Matricaria chamomilla L. is a popular medicinal herb that is used for healing various diseases and is widely distributed worldwide in temperate climate zones, and even in the subtropical climate of Southern and Western Iran. This study was aimed at comparing the volatile oil constituents, along with antiradical potential and HPLC analysis of methanolic extracts from twelve plant samples growing in Iran. The present research was carried out for the first time on these populations. Among seventeen identified volatile chemicals evaluated by GC/MS and GC/FID, representing 92.73–97.71% of the total oils, α-bisabolone oxide A (45.64–65.41%) was the major constituent, except in case of “Sarableh” as a new chemotype, where (E)- and (Z)-γ-bisabolene  were the predominant components. Oxygenated sesquiterpenes (53.31–74.52%) were the most abundant compounds in the samples excluding “Sarableh” with 91.3% sesquiterpene hydrocarbons. “Sarableh” also exerted the most potent antioxidant capacity with EC50 = 7.76 ± 0.3 µg/mL and 6.51 ± 0.63 mmol TE /g. In addition, populations “Lali” and “Bagh Malek” contained the highest amounts of apigenin and luteolin with 1.19 ± 0.01 mg/g and 2.20 ± 0.0 mg/g of plant material, respectively. Our findings depict a clear correlation between phytochemical profiles and antiradical potential of M. chamomilla and geographical factors. Matricaria chamomilla L. , belonging to the Asteraceae family, is one of the well-known medicinal plant species which has been widely used for centuries. The herb is currently consumed around the world as herbal tea, with more than 1 million cups daily [M. chamomilla is used in the treatment of many ailments and disorders; internally to facilitate digestion and as antispasmodic, externally to treat minor wounds. In folk medicine, its use spreads from the relief of various pains such as headaches and toothaches to the facilitation of menstruation [ps daily . M. chamtruation . Chamomitruation ,5.M. chamomilla EO. Spathulenol (12.50%) [E)-β-farnesene (42.59%) [trans-β-farnesene [Several papers report sesquiterpenes as the most dominant constituent of 12.50%) , α-bisab(12.50%) ,8,9,10, (12.50%) , (E)-β-f.50% , α-(42.59%) , trans-βarnesene ,15, chamarnesene , and α-barnesene  were recM. chamomilla have also been investigated. The major phytochemicals comprise polyphenols, particularly the flavonoids apigenin, patuletin, quercetin, luteolin and their glucosides [Non-volatile compounds of ucosides ,19,20; aucosides .M. chamomilla populations collected from diverse regions were qualitatively and quantitatively assessed. Since the main radical scavengers are phenolics, flavonoids being the major group within phenolics [In the present study, EO compositions of twelve Iranian henolics , our stuhenolics ,23, by uAs shown in Seventeen compounds were detected in these twelve populations. The sesquiterpene α-bisabolone oxide A (45.64–65.41%) was the major EO constituent in the samples except “B” and “S”, whereas its concentration was the highest in population “Mu” .The cultivated sample “B” was rich in α-bisabolol oxide B (21.88%) and chamazulene (19.22%), while the percentages of these compounds were lower under the wild growth conditions. The blue colour of EOs in all samples except “S” represented their chamazulene contents, whereas, “S” due to lack of this compound showed a yellowish green colour. Accordingly, oxygenated sesquiterpenes (53.31–74.52%) were the predominant chemical group of EO constituents in all studied samples, excluding “S”, which was rich in sesquiterpene hydrocarbons .Methanolic extracts of samples “L” and “A” contained the highest amounts of apigenin, with 1.19 ± 0.01 mg/g and 1.02 ± 0.01 mg/g, respectively. Luteolin was present in higher concentrations in “BM” (2.20 ± 0.0 mg/g) and “A” (1.01 ± 0.02 mg/g) extracts .M. chamomilla populations, their EO compositions and main flavonoids (apigenin and luteolin) were submitted to cluster analysis (CA) and principal component analysis (PCA). As shown in M. chamomilla populations into three main groups, each representing a distinct chemotype.To characterize and identify the different chemotypes of Iranian E)-β-farnesene, α-bisabolone oxide A and (E)-spiroether, and negative correlation with (Z)-γ-bisabolene and (E)-γ-bisabolene. The second PC (PC2), with 24.98% of variance, demonstrated positive correlation with chamazulene, α-bisabolol oxide B, α-bisabolone oxide A and the EO content. Furthermore, PC3 represented positive correlation in the case of apigenin and luteolin, which accounted for 12.02% of the total variance . PC1, PC2, and PC3 showed the highest variation of phytochemicals among the studied populations. PC1 explained 41.57% of total variation and had a positive correlation with α-bisabolol oxide A,  and (E)-γ-bisabolene.In accordance with the CA, the populations “I”, “DS”, “Mo”, “MS”, “Mu”, “G”, “SS”, “L”, “A” and “BM” were classified into the same category, while, “B” and “S” were grouped into the individual subclasses. The first group possessed α-bisabolone oxide A and α-bisabolol oxide A as the major constituents as well as apigenin and luteolin (chemotype I). The second chemotype (II), was characterized by high amounts of chamazulene and α-bisabolol oxide B. The chemotype (III) was the richest in  was applied based on a matrix of three environmental factors including altitude, mean annual temperature (MAT), and mean annual precipitation (MAP)  and majoAccording to the CCA, phytochemicals of populations in first group were significantly affected by ecological parameters  while the main oil composition and flavonoids of “Sarableh” and “Boldgold” were changed by genetic factors. Therefore, the “Sarableh” population can be introduced as a new chemotype.50 = 7.76 ± 0.3 µg/mL and 6.51 ± 0.63 mmol TE/g measured by DPPH and ORAC assays, respectively. However, the extracts showed lower activity compared to ascorbic acid (EC50 = 0.3 ± 0.02 µg/mL) in the DPPH and rutin (20.22 ± 0.63 mmol TE/g) and EGCG (11.97 ± 0.02 mmol TE/g) in the ORAC assay  highly impact the phytoconstituents variation, and/or biotic factors remarkably influenced the terpene biosynthesis pathways and chemotype profiles . HoweverMatricaria chamomilla L. samples, which corroborates previous reports. The wild populations were significantly different compared with the cultivated sample (Bodgold) in EO composition. α-Bisabolone oxide A was mostly the major EO constituent in the populations.In accordance with our findings, oxygenated sesquiterpenes (53.31–74.52%) are the most dominant EO compounds of the selected M. chamomilla by different groups; for instance, the EO, diethyl ether and dichloromethane fractions of EO contained α-bisabolone oxide A, with 47.7, 57.7 and 50.5%, respectively [M. chamomilla sample was characterized by high bisabolone oxide A content (13.9%) [M. chamomilla grown in Italy (9.2–11.2%) [M. chamomilla EO revealed that the highest amount can be between 16:00–18:00 (55.41%) [In the literature, α-bisabolone oxide A was previously identified from ectively ; whereas 13.9%) . This co2–11.2%) , Iran (52–11.2%) , India (2–11.2%)  and Turk(55.41%) . Moreove% . This It is noteworthy, that the sample “Sarableh”  as a new chemotype was the richest in two geometric isomers of γ-bisabolene (82.84%), whilst these sesquiterpenes were not markedly detected in the other plant populations. This population also demonstrated the most potent antiradical capacity compared with those samples.Pimpinella pruatjan Molk. [Ocimum africanum Lour. was also rich in (E)-γ-bisabolene (2.6–9.5%) [Ziziphus jujuba var. inermis. [M. chamomilla harvested in Iran were identified as α-bisabolol (7.27%), -farnesol (39.70–66.00%) [E)-β-farnesene (24.19%) [γ-Bisabolene was previously specified as the major characteristic constituent of an Molk. . EO of O.6–9.5%) . This seinermis. . The mai–66.00%) , (E)-β-f(24.19%)  and α-bi(24.19%) . EOs con(24.19%) ,37,38,39According to the results, the abundance of luteolin was much higher than the apigenin content. The lack of luteolin and apigenin as the selected flavones was reported in populations “MS” and “Mo”. These samples are most probably rich in other phenolic natural products.M. chamomilla extract were 37.51 and 21.72 mg/g of dry weight, respectively; this report was formerly accomplished by using HPLC-DAD [M. chamomilla extract [The total phenolic and flavonoid contents of a HPLC-DAD . Moreove extract .As apigenin and luteolin were present in “Sarableh” in low concentrations, the high free radical scavenging capacity of this sample is obviously related to other polyphenolic compounds.M. chamomilla yielded from different samples indicated more potent antiradical activity than EOs analysed by the DPPH test, due to polyphenols present in extracts; although, they possessed a moderate effect in comparison with the standards [In a similar study, methanol extracts of tandards .M. chamomilla extracts, along with its apigenin and apigenin-7-glycoside contents were previously characterized, with IC50 of 18.19 ± 0.96 µg/mL, 2.0 ± 0.1 mg/g and 20.1 ± 0.9 mg/g, respectively [M. chamomilla. Moreover, free radical scavenging activity (DPPH assay) of M. chamomilla volatile oil and its major components were formerly recorded in the following order: chamazulene > α-bisabolol oxide A > chamomile EO > (E)-β-farnesene > α-bisabolol [Furthermore, in vitro antioxidant activities of ectively , confirmM. chamomilla flowers (300 g/sample) were individually collected from different regions of Iran in the flowering period, in spring (April) 2017. The harvested populations “Izeh”, “Bagh Malek”, “Lali”, “Masjed Soleyman”, “Mollasani”, “Gotvand” and “Saleh Shahr” from Khuzestan and “Murmuri”, “Abdanan”, “Darreh Shahr” and “Sarableh” from Ilam province were compared with “Bodgold”, which was cultivated at the botanical garden of Department of Horticultural Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran.The plants were identified by Dr. Mehrangiz Chehrazi affiliated with the same department, and a voucher specimen of each sample was deposited in the herbarium of the department. The voucher’s codes and geographic coordinates including the latitude, longitude, altitude using the Global Positioning System (GPS), along with mean annual temperatures of the studied populations are given in ®) , fluorescein ; ascorbic acid, rutin and Na2SO4 ; epigallocatechin gallate (EGCG), apigenin (≥99%) and luteolin (≥97%)  were purchased. Furthermore, all solvents of analytical grade were provided by the Merck company (Germany). Spectrophotometric measurements were carried out by a UV-VIS spectrophotometer .Analytical grade 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azobis-2-methylpropionamidine dihydrochloride (AAPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid  for 3 h. The obtained EOs were dried over anhydrous sodium sulphate and stored in refrigerator at 4 °C until analysis. Yields of extracted EOs were calculated by Equation (1):Diethyl ether was used to elute the whole amount of EOs from the apparatus, and the weighing process was performed after evaporating the solvent.In the case of GC-FID analysis, the EOs were analyzed by Shimadzu GC-17A  equipped with an FID detector and SGE™ BP5 capillary column  (30 m × 0.25 mm column with a 0.25 μm film thickness). The split mode in GC was a ratio of 1:100. Injector and FID detector temperatures were set at 280 and 300 °C, respectively. The oven temperature was kept at 60 °C for 1 min and then raised to 250 °C at 5.0 °C/min and held for 2 min, while the ambient oven temperature range was +4 to +450 °C. Helium gas was used at a flow rate of 1 mL/min as a carrier gas.Analysis of the samples was carried out using an Agilent 7890B gas chromatograph  equipped with a 5977B mass spectrometry detector. The GC instrument was equipped with a split inlet, working in a split ratio of 100:1 mode with a 30 m × 0.25 mm HP-5MS capillary column with a 0.25 μm film thickness and 0.25 μm particle size (temperature range: −60 to +320/340 °C). The injection port temperature was 250 °C. The oven temperature was kept at 60 °C for 1 min and then programmed from 60 °C to 250 °C at 5 °C/min; then, the temperature was kept at 250 °C for 2 min. Helium (99.999%) was used as a carrier gas with a flow rate of 1 mL/min and inlet pressure 35.3 kPa. The mass spectrometer was operated in the electron impact mode at 70 eV, and the inert ion source (HES EI) temperature was set to 350 °C; the temperature of the quadrupole was set at 150 °C, while the MS interface was set to 250 °C. A scan rate of 0.6 s (cycle time: 0.2 s) was applied, covering a mass range from 35 to 600 amu.n-alkanes (C8–C24) [TM  and XcaliburTM software , respectively.Most of the compounds were identified using two different analytical approaches: (a) comparison of Kovats indices of (C8–C24)  and (b) Five g of each sample was individually extracted with MeOH (3 × 75 mL) in an ultrasonic bath (VWR-USC300D) for 10 min, at 40 °C under power grade 9.After removing the solvent under reduced pressure at 50 °C , the concentrated extracts were subjected to evaluate the antiradical assays and HPLC analysis.2O was applied as the mobile phase with a gradient system, increasing MeOH from 30% to 70% within 40 min, with a flow rate of 1 mL/min, at ambient temperature. Analysis was performed using SAS software, version 9.2 .Twenty μL of each extract (1 mg/mL) was separately injected into an analytical high-performance liquid chromatography system (HPLC)  by using an end capped Eurospher II 100-5 C18, Vertex Plus Column  (250 × 4.6 mm with precolumn) with particle size: 5 µm, pore size: 100 Å and temperature 30 °C; coupled to UV detector  at a wavelength range 190 to 500 nm (quantification at 330 nm), while MeOH/H0 is the absorbance of the control and A1 is the absorbance of the standard sample. Anti-radical activity of the samples was expressed as EC50 (concentration of the compounds that caused 50% inhibition). EC50 (µg/mL) values were calculated using GraphPad Prism software version 6.05 . Each sample was measured in triplicate.Free radical scavenging activity of the plant extracts was assessed by DPPH assay . The mea® was used. Activity of all samples was compared with rutin and EGCG as positive controls. Antioxidant capacities were reported as μmol TE /g of dry matter.Twelve extracts were subjected to ORAC assay  with slip < 0.05).All the experiments were done in triplicate, and the results are expressed as means ± SD. The data were assessed with one-way analysis of variance (ANOVA) using SAS software  and GraphPad Prism version 6.05. The means were compared using Duncan’s comparisons test (Matricaria chamomilla populations were highly affected by a variety of ecological conditions. To acquire the best yield with a good profile of active principles, it is crucial to combine a good genotype with optimal environmental circumstances.According to our preliminary study, the chemo-diversity and antiradical potential of twelve studied Matricaria chamomilla samples. Almost all plant populations contained apigenin and luteolin. Since the plant sample “Sarableh” indicated the most potent capacity to scavenge free radicals and its apigenin and luteolin contents were insignificant, its activity undoubtedly refers to the presence of other polyphenolic compounds. Due to high amounts of apigenin and luteolin, populations “Lali” and “Bagh Malek” can be considered as a rich source of these compounds. Our results confirm the effects of growth conditions on the quantity and quality of aromatic phytochemicals. More investigations are required to study the amounts of other polyphenolic compounds in diverse populations and the correlations between secondary metabolite contents, bioactivities and ecological effects.In accordance with our findings, oxygenated sesquiterpenes are the most dominant EO compounds of the selected


Pseudomonas mendocina strain GL6. It exhibited efficient aerobic denitrification ability, with the nitrate removal rate of 6.61 mg (N)·L−1·h−1. Sequence amplification indicated that the denitrification genes napA, nirK, norB, and nosZ were present in strain GL6. Nitrogen balance analysis revealed that approximately 74.5% of the initial nitrogen was removed as gas products. In addition, the response surface methodology experiments showed that the maximum removal of total nitrogen occurred at pH 7.76, C/N ratio of 11.2, temperature of 27.8 °C, and with shaking at 133 rpm. Furthermore, under the optimized cultivation condition, strain GL6 was added into wastewater treatment plant effluent and the removal rates of nitrate nitrogen and total nitrogen reached 95.6% and 73.6%, respectively. Thus, P. mendocina strain GL6 has high denitrification potential for deep improvement of effluent quality.To remove nitrate in wastewater treatment plant effluent, an aerobic denitrifier was newly isolated from the surface flow constructed wetland and identified as  Nitrate is the primary source of dissolved inorganic nitrogen in wastewater treatment plant effluent . High niThe main methods for nitrate removal include ion exchange, adsorption, reverse osmosis, electrodialysis, catalysis, and biological denitrification . The maiPseudomonas stutzeri strain T1 [Vibrio diabolicus SF16 [Pseudomonas stutzeri YHA-13 [Enterobacter cloacae strain HNR [Aerobic denitrification that can simultaneously make use of nitrate and oxygen as electron acceptors was first reported in the early 1980s [cus SF16 , Pseudomi YHA-13 , and Entrain HNR . Howeverrain HNR ,13,14. Hrain HNR . TherefoPseudomonas mendocina. The capability of aerobic denitrification by strain GL6 was evaluated, and the functional genes related to nitrogen transformation were amplified. In addition, according to response surface methodology (RSM) analysis, the factors that influence the performance of strain GL6 were comprehensively evaluated. The application of strain GL6 in wastewater treatment plant effluent was conducted to investigate its potential for use in bioaugmented treatment. The results of the current study may contribute to the development of enhanced bioremediation methods for wastewater treatment plant effluent.In this study, an aerobic denitrifier, strain GL6, was isolated from surface flow constructed wetland located in a wastewater treatment plant and identified as 3, 0.720; sodium citrates, 4.08; K2HPO4, 1.00; KH2PO4, 1.00; MgSO4·7H2O, 0.200; and trace elements solution (2.00 ml/L). The final pH of the medium was adjusted to 7.20. The trace element solution contained (g/L): Na2EDTA, 57.1; ZnSO4·7H2O, 3.90; FeSO4·7H2O, 5.00; CaCl2·2H2O, 7.00; MnCl2·4H2O, 5.10; (NH4)6Mo7O24·4H2O, 1.10; CuSO4·5H2O, 1.60; and CoCl2·6H2O, 1.60 (pH = 6.00). LB (Luria–Bertani) medium included (g/L): yeast extraction, 5.00; Peptone, 10.0; and NaCl, 10.0, which was used for preculture.The sources of aerobic denitrifier isolation were sediment samples from the surface flow constructed wetland located in a wastewater treatment plant in Wuxi city, Jiangsu Province, China. The aerobic denitrification (AD) medium consisted of (g/L): KNOFresh sediment sample (10 mL) was inoculated into 100 mL of sterile AD medium in the 250 mL Erlenmeyer flask, and incubated at 30 °C and 160 rpm to enrich for denitrifying consortium. After three days of culture, the bacterial suspensions were transferred into fresh AD medium with 10% inoculation ratio. This process of subculture was repeated three times. The enriched bacterial suspensions were diluted in a gradient and spread on the AD medium agar plates, then cultivated at 30 °C for about 48 h. The colonies with different apparent features were picked and purified via repeated streaking on the fresh agar plates. At last, the isolates were inoculated in the AD medium to check their denitrifying activity. The strain with high nitrogen removal ability was stored in 20% glycerol solution at −80 °C.http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) [https://www.megasoftware.net/) using the neighbor-joining method with 1000 bootstrap replicates [The selected isolate GL6 with the highest TN  removal efficiency was characterized and identified using standard biochemical tests in Bergey’s Manual of Determinative Bacteriology . Moreoveast.cgi) . A phyloplicates .napA, nirK, nirS, norB, and nosZ amplification are shown in napA, norB, at 55 °C for nirS, nirK, nosZ; extension at 72 °C for 60 s; the last step was extension at 72 °C for 5 min. All PCR products were analyzed with electrophoresis on a 1.5% agarose gel and sequenced as the 16S rRNA amplicon.The total genomic DNA of strain GL6 was extracted using the Genomic DNA Extraction Kit  in accordance with the manufacturer’s instructions. The primers used for 3−-N (nitrate nitrogen) concentration was maintained at 100 mg/L. Strain GL6 was precultured overnight in LB medium. Then, 2 mL cell suspension was inoculated in a 500 mL flask containing 200 mL AD medium. The cultures were cultivated at 30 °C and 160 rpm. NO3−-N concentration and OD600  in the cultures were determined after 48 h incubation. The above experiments were conducted in triplicate.To seek the optimal carbon source for strain GL6, sodium acetate, sodium succinate, potassium sodium tartrate, glucose, sucrose, ethanol, and methanol were selected separately as the sole carbon source instead of sodium citrate in the AD medium, to keep a C/N ration 10. The initial NOg). An inoculum concentration of 1% (v/v) suspension cells was aseptically transferred into the AD medium. The culture was cultivated at 30 °C and 160 rpm for 48 h. Cultures were sampled at 3 h intervals to determine OD600, the concentrations of NO3−-N (nitrate nitrogen), NO2−-N (nitrite nitrogen), NH4+-N (ammonium nitrogen), and TOC . Each experiment was performed in triplicate.Strain GL6 was precultured at 30 °C and 160 rpm using LB liquid medium for 12 h. The biomass was harvested by centrifugation . An inoculum concentration of 1% (v/v) suspension cells was aseptically transferred into the AD medium in the flasks. After 48 h of culture, the samples were taken to determine the TN removal efficiency. Design-Expert 10.0 program was utilized to analyze the experimental results statistically.The response surface methodology (RSM) is a statistical technique that establishes multiple quadratic regression equations to fit the functional relationship between factors and response values, and seeks the optimal process parameters for desirable responses through the analysis of regression equation. The TN removal rate was optimized with RSM using the Box–Behnken design. There are four independent variables with three levels: temperature , pH value , C/N ratio , and shaking speed  . A total3−-N, 11.94 ± 0.63; NH4+-N, 0.13 ± 0.06; COD , 15.26 ± 2.3; TN, 12.78 ± 0.85. Strain GL6 was cultured in LB medium for 12 h, then it was inoculated into the 250 mL flask containing 100 mL filter sterilized wastewater with 10% inoculation ratio. Culture was supplemented with sodium citrate (at optimized concentration) as carbon source and cultivated under the optimal conditions above. Concentrations of NO3−-N, NH4+-N, and TN were determined periodically. Raw wastewater without the addition of carbon source and bacterial inoculum served as control. All experiments were performed in triplicate.The wastewater used in the current experiment was the effluent of a municipal wastewater treatment plant (A2/O process) in Wuxi, China. The wastewater contained (mg/L): NO3−-N, NH4+-N, NO2−-N, and TN were detected by the standard methods [g) from the TN of noncentrifuged medium.The growth of GL6 was measured using a spectrophotometer at a wavelength of 600 nm . The concentrations of NO methods . TOC wasP. mendocina NK-01 with a similarity of 99%. A neighbor-joining phylogenetic tree was constructed based on 16S rRNA of strain GL6 and some other phylogenetically related strains (P. mendocina were in the same group. The strain is gram-negative and rod-shaped (approximately 0.4–0.5 × 1.5–2.0 μm)  of strain GL6 was obtained and submitted to the GenBank under the accession number MF784388. The BLAST results indicated that strain GL6 was closely related to  strains a. Result–2.0 μm) b. The conapA gene was amplified successfully from strain GL6, which proved the ability for nitrate reduction under aerobic conditions. The BLAST results indicated that the napA gene sequence similarity reached 99% between strain GL6 and Pseudomonas mendocina NK-01 (CP002620.1). This result further confirmed that the amplified gene fragment was napA. The major biological denitrification pathway was reduction of NO3−-N to N2 in the chemical steps of NO3−-N→NO2−-N→NO→N2O→N2, catalyzed by nitrate reduction (NR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (NOS), respectively [napA gene encodes the large catalytic subunit of NAP (periplasmic nitrate reductase). NAP can function under aerobic or anaerobic conditions but is primarily expressed under aerobic conditions. NAP is not dependent on nitrate transport through the cytoplasmic membrane and can catalyze the reduction of nitrate to nitrite in aerobic denitrification [Thiosphaera pantotropha [Pseudomonas stutzeri strain T1 [Klebsiella pneumoniae CF-S9 [Pseudomonas aeruginosa PCN-2 [napA gene. Hence, napA is usually employed as a functional marker to identify aerobic denitrifying bacteria [As shown in K-01 CP0020.1. Thification . It is rtotropha , Pseudomae CF-S9 , and Psesa PCN-2  express bacteria .1-containing nitrite reductase encoded by the genes nirK and nirS, respectively [nirK was successfully amplified while the amplification of nirS failed. The BLAST results also showed that the nirK gene sequence of strain GL6 was closely related to that of Pseudomonas mendocina S5.2 (CP013124.1) (similarity 99%). The results suggest that strain GL6 could only express copper nitrite reductase. This was in agreement with the claim that the two nitrite reductases could not be expressed simultaneously in the same bacteria. [norB gene and a 250 bp length fragment of the nosZ gene were amplified from strain GL6  (similarity 99%). The last denitrification step is the reduction of nitrous oxide to nitrogen, which is catalyzed by NOS. The presence of the nosZ gene in strain GL6 may reduce the accumulation of greenhouse gas N2O. The BLAST results indicated that nosZ from strain GL6 and Pseudomonas mendocina strain NCTC 10897 (LR134290.1) may have a relatively close phylogenetic relationship.The ectively . NOR catnapA, nirK, norB, nosZ genes provided additional proof of aerobic denitrification by strain GL6, and further demonstrated that the nitrate removal of strain GL6 could be accomplished by the complete aerobic denitrification pathway.The successful amplification of the Carbon source is one of the important factors influencing cell growth and aerobic denitrification ability, for it serves as energy and electron donor source . Thus, d600, and TOC with time. Herein, OD600 was employed as an indicator for the bacterial biomass, and TOC for the consumption of carbon sources. Within 6 h after inoculation, the OD600 barely increased and kept low values. This phenomenon indicated that bacteria grew slowly and acclimated to the liquid medium. This stage is the lag phase of strain GL6. From 6 to 18 h, the biomass (indicated by OD600) increased exponentially. Meanwhile, the nitrate-N concentration decreased significantly, as did the TN and TOC concentrations. After 18 h, the strain entered the stationary phase. OD600 reached the peak value of 0.741 and subsequently decreased slightly. TOC concentration decreased from 931.2 to 49.5 mg/L within 24 h with a removal efficiency of 94.7%, and TN removal efficiency was 74.4% at the same time. The results showed that the strain could remove nitrate and organic carbon simultaneously through heterotrophic aerobic denitrification.The ability of aerobic denitrification by strain GL6 was evaluated with potassium nitrate as the unique nitrogen source. −1·h−1. The removal rate was higher than that of Enterobacter cloacae HNR with 4.58 mg (NO3−-N)·L−1·h−1 [Klebsiella pneumoniae CF-S9 with 2.20 mg (NO3−-N)·L−1·h−1 [Vibrio diabolicus SF16 with 2.83 mg (NO3−-N)·L−1·h−1 [Enterobacter sp. strain FL with 31.0 mg/L [Pseudomonas stutzeri strain XL-2 with 47.7 mg/L [From 18 to 24 h, the rate of decrease in nitrate-N concentration turned down. Nitrate-N concentration was basically unchanged after 24 h. The same trend in TN and TOC concentrations was observed. Strain GL6 removed 97% of nitrate-N within 15 h with an average removal rate of 6.61 mg (N)·L·L−1·h−1 , Klebsie·L−1·h−1 , and Vib·L−1·h−1 . Consequ1.0 mg/L  and Pseu7.7 mg/L . Ammoniu4+-N, NO2−-N, and organic nitrogen. The comparison between the initial and final nitrogen concentrations can be calculated such that 20.2% of initial nitrate-N was converted into intracellular-N, and 5.37% was transformed into other nitrogen compounds . Therefore, it is concluded that approximately 74.4% of the initial nitrogen was lost, probably converted into nitrogenous gases by aerobic denitrification. This result was consistent with the previous studies [Nitrogen balance was conducted with nitrate as the sole nitrogen source. As shown in  studies ,31. Ther1, X2, X3, and X4 are the coded values of C/N ratio, pH, shaking speed, and temperature, respectively.It was found that aerobic nitrate removal of strain GL6 was greatly affected by four factors: C/N ratio, pH, shaking speed, and temperature. Moreover, the interactive effects of these factors were determined by RSM with a Box–Behnken design. The experimental tests and corresponding results are shown in 2) of this regression model was 0.864 and the probability value (P) was 0.0007 . As shown in 3−-N concentration decreased from 11.94 to 0.52 mg/L within 24 h with a removal efficiency of 95.6%, and TN removal efficiency was 73.6% at the same time. Meanwhile, NO2−-N could not be detected and NH4+-N concentration was insignificant in the whole experimental period. In the controls, there was almost no removal of NO3−-N and TN during the assay. Therefore, strain GL6 has potential application in the advanced treatment of wastewater treatment plant effluent. Nevertheless, subsequent experiments should be carried out to discuss the strategies of bioaugmentation and the survival capability of the strain GL6 in the practical wastewater treatment system [t system .Pseudomonas mendocina strain GL6 was newly isolated from the surface flow constructed wetland. The denitrification genes napA, nirK, norB, and nosZ were amplified. The optimum conditions for aerobic denitrification by strain GL6 were sodium citrate as the carbon source, C/N ratio of 11.2, pH of 7.76, shaking speed of 133 rpm, and temperature of 27.8 °C. Moreover, strain GL6 was inoculated into wastewater treatment plant effluent and showed the nitrate nitrogen removal rate of 95.6% and the TN removal rate of 73.6%. Thus, P. mendocina strain GL6 exhibited potential applications for the bioremediation of polluted water.


Oscillatoria spp. and other harmful cyanobacteria, but its effector mechanism and algicidal threshold are unknown. In this study, we examined the algicidal effect of CZBC1 on O. chlorina, O. tenuis, and O. planctonica by microscopic enumeration and scanning electron microscopy observation. Then, we examined the alginolytic effects of CZBC1 (concentrations 103–106 colony forming units (cfu)/mL) on these three species (concentrations 103–106 cells/mL) to determine the effective concentrations of CZBC1 for Oscillatoria spp. alginolysis. Results showed that CZBC1 can directly lyse O. chlorina and O. tenuis but indirectly lyse O. planctonica. When the initial concentration of CZBC1 was 106 cfu/mL, alginolytic effects were high for all three species at all concentrations, and the alginolytic rate could reach 100% in 3–9 days. When the initial concentration of CZBC1 was lower (103 cfu/mL), its inhibitory effects were delayed by 2–5 days, but the cell counts were significantly decreased compared with the control, evidencing significant alginolysis. In addition, the higher the concentration of the algicidal bacteria suspension, the more significant the alginolytic effects. Our results indicate that CZBC1 has different alginolytic mechanisms for O. chlorina, O. tenuis, and O. planctonica, and that different initial concentrations of CZBC1 have different alginolytic effects on these algal species.Frequent harmful cyanobacteria blooms limit the sustainable development of aquaculture. Algicidal bacteria can efficiently control harmful algae without secondary pollution. The algicidal bacteria CZBC1 can lyse  Oscillatoria species are dominant cyanobacteria in prawn aquaculture ponds /mL of the bacterium Alcaligenes denitrificans could lyse Microcystis aeruginosa. Peng et al. , and a national invention patent was issued  and O. planctonica (FACHB-708) were obtained from the freshwater algal species repository of the Institute of Hydrobiology, Chinese Academy of Sciences.We selected g for 2 min. The resulting supernatant was separated from the pellet and used as a sterile filtrate, while the pellet was washed with sterile water and centrifuged again at 9391×g for 2 min; the resulting supernatant was then discarded. This process was repeated three times. The collected pellet was resuspended in sterile water to the required concentration of bacterial suspension for subsequent experiments.A small amount of CZBC1 stored in preserving solution was inoculated to a nutrient agar plate under aseptic conditions and then activated at 28 °C for 24 h. A few activated CZBC1 colonies were inoculated into a 250-mL sterile conical flask containing 100 mL of sterile nutrient broth, and the flask was placed in a shaking incubator at 30 °C for 16 h. The CZBC1 bacterial suspension was collected and centrifuged at 9391×Oscillatoria chlorina, O. tenuis, or O. planctonica was inoculated aseptically into a sterile 1000-mL conical flask containing 600 mL of sterile BG11 culture broth and placed in an illuminated incubator. Expansion culture was carried out under 28 °C, 2500 lx light intensity, and a light/dark cycle of 12/12 h. The culture flasks were periodically shaken throughout the day to prevent the algal cells from attaching to the walls, and the culture was carried out until the algae reached the logarithmic growth phase.O. chlorina, O. tenuis, or O. planctonica in the logarithmic growth phase. The volume of the co-culture system was 100 mL. The initial concentration of O. chlorina, O. tenuis, and O. planctonica was 6.8 × 106 cells/mL, 1.21 × 107 cells/mL, and 3.87 × 106 cells/mL, respectively. In the culture system, the concentration of CZBC1 was 107 cfu/mL and an equal volume of sterile water was added to the control group. Parallel triplicates were set up for each group and placed in an illuminated incubator. The flasks were cultured at 28 °C, 2000 lx light intensity, and a light/dark cycle of 12/12 h for 7 days. After the bacterial suspension and sterile filtrate were added to the algal solution, sampling was carried out immediately and then once every 24 h. The samples were fixed with formaldehyde and optical microscopy was used to observe algal cell morphology and to enumerate algal cells.Next, the CZBC1 suspension or the sterile filtrate (10 mL) was added to a sterilized 250-mL conical flask containing 90 mL of g for 10 min to collect algal cells, which were fixed with 2.5% glutaraldehyde for more than 3 h. The algal cells collected by centrifugation were washed 4–6 times with 0.1 M phosphate buffer for 20 min/wash. Then, ethanol dehydration was performed using 30%, 50%, 70%, and 90% for 15 min each, followed by three anhydrous ethanol dehydration steps for 15 min each time. The cells were placed in tert-butanol three times, 15 min each time, and, after collecting algal cells, tert-butanol was added, and the cells were added to a slide for carbon dioxide critical point drying, metal spraying, and observation by scanning electron microscopy.After the CZBC1 suspension or the sterile filtrate was added to the algal solution, sampling was carried out immediately and then once every 6 h. The volume of each sample was 5 mL. The samples were centrifuged at 438×3 cfu/mL (J-3 group), 104 cfu/mL (J-4 group), 105 cfu/mL (J-5 group), and 106 cfu/mL (J-6 group). An equal volume of sterile water was added to the control group. Parallel triplicates were set up for each group . In the culture system, the concentrations of O. chlorina, O. tenuis, and O. planctonica were 103 cells/mL, 104 cells/mL, 105 cells/mL, and 106 cells/mL. Different concentrations of CZBC1 suspension (10 mL) were collected and added to 90 mL of algal solution at different concentrations. The flasks were cultured at 28 °C, 2000 lx light intensity, and a light/dark cycle of 12/12 h for 9 days. Sampling was carried out immediately after adding the bacterial suspension, and then once every 24 h.The initial concentrations of CZBC1 used were 10P < 0.05. Alginolysis rate (%) was calculated as (1 − Nt/N0) × 100, where Nt is the algal cell count at time t, and N0 is the initial algal cell count  × 100, where NJt is the algal cell count at time t in the group to which the bacterial suspension was added and NCt is the algal cell count at time t in the control group.SPSS 20.0 software (IBM) was used to carry out one-way analysis of variance (ANOVA) followed by Duncan’s test. The significance level was set as Oscillatoria chlorina and O. tenuis showed the same trend. In the control groups, O. chlorina (Oc-C) and O. tenuis (Ot-C) showed logarithmic growth, and the algal cell counts on day 7 were 3.79 × 107 cells/mL and 2.62 × 107 cells/mL, respectively. The cell counts of both species started to decrease in the bacterial suspension groups (Oc-B and Ot-B), where O. chlorina cells died by day 4 and O. tenuis cell count on day 7 was 5 × 105 cells/mL. Microscopy analysis showed alginolysis of O. chlorina and O. tenuis cells, and flocculation could be observed at the bottom of the conical flasks. In the sterile filtrate groups (Oc-F and Ot-F), the cells increased to 3.11 × 107 cells/mL and 2.48 × 107 cells/mL on day 7, which was not significantly different from that observed for the control group  showed logarithmic growth, and its cell count on day 7 was 2.19 × 107 cells/mL. In the bacterial suspension group (Op-B), O. planctonica cell count increased in the first 2 days, reaching 5.83 × 106 cells/mL on day 2. Subsequently, algal cell count decreased, and all cells died by day 7. In the sterile filtrate group (Op-F), O. planctonica started to decrease when the sterile filtrate was added, and all algal cells died by day 5 . In the J-3 and J-4 groups, the cell count increased in the first 2 days reaching 1.47 × 104 cells/mL and 1.27 × 104 cells/mL, respectively. Subsequently, the cell count decreased. On day 3, the alginolysis rate was 76.39% and 84.72%, respectively, and by day 6 all O. chlorina cells of both groups had died. In the J-5 and J-6 groups, the cells started to decrease after the bacterial suspension was added. On day 1, the alginolysis rate was 75.69% and 86.11%, respectively, and all O. chlorina algal cells had died by day 5 and day 4, respectively.When the initial cell count of /mL Fig. a, cells O. chlorina was 104 cells/mL . The cell count increased in the J-3, J-4, and J-5 groups after the bacterial suspension was added, reaching a maximum of 5.46 × 104 cells/mL, 6.33 × 104 cells/mL, and 2.3 × 104 cells/mL, respectively. Subsequently, the cell count of the three groups started to decrease, and all algal cells had died by days 7, 7, and 5, respectively. In the J-6 group, the alginolysis rate on day 1 was 78.30% and all algal cells had died by day 5.When the initial cell count of /mL Fig. b, cells O. chlorina was 105 cells/mL . The cell count increased in the J-3, J-4, and J-5 groups after the bacterial suspension was added, reaching a maximum of 5.83 × 105 cells/mL, 5.54 × 105, and 3.53 × 105 cells/mL, respectively. Subsequently, the cell count of the three groups started to decrease and all algal cells had died by days 9, 9, and 8, respectively. In the J-6 group, the alginolysis rate on day 1 was 79.55% and all algal cells had died by day 6.When the initial cell count of /mL Fig. c, cells O. chlorina was 106 cells/mL . On day 6, cell count was 2.43 × 107 cells/mL in the J-4 group. The cell counts of the J-3 and J-4 groups significantly decreased, and on day 9 of the experiment, the algal cell count reduction rates of the J-3, J-4, and J-5 groups were 72.73%, 97.02%, and 96.76%, respectively. The alginolysis rate in the J-6 group on day 5 was 84.59%, and all algal cells had died by day 9.When the initial cell count of /mL Fig. d, cells O. tenuis was 103 cells/mL . In the J-3 and J-4 groups, the cell count increased on the first day and reached 2.67 × 104 cells/mL and 1.66 × 104 cells/mL, respectively, before decreasing. On day 3, the alginolysis rate for O. tenuis was 76.39% and 84.06% in the J-3 and J-4 groups, respectively. By day 4, all O. tenuis in both groups had died. In the J-5 and J-6 groups, the cell count started to decrease after the bacterial suspension was added, and all algal cells from these two groups had died by day 3.When the initial cell count of /mL Fig. a, cells O. tenuis was 104 cells/mL . In the J-3 and J-4 groups, the cell count increased after the bacterial suspension was added, and the maximum cell count was 2.29 × 105 cells/mL and 1.25 × 105 cells/mL, respectively. Subsequently, algal cell counts in these two groups decreased, and all algal cells had died by day 6. In the J-5 and J-6 groups, the cell count started to decrease after the bacterial suspension was added, and all algal cells from these two groups had died by day 3.When the initial cell count of /mL Fig. b, cells O. tenuis was 105 cells/mL . The cell count increased in the J-3, J-4, and J-5 groups after the bacterial suspension was added, reaching a maximum of 1.23 × 106 cells/mL, 1.03 × 106 cells/mL, and 7.15 × 105 cells/mL, respectively. Subsequently, the O. tenuis cell count of the three groups started to decrease and all algal cells had died by days 8, 7, and 7, respectively. In the J-6 group, algal cell count started to decrease after the bacterial suspension was added. The alginolysis rate on day 3 was 98.02%, and all algal cells had died by day 4.When the initial cell count of /mL Fig. c, cells O. tenuis was 106 cells/mL . The cell count first increased before decreasing in the J-3, J-4, and J-5 groups, after the bacterial suspension was added, and peaked on day 4, day 4, and day 3, respectively, which was later than that observed for O. chlorina and O. tenuis. The cell count on these days was 1.30 × 105 cells/mL, 1.97 × 105 cells/mL, and 3.50 × 104 cells/mL, respectively. Subsequently, the cell count of the three groups started to decrease and all algal cells had died by day 8, day 7, and day 6, respectively. In the J-6 group, algal cell count started to decrease after the bacterial suspension was added, and all algal cells had died by day 3.When the initial cell count of /mL Fig. a, cells O. planctonica was 104 cells/mL . The cell count increased in the J-3, J-4, and J-5 groups after the bacterial suspension was added, reaching a maximum of 1.72 × 105 cells/mL, 1.50 × 105 cells/mL, and 8.13 × 104 cells/mL, respectively. Subsequently, the O. planctonica cell count of the three groups started to decrease, and all algal cells had died by day 8, day 7, and day 6, respectively. In the J-6 group, algal cell count started to decrease after the bacterial suspension was added, and all algal cells had died by day 4.When the initial cell count of /mL Fig. b, cells O. planctonica was 105 cells/mL . The cell count increased in the J-3, J-4, and J-5 groups after the bacterial suspension was added. In the J-3 group, cell count peaked on day 5 with a cell count of 6.44 × 105 cells/mL, in the J-4 group it peaked on day 4 with a cell count of 4.29 × 105 cells/mL, and in the J-5 group it peaked on day 3 with a cell count of 2.22 × 105 cells/mL. Subsequently, the cell count of the three groups started to decrease, and all algal cells had died by days 8, 7, and 6, respectively. In the J-6 group, algal cell count started to decrease after the bacterial suspension was added. The alginolysis rate on day 1 was 83.71%, and all algal cells had died by day 5.When the initial cell count of /mL Fig. c, cells O. planctonica was 106 cells/mL  of Lactobacillus paraplantarum might be a prerequisite for achieving successful termination of natural blooms. And Shao et al. , its inhibitory effects on the different initial concentrations of O. chlorina, O. tenuis, and O. planctonica were delayed by 2–5 days. However, the cell counts significantly decreased compared with those of the control group, showing significant alginolysis.The alginolytic effects of algicidal bacteria on algae are associated with bacterial concentration. Su et al.  and Huano et al.  reportedn et al.  studied O. chlorina and O. tenuis and indirect alginolytic effects on O. planctonica, and we directly determined algal cell count to more accurately assess alginolytic effects. Comparisons performed in the present study revealed that when the initial algal cell count was 103–105 cells/mL, the alginolytic effects of CZBC1 appeared earlier on O. chlorina and O. tenuis than on O. planctonica, and direct alginolysis was used to rapidly achieve alginolysis.Most studies conducted so far have considered chlorophyll content to assess the alginolytic effects of algicidal bacteria  once every 7–10 days. After cyanobacteria blooms, CZBC1 application should be increase to 106 cfu/mL 2–3 times to control Oscillatoria and other cyanobacteria.The cell count of ases Cao . AccordiOscillatoria species and compared the alginolytic effects of different initial concentrations of CZBC1 on different cell counts of O. chlorina, O. tenuis, and O. planctonica. Our study showed that CZBC1 can directly lyse O. chlorina and O. tenuis and indirectly lyse O. planctonica. The higher the concentration of this algicidal strain, the more significant its alginolytic effects against the three Oscillatoria species. We also determined the effective application concentration of CZBC1. We will carry out further studies on the transformation routes of algal debris after cyanobacteria lysis by algicidal bacteria, and the residual form and attenuation effects of cyanobacterial toxins. At the same time, the physiological and molecular mechanisms of the alginolytic effects of CZBC1 will also be studied.In summary, our study examined the alginolytic effects of the algicidal strain CZBC1 on three


A total of four metabolites of sibirioside A were identified: SM1, SM2 and SM3 which were known as new compounds. A total of 25 metabolites were detected for angoroside C: AM4, AM5, AM6, AM7, AM16, AM17, AM20, AM21, AM22, AM23 and AM25 which were identified to be new compounds. The main metabolic reactions were hydrolysis, reduction, hydroxylation, methylation, sulfation, and gluconylation. The prototype of sibirioside A was widely distributed in tissues found in the heart, liver, spleen, lung, kidney, stomach and small intestine of rats, and mainly distributed in the stomach, small intestine, kidney and liver. But for angoroside C, nothing was found in the viscera except the stomach and small intestine. The metabolites of sibirioside A were mainly eliminated from feces, while it was urine for the metabolites of angoroside C. Furthermore, 19 metabolites were likely to have bioactivities based on the ‘PharmMapper’ analysis, which roughly matched the known pharmacological activities of Scrophulariae Radix (SR) and the prototypes. One of the main pharmacological activities of SR in traditional Chinese medicine is anti-diabetes, and the predicted results showed that SM1, SM2, SM3, AM2, AM4, AM5, AM6, AM9, AM10, AM11, AM12, AM13, AM15, AM18, AM19, AM24, and AM25 might be used to cure diabetes. These findings provide a reference for studying the metabolism, distribution and pharmacological actions of phenylpropanoid glycosides in vivo.Sibirioside A and angoroside C are two important phenylpropanoid glycosides of the traditional Chinese medicine Scrophulariae Radix. High performance liquid chromatography, coupled with an ion trap time-of-flight multistage mass spectrometry equipped with electrospray ionization source (HPLC-ESI-IT-TOF-MS Scrophularia ningpoensis Hemsl. −) and m/z 471.1414 ([M − H]−). Its MS2 spectra fragments m/z 323.0918 ([M-cinnamic acid − H]−), m/z 309.0991 ([M − fructosyl − H]−), m/z 179.0523 ([glucose − H]− or [fructose − H]−), m/z 161.0401 ([glucose − H2O − H]− or [fructose − H2O − H]−). The quasimolecular ion peak of angoroside C was m/z 783.2513 ([M − H]−), and the MS2 fragments were shown as m/z 607.1993, m/z 475.1826, m/z 461.1514, m/z 443.1411, m/z 329.1078, m/z 325.0911. The fragmentation processes of sibirioside A and angoroside C were shown in Aqueous solutions of the reference substances, sibirioside A and angoroside C, were directly injected for analysis to obtain the fragmentation characteristics of the parent compounds. Several characteristic fragments of sibirioside A and angoroside C were observed  in the nIn total, four sibirioside A metabolites and 25 angoroside C metabolites were identified, and the Liquid Chromatography Mass Spectrometry (LC-MS) results are shown in SM1 at m/z 229.0153 ([M − H]−) was detected in feces and urine samples, and speculated as C9H10O5S. Its degrees of unsaturation were five, meaning there was only one unsaturated bond except for the phenyl group in the molecule. The MS2 spectrum showed fragment at m/z 147.0459 (speculated to be [C9H8O2 − H]−), and its MS3 fragment ion at m/z 103.0348 ([C9H8O2 − CO2 − H]−) indicated that C9H8O2 might be cinnamic acid. Cinnamic acid was the product of SM1 with 82 Da lost, which was considered to be H2SO3. It is well-known that the sulphated compounds could easily lose 80 Da radical group (SO3) instead of 82 Da, but the neutral molecule H2SO3 might be lost if there was active-H in the phenylpropionic acid structure. Then we could conjecture SM1 as phenylpropionic acid sulphate. The cinnamic acid first reacted with glutathione and then hydrolyzed into the cysteine adduct by peptidase, and the cysteine β-liase gave a thiol immediately after, and finally oxidized to be SM1.The negative quasi-molecular ion of SM2 detected in feces samples showed quasi-molecular ion peak at m/z 259.0293 ([M − H]−), indicating C10H12O6S as the predicted formula. The characteristic fragment ion peaks were shown as m/z 177.0540 ([M − H2SO3 − H]−) and m/z 133.0983 ([M − H2SO3 − CO2 − H]−). Its trends of neutral loss conformed with those of SM1, and all the fragment ion peaks were 30 Da (predicted to be CH2O) larger than the correspondent peaks of SM1, which suggested the substitution of methoxyl group. The methoxyl group was the best possibility to substitute at the aromatic ring owing to the absence of methyl (14 Da) or methoxyl (30 Da) loss in MS2 spectrum. Then it could be speculated that SM2 might be 4-methoxyphenylpropanoic acid sulphate in consideration of the substitutive resistance.SM3 was observed in feces samples, and showed quasi-molecular ion peak at m/z 393.0755 ([M − H]−). The fragment ion at m/z 311.1019 ([M − H2SO3 − H]−) suggested SM3 to be a sulphate, and it could be dissociated to get m/z 163.0583 and m/z 147.0454. There was no fragment ion at m/z 103.03 rooted from m/z 147.0454, which indicated that the ion at m/z 147.0454 was not cinnamic acid. According to the elemental composition C9H8O2, this fragment was most likely to be β-oxobenzenepropanal. In accordance with the above analysis, SM3 might be β-hydroxybenzenepropanoic anhydride sulphate.The different chromatographic peak of SM4 detected in stomach samples was predicted to be C27H38O17 according to the quasi-molecular ion peak at m/z 633.2014 ([M − H]−). Its MS2 spectrum showed fragment ion peaks at m/z 485.1416 ([M − cinnamoyl − H]−) and m/z 323.0932 ([sucrose − H]−). Neutral loss of 148 Da (cinnamic acid) occurred from the parent ion to obtain m/z 485.1416, then neutral loss of 162 Da (glucosyl) occurred from m/z 485.1416 to get m/z 323.0932, which was the main fragment ion of sibirioside A predicted to be [sucrose − H]−. To sum up, SM4 was conjectured to be a sibirioside A glucose conjugation.The molecular formula of The metabolites and metabolic pathways of sibirioside A in rats are shown in AM1 showed [M − H]− ion peak at m/z 233.0119 ([M − H]−) in the negative MS1 spectrum, which was predicted as C8H10O6S. The fragment ion peaks at m/z 153.0583 ([M − SO3 − H]−) and m/z 123.0492 ([M − SO3 − CH2O − H]−) were observed in negative MS2 spectrum, and m/z 153.0583 should be the phenylethanoid unit derived from angoroside C. Therefore, AM1 was speculated to be 4-(2-hydroxyethyl)-1,2-benzenediol sulphate.AM2 was determined to be C9H8O6S based on its quasi-molecular ion peak at m/z 242.9965 ([M − H]−). The characteristic fragment ion peaks were observed as m/z 163.0415 ([M − SO3 − H]−) and m/z 119.0532 ([M − SO3 − CO2 − H]−). Therefore, the ion at m/z 163.0415 was thought to be the demethoxylation product of ferulic acid, which was the hydrolysate of angoroside C. And AM2 was thus determined as p-coumaric acid sulphate. It was easy to confirm that AM3 was 4-hydroxybenzenepropanoic acid sulphate, the hydrogenation product of AM2 based on the quasi-molecular ion peak at m/z 245.0109 ([M − H]−) and its MS2 fragment ion peaks at m/z 165.0574 ([M − SO3 − H]−) and m/z 121.0680 ([M − SO3 − CO2 − H]−).The formula of AM4, AM5 and AM6, the exact formula C9H12O6S was calculated based on the quasi-molecular ion peak at m/z 247.03 ([M − H]−). The ion at m/z 167.07 ([M − SO3 − H]−) was detected in MS2 spectrum, which suggested that they were 3,4-hydroxyphenylpropanol sulphate. And there were three reaction sites in 3,4-hydroxyphenylpropanol for sulphating, so the three metabolites were considered as isomers.For AM7 at m/z 259.0271 ([M − H]−) suggested the formula as C10H12O6S. And the fragment ion peaks at m/z 179.0726 ([M − SO3 − H]−), m/z 163.0385 ([M − SO3 − CH2 − H]−), m/z 147.0473  and m/z 119.0507 ([M − SO3 − CH2 − CO2 − H]−) indicated the metabolite might be methoxybenzenepropanoic acid sulphate, which was a isomer of SM2.The quasi-molecular ion peak of AM8 and AM9 were speculated to be C9H10O7S owing to the quasi-molecular ion peak at m/z 261.01 ([M − H]−) in negative ion mode MS1 spectrum. The multistage fragment ion peaks at m/z 217.0175 ([M − CO2 − H]−), m/z 181.0522 ([M − SO3 − H]−) and m/z 137.0633 ([M − SO3 − CO2 − H]−) indicated that they were 3,4-dihydroxybenzenepropanoic acid-O-sulphate.m/z 275.02 ([M − H]−) observed in the negative MS1 spectrum indicated the formula C10H12O7S. Then, it was speculated that AM10 was 3-methoxyl-4-hydroxybenzenepropanoic acid sulphate and that AM11 was isomer of it owing to the fragment ion peaks at m/z 195.0665 ([M − SO3 − H]−), m/z 151.0799 ([M − SO3 − CO2 − H]−) and m/z 177.0777 ([M − SO3 − H2O − H]−) in the MSn spectrum.The quasi-molecular ion peak at − peaks of AM12, AM13 and AM14 were observed at m/z 329.09, suggesting the formula as C14H18O9. The parent ion was cleaved into two parts m/z 175.0282 ([glucuronic acid − H2O − H]−) and m/z 153.0568. And the ion at m/z 153.0568 was determined to be 3,4-dihydroxyphenylethanol similar to AM1, which has three reaction sites for gluconylation. The above data suggested AM12, AM13 and AM14 to be 4-(2-hydroxyethyl)-1,2-benzenediol glucuronide.The [M − H]AM15, speculated to be C15H18O9, was detected in urine samples with quasi-molecular ion peak at m/z 341.0848 ([M − H]−). The MS2 fragment ion peaks at m/z 165.0520 ([M − glucuronic acid − H]−) and m/z 121.0628 ([M − glucuronic acid − CO2 − H]−) were just similar to AM3, and in combination with m/z 175.0244, AM15 was determined to be 4-hydroxybenzenepropanoic acid glucuronide. And the MS1 chromatographic peaks of AM16 and AM17 were extracted in urine sample at m/z 343.1017 ([M − H]−). Their fragment ion peaks at m/z 175.0265 and m/z 167.0713 indicated that they were 3,4-hydroxyphenylpropanol glucuronide, the reduzates of AM15.− peak of AM18 at m/z 369.0794 (C16H18O10) was detected in urine samples. The MS2 fragment ion at m/z 175.0169 suggested the existence of glucuronic acid, and m/z 193.0508 (C9H10O4) was consistent with the phenylpropanoid unit of angoroside C. Therefore, AM18 might be 3-methoxyl-4-hydroxycinnamic acid glucuronide. And the quasi-molecular ion peak at m/z 371.0988 ([M − H]−) and the MS2 fragment ion peaks at m/z 175.0265 and m/z 195.0658 of AM19 suggested that it could be the hydrogenation product of AM18, namely 3-methoxyl-4-hydroxybenzenepropanoic acid glucuronide.The [M − H]m/z 637.21 were the quasi-molecular ions of AM20 and AM21, which was the characteristic fragment ion of angoroside C. And the MS2 fragment ion peaks at m/z 461.1636, m/z 311.1151, m/z 193.0548, m/z 167.1269 and m/z 137.0759 were also in conformity with angoroside C. Then they might be the isomers of deglycosylated angoroside C. For AM22 and AM23, the quasi-molecular ion peaks at m/z 959.30 and main MS2 fragment ion peak at m/z 783.26 ([angoroside C − H]−) were observed, which indicated AM22 and AM23 to be isomers of angoroside C glucuronide.The ion peaks at AM24 detected in feces samples was predicted to be C20H30O12 based on the quasi-molecular ion peak at m/z 461.1701. The fragment ion peaks at m/z 443.1711 and m/z 329.1180 could also be observed in the MS2 spectrum of angoroside C. So it could be speculated that AM24 was the hydrolysate of deferuloyl-angoroside C, namely 2-(3-hydroxy-4-methoxyphenyl)ethyl 6-O-arabinopyranosyl glucopyranoside.The formula of AM25 at m/z 247.0266 ([M − H]−) was detected in feces and plasma samples, and speculated as C9H12O6S. The fragment ion peaks at m/z 167.0750 ([M − SO3 − H]−), m/z 137.0627 ([M − SO3 − CH2O − H]−) and m/z 121.0264 ([M − SO3 − CH2O − CH4 − H]−) suggested that AM25 was 4-(2-hydroxyethyl)-1,2-benzenediol sulphate.The negative quasi-molecular ion of The metabolites and metabolic pathways of angoroside C in rats are shown in m/z 471.14 and m/z 783.25 were extracted in negative MS1 spectrum. The extract ion flow chromatography (EIC) of different biological samples are shown in To investigate the distribution of the prototypes of sibirioside A and angoroside C, the corresponding quasi-molecular ion peaks at SM1, SM2, SM3, AM2, AM4, AM5, AM6, AM9, AM10, AM11, AM12, AM13, AM15, AM18, AM19, AM24, AM25 might be used to cure diabetes, which was one of the main pharmacological activities of SR in traditional Chinese medicine. In addition, SM3, AM2, AM5, AM8, AM9, AM10, AM11, AM14, AM15, AM18, AM19, AM24 and AM25 may be available to target chronic leukemia; AM2, AM4, AM5, AM6, AM8, AM9, AM10, AM11, AM14 and AM24 may have osteogenic actions. The predicted outcomes were in good agreement with the actual activities reported in the literatures [The bio-activities of the metabolites of sibirioside A and angoroside C were predicted through “PharmMapper Server”, which based on a massive pharmacophore database containing relatively well-developed target information over 7000 pharmacophore models recorded in TargetBank, BindingDB, DrugBank, and other drug target databases ,31,32. IThe metabolites of sibirioside A and angoroside C have a variety of potential pharmacological actions, which require further research.SM1 was in urine, SM1–SM3 in feces, and SM4 in the stomach. A total of 25 metabolites of angoroside C were found and identified, of which AM1–AM23 were in urine, AM24 and AM25 were in feces, and AM25 in plasma. For sibirioside A, the main metabolic reactions were hydrogenation, hydroxylation, methylation, sulfation, glycosylation, and dimerization, while the metabolic reaction of angoroside C included hydrolyzation, hydrogenation, sulfation, gluconylation, demethylation, and dehydroxylation. The hydrolyzation reactions occurred in the sugar ester bonds, while the hydrogenation reactions were mainly in double bonds or carboxyl. The sulfation reactive site was the carboxyl group for sibirioside A, but it was the hydroxyl group for angoroside C. The results showed that gluconylation was one of the prime and representative metabolic reactions for angoroside C.A total of 4 metabolites of sibirioside A were found in biological samples, of which Sibirioside A is an ester glycoside consisting of cinnamic acid and sucrose, and there were less metabolites of it due to the simple molecular structure. The relatively large polarity allowed it to be easily excreted from the urine without the need for complicated metabolic processes. Catalyzed by a variety of enzymes, a portion of sibirioside A was transformed into the metabolites which were mainly sulfated products, then discharged from feces through the bile. The prototype of sibirioside A was detected in all the selected samples except for feces and plasma, which indicated that the rate of sibirioside A clearance was relatively fast in the blood, and it was mainly distributed in the heart, liver, spleen, kidney, stomach, and small intestine in the form of prototype. It was conjectured that sibirioside A might work in the original form and could be preserved for a long time in the viscera in order to extend the time for drug concentration.Angoroside C is a phenylpropanoid glycoside consisting of three monosaccharide, a phenylpropanoid unit, and a phenylethanoid unit. For angoroside C, total of 25 metabolites were identified, much more than that of sibirioside A, of which 22 were sulfate and glucuronide conjugates. The prototype of angoroside C was detected in urine, feces, stomach, and small intestine samples, but not observed in plasma. It was speculated that the clearance rate of angoroside C in plasma might be fast like sibirioside A. A total of 22 metabolites derived from urine samples had covered all types of metabolic reactions found in this study, and most of the prototype and metabolites were rapidly excreted from the urine.Sibirioside A  and angoroside C  were purchased from Chendu Must Bio-Technology Co., Ltd. . Acetonitrile  and formic acid  were of HPLC-grade, and ultrapure water was prepared by Milli-Q water purification system .Nine male Sprague-Dawley rats weighing around 250 g were obtained from the Experimental Animal Center of Peking University’s Health Science Center . The rats were housed in metabolic cages  kept in an environmentally controlled breeding room for one week for acclimation and had unrestricted access to laboratory-made chow and distilled water to reduce the biological background interferences. Nine rats were divided into three groups, namely three rats in each group . The standards of sibirioside A and angoroside C were dissolved in ultrapure water and orally administrated respectively to group B and group C at the dose of 200 mg/kg for six continuous days. The rats in group A were orally treated with the same volume of water. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. Experiments were reviewed by the Biomedical Ethical Committee of Peking University . During the continuous six days of administration, the feces and urine samples were dividually collected using the metabolic cages, and the heparin anticoagulant blood sample was collected by heart puncture under anesthesia 1 h after the last administration. The hearts, livers, spleens, lungs, kidneys and small intestines were quickly scissored and lavaged by normal saline, then the tissue samples were stored at −80 °C.Blood samples were centrifuged at 5000 rpm  for 15 min to obtain plasma samples. The plasma samples were precipitated with four times methanol, then centrifuged at 10,000 rpm for 15 min. The supernatant was dried under reduced pressure at room temperature. The residue was redissolved in one-twelfth of methanol and centrifuged at 15,000 rpm for 20 min. The supernatant was collected for analysis.Urine samples were dried under reduced pressure at 50 °C ; the residue was ultrasonically extracted in 50 mL methanol for 30 min. The suspension was centrifuged at 5000 rpm for 15 min, and the supernatant was dried under reduced pressure at 40 °C. The residue was redissolved in five times of methanol, and filtered through a 0.22 µm nylon filter.Feces samples were dried in a thermostat oven  at 50 °C, and then pulverized to get powders. The feces powders were ultrasonically extracted for 30 min in five times of 50% methanol, repeated three times. The extracts were merged and dried under reduced pressure at 40 °C, and the residue was redissolved in 10 times of methanol, then filtered through a 0.22 µm nylon filter.Organ tissues were homogenized by an ultra-turrax T10 electric homogenizer  with twice as much water, and five times of methanol was added into the homogenate. The mixture underwent ultrasonic processing for 30 min, thencentrifuged at 15,000 rpm for 20 min. The supernatant was dried under reduced pressure at room temperature. The residue was redissolved in methanol and centrifuged at 15,000 rpm for 20 min. The supernatant was collected for analysis.18 column  protected by an Agilent Zorbax SB-C18 column. A 10 μL aliquot was injected for each sample. The thermostatted autosampler was maintained at 15 °C and the column oven temperature was kept at 30 °C. The column was eluted with a gradient mobile phase consisting of 0.1% formic acid aqueous solution (solvent A) and acetonitrile (solvent B) with a flow rate of 1.0 mL/min. For the sibirioside A group, the gradient program was adopted in the following manner: 5–18% B at 0–20 min, 18–37% B at 20–40 min, 37–43% B at 40–43 min, 43–56% B at 43–63 min, 56–68% B at 63–65 min, 68–81% B at 65–75 min, 81–100% B at 75–95 min and 100% B at 95–140 min. For the angoroside C group, the gradient program was adopted in the following manner: 3% B at 0–8 min, 3–5% B at 8–10 min, 5% B at 10–20 min, 5–8% B at 20–23 min, 8–19% B at 23–38 min, 19% B at 38–45 min, 19–27% B at 45–50 min, 27% B at 50–63 min, 27–55% B at 63–73 min, 55% B at 73–83 min, 55–100% B at 83–93 min and 100% B at 93–140 min. High-resolution mass spectra were recorded on an IT-TOF mass spectrometer . The ESI source was operated in negative ion mode. The mass spectrometer was programmed to carry out a full scan over m/z 100–1000 Da (MS1) and m/z 50–1000 Da (MS2 and MS3). The other parameters were set as follows: flow rate, 0.2 mL/min (split from 1.0 mL/min HPLC effluent); heat block and curved desolvation line temperature, 200 °C; nebulizing nitrogen gas flow, 1.5 L/min; interface voltage: (+), 4.5 kV; (−), −3.5 kV; detector voltage, 1.70 kV; relative collision-induced dissociation energy, 50%. All data were recorded and processed using Shimadzu software LCMS solution version 3.60 and Formula Predictor version 1.2 .HPLC analysis was performed on a Shimadzu HPLC system equipped with two LC-20AD pumps, a CTO-20A column oven, an SIL-20 AC autosampler, and a CBM-20A system controller. Chromatographic separation was carried out on an Agilent Zorbax SB-CA total of four metabolites of sibirioside A were detected and identified, three of which were new compounds. The main metabolic reactions included hydrogenation, hydroxylation, methylation, sulfation, glycosylation, and dimerization, and the different types of reactions usually occurred simultaneously. Sibirioside A was mainly excreted from urine in the form of prototype. The in vivo metabolism might be carried out in the liver, and then excreted from feces through bile. It was speculated that sibirioside A might take effects in prototype form due to its wide distribution in the heart, liver, spleen, lung, kidney, stomach, and small intestine. There were 25 metabolites identified in vivo for angoroside C, 11 of which were new compounds. The prime metabolic reactions were hydrolyzation, hydrogenation, sulfation, glucuronidation, demethylation, and dihydroxylation. A total of 22 of the metabolites were sulfated or glucuronidization products and mainly excreted from urine just like the prototype, which was concentrically distributing in the stomach and small intestine.In total, 29 targets were identified including INSR, RASH, KPYR, ANGI and so on, which might be related to anti-diabetes, anti-cancer, anti-anemia, neuroprotection, anti-leukemia, anti-coagulopathy, and osteogenic activities. The predicted outcomes indicated that the metabolites of sibirioside A and angoroside C have a variety of potential pharmacological activities similar to the prototypes, which require further research.Phenylpropanoid glycosides are a large class of compounds with a simple structure and extensive pharmacological activities. Their metabolic processes in vivo are simple and predominantly they might distribute and take effect in the form of prototypes.


We have recently demonstrated that dietary nitrate, a source of nitric oxide via the enterosalivary pathway, can improve muscle contractile function in healthy older men and women. Nitrate ingestion has also been shown to reduce blood pressure in older individuals. However, the optimal dose for eliciting these beneficial effects is unknown. We therefore performed a randomized, double-blind, crossover study to determine the effects of ingesting 3.3 mL/kg of beetroot juice (BRJ) containing 0, 212, or 425 µmol/kg of nitrate in six healthy older (age 69±3 y) subjects. Maximal knee extensor speed (Vmax) and power (Pmax) were measured 2 h after BRJ ingestion using isokinetic dynamometry; blood pressure was monitored periodically throughout each study. Mean arterial pressure (in mmHg) was lower (P<0.05) after the high (80±4) vs. the low (84±3) or placebo (88±2) doses. Vmax (in rad/s), however, was higher (P<0.05) after the low dose (11.7±0.8), but not the high dose (10.8±1.0), compared to the placebo (10.5±1.0). Pmax (in W/kg) also tended to be higher (P=0.11) in the low (3.9±0.5) compared to the placebo (3.7±0.5) or high (3.7±0.5) trials. Five out of six subjects achieved a higher Vmax and Pmax after the low vs. the high dose. We conclude that dietary nitrate has differential effects on muscle function and blood pressure in older individuals. A high dose of nitrate intake further lowers blood pressure but does not enhance muscle contractility as much as a lower dose. Supported by Indiana University Purdue University Indianapolis and by the NIA (R21 AG053606)


Only MD001 functions as a PPARα/γ dual agonist in vitro. MD001 increased the transcriptional activity of PPARα and PPARγ, resulting in enhanced expression of genes related to β-oxidation and fatty acid and glucose uptake. MD001 significantly improved blood metabolic parameters, including triglycerides, free fatty acids, and glucose, in db/db mice. In addition, MD001 ameliorated hepatic steatosis by stimulating β-oxidation in vitro and in vivo. Our results demonstrated the beneficial effects of the novel compound MD001 on glucose and lipid metabolism as a PPARα/γ dual agonist. Consequently, MD001 may show potential as a novel drug candidate for the treatment of metabolic disorders.Peroxisome proliferator-activated receptor (PPAR)-α/γ dual agonists have been developed to treat metabolic diseases; however, most of them exhibit side effects such as body weight gain and oedema. Therefore, we developed a novel PPARα/γ dual agonist that modulates glucose and lipid metabolism without adverse effects. We synthesised novel compounds composed of coumarine and chalcone, determined their crystal structures, and then examined their binding affinity toward PPARα/γ. We investigated the expression of PPARα and PPARγ target genes by chemicals in HepG2, differentiated 3T3-L1, and C2C12 cells. We examined the effect of chemicals on glucose and lipid metabolism in  The increase in free fatty acids (FFA) gives rise to insulin resistance by desensitizing insulin-mediated signal transduction, resulting in aberrant lipid metabolism associated with metabolic diseases including type 2 diabetes, obesity, hyperglycaemia, hyperlipidaemia, hepatic steatosis, atherosclerosis, and cardiovascular diseases2. Over the last few decades, a variety of small molecules have been developed to improve abnormal lipid metabolism.Energy metabolism is maintained by three major organs - adipose tissue, liver, and muscle - and crosstalk among them is essential for homeostasis. Surplus energy due to excess nutrient ingestion, reduced energy consumption in the cell, or both induces over-production of triglycerides (TG), which are stored in adipose tissue, skeletal muscle, and the liver3. The activation of PPARα or PPARβ/δ stimulates fat consumption by inducing the expression of genes associated with beta-oxidation, resulting in the improvement of hyperlipidaemia5. PPARγ, on the other hand, promotes the mobilisation of fatty acid into adipocytes by encouraging adipogenesis and upregulating the expression of genes related to fatty acid transport, including those for CD36 and adipocyte fatty acid-binding protein (FABP), to reduce lipotoxicity6.Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family comprised of PPARα, PPARβ/δ, and PPARγ, are known to be critical regulators of lipid metabolism8. Fibrate, a selective PPARα agonist, relieves hyperlipidaemia by promoting β-oxidation and induces a minor reduction in body weight9. However, fibrates are known to cause severe side effects, including gallstone formation and hepatotoxicity11. Thiazolidinedione (TZD), a synthetic PPARγ agonist, has been approved to reduce blood glucose levels and lipotoxicity by improving insulin resistance. TZD upregulates PPARγ target genes involved in lipid and glucose metabolism, insulin signal transduction, and adipocyte differentiation. For instance, PPARγ activation by TZD increases the expression of CD36 and FABP for lipid storage and the expression of glucose transporter for glucose uptake in adipocytes, liver, and skeletal muscle, thereby reducing the amount of fatty acid and glucose levels in blood by increasing insulin sensitivity. However, since TZD induces storage of fatty acids and glucose rather than consumption in the cell, it has been associated with several concerns, including body weight gain and myocardial infarction14. Therefore, the dual activation of PPARα and PPARγ is proposed to be more beneficial with limited adverse effects17. A number of PPARα/γ dual agonists have been developed and tested19. However, a majority of these drugs showed severe adverse effects, including heart failure, renal failure, urinary cancer, body weight gain, stroke, and anemia22, necessitating the development of novel PPARα/γ dual agonists without side-effects.Peroxisome proliferator-activated receptor agonists have been widely used to treat metabolic diseases such as hyperglycaemia and lipid dysregulationCyclosorus terminans, and showed its stimulatory effect on adipogenic differentiation and glucose consumption23. In the current study, we tried to synthesise various constitutional isomers of Int B to find a novel agonist targeting PPARs and investigate its ability to improve metabolic disorders in vitro and in vivo.In a previous study, we isolated interruptin B (Int B) from 1)24 and ethyl benzoyl acetate to afford a roughly 1:1 mixture of 2a and 2b owing to the presence of two isomeric ortho and para hydroxyl groups to the methyl group. Compounds 2a and 2b proved to be labile during SiO2 column chromatography and insoluble in various solvents probably due to the 1,3-dihydroxy group. Thus, the mixture of 2a and 2b was methylated in the presence of K2CO3 and Me2SO426 to give rise to 3a and 3b, pleasingly, which were readily dissociable and stable during the separation process. The structure of 3b was confirmed by the X-ray crystallography  . The structure of 4a was verified from by X-ray crystallography  and 5b (37%) respectively29.Our synthetic procedure is summarised in Fig. 5a (MD001): yield −41%; yellow solid; m.p. 274–278 °C; 1H-NMR : δ 9.31 , 8.58 , 7.55–7.54 , 7.53 , 7.34–7.31 , 7.24–7.20 , 7.20–7.14 , 6.72 , 6.59 , 6.30 , 2.16 ; 1H NMR : δ 10.46 , 9.82 , 7.63 , 7.49 , 7.39–7.37 , 7.22–7.16 , 6.63 , 6.27 , 2.11 ; 13C-NMR : δ 193.5, 171.5, 161.2, 159.9, 156.6, 155.4, 153.8, 146.6, 138.6, 136.1, 131.8, 131.1, 129.7, 129.2, 128.9, 128.8, 128.5, 122.9, 104.6, 102.8, 100.4, 8.3; IR (ATR) νmax 3373, 2931, 1716, 1604, 1361, 1138, 1099 cm−1; HRMS (ESI) m/z calculated for C25H19O5 (M + H)+ 399.1232, detected 399.1223.Representative spectral data for 5a (MD001) promoted simultaneous transcriptional activity of PPARα and PPARγ  and PPARγ (KD = 0.14  ± 0.03 μM), but not PPARβ/δ. In addition, we compared the stimulatory effect of MD001 on PPARα and PPARγ with that of WY14643 and rosiglitazone, agonists of PPARα and PPARγ, respectively. A luciferase activity assay using PPRE in HEK293 cells showed that MD001 significantly increased transcription via PPARα and PPARγ activation , carnitine-palmitoyl transferase (CPT), and middle-chain acyl-CoA dehydrogenase (mCAD); the siRNA–mediated knockdown of PPARα showed the opposite effect , and fatty acid transporter (FATP) in HepG2, and ACOX and CPT in both 3T3-L1 and C2C12 cells  and glucose-6-phosphatase (G6Pase) in the livers of db/db mice. TZDs, including rosiglitazone, pioglitazone, and troglitazone, are known to induce severe body weight gain as an adverse effect both in animals and humans32. Therefore, we evaluated the effect of MD001 on body weight. As shown in Fig. db/db mice compared to the vehicle control; on the contrary, MD001 and WY14643 showed no induction of body weight gain in both wild type C57BL/6J and db/db mice without changes in food ingestion  levels , and PPARγ   Fig. , but dec36) Fig. . Thus, Mdb/db mice, as the progression of obesity induces increases in cell size and adipocyte number. Adipocyte size is one of the indicators of metabolic stresses such as inflammation, insulin resistance, and hyperlipidaemia38. While rosiglitazone and WY14643 significantly increased the number of fat cells below 100 μm in diameter, they significantly reduced the number of fat cells above 150 μm in diameter , indicating that MD001 may increase glucose and fatty acid metabolism in skeletal muscle  and a similar chemical structure, it failed to enhance the transcriptional activity of PPARα and PPARγ, which may be attributable to the different location of its methyl group. Further studies on the relevant structure-activity relationships (SARs) may reveal the mechanism involved.Type 2 diabetes and its related complications, including hypertension, arteriosclerosis, and diabetic retinopathy are recognised as serious problems in Westernized societies. The advantages of PPARα and PPARγ agonists in the treatment of metabolic syndrome have led to the development of PPARα/γ dual agonists. In this study, we synthesised derivatives of Int B and examined their potential roles as PPARα and PPARγ agonists. Of these, only MD001 enhanced the transcriptional activity of PPARα and PPARγ In vitro binding assays revealed that MD001 exhibited a significantly higher binding affinity for PPARγ than PPARα . Methyl phloroglucinol , ethyl benzoyl acetate , trifluoroacetic acid (2 mL), and AcOH (40 mL) were added to a round-bottomed flask and the reaction mixture was heated to reflux for 12 h. The reaction mixture was evaporated under reduced pressure and water (20 mL) was added. The aqueous layer was extracted with EtOAc (50 mL) three times. The complete protocol for the synthesis of Int B isoform is described in Supplementary Information. After synthesis, each compound was dissolved in a solution of 10% diethylene glycol monoethyl ether , and 20% polyoxyl 15 hydroxystearate .2. HepG2 cells were cultured in Minimum Eagle’s medium (MEM) supplemented with 10% FBS and 1% penicillin and streptomycin. HEK293, C2C12, and 3T3-L1 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS and 1% penicillin and streptomycin. Adipocyte differentiation was induced as previously described45. C2C12 cells were differentiated as previously described46.Cells were maintained at 37 °C in a humidified chamber containing 5% CORenilla vector for 24 h. Cells were treated with MD001, rosiglitazone , or WY14643 (Sigma-Aldrich/Millipore) at indicated concentrations for 24 h. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System  and quantified using GloMax  according to the manufacturer’s protocol. Luciferase activity was normalised to Renilla luciferase activity. For the knockdown experiment, HepG2, 3T3-L1, and C2C12 cells were transfected with 20 nM of siRNA targeting PPARα and PPARγ  using lipofectamine 2000 (Invitrogen/Thermo Fisher Scientific) for 48 h. Following transfection, cells were treated with vehicle, rosiglitazone, WY14643 or MD001 for 24 h.For transient transfection, cells were transfected with plasmid DNA constructs by polyethylenimine  according to the manufacturer’s instructions. Transcription activity assays of PPARα and PPARγ in HEK293 cells were performed by co-transfection of HA-PPARα, HA-PPARγ, and peroxisome proliferator-response element (PPRE) reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) with Total RNA was isolated using an RNeasy kit  according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix  on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific). GAPDH was used as an endogenous control. Primer sequences are summarized in Supplementary Table 3H]palmitate, 5 μci/mL)  and 1% bovine serum albumin for 24 h. Following incubation, the medium was harvested and precipitated with 10% trichloroacetic acid (Sigma-Aldrich/Millipore), vortexed, and incubated for 20 min at room temperature, then centrifuged at 16000 × g for 10 min at 4 °C. The supernatants were transferred to a new tube and incubated in a scintillation vial containing 0.5 mL water at 60 °C for 12 h. After incubation, the vial was removed and radioactivity (3H2O in water) was quantified using a liquid scintillation counter .For the fatty acid oxidation assay, cells were incubated in α-MEM  containing 0.1 mM palmitate .The glucose consumption assay was performed as previously described23 and was immobilised on CMDH chips (Reichert Technologies) according to the manufacturer’s instructions. MD001 was loaded in a dose-dependent manner  for the SPR assay. The dissociation constant (KD) value was determined using the Scrubber2 program .SPR analysis was performed using a Reichert SR7500DC . HA-PPARα, HA-PPARβ/δ, and HA-PPARγ were purified as previously describedLeprdb/Leprdb or wild type C57BL/6J male mice were purchased from Orient Bio, Inc.  and acclimatised for one week. Mice were randomly grouped and orally administered with vehicle, WY14643 (20 mg/kg), rosiglitazone (20 mg/kg), or MD001 (5 mg/kg or 20 mg/kg) once a day for two months. For the oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT), mice were fasted for 12 h and were treated with sterile glucose  or human insulin . Blood glucose levels were measured at the indicated time point using an OneTouch Ultra Blood Glucose Monitoring System . For the toxicity study, wild type C57BL/6J male mice were randomly grouped and orally administered with vehicle and MD001 (50 mg/kg or 100 mg/kg) once a day for two month. Blood cells, body weight change, and blood and urine metabolites were analysed for toxicity.The Ajou University Animal Care and Use Committee approved all animal studies (IACUC2015-0001), and the experiment conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. All experiments were performed in accordance with relevant guidelines and regulations. Six-week-old C57BLKS/J-47. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels using WAKO reagents  were measured for analysis of hepatotoxicity.Livers and blood samples were collected from mice in each group. Serum and liver TG, FFA, and total cholesterol were measured using WAKO reagents  using Hitachi Clinical Analyzer 7180 . Serum concentrations of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were determined using Sekisui reagents Tissues specimens including liver, perigonadal adipose tissue, skeletal muscle, spleen, kidney, and heart, were collected from mice in each group, fixed in 10% formalin, and embedded into paraffin. Tissue sections (5 μm) were stained with haematoxylin and eosin (H&E).P < 0.05 was considered statistically significant.All data were analysed using GraphPad Prism 5.0 software . The results are expressed as mean ± SD or mean ± SEM. Statistical significance was calculated using one-way or two-way analysis of variance (ANOVA) with a post hoc Bonferroni multiple comparison test to compare the differences between groups. Supplementary information


The synthesized nanocubes (NCs) are labeled as CeO2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, corresponding to 0.5, 1.0, and 2.0 g of urea, respectively. The synthesized NCs were characterized by FT-IR, UV-visible, XRD, XPS, SEM and HR-TEM analysis. The synthesized NCs were cubic in shape with average sizes of 12, 12, and 13 nm for the CeO2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, respectively, obtained by the XRD analysis. The catalytic activity of the CeO2 NCs was studied for the purpose of obtaining the reduction of malachite green (MG) in the presence of sodium borohydride (NaBH4) at room temperature.This study describes a simple, high-yield, rapid, and inexpensive route for the synthesis of cubic shape-like cerium oxide nanocubes (CeO Dyes are major pollutants that are released from textile industrial effluent2. Malachite green  is a water-soluble cationic dye that is available in two forms malachite green chloride (C23H25ClN2) and malachite green oxalate (C52H54N4O12) , lanthanide series, and exists as a free metal or oscillates between in the Ce2 is an effective catalyst for the removal of organic dyes from effluents because ceria also hops between Ce3+ and Ce4+ valence states containing oxygen vacancies that allow NCs to act as regenerative catalysts32. Various methods have been reported in the literature for the synthesis of ceria37, including sol-gel38, microwave combustion39, flame spray pyrolysis40, solvothermal41, microemulsion42, spray drying system43, hydrothermal44, and thermal decomposition45. Among these, the hydrothermal method is simple, which can produce high product yields46.CeO2 NCs using urea (CH4N2O), as capping and reducing agent, urea was also known as carbamide and it is easily soluble in water. It has been used as a nitrogen source for the synthesis of environmentally benign nanoparticles. It has been widely used in many industries such as in agriculture, plastic, drug, soap, and detergent industries. However, excess concentrations urea in soil or in water causes to soil acidification and eutrophication and toxic effects to aquatic organisms, animals and humans48. The ceria nanocubes (CeO2 NCs) were synthesized via the one-pot hydrothermal method using different concentrations of urea. The CeO2 NCs are applied as a catalyst for the degradation of MG in the presence of NaBH4 in an aqueous medium using UV-visible spectroscopy.In this study we synthesized CeO2 NCs and dye degradation studies.Analytical grade cerium nitrate hexahydrate and urea were purchased from Alfa-Aesar, South Korea and used directly. Malachite green dye was obtained from Daejung Chemicals, South Korea. Deionized (DI) water was used throughout the study for the synthesis of CeO2 NCs-0.0).Cerium nitrate hexahydrate, 0.01 M was prepared at a volume of 70 mL in DI water. Different quantities of urea  were added and stirred until dissolved. This reaction mixture was transferred to an autoclave at a temperature of 180 °C for 6 h. The reaction solution was naturally cooled down to room temperature. The reaction mixture was carefully collected by centrifuge and wished with DI and ethanol to remove the unreacted substance, before drying at 80 °C overnight. This was later calcined at 400 °C for 4 h and for comparison we prepared ceria without urea  using a Cu Kα source (λ = 1.5405 Å). FT-IR  was used to identify the vibrational modes by an ATR mode. The morphology of the samples was investigated by scanning electron microscopy  and high-resolution transmission electron microscopy  with a 200 kV field emission gun in high brightness Schottky mode FEG (X-FEG). UV-visible absorption was measured on a Neogen (NEO-D3117). The elemental composition was quantitatively compared using an X-ray photoelectron spectrometer  with Al Kα radiation (1486.6 eV).2 NCs (2 mg/mL) and 0.2 mL of (0.2 M) freshly prepared NaBH4 were added to all the samples. The degradation efficiency was monitored by UV-visible spectroscopy . The degradation efficiency was calculated as a function of time for Co/Ct and ln Co/Ct, where Co is initial concentration and Ct is the final concentration of the MG dye. Blank control experiments were performed without catalyst (MG + NaBH4) or NaBH4 (MG + CeO2 NCs) to study the catalyst efficiency.The synthesized samples were used to check the catalytic ability using the MG dye. For the catalytic reactions, 2.5 mL of the MG dye, 25 µL of well-dispersed CeO2 NCs-0.5); 209 and 310 nm (CeO2 NCs-1.0); and 209 and 314 nm (CeO2 NCs-2.0). These peaks correspond to the characteristic absorption peaks of Ce3+ and Ce4+ of the CeO2 NCs. These results are similar to those obtained by Nurhasanah49et al. who observed absorption peaks at 207 and 303 nm for CeO2 NCs .The UV-visible absorbance spectra of the ceria nanocubes are shown in Fig. 2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0). The features near 1540 and 1630 cm−1 related to (ν C=O) and (N-H stretching) bond stretch51. The remaining observed peaks are as follows: near 3362 cm−1 (O-H stretching vibrations)52; near 2980 cm−1 (asymmetric C-H stretching)53; sharp band near 1340 cm−1 (N-O stretch due to the presence of nitrate)54; 1071 cm−1 (C-O stretching vibration)55; 850 cm−1 56; and below 700 cm−1 (O-Ce-O stretching vibrations)57.Fig. 2 NCs. The XRD revealed sharp peaks which located at 2θ = 28.5°, 32.9°, 47.5°, 56.3°, 59.0°, 69.3°, 76.7°, 78.8°, and 88.4°, are corresponding to (111), (200), (220), (311), (222), (400), (331), (420) and (422) planes, respectively. No extra peaks were observed, which supported the assumption that urea-based NCs are pure with cubic fluorite structure (JCPDC: 034–0394), similar peaks were also observed in CeO2 NCs-0.0 and results are shown in Fig. 59:β is the broadening in the full-width at half maximum (FWHM), λ is the X-ray wavelength (1.5406 Å), and θ is the Bragg diffraction angle. The microstrain, ε, of the NCs is evaluated byFig. δ, of the NCs is defined as the length of dislocations lines per unit volume and given byThe dislocation density 61.The lattice strain (LS) of the NCs was calculated using the following relation2 NCs-0.5 and CeO2 NCs-1.0 were showing similar value (12 nm), and for CeO2 NCs-2.0 showing slightly bigger size (13 nm), whereas CeO2 NCs-0.0 showing much higher than (31 nm) urea mediated CeO2 NCs, this MGS analysis clearly indicating the urea was acted as capping and reducing agent to control the size of the NCs, meanwhile, the dislocation density decreased with increasing urea concentration, but for higher concentrations (CeO2 NCs-1.0 and CeO2 NCs-2.0) dislocation density was constant, in CeO2 NCs-0.0 too low than the urea mediated CeO2 NCs. The microstrain was constant with increasing urea concertation but without urea CeO2 NCs showing lower than other CeO2 NCs, however, lattice strain (LS) was constant for the first two concentrations (CeO2 NCs-0.5 and CeO2 NCs-1.0), and for the higher concertation (CeO2 NCs-2.0), it was slightly lowered compared with those of the lower concentrations, whereas in CeO2 NCs-0.0 showing very lower values, based on these results urea playing vital role as capping and reducing agent in the formation of CeO2 NCs.From Table 2 NCs, we performed the XPS analysis  and the results are presented in Fig. 3+ and Ce4+ oxidation states. The binding energies (BE) at 882.9, 901.2 and 917.2 eV are attributed to Ce4+, and the peaks at 885.5, 899.1 and 908.3 eV are the characteristic peaks of Ce3+. These results suggest the presence of mixed oxidations and a valence state for the Ce ions in the NCs63 and Deshpande64et al. reported that Ce3+ ions create oxygen vacancies in the CeO2 lattice that are defects. There were three peaks in the high-resolution O 1 s deconvoluted spectrum in Fig. 2 NCs, not doped in CeO2 NCs, which are similar to the values in literature67.To identify the chemical composition on the surface of the CeO2 NCs was obtained by SEM, and the images are revealed in Fig. 2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, SEM images show that aggregates were formed by the accumulation of nanocubes. SEM images of CeO2 NCs-0.0 was shown in Fig. 2 NCs-0.0 were more in aggregation and size also higher than the urea mediated CeO2 NCs. The details of CeO2 NCs were further confirmed by HR-TEM studies. Fig. 2 NCs synthesized with different concentrations of urea. The TEM images show that CeO2 NCs were well dispersed, almost polydispersed, consisting of cubic shapes. The CeO2 NCs obtained with 0.5 urea-based NCs were smaller in size and less aggregated compared with the 1.0 and 2.0 urea-based NCs. To confirm the phase formation of the NCs, HR-TEM images were analyzed using the Gatan software, and the results are shown in Fig. 2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, respectively. The lattice fringe distance values were similar, 0.307 nm for CeO2 NCs-0.5, 0.307 nm for CeO2 NCs-1.0 and 0.306 nm CeO2 NCs-2.0, and were assigned to the (111) plane of the CeO2 NCs. The corresponding selected area electron diffraction (SAED) pattern  shows well-defined diffraction rings that confirm the polycrystalline nature of CeO2 NCs. These results were in good agreement with the XRD results. Therefore, the HR-TEM images revealed a good crystalline nature of the urea assisted NCs.The morphology of CeO2 NCs in the presence of NaBH4 as the reducing agent under ambient conditions. The results are presented in Fig. 2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, respectively. Fig. 4. The MG maximum absorption peak intensity was at~617 nm. The intense green color of the MG dye solution slowly faded becoming colorless, during the catalytic dye degradation process. A plot of Co/Ct versus degradation, time is shown in Fig. Co is initial concentration and Ct is concentration at time ‘t’.MG dye was selected as a model pollutant to examine the catalytic activity of CeO68:−1) of the dye degradation reaction, Co is the initial concentration and Ct is the concentration of the dye solution after time ‘t’ in minutes, respectively. The results are shown in Fig. 4 + CeO2 NCs and in the second experiments, we performed the reaction between dye (MG) + NaBH4, respectively. In both these control experiments, there was no observed degradation, based on these control experimental results we confirmed that the catalyst (CeO2 NCs) playing a vital role in the dye decolorization, and when both the catalyst + NaBH4 were together, the degradation materialized rapidly; the results are shown in Fig. 2 NCs-0.5 exhibited an excellent dye degradation ~90%, while the CeO2 NCs-1.0 and CeO2 NCs-2.0 exhibited ~70% MG dye degradation within 21 min. The possible mechanism of dye reduction by the catalyst is explained by the electron relay system. The NCs start the catalytic reduction by relaying electrons from the donor et al. described hydrogen species generated from 26. This reaction leads to a reduction in the MG dye, which is generally leuco MG (colorless). The CeO2 nanocatalysts show slope of −0.127, −0.061 and −0.064 for CeO2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, respectively. The rate constants for dye degradation were 0.127 min−1, 0.061 min−1, and 0.064 min−1 for CeO2 NCs-0.5, CeO2 NCs-1.0, and CeO2 NCs-2.0, respectively (Fig. 2 NCs-0.5 exhibited the highest dye degradation due to their size and high dispersion. We evaluated the catalytic activity of the CeO2 NCs-0.0, the rate constant was 0.03 min−1, as shown in Fig. 2 NCs-1.0 and 2.0 that of counterparts due to bigger size and aggregation of particles.Langmuir-Hinshelwood pseudo-first order kinetics was applied to determine the first-order rate constant for dye degradation using the following relationely Fig. . CeO2 NC2 NCs using carbamide by one-pot hydrothermal method. The HR-TEM images revealed the synthesized NCs was cubic in shape. XRD results show the synthesized nanoparticles MGS of 12, 12, 13 and 31 nm for CeO2 NCs-0.5, CeO2 NCs-1.0, CeO2 NCs-2.0 and CeO2 NCs-0.0, respectively. These results were indicating that urea acting as capping and reducing agent. The synthesized CeO2 NCs also acted as efficient catalysts in the degradation of the MG dye.We developed cost-effective CeOSupplementary Information