-mass spectrometry, using a previously published method with slight modifications, to determine the bonding structure of GFPA . A gas c2O several times and dissolved in D2O (0.5 mL) containing the internal standard trimethylsilylpropanoic acid. One- and two-dimensional nuclear magnetic resonance (NMR) spectra were recorded in Fourier transform mode using an NMR spectrometer  equipped with a 13C/1H dual probe . Lyophilized samples were affixed to a specimen holder with conductive tape and sputtered with gold in a vacuum sputter coater. The samples were then observed at an accelerating voltage of 0.02-30 kV .ad libitum access to food. Either an HFD  or a normal chow diet  was fed to the mice for 10 weeks. The DIO mice were fed an HFD ad libitum during the entire experimental period and were randomly divided into four groups (n = 6/group). They were then treated with intragastric administration of 5 mL/kg normal saline (vehicle-treated HFD-fed mice), 3 mg/kg SV (SV-treated HFD-fed mice) or GFPAs at 50 or 100 mg/kg (GFPA-treated HFD-fed mice) once a day for a further 8 weeks. SV, a commercially available statin drug, has use as the positive control drug in many literatures. Therefore, we choose SV as the positive control drug. According to the doses of G. frondosa usage in our previous study . Thirty male C57BL/6JGpt mice aged 5 weeks  were maintained at a temperature of 23 ± 1°C and a humidity of 40% to 60%, with a 12-h light/dark cycle and us study  and the As previously described , enzyme-As described in our previous study , three tLiver sections were incubated in 3.7 mM Oil red O solution at 25°C for 10 min and then incubated in hematoxylin at 25°C for 5 min. The stained sections were coated with malinol and observed under a microscope as previously described  containing 1% protease inhibitor cocktail  and 2% phenylmethanesulfonyl fluoride . After determining the protein concentration, 40 μg of protein was separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes . After blocking with 5% bovine serum albumin at 4°C for 4 h, the membranes were incubated with primary antibodies at 4°C for 12 h and subsequently incubated with the appropriate secondary antibodies at 4°C for 4 h Table S1post-hoc multiple comparisons (Holm-Sidak) test, was used to analyze the data using DSS Statistics 25 . Statistical significance was defined at a p value less than 0.05.All values are expressed as the mean ± standard error of the mean (S.E.M.). A one-way analysis of variance, followed by a Four polysaccharide fractions, named GFP1, GFP2, GFP3 and GFP4, were obtained using a DEAE-52 anion exchange column S1. Due -1) of GFPA is depicted in −1. A peak at 3,362.01 cm-1 corresponded to the O-H stretching vibration, which is a characteristic peak of sugars  in the 5.89 ppm –30.Using the NOESY atlas  (p < 0.05) (p < 0.01) (p < 0.05)  (p < 0.001) and the low concentration of HDL-C (p < 0.01) in the liver of HFD-fed mice were noted, but these were regulated by over 29.7%  (p < 0.01) (p < 0.01)  (p < 0.05) except serum HDL-C concentration (Significant increases in body weight and plasma Glc concentration ( < 0.05) Table S2p < 0.001) (p < 0.05) (p < 0.01) (p < 0.01)  (p < 0.05) (p < 0.01) (p < 0.05) (p < 0.05) (Significant increases in ALT (p < 0.05)  (p < 0.01) (p < 0.05) (p < 0.05) (p < 0.05)  (p < 0.01) (p < 0.01) (p < 0.05) (p < 0.01) (p < 0.01) (p < 0.01) (p < 0.01) (p < 0.05) except serum TLR4 concentration and hepatic TNF-α and IL-1β concentrations , MyD88 , TRAF6 , phosphorylated (p)-inhibitor of nuclear factor kappa-B kinase (IKK) (α+β) , p-inhibitor of nuclear factor kappa-B α , p-NF-κB (p < 0.05), TNF-α (p < 0.001), IL-6 (p < 0.01) and IL-1β (p < 0.001) in the livers of HFD-fed mice were significantly downregulated by GFPA treatment -α-D-Glcp backbone and a small number of terminal β-D-Galp residues, was isolated from G. frondosa .DW designed the experiments. XJ, JH, YFZ and ZJL performed the experiments. XJ processed the data. XJ wrote the paper. DW, LRT, LZL, YLZ and YL revised the paper. All authors contributed to the article and approved the submitted version.This work was supported by the Science and Technology Development Project in Jilin Province, China (20200708068YY and 20210401169YY), the National Key Project of “Science and Technology for Economy 2020” (2020YFF0414043), the Science and Technology Research Project from Education Department of Jilin Province in China (JJKH20211227KJ), and the China Agriculture Research System of MOF and MARA (CARS-20-08B).We thank Prof. Zhang Bo from the Jilin Agricultural University for the fungus appraisal.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Persicaria hydropiper L. leaves and evaluate their in vitro and in silico antidiabetic potentials.  Natural products are among the most useful sources for the discovery of new drugs against various diseases. Keeping in view the ethnobotanical relevance ethnopharmacological significance of Polygonaceae family in diabetes, the current study was designed to isolate pure compounds from 1H-NMR, 13C-NMR, and mass spectrometry techniques. Isolated phytochemicals were subjected to in vitro antidiabetic studies, including α-glucosidase, α-amylase inhibition, and DPPH, and ABTS antioxidant studies. Furthermore, the in silico binding mode of these compounds in the target enzymes was elucidated via MOE-Dock software.  Six compounds were isolated from the chloroform-ethyl acetate fractions using gravity column chromatography and were subjected to structure elucidation process. Structures were confirmed using α-glucosidase enzyme. Ph-1 and Ph-2 were most potent with 81.84 and 78.79% enzyme inhibitions at 1000 µg·mL−1, respectively. Ph-1 and Ph-2 exhibited IC50s of 85 and 170 µg·mL−1 correspondingly. Likewise, test compounds showed considerable α-amylase inhibitions with Ph-1 and Ph-2 being the most potent. Tested compounds exhibited considerable antioxidant potentials in both DPPH and ABTS assays. Molecular simulation studies also revealed top-ranked confirmations for the majority of the compounds in the target enzymes. Highest observed potent compound was Ph-1 with docking score of −12.4286 and formed eight hydrogen bonds and three H-pi linkages with the Asp 68, Phe 157, Phe 177, Asn 241, Glu 276, His 279, Phe 300, Glu 304, Ser 308, Pro 309, Phe 310, Asp 349, and Arg 439 residues of α-glucosidase binding packets. Asp 68, Glu 276, Asp 349, and Arg 439 formed polar bonds with the 3-ethyl-2-methylpentane moiety of the ligand.  The isolated compounds revealed concentration-dependent inhibitions against  The isolated compounds exhibited considerable antioxidant and inhibitory potentials against vital enzymes implicated in T2DM. The docking scores of the compounds revealed that they exhibit affinity for binding with target ligands. The enzyme inhibition and antioxidant potential of the compounds might contribute to the hypoglycemic effects of the plant and need further studies. It is asorption , 4. Inhisorption . This issorption , 7. ConsGalega officinalis L. from Fabaceae family was the first-ever medicinal plant with a clear antidiabetic profile and has a long history of use as a complementary antidiabetic therapy. Later, an important antidiabetic compound galegine was isolated from this plant that has almost similar structure with its synthetic counterpart metformin [Pharbitis nil L. and Ipomoea batatas L. significantly reduce postprandial hyperglycemia via inhibition of α-glucosidase enzyme [Pinus pinaster leads to the discovery of an important α-glucosidase and competitive α-amylase inhibitor pycnogenol [α-glucosidase and α-amylase inhibitors isolated from microbial origin [Preclinical and clinical antidiabetic studies on medicinal plants revealed their greater potential for drug discovery against diabetes. etformin , 9. Plane enzyme . Likewiscnogenol , 11. Addl origin . NumerouPersicaria hydropiper L. belong to family Polygonaceae (smartweed), which consist of about fifty genera and twelve hundred species [Antigonon leptopus Hook. & Arn., a member of Polygonaceae, is used in traditional medicine to treat hyperglycemia and is scientifically validated in animal models [P. hydropiper L. ethanolic extract revealed antidiabetic potentials in oral glucose tolerance test [Polygonum senegalensis, Muehlenbeckia tamnifolia (Kunth) Meisn, Rheum ribes L., Polygonum glabrum, and other species are validated for antidiabetic potentials [P. hydropiper exhibit neuroprotective, gstroprotective, cytotoxic, and antimicrobial properties [P. hydropiper and were subjected to enzyme inhibition and molecular docking studies for potential applications in T2DM. species . Polygonl models , 15. Prence test . Polygontentials –20. Our operties –29. BaseP. hydropiper was selected for antidiabetic studies. The plant was collected in 2013 from Talash (Dir KPK), after identification by plant taxonomist Dr. Gul Rahim. A dried sample of the same was secured in the herbarium of University of Malakand, Chakdara (Voucher no. H.UOM.BG.107). The study was approved by Departmental Research Ethics Committee via reference no. DREC/20160502/01. Some initial pilot studies were performed on various fractions of the plant including crude methanolic extract, chloroform, ethyl acetate, n-hexane, butanol, and aqueous fractions [n-hexane only (100%). After some time, we gradually increase the polarity with the addition of 2% ethyl acetate each time . We visualize the TLC each time and change the polarity accordingly. Thereafter, on the bases of fractions quantity, we selected large-size gravity columns, packed with flash silica slurry and loaded with the selected fractions. Elution was started from nonpolar n-hexane towards increase in polarity via addition of ethyl acetate. Partially pure fractions (approximately more than 80%) were collected, run on TLC plates to combined co-elution fractions based on Rf values and were further purified via silica-packed pencil columns. These columns were again carefully eluted with n-hexane and chloroform solvent systems for the purification of target compounds. The compounds Ph-1 and Ph-2 were isolated from chloroform fraction, while compounds Ph-3 to Ph-6 were isolated from ethyl acetate bioactive fraction. The compounds Ph-1, Ph-2, Ph-3, Ph-4, Ph-5, and Ph-6 were purified in 250, 275, 122, 145, 270, and 265 mg, respectively.Keeping in view the ethnobotanical importance of Polygonaceae family in diabetes, whole aerial plant of ractions . Based o1H NMR analysis was performed to confirm about the compound structure and compared it with the literature. Further, 13C NMR analysis was performed to identify the carbon-skeleton, whereas further confirmations were done via mass spectrometry [The purified compounds were subjected to the removal of trace solvents using rotary evaporator. Subsequently, trometry , 32.µL was mixed with same volume (100 µL) of test samples in 96-well plates, followed by incubation for about thirty min in a dark place 25 ± 3°C. Solutions of the provided samples were prepared in concentrations 1000-62.5 µg·mL−1. Likewise, Trolox methanolic solution (100 µM) was prepared and subsequently its five concentrations/dilutions 1000, 500, 250, 125, and 62.5 µM were prepared. Subsequent to incubation, a decline in DPPH color was observed and absorbance was recorded at 540 nm using microplate reader [DPPH antiradicals study was conducted in accordance with our previously published protocol , 34. Inie reader . PercentµL of 140 mM potassium persulphate and the mixture was left in the dark for 24 hours. Subsequently, 1 mL of the reaction mixture was diluted to 50 mL using methanol to obtain the final ABTS dilution used in the assay. Consequently, 190-microliter ABTS solution was added to 10 µL sample solutions and in 96-well plates and were incubated for 2 hrs in dark place at room temperature. After the incubation, decline in color intensity of ABTS was measured at 734 nm via microplate reader. Solutions of positive control were prepared in at the concentrations of 1000-62.5 µM. Percent ABTS scavenging was determined using equation:ABTS antiradicals' study was performed following our earlier reported protocol , 37. Forα-glucosidase inhibition assay, 50 µL solution containing 0.5 U·mL−1 was added to 50 µL phosphate buffer (6.8 pH) and was added to various concentrations of test samples. The resultant mixture was properly mixed and stored at 37°C for fifteen min. After incubation, 100 µL (3 mM) pNPG solution was added to it. The reaction mixture was again incubated for 10 min at 37°C and finally 0.1 M Na2CO3 was added to stop reaction [For reaction . AbsorptµL of 1% starch solution prepared in 29 mM phosphate buffer (pH 6.9) was mixed with 100 µL of sample solution (1000-62.5 µg·mL−1) and stored for about 10 min at 25°C in air-tight tubes. Subsequently, 100 µL of 0.5 mg·mL−1α-amylase solution was added to the mixture and again incubated for 10 min at 25°C. Reaction was stopped via the addition of 200 µL of dinitrosalicylic acid was added and incubated for an additional 5 min in boiling water bath and allowed to cool again. Finally, 50 µL solutions was transferred to 96-well microplate reader and further diluted to 200 µL via the addition of distilled water. Absorbencies were recorded at 540 nm and percent inhibitions were calculated using the equationUsing this assay, a previously reported protocol was followed . In brieIn silico studies involving the interactions of test drugs with its molecular targets are an important tool in drug discovery [α-glucosidase and α-amylase enzymes. As the α-glucosidase enzyme crystal structure is available, so previously reported homology model [α-amylase enzyme (4W93) was obtained from Protein Data Bank. Prior to molecular docking, the crystal structures were devoid of water molecules or any ions via Molecular Operating Environment software. Using 3D protonation, hydrogen atoms were added to the proteins and the energy was minimized via MOE parameters .iscovery , 40. MOEgy model  was usedStructures of our test samples were initially built using MOE and the energy was reduced. Subsequently, both enzymes were allowed to interact and dock with test samples through MOE preset parameter including placement at Triangle Matcher and rescoring with London dG. Ten confirmations were generated for every compound and top-rank confirmations of the compounds were selected for further analysis. Following molecular docking, the best poses with polar, arene-arene, H-pi, and pi-H interactions were assessed by PyMOL software.All tests were done three times and results were presented as Mean ± SEM of the collected data. One-way ANOVA coupled with multiple-comparison Dunnett's test was applied for the statistical assessment of the test samples to the control groups.1H-NMR, 13C-NMR as well as mass-spectrometry assessments. Compounds spectral data is available as supporting information.In this research, we have isolated six bioactive compounds (Ph-1 to Ph-6) as shown in The compound Ph-1 was isolated as pure white solid. The molecular weight was confirmed as 414 by mass analysis. The compound Ph-2 was also isolated in white solid form. The observed molecular weight of Ph-2 was 212. In both of these steroidal compounds, majority of the protons are giving splitting in the upfield region due to the presence of aliphatic skeleton. The splitting of protons associated with the double bond or the hydroxy group is showing up in the downfield area. In both steroidal compounds, the methyl groups are majorly showing up signals between chemical shifts 1-2. The methylene and methyne unit are slightly downfield as compared to the methyl groups. The hydroxyl proton in both of the compounds is showing up between chemical shifts 3 and 3.5. Similarly, the alkene protons appeared in the downfield area of 5.2. We have previously reported both Ph-1 and Ph-2 with anti-Alzheimer and anti-cancer activities , 42.The compound Ph-3 is a furan-type derivative isolated as yellowish semisolid form. This derivative contains dominant groups like ester, ketone, and methyl. The compound spectra are shown in the supporting information. The methyl group attached to the carbonyl is giving up a singlet in around 2 chemical shifts. The second methyl group attached to the furan moiety is giving up splitting at upfield area of three protons. Similarly, all the ten protons in the structure are shown in the proton NMR. The compound Ph-4, a phenolic type of derivative, was isolated as brownish oil. The two distinct methoxy groups (each with three protons) appeared slightly upfield to chemical shift 4. Similarly, the phenolic hydroxyl group appeared as a singlet at 5.18 chemical shift. The three aromatic protons appeared in the range of 7.15 to 7.30. The molecular weight of compound Ph-4 was confirmed as 182 by mass analysis as shown in the spectrum in supporting information. The compound Ph-5 was isolated in small amount as turbid semisolid. The ethyl substituent gave two splittings . The triplet appeared at 1.08 while the quartet, due to the phenoxy attachment, appeared at 4.63. The methoxy group appeared at 3.95. The methyl group appeared as a singlet at 2.10. The aromatic protons appeared in the downfield region of 7.26 to 7.81. The compound Ph-6 appeared as dominant spot on TLC plate due to the presence of aromatic-conjugated system. The methoxy group with three protons appeared at 3.85. Two singlets (each of one proton) appeared at 5.28 and 5.82. A multiplet of two protons was also observed at 6.50 to 6.60. The phenyl group appeared in the range of 7.24 to 7.50 as multiplets of five protons.µg·mL−1 showed 17.51, 39.49, 51.38, 66.28, 70.62, and 74.19% scavenging effect, respectively. The IC50 value for Ph-1 was 140 µg·mL−1. Likewise, percent scavenging potentials of Ph-2 was observed to be 11.83, 21.33, 34.16, 59.66, 68, and 79.33 at the same tested concentrations correspondingly with IC50 of 200 µg·mL−1. Other compounds were comparatively less potent and exhibited a concentration dependent scavenging effect of 9.2, 18.5, 29.66, 33.33, 42, and 56, respectively, by Ph-3 at the same tested concentrations. Furthermore, Ph-4 showed percent antiradicals potentials of 7.6 (31.25 µg·mL−1), 12.06 (62.50 µg·mL−1), 19.85 (125 µg·mL−1), 27.48 (250 µg·mL−1), 48.73 (500 µg·mL−1), and 60.26 (1000 µg·mL−1) correspondingly, whereas the highest inhibitory activities of Ph-5 and Ph-6 were 58.33 and 44% at 1000 µg·mL−1 correspondingly. IC50 µg·mL−1 for Ph-3 (550 µg·mL−1), Ph-4 (650 µg·mL−1), Ph-5 (390 µg·mL−1), and Ph-6 (910 µg·mL−1) was calculated. Positive control (ascorbic acid) showed 45, 56.83, 62.33, 72.85, 83.33, and 91.80% free radicals scavenging potentials at 31.25, 62.50, 125, 250, 500, and 1000 µg·mL−1 correspondingly and an IC50 of 40 µg·mL−1.In DPPH antiradicals assay, Ph-1 exhibited considerable scavenging effect against DPPH radicals . Ph-1 atµg·mL−1 showed 83.44, 75.22, 68.47, 47.34, 32.58, and 25.25% inhibitions, respectively, against ABTS radicals. The IC50 value was 120 µg·mL−1 at the tested concentrations. Likewise, Ph-2 was comparably effective against ABTS radicals with percent inhibitions of 80.63, 72.68, 60.56, 42.89, 27.88, and 17.44, respectively, at the same concentrations. The IC50 of Ph-2 against ABTS radicals was 160 µg·mL1. The percent inhibitions of Ph-1 and Ph-2 were very comparable with the positive control which showed 94% inhibition at the highest tested concentration (1000 µg·mL−1) and an IC50 of 20 µg·mL−1 (µg·mL−1) with IC50 of 320 µg·mL−1. Furthermore, Ph-4 (53.66%), Ph-5 (69.16%), and Ph-6 (61%) inhibitions were observed at 1000 µg·mL−1, respectively, whereas the lowest concentrations were less effective , Ph-5 (350 µg·mL−1), and Ph-6 (550 µg·mL−1).In this assay, Ph-1 exhibited considerable free radicals scavenging effect. Ph-1 at the tested concentrations of 1000, 500, 250, 125, 62.5, and 31.25  µg·mL−1 . Other cffective . The IC5α-glucosidase inhibition studies are summarized in α-glucosidase enzyme. Compounds Ph-1 and Ph-2 were most potent with 81.84 ± 0.32 and 78.79 ± 0.63% enzyme inhibitions at 1000 µg·mL−1, respectively. Ph-1 and Ph-2 exhibited IC50s of 85 and 170 µg·mL−1 correspondingly. Inhibitory results of both compounds were very comparable with positive control group. Among the other samples, Ph-3, Ph-4, Ph-5, and Ph-6 exhibited 55.44 ± 0.09, 64.36 ± 0.61, 67.37 ± 0.68, and 58. 91 ± 1.54% enzyme inhibition at maximum used concentration of 1000 µg·mL−1, respectively. The IC50 values for Ph-3, Ph-4, Ph-5, and Ph-6 were 474.83, 455.94, 228, and 337.94 µg·mL−1, respectively.Results of µg·mL−1, respectively. Likewise, Ph-2 displayed 71.62, 63.86, 44.48, 37.54, and 31.74% inhibitions at the same tested concentrations, respectively. IC50s for Ph-1, Ph-2 were 69.48 and 252.01 µg·mL−1 correspondingly as shown in 50 of 45 µg·mL−1. Among the other compounds, Ph-3, Ph-4, Ph-5, and Ph-6 exhibited 57.33, 72.51, 64.23, and 61.33% inhibitions, respectively, at 1000 µg·mL−1. The IC50 values for these compounds were Ph-3 (332.19 µg·mL−1), Ph-4 (333.25 µg·mL−1), Ph-5 (370 µg·mL−1), and Ph-6 (271.24 µg·mL−1), respectively.In this study, Ph-1 and Ph-2 exhibited considerable enzyme inhibitory potentials in comparison to standard drug. Percent enzyme inhibitions observed for Ph-1 were 75.83, 69.54, 64.36, 55.84, and 48.8 at the tested concentrations of 1000, 500, 250, 125, and 62.50 α-glucosidase and α-amylase is among the most useful strategies to reduce postprandial hyperglycemia in T2DM patients. Natural products are proved to be among the vital sources for drug discovery against DM. Numerous plants have been proved to act as antihyperglycemic agents alone or as complementary therapies with other antidiabetic agents. Medicinal plants and their fruits/seeds including cinnamon, aloe, gymnema, bitter melon, coffee, guava, cocoa, green tea, nettle, garlic, sage, caper, soybean, turmeric, yerba mate, and walnut have got tremendous focus and have scientifically proved antidiabetic potentials. Several isolated compounds from natural origin including phenolics, galegine, pycnogenol, miglitol, voglibose, and acarbose are extensively employed for the management of T2DM [Inhibition of  of T2DM . In the α-glucosidase enzyme and majority of the samples were properly accommodated in the enzyme active site as summarized in α-glucosidase as shown in As obvious from the results in We found Ph-2 as another highly potential compound with docking score of −11.9020. The compound showed five polar interactions with the active residues of the target enzyme . It was in silico results in comparison to positive control (docking score = −5.3602). The control, acarbose, makes seven hydrogen bonds with the active site residues . Ph-1 exhibited docking score of -6.9473 and the test compound produced eight H-bonds at the enzyme active sites. Ph-1, showing that the hydroxyl group interacts with His 101 and Tyr 62 and forms H-acceptor contacts while with the Asp 197 residue a H-donor interaction was observed. Asp 197 showed H-donor interaction with the carbon atom of the cyclohexanol moiety of the ligand. The carbon atoms of the 1-(5-ethyl-6-methylheptan-2-yl)-7a-methyloctahydro-1H-indene moiety of the inhibitor contacts with the Glu 240 and Asp 300 via hydrogen bonding. Inhibitory potential of the compound can be attributed to the presence of electron donating group (-OH) as well the electronic-cloud system might be implicated in the high enzyme inhibitory property of the test compound.Our docking results against α-amylase drug target as shown in α-amylase.Among the series of six isolated compounds, the second one good inhibitor was compound Ph-2 (docking score = −6.6032) according to the docking scores . The comP. hydropiper followed by identification via different techniques. The compounds were tested using antidiabetic and antioxidant studies. All tested compounds exhibited considerable antioxidant (DPPH/ABTS) and enzymes inhibition potentials. Among the tested compounds, Ph-1 and Ph-2 showed highest activity against free radicals and target enzymes. Antioxidant and α-glucosidase as well as α-amylase enzymes might be important in the antidiabetic potentials of the samples. The binding affinities of the test samples were further confirmed by their interactions at the active sites of the enzyme and low binding energies during the molecular docking studies.In an activity-guided isolation process, six compounds were isolated from the most active fractions of


Specifications for the HTIP chelating ligand were successfully determined using many analytical techniques, 13C–NMR, 1H–NMR, FTIR, EDX, and GC–MS analyses, which assured a reasonable synthesis of the HTIP ligand. The ability of HTIP to retain U(VI) ions was investigated. The optimum experimental factors, pH value, experimental time, initial U(VI) ion concentration, HTIP dosage, ambient temperature, and eluents, were attained with solvent extraction techniques. The utmost retention capacity of HTIP/CHCl3 was 247.5 mg/g; it was achieved at pH = 3.0, 25 °C, with 30 min of shaking and 0.99 × 10−3 mol/L. From the stoichiometric calculations, approximately 1.5 hydrogen atoms are released during the extraction at pH 3.0, and 4.0 moles of HTIP ligand were responsible for chelation of one mole of uranyl ions. According to kinetic studies, the pseudo–first order model accurately predicted the kinetics of U(VI) extraction by HTIP ligand with a retention power of 245.47 mg/g. The thermodynamic parameters ΔS°, ΔH°, and ΔG° were also calculated; the extraction process was predicted as an exothermic, spontaneous, and advantageous extraction at low temperatures. As the temperature increased, the value of ∆G° increased. The elution of uranium ions from the loaded HTIP/CHCl3 was achieved using 2.0 mol of H2SO4 with a 99.0% efficiency rate. Finally, the extended variables were used to obtain a uranium concentrate  with a uranium grade of 69.93% and purity of 93.24%.A new synthetic chelating N–hydroxy–N–trioctyl iminophosphorane (HTIP) was prepared through the reaction of trioctylphosphine oxide (TOPO) with N–hydroxylamine hydrochloride in the presence of a Lewis acid (AlCl The results of the entire investigation indicated that the HTIP ligand could be synthesized successfully. Gas chromatography–mass spectrometry (GC–MS) is a powerful and effective tool for predicting chemical formulas and purity; the more stable fragment, [m/z]1H–NMR analysis with a 500.15 MHZ energy and CDCl3 as a diluent is a useful and efficient technology that provides important information about protons in the produced substance and aids in structure estimates. The primary δ (ppm) assignments were 7.259, 0.815–0.842, and 1.222–1.637 ppm, which corresponded to the protons of OH, methyl, and methylene, respectively. The assignments of the OH proton (δ = 7.259 ppm) were more deshielded than the assignments of the –CH2 and –CH3 protons, whereas –CH2 (δ = 1.222–1.637 ppm) was more deshielded than –CH3 (δ = 0.815–0.842 ppm). 1H–NMR characterization of the HTIP chelating ligand.13C–NMR analysis with a 125.76 MHZ energy and CDCl3 as a diluent is a useful method for determining the number of carbon atoms in an HTIP ligand. The major δ (ppm), which is connected to alkyl carbon, occurred at 14.126–31.848 ppm  complex by EDX spectrum is presented in 22+, UO2(OH)+, and (UO2)2(OH)22+, whilst UO2SO4, UO3.2H2O, UO2(OH)3–, and UO2(OH)42– are neutral and anionic species that are identified till pH 12.0.The role of pH was very important in the retention of U(VI) on the ligand’s active sites because it impacted the form of uranium in aqueous media as well as the features of the active sites of the HTIP ligand. −4 mol/L U(VI) solution (100 mg/L) and 25.0 mL of 0.99 × 10−4 mol/L HTIP/CHCl3 (10 mg of HTIP in 25.0 mL CHCl3) at pH ranges from 0.25–6.0 and room temperature for 30 min. eq = 12.5 mg/g) to pH 3.0 (eq = 247.5 mg/g) and remained constant up to pH 6.0. It was noticed that in highly acidic medium, pH 0.25–1.0, there was a small variation in HTIP retention due to the high conc. of hydrogen ions, which may compete with uranyl ions and protonated HTIP ligand. The ultimate retention of U(VI) was detected at pH 3.0 (eq = 247.5 mg/g) because UO22+, UO2(OH)+, and (UO2)2(OH)22+ species are predominant in this range. Therefore, pH 3.0 has been proposed as the best pH value for U(VI) ion retention on HTIP/CHCl3, with eq = 247.5 mg/g retention capacity (99.0%). A graph of logD versus pH shows a straight line with a slope of 1.5 and an intersection of 2.7157 in linear regression analysis , as displayed in The retention of U(VI) on HTIP was examined using 25.0 mL of a 4.2 × 10Lastly, based on the preceding analysis, the suggested complex structure and chelation mechanism are presented in −4 mol/L (10 mg/25.0 mL) HTIP/CHCl3 and a 25.0 mL aqueous uranium(VI) ion solution with a concentration of 0.42 ×10−3 mol/L at pH 3.0. U(VI) ion retention rose with rising equilibrium time and reached an ultimate value  after 30 min, which remained roughly constant for the next 120 min, as shown in Contact time is one of the highly essential factors on the financial side. The effect of equilibrium time on U(VI) retention was examined using 9.96 × 103, providing crucial information for the design and modeling of the extraction process. Both pseudo first order (PFO) and second order (PSO) kinetic models were employed to identify the mechanism of U(VI) ion entrapment using HTIP/CHCl3 and the rate constant of the process. The PFO model is presented by the equation below [eq and tq (mg/g) correspond to the quantity of U(VI) ions entrapped per unit mass at equilibrium and time t, respectively, and k1 (min−1) denotes the rate constant. e − qtq) against t; the values of both k1 and eq were calculated from the slope and intercept, respectively. The calculated value of eq was 245.47 mg/g, and of k1, 0.1648 min−1, with R2 = 0.9586. It is obvious that the calculated value of eq was extremely close to the practical retention capacity of 247.5 mg/g (k2 (g/mg.min) is in agreement with the rate constant. tt/q vs. t; it has a slope of 1/eq and an intercept of 1/k2qe2. The PSO model was found to be ineffective in explaining the practical data. eq as 277.47 mg/g, which was somewhat greater than the experimental retention capacity, whereas the value of k2 was 0.001 min−1 with R2 equal to 0.9991. Therefore, the PFO kinetic model was more reliable in characterizing the extraction process of U(VI) ions using HTIP/CHCl3, as it was appropriate for the experimental data.Kinetic studies were used to express the rate of uranyl ion entrapment using HTIP/CHClon below :(5)log(q7.5 mg/g . Nonethe3 is 247.5 mg/g (99.0%).The impact of the initial concentration of U(VI) ions on extraction efficiency is crucial to investigate since it helps us to anticipate retention power. A diagram of retention power against different initial U(VI) concentrations is shown in 3 on U(VI) retention efficiency was investigated. 3 improved U(VI) ion retention efficiency, followed by retention diminishing from 0.025 g to 0.1 g/25.0 mL CHCl3 because the number of HTIP incorporation sites surpassed the number of uranyl ions. The retention capacity of 0.01 g/25.0 mL HTIP/CHCl3 was 247.5 mg/g with a 99.0% removal efficiency. As a consequence, the best concentration for consequent extraction studies was 0.01 g/25.0 mL HTIP/CHCl3. It is obvious that the retention efficiency of HTIP is higher than that of other materials reported in earlier studies, as shown in The HTIP amount is important for better uranyl ion sequestration because it impacts the equilibrium of the system. The effect of HTIP doses varying from 0.0025 to 0.1 g/25.0 mL CHClR2 = 0.964 when plotting logD versus logHTIP. The linear regression analysis  can elaborate the stoichiometry mechanism between HTIP chelating ligand and U(VI) ions, which suggests that 1.0 mole of U(VI) ions is chelated by 4.0 moles of HTIP.The regression analysis was studied to guarantee the configuration of the HTIP–U(VI) complex formed. −4 mol/L, HTIP/CHCl3 conc. 0.99 × 10−3 mol/L (10 mg/25.0 mL), 3.0 pH value, and A/O phase ratio 1:1 for 30 min. It was observed that two theoretical extraction phases are required to extract nearly all of the U(VI) ions. Furthermore, the elution of U(VI) ions from the HTIP–U(VI) complex was conducted using 2.0 M of H2SO4. The HTIP/CHCl3 organic phase had a concentration of U(VI) ions of 95.0 mg/L. 3 was investigated by mixing 25.0 mL of 0.99 × 10−4 mol/L (10 mg/25.0 mL) HTIP/CHCl3 and 25.0 mL of uranium(VI) ion solution with a concentration of 4.2 × 10−4 mol/L (100 mg/L) for 30 min at pH 3.0 and agitation temperature varying from 25–65 °C. The uptake efficiency of HTIP/CHCl3 for U(VI) ions dropped from 247.5 mg/g to 200 mg/g when the temperature was elevated from 25 to 65 °C,  ions using HTIP/CHClo 65 °C, a. The paG° (kJ/mol) points to Gibbs free energy, ∆H° (kJ/mol) is attributed to enthalpy change, ∆S° (J/mol.K) is defined as entropy change, T (K) stands for the temperature, and R (8.314 J/mol.K) corresponds to the universal gas constant. dK versus 1/T graph with R2 = 0.9974; ∆H° and ∆S° were generated by the slope and intercept, respectively.Thermodynamic analysis was also carried out to characterize the extraction mechanism using the Gibbs free energy formula ,83,84:(7G° is negative implies that the retention of U(VI) on HTIP is thermodynamically spontaneous and attainable  ion retention on HTIP is an exothermic process, suggesting that heat is produced during the extraction. Finally, the negative value of ∆S° affirms that the capture of U(VI) ions on HTIP is more practicable and less disorganized.The fact that ∆tainable . In addiEa) of entrapped U(VI) ions at various temperatures using the slope of the straight line in aE (kJ/mol) corresponds to the activation energy of extraction and A correlates with the pre-exponential factor, which is independent of temperature. According to calculations, the extraction of U(VI) ions on HTIP ligand requires an activation energy of −8.261 kJ/mol; this implies that the extraction procedure occurs spontaneously and exothermically at room temperature, with no need for activation energy.The Arrhenius equation was employed to predict the activation energy (−4 mol/L (100 mg/L) U(VI) ion solution. Each ion was identified as an interfering ion when the extraction efficiency differed by more than ±5.0%. Thus, the co-ion concentration that produced a ±5.0% error in uranyl extraction efficiency was used to set the tolerance limit. The investigated co-ions were identified as co-ions accompanied by U(VI) during the leaching process. The impact of co-existing ions was examined separately under optimal extraction conditions by introducing each one into 25.0 mL of a 4.2 × 103, or 2.0 M H2SO4. Subsequently, the eluted solution was precipitated with 30.0% NaOH solution until pH 7.0–8.0 was attained, where uranium precipitated as sodium diuranate (Na2U2O7), yielding a final uranium concentrate (yellow cake). The precipitate was left to settle for 24.0 h and filtered. Lastly, the precipitate was dried for 3.0 h at 110 °C in an electrical oven to obtain the uranium concentrate as a final product.Three types of acids with different concentrations ranging from 0.025–2.0 M were utilized as stripping agents for stripping of U(VI) ions from the HTIP–U(VI) complex; the experiments were carried out using 10.0 mL acid volume for 25.0 mL of HTIP–U(VI) complex, shaking well for 10 min at 25 °C. According to the data listed in 3 (0.99 × 10−3 mol/L) with 1.0 L of leach liquor under the previously defined optimum conditions . According to the study, the extraction efficiency of U(VI) reached 99.0%. In addition, it was affirmed that the released U(VI) ions from the HTIP–U(VI) complex could be easily eluted in 10 min using 2.0 M H2SO4.The previous data suggest that the HTIP chelating ligand can extract uranium from leachates of geological ores. Accordingly, this hypothesis was investigated using G. Gattar granite leach liquor ,86. Uran2U2O7 by adjusting the pH to 7.0–8.0. The uranium concentrate was characterized using XRD and EDX, in addition to ICP-OES analysis techniques in order to determine the U(VI) content alongside other associated metal ions. The results are given in After the elution process, 30.0% NaOH was used to precipitate U(VI) ions as sodium diuranate precipitate Na1H–NMR, 13C–NMR, FTIR, EDX, and GC–MS analyses. The uranium sequestration procedures were optimized by mixing 25.0 mL of U(VI) ion solution containing 0.42 × 10−4 mol/L with 0.99 × 10−3 mol/L HTIP/CHCl3 at pH 3.0, 1:1 A/O phase ratio, at 25 °C for 30 min. The utmost retention capacity of HTIP/CHCl3 was 247.5 mg/g. From the stoichiometric calculations, approximately 1.5 hydrogen atoms were released during the extraction at pH 3.0, and 4.0 moles of HTIP ligand were responsible for chelation of 1 mole of uranyl ions. The kinetic modeling data were well-suited to the pseudo first-order model. Furthermore, thermodynamic study demonstrated a negative ΔS° value, indicating that the uptake process is less disordered. Moreover, the rise in ΔG° value pointed to the spontaneousness and possibility of removing U(VI) ions at low temperatures. The elution of uranium loaded on HTIP was achieved using 2.0 M of H2SO4 as uranyl sulfate with 99.0% efficiency. Lastly, uranium concentrate  with a purity of 93.24% was obtained by adding 30.0% NaOH to the elution and adjusting the pH to 7.0–8.0 with continuous stirring for 2 h.A novel synthetic chelating agent, N–hydroxy–N–trioctyl–iminophosphorane (HTIP), was synthesized using an effective substitutional technique compared to the conventional methods and was utilized to uptake U(VI) ions from leach liquor of G. Gattar, North Eastern Desert, Egypt. Characterization was performed successfully using numerous analytical techniques, including


Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco ( Hanke. Flavodoxin genes were amplified by PCR with overhangs and cloned into pET14b vector using NEB HiFi DNA assembly master mix (New England Biolabs). The IsiB gene from B49/hNPR . After aE. coli generated from a DNA shuffling library using CYP2A5, CYP2A6, and CYP2A13 isoforms . The Ubi.U4 promoter was amplified from N. tabacum genomic DNA. After assembly, colony PCR positive clones were confirmed by sequencing. pEAQ-HT vectors with AroG*, AtFd1, IsiB6803, HsCPR62-241 and HsCPRfl inserts were synthesized by Twist Bioscience . Nucleotide sequences of the genes, promoters and terminators used are given in the supplementary materials  or 50 µg mL-1 kanamycin (pEAQ) and grown with shaking (200 rpm) for 2 d at 28°C. Cells were harvested and suspended in infiltration buffer  to the desired OD and incubated at RT with gentle agitation for 2 h. Leaves of 4-6 week old greenhouse-grown tobacco plants were infiltrated on the abaxial leaf surface. Plants were allowed to dry before returning to greenhouse.Colonies of N. benthamiana plants 3-5 days post inoculation (dpi), snap frozen in liquid N2 and homogenized using a Retsch Tissue-Lyzer mixer mill . The homogenized material was extracted with 0.5 mL 80% methanol for 30 min at 4°C. Samples were centrifuged  and supernatant diluted 4-fold into milliQ water containing 1 mg L-1 Leu-enkephalin as internal standard. LC-MS/MS was performed on a Dionex UltiMate 3000 Quaternary Rapid Separation UHPLC+ focused system  using a Kinetex XB-C18 column . Formic acid (0.05% v/v) in water and acetonitrile (supplied with 0.05% v/v formic acid) were employed as mobile phases A and B, respectively. Gradient conditions were as follows: 0.0-0.5 min 5% B; 0.5−.0 17.5 min 5−45% B, 17.5-23.0 min 45-75% B, 23.0-25.0 min 75-100% B, 25.0−26.95 min 100% B, 26.95-27.0 min 100−5% B, and 27.0−30.0 min 5% B. The flow rate of the mobile phase was 300 μL min-1. The column temperature was maintained at 30°C. UV spectra for each sample were acquired at 214, 260, 280, and 300 nm. The UHPLC was coupled to a Compact micrOTOF-Q mass spectrometer  equipped with an electrospray ion source (ESI) operated in positive or negative ion mode. The ion spray voltage was maintained at +4500 V and -3900 V in positive and negative ion mode, respectively. Dry temperature was set to 250°C, and nitrogen was used as the dry gas (8 L min-1), nebulizing gas (2.5 bar), and collision gas in both ion modes. Collision energy was set to 10 eV and 15 eV in positive and negative ion mode, respectively. MS spectra were acquired with a sampling rate of 2 Hz in an m/z range from 50 to 1000 and MS/MS spectra in a range of m/z 100-800 in positive ion mode while in negative ion mode MS spectra were acquired with a sampling rate of 3 Hz in an m/z range from 50 to 1400 and MS/MS spectra in a m/z range from 100-1000. Na-formate clusters were used for mass calibration in both ion modes. Positive ion mode was used for untargeted metabolomics and negative ion mode was used to detect indican. Indole did not provide detectable MS signal and was instead quantified using its UV absorbance at 214 nm against a standard curve containing 0.75-100 µM indole in 20% methanol.Samples (80-120 mg) were taken from https://mona.fiehnlab.ucdavis.edu/) using MS1 and MS2 tolerances of 0.01 Da and 0.025 Da, respectively. After alignment, data was normalized to the peak area of the Leu-Enk internal standard. PCA analysis was performed on log10-transformed and auto-scaled data using only reference matched features. Normalized peak areas of compounds of interest were extracted directly from MS-DIAL and plotted using GraphPad Prism.Data analysis was performed using MS-DIAL . A MS1 t-1p-hydroxybenzaldehyde as internal standard and subjected to LC-MS analysis. Chromatography was performed on a 1290 Infinity II UHPLC system (Agilent Technologies), using a Kinetex XB-C18 column . Formic acid  in water and acetonitrile (supplied with 0.05% v/v formic acid) were employed as mobile phases A and B, respectively. The elution profile for indican was: 0.0-2.5 min, 5-65% B; 2.5-3.0 min 65-100% B, 3.0-4.0 min 100% B, 4.0-4.1 min, 100-5% B and 4.1-5.0 min 5% B. The mobile phase flow rate was 400 µL min-1. The column temperature was maintained at 40°C. The liquid chromatography was coupled to an Ultivo Triplequadrupole mass spectrometer (Agilent Technologies) equipped with a Jetstream electrospray ion source (ESI) operated in negative ion mode. The ion spray voltage was set to -3000 V. Dry gas temperature was set to 325°C and dry gas flow to 9 L min-1. Sheath gas temperature was set to 325°C and sheath gas flow to 12 L min-1. Nebulizing gas was set to 40 psi. Nitrogen was used as dry gas, nebulizing gas and collision gas. Multiple reaction monitoring (MRM) was used to monitor precursor ion → fragment ion transitions  at a concentration of 10 µg*mL-1 either directly or after 10-fold dilution with water and analyzed by LC-MS. The analysis was performed as described in  equipped with an electrospray ionization source (ESI). Instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at 3000 V in positive ion mode. Cone temperature was set to 300°C and cone gas flow to 20 psi. Heated probe temperature was set to 400°C and probe gas flow set to 50 psi. Nebulizing gas was set to 60 psi and collision gas to 1.6 mTorr. Nitrogen was used as both cone gas and nebulizing gas and argon as collision gas. Analyte precursor ion → fragment ion transitions were monitored by MRM, with transitions chosen as described by Jander et al., with additions from Docimo et al. for Arg and Lys  with ribed in  with cha and Lys . Both Q1r of 1.0 .E. coli DH5α after inducing cells with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD=1.0 and growing the cells overnight at 37°C with 200 rpm shaking. Purification was performed essentially as previously described (E. coli BL21 (DE3) after inducing cells with 1 mM IPTG at OD=0.5-0.6 and growing the cells overnight at 30°C (IsiB6803) or 25°C (HsCPR62-241). Cells were and harvested by centrifugation  and stored at -20°C. Cells were lysed by incubation for 30 min on ice with stirring in lysis buffer  before sonicating for 15 cycles  using a Branson Sonifier 450 sonicator. Lysates were clarified by centrifugation  followed by filtering at 0.45 µm. Purification was performed using a Cytiva HisTrap HP column at 1 mL min-1 on an ÄKTA Start purification system (Cytiva). The column was washed  prior to eluting with a linear gradient of 5-250 mM imidazole over 30 column volumes. Yellow fractions were pooled, desalted into 50 mM Tris-HCl (pH 7.5) and checked for purity using SDS-PAGE.AtFd1 was expressed in escribed . Flavodo6803 and HsCPR62-241). FMN incorporation was typically >95%. Flavodoxin extinction coefficients were determined by adapting established protocols , based on spectrophotometric measurements of FMN extracted from flavodoxins and holoflavodoxins measured in UV-clear microtiter plates using a BioTek Synergy H1 plate reader and corrected for buffer background. Coefficients of 8.7 and 10.1 mM-1 cm-1 were obtained, respectively, for IsiB6803 and HsCPR62-241.Flavodoxins (~1-2 mM) were reconstituted with 10 mM FMN on ice overnight and desalted into 50 mM tris-HCl (pH 7.5). FMN incorporation was estimated by comparing with calculated absorbance ratios for FMN and protein at 280 nm .Isothermal titration calorimetry was performed on a MicroCal PEAQ-ITC calorimeter . Titrations were performed by injecting FMN  into a solution of pure IsiB2, 100 mM sodium ascorbate, 5 mg mL-1 bovine serum albumin) using a blender. The homogenate was filtered centrifuged for 10 min at 5,000 g, 4°C. Pelleted chloroplasts were resuspended in 5 mM Tricine pH 7.9 and left to rupture osmotically on ice for 15 min. Thylakoid membranes were pelleted at 11,200 g, 4°C for 10 min and resuspended in storage buffer . Chlorophyll concentration of preparations was measured according to Lichtenthaler  and redox partners (0.1-30 µM). Reactions were maintained at 25°C and were started by illuminating assays in black 96-well microtiter plate using a rectangular LED array connected to a laboratory power supply and adjusted to deliver a photon flux of 170 µmol m-2 s-1 light intensity. Reactions were stopped after 2-10 min by addition of 0.33 µM methyl viologen and switching off the light. Plates were centrifuged  and supernatants were filtered through 0.45 µm filters to remove thylakoid membranes. Umbelliferone fluorescence was measured against authentic standards (5-100 nM) dissolved in 50 mM tricine (pH 7.5), using excitation and emission wavelengths of 370 and 450 nm, respectively. Enzymatic rates were corrected for background rates without redox partner and the data fitted to the Michaelis-Menten equation to obtain kinetic parameters using GraphPad Prism.The activity of CYP2A_B49 was measured after isolating thylakoid membranes from pN-CYP2A_B49-infiltrated tobacco plants. Assays contained 50 mM tricine (pH 7.5), 100 mM NaCl, 10 µM coumarin, CYP2A_B49-containing thylakoids (100 µg chl mLc reduction was measured using a Shimadzu UV-2550 double beam spectrophotometer fitted with a light fiber and 550 nm band pass filters on the measuring beam windows. Temperature was controlled at 25°C by thermostat. Assays contained 50 mM Tricine (pH 7.5), 100 mM NaCl, 200 µM cytochrome c, thylakoids (10 µg chl mL-1) and 0.01-4 µM redox partners. The assay was initiated by switching on the light source (Schott KL-1500 fitted with red-light bandpass filters) and the activity was followed for 60 s. Initial rates of cytochrome c reduction were calculated by taking a reduced-oxidized extinction coefficient of 18.5 mM-1 cm-1  and electron carriers . Absorbance was measured at 550 nm for 2 min using a BioTek Syntergy H1 plate reader. Initial rates of cytochrome c reduction were calculated taking a reduced-oxidized extinction coefficient of 18.5 mM-1 cm-1 (c reduction rate in assays without electron carriers. The data was fitted to the Michaelis-Menten equation using GraphPad Prism.Assays were carried out at RT in 96-well microtiter plates by adding 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgClM-1 cm-1  and corrNicotiana benthamiana, and used the hydroxylation of indole as a readout for P450 activity (E. coli L-tryptophan indole lyase (tnaA), which hydrolyses tryptophan to indole, also with chloroplast targeting to provide indole precursor for the P450s. We used the chloroplast transit peptide from Arabidopsis ferredoxin 2 (TPFd2), which can direct both membrane targeted P450s and soluble enzymes to chloroplasts in tobacco  or N. tabacum polyubiquitin U4 promoter , both with the Arabidopsis Hsp18.2 terminator  that contained both tnaA and CYP2A_B49 genes driven by different promoters  after transient expression of a feedback insensitive variant of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, AroG6N/A202T  - denote6N/A202T Table 1.We wanted to test whether increasing Trp content would yield a corresponding increase in indican accumulation. An added benefit of the pBJ1-1 and -2 constructs is that we can co-express additional proteins by swapping them into the empty cloning site on the pEAQ-EV vector we co-infiltrate to deliver P19  almost double that of AtFd1 (420), and nearly four times that of HsCPR62-241 (198), though all carriers showed sub-µM KM values (max/KM = 13.6 for HsCPR62-241 was about double that of AtFd1 (5.4), and 20-times higher than that of IsiB6803  compared to the flavodoxins (6803 (Vmax/KM = 1.9) and 22 times higher catalytic efficiency than HsCPR62-241 Vmax/KM = 0.6). Surprisingly, despite deriving from the native human CPR and reduced quite efficiently by photosystem I, HsCPR62-241 was the worst redox partner for light-driven CYP2A_B49 activity. To go further and investigate how the electron carrier proteins would support CYP2A_B49 activity in the presence of a competing electron sink measured its light-driven activity in the presence and absence of the competing enzyme FNR. FNR readily oxidizes ferredoxins and flavodoxins to reduce NADP+ to NADPH and consumes most of the photosynthetic reducing power in chloroplasts  in a transient expression system (FW-1) are approximately equal to that of plants stably expressing CYP2A6  remained lower than those of human P450s (-1) and KM values (140 µM), and resultant much improved catalytic efficiency of 47.9*103 M-1 s-1 (Pseudomonas accession (sp. 19-rlim) and is a soluble ferredoxin-dependent P450 whose kinetic characteristics have not been described. Both it and P450s BM3 variants may thus be interesting candidates for targeting to chloroplasts.Only a handful of P450s are currently known to perform indole hydroxylation at high rates. Early indole hydroxylating variants of P450 BM3 had Kan P450s . Since,  M-1 s-1 . A bacte M-1 s-1 . This enPolygonum tinctorium  are NADPH-dependent enzymes, some of which have been found to produce indigo from indole , and havnctorium  could benctorium  or increnctorium . Some banctorium . The NDO domains  and as s domains , and theThis study shows the complexities one encounters when optimizing plant-based metabolic engineering to improve productivity. Though the tools and approaches in plant metabolic engineering grow increasingly sophisticated, the complex genetic and metabolic context found in plant hosts complicates engineering and makes highly parallelized experimental workflows difficult to implement. Systematic studies of genetic and metabolic pathway designs, and studies aimed at increasing the design toolbox would facilitate knowledge-based optimization in the future. In addition, integrating modelling, omics and targeted analytical workflows for accurate assessment of enzyme and metabolite levels, by now relatively routine in microbial metabolic engineering studies  would fain vitro investigation shows that ferredoxin provides optimal coupling of photosynthetic electron transport and P450 activity. Our results demonstrate that plant chloroplasts provide a viable membrane and redox environment for animal P450s. Thus, transient expression with chloroplast targeting can be used to generate active P450s that do not require additional redox partners and has applications for metabolic engineering or could be used to elucidate metabolite profiles of human P450s.We demonstrate chloroplast-based production of indican from tryptophan using photosynthesis-P450 hydroxylation. We show that indican productivity can be improved by combining both tryptophanase and P450 on one T-DNA. Further, we boost tryptophan accumulation by co-expressing feedback insensitive mutant DAHP synthase, which increases indole but not indican yield. Co-expressing additional redox partners also leads to unaltered indican yield. Further The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SM, JB, and MP designed experiments. SM, JB, JI, and CC performed experimental work. SM, JB, CC, TL, EG, and MP analyzed the data. SM wrote the manuscript with help and feedback from JB, JI, CC, TL, EG, and MP. All authors contributed to the article and approved the submitted version.


In vitro experiments using BM-isolated neutrophils showed significant downregulations in their activities, such as phagocytosis, reactive oxygen species (ROS) production, interleukin (IL)-1β production, neutrophil extracellular trap (NET) formation, and migration as well as bacterial clearance, in the aged mouse neutrophils compared to those of young mice counterparts. Interestingly, the magnitudes of functional parameters were strongly correlated with the Ly-6G expression in the neutrophils. Thus, our results suggest that downregulation of Ly-6G reflects the age-associated functional attenuation of the neutrophils.Immunological aging is a critical event that causes serious functional impairment in the innate immune system. However, the identification markers and parameters are still poorly understood in immunological aging of myeloid lineage cells. Here, we show that a downregulation of lymphocyte antigen 6 complex locus G6D (Ly-6G) observed in aged mouse neutrophils could serve as a novel marker for the prediction of age-associated functional impairment in the neutrophils. Ly-6G expression was significantly downregulated in the bone marrow (BM) neutrophils of aged mice compared to young mice confirmed by flow cytometry analysis Aging is an unavoidable physiological event and affects various biological processes including immunity . Both thin vitro experimental system is required to assess the function.Neutrophils are frequently studied due to their central role in the innate immune system, specifically their involvement in antibacterial immunity . Under bLeishmania major-infected site  in myeloid lineage cells determined by CD11b expression in mice . Alternated site . In thisted site . Thus, tIn this report, we show that Ly-6G expression is downregulated on the neutrophils originating from aged mice compared to those of young mice. Furthermore, we found a strong correlation between the expression level of Ly-6G and the grade of immunological parameters in antibacterial immunity, which are attenuated by aging, in the neutrophils. Thus, we propose that Ly-6G downregulation is a novel marker for age-associated functional impairment of neutrophils.Both young and aged C57BL/6J mice were obtained from CLEA Japan, Inc. . The mice were maintained in a specific pathogen-free (SPF) condition with 12-h day/night cycles and were allowed free access to food and water. The mice were prepared for young (10 to 20 weeks) and aged (20 to 22 months) groups. Since we had experienced the difficulty to keep enough number of aged male mice in our colony, we decided to use female mice in this study. All aged mice were screened for their condition prior to use for experiments, and the mice suspected to have abnormalities  were excluded from the subject. The data of body weight (g), spleen weight (mg), and body temperature (°C) were shown for all mice, as shown in In vivoGen . Fluorescein isothiocyanate (FITC)-labeled E. coli (K-12 strain) BioParticles, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), carboxyfluorescein succinimidyl ester (CFSE), and SYTOX™ Green were purchased from Thermo Fisher Scientific . Anti-CD45 (30-F11), anti-CD11b (M1/70), anti-Ly-6G (1A8), anri-Ly-6C (HK1.4), anti-CD18 (M18/2),anti-CD11a (M17/4), anti-CD16/CD32 (2.4G2), anti-CD64 (S18017D), anti-CD21/CD35 (7E9), anti-CXC chemokine receptors 2 (CXCR2) (SA044G4), anti-CXCR4 (12G5), and 7-amino-actinomycin D (7-AAD) were all purchased from BioLegend . Recombinant mouse C–X–C motif chemokine 1 (CXCL1) and C–X–C motif chemokine 5 (CXCL5) were purchased from PeproTech .Phorbol 12-myristate 13-acetate (PMA) was purchased from Merck/Millipore Sigma . Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)) were purchased from The neutrophils were isolated from the bone marrow (BM) by the following protocol described in the previous reports , 18. BriEscherichia coli (DH5α) was purchased from the American Type Culture Collection (ATCC). The frozen stock was thawed on ice, and the bacteria were cultured in LB medium following the product manual. The bacterial colony-forming unit (CFU/mL) was calculated by following the previous reports  were seeded in a 96-well plate (round bottom) in RPMI complete medium. The samples were treated with K-12 BioParticles (10 μg/ml) at 37°C for 60 min. The phagocytosis activity was measured by flow cytometry.Neutrophils (1.0 × 107/ml) were seeded in a 96-well plate (round bottom) in RPMI complete medium containing DCFDA (500 nM). The samples were treated with LPS (1 μg/ml), live E. coli (1.0 × 107 CFU/ml), or vehicle control (PBS) at 37°C for 60 min without direct exposure to light. After incubation, ROS production was analyzed by flow cytometry.Neutrophils (1.0 × 107/ml) were seeded in a 96-well plate (flat bottom) and then were stimulated with LPS (1 μg/ml), poly(I:C) (10 μg/ml), or live E. coli (1.0 × 107 CFU/ml). The control samples were treated with vehicle control (PBS). The cultures were incubated at 37°C overnight, and then the plate was immediately frozen at -80°C until use. For measuring the IL-1β concentration in the cultured medium, the plate was centrifuged at 300 g for 5 min and supernatant was collected for using enzyme-linked immunosorbent assay (ELISA).Neutrophils (1.0 × 106/ml) were seeded in a transmigration well  in RPMI 1640 medium. The wells were placed on a 96-well plate which contains RPMI 1640 medium supplemented with CXCL1 (1 μg/ml) or CXCL5 (1 μg/ml), and then the plate was incubated at 37°C for 2 h. After the incubation, the fluorescence originated from the migrated cells was measured in the plate by a microplate reader .BM-isolated neutrophils were labeled with CFSE (5 μM) in PBS at 37°C for 15 min, then washed with PBS. The CFSE-labeled neutrophils (1.0 × 105/ml) were seeded in a 96-well plate  in RPMI complete medium containing PMA (50 nM) and incubated at 37°C for 4 h. After incubation, the cultured medium was collected for cell-free DNA (CFD), citrullinated Histone H3 (CitH3), and myeloperoxidase (MPO)–DNA complex assays, and the cells were used for NET staining. For the CFD assay, the released DNA in the cultured medium was stained with staging with SYTOX™ Green (500 nM) at 37°C for 15 min. CitH3 and MPO–DNA complex assays were performed by using the Citrullinated Histone H3 ELISA Kit  and laboratory homemade ELISA system, respectively. The fluorescence and absorbance were measured by a microplate reader . For NET staining, the cells were treated with PBS containing SYTOX™ Green (500 nM) at 37°C for 15 min followed by washing with PBS and fixation with 1% PFA. NETosis was observed using a fluorescence microscope .NETosis activity was assessed by following a method described in the previous report . Neutrop7/ml) were seeded in a 96-well plate (flat bottom) in RPMI complete medium. The samples were exposed to live E. coli (1.0 × 107 CFU/ml) at 37°C for 6 h. The cultured medium was harvested and diluted with PBS prior to seeding on an LB agar plate. The bacterial CFU was calculated in each sample. The efficiency of bacterial clearance was calculated by the following formula: Bacteria clearance (%) = E. coli-CFU/mL after incubation/Loaded E. coli-CFU/mL × 100.Neutrophils . For the surface marker staining, the cells were first incubated with an FcR blocker (anti-CD16/CD32) at 4°C for 10 min followed by incubation with the antibodies in PBS/2% FBS at 4°C for 30 min. All data were analyzed by FACSDiva (BD Biosciences) and FlowJo (BD Biosciences). All flow cytometry analyses were performed by following a gating strategy shown in The IL-1β concentration in cultured medium was measured using the mouse IL-1β DuoSet ELISA Kit . All procedures were performed by following the manufacturer’s instruction.t-test was used to analyze the data for significant differences.Statistical analyses were performed by using GraphPad Prism . Student’s vs. Aged 92.46 ± 0.6 SEM, Number (×107): Young 4.87 ± 0.1 SEM vs. Aged 2.73 ± 0.09 SEM) (vs. Aged 65 ± 0.65 SEM), neutrophils , and polymorphonuclear leukocytes  were all significantly increased in aged mice compared to the counterparts of young mice, while the cell numbers were decreased in those populations affected by decreased total BM leukocyte (CD45+) cells in the aged mice (Myeloid (×107): Young 2.96 ± 0.08 SEM vs. Aged 1.78 ± 0.07 SEM, Neutrophils (×107): Young 1.97 ± 0.05 SEM vs. Aged 1.23 ± 0.04 SEM, PMNs (×107): Young 1.89 ± 0.05 SEM vs. Aged 1.21 ± 0.05 SEM) (vs. Aged 14.04 ± 0.55 SEM), while the cell numbers were decreased in aged mice similar to other subsets (Numbers (×106): Young 4.33 ± 0.16 SEM vs. Aged 2.46 ± 0.1 SEM)  and elementally aged mice (20–22 months); therefore, we also have to investigate Ly-6G expression and the functions of the neutrophils at the intermediate age . This is an important point to understand when the Ly-6G downregulation becomes obvious, and the expression level starts to show a strong correlation with the age-associated dysfunction of the neutrophils. In addition, this study used female mice. It has been introduced gender difference-based functional alterations in neutrophils , 26; theThere are still some required points to reveal the details in our findings; however, we try to propose that Ly-6G downregulation is a novel marker for the prediction of age-associated functional impairment in mouse neutrophils.The original contributions presented in the study are included in the article/This study was reviewed and approved by Advanced Industrial Science and Technology  and CLEA Japan, Inc. (AE2157-121).SS and NMT established the concept in this study. SS, AO, TM, RK, OY, NT and MI performed experiments. SS established analysis methodologies and performed data analyses. NMT, OY, NT, MI and YK had responsibility in animal resources. SS wrote the original manuscript. SS and NMT reviewed and finalized the manuscript. All authors participated in the discussion. SS and NMT supervised this study. All authors contributed to the article and approved the submitted version.This study was supported by the AIST- CLEA-Japan Joint Research Fund , Japan Society for the Promotion of Science (21K15958 (SS)), Japan Society for the Promotion of Science (19H04042 (NMT)), and Mishima-Kaiun Memorial Fund (SS). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.We thank Yasaman Bahojb Habibyan M.sc and Maria Alexiou Ph.D for providing support in manuscript writing. We also thank Dr. Yohichi Shimma for providing a support in project administration.OY, NT, MI and YK are employees in CLEA Japan, Inc. TM is an employee in iFoodMed, Inc. RK and NMT are scientific advisors in iFoodMed, Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The title compound represents the first instance of a 1,4-phenylene bis(carbonate) derivative characterized by X-ray diffraction. 14H14O6, is based on a benzene core di-substituted by allyl carbonate groups in the para positions. The molecule is placed on an inversion centre, and the substituents are twisted with respect to the central benzene ring plane. The crystal structure does not include significant intermolecular interactions other than weak C—H⋯O contacts between CH groups in the benzene ring and carbonate O atoms.The title molecule, C Nair et al., 2010et al., 2009et al., 1997et al., 2010p-allylcarbonate)benzoate]  and a diol affords mono allyl carbonate and diallyl carbonate derivatives. The reaction of ACF with hydroquinone gives allyl-4-hydroxyphenyl carbonate °, and the dihedral angle between the carbonate group and the allyl group C8/C9/C10 is 51.1 (2)°. This twisted conformation was previously observed for the four reported X-ray structures bearing a benzene ring substituted by an allyl carbonate group , previously dissolved in 5 ml of distilled water, was added. Then, the reaction flask was placed in an ice bath and allyl chloroformate  was slowly added dropwise, maintaining the agitation. After complete addition, the reaction was left for 5–10 minutes at 273 K, and then at room temperature for 2 h. After completion of the reaction, the products were extracted in a separation funnel using CH2Cl2, and dried over anhydrous Na2SO4. The reaction mixture was filtered and concentrated. The resulting concentrated solution was precipitated into hexane. The precipitate was collected, washed with hexane, and dried in vacuo . Transparent prismatic single crystals were recovered from this material for X-ray study , δ (p.p.m.): 4.75 , 5.34 , 5.44 , 5.90 , 7.21 ; 13C NMR , δ (p.p.m.): 69.3 (–CH2–), 119.7 (=CH2), 122.0 (benzene), 131.1 (=CH), 148.6 (benzene), 154.3 (C=O); FTIR : 3082 (Csp2—H), 2960 (Csp3—H), 1757 (C=O), 1649 , 1602 , 770 (aromatic ring), 730 (Csp3—H).To a three-neck round-bottom flask connected to an addition funnel, hydroquinone  was added and dissolved in 20 ml of THF under an argon atmosphere. After continuous agitation, a homogeneous phase was observed in the reaction flask, and NaHCOCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623001335/bv4045sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2414314623001335/bv4045Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623001335/bv4045Isup3.cmlSupporting information file. DOI: 2241801CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


TCPP also promoted the generation of intracellular reactive oxygen species (ROS) and triggered DNA damage, evidenced by an increase of phosphorylated histone H2A.X (γH2A.X) in the nucleus. Furthermore, the cell cycle was arrested at the G1 phase at 100 μg/mL by upregulation of the mRNA expression of p53 and p21 and downregulation of cyclin D1 and CDK4 expression. Additionally, both the senescence-associated-β-galactosidase activity and related proinflammatory cytokine IL-1β and IL-6 were elevated, indicating that TCPP exposure caused cellular senescence may be through the p53-dependent DNA damage signal pathway in HaCaT cells. Taken together, our data suggest that flame-retardant exposure may be a key precipitating factor for human skin aging.Tris (1-chloro-2-propyl) phosphate (TCPP) is one of the most frequently detected organophosphorus flames in the environment. Continuous daily exposure to TCPP may harm human skin. However, little is known about the adverse effects of TCPP on human skin. In this study, we first evaluated the detrimental effects and tried to uncover the underlying mechanisms of TCPP on human skin keratinocytes (HaCaT) after 24 h exposure. We found that TCPP caused a concentration-dependent decrease in HaCaT cell viability after exposure to 1.56–400 μg/mL for 24 h, with an IC Tris 1-chloro-2-propyl) phosphate (TCPP) as an organophosphorus flame retardant (OPFR) has been widely used in various consumer products, building materials, and baby products -propyl p. In addi2 and weighing about 5 kg in adult people [The human skin is the largest body organ of the integumentary system, with a surface area of about 2 mt people . Howevert people . Skin agt people . The extt people . Environt people  and may t people . In addit people ,14.Il-6) in senescent cells is known as senescence-associated secretory phenotype (SASP), and those bioactive molecules play an important role in the progress of senescence [Mounting evidence demonstrated that flame retardant exposure may induce cellular senescence. Behnia et al. 2015) found that exposure to polybrominated diphenyl ethers (PBDEs) flame retardant could induce primary amnion cell senescence  found th. In addinescence . HoweverIn this study, to better understand its toxicity, normal human skin keratinocytes (HaCaT) were employed, and the changes of cell viability, morphology, reactive oxygen species (ROS), DNA damage factor Phosphorylated histone H2A.X (γH2A.X), cell cycle arrest, and senescence-associated-β-galactosidase activity  were determined after TCPP exposure. Additionally, the mRNA and protein expression of senescence markers was detected to uncover the underlying mechanism of TCPP-induced of skin aging.50 concentrations of TCPP at 275 μg/mL , leading to cell cycle arrest [p53/p21 mRNA expression may be an important mechanism for contaminant-induced cellular senescence in human cells, including HaCaT cells. In this study, TCPP did not affect the mRNA expression of p53 and p21 at ≤25 μg/mL; however, when concentration increased to 100 μg/mL TCPP, the expression of p53 and p21 was increased to 3.4 and 2.2 folds, while cyclinD1 and CDK4 were decreased to 0.45 and 0.59 folds  . CellulaTris (1-chloro-2-propyl) phosphate  was from Dr. Ehrenstorfer GmbH . The cell counting Kit-8 kit was purchased from GlpBio Technology, Ltd. . The cell cycle testing kit, apoptosis detection kit, SYBR green qPCR master mix, and Total RNA Extraction Reagent were from Yi Fei Xue Biotech Co. . The cDNA synthesis kit was from TaKaRa Biotech, Ltd. . Senescence β-Galactosidase Staining Kit was from Beyotime Biotechnology , DAPI was from Sigma Aldrich, and Phosphorylated histone H2A.X (γH2A.X) antibody was from Abcam . Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were from Procell Life Science & Technology Co., Ltd. ; Penicillin-streptomycin (PS) and 0.25% trypsin-EDTA were from HyClone .2. HaCaT cells were subcultured twice a week. Before exposure to TCPP, HaCaT cells were seeded in 96-well or 6-well plates overnight. The cells were then treated with 1.56, 6.25, 25, 50, 100, 200, or 400 μg/mL of TCPP for 24 h. TCPP was dissolved in dimethyl sulfoxide , and the final concentration of DMSO in each treatment was ≤0.1% (v/v), with 0.1% DMSO solution being set as the control.The human skin keratinocytes (HaCaTs) were from American Type Culture Collection and cultured with RIPM1640 medium supplemented with 10% FBS and 1% PS in an incubator at 37 °C and 5% CO4 cells/100 μL/well. Following overnight incubation, the cells were treated with different concentrations  of TCPP for 24 h. Then cells were incubated with 10 μL of CCK-8 solution for 2 h at 37 °C in a 5% CO2 incubator. The absorbance at 450 nm was recorded using a microplate reader . Additionally, the cell morphology was observed by an inverted microscope . The effect of TCPP on cell viability was measured using the CCK-8 assay. Briefly, the HaCaT cells were seeded to 96-well plates at the density of 1 × 102 incubator at the density of 1 × 106 cells/mL. Based on the median inhibitory concentration (IC50) at 275 μg/mL, the cells were treated with TCPP at 1.56–100 μg/for 24 h. Then, the cells were incubated with 10 μM DCFH-DA-containing serum-free medium for 20 min at 37 °C. Cells were washed three times with PBS. Subsequently, the level of intracellular ROS was detected using a flow cytometer . Data were collected from 10,000 cells and analyzed by the FlowJo v10.6.2 software .The level of intracellular ROS was examined using a ROS assay kit  according to the manufacturer’s instructions. Briefly, the HaCaT cells were seeded to 6-well plates and incubated overnight at 37 °C in a 5% CO4 cells/mL overnight; then, the cells were exposed to 1.56–100 μg/mL TCPP for 24 h at 37 °C. Subsequently, HaCaT cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 15 min. After being blocked with 1% BSA for 1 h at room temperature, samples were incubated with γH2AX primary antibody  overnight at 4 °C. Cells were washed 3 times with ice-cold PBS and incubated with secondary antibodies  for 1 h at room temperature. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole . The fluorescence images were captured with an inverted microscope . The nuclear γH2AX fluorescence intensity was quantified using Image J software .For immunofluorescence staining, the HaCaT cells were seeded on 24-well plates and cultured at the density of 5 × 106 cells/mL. Following overnight incubation, the cells were exposed to 1.56–100 μg/mL TCPP for 24 h, and then they were detected [®Cube 6 flow cytometer . Data were collected from 10,000 cells and analyzed by the FlowJo v10.6.2 software .For cell cycle analysis, the HaCaT cells were seeded to 6-well plates at the density of 1 × 10detected . Briefly5 cells/well. After exposing them to TCPP for 24 h, they were washed with PBS and fixed in 1 mL β-galactosidase stain for 15 min at room temperature. Then, they were washed three times with PBS and subsequently incubated overnight with 1 mL staining solution mix  at 37 °C. Then, the stained positive cells (blue color) were observed via inverted microscopy . The number of SA-β-Gal positive cells was counted using Image Pro-Plus 6.0 software . The percentage of senescent cells was calculated from five individual fields.Senescence β-Galactosidase Staining was performed using the Senescence β-Galactosidase Staining Kit  according to the manufacturer’s instructions. Briefly, HaCaT cells were seeded to 6-well plates overnight at 5 × 10β-Actin gene served as an internal control. The relative expression level was calculated by the 2−ΔΔCT method, with primers listed in After exposure to TCPP for 24 h, the total RNA of HaCaT cells was isolated using TRIzol reagent . First-strand cDNA was synthesized with Takara PrimeScript™ RT Master Mix . The qRT-PCR analysis was performed using an SYBR Green qPCR Master Mix and Roche LightCycler 480II Real-Time PCR system. The amplification conditions were as follows: pre-denaturation at 95 °C for 15 min, followed by 40 cycles of 2-step amplification (95 °C for 15 s and 60 °C for 1 min). The The protein levels of IL-1β and IL-6 in cell supernatants were assayed with human IL-1β and IL-6 ELISA Kits  following the manufacturer’s instructions. The absorbance at 450 nm was recorded using a microplate reader . The results of IL-1β and IL-6 were expressed as concentrations as ng/L.p < 0.05 being significant.All experiments were repeated at least three times. Data are presented as the mean ± SEM. All results were analyzed with GraphPad Prism 5.0 statistical software . Differences in different groups were assessed by one-way ANOVA, with 50 of 275 μg/mL, and induced obvious DNA damage and cell senescence. In addition, TCPP induced cell cycle arrest in the G1 phase at 100 μg/mL by upregulation of the mRNA expression of p53 and p21, while the expression of cyclin D1 was suppressed. Meanwhile, SASP proinflammatory cytokines IL-1β and IL-6 were also enhanced at both mRNA and protein levels. Taken together, our results indicate that TCPP exposure caused cellular senescence may be through the p53/p21 pathway in HaCaT cells, which may provide a new perspective on skin aging.In this study, we found that TCPP caused a concentration-dependent decrease in HaCaT cell viability after exposure to 1.56–400 μg/mL for 24 h, with an IC