Bidens pilosa Linn. var. Radiata (Asteraceae) is well known for its folkloric medicinal use against various diseases from many decades. Mizoram, North East India, has high plant diversity and the use of this plant as herbal medicine is deep rooted in the local tribes. The present study was executed to understand the pharmacological potential of B. pilosa leaves extract.Research of natural products from traditionally used medicinal plants to fight against the human ailments is fetching attention of researchers worldwide. C. quinquefasciatus using dose response and time response larvicidal bioassay. Additionally, the major phenolic and volatile compounds were determined using UHPLC-QqQLIT-MS/MS and GC/MS respectively.The antimicrobial potential was determined using agar well diffusion and broth microdilution method against bacterial and yeast pathogens. Cytotoxicity was evaluated using MTT and apoptotic DNA fragmentation assays. Further, the antioxidant ability of the extract was analysed using DPPH and ABTS free radical scavenging assay. Mosquitocidal activity was evaluated against third in-star larvae of E. coli (MIC 80 μg/mL and IC50 110.04 μg/mL) and showed significant cytotoxicity against human epidermoid carcinoma (KB-3-1) cells with IC50 values of 99.56 μg/mL among the tested cancer cell lines.We found that the extract showed highest antimicrobial activity against 50 values for scavenging DPPH and ABTS was 80.45 μg/mL and 171.6 μg/mL respectively. The extract also showed the high phenolics (72 μg GAE/mg extract) and flavonoids (123.3 μg Quercetin /mg extract). Lastly, five bioactive and six volatile compounds were detected using UHPLC-QqQLIT-MS/MS and GC-MS respectively which may be responsible for the plant’s bioactivities. An anticancerous compound, Paclitaxel was detected and quantified for the first time from B. pilosa leaves extract, which further showed the anticancerous potential of the tested extract.The ICB. pilosa might be a good candidate for the search of efficient environment friendly natural bioactive agent and pharmaceutically important compounds.On the basis of the present investigation, we propose that the leaf extract of  Bidens pilosa Linn. var. Radiata (Spanish needles or beggar ticks) from the family Asteraceae is an annual weed widely distributed throughout the tropical and sub-tropical regions of the world  were selected and screened against the obtained extract as described earlier .−4 cells/well in 100 μl of medium on 96-well plates and incubated for 24 h at 37 °C in 5% CO2 incubator chamber. 5% methanolic plant extracts (1–200 μg/mL) were added to the plates. Cells incubated with 5% methanol were used as blank while untreated cells represented positive control. Experiment was performed in triplicate. After the incubation of 72 h, the culture medium was replaced with 20 μl of MTT in each well and again incubated for 4 h. of incubation. DMSO was added to each well and absorbance was recorded at 570 nm. The percentage of cell viability was calculated as previously described  assay was employed to evaluate the cytotoxicity activity against three cancer cell lines: human epithelial carcinoma (HeLa), human hepato carcinoma (HepG2) and human epidermoid carcinoma (KB-3-1). The ICB. pilosa. All the cells were grown and were treated by the IC50 concentration of the extract for 72 h. Further, DNA was extracted from the treated cells using 2.0% agarose gel electrophoresis. A typical ladder like pattern was observed which shows the internucleosomal fragmentation. The findings suggested that the methanolic leaf extract of B. pilosa is a potent inducer of apoptosis in HeLa, HepG2, and KB-3 cells  were from 0.9996 to 1.0000 within test ranges. LOD and LOQ of reference analytes was 0.01 to 0.20 ng/ml and 0.03 to 0.61 ng/ml respectively.Calibration curves of standard compounds were established using different concentrations of reference analytes. LOD and LOQ were determined using diluted standard compounds when the signal-to-noise rations of reference analytes were about 3 and 10, respectively. The obtained results are listed in Table B. pilosa leaves extract has good accuracy, with recovery ranges from 94.87% to 105.19% for all analytes (Table Relative standard deviation (RSD) was used to measure precision and intra-day and inter-day variations were evaluated by using six replicates and repeating the experiments for 3 days. The intra-day and inter-day precision was found to be less than 1.21%. Stability was also measured by replicating the injections at 0, 2, 4, 8, 12 and 24 h. The percentage of RSD of five standard analytes was found to be 2.55. The method developed for evaluation of bioactive compounds from LIT-MS/MS method was applied to five quantitative reference compounds. Quantitative results are listed in Table B. pilosa. The findings of the study prove the existence of variations among the tested reference analytes in B. pilosa. The MRM, extracted ion chromatogram and MS/MS spectra of five mixed standards are shown in Figs. In this study, the UHPLC-QqQB. pilosa, shown in Table B. pilosa including 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-; 1H-3A, 7-Methanozulene, Octahydro-1,4,9,9-tetramethyl; 9H–Fluorene, 9-Diazo; 1-Octadecyne; N-Hexadecanoic acid and 3,7,11,15-Tetramethyl-2-Hexadecen-1-ol. The spectrum profile of GC-MS analysis showing six components individual fragmentation pattern with retention time 14.05, 16.18, 17.80, 18.90, 20.16 and 21.62 is demonstrated in Fig. GC-MS analysis of compounds was performed in methanolic leaf extract of C. quinquefasciatus treated with methanolic extracts of B. pilosa is illustrated in Table B. pilosa was highest at 1000 ppm concentration at different time intervals (P < 0.05) at 24 and 48 h of exposure  of the third instar larva of 50) values of the larvicidal assay after 24 and 48 h of B. pilosa. The highest larvicidal activity was found in methanolic extract of B. pilosa (LC50= 148.7) after 24 h and (LC50= 101.7) after 48 h. Chi-square value was highly significant at P < 0.045 to 0.0004 levels in B. pilosa plant extract. The result of one way ANOVA of methanolic extract of B. pilosa at different concentrations (50–1000 ppm) and at different time intervals (24 and 48 h) also exhibited significant difference in larval mortality (P < 0.0004). Higher slope value  and lower and upper limits at 95% confident level of LC50  were observed for methanolic extract of B. pilosa. The regression analysis showed a positive correlation among the mortality rate (Y) and the concentration of exposure (X) having a regression coefficient (R2) of 0.96 and 0.99 respectively. Time response larvicidal bioassay was carried out in methanolic extract of B. pilosa at different concentrations (50–1000 ppm) for 48 h against C. quinquefaciatus. Methanolic extract of B. pilosa has taken minimum lethal time (LT50 = 6 h) to kill 50% of C. quinquefasciatus at 500 ppm , higher slope value (2.212 ± 0.101 at 300 ppm) and lower and upper limits at 95% confident level of LT50 (4.28 at 500 ppm & 29.98 at 50 ppm) were also observed in methanolic extract of B. pilosa.Table B. pilosa (38.1 mg of GAE/g of DW). The total flavonoids content was found to be 123.3 μg Quercetin/mg of DW. The findings were in support of Lee et al. [B. pilosa have highest TPC and TFC contents as compared to other parts. The main role of flavonoids in the plants is to protect plants from sun radiation and scavenge free radicals. Hence, it is quite expected that the plant parts exposed to sunlight are high in the TFC [Phenolics are one of the vital groups of secondary metabolites present in plants. Rose and Kasum,  suggestee et al. . Cortés-e et al.  suggeste the TFC .B. pilosa methanolic extract was tested for free radical scavenging ability using DPPH and ABTS method. In our study, we found that DPPH IC50 value of 80.45 μg/ml in methanolic extract of B. pilosa. Adedapo et al. [50 value of 94.2 mg/mL which was higher than our reported value. Deba et al. [B. pilosa and showed that leaves and flowers essential oil having DDPH IC50 value of 47 and 50 μg/ml respectively which further proved that leaves has the highest antioxidant potential as compared to other parts of the selected plants. ABTS, A more appropriate decolorization technique assay in which the radicals are generated directly in a stable form prior to reaction with putative antioxidants [50 value of 171.6 μg/ml which is higher than the previously reported by Adedapo et al. [50 value of ABTS as 0.75 mg/mL.Free radicals are well known to play quiet effective role in pathological symptoms . Antioxio et al.  reporteda et al.  reportedoxidants . In our B. pilosa have significant antimicrobial potential against four human bacterial pathogen  and yeast C. albicans which are the most common cause of different food borne diseases. In this study, methanolic extract of B. pilosa exhibited significant inhibitory effect against gram-negative bacteria (18.1 mm diameter zone of inhibition) than the gram positive bacteria (14.6 mm diameter zone of inhibition) which is compared to standard known antibiotics ampicillin (50 μg/disc). The highest zone of inhibition was found against E. coli (18.2 mm). The findings of zone of inhibition was slightly higher than a study reported by Falowo et al. [B. pilosa showed zone of inhibition against E. coli (16.0 mm).The antimicrobial activity showed that o et al.  who statB. pilosa leaves extract exhibited significant antibacterial activity against S. aureus (15.6 mm). This result was similarly reported by Ashafa and Afolayan, [B. pilosa have suppressed the growth of Gram positive bacteria S. aereus (5.0 mm). According to some previous researchers, methanolic extract of B. pilosa was inactive against P. aeruginosa and S. aureus [We found that folayan,  who demo. aureus , 47. As . aureus , 50.B. pilosa against selected bacterial pathogens is represented in Table B. pilosa inhibited bacterial and yeast pathogen with MIC ranging from 80 to 380 μg/mL. B. pilosa showed highest activity against E. coli (80 μg/mL) followed by S. aureus (110 μg/mL) and P. aeruginosa (220 μg/mL). Previous reports also showed that the methanolic leaves extract was more active which indicates that the methanolic leaves extract has the potential antimicrobials [B. pilosa extract showed significant inhibitory activity against bacterial and yeast pathogen which suggest as an exploitable source for the discovery of antimicrobial agents [The minimum inhibitory concentration (MICs) of methanolic extracts of crobials . The B. l agents , 51.B. pilosa have anticancer activities against various types of cancer. According to Kviecinski and Felipe, [B. pilosa possess anti-tumor activity which has assessed using brine shrimp, hemolytic, MTT, and neutral red uptake (NRU) assays. In present study, the methanolic extract of B. pilosa inhibited the growth of three cancer cell lines KB-3-1, HepG2 and HeLa with IC50 values of 99.56 μg/mL, 210.8 μg/mL and 179.3 μg/mL respectively. Percentage of inhibition was found significantly high than the previous studies reported by Sundararajan et al. [B. pilosa showed anticancer activity against HeLa, HepG2 and KB cells with IC50 values of 965.2 μg/mL, 119.55 μg/mL and 586.2 μg/mL respectively. Steenkamp and Gouws, [Asteraceae family such as B. pilosa showed cytotoxic activity on some tumor cell lines. Furthermore, Kumari et al. [B. pilosa has antiproliferating effect against human oral, liver, colon, and breast cancer cell lines with IC50 values of 8.0 ± 0.01, 0.49 ± 0.45, 0.7 ± 0.01and 10 ± 0.01 μg/mL respectively. Further, DNA fragmentation was observed in HeLa, HepG2, and KB-3 cells treated with B. pilosa extract, thereby indicating the onset of apoptotic cell death. Thus, the results obtained in this study suggest that the methanolic extract of B. pilosa might have an apoptosis-inducing property, isolated from the leaves of B. pilosa can act as potential anticancer agents in cancer chemotherapy.Previous reports have stated that isolated new compounds from  Felipe, , differen et al.  who statd Gouws,  reportedB. pilosa plant. Kaempferol, phenolic compound was also reported first time from B. pilosa which is used for the treatment of various types of cancers [B. pilosa using HPLC system. Paclitaxel, brand name taxol is a chemotherapy medication which was reported first time from B. pilosa. This compound was isolated first time from the bark of the Pacific yew, Taxus brevifolia and its given name “taxol” [A few phenolic compounds like gallic acid, Kaempferol, Catechin, Paclitaxel and Ferulic acid was detected for the first time from methanolic extract of  cancers , 55. Fer cancers  who has  “taxol” .B. pilosa showed the presence of six volatile compounds i.e. 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-; 1H-3A, 7-Methanozulene, Octahydro-1,4,9,9-tetramethyl; 9H–Fluorene, 9-Diazo; 1-Octadecyne; N-Hexadecanoic acid and 3,7,11,15-Tetramethyl-2-Hexadecen-1-ol. These compounds are responsible for numerous pharmacological actions like antimicrobial activities useful in a treatment of variety of diseases and anticancer activities against various cancers [Adiantum capillus-veneris L which has similarly reported in our study from methanolic extract of B. pilosa plant. To best our knowledge, this is first time reported six compound 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-; 1H-3A, 7-Methanozulene, Octahydro-1,4,9,9-tetramethyl; 9H–Fluorene, 9-Diazo; 1-Octadecyne; N-Hexadecanoic acid and 3,7,11,15-Tetramethyl-2-Hexadecen-1-ol from methanolic extract of B. pilosa.GC-MS analysis of the methanolic extract of  cancers , 59. RecC. quinquefasciatus last several years in Northeast India. The use of DDT is stopped in several places of India due to development of resistance in vector populations. Though, this chemicals are still used for control of Kala-azar vector and malaria vectors of different parts of North-eastern India especially Mizoram [B. pilosa showed larvicidal effect against third instar larva of C. quinquefasciatus. The methanolic extract of B. pilosa exhibited 100% mortality rate after 12 h of incubation at the concentration of 1000 ppm. Similarly, Macêdo et al. [Bidens pilosa showed larvicidal effect against fourth instar larva of Aedes fluviatilis who stated that 12.2% of mortality at 100 mg/L concentration. To best our knowledge, this is first time reported that methanolic extract of B. pilosa exhibited larvicidal activity against third instar larva of C. quinquefasciatus.Larvae are mainly killing by using different synthetic chemicals like – organochlorine (DDT), organophosphates , synthetic pyrethroids (deltamethrin), insect growth regulators (diflubenzuron and methoprene) etc. A high amount of DDT and Malathion resistance was used in  Mizoram . Since i Mizoram –64. The o et al.  checked B. pilosa and the ecological significance of human well being. The results obtained bring up supporting data for future investigation of the studied plant which could lead to their use in cancer, oxidative stress and antimicrobial therapy.The overall findings of our study provide evidence for the bioactive potential of methanolic leaves extract of


Porphyridium purpureum within Rhodophyta abundantly produces several valuable proteins, polysaccharides, pigments and long-chain polyunsaturated fatty acid; it is especially effective in accumulating arachidonic acid (ARA). However, this high ARA yield is always achieved in conditions unfavourable for cell growth. In this study, we present a method for obtaining desirable ARA levels from P. purpureum while simultaneously promoting cell growth using appropriate concentrations of the growth hormone 5-Aminolevulinic acid (5-ALA).The microalga P. purpureum were enhanced by stimulation with 20 mg/L 5-ALA, leading to an optimal ARA yield of 170.32 mg/L—a 70.82% increase compared with control conditions. This ARA yield is the highest ever reported for microalgae. Based on variations in the fatty acid composition, total lipids, total proteins, total carbohydrates and pigment content during the cultivation period, we propose that the accumulation of ARA stimulated by 5-ALA occurs at the expense of other UFAs and total proteins, which may be related to decreased zeaxanthin. Lipidomic analysis revealed that triacylglycerols (TAGs) accounted for 47.5 ± 3.6% of all detected lipids, followed by phosphatidylglycerol (PG) and digalactosyldiacylglycerol (DGDG). As the levels of the most abundant TAGs increased under 5-ALA promotion and because 78.1 ± 3.4% (by weight) of detected TAG-branched chains contained ARA, the increase of ARA was mainly caused by TAG accumulation.Both the biomass and the ARA content of P. purpureum by introducing a small amount of 5-ALA. These results are helpful for understanding the microalgae metabolic pathways affected by phytohormones and for guiding the development of bioproducts from microalgae.This work demonstrated a simple and effective strategy to promote both biomass and ARA yield in  Arachidonic acid ARA, C20:4ω6), a long-chain polyunsaturated fatty acid (LC-PUFA), is of great nutritional importance because it is one of the major components of brain membrane phospholipids and a precursor of numerous eicosanoids. Mammals, including humans, cannot synthesize ARA directly . Major A0:4ω6, a Porphyridium purpureum is the only microalga reported to produce ARA in significant quantities  (m/z 236) was used for precursor ion scanning to identify betaine lipid diacylglycerol-O--homoserine (DGTS). Sequential neutral loss scanning +) of MGDG, DGDG and TAG were analysed by single-stage mass spectrometry (MS). The deprotonated forms of the anionic glycerolipids PG, PI and SQDG were analysed by MRM in a negative mode. The MS parameters were set as follows: nebulizing gas (nitrogen), 40 psi; dry gas (nitrogen), 4 L/min at 200 °C; spray capillary voltage, 4000 V for positive ion mode and 3500 V for negative ion mode; gas temperature, 250 °C; gas flow, 5 mL/min and sheath gas temperature, 350 °C. Prior to MS analysis, lipid extracts were separated on a ZORBAX SB C18 column  for the positive mode or on an Extend C18 column  for the negative mode. The mobile phase for the positive mode was composed of A, methanol:acetonitrile:H2O , and B, isopropanol; both A and B contained 10 mM ammonium acetate and 0.1% (w/v) formic acid. The following gradient was used: 0-5 min, 90% A, 10% B; 25 min, 60% A, 40% B; 45 min, 40% A, 60% B; 55 min, 20% A, 80% B; 57 min, 20% A, 80% B; 60 min, 90% A, 10% B and 70 min, 90% A, 10% B. The mobile phase for the negative mode was composed of A, methanol:acetonitrile:water  and B, isopropanol. Both A and B contained 0.025% (w/v) ammonium hydroxide. The following gradient was used: 0 min, 95% A, 5% B; 15 min, 85% A, 15% B; 25 min, 50% A, 50% B; 30 min, 37% A, 63% B; 33 min, 20% A, 80% B; 53 min, 20% A, 80% B; 53.01 min, 95% A, 5% B and 60 min, 95% A, 5% B. For each sample, the column was re-equilibrated with A for 10 min before gradient elution. The temperature of the columns was maintained at 40 °C, and the flow rate was 0.2 mL/min.Lipids were extracted with chloroform:methanol  from ~0.1 g lyophilized algal cells. Lipidomic analysis was performed with LC-Mass/Mass Spectrometry (LC–MS/MS) using an Agilent 6490 QQQ system equipped with an Agilent 1290 Infinity LC system according to the method of Wang et al. . Specifiscanning  and prodFor absolute quantification, lipid extracts were mixed with internal standards (ITSDs), including TAG 17:0/17:0/17:0 , PE 17:0/17:0 (Avanti Polar Lipid) and PG 17:0/17:0 (Avanti Polar Lipid), PC 17:0/17:0 (Avanti Polar Lipid). Among these, PC 17:0/20:4 was used as an ITSD for both PC and DGTS quantification, and PG 17:0/17:0 was used for PG, PI and SQDG quantification. TAG 17:0/17:0/17:0 was used for MGDG and DGDG quantification. The external standards (ETSDs) for calibration included TAG 16:0/16:0/16:0 , TAG 16:0/18:1/16:0 (for TAG C50), TAG 18:1/16:0/18:1 (for TAG C52), TAG 18:1/18:1/18:1 (for TAG C54) (Sigma-Aldrich) and TAG 20:4/20:4/20:4 (for TAG C60) (Sigma-Aldrich). MGDG 16:3/18:3 , DGDG 18:3/18:3 (Matreya), PE 18:1/18:1 (Avanti Polar Lipid), PG 18:1/18:1 (Avanti Polar Lipid), PC 18:1/18:1 (Avanti Polar Lipid), DGTS 16:0/18:0 (Avanti Polar Lipid) and SQDG 16:0/18:3 (Avanti Polar Lipid) were used as ETSDs for the corresponding classes of membrane lipids.P. purpureum biomass of the control culture in the absence of 5-ALA was higher on the 4th day than the biomass of cultures including 5-ALA, but the opposite was seen beginning on the 8th day. These results suggested that P. purpureum benefits from 5-ALA after a short period of adaptation for the range of concentrations 10–50 mg/L and that this adaptation leads to higher final biomass yields. The highest biomass yield of 18.81 g/L was obtained with 10 mg/L 5-ALA , whereas no significant effect was found at the higher 5-ALA concentrations. Our results are consistent with those of a previous report detailing the responses of Scenedesmus abundans and Chlorella ellipsoidea to 2,4-dichlorophenoxyacetic acid  and/or gibberellic acid (GA3) addition [The cell growth curve Fig.  shows thaddition . The impaddition .Fig. 1PP. purpureum was non-linear. Among the 5-ALA addition groups, the highest TFA content (46.76 mg/g) was obtained with 20 mg/L 5-ALA  content was decreased by 5-ALA addition in all cases. A maximum TFA content of 51.61 mg/g was obtained in the control group at the late exponential stage . ARA content reached its maximum of 10.61 mg/g in the 20 mg/L 5-ALA group at the 18th day and exceeded that of the control , C18:2 (linoleic acid), C20:4 (ARA) and C20:5 (EPA) , zeaxanthin and β-carotene were detected by HPLC  chlorophyll PLC Fig. ; the leverythrin . No appatentious , our resP. purpureum. Based on the LC–MS/MS analysis, TAG was the most dominant lipid in P. purpureum at the 18th day, accounting for 47.5 ± 3.6% of all the detected lipids  . In thisP. purpureum and its biochemical compounds and was the first to analyse the lipidomics in P. purpureum by LC–MS/MS. The results demonstrated that with 20 mg/L 5-ALA, P. purpureum grew significantly better than under control conditions, leading to a biomass content enhancement of up to 43.18%. Under these conditions, the maximum fatty acids yield (750.29 mg/L), ARA yield (170.32 mg/L), cellular ARA content (10.61 mg/g) and ARA:TFA ratio (2.51) were obtained, which correspond to 29.76, 70.82, 19.21 and 99.21% increases, respectively, compared with the control. This higher ARA accumulation was associated with enhanced TAG accumulation and decreased levels of other UFAs, total protein and zeaxanthin. Zeaxanthin may play an important role in regulating the biosynthesis of metabolic products in P. purpureum; these pathways are distinct in stress and non-stress conditions. Further investigations should focus on the underlying mechanisms to further enhance the yields of valuable PUFAs by P. purpureum.This study explored the potential effects of 5-ALA on the growth of


The in vitro hydrolysis data confirmed that synthesized prodrugs 5a–c were stable in stomach while undergo significant hydrolysis in 80% human plasma and thus release free dexibuprofen. The minimum reversion was observed at pH 1.2 suggesting that prodrugs are less irritating to stomach than dexibuprofen. The anti-inflammatory activity of 5c (p < 0.001) is more significant than the parent dexibuprofen. The prodrug 5c produced maximum inhibition (42.06%) of paw-edema against egg-albumin induced inflammation in mice. Anti-pyretic effects in mice indicated that prodrugs 5a and 5b showed significant inhibition of pyrexia (p < 0.001). The analgesic activity of 5a is more pronounced compared to other synthesized prodrugs. The mean percent inhibition indicated that the prodrug 5a was more active in decreasing the number of writhes induced by acetic acid than standard dexibuprofen. The ulcerogenic activity results assured that synthesized prodrugs produce less gastrointestinal adverse effects than dexibuprofen. The ex vivo antiplatelet aggregation activity results also confirmed that synthesized prodrugs are less irritant to gastrointestinal mucosa than the parent dexibuprofen. Molecular docking analysis showed that the prodrugs 5a–c interacts with the residues present in active binding sites of target protein. The stability of drug–target complexes is verified by molecular dynamic simulation study. It exhibited that synthesized prodrugs formed stable complexes with the COX-2 protein thus support our wet lab results. It is therefore concluded that the synthesized prodrugs have promising pharmacological activities with reduced gastrointestinal adverse effects than the parent drug.Dexibuprofen–antioxidant conjugates were synthesized with the aim to reduce its gastrointestinal effects. The esters analogs of dexibuprofen  R,S-2-(4-isobutylphenyl)-propionic acid) a non-steroidal anti-inflammatory drug exists in two enantiomeric forms which are present in the racemate [S-(+)-ibuprofen is pharmacologically more active than racemic ibuprofen which has equal quantities of R-(−) and S-(+) enantiomers [Ibuprofen,  including dexibuprofen are stomach ulceration, bleeding, perforation and gastrointestinal (GI) irritations ,6. The f5a–c) was carried out in aqueous buffers and in 80% human plasma pH 7.4 for ensuring their stability. Pharmacological screenings like anti-inflammatory, analgesic, antipyretic and ulcerogenic activities of these prodrugs were evaluated and results were compared with standard dexibuprofen. The ex vivo platelet aggregation activity of the synthesized prodrugs was also carried out. Molecular docking and simulation studies were performed to predict the binding affinity of synthesized prodrugs with COX-2.A number of prodrugs of ibuprofen have been reported ,15,16 bu2a–c to afford their chloroacetyl chloride derivatives 3a–c. Dexibuprofen was then reacted with derivatives 3a–c to afford mutual prodrugs 5a–c  was found to be 69.4%, 61.5% and 64.7% in prodrugs 5a–c, respectively. All of the mutual prodrugs showed very encouraging hydrolysis rate in 80% human plasma (pH 7.4) and the regeneration of active drug was found to be 61% to 92%. The In vitro hydrolytic patterns of the ester prodrugs 5a–c in SIF (pH 7.4) and in 80% human plasma (pH 7.4) are presented in The in vitrohydrolysis of the synthesized prodrugs to free dexibuprofen was performed at pH 1.2 and 7.4 to mimic the stomach and intestinal environment respectively. The release of free drug by hydrolysis of the prodrugs was also studied in 80% human plasma at pH 7.4 to mimic the pH of blood. The prodrugs at gastric pH  are not hydrolyzed to free dexibuprofen while at higher (SIF pH 7.4) hydrolysis of the prodrugs is higher resulting in increase the amount free dexibuprofen. The dexibuprofen produced on hydrolysis after 1 h  of prodrugs 5a–c produced a significant reduction in edema formation. The prodrug 5c exhibited anti-inflammatory effects by significantly (p < 0.001) inhibiting the paw-edema at 4 h of post carrageenan induced inflammation with percentage inhibition of (28.99%). However, prodrugs 5a and 5b produced comparatively less anti-inflammatory response (p < 0.01) than standard dexibuprofen (p < 0.001) in second phase of induction of edema. Thus, this effect could be due to inhibitory effect on cyclooxygenase enzyme that leads to decreased production of Prostaglandins. The anti-inflammatory effect of standard dexibuprofen is also due to inhibition of both COX-1 and COX-2 [Anti-inflammatory activity results confirmed that there was a gradual increase in paw volume of mice in the control group. However, in the treated groups, all prodrugs buprofen . Carragend COX-2 .5a–c in egg induced inflammation also showed significant (p < 0.001) anti-inflammatory effects at 3 h post albumin induced inflammation with maximum inhibition (42.06%) produced by 5c. The standard drug dexibuprofen also produced significant (p < 0.001) anti-inflammatory effects at 3 h after the administration of egg albumin. Anti-inflammatory effects exhibited by prodrugs were comparable to parent dexibuprofen. Previously, it has been reported that injection of egg albumin caused release of histamine and 5-HT which lead to inflammation. Histamine is a vasodilator and causes increase in vascular permeability [Prodrugs eability ,24. The eability .5a–c exhibited a significant (p < 0.001) reduction in writhings induced by acetic acid. The mean percent inhibition indicated that the prodrug 5a was more active in decreasing the number of writhes induced by acetic acid than standard dexibuprofen. On the other hand, 5b and 5c showed comparable analgesic activity to dexibuprofen. On the contrary, all prodrugs displayed higher analgesic activity than standard dexibuprofen in formalin induced licking in mice. The analgesic response of synthetic prodrugs in formalin induced licking in mice was in order of 5a > 5b > 5c  reduced licking time in mice. The analgesic activity of prodrugs 5a–c was evaluated on the first and second phase of formalin-induced paw licking test. The results verified that these analgesic effects may be due to its central action as proposed previously by Ghannadi et al., 2005 [In acetic acid induced writhing model, abdominal constriction response is due to peritoneal receptors . It is rl., 2005 .5a and 5b showed significant inhibition of pyrexia (p < 0.001). Prodrug 5c showed significant inhibition of pyrexia after 2 h induction of pyrexia while standard dexibuprofen showed less significant (p < 0.05) inhibition of pyrexia. It has been well established that yeast induces pyrexia by increasing the production of prostaglandins (PGE2), which raises the set point of thermoregulatory center in hypothalamus [5b may likely to reduce pyrexia by decreasing prostaglandin E2 concentration in brain particularly in the hypothalamus [5a–c.Anti-pyretic effects in mice indicated that prodrugs thalamus . Therefothalamus  or by inthalamus . Antipyr5a–c was performed. The ulcer index (UI) of animals administered with dexibuprofen prodrugs 5a–c were compared with that of dexibuprofen and results were evaluated using one way ANOVA (followed by Tukey) test  test . Ulcer i5a–c) significantly inhibited platelet aggregation of rat platelet rich plasma induced by ADP or collagen. 5a–c. All of the synthesized prodrugs 5a–c having higher potential to inhibit the platelet aggregation than the positive control ASA. The prodrug 5c exhibited most potent inhibitory effects compared to other synthesized prodrugs.Dexibuprofen–antioxidant mutual prodrugs , comprising 552 residues. The crystal structure of COX-2 consists of 44% helices (248 residues), 11% β sheet (64 residues) and 43% coil (240 residues). The X-ray diffraction study confirmed its resolution 2.19 Å, R-value 0.255 and unit cell crystal dimensions like length and angles of coordinates. The unit cell length values and angle dimensions were observed for a = 180.27 Å, b = 134.27 Å and c = 122.61 Å with α, β and γ angles 90°, 90° and 90° respectively. The reliability and efficacy of cox-2 3D structure was confirmed by Ramachandran plots. The Ramachandran plots indicated that the 97.3% of all residues were present in favored regions and 100.0% residues were in allowed regions .5a–c were analyzed computationally to predict their basic molecular and chemo-informatic properties (3), molecular weight (160 to 480 g/mol) and PSA (<89 Å2) [5a–c results showed that MR  and PSA  predicted values are much effective compared to standard values. Lipinski′s rule of five (RO5) evaluations also presented their good therapeutic potential of ligands. It has been studied that that poor permeation is observed in the presence of greater values  of HBA and HBD, respectively [5a–c have<10 HBA and <5 HBD and comparable values for molecular weight (g/mol) and logP with standard values (<500 g/mol and <5). The RO5 also rationalizes that molecules having poor absorption are the results of >5HBD, >10HBA, MWT over 500, and logP more than 5. However, there are few existing drugs that violate the RO5 rule and have therapeutic potential [5b showed good drug likeness value (1.22) as compared to 5a (0.83) and (5b) (1.04). Overall, these predicted values justify the significance of 5a–c compounds as potential drug candidates.The prodrugs operties . Literat(<89 Å2) ,35. The ectively . The comotential ,38. 5b s5a–c were analyzed and evaluated on minimum energy  value as mentioned in 5b showed lowest binding energy value  as compared to others. Moreover, 5a and 5c also possess good binding energy values . The binding energy value of the parent dexibuprofen is −8.60 kcal/mol. Based upon the docking energy values it is proposed that all of synthesized prodrugs have comparable therapeutic potential. The binding energy values of all the docked complexes are also shown in the The prodrugs docked complexes of dexibuprofen and 5a–c directly interact with the binding pocket residues of targeted protein. 5a and 5b interact with same residues GLN189 and HIS193 in the active binding site of targeted protein. The ester carbonyl oxygen and phenoxy oxygen in 5a formed hydrogen bonds with GLN172 and HIS176 at a bonding distances 2.40 and 3.00 Å respectively. Similarly, the ester and methylene oxygen in 5b interact through hydrogen bonds with same residues GLN189 and HIS193 at bonding distances 2.44 and 3.60 Å, respectively. The comparative analysis showed that both produgs 5a and 5b confined at the same position in the active region of target proteins. The binding energy values  and strong binding interactions assure their greater affinity to bind at active binding site of COX-2. The 5c-receptor docked complex showed the conformational state of prodrug molecule with hydrogen bonds interactions near the receptor binding pocket (5c) docking result showed that three hydrogen bonds were observed at HIS374 and HIS372 residues position in the target protein. The coumarin lactonic oxygens forms two hydrogen bonds with HIS374 having bond length 3.64 and 3.54 Å. Similarly, the ester oxygen of same compound also forms hydrogen bond with HIS355 with bond distance 3.68 Å. The dexibuprofen docked complex with target protein is presented in The hydrogen bonding analyses showed that dexibuprofen and synthesized prodrugs g pocket . The (5c5a–c docking complexes in Gromacs 4.5.4 and performed MD at 5 ns. The Root Mean Square Deviation (RMSD) played a significant role in protein stability [5a–c depicted the backbone flexibility of the targeted protein  of the docked dexibuprofen structure showed the efficient binding within the active binding site. It has been reported that the higher RMSD value showed more deviation from the template structure [To evaluate the residual flexibility and structural behavior of target protein, we incorporated tability . The gen protein . Furing tructure .5a–c docking complexes were observed by Root Mean Square Fluctuation (RMSF). The predicted RMSF graphs of C-α atom of all three complexes were plotted against the interacted residues based on the trajectory period of MD simulation. All the residues in docking complexes were fluctuated around 0.1–0.3 nm in the simulation period. All of the prodrugs showed little changes in residual fluctuation at different loop regions. The RMSF results also reflect that N-terminal lobe of COX-2 is exhibited more fluctuations than C-terminus (Rg). The predicted results of COX-2 justified that Rg value is little fluctuated between 2.3 and 2.4 nm throughout the simulation time 0–5000 ps. The comparative results showed that residual backbone and folding of COX-2 was steadily stable after binding the inhibitor 5b compared to others , Carrageenan , Dried yeast , and Normal saline (0.9% NaCl) were used.1H-NMR and 13C-NMR spectral analysis were performed as CDCl3 solutions. The Agilent 6460 Series Triple Quadrupole instrument  was used to determine the molecular masses of the synthesized prodrugs. The electrospray ionization technique in the positive ion mode (ESI+) was employed for ionization. The purity of the final products was ascertained by preparative thin layer chromatography (TLC) on silica gel HF-254.The digital Gallenkamp  melting point apparatus model MPD BM 3.5 was used to determine the melting points. The FTS 3000MX and Bruker AM-300 spectrophotometers were used for FTIR and NMR spectral analysis. The 3a–c were synthesized starting with natural antioxidants 2a–c by employing this general procedure. The synthesized compounds 3a–c were purified by recrystallization in petroleum ether and ethyl acetate mixture (2:1).Antioxidants (0.01 mol) and triethylamine  were dissolved in anhydrous dichloromethane (25 mL) at 0 to −5 °C. To this solution, chloroacetyl chloride  in dry dichloromethane was added drop wise over a period of 1 h maintaining the temperature constant. The reaction mixture was stirred for 5 h at room temperature. After the completion of reaction the products were extracted with ethyl acetate and washed with 5% HCl, 5% sodium hydroxide and with saturated NaCl solution. The ethyl acetate layer was rotary evaporated to afford the antioxidant chloroacetyl derivatives. All of the antioxidant chloroacetyl derivatives Synthesis of 2-isopropyl-5-methylcyclohexyl chloromethanoate3a. Yield 71%, solid, m.p. 38–40 °C, Rf 0.55 (pet. ether:ethyl acetate 3:1), IR νmax cm−1 (KBr): 2961 (C–H), 1739 (C=O ester), 1511 (C=C), 1196 (C–O), 788 (C–Cl).Synthesis of 3,4-(methylenedioxy)phenyl chloromethanoate3b. Yield 82%, solid, m.p. 46–48 °C, Rf 0.6 (pet. ether:ethyl acetate 3:1), IR νmax cm−1 (KBr): 2941 (C–H), 1767 (C=O ester), 1510 (C=C), 1165 (C–O), 837 (C–Cl).Synthesis of 2-oxo-2H-chromen-7-ylchloroethanoate3c. Yield 81%, solid, m.p. 36–38 °C, Rf 0.59 (pet. ether:ethyl acetate 3:1), IR νmax cm−1 (KBr): 2945 (C–H), 1767 (C=O ester), 1511 (C=C), 1154 (C–O), 788 (C–Cl).4; 2.06 g, 0.01 mol) were dissolved in dimethylformamide DMF (25 mL). Triethyl amine  and potassium iodide  were added to the reaction mixture and stirred it overnight at room temperature. After the completion of reaction the mixture was poured into ice cold water and extracted with chloroform (4 × 25 mL). The combined organic layer was washed with 2% sodium thiosulfate, 5% HCl, 5% sodium hydroxide and finally with brine solution. The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure to obtain the corresponding mutual prodrugs. The title mutual prodrugs 5a–c were synthesized by following the above-mentioned general procedure starting with different antioxidants chloroacetyl derivatives 3a–c (Antioxidant chloroacetyl derivatives (0.01 mol) and dexibuprofen (ves 3a–c . The proSynthesis of (S)-((2-isopropyl-5-methyl-5-methylcyclohexyl) carbonyl) methyl 2-(4-isobutylphenyl) propanoata5a. Yield 76.5%, semisolid, Rf 0.64 (pet. ether:ethyl acetate 3:1), IR νmax cm−1 (KBr): 2953 (C–H), 1745 (C=O ester), 1138 ; 1H-NMR : 1.01 , 1.45 , 1.50 , 1.52 , 1.55 , 1.72 , 1.76 , 1.78 , 1.82 , 2.25 , 2.43 , 2.88 , 2.95 , 3.24 , 4.55 , 7.06 , 7.24 ; 13C-NMR : 26.15 (C-1), 31.65 (C-2), 44.63 (C-3), 25.89 (C-4), 40.63 (C-5), 167.38 (C-6), 50.18 (C-7),174.04 (C-8), 25.89 (C-9), 30.13 (C-10), 23.41 (C-11), 140.05 (C-1′), 127.23 (C-2′), 129.35 (C-3′), 137.17 (C-4′), 61.11 (C-1′′), 34.15 (C-2′′), 31.43 (C-3′′), 34.58 (C-4′′), 31.37 (C-5′′), 46.83 (C-6′′); ESI-MS: m/z 425 (43%) (M + 23) (M + Na), 247 (23%), 189 (100%), 161 (62%), 155 (43%), 133 (31%).d)-dioxol-6-yloxy) carbonyl) methyl-2-(4-isobutylphenyl) propanoateSynthesis of (S)-, IR νmax cm−1 (KBr): 2957 (C–H), 1735 (C=O ester), 1147 , 1105 (C–O ether); 1H-NMR : 1.05 , 2.11 , 2.20 , 2.67 , 3.78 , 4.89 , 5.80 , 6.40 , 6.53 , 6.72 , 7.15 , 7.24 ; 13C-NMR : 20.81 (C-1), 23.43 (C-2), 44.62 (C-3), 18.24 (C-4), 43.25 (C-5), 61.90 (C-6), 157.64 (C-7), 160.90 (C-8), 99.82 (C-9), 137.21 (C-1′), 128.00 , 128.35 , 131.01 (C-4′), 143.70 (C-1′′), 112.29 (C-2′′), 113.74 (C-3′′), 144.81 (C-4′′), 148.92 (C-5′′), 105.43 (C-6′′); ESI-MS: m/z 407 (39%) (M + 23) (M + Na), 247 (25%), 189 (100%), 161 (54%), 137 (37%), 133 (26%).Synthesis of (S)-((2-oxo-2H-chromen-7-yloxy) carbonyl) methyl 2-(4-isobutylphenyl) propanoate5c. Yield 68%, semisolid, Rf 0.75 (pet. ether:ethyl acetate 3:1), IR νmax cm−1 (KBr): 2957 (C–H), 1730 (C=O ester), 1142 ; 1H-NMR : 1.49 , 1.53 , 2.22 , 2.66 , 4.25 , 5.13 , 6.15 , 6.80 , 6.83 , 7.24 , 7.38 , 7.39 , 7.44 ; 13C-NMR : 20.81 (C-1), 25.34 (C-2), 49.62 (C-3), 16.50 (C-4), 40.21 (C-5), 173.15 (C-6), 69.29 (C-7), 174.44 (C-8), 137.73 (C-1′), 128.12 (C-2′), 129.39 (C-3′), 131.42 (C-4′), 150.01 (C-1′′), 116.34 (C-2′′), 125.21 (C-3′′), 117.08 (C-4′′), 140.64 (C-5′′), 109.50 (C-6′′), 160.92 (C-7′′), 147.64 (C-8′′), 108.28 (C-9′′); ESI-MS: m/z 431 (36%) (M + 23) (M + Na), 247 (30%), 189 (100%), 161 (63%), 134 (49%), 133 (32%).5a–c were performed in simulated fluid (pH 1.2 and 7.4) and in 80% human plasma (pH 7.4). These simulated fluids i.e., simulated gastric fluid (SGF pH 1.2) and simulated intestinal fluid (SIF pH 7.4) are extensively utilized in pharmaceutical industries for dissolution tests. Hydrochloric acid solution and potassium phosphate monobasic were used to prepare these media. The simulated gastric fluid is prepared by mixing the 0.2 M aqueous potassium chloride with a 0.2 M hydrochloric acid solution. The SGF was degassed after adjusting the final pH. The monobasic potassium phosphate buffer was prepared in water and final pH 7.4 was adjusted by slow addition of potassium hydroxide solution. To prepare the stock solution, 10 mg of each prodrug was mixed in 90 mL of SGF (pH 1.2) or SIF (pH 7.4). The 15 mL of this stock solution was taken repeatedly and kept in test tubes maintained at 37 ± 0.5 °C. From these test tubes some solution was taken after a fixed time interval  and was transferred to micro centrifuge tubes. The solution was then centrifuged at high speed (3000 rpm) for 5 min and clear supernatant was obtained. The amount of dexibuprofen released was then measured on UV spectrophotometer  after hydrolysis in buffer solutions at 230–215 and 280–260 nm respectively.The in vitrohydrolytic studies of mutual prodrugs The animals used for this study were 20–30 g weight mice either male or female and were housed at animal house department of Pharmacology, University of Sargodha. Animals were provided standard diet and water at libitum. All animals were treated according to instructions of National Institute of Health (NIH) guidelines. The study protocol was approved by the ethical Committee, Faculty of Pharmacy, University of Sargodha in the month of June 2014 vide project approval number PHRM-UOS-125.The temperature range of animal house was 26–34 °C and relative humidity was kept in a controlled range of 30% to 70%.The proper ventilation was provided to animals with quality air and stable environment. Animals were avoided from direct exposure to high velocity air (drafts) during the experiment. Heating, ventilation, and air conditioning (HVAC) systems were used to maintain the ventilation rates in accordance with heat load and other variables. Ten to fifteen fresh air changes per hour in animal house were acquired to maintain macro-environmental air quality by constant volume systems and may also ensure micro-environmental air quality.The solutions/suspensions of drugs were administered to the animals by either oral or subcutaneous routes. For oral administration oral gavage tube was used while for subcutaneous administration syringe was used.5a–c was determined by following the already reported method by slight modification against carrageenan induced paw edema in mice [5a–c, respectively, as 20 mg/kg p.o. The inflammation was induced after 1 h administration of prodrugs by injecting 0.1 mL freshly prepared carrageenan suspension (1%) in normal saline into the sub planar surface of the right hind paw. The paw volume was measured at 0, 1, 2, 3 and 4 h after the administration of carrageenan by using vernier caliper.X = mean increase in paw thickness of treated mice; and Y = mean increase in paw thickness of control mice.Anti-inflammatory activity of mutual prodrugs  in mice . The ani5a–c against egg albumin induced inflammation was also determined for further verification by following the previously reported method [5a–c were given to Groups III–V, respectively, as 20 mg/kg p.o. After 1 h of treatment, 0.1 mL of fresh egg albumin was injected into the paw for the induction of inflammation and the paw volume was calculated at 0, 1, 2 and 3 h after the administration of inflammatory agent.Anti-inflammatory potential of synthesized prodrugs d method . The ani5a–c [5a–c (20 mg/kg p.o.) were given to Groups III–V, respectively. For induction of writhings, acetic acid solution  was given to every mouse post 30 min of treatment. After 5 min of injection of this solution, the number of writhings was counted up to 15 min. The analgesic activity of synthesized mutual prodrugs 5a–c and dexibuprofen was determined by comparing the results with those of the animals in the control group.Koster et al., reported method was adopted for the determination of analgesic activity of the mutual prodrugs 5a–c . The mic5a–c for further verification [5a–c (20 mg/kg p.o.) were administered to Groups III–V, respectively. Formalin  was injected into sub-planter of right hind paw and responses were observed for 30 min.Formalin induced licking in mice was also used to determine analgesic activity of prodrugs fication . The ani5a–c was evaluated against yeast induced pyrexia in mice according to Sakande et al., reported method [5a–c (20 mg/kg p.o.) were administered to Groups III–V, respectively. Then animals were treated as described earlier with control, standard and prodrugs 5a–c. Rectal temperature of animals was recorded at 1, 2, 3 and 4 h after drug administration.Antipyretic activity of prodrugs d method . Aqueous5a–c were determined by following the fasted rat model. The animals were categorized into 6 groups, each has 5 animals. Group 1 was control and given 0.5% carboxymethyl cellulose (CMC) suspension. The standard dexibuprofen at a dose of 20 mg/kg p.o. was given to group II. The prodrugs 5a–c (20 mg/kg p.o.) were given to Groups III–V, respectively. Food and water was removed from animal cages 24 h before starting the experiments. After 4 h administration of dose, the animal′s stomach was removed by sacrificing the animal. Stomach was kept on saline soaked filter paper for inspection. The stomach was opened along larger curvature and cleaned with distilled water. The mucous was removed and the area of ulcers and stomachs were noted [The ulcerogenic potential of dexibuprofen and synthesized mutual prodrugs re noted ,48.UI) [Following formula was used for calculation of Ulcer index (UI) ;(2)UI=A5a–c was investigated by following the already reported method with some modifications [g for 15 min and further centrifuged at 850× g for 15 min to prepare platelet-poor plasma (PPP). Platelet aggregation was induced by 32.8 µg/mL of collagen or 1.3 µmol/L of adenosine diphosphate (ADP) in 300 µL of PRP. The PRP (300 µL) was incubated at 37 °C for 2 min in the aggregometer with stirring at 1000 rpm and then stimulated with ADP, collagen, epinephrine and A23187. The changes in light transmission were recorded for 5 min after stimulation. The synthesized mutual prodrugs 5a–c were added to the platelet suspension 3 min before the addition of the aggregating agents. The extent of inhibition of platelet aggregation is expressed as percent inhibition (X) using the following formula:A = maximal aggregation of the control; and B = maximal aggregation of sample-treated PRP.The antiplatelet activity of synthesized mutual prodrugs ications . Male Sppost-test were applied to assess the data. Values of p < 0.05 were considered as statistically significant.The results are expressed as means ± SEM and Parametric data were compared to control group. One-way ANOVA followed by Dunnet′s test and two way ANOVA followed by Bonferroni The three dimensional crystal structure of mice cyclooxygenase-2 (COX-2) was retrieved from the Protein Data Bank (PDB) with PDBID 3NTG. The overall stereo-chemical properties of cyclooxygenase-2 structure and Ramachandran graph and values  were ass5a–c were sketched in ACD/ChemSketch tool. The designed structures were further visualized and minimized by UCSF Chimera 1.10.1 [http://www.molinspiration.com/) and Molsoft (Available online: http://www.molsoft.com/) online computational tools were applied to predict the drug-likeness and basic biological properties of synthesized prodrugs. Moreover, Lipinski′s rule of five was analyzed using Molsoft and Molinspiraion tools. The number of rotatable bonds, H-bond acceptors (HBA) and H-bond donors (HBD) were also confirmed by PubChem (Available online: https://pubchem.ncbi.nlm.nih.gov/).The parent dexibuprofen and prodrugs a 1.10.1 . The MolX = 28.6432, Y = 28.6975 and Z = 9.4725, while, size of X = 59.75, Y = 77.94 and Z = 63.86, respectively, with spacing 1.0 Å and exhaustiveness values were adjusted to attain the finest binding conformational poses of ligand molecules. The maximum docking poses (100 runs) for each docking were adjusted to get the best docking complex with good conformational pose. All the synthesized mutual prodrugs 5a–c and dexibuprofen were docked separately against crystal structure of COX-2 and the obtained docked complexes were further evaluated on lowest binding energy  values. Docking analysis was employed by Discovery Studio (2.1.0) [The structure of target enzyme COX-2 was prepared and optimized to perform molecular docking studies. Hydrogen atoms were added to the target enzyme and bond order was also assigned. Molecular docking was carried out and different docked complexes were visualized by PyRx tool (vina wizard) . The gri (2.1.0)  and UCSFhttp://plasma-gate.weizmann.ac.il/Grace/). The best docked energy  complexes were selected for molecular dynamics (MD) simulation through Groningen Machine for Chemicals Simulations (GROMACS) 4.5.4 package  with GRO5a–c. The spectroscopic data of these prodrugs confirmed their structures. All synthesized mutual prodrugs showed encouraging hydrolysis rate in SIF + 80% human plasma probably due to the presence of esterase enzymes in plasma. The synthesized prodrugs are stable in gastric mucosa at pH 1.2 and are not hydrolyzed to free dexibuprofen. On the other hand, in simulated intestinal fluid pH 7.4, 18% of the prodrugs are hydrolyzed to free drug. The prodrugs synthesis also prevents the accumulation of drug in gastric mucosa, resultingin decreased GI irritation without the loss of pharmacological response. The molecular docking and simulation studies also proved that prodrugs formed stable complexes with the Cox-2 protein, thus supporting our wet lab results. The ulcerogenic activity and ex vivo antiplatelet aggregation activity results confirmed that synthesized prodrugs are less irritant to gastrointestinal mucosa than dexibuprofen. It is concluded, therefore, that the synthesized prodrugs have promising pharmacological activities with reduced gastrointestinal adverse effects compared to the parent drug.The present work utilizes safer promoieties, i.e., natural antioxidants, for the synthesis of dexibuprofen prodrugs


Hordeum vulgare L.). Breeding barley cultivars with high GPC has practical value for feed and food properties. The aim of the present study was to identify quantitative trait loci (QTLs) for GPC that could be detected under multiple environments.Grain protein concentration (GPC) is a major determinant of quality in barley  that linked in coupling phase were firstly identified. Moreover, the genetic effects of stable QTLs on chromosomes 2H, 6H and 7H were validated using near isogenic lines (NILs).A population of 190 recombinant inbred lines (RILs) deriving from a cross between Chinese landrace ZGMLEL with high GPC (> 20%) and Australian cultivar Schooner was used for linkage and QTL analyses. The genetic linkage map spanned 2353.48 cM in length with an average locus interval of 2.33 cM. GPC was evaluated under six environments for the RIL population and the two parental lines. In total, six environmentally stable QTLs for GPC were detected on chromosomes 2H (1), 4H (1), 6H (1), and 7H (3) and the increasing alleles were derived from ZGMLEL. Notably, the three QTLs on chromosome 7H  contains supplementary material, which is available to authorized users. Hordeum vulgare L.) is one of the earliest domesticated crops in the world. Approximately 25% of its production has relatively lower GPC and is suitable for malting and brewing, while the remaining 75% with relatively higher GPC is used for feed and food (http://faostat.fao.org/). Hence, there is increasing need for breeding barley cultivars with high GPC, but this has been hindered by the relatively low heritability of GPC due to the significant interaction between environmental and genetic factors [Protein is an essential nutrient for the survival of humans and animals , 2. Prot factors . Based oBmac0096-Bmag0323 and ABG458-HVM74 intervals, respectively, have been repeatedly detected [HvNAM1 and HvNAM2) on chromosomes 6H and 2H in barley, which were suggested to be orthologous to TtNAM-B1, contributed a substantial effect on GPC [HvNAM2 was associated with GPC, which is useful in developing high quality barley cultivars [To date, numerous studies have been conducted on dissecting the genetic basis of GPC, and QTLs have been mapped on all seven barley chromosomes. In particular, several consensus QTLs mapped on chromosomes 2H, 4H, 5H, 6H, and 7H have been repeatedly detected by multiple studies –15. For detected –12, 16. detected –19. In at on GPC , 20. Notultivars . Althougultivars , 22.A saturated genetic linkage map will improve the precision of QTL localization and estimation of phenotypic variance, especially for some small and medium-sized QTLs . Due to 9 to F11) containing 190 RILs derived from two spring barley varieties, ZGMLEL and Schooner, was employed to identify QTLs controlling for GPC. ZGMLEL is a hull-less landrace with high GPC, while Schooner is a hulled cultivar with low GPC. All the RILs and their parental lines were kindly provided by Dr. Yawen Zeng .A RIL population  and 7H  were linked in coupling phase, the QTL clusters were introgressed into the Schooner background as a unity, respectively. After three backcross generations (BC3), individuals that solely exhibited heterozygosity at one QTL region were self-pollinated to produce its corresponding BC3F2 populations. Finally, three NIL populations, that is BC3F2-I (region 2H), BC3F2-II (region 6H) and BC3F2-III (region 7H), were developed for the validation their corresponding QTLs. The number of progenies in BC3F2-I, BC3F2-II and BC3F2-III populations were 249, 205 and 213, respectively.For NIL development, one RIL line (RIL7) was crossed with the recurrent parent (Schooner). Because the QTLs on chromosomes 6H  in Beijing, Wangtaibao Experiment Station of NAAFS (Ningxia Academy of Agriculture and Forestry Sciences) in Ningxia Hui Autonomous Region, and Dishang Experiment Station of HAAFS (Hebei Academy of Agriculture and Forestry Science) in Hebei Province. The RIL population and the two parents were grown during three growing seasons from 2013 to 2015, providing data for six environments. Location-year information and corresponding weather data are presented in Additional file 3F2 populations for QTL validation were planted in Beijing (2016). Individuals were grown in 2-m-long rows with a 0.25-m row spacing. Within each row, 15 plants were evenly sown. At maturity, all the panicles were harvested from single-plant and sun-dried. Grain protein concentration (GPC), grain yield (GY) and thousand grain weight (TKW) were scored on a single-plant basis.Three BCThe field experiments were in accordance with local practice. All the trails were conducted under optimum irrigation. Nitrogen (N) was supplied at a rate of 220 kg/ha, including 70 kg/ha of N as diammonium phosphate and 80 kg/ha of N as urea applied before sowing. In addition, 70 kg/ha of N as urea was applied at booting stage.3F2 populations were ground to a powder using a Cyclotec 1093 sample mill . Then, the ground powder was dried to a constant mass in an 80 °C oven. The total nitrogen content was determined using the Kjeldahl method with a FOSS Kjeltec ™ 2300 and then the GPC was calculated using a factor of 5.83 [Mature grains of RIL population and BC® V.8 (SAS Institute Inc. 2000) with the PROC MIXED procedure. Under the random-effect model, environments were treated as fixed, and genotype and genotype-environments interactions were considered as random factors. The broad sense heritability (hB2) on a family basis was calculated using SAS® V.8 (SAS Institute Inc. 2000) with the PROC GLM procedure, which was calculated according to the following formula: hB2 = óg2/ (óg2 + óge2/n + ó2/nr) where óg2 = genotypic variance, óge2 = genotype by environmental variance, ó2 = the residual error variance, n = the number of environments, and r = number of replicates.The basic statistical analysis was performed using SPSS version 20.0 . The Shapiro-Wilk test was conducted using R software (V. 3.2.2) for the normality test. The best linear unbiased prediction (BLUP) for GPC across the six environments was calculated using SAShttp://wheat.pw.usda.gov/GG3/. Only markers with less than 5% missing data were selected for map construction. The genetic linkage map was constructed using RECORD 2.0 [The RIL population was genotyped using the barley 9 K SNP chip developed from the RNA-seq data of barley varieties . AdditioCORD 2.0  and JoinCORD 2.0 . MarkersP ≤ 0.05, and these QTLs were considered “identified QTLs”. A 2-LOD support with a 99% confidence level was chosen for each identified QTL. The identified QTLs detected in different environments with overlapping confidence intervals were regarded as the same in this study. The QTLs were named following the rules of Blake and Blake [The average GPC data in each environment and BLUP values across six environments were collected for QTL analysis. WinQTLCart2.5 software with the composite interval mapping (CIM) method was used to identify QTLs for GPC. The walking speed was set to 1 cM. Model 6 was chosen for QTL analysis, with 5 control markers and 10 cM window size defaults. The LOD threshold was set via 1000 permutations at nd Blake .Brachypodium) were used as queries to blast against the database “assembly_WGSMorex” at IPK Barley BLAST Server (http://webblast.ipk-gatersleben.de/barley_ibsc/). The Morex contigs with best hit were employed to search for simple sequence repeat using the SSR Hunter software. Finally, the selected sequences were used to design SSR markers using the Primer3 software (http://frodo.wi.mit.edu/primer3/).The flanking markers of the QTLs were employed to define the target region, which could be used to compare to the barley Genome Zipper developed by Mayer et al. . Gene seP < 0.01) than that of Schooner (16.35–17.20%) (Table hB2) for the GPC of the RILs was 80.67%, indicating that the GPC variance was mostly determined by genetic factors.The basic statistics of minimum, maximum, mean standard deviation and coefficient of variation for GPC in six environments are listed in Table 16.35–17.0%  were polymorphic between ZGMLEL and Schooner. After removing 53 SNPs with over 5% missing data, we used 1473 SNP markers and 21 polymorphic SSR markers to construct the linkage map. The resultant linkage map comprised nine linkage groups that contained 1011 unique loci and spanned 2353.48 cM. These linkage maps had an average locus interval of 2.33 cM  were successfully assigned to the barley genome  . We calcQGpc.ZgSc-6H.1, ZGMLEL contributed effects for increased GPC at other 16 QTLs. These 16 significant QTL had LOD values ranging from 2.51 to 15.51 and explained the GPC variation from 2.4% to 19.86%. Schooner contributed effects for increased GPC at the locus of QGpc.ZgSc-6H.1, which had a LOD value of 3.37 and accounted for 5.70% of GPC variation. This indicated that the favorable alleles for increased GPC were mainly inherited from the feed barley ZGMLEL. In the present study, significant QTLs that could be detected in no less than three environments as well as in the combined analysis were defined as environmentally stable QTLs. According to this criterion, 6 of 17 significant QTLs were environmentally stable QTLs, which were identified on chromosomes 2HL (1), 4HS (1), 6HL (1), and 7HS (3).A total of 17 QTLs were detected for GPC, which are randomly distributed among chromosomes 2H 3 QTLs), 4H (3 QTLs), 5H (3 QTLs), 6H (4 QTLs), 7H (4 QTLs) ; Table 3 QTLs, 4HSCRI_RS_171032 and BOPA2_12_30901, QGpc.ZgSc-2H.1, was considered being environmentally stable, which could be detected under three environments. QGpc.ZgSc-2H.1 had a LOD value of 3.78 and explained 4.47% of the GPC variation for the combined analysis. The other two putative QTLs, QGpc.ZgSc-2H.2 and QGpc.ZgSc-2H.3, were observed in one environment and explained 5.69–8.58% of GPC variation.Three QTLs associated with GPC were detected on chromosome 2HL. Only the one flanked by QGpc.ZgSc-4H.1. This QTL had a minor effect, with a LOD value of 3.55, and explained 4.20% of the GPC variation for the combined analysis. QGpc.ZgSc-4H.2 and QGpc.ZgSc-4H.3 could be detected only in one environment, which had LOD and R2 values ranging from 4.58 to 5.59 and 5.68% to 6.10%, respectively.Among three significant QTLs for GPC detected on chromosome 4H, one stable QTL was identified on chromosome 4HS and designated QGpc.ZgSc-5H.3, QGpc.ZgSc-5H.1, and QGpc.ZgSc-5H.2. QGpc.ZgSc-5H.3 had a LOD value of 3.31 and explained 3.37% of the GPC variation for the combined analysis. The other two QTLs had the LOD values ranging from 4.13 to 4.71, and explained the GPC variation from 6.56% to 7.08%.Chromosome 5HL carried three putative QTLs significantly associated with GPC, which were designated QGpc.ZgSc-6H.1, QGpc.ZgSc-6H.2, QGpc.ZgSc-6H.3, and QGpc.ZgSc-6H.4. Schooner contributed the effects for increasing GPC at QGpc.ZgSc-6H.1, and ZGMLEL contributed increased GPC at the other three loci. QGpc.ZgSc-6H.3 was the most stable QTL for GPC, which could be detected in all six environments and explained as much as 17.90% of the GPC variation for the combined analysis. Two putative QTLs, QGpc.ZgSc-6H.2 and QGpc.ZgSc-6H.4, were identified in two environments and explained 8.45–18.78% of GPC variation.Four significant QTLs were identified on chromosome 6HL and were designated QGpc.ZgSc-7H.1, QGpc.ZgSc-7H.2, and QGpc.ZgSc-7H.3. These three stable QTLs were detected in three to five environments, explaining 12.37–13.29% of GPC variation for the combined analysis. The last putative QTL, QGpc.ZgSc-7H.4, was detected at E6 and accounted for 7.48% of GPC variation.Four QTLs were found to be significantly associated with GPC on chromosome 7HS. Among these significant QTLs, three were environmentally stable QTLs, and they were designated 3F2 populations were developed and named BC3F2-I, BC3F2-II and BC3F2-III, respectively. Accordingly, three sets of SSR markers were used for foreground selection, i.e., 2L10, 2L11 and 2L12 for BC3F2-I, 6L89, 6L155 and 6L147 for BC3F2-II, and 7S40, 7S69, 7S87 and 7S89 for BC3F2-III , two homozygous groups, namely, ZGMLEL homozygous (ZZ) and Schooner homozygous (SS) were classified in each NIL population. The evaluation results for GPC showed that plants with ZZ genotype in BC3F2-I, BC3F2-II and BC3F2-III had an average GPC of 13.82, 14.18 and 14.20%, respectively. In contrast, plants with SS genotype in BC3F2-I, BC3F2-II and BC3F2-III had an average GPC of 13.15, 13.19 and 13.48%, respectively, which is similar to the recurrent parent, Schooner (13.32%)   appeared to be novel  locus, which is obviously different from QGpc.ZgSc-4H.1  were detected on chromosome 7HS. QTLs for GPC on chromosome 7H have been extensively reported [nud locus on chromosome 7HL [Remarkably, three neighboring QTLs , which might be caused by the special materials used in our study.An efficient method to introgress favorable alleles into elite germplasm is to select consensus QTLs that steadily affect GPC in different genetic backgrounds and environments . For exao et al. . Anotherpulation . HoweverHvNAM1 and HvNAM2 associating with GPC on the short arm of chromosomes 6H and 2H, respectively, have been widely studied [HvNAM1 and HvNAM2 markers were associated with GPC in barley. In the present study, two identified QTLs, QGpc.ZgSc-6H.3 and QGpc.ZgSc-2H.1 associated with GPC were also detected on chromosomes 6H and 2H, respectively, while they were both located on the long arms, demonstrating that HvNAM1 and HvNAM2 were obviously different from the QTLs detected in this study.To date, two homologous genes,  studied , 48, 49. studied  performeQGpc.ZgSc-6H.3) and two putative QTLs (QGpc.ZgSc-6H.2 and QGpc.ZgSc-6H.4). These three QTLs with favorable alleles from one parent (ZGMLEL) were in coupling phase. A shadow QTL, significant but false, is caused by a real QTL in an adjacent marker interval [QGpc.ZgSc-6H.2 and QGpc.ZgSc-6H.4 were located close to the stable QTL QGpc.ZgSc-6H.3, it is difficult to determine whether these two loci were shadow or genuine QTLs. Similarly, region 7H also contains three linked QTLs that were in coupling phase. Unlike the region 6H, region 7H harbored three environmentally stable QTLs and showed similar effects on GPC. Further studies are needed to dissect these two complex regions using advanced population.Neighboring QTLs associated with many important traits, such as yield and quality, that are linked in coupling phase are commonly observed in primary QTL analysis , 51. Preinterval . Since QQGpc.ZgSc-2H.1, QGpc.ZgSc-6H.3, QGpc.ZgSc-7H.1, QGpc.ZgSc-7H.2, and QGpc.ZgSc-7H.3 were identified and had additive effects of 0.23, 0.46, 0.38, 0.41, and 0.39% in the RIL population, respectively. Of these loci, three located on chromosome 7HS were linked together and resided in one genomic region. Three NIL populations, BC3F2-I, BC3F2-II and BC3F2-III, were developed according to a standard process of consecutive backcross, which targeted the genomic regions of chromosomes 2H, 6H and 7H, respectively. These stable QTLs were validated in the corresponding NIL population and their contribution to GPC could be directly compared between two homozygous groups in a similar genetic background. In populations I, II and III, the average GPC of plants carrying homozygous ZGMLEL were 0.66, 0.99 and 0.71% higher than that of plants carrying homozygous Schooner, respectively, providing further evidence for the reliability of these stable QTLs. Interestingly, the locus on chromosome 2HL (QGpc.ZgSc-2H.1) exhibited a strong potential increased in GPC in the Schooner background, which illustrated the conclusion that near-isogenic lines could be used to identify a quantitative locus even though it showed a relatively small effect on the phenotype [QGpc.ZgSc-2H.1, is feasible for cloning using NILs with least genetic noise. The estimation of the combination of three loci  in BC3F2-III, however, was somewhat lower than expected by the sum of the individual effects of three loci, which might be caused by QTL × QTL interactions, QTL × environment interactions or QTL × new genetic background interactions.QTL effect was generally not precisely estimated in primary QTL analysis due to the genetic noise in mapping populations –62. In vhenotype . Thus, t3F2 populations (11.66–16.82%) grown in Beijing during year 2016 was observed as compared with the RIL population grown in Beijing during year 2013 (16.08–23.36%) or 2014 (14.08–23.75%). This provided further evidence that GPC is largely modified by environmental conditions, which may be due to the alteration of weather condition in 2016. Previous studies indicated high temperature during grain filling stage could result in enhanced GPC [A relatively lower GPC of BCtions 11.–16.82% gnced GPC . InteresQGpc.ZgSc-2H.1), 6HL (QGpc.ZgSc-6H.3) and 7HS  using three NIL populations. In many cases, improvement of GPC is always accompanied by a significant reduction in grain yield [2L10-2L12), 6H (6L89-6L147) and 7H (7S87-7S40) may provide favorable regions for marker-assisted introgression into the elite barley germplasm.Since grain protein concentration is greatly influenced by environmental factors, breeding high-GPC cultivars only through phenotypic evaluation has been proved to be less effective , 69. Henin yield , 71. NotBased on genotyping 190 RILs in a genome-wide scale and measuring of GPC collected from six environments, six environmentally stable QTLs were significant associated with GPC, among which four QTLs on chromosomes 4H and 7H were firstly identified in the present study. Furthermore, three genomic regions harboring five stable QTLs on chromosomes 2H, 6H and 7H were validated using NIL populations, suggesting the reliability of QTLs detected in primary population. The markers linked to the stable QTLs would be valuable for MAS in barley breeding.Additional file 1: Table S1.Location-year information and climate data for field trails. (DOC 39 kb)Additional file 2: Table S2.Phenotypic data of two parental lines and 190 RILs under six individual environments and the combined analysis. (XLSX 31 kb)Additional file 3: Table S3.Map locations of all markers and genotypic data of 190 RILs. (XLSX 1050 kb)Additional file 4: Figure S1.Illustration of nine linkage groups constructed using 190 RILs. Notes: A centiMorgan (cM) scale is shown on the left. Black ellipses represent the approximate position for chromosome centromeres. The marker names are not shown. Detail information of the linkage group is provided in Additional file Additional file 5: Table S4.Alignments of SNP markers to barley reference sequence. (XLSX 76 kb)Additional file 6: Figure S2.New genetic linkage maps in the target region of the stable QTLs detected in ZGMLEL × Schooner RIL population. (A) chromosome 2HL, (B) 6HL, and (C) 7HS. New SSR markers are showed in bold and underlined. (DOC 299 kb)Additional file 7: Table S5.Markers developed to select the target regions of stable QTLs on chromosomes 2H, 6H and 7H. (DOC 38 kb)Additional file 8: Table S6.Phenotypic data of two homozygous groups in each NIL population. (XLSX 36 kb)Additional file 9: Table S7.a the consensus map reported by Close et al. [b the consensus map reported by Muñoz-Amatriaín et al. [Genome and chromosome size comparisons of this genetic map with previously reported maps. Notes: e et al. ; b the cn et al. .  in the ZGMLEL × Schooner population. Notes: QTLs in bold represent the environmentally stable QTLs. (DOC 35 kb)


N-Acetylneuraminic acid lyase  is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the equilibrium favoring Neu5Ac cleavage, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a “GRAS”  strain C. glutamicum ATCC 13032 . Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor Neu5Ac synthesis the most. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved . All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition. N-acetylneuraminic acid (Neu5Ac) is the most ubiquitous form2Sialic acids are a family of 9-carbon amino sugars involved in the modulation of various biological processes acid NeuAc is theN-acetylneuraminic acid lyase  has been employed in industrial production of Neu5Ac16N-acetyl-D-mannosamine (ManNAc) and pyruvate 1516Haemophilus influenzae NAL (PDB ID: 1F5Z)Pasteurella multocida NAL 8 barrel profile, and activated as a homotetramer82014trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase (HBPHA), trans-2′-carboxybenzalpyruvate hydratase-aldolase (CBPHA) and 2-keto-3-deoxygluconate aldolase (KDGA)182223E. coli NAL (L142R) to shift NAL's activity towards DHDPSIn order to understand its function and reaction mechanism, crystal structures of in vitro chemoenzymatic synthesis of Neu5Ac and its derivatives, recombinant NAL has been broadly applied with either E. coli1113142728As an important biocatalyst for Corynebacterium glutamicum ATCC 13032, which shows dual functions as NAL and DHDPS. More importantly, as NAL, CgNal shows a unique property of favoring Neu5Ac synthesis in the reversible reaction. In addition, the recombinant CgNal obtained a high expression level and achieved high production yield of Neu5Ac. These characteristics make CgNal a promising biocatalyst in industrial Neu5Ac biosynthesis processes.In this study, we report a novel NAL  from a “GRAS” organism C. glutamicum ATCC 13032 (GenBank accession: NP_601846.1) encoding a 939 bp open reading frame, which corresponds to 312 amino acids, was cloned into pET-28a (+) vector with an N-terminal His6-tag . The E. coli strain harboring pET28a-CgNal exhibited the highest CgNal activity when OD600 reached 0.5 with 0.2 mM IPTG as inducer at 30°C (E. coli Rosetta (DE3) pLysS culture, which is 2.23 fold higher than the highest expression level reported for LpNal (215 mg/L cell culture)CgNal gene of  at 30°C . Culture at 30°C . When cu at 30°C . Up to 4E. coliP. multocidaC. perfringensS. carnosus TM300L. plantarum WCF51CgNal catalyzes a reversible aldo-cleavage/synthesis reaction. The effects of pH on CgNal were determined considering both Neu5Ac cleavage and synthesis activities. Interestingly, CgNal prefers more alkaline conditions  than mosIn contrast to other NALs, CgNal is much more pH sensitive. For both Neu5Ac synthesis and cleavage reactions, CgNal showed sudden enhancement in its activities when the pH increased from 8 to 8.5. And the Neu5Ac synthesis activity was even higher than Neu5Ac cleavage activity when pH reached pH 8.8. This is the first time to report a NAL that favors aldo-condensation rather than cleavage. Considering this sharp change of CgNal's activity above pH 8, many CgNal characters were assayed at both pH 7.5 and pH 8.5.C. perfringens NALS. carnosus TM300 NALL. plantarum WCFS1 NALThe optimum temperature of CgNal was measured over a broad temperature range (30°C–65°C) at pH 7.5 and pH 8.5 for both Neu5Ac synthesis and cleavage directions . At pH 7The above results are in agreement with the following thermo stability studies . When Cg2+, activated the Neu5Ac synthesis at pH 7.5, while all metal ions inhibited the Neu5Ac synthesis at pH 8.5. There was no trend for the detergents' effects. EDTA showed activation activity at pH 7.5. TritonX-100 activated the Neu5Ac synthesis by 49.35% at pH 8.5. CTAB and SDS inhibited the synthesis activity on both pH. For Neu5Ac cleavage, metal ions can barely affect CgNal's enzymatic activity glutamicum ATCC 13032 DHDPS (PDB ID: 3CPR)In spite of its high NAL activity, the protein sequence of CgNal was annotated in GenBank (accession: NP_601846.1) as a putative DHDPS. Actually, CgNal showed low sequence identities with either EcNal (23.7%) 15168-barrel and the C-terminal extension of several α-helices, which are usual in the NAL family, includingNAL, DHDPS, KDGDH, HBPHA and other related enzymesAlthough, the identity of primary structure between CgNal and other NALs was fairly low, the predicted CgNal secondary structure showed much higher similarity with that of EcNal . CgNal sPseudomonas putida (GenBank accession: P42233.1) defined as the outgroup According to the primary sequence alignment, CgNal obtains 26.4% identity with EcDHDPS and 23.7% identity with EcNal. The phylogenetic analysis shows that CgNal is evolutionarily more related to DHDPS . But thecatk values of CgNal for Neu5Ac and ManNAc are similar to other NALs, but catk for pyruvate is about 3 times to others. At pH 8.5, the turnover rate increases dramatically for all substrates. The catk values for Neu5Ac, ManNAc and pyruvate are 44.2 s−1, 40.7 s−1 and 42.6 s−1, which are 3~20 times to those of other NALs. CgNal exhibits low affinity to Neu5Ac, which is the substrate for cleavage reaction. Km values of CgNal are 33.5 mM and 87.7 mM at pH 7.5 and pH 8.5, respectively. It is an order of magnitude higher than the Km values of NALs, which are 3.5 mM, 4.9 mM, 3.2 mM, 2 mM and 1.8 mM from E. coli, P multocida, C. perfringens, S. carnosus TM300 and L. plantarum WCFS1 respectively   . In EcDHAlthough there is no three-dimensional structure for either CgNal or CgNal-substrate complex available to confirm our hypothesis, the comparison among enzyme activities with different substrates of NAL (Neu5Ac synthesis/cleavage) and DHDPS supports our assumption. CgNal activities towards the substrates of both NAL (ManNAc and Neu5Ac) and DHDPS (ASA) were assayed with EcNal and EcDHDPS as controls. CgNal, EcNal and EcDHDPS all showed detectable activities towards substrates of NAL and DHDPS , possiblAs a unique enzyme showing bi-function as NAL and DHDPS, CgNal illustrated a distinctive set of key residues for substrates recognition. Moreover, the outstanding Neu5Ac synthesis activity of CgNal made it an interesting enzyme for enzymatic research and a promising catalyst in industrial synthesis of Neu5Ac. Due to the limited information we can extract from sequence alignment, further elucidation of the catalysis mechanism of CgNal can be achieved by resolving the crystal structure of CgNal itself and CgNal-substrate complex in our future study.C. glutamicum ATCC 13032 strain was obtained from ATCC . E.coli DH5α and E.coli Rosetta (DE3) pLysS strains were from Promega . pET-28a(+) vector were from Novagen . Restriction enzymes, DNA polymerase, genomic DNA extraction kit, DNA markers, protein markers were from Takara . CloneExpress one-step cloning kit was from Vazyme . N-acetyl-D-mannosamine and N-acetylneuraminic acid were from Sigma-Aldrich . All other chemicals were of analytical grade and commercially available.C. glutamicum ATCC 13032 and E. coli Rosetta (DE3) pLysS were isolated as PCR template, respectively, using Takara genomic DNA extraction kit. The CgNal gene sequence (GenBank accession: NP_601846.1), which was deposited in NCBI database as a putative DHDPS, was amplified from C. glutamicum genomic DNA with primers specific for its 5′ and 3′ ends. The E. colidapA gene, encoding a DHDPS, and dapB gene, encoding a dihydrodipicolinate reductase (DHDPR) protein, were amplified from E. coli genomic DNA with primers specific for each 5′ and 3′ ends, incorporating EcoRI and HindIII restriction sites, respectively. All primers (E. coli Rosetta (DE3) pLysS cells to obtain recombination strains pET28a-CgNal-Rosetta, pET28a-EcDHDPS-Rosetta and pET28a-EcDHDPR-Rosetta.Genomic DNA of  primers  were desE. coli was constructed in our previous research−1 kanamycin and 34 μg·ml−1 chloromycetin) overnight at 37°C, 220 rpm, and were induced at optical density (OD600) of 0.6–0.8, with 0.5 mM β-D-1-thiogalactopyranoside (IPTG) at 30°C overnight. Cells were harvested by centrifugation at 4000 g for 10 min at 4°C . Prior to sonication, the cell pellets were washed twice with lysis buffer . Resuspended with 10 ml of lysis buffer, cells were sonicated at 200 W with 3 s on and 5 s off for 60 times, using Ultrasonic Disruptor JY92-11 . Clarified by centrifugation at 9000 g for 20 min at 4°C, the supernatants were loaded onto Ni–NTA Agarose column . After washing the column with 40 ml of washing buffer , enzymes were eluted with 2 ml of eluting buffer . Purified CgNal, EcNal, EcDHDPS and EcDHDPR were dialyzed in 100 mM Tris-HCl, pH 7.5, for 24 h, at 4°C prior to any assay. Purities of enzymes were estimated to be >90% by Coomassie Blue G-250 stained 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The recombinant strain pET28a-EcNal-Rosetta harboring Nal cloned from 600) before induction, β-D-1-thiogalactopyranoside (IPTG) concentration and induction temperature on CgNal expression were examined. It was performed by changing OD600 between 0.1–1.5, IPTG concentrations between 0.1–0.8 mM and induction temperature between 20°C–40°C, respectively, while other conditions were fixed. Crude enzyme activities  after centrifugation and sonication were assayed to determine the effects.Effects of induction conditions, including optical density  as well as its ability to cleave Neu5Ac into ManNAc and pyruvate (Neu5Ac cleavage). The Neu5Ac synthesis reaction mixture consisted of 0.1 M pyruvate, 0.05 M ManNAc and 0.1 M Tris-HCl (pH 7.5 or 8.5) and the Neu5Ac cleavage reaction mixture contained 0.05 M Neu5Ac and 0.1 M Tris-HCl (pH 7.5 or 8.5). Protein concentration was determined by Bradford methodThe effect of pH on CgNal was determined using the following buffers: 0.1 M Tris-HCl (pH 7–pH 8.8) and 0.1 M glycine-NaOH (pH 9). Optimum pH for CgNal was determined by measuring CgNal activities in reaction systems with different pH. Optimum temperature of CgNal was determined by measuring CgNal activities (at pH 8.5) under different reaction temperatures (30°C–65°C). CgNal stability was determined by measuring residual Neu5Ac activities after incubating the enzyme at pH 8.5, 40°C for 48 h.2, NaCl, BaCl2, FeCl3, KCl, ZnCl2, CoCl2, MgCl2, NH4Cl, NiSO4, EDTA, TritonX-100, CTAB and SDS. Reaction mixture without salts and detergents was used as control.Effects of salts and detergents on CgNal were determined by measuring Neu5Ac synthesis and cleavage activities of CgNal in 100 mM Tris-HCl buffer (at pH 7.5 and pH 8.5) in the presence of 5 mM salts and detergents, including CaClm, Vmax) for CgNal and EcNal were assayed by measuring their activities at the presence of various concentrations of substrates. For Neu5Ac synthesis reaction, ManNAc concentration was fixed at 50 mM with pyruvate concentrations varied (20 mM–450 mM) to determine kinetic parameters for pyruvate; and pyruvate concentration was fixed at 100 mM with ManNAc concentration varied (20 mM–450 mM) to determine kinetic parameters for ManNAc. As for Neu5Ac cleavage reaction, Neu5Ac concentration was varied from 1 mM to 200 mM to determine kinetic parameters for Neu5Ac. Km and Vmax were calculated with GraphPad Prism 5.0 .Kinetic parameters  of NCBI was used to identify proteins homologous to CgNal, and MEGA 5.2CgNal's activity towards the substrates of both NAL (ManNAc and Neu5Ac) and DHDPS (ASA) were assayed with EcNal and EcDHDPS as controls. The activities of CgNal with ManNAc and Neu5Ac as substrate were assayed as mentioned in Enzyme assay of CgNal section. The activity of CgNal with ASA as substrate was measured using a coupled enzymatic assay as described by Yugari and GilvargCharacterization of a novel N-acetylneuraminic acid lyase favoring N-acetylneuraminic acid synthesis