The lignin can compete for binding cellulase enzymes with cellulose fibers and decrease the accessibility of enzymes to carbohydrates. The competitive adsorption of cellulase to lignin mainly depended on the chemical structure of lignin. The post-pretreatment can decrease the lignin content and modify the lignin structure of pretreated substrates, which reduced the lignin inhibition on enzymatic saccharification. Therefore, the post-treatment by modifying the lignin structure would attract considerable attention for weakening the cellulase–lignin interactions.P. amarus, 15.4% for AO-P. amarus and 21.4% for AC-P. amarus, respectively, which were 1.4–3.5 times of alkali pretreated P. amarus.Three modified lignins, including sulfonated lignin (SL), oxidized lignin (OL), and carboxylated lignin (CL), were prepared from alkali lignin (AL) and their structures and physicochemical properties were characterized using FTIR, NMR, XPS analysis, zeta potential, and contact angle, respectively. Compared to AL, three modified lignin preparations exhibited the decrease in contact angle by 61–70% and phenolic hydroxyls content by 17–80%, and an obvious increase of negative charges by about 21–45%. This was mainly due to the drop of condensation degree and the incorporation of carboxylic and sulfonic acid groups into modified lignins. Langmuir adsorption isotherms showed that the affinity strength between cellulase and modified lignins significantly reduced by 54–80%. Therefore, the 72 h hydrolysis yield of Avicel with SL, OL, and CL was 48.5, 51.3, and 49.4%, respectively, which was increased 8–15.3% than that of Avicel with AL, 44.5%. In the enzymatic hydrolysis of bamboo biomass, the glucose yield at 5 d was 38.5% for AS-The post-treatment can weaken the nonproductive adsorption between lignin and cellulase proteins and improve the enzymatic saccharification efficiency. This study will provide a conceptual combination of pretreatment technologies and post-pretreatment by modifying lignin structure for reducing the cellulase–lignin interaction. Biochemical conversion of lignocellulosic biomass to sugar-based compounds has been devoted for the production of biofuels and platform chemicals such as 5-hydroxymethylfurfural and furfural . A crucip-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, through the linkage of various interunit linkages, such as β-O-4′, β-5′, β-β′, 5-5′, 5-O-4′, and β-1′ [Lignin is a cross-linked polymer and consists of different phenylpropane, such as and β-1′ . In addiand β-1′ . Therefoand β-1′ , but alsand β-1′ , 9. Alth2+ and Mg2+, might also occupy the active sites of lignin and form lignin–metal complex, which blocked the interaction between cellulase and lignin [How to reduce the cellulase–lignin interaction for enhancing the enzymatic saccharification efficiency has been widely studied. The anionic surfactant and hydrophobic proteins, as lignin blocker, had been reported to be effective to weaken or eliminate the nonproductive adsorption by blocking the exposed sites of lignin surfaces, thereby decreasing the cellulase activity loss , 11. Ligd lignin , 14. Thed lignin , 16. It d lignin . Lignin d lignin , 19. In d lignin –22. ThesThe primary goal of this work is to modify the surface properties and structure of residual lignin through introducing functional groups, which have potential for reducing the cellulase–lignin interaction. Modified lignin preparations, including sulfonated lignin (SL), oxidized lignin (OL) and carboxylic lignin (CL), were prepared from alkali lignin (AL), and their structural properties were thoroughly investigated by FTIR, 13C, 2D HSQC NMR, and XPS analysis. The phenolic hydroxyl group content, the amount of negative charges and contact angle of lignin preparations were measured, respectively. The Langmuir adsorption isotherm was used to characterize enzyme affinity to lignin preparations. Finally, the effect of modified lignins on the enzymatic digestion of cellulose were evaluated and compared; whether the lignin modification as post-treatments can work on lignocellulosic biomass was also investigated.13C and 2D HSQC NMR were used to characterize the chemical structure of lignin preparations. The fingerprint region (800–1800 cm−1) of FTIR spectra, corresponding to the stretching vibrations of different groups in lignin preparations, is displayed in Fig. −1 observed in all samples were the aromatic skeletal vibrations, implying that the core of lignin structure was not altered significantly during the modified process. However, the spectra also showed some changes in the peaks and the absorption intensities. The peak at 1708 cm−1 characteristic of non-conjugated carbonyl groups is much more intense in AL than OL. And even such a band was not present in SL and CL. It could be due to the carboxyl or ester linkage of Cγ in AL side chain was partially cleaved during the modification process. The absorption band of 1649 cm−1, assigned to conjugated carbonyl groups, was identified in SL, OL, and CL. The intensity of 1272 cm−1 corresponding to the C–O stretching of G type lignin had not been significantly changed after modifications. The signal intensity of p-hydroxyphenyl units (H unit) at 834 cm−1 was reduced in modified lignin, compared with that of AL, indicating the dissolving of H-type lignin. As compared to AL, OL, and CL, the successful introduction of the sulfonic groups into SL can be justified by the stronger bands at 1043 cm−1 (S=O stretching vibration) and 536 cm−1 (C–S stretching vibration).The FTIR, 13C NMR spectra  units occurred as two signals at 128.0 and 129.2 ppm (C-2/C-6); and the lignin sulfonation, oxidation and carboxylation can reduce the level of H-lignin unit. The transformation of above signals indicated that lignin polymer was dissociated and the modification can improve the hydrophilicity and solubility of lignin due to altering the relative proportions of lignin subunit . Moreover, the bands of condensed aromatics (140–123 ppm) became weak and narrow in SL, OL, and CL compared to those in AL, implying that fewer condensation reactions could occur between free lignin units during the modification. The signal intensities between 50 and 90 ppm corresponding to lignin interunit linkages had no changes, suggested that the modification could occur on the surface of lignin moieties. The decreasing signal intensities for –OCH3 can be seen at 56.5 ppm, implying that the modification reactions could cause demethoxylation and demethylation in AL. In addition, the aliphatic COOR at 178–168 ppm are more abundant in CL, followed by OL and SL. In the carboxylation reaction, the H atom in lignin hydroxyl was substituted by the –OCH2COOR, which could be responsible for the most abundant aliphatic COOR.In the tra Fig.  and the tra Fig.  of ligniositions , 26. As δC/δH 50–95/2.5–6.0) and aromatic regions (δC/δH 95–160/5.5–8.50) and correlation signals are assigned in Table To further structural investigation, the 2D HSQC NMR was used to analyze the structural units and different interunit linkages of lignin polymers. The HSQC spectra consisted of the side chain (O-4′ aryl ether (A) and methoxyl groups (δC/δH 55.4/3.72) are the most prominent signals observed in all spectra. The signals at δC/δH 71.6/4.88 (Aα), δC/δH 82.8–85.7/4.13–4.46 (Aβ(G) and Aβ(S)), and δC/δH 59.5–59.7/3.40–3.63 (Aγ) belong to the Cα-Hα, Cβ-Hβ, and Cγ-Hγ correlations of the β-O-4′ ether substructures, respectively. As shown in Fig. Aα and Aβ were still detected and no changes occurred in all samples. However, it was interesting that by comparison with AL, three modified lignins exhibited the absorption of intense signals at δC/δH 59.5–63.5.7/3.40–3.83 (Aγ). This phenomenon suggested that the AL has been modified at the acylated γ-carbon in β-O-4′ aryl ether linkages of the side chains. And β-β′, β-1′and β-5′ substructures were not also observed in the spectra of modified lignin, suggesting that the surface modification of lignin could not cause the condensation between lignin units. Aromatic regions of the HSQC spectra can give basic correlation signals of the p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin units. Specifically, the C2,6-H2,6 correlations signals of S and Cα-oxidized S (T′) units were shown at δC/δH 103.8/6.69 and δC/δH 106.4/7.31. In Fig. 2,6-H2,6 in S unit tended to reduce in the modified samples compared to that of the control AL, indicating a decrease of S units in the surface of modified lignins, which was due to the cleavage of β-O-4 linkage. Similarly, the C5-H5, C2-H2, and C6-H6 correlations in G units, at δC/δH 115.1/6.72, δC/δH 111.0/7.02, and δC/δH 119.0/6.77, respectively, were observed in very low intensities for three modified lignins. It is worth mentioning that p-hydroxyphenyl (H) units exhibited the strong signal for C2,6-H2,6 at δC/δH 127.5/7.23 in others lignin, while it was weak in OL due to that oxidation treatment can result in less H units than that of other lignin samples. The above findings to aromatic ring units of lignin are consistent with FTIR analyses. In addition, the C2-H2, C6-H6, and C7-H7 correlations signals of ferulic acid (FA) were clearly observed at δC/δH 110.8/7.35, 123.1/7.16, and 144.4/7.42 in the spectra. Alkali lignin showed a higher proportion of FA, but the intensity of FA signals could be weakened after the oxidized and carboxylic reaction, and it disappeared after sulfonated reaction. Simultaneously, the relatively strong signals at the δC/δH 129.8/7.52, 143.9/7.51, and 115.0/6.24, corresponding to p-coumarates (pCA) were presented in all lignin samples except for SL; however, in comparison with AL, pCA units of oxidized (OL) and carboxylic lignin (CL) samples showed a slightly decrease. It had suggested that p-coumaric and acid ferulic acid are associated with the lignin monomer through either C–C or ether linkages and the carboxylic groups were not in free but in ester form [In the side-chain regions, the β-ter form –31. Thister form .Fig. 32DThe XPS analysis can provide some knowledge about the elemental composition and functional group abundances on the near-surface regions of materials , 34. As p-coumaric, acid ferulic acid, sulfonate groups, and aliphatic COOR, and a decrease of condensation degree and S-lignin, G-lignin unit in the modified lignins. The results were further indication that the sulfonated, oxidized, and carboxylation modification of lignin could be used to adjust lignin chemical functionality and surface hydrophilicity. Zeta potential values of all lignin samples were measured and are compared in Table The total concentration of free phenolic groups in all lignins using the F–C method is listed in Table R = K × Γm) of cellulase on AL that represented the binding strength of lignin to cellulase enzymes was 0.253 L/g lignin, which was two to threefolds higher than those on SL (R = 0.083 L/g), OL (R = 0.089 L/g) and CL (R = 0.104 L/g). It was noticed that the binding strength of AL (R = 0.253 L/g) was even higher than that of Avicel (R = 0.133 L/g), suggesting that AL would get more chance to adsorb cellulase enzyme than cellulose. Consequently, after modification reaction, the maximum adsorption capacity of lignin toward enzymes was significantly reduced. Specifically, AL (Γm = 42.78 mg/g lignin) had stronger adsorption capacity, which can adsorb about 7.9–24% more protein than modified lignins. And the maximum adsorption capacity of modified lignins decreased in the order OL > CL > SL, among which sulfonic acid groups caused greater decrease in the binding strength than the oxidized and carboxylate groups. This was probably because the introduction of sulfonic groups could consume more phenolic hydroxyls and bring more negative charges in lignin moieties to some extent.To examine how the modification in AL interfered the cellulase–lignin interaction, the cellulase adsorption on lignin samples and Langmuir adsorption isotherms of cellulase enzymes were analyzed and are summarized in Fig. −1), thus the zeta potential values of modified lignin were increased from 3.5 eV (AL) to − 21.5 eV (OL), − 27.3 eV (CL) and − 45.9 eV (SL), which increased the electrostatic repulsion between enzyme and lignin and prevented the nonproductive binding. And with the consumption of phenolic hydroxyl groups in the sulfonation, oxidation, and carboxylation, the content of phenolic hydroxyls decreased by 49, 17, and 80%, respectively, comparing to AL. This meant the decrease of binding strength between cellulase and modified lignins, because the cellulase adsorption on lignin correlated positively with phenolic hydroxyl content [We believe that the decrease of cellulase–lignin interaction was related to the structure and physicochemical properties of modified lignins. As well known, the hydrophobic, electrostatic, and hydrogen-bonding interactions could mediate the nonproductive adsorption of cellulase onto lignin. Alkali lignin had a contact angle of 98.7° and the binding strength of 0.253 L/g lignin. The modification led to a decrease in contact angle of lignin by 61–70%, due to the drop in the degree of condensation and lignin subunits, such as syringyl (S) and guaiacyl (H), and the more exposed carboxylic groups. This can make the lignin more hydrophilic, thereby decreasing the nonproductive binding for cellulase. In addition, the functional groups introduced into AL also had a much greater impact on the surface charge of lignin moieties and the formation of hydrogen bonds. According to XPS analysis  and C13 NMR 178–168 ppm), a low content of carboxylic groups was introduced to the CL and OL surface, and the abundant of sulfonic acid groups also was incorporated into SL in the FTIR  was extracted with 10% (w/w) sodium hydroxide at 70 °C for 3 h, followed by sulfonation, oxidation and carboxylation post-treatment. As seen in Fig. P. amarus almost was not degraded by enzymatic cellulase due to the strong recalcitrance. The 5 days hydrolysis of alkali pretreated P. amarus (A-P. amarus) only obtained the glucose yield of 11.1%. However, when alkali pretreatment was followed by the sulfonation, oxidation and carboxylation post-pretreatment, glucose yield was increased to 38.5% (AS-P. amarus), 15.4% (AO-P. amarus), and 21.4% (AC-P. amarus), respectively. The corresponding xylose yield was also increased from 26.2 to 53.9, 29 and 30.9%, respectively. And the enhancing effects caused by sulfonation post-treatment were greater than oxidation and carboxylation post-pretreatment. The results suggested that the post-pretreatment by modifying lignin can enhance glucose and xylose release in the enzymatic hydrolysis of lignocellulosic biomass. We believe that the increase of glucose and xylose yield after post-treatment can most likely be attributed to the decrease of lignin inhibition on enzymatic hydrolysis. In addition, it is important that alkali pretreatment is still considered as the appropriate method for developing the combined pretreatment technologies for reducing lignin inhibition on enzymatic hydrolysis. Because alkali pretreatment can decrease the lignin content, the sulfonation, oxidation, and carboxylation post-treatment can reduce the nonproductive adsorption of cellulase on lignin, which could contribute to the decrease of lignin inhibition together.To verify whether lignin modifications as post-treatment technologies can work on lignocellulosic biomass, bamboo  was obtained from Yunnan, China and its glucan, xylan, and lignin contents were 41.7, 15.7 and 37.5%, respectively. Cellulase (UTA-8) from Trichoderma reesei with the filter paper activity of 100 FPU/mL and the β-glucosidase activity of 71 IU/mL, was kindly donated by Youteer Biochemical Co., Ltd. (Hunan) and used in subsequent enzymatic saccharification of cellulose. Celluclast 1.5 L (Sigma 2730) from Sigma-Aldrich (Shanghai) exhibited the protein content of 40 mg/mL and was employed in experiments of cellulase adsorption.Alkali Lignin, a mixture of different herbaceous plants, such as corn stover, bamboo and straw, were supplied by Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai), which was obtained by NaOH pretreatment and then purified by an acidulation precipitation method. The chemical composition of AL was as follows (dry weight basis): 90.9% acid insoluble lignin, 3.67% acid soluble lignin, 0.1% glucan, 0.05% xylan, 2.19% ash, and 2.79% others. Microcrystalline cellulose  was purchased from Sigma-Aldrich (Shanghai). The bamboo (2O2 of 2 mL/g lignin and FeSO4 (16 mM) were added in the solution and the mixture was reacted at room temperature (22 ± 1 °C) and 80 rpm in a water bath for 24 h. Carboxylated Lignin (CL) was prepared through the reaction between hydroxyl groups and sodium chloroacetate, as described previously [Three modified lignins were prepared from AL. Sulfonated Lignin (SL) was obtained by treating 10.0 g AL with 30 mL NaOH solution (0.8 M) and 1.5 mL formaldehyde in 70 °C for 90 min. Sodium sulfite of 2 g was mixed with the above solution to carry out sulfonation reaction at 95 °C and 3 h . Oxidizeeviously . The sol13C and HSQC NMR spectra of lignins were acquired with a Bruker AVIII 400 MHz spectrometer. The sample (80 mg) was placed in NMR tubes with 0.5 mL dimethyl sulfoxide-d6. 2D HSQC spectra were recorded on the same spectrometer using 128 scanning time, a 2.6-s delay time between transients, and a 1.5-s relaxation time. X-ray photoelectron spectroscopy (XPS) analyses was conducted on an Escalab 250Xi spectrometer , with non-monochromatic Al K alph , under a high vacuum of 2 × 10−9 mbar and at room temperature. The content of free phenolic group in lignin samples were estimated by the Folin–Ciocalteu (F–C) method using phenol as standard [θ) values were obtained from measurements at three points in each lignin surface. The measurements of Zeta potentials were analyzed using a Malvern Zetasizer Nano ZS instrument. Lignin preparations  were incubated in 0.05 M citrate buffer (pH 4.8) for 1 h, followed by determination of zeta potential. The reported result of each sample was the average of three trials.The lignin preparations were analyzed using a 710 FTIR spectrophotometer . The standard , 40, 41.Γm, mg/g lignin) and affinity constant were estimated by nonlinear regression of adsorption data according to the Langmuir isotherm equation (Γ = ΓmKC/(1 + KC)), where Γ is the amount of adsorbed enzyme (mg/g substrate), C the amount of free enzyme in solution (mg/mL) and K the Langmuir constant (mL/mg enzyme). And the distribution coefficient (R = Γm × K L/g), represented binding strength, was also analyzed.The adsorption isotherm of enzyme on lignin preparations was studied with different concentration cellulases from 0.01 to 2 mg/mL . Lignins2SO4 as eluent at the flow rate of 0.6 mL min−1, as described before. The glucose yield was defined as the percentage of the glucose content in hydrolysate based on the theoretical glucose available in the cellulose. And protein concentration in supernatant was determined by Bradford assay, and calculated based on the total protein concentration. The relative enzyme activity at 72 h of enzymatic hydrolysis was also measured using the filter paper assay [Enzymatic hydrolysis was conducted at 50 °C and 150 rpm in the 0.05 M sodium acetate buffer (pH 4.8) for 72 h, with a solid substrate loading of 2% (w/v) Avicel. The dosage of cellulase (UTA-8) was 10 FPU/g glucan. To investigate the effects of lignin compounds  on enzymatic hydrolysis, the 4 g/L lignin sample  or the mixture of 2 g/L AL with 2 g/L  were added into pure cellulose, respectively, prior to the addition of cellulase. Enzymatic saccharification with addition of AL only was used as the control. Aliquots (0.2 mL) were withdrawn from the supernatant at 2, 4, 6, 10, 24, 48 and 72 h of hydrolysis. Quantitative analyses of glucose were measured by an Agilent 1260 series HPLC equipped with Aminex HPX-87H column at 60 °C column temperature, using 5 mM Her assay .P. amarus was extracted at 70 °C for 3 h with 10% NaOH (w/w) based on the dry weight of biomass, in a solid to liquid ratio of 1:10 (w/w). After washing with water, the solid residue was subjected to sulfonation, oxidation and carboxylation post-treatment; the post-treatment condition was the same as that of the AL. The effects of combination of alkali pretreatment and post-treatment were evaluated based on the subsequent glucose and xylose release in the hydrolysate. Enzymatic hydrolysis of pretreated substrates was conducted at 50 °C, pH 4.8 and 150 rpm for 5 days, with a substrate loading of 5% (w/v) and a cellulase (UTA-8) loading of 20 FPU/g glucan. The glucose and xylose yield were defined as weight percent yield of glucose and xylose with respect to the total glucan and xylan in the bamboo biomass. The compositions of alkali pretreated P. amarus (A-P. amarus) were 51.8% glucan, 17.5% xylan, and 35.1% lignin. The combination with alkali pretreatment and sulfonation, oxidation and carboxylation post-treatment of P. amarus was defined as AS-P. amarus and AO-P. amarus and AC-P. amarus, respectively. After post-treatment, AS-P. amarus contained 50.9% glucan, 13.1% xylan, and 23.4% lignin; and AO-P. amarus 54.4% glucan, 15% xylan, and 27.1% lignin; and AC-P. amarus 60.9% glucan, 14.8% xylan, and 21.6% lignin, respectively.


Its detection threshold was determined by Three-Alternative Forced-Choice (3-AFC) and curve fitting (CF), which was 0.740 mg∙L−1 in 38% ethanol solution. The contribution of propyl lactate on the aroma of these distillate drinks was evaluated by their odor activity values (OAVs), which varied from 0.066 to 4.440. The OAVs of propyl lactate were found to exceed 1 in 13 Chinese Baijius, including 50° Jingzhi Guniang 5 years (4.440), 52° Jingzhi Guniang 10 years (3.024), Jingyanggang (2.568), Xianghe Ronghe Shaofang (2.313), and 1956 Laolang (1.431), which indicated that propyl lactate was one of odor-active components in these Chinese Baijius.As one of the oldest distillates in the world, flavor compounds of Chinese Baijiu (Chinese liquor) were extremely complex. Propyl lactate was firstly detected by direct injection and gas chromatography-mass spectrometry (GC-MS) in 72 Chinese Baijius. The objectives were to detect the contents of propyl lactate and evaluate its contribution to the aroma of Chinese Baijiu based on odor activity values (OAVs). The levels of propyl lactate in these distillates were determined by internal standard method and selective ion monitoring (SIM), which ranged from 0.050 to 1.900 mg∙L Chinese Baijiu (Chinese liquor) is one of the oldest distillates in the world, and it is the most popular spirits in China with the annual production of about 4 million metric tons. The general process for the production of Chinese Baijiu is as follows. At first, the grain raw materials, such as wheat, sorghum, corn, rice or glutinous rice, are cooked with steam. And then some saccharification and fermentation agents (“DaQu” or “XiaoQu”) are added into the cooked grain matrix. Finally, the liquors are distilled out with steam from the fermentation products after several months or years of fermentation . The freFlavor compounds of Chinese Baijiu are extremely complex due to different raw materials, various microorganisms and diverse procedures in different production regions, and a great number of compounds have been studied extensively ,4,5,6, sThe mass spectra of an unknown compound shown at 17.4 min in sample 33 and propyl lactate standard were presented as −1 and 0.050 mg∙L−1, respectively. The internal standard curve equations included three equations in different concentration ranges, including y = 4.4587x + 0.0112 (0.050 < y < 0.60), y = 1.9987x + 0.2673 (0.60 < y < 1.00) and y = 2.1972x + 0.0569 (1.00 < y < 4.50). The corresponding correlation coefficients (R2) were 0.9994, 0.9917 and 0.9993, respectively. The quantitative results of propyl lactate were shown in The concentrations of propyl lactate were determined in these spirit drinks by the internal standard method with the selected ions monitoring mode of GC-MS. The LOD and the LOQ of the method were 0.025 mg∙L−1 (LOQ) and 0.025 mg∙L−1 (LOD). The top 5 distillate samples were Jingzhi Guniang 5 years (50°) (3.286 mg∙L−1), Jingzhi Guniang 10 years (52°) (3.024 mg∙L−1), Gujing Yuanjiang (65°) (2.237 mg∙L−1), “Jingyanggang” (38°) (1.900 mg∙L−1), and “Xianghe Ronghe Shaofang” (53°) (1.712 mg∙L−1).As shown in The determination of detection threshold of propyl lactate was conducted by the statistical analyses and the Curve Fitting, and the result was shown in y = 0.1623x + 0.6877, and the correlation coefficient R2 = 0.9663. The threshold was the corresponding X value (0.740 mg∙L−1) when Y = 66.67% [The calibration curve equation was = 66.67% .The unknown compound shown at 17.4 min in sample 33 was identified to be propyl lactate based on the comparison of mass spectra and retention time with the standard. Propyl lactate was also discovered in all other samples under investigation by the same method.Propyl lactate was possibly formed through the esterification of lactic acid with propanol, both of which have been reported during the fermentation. There were a great number of lactobacilli during the fermentation process of Chinese spirit drinks, which could convert sugars to lactic acid ,19. HighThe odor activity values (OAV) equaled to the ratio of the concentration of propyl lactate and its detection threshold value. If a compound has an OAV > 1.0, then it would contribute to the flavor of a product . The OAVChemicals and standards were GC grade with a high purity (>99.0%).The water was boiling for at least 0.5 h and redistilled twice before use. Methyl lactate (PubChem CID: 11040), used as internal standard (IS), and propyl lactate (PubChem CID: 92821) were obtained from Tokyo Chemical Industry CO., Ltd. . Absolute ethanol (PubChem CID: 702) was obtained from Merck .A total of 72 spirit drinks, shown as 1.0 µL of Chinese spirit drinks was injected and analyzed by GC-MS with the full scan mode, and the occurrence of propyl lactate was confirmed by comparing its retention time and mass spectrum with the standards.−1, 2.500 mg∙L−1, 1.250 mg∙L−1, 0.625 mg∙L−1, 0.313 mg∙L−1, 0.156 mg∙L−1, 0.078 mg∙L−1, 0.039 mg∙L−1 and 0.020 mg∙L−1, were prepared with absolute ethanol and analyzed by GC-MS. Then 1.0 mL of each distillate drink sample with 10.0 µL of methyl lactate solution (100.000 mg∙L−1) were placed in 72 tightly closed sample vials, numbered 1 to 72, for GC-MS analysis. Finally, the concentrations of propyl lactate were calculated by the software of GC-MS. The conditions of GC-MS  were as follows.The concentrations of propyl lactate in these distillates were determined by the internal standard method with the selected ions monitoring mode of GC-MS. At first, a series of the standard solutions, such as 5.000 mg∙L−1; The oven temperature was programmed at 50 °C for 2 min, then raised to 100 °C at 6 °C∙min−1, then raised to 170 °C at 3 °C∙min−1 for 2 min, and then raised to 200 °C at 10 °C∙min−1 for 2 min, and finally raised to 230 °C at 15 °C∙min−1 for 5 min; inlet temperature, 250 °C; transfer line temperature, 250 °C; injection volume, 1 µL; split ratio, 20:1.GC conditions: DB-FFAP capillary column ; carrier gas, helium, 99.9995%; flow rate, 1.0 mL∙minMS conditions: electron ionization source, 70 eV; ion source and quadruple temperatures, 230 °C and 150 °C, respectively; The monitored ions and other parameters of selected-ion-monitoring (SIM) mode were listed in 3-AFC test was recommended as the national standard method for determination of Chinese Baijiu flavors thresholds by GB/T 22366-2008 , which i−1 (A1), 0.412 mg∙L−1 (A2), 1.235 mg∙L−1 (A3), 3.704 mg∙L−1 (A4), 11.111 mg∙L−1 (A5) and 33.333 mg∙L−1 (A6).Most concentrations of alcohol in the distillate samples under investigation were 38 vol% or nearby, so 38% ethanol solution was used as the benchmark. A series of propyl lactate solutions were prepared for sensory evaluation with 38% ethanol solution, such as 0.137 mg∙LThere were eighteen samples for sensory evaluation, which were equally divided into six groups. All these samples were placed in 10 mL tulip-like glass wine cups. Two samples of each group were the control samples and the remaining one had different concentration of propyl lactate in it. Each sample was marked in a random four-digit number. A group of 30 untrained and normal olfaction assessors were invited to determine propyl lactate detection threshold. All samples were assessed at room temperature. For each test, the assessors needed to pick out the one which was “very different from the references”, and wrote down the number. Each test was replicated 3 times, so that 90 responses were obtained for each testing concentration. Then the Curve Fitting (CF) was conducted by the statistical analyses, and the curve was drawn. When Y = 66.7%, the corresponding X value was the detection threshold value of propyl lactate.−1 in 72 Chinese Baijius. The detection threshold of propyl lactate in 38% ethanol solution was 0.740 mg∙L−1. Based on OAV analysis in this research, propyl lactate had much contribution to the aroma of 13 Chinese Baijius , including 50° Jingzhi Guniang 5 years (4.440), 52° Jingzhi Guniang 10 years (3.024), Jingyanggang (2.568), Xianghe Ronghe Shaofang (2.313), and 1956 Laolang (1.431).In summary, this work reported the occurrence of propyl lactate in 72 Chinese Baijius for the first time. The concentration of propyl lactate ranged from 0.050 to 1.900 mg∙L


The polymers exhibited low critical solution temperature (LCST) characteristics in aqueous solutions, which is essential for recovering a draw solute from pure water. The LCSTs of the 20 wt% aqueous solutions of PSSBP8, PSSBP6, and PSSBP4 were confirmed to be approximately 30, 38, and 26 °C, respectively, which is advantageous in terms of energy requirements for the recovering draw solute. When the concentration of the PSSBP4 draw solution was 20 wt%, water flux and reverse solute flux were approximately 1.61 LMH and 0.91 gMH, respectively, in the active layer facing the draw solution (AL-DS) system when the feed solution was distilled water. The PSSBP# thermal-responsive draw solute has considerable potential for use as a next-generation draw solute because of its excellent osmotic performance and efficient recovery. Therefore, this study provides inspiration for novel ideas regarding structural transformations of polymers and their applicability as draw solutes.The thermal-responsive polymers, poly(alkane-1,#-diylbis(tri- Water scarcity is a critical problem facing humanity, as it is essential to supply clean water to societies worldwide. To date, technologies have been developed to obtain pure water from saline, waste, and agricultural water mainly in the form of water separation technologies, which can be classified as distillation or membrane methods . The dis4HCO3) draw solute derived from ammonia (NH3) and carbon dioxide (CO2) in the aqueous phase : 0.91–0.98 ), 1.41–1.81 , (P+–CH2–(CH2)2–CH2–P+)), 2.17–2.34 ).n-butylphosphonium) 4-vinylbenzenesulfonate (SSBP#) series and tetrabutylphosphonium 4-vinylbenzenesulfonate (SSMP) were prepared according to a previously reported method : 0.86–0.94 ), 1.35–1.53 , (P+–CH2–(CH2)4–CH2–P+)), 1.99–2.11 ), 5.37–5.43 ), 5.87–5.96 ), 6.74–6.84 ), 7.54–7.79 ).1H-NMR of SSBP4 : 0.83–0.93 ), 1.30–1.64 , (P+–CH2– (CH2)2–CH2–P+)), 2.10–2.31 ), 5.22–5.29 ), 5.78–5.86 ), 6.66–6.76 ), 7.37–7.58 ).1H-NMR of SSMP : 0.87–0.95 ), 1.36–1.53 ), 2.01–2.11 ), 5.39–5.44 ), 5.90–5.97 ), 6.76–6.86 ), 7.57–7.80 ).n-butylphosphonium) 4-vinylbenzenesulfonate) (PSSBP#) series and poly(tetrabutylphosphonium 4-vinylbenzenesulfonate) (PSSMP), which is a polymeric draw solute of the PSSBP# series, were synthesized via free radical polymerization. SSBP8  and AIBN  were mixed into 20 mL of DMF and stirred for 24 h at 90 °C under a nitrogen atmosphere. The resulting polymer was dialyzed thoroughly against DMF and other impurities using a dialysis tube (molecular weight cut-off = 3.5 kDa) for 3 days. After evaporating the remaining distilled water, the product was dried in a freeze dryer for 24 h. Other members of the PSSBP# series, i.e., PSSBP6 and PSSBP4, were prepared using the same procedure and amounts of materials. PSSMP was synthesized using same procedure, except a different amount of AIBN  was used. 1H-NMR spectroscopy was used to identify and confirm the PSSBP# series and PSSMP, and the associated spectra are shown in Figure 3.The poly), 1.06–1.17 ), 1.18–1.49 , (CH3–CH2–CH2–CH2–P+–), (P+–CH2–(CH2)6–CH2–P+)), 1.89–2.16 ), 6.09–7.06 ), 7.34–7.93 ).1H-NMR of PSSBP6 : 0.80–0.87 ), 1.28–1.48 , (CH3–CH2–CH2–CH2–P+–), (P+–CH2–(CH2)4–CH2–P+)), 1.99–2.12 , (–CH2–P+–)), 6.16–7.02 ), 7.38–7.88 ).1H-NMR of PSSBP4 : 0.71–0.99 ), 1.23–1.53 , (CH3–CH2–CH2–CH2–P+–), (P+–CH2–(CH2)2–CH2–P+)), 1.62–1.80 ), 2.01–2.33 ), 6.15–6.97 ), 7.37–7.87 ).1H-NMR of PSSMP : 0.74–0.90 ), 1.27–1.50 , (CH3–CH2–CH2–CH2–P+–)), 1.97–2.14 , (–CH2–P+–)), 6.18–7.93 ).Water flux is a key property of the FO process and was investigated herein. The water flux was measured using a small-scale FO system connected to custom-made, cap-fixed L-shaped glass tubes. A membrane  was placed in a channel (diameter: 2.3 cm) between two L-shaped glass tubes. Draw solutes were added to one tube facing the active layer of the membrane, and distilled water as a feed solution was added into the other tube. The glass tubes were placed under an air atmosphere at room temperature. Measurement of water flux was performed in the active layer facing draw solution (AL-DS) mode. Depending on the orientation of the FO membrane, the degree of internal concentration polarization (ICP) phenomenon, which is one of the factors that reduces water flux, is changed. Particularly, it is well known that AL-DS mode exhibits less ICP phenomenon than active layer facing feed solution (AL-FS) mode. Thus, we selected AL-DS mode ,40,41,42wJ, ] was calculated from the volumetric change of the draw solution using Equation (1).t, and A is the effective membrane area (4.15 × 10−4 m2).The draw and feed solutions were stirred using a magnetic bar and a solenoid  simultaneously. Water flux was determined by measuring the height difference of the solution before and after FO. The water flux  and alkylene groups  were identified, and the integral ratio of each region was in agreement with the ratio of the predicted number of proton atoms. Based on these results, we concluded that the dicationic phosphonium bromide (BP#Br2) was successfully synthesized. The monomers, i.e., SSBP8, SSBP6, SSBP4, and SSMP, and the monomer from PSSMP as a referential polymer for PSSBP# polymers were obtained by solvent extraction using distilled water and dichloromethane. This procedure utilizes the partition coefficient of the solute between water as a polar solvent and dichloromethane as a nonpolar solvent. Thus, the resultant monomers, SSBP# series, and SSMP were dissolved in the nonpolar dichloromethane layer, which was removed to obtain the desired monomer series. Structural analysis of the corresponding pure monomers was performed by 1H-NMR spectroscopy ] and aryl groups [δ = 7.38–7.79 (peak f)] whose peaks were unidentified in the 1H-NMR spectra shown in troscopy . The bro1H-NMR  .(4)D=kTThe osmotic pressure in an aqueous solution containing nonionic or ionic materials is closely related to the diffusion of the materials . Higher The separation of draw solutes from the pure water and draw solution mixture during recovery in the FO process is essential for the recycling of draw solutes. Among the recovery methods of thermal-responsive draw solute, the LCST is the critical temperature at which, when the temperature is raised, the draw solute and water changes from homogeneous to heterogeneous phase. To measure the LCST of the PSSBP# series and PSSMP in an aqueous solution using UV-Vis spectrophotometry, the temperature of the phase change must be determined using a temperature versus transmittance curve. The monomers of the PSSBP# series, e.g., SSBP8, SSBP6, and SSBP4, showed LCST properties. However, the LCSTs of the monomers were too low to be measured at values close to 0 °C (data now shown). Consequently, we concluded that the monomers cannot be utilized as draw solutes in FO. To optimize the FO process, the bidirectional diffusion of water and draw solute must be considered . Thus, tA draw solute series with dumbbell-shaped dicationic structures, PSSBP#, where # is the number of carbon atoms in the central bridge structure of dicationic phosphonium moiety, was synthesized via free radical polymerization to examine its feasibility for use in FO. In aqueous solutions, the PSSBP# series showed LCSTs, which is essential for recovery in FO. The LCSTs of PSSBP8, PSSBP6, and PSSBP4 were approximately 30, 38, and 26 °C, respectively. The respective water fluxes were identified in AL-DS mode as 1.84, 1.61, and 1.61 LMH at 20 wt%, and the respective reverse solute fluxes were 1.12, 0.61, and 0.91 gMH the under same conditions. PSSBP#, a thermal-responsive homopolymer series containing a dumbbell-shaped dicationic phosphonium moiety, can surmount the typical trade-off relationship, which is a chronic problem of copolymer systems. Furthermore, this series exhibiting LCST characteristics has great potential in terms of draw solute recovery because its recovery temperature is close to room temperature, reducing the amount of energy required for recovery compared to those of another draw solute. This study provides inspiration in terms of structural modification for controlling recovery temperature in FO and a novel methodology for developing draw solutes.


This study aimed to determine early postoperative changes of serum sphingomyelin (SM) and ceramide (CER) species following laparoscopic sleeve gastrectomy (LSG).2] underwent LSG and normal weight control patients  underwent laparoscopic cholecystectomy. Fasting blood samples were collected prior to surgery, at day 1 and day 30 after surgery. Circulating levels of C16-C24 SMs, C16-C24 CERs and sphingosine-1-phosphate (S1P) were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of neutral sphingomyelinase (N-SMase) was assayed by standard kit methods, and ceramide-1-phosphate (C1P) levels were determined by enzyme-linked immunosorbent assay (ELISA). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods. Insulin sensitivity was evaluated using homeostatic model assessment for insulin resistance (HOMA IR).Twenty obese patients [mean body mass index (BMI) 45,64 ± 6,10 kg/mA significant decrease was observed in serum levels of very-long-chain C24 SM, very-long-chain C22-C24 CERs, HOMA-IR, N-SMase and C1P in LSG patients after postoperation day 1 and day 30 compared to preoperation levels. At 30 days postsurgery, BMI was reduced by 11%, fasting triglycerides were significantly decreased, and insulin sensitivity was increased compared to presurgery values. A significant positive correlation was found between HOMA-IR and serum levels of C22-C24 CERs in LSG patients.We conclude that very long chain CERs may mediate improved insulin sensitivity after LSG. Morbid obesity has reached epidemic proportions in many societies . ObesityThe lipid pathways and the molecular mechanisms that control obesity-induced comorbidities are still subjects of intense research . Studiesprotein kinase B (PKB), also known as Akt [Ceramides (CERs) serve as precursors for SMs, which are bioactive hydrophobic backbones of SMs . Plasma n as Akt . Formatin as Akt . During n as Akt . Upreguln as Akt . Inhibitn as Akt .In connection with their possible involvement in insulin resistance and inflammation, changes in plasma sphingolipid levels after LSG, may serve as a biomarker of insulin resistance and inflammation. The aim of this study was to quantify individual SMs and CERs in the circulation of severely obese patients undergoing LSG; at baseline, day 1 and day 30 postoperatively. We also examined the relationship between plasma ceramide levels and insulin resistance as well as circulating N-SMase activity, C1P, S1P at all time-points.2. All patients went through a clinical, biochemical, and pre-anesthetic evaluation, and subjects with apparent history of stroke, coronary heart disease, arrhythmia, peripheral artery disease, severe kidney dysfunction, liver disease, thyroid dysfunction, and infectious disease were excluded. All patients met the surgical indication criteria in the inter-disciplinary European guidelines on surgery of severe obesity [2 and all were non-smokers. Fasting blood samples were collected from the control groups prior to surgery, at day 1 and day 30 after surgery. All sleeve gastrectomy patients and control group subjects gave written informed consent prior to entry. This study was approved by the Institutional Review Board of Antalya Research and Education Hospital and was performed in accordance with the Declaration of Helsinki.The sleeve gastrectomy group included 20 obese female patients who were admitted to Antalya Research and Education Hospital, Surgery Clinic. The mean body mass index (BMI) of patients was 45,64 ± 6,10 kg/m obesity . FastingHemoglobin (Hb) levels were measured on LH 780 Hematology Analyzer . Serum glucose, insulin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine phosphokinase (CPK), creatinine, uric acid, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triacylglycerol (TG) were assayed by standard kit methods using an auto-analyzer . Low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) levels were calculated via the Friedewald formula . Insulinv/v/v) with 10 mM ammonium formate and solvent B was acetonitrile–2-propanol . Gradient program was solvent B, 65% (0–2 min), 90% (2.01–13 min), 100% (13.01–20 min) and 65% (20.1–23 min). MRM transitions and responses were automatically optimized for individual compounds in positive electrospray ionization (ESI). In the positive ESI-MS mode the precursor and product m/z values for all analyzed sphingolipids were as follows: S1P, precursor m/z: 380.10, product m/z: 264.40; C16 SM, precursor m/z: 703.30, product m/z: 184.20; C18 SM, precursor m/z: 731.40, product m/z: 184.20; C24 SM, precursor m/z: 815.50, product m/z: 184.20; C16 CER, precursor m/z: 538.50, product m/z: 264.40; C16 CER *IS, precursor m/z: 554.30, product m/z: 264.30; C18 CER, precursor m/z: 566.30, product m/z: 264.40; C20 CER, precursor m/z: 594.60, product m/z: 264.50; C22 CER, precursor m/z: 622.60, product m/z: 264.40; C24 Ceramide, precursor m/z: 650.40, product m/z: 264.30. Responses to analyzed sphingolipids were optimized to a linear calibration range from 39 to 625 ng/ml and a sample analysis time of 23 min.An optimized MRM method was developed using UFLC coupled with MS/MS as previously described . Standarv/v) diluted in distilled water. Samples were briefly vortexed and 375 μl of chloroform/methanol  was added. The samples were sonicated for 30 sn and 100 μl of water was added. The mixtures were vortexed for 5 min and stood at room temperature for 30 min. After centrifugation at 2000 g for 5 min, the supernatant was taken and 125 μl of chloroform and 125 μl of water was added. The samples were vortexed and stood for 30 min for phase separation. The upper organic layer was transferred to glass tubes and evaporated at room temperature under a constant stream of nitrogen with height adjustable gas distribution unit . The dried residue was dissolved in 100 μl of methanol and 10 ul was injected into the column.Serum sphingolipid extraction was done as previously described . Two μl Ceramide-1-phosphate levels were measured in serum via an ELISA kit . Serum C1P captured by a solid phase human monoclonal antibody was detected with a biotin-labeled polyclonal antibody. An avidin-peroxidase conjugate was then added to bind the biotinylated antibody. A TMB substrate was added and the yellow product was measured at 450 nm. A standard curve of absorbance values of known C1P standards was plotted as a function of C1P standard concentrations using the GraphPad Prism Software program for windows version 5.03. (GraphPad Software Inc.). The amount of C1P in the samples were calculated from their corresponding absorbance values via the standard curve.Neutral-SMase activity was measured in serum via a SMase assay kit . The kit uses a colorimetric probe to quantify phosphocholine produced from the hydrolysis of SM. The generated color is measured colorimetrically at 655 nm. A standard curve of absorbance values of known amounts of SMase standards was generated. Sphingomyelinase activity in the samples (mU/dL) was calculated from their corresponding absorbance values via the standard curve.p value of < 0,05 was considered statistically significant.Statistical analysis was performed using SigmaStat statistical software version 2.0. Statistical analysis for each measurement is described in the figure and table legends. A 2, respectively. The control group was also composed of women. The mean ± SD of age, body weight, and body mass index was 39,4 ± 12,7 years, 75,2 ± 16,2 kg, and 31,0 ± 6,2 kg/m2, respectively. Body mass index in the LSG group was significantly greater compared to the control group at preoperation and 30 days after operation . This decrease caused a significant difference in Hb levels between LSG and control group patients at post operation day 1 . Hemoglobin levels were significantly restored in LSG patients at post operation day 30. Homeostasis model assessment of insulin resistance showed a significant decrease in LSG patients at post operation day 1 and day 30, compared to preoperation levels . The HOMA-IR levels in LSG patients at post operation day 30 were also significantly lower than the control group . ALT levels showed a significant increase in LSG patients at post operation day 1 compared to preoperation levels . Preoperation levels of CPK was significantly greater in LSG patients compared to the control group . Creatine phosphokinase levels significantly increased in the control group following cholecystectomy . Preoperation and post operation day 30 levels of uric acid were significantly higher in LSG patients compared to the control group . Uric acid levels significantly increased in LSG patients at post operation day 30 compared to both preoperation and post operation day 1. 25-(OH)-vitamin D levels significantly increased following LSG. Post operation day 30 levels of 25-(OH)-vitamin D were significantly greater than both preoperation and post operation day 1. . HDL-C levels significantly decreased in LSG patients at postoperation day 1 compared to preoperation levels . A significant decrease was observed in TG and VLDL-C levels in LSG patients at postoperation day 30 compared to both preoperation and post operation day 1 .Laboratory values measured in LSG patients and control group are shown in Table p < 0,05) . C16 SM levels were restored in LSG patients at post operation day 30. Preoperation and post operation day 30 levels of C18 SM were also significantly higher in LSG patients compared to the control group  . Unlike the LSG group, C24 SM levels increased in the control group following cholecystectomy. This increase was significant at postoperation day 30 .Serum levels of SMs are given in Table 05) Fig.  a. A sigp < 0,05). Likewise, postoperation day 1 and day 30 C22- and C24- CER levels were significantly lower in LSG patients compared to controls . A significant positive correlation was found between HOMA-IR and serum levels of C22-C24 CERs in LSG patients  and sleeve gastrectomy patients (n = 20) at preoperation were 4,89 ± 0,34 and 4,25 ± 0,45 μg/ml respectively; at postoperation day 1 were, 5,99 ± 0,77 and 3,84 ± 0,60 μg/ml, respectively; and at postoperation day 30 were, 6,75 ± 1,02 and 4,41 ± 0,41 μg/ml, respectively. A significant decrease was observed in serum levels of S1P in LSG patients at postoperation day 1 and day 30 compared to controls   measured in both control group (n = 15) and sleeve gastrectomy patients (n = 20) at preoperation were 7,43 ± 0,49 and 7,96 ± 0,36 ng/ml respectively; at postoperation day 1 were, 7,82 ± 0,49 and 7,97 ± 0,47 ng/ml, respectively; and at postoperation day 30 were, 5,96 ± 0,50 and 4,87 ± 0,83 ng/ml, respectively. A significant decrease was observed in serum levels of C1P in LSG patients and controls at postoperation day day 30 compared to preoperation levels and postoperation day 1   measured in both control group   showed no significant difference between the control group (n = 15) and sleeve gastrectomy patients (n = 20) . A significant decrease was observed in serum levels of N-SMase in LSG patients  compared to controls  at postoperation day 1 . Neutral SMase activity was significantly decreased in LSG patients after postoperation day 1 and day 30 compared to preoperation levels  at 30 days following LSG. Our results are in agreement with recent studies that reported 23,9 ± 9.6% [ surgery . We have surgery  and 30 d surgery . Improve surgery . It is aPreoperation levels of C16 and C18 SM were significantly greater in LSG patients compared to the control group (Table We have observed a significant decrease in serum levels of very-long-chain C24 SM and very-long-chain C22-C24 CERs in LSG patients after postoperation day 1 and day 30 compared to preoperation levels (Table It is known that bariatric surgery produces a marked decline in inflammatory cytokines secreted by adipose tissue such as tumor necrosis factor (TNF)-α, interleukin-1 (IL-1) and IL-6  which inIt is important to acknowledge that the amount of circulating and tissue CERs depend on the equilibrium between production and degradation. Ceramide generation may result from hydrolysis of SM by SMase. Five types of SMase have been identified based on their cation dependence and pH optima of action . Among tp < 0,05)  to yield free fatty acid (FFA) and sphingosine (SPH). SPH may be further phosphorylated to S1P by the enzyme SPH kinase. Formation of CERs also lead to the production of metabolites such as C1P, SPH and S1P which are key regulators of inflammation . Previou05) Fig. . We founprotein kinase B (PKB) [We found a significant positive correlation between HOMA-IR and serum levels of C22-C24 CERs in LSG patients Fig. . Recentl B (PKB) . Several B (PKB) . An addi B (PKB) . The sig B (PKB) . Likewis B (PKB) . The eff B (PKB) . Our datIn summary, we have observed that decreased long chain CER levels following LSG is likely to contribute to the rapid decrease of insulin resistance observed in bariatric patients post-operatively. Referenced studies and the findings reported herein reinforce that circulating ceramide levels may be an important mediator of insulin sensitivity in peripheral tissues. The restoration in insulin sensitivity, probably secondary to reduced ceramide levels, emphasize one of the most important benefits of LSG in reducing comorbidities associated with obesity. It provides additional evidence to support the development and application of surgical techniques for the treatment of obesity.


Their porosity and morphology were strongly affected by the structure of the precursors, and partial oxidative cleavage of Si-Si bonds occurred during the sol–gel process. The new condensation polymer precursors are expected to expand the choice of approaches for new polysilane–polysiloxane.The sol–gel polymerization of alkoxysilanes is a convenient and widely used method for the synthesis of silicon polymers and silicon–organic composites. The development of new sol–gel precursors is very important for obtaining new types of sol–gel products. New condensation polymer precursors containing consecutive silicon atoms—decaisopropoxycyclopentasilane (CPS) and dodecaethoxyneopentasilane (NPS)—were synthesized for the preparation of polysilane–polysiloxane material. The CPS and NPS xerogels were prepared by the sol–gel polymerization of CPS and NPS under three reaction conditions . The CPS and NPS xerogels were characterized using N We eIn this report, we describe the synthesis of new condensation polymer precursors containing consecutive silicon atoms—decaisopropoxycyclopentasilane (CPS) and dodecaethoxyneopentasilane (NPS)—as well as their sol–gel reactions and structures of the resulting CPS and NPS xerogels, representing the first sol–gel study of peralkoxyoligosilane.n-hexane were distilled from Na/Ph2CO. EtOH and iPrOH were distilled from calcium hydride. Other starting materials were purchased as reagent grade and were used without further purification. Glassware was flame-dried with nitrogen or argon flushing prior to use. All of the manipulations were performed using the standard Schlenk techniques in nitrogen or argon atmosphere and using a glove box .In all of the reactions in which air-sensitive chemicals were used, the reagents and solvents were dried prior to use. Diethyl ether, triethylamine, and 1H, 13C, and 29Si NMR spectra were recorded using a Bruker Avance II+ BBO 400 MHz S1 spectrometer . The chemical shifts were referenced to internal C6D6 or CDCl3, or external tetramethylsilane. All of the solid-state NMR experiments were conducted with NMR instruments  using 5 and 2.5 mm double resonance MAS probe heads for 29Si and 1H at 14.1 T  at room temperature. Mass spectra were recorded using a low-resolution  EI (electron ionization) mass spectrometer and a high-resolution  instrument. The morphology of the xerogels was monitored by scanning electron microscopy . High-resolution transmission electron microscopy (HRTEM) was performed with a JEOL JEM2100F (200 kV) instrument  using a carbon-coated 200-mesh copper grid. Nitrogen adsorption–desorption isotherms were obtained using a surface area analyzer . Thermogravimetric analysis (TGA) curves were recorded using a simultaneous thermal analyzer  in N2 (50.0 mL/min) atmosphere with heating from 30.0 to 1000.0 °C (10.0 °C/min).iPrOH  and Et3N  dissolved in Et2O (150 mL) were slowly added to decachlorocyclopentasilane [2O (350 mL) for 1 h at −78 °C. After the mixture was stirred for 6 h at −78 °C and slowly warmed to room temperature, the solution was stirred for 12 h at room temperature. The Et3N+Cl− salt was removed by filtration and washing with n-hexane in argon atmosphere in a glove box, and volatiles were distilled using vacuum distillation. Solid CPS  was obtained at 88% yield. Si5(OiPr)10 (CPS): 1H NMR : δ 4.58 2), 1.40 2). 13C NMR : δ 26.79 2), 69.17 2). 29Si NMR : δ −14.41 (s). HRMS (high resolution mass): C27H63O10Si5 687.3268 , 687.3267 (found).tasilane ,24  dissolved in Et2O (150 mL) were slowly added to the dodecaethoxyneopentasilane [2O (350 mL) for 1 h at −78 °C. After the mixture was stirred for 6 h at −78 °C and slowly warmed to room temperature, the solution was stirred for 12 h at room temperature. The Et3N+Cl− salt was removed by filtration and washing with n-hexane in argon atmosphere in a glove box, and volatiles were distilled using vacuum distillation. NPS  was obtained at 82% yield as an oil. Si5(OEt)12 (NPS): 1H NMR : δ 3.87 , 1.16 . 13C NMR : δ 18.90 , 58.85 . 29Si NMR : δ −40.52 3)4), −152.16 3)4). HRMS: C24H61O12Si5 681.3009 , 681.3030 (found).EtOH  and Ettasilane   was added to the vial. The solutions were sealed and shaken at room temperature. Gelation was determined at a point where the solution did not flow as a liquid. After gelation, the monoliths were aged for 2 weeks at 30 °C prior to further processing. The gels were crushed in water (100 mL), and filtered and washed with water (2 × 100 mL) and alcohols . After drying overnight in air, the xerogels were prepared by vacuum drying at 50 °C for 24 h.The sol–gel polymerization of CPS and NPS were carried out at 0.4 M concentration in EtOH or iPrOH (4.5 mL) to adjust to 0.4 M concentration in an oven-dried vial (25 mL). A prepared aqueous acid catalyst, namely, 0.01 wt % HCl dissolved in 10 eq of H2O , was added to the vial, and then the vial was sealed and shaken. Gelation occurred within 5 min. After aging for 2 weeks at 30 °C, the wet gel was crushed in distilled H2O (100 mL) and filtered. The solids were washed twice with distilled H2O (100 mL) and anhydrous iPrOH (100 mL). After drying for 12 h in air, a white solid CPSH xerogel (0.54 g) was obtained by vacuum drying for 24 h at 50 °C. CPSH: 1H CP/MAS NMR (600 MHz): δ 0.975, 1.138, 5.126. 13C CP/MAS NMR (150 MHz): δ −1.862, 23.385, 47.661, 65.787. 29Si CP/MAS NMR (120 MHz): δ −29.062 (D), −70.335 (T), −100.860 (Q). N2 physisorption surface area  [2/g, N2 absorption mean pore diameter : 28, 50, 90 nm. Total weight loss : 19.3%.CPS  was diluted in anhydrous er; BET) : 317 m2/2O , instead of the acid catalyst. Gelation occurred within 3 min. After aging, washing and drying were carried out in the same manner as described above. A white solid CPSOH xerogel (0.51 g) was obtained. CPSOH: 1H CP/MAS NMR (600 MHz): δ 0.690, 0.975, 1.138, 5.329. 13C CP/MAS NMR (150 MHz): δ 22.738, 65.657. 29Si CP/MAS NMR (120 MHz): δ −26.175 (D), −69.782 (T), −101.229 (Q). N2 physisorption surface area (BET): 408 m2/g, N2 absorption mean pore diameter (BJH): 24, 43, 76 nm. Total weight loss : 17.8%.Similarly to the procedure for the preparation of CPSH, CPSOH was prepared using an aqueous base catalyst, namely, 0.01 wt % NaOH dissolved in 10 eq of H2O  without any acid or base catalyst. Gelation occurred within 10 min. After aging, washing and drying were carried out in the same manner as described above. A white solid CPSN xerogel (0.53 g) was obtained. CPSN: 1H CP/MAS NMR (600 MHz): δ 0.120, 0.527, 4.190. 13C CP/MAS NMR (150 MHz): δ 0.534, 24.615, 30.182, 65.978. 29Si CP/MAS NMR (120 MHz): δ −32.076 (D), −71.382 (T), −100.984 (Q). N2 physisorption surface area (BET): 150 m2/g, N2 absorption mean pore diameter (BJH): 32, 50, 76 nm. Total weight loss : 16.2%.Similarly to the procedure used for the preparation of CPSH, CPSN was prepared using 10 eq of distilled H2O , was added to the vial and then the vial was sealed and shaken. Gelation occurred within 4 days. After aging for 2 weeks at 30 °C, the wet gel was crushed in distilled H2O (100 mL) and filtered. The solids were washed twice with distilled H2O (100 mL) and anhydrous EtOH (100 mL). After drying for 12 h in air, a white solid NPSH xerogel (0.63 g) was obtained by vacuum drying for 24 h at 50 °C. NPSH: 1H CP/MAS NMR (600 MHz): δ 0.080, 0.487, 3.905. 13C CP/MAS NMR (150 MHz): δ 1.052, 17.688, 29.987, 59.117. 29Si CP/MAS NMR (120 MHz): δ −80.472 (T), −101.721, −109.951 (Q). Total weight loss : 14.9%.The NPS  was diluted in anhydrous EtOH (4.3 mL) to adjust to 0.4 M concentration in an oven-dried vial (25 mL). A prepared aqueous acid catalyst, namely, 0.01 wt % HCl dissolved in 12 eq of H2O , instead of the acid catalyst. Gelation occurred within 1 day. After aging, washing and drying were carried out in the same manner as described above. A white solid NPSOH xerogel (0.67 g) was obtained. NPSOH: 1H CP/MAS NMR (600 MHz): δ 0.202, 2.806, 6.306. 13C CP/MAS NMR (150 MHz): δ 17.365, 30.117, 59.894. 29Si CP/MAS NMR (120 MHz): δ −91.158 (T), −101.721, −110.688 (Q). N2 physisorption surface area (BET): 7 m2/g, N2 absorption mean pore diameter (BJH): 66, 90, 119 nm. Total weight loss : 27.4%.Similarly to the procedure used for the preparation of NPSH, NPSOH was prepared using an aqueous base catalyst, namely, 0.01 wt % NaOH dissolved in 12 eq of H2O  without any acid or base catalyst. Gelation occurred within 2 days. After aging, washing and drying were carried out in the same manner as described above. A white solid NPSN xerogel (0.62 g) was obtained. NPSN: 1H CP/MAS NMR (600 MHz): δ 0.527, 3.986. 13C CP/MAS NMR (150 MHz): δ 1.700, 17.753, 30.052, 58.793. 29Si CP/MAS NMR (120 MHz): δ −80.717 (T), −101.844, −110.442 (Q). N2 physisorption surface area (BET): 28 m2/g, N2 absorption mean pore diameter (BJH): 21, 43, 120 nm. Total weight loss : 17.9%.Similarly to the procedure for the preparation of NPSH, NPSOH was prepared using 12 eq of distilled H5(OiPr)10—and dodecaethoxyneopentasilane (NPS)—Si(Si(OEt)3)4—were synthesized by the alcoholysis of decachlorocyclopentasilane and dodecachloroneopentasilane, respectively  was removed by filtration without contact with air and moisture. The CPS cyclic compound was obtained as a solid at 88% yield. Using a method similar to the method described above, branched precursor NPS was obtained as an oil at 82% yield. The two products were characterized by 1H NMR —Siectively . The alcy 1H NMR , 13C NMR29Si NMR , and HRM29Si NMR . The 29S2O), and basic (0.01 wt % NaOH per H2O) conditions. For the hydrolysis, water was used with the number of moles of the equivalent of alkoxy groups of each precursor (10 eq for CPS and 12 eq for NPS). The CPS solution became a viscous gel within a few minutes, while the gelation of NPS required a few days. After gelation, all of the gels were aged for 2 weeks at room temperature to obtain condensation that was as complete as possible prior to further processing. The wet gels were washed with water and the corresponding solvent to remove the catalyst. Xerogels were obtained as opaque white brittle solids by vacuum drying for 24 h at 50 °C. The xerogels were ground into fine powder for analyses  were carried out at room temperature in isopropanol or ethanol according to the substituent groups of the precursors. Hydrolysis and condensation reactions were carried out in neutral, acidic  of 0.74 cm3·g−1. CPSH showed a quite large surface area of 317 m2·g−1 and the largest total pore volume of 0.83 cm3·g−1. By contrast, the NPS xerogels were either non-porous or exhibited negligible porosity. These results indicated that the designed structure of the precursors affected the surface area and the porosity of the xerogels. Considering that the core size of CPS is larger than that of NPS, it was assumed that the higher porosity of CPS may be due to the difference in the core size. Usually, bridged polysilsequioxanes, which are large-core silsesquixoanes, are highly porous and have high surface areas [The porosity of the xerogels was determined by Nce areas ,30,31,321H, 13C, and 29Si CP/MAS NMR spectroscopy. Shorthand notations have been commonly used in the organosilicon literature for the Si–O bonding configurations: The structures of (SiO)1Si, (SiO)2Si, (SiO)3Si, and (SiO)4Si are referred to as M, D, T, and Q, as this relates to the number of O atoms bonded to a Si atom, respectively. 29Si CP/MAS NMR spectra of the CPS xerogels show the presence of D (R2SiO2) (δ from −20 to −40 ppm), T (RSiO3) (δ from −60 to −80 ppm), and Q (SiO4) units (δ from −90 to −120 ppm) [29Si CP/MAS NMR spectrum of CPS xerogels, the Q unit is also found in the spectra of NPS xerogels. Without Si–Si bond breaking of NPS during the sol–gel process, only the T unit should be observed. The resonances at the T and Q units of NPS xerogels observed in The xerogels were characterized by solid-state 120 ppm) ,35,36,37120 ppm) ,42,43,44The morphology and pore texture of xerogels were investigated by scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM). For CPS xerogels, The morphologies of NPS xerogels were observed to be significantly different from those of the CPS xerogels. NPSH and NPSN are flat plates connected to each other as stacked films, and NPSOH is an irregularly wrinkled plate. In the TEM images, the textures consist of highly crystalline composites with no pores .29Si CP/MAS NMR study of the xerogels indicated that a partial oxidative cleavage of the Si–Si bonds in cyclopentasilane and neopentasilane occurred during the sol–gel process. The structural features—a hybrid of Si–Si and Si–O–Si bonds—might provide a new optical property since consecutive silicon bonds absorb UV-visible light [The new condensation polymer precursors containing consecutive silicon atoms—CPS and NPS—were successfully synthesized at high yields. CPS and NPS xerogels were prepared by sol–gel polymerization under three reaction conditions  using the new precursors. The characterizations of CPS and NPS xerogels using BET, BJH, and microscopy showed that their porosity and morphology were strongly affected by the structure of precursors. The CPS xerogels exhibited relatively large surface areas and total pore volumes, while the NPS xerogels exhibited nonporosity or negligible porosity. The relatively high porosity of CPS xerogels was achieved without any organic substituent, template, or porogen, and they showed 16~19% of weight loss in TGA analysis. Bridged polysilsesquioxanes exhibited higher porosity, however, the weight loss reached around 45% ,29. 29Sile light , and por


Anoect chilus, has diverse pharmacological actions, and it is considered to be a promising antihyperlipidemic drug candidate. This study evaluates the effects of kinsenoside on CYP enzyme-mediated drug metabolism in order to predict the potential for kinsenoside-drug interactions. Kinsenoside was tested at different concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 µM in human liver microsomes. The c Cktail probe assay based on liquid chromatography-tandem mass spectrometry was conducted to measure the CYP inhibitory effect of kinsenoside. Subsequently, the metabolism profiles of amlodipine and lovastatin in human liver microsomes were analyzed following co-incubation with kinsenoside. The concentration levels of the parent drug and the major metabolites were compared with the kinsenoside-cotreated samples. The effect of kinsenoside was negligible on the enzyme activity of all the CYP isozymes tested even though CYP2A6 was slightly inhibited at higher concentrations. The drug-drug interaction assay also showed that the concomitant use of kinsenoside has a non-significant effect on the concentration of lovastatin or amlodipine, and their major metabolites. So, it was concluded that there is almost no risk of drug interaction between kinsenoside and CYP drug substrates via CYP inhibition.Kinsenoside, the herb-derived medicine isolated from the plant  R-3-β-d-glucopyranosyloxybutanolide) is the principal bioactive constituent of Anoect chilus formosanus, an important ethnomedicinal plant in Asian countries, primarily Taiwan and China [Kinsenoside  and stroke, both of which are major causes of death in developed countries . ClinicaKinsenoside, which is an herb-derived medicine considered a promising new drug candidate, should be investigated for the inhibitory effects on CYP enzyme activities in order to predict its possible interaction with CYP drug substrates via CYP inhibition. No previous study has shown the kinsenoside CYP activity. Because pharmacological studies have revealed that kinsenoside is a promising antihyperlipidemic drug candidate, it should be directly evaluated for CYP-mediated drug interactions towards antihyperlipidemic and antihypertensive classes of drugs. Therefore, this study, based on liquid chromatography/tandem mass spectrometry (LC/MS/MS), examined the inhibitory effects of kinsenoside on the CYP450-mediated drug metabolism in human liver microsomes, and further investigated the qualitative and quantitative profile of CYP-mediated metabolites of commonly used antihyperlipidemic and antihypertensive drugs, lovastatin and amlodipine, respectively, following the treatment with kinsenoside.i.e., 7-OH-coumarin) was slightly inhibited in a concentration-dependent manner. To confirm the inhibition of CYP2A6 by kinsenoside, we further investigated with cDNA-expressed recombinant CYP2A6 isozyme. Kinsenoside also exhibited a slight inhibitory effect on recombinant CYP2A6 isozyme. However, the effect was shown to reach saturation at higher concentrations and remained inhibited as approximately 60% of control even at a concentration of 100 µM . Kinsenoside purity was determined by a high performance liquid chromatography with an evaporative light scattering detector (ELSD), which was >98% . The HPL+, glucose-6-phosphate dehydrogenase, phenacetin, coumarin, diclofenac, mephenytoin, dextromethorphan, midazolam, ketoconazole, and terfenadine were obtained from Sigma Chemical Co. . Amlodipine and lovastatin were purchased from Sigma-Aldrich . All other solvents used were of HPLC grade and were purchased from J. T. Baker . Distilled water was prepared using a Milli-Q purification system . All standard solutions and mobile phases were passed through a 0.22 µm membrane filter before use.Pooled human liver microsomes and recombinant CYP2A6 were purchased from BD Gentest . Glucose-6-phosphate, β-NADP−1), kinsenoside at different concentrations in distilled water (DW) , an NADPH-generating system (NGS) containing β-NADP+ (10 mg·mL−1), glucose-6-phosphate (0.1 M) and glucose-6-phosphate dehydrogenase (1.0 U·mL−1), and a mixture of substrates  in 200 µL of potassium phosphate buffer . The reaction mixture without NGS was pre-incubated at 37 °C for 5 min and then further incubated with NGS for 30 min in a water bath. For the positive control, 5 µM ketoconazole, 50 µM sulfaphenazole, 10 µM furafylline, 10 µM methoxsalen, 30 µM quercetin, 50 µM quinidine, and 20 µM ticlopidine were tested. After incubation, the reaction was stopped by adding 400 µL of internal standard terfenadine (0.16 µM) in 0.1% acetic acid (cold ice). For the test with a recombinant CYP2A6 isozyme, cDNA-expressed CYP2A6 supersomes and a single substrate (coumarin) were used instead of human liver microsomes and the substrate mixture, respectively. The rest of the procedure was the same as described above.The reaction mixtures used in this study consisted of human liver microsomes (0.5 mg·mL−1), amlodipine or lovastatin at a concentration of 10 µM, kinsenoside at a concentration of 10 µM, and an NADPH-generating system (NGS) containing β-NADP+ (10 mg·mL−1), glucose-6-phosphate (0.1 M), and glucose-6-phosphate dehydrogenase (1.0 U·mL–1) in 200 µL of potassium phosphate buffer . For the control sample, kinsenoside was replaced by DW. The reaction mixture without NGS was pre-incubated at 37 °C for 5 min and then further incubated with NGS for 60 min in a water bath. After one hour, the reaction was stopped by adding 400 µL of 0.1% acetic acid (ice cold), and the sample was placed in ice until solid-phase extraction (SPE).Qualitative and quantitative analysis of CYP-mediated metabolites of amlodipine and lovastatin were conducted. In this study, the reaction mixtures consisted of human liver microsomes  were activated with 1 mL of methanol followed by 1 mL of 0.1% acetic acid in water. The incubation mixtures were loaded into the cartridge, and then washed twice with 1 mL of 0.1% acetic acid in water under vacuum. The analytes were eluted with 1 mL of methanol. The eluate was evaporated under nitrogen gas; the residue was reconstituted in 100 µL of the mobile phase. A 5 µL aliquot was injected into the respective mass spectrometry systems for analysis.−1. The solvent B composition was changed as follows: 0–3.0 min, 85% ; 3.0–4.5 min, 85% (maintained); 4.5–4.6 min, 15%; and 4.6–8.0 min, 15% (re-equilibrium). The total run time was 8.0 min, and the injection volume was 5 µL. Mass detection was performed in positive ion mode with multiple reaction monitoring (MRM). The MRM modes used for the precursor-product ion pairs (Q1/Q3) are shown in An Agilent 1260 binary pump HPLC system, with the Agilent 6460 Triple Quadrupole mass spectrometer  equipped with an electrospray ionization (ESI) source were used as the Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) system. The Fortis C8 column  was used for the chromatographic separation. The column temperature was maintained at 40 °C. The high-performance liquid chromatography (HPLC) mobile phases consisted of solvent A, 0.1% formic acid in DW and solvent B, 90% acetonitrile in solvent A. A gradient elution was used with an initial concentration of 15% of solvent B and a flow rate of 0.2 mL·min−1 for amlodipine. The solvent B composition was changed as follows: 0–20 min, 10%–70% ; 20–21 min, 70%–10%; and 21–30 min, 10% (re-equilibrium). For lovastatin, the gradient elution was: initial, 30% B; 0–15 min, 30%–80%; 15–18 min, 80%; 18–18.1 min, 80%–30%; and 18.1–25 min, 30%. The injection volume was 5 µL for both amlodipine and lovastatin. A mass detection scan was performed in the positive ion mode.For CYP metabolite analysis of amlodipine and lovastatin, an Agilent 1260 binary pump HPLC system with an Agilent 6530 LC-QTOF MS system (Agilent Technologies) was used. The chromatographic separation was performed using a Thermo Hypersil GOLD column , with the column temperature maintained at 40 °C. The HPLC mobile phases consisted of solvent A, 0.1% formic acid in DW and solvent B, 90% acetonitrile in solvent A. A gradient elution was used with an initial concentration of 10% of solvent B and a flow rate of 0.3 mL·minin vitro metabolic profile of amlodipine (an antihypertensive drug) and lovastatin (an antihyperlipidemic drug) after treatment with kinsenoside were investigated. The results showed that there is almost no risk of potential drug interactions between kinsenoside and CYP drug substrates via CYP inhibition. Therefore, considering our findings, the drug-drug interaction between kinsenoside and other conventional drugs via CYP inhibition would be negligible.Kinsenoside is a promising drug candidate, however, despite its pharmacological importance no study has yet shown the effects of kinsenoside on CYP enzyme activities. For the first time, this study investigated the inhibitory effect of kinsenoside on such enzymes. To predict possible drug-drug interactions, the


F. viridissima by UHPLC-ESI-QTOF-MS. Lignan glycosides (1 and 2), lignans (3 and 4), and lignan dimers (5–10) were identified by analysis of their exact masses and MSe spectra along with the characteristic mass fragmentation patterns and molecular formulas. We further investigated NO inhibitory effects of F. viridissima fractions and their major lignan derivatives to evaluate those anti-inflammatory effects. The methylene chloride fraction of F. viridissima as well as compounds 8 and 10 showed potent dose-dependent NO inhibitory effects on RAW 264.7 cells. Corresponding to the NO inhibition by compounds 8 and 10, lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression was notably reduced by both compounds. Our combined data with the bioactive results and the component analysis by UHPLC-ESI-QTOF-MS suggest that the methylene chloride fraction of F. viridissima roots could be potential anti-inflammatory agents and these are related to major lignans including dimeric dibenzylbutyrolactone lignans.Lignans are known to be an important class of phenylpropanoid secondary metabolites. In the course of our studies on the chemodiversity of lignans, the necessity arose to develop a method for the fast detection and identification of bioactive lignan subclasses. In this study, we detected 10 lignan derivatives of different extracts of  Viridissimaol A (6), the protonated ion peak and the addition of the sodium ion were detected at m/z 729.2905 and 751.2716, respectively, and loss of one or two water molecules corresponded at m/z 711.2792 or 693.2686 (7) were detected at m/z 729.2896 and 751.2712, which exhibited [M + H]+ and [M + Na]+ (m/z 137 and 151. Both viridissimaol A (693.2686 . SimilarM + Na]+ . The dau8) is a dimer associated with arctigenin and -7,8-didehydroarctigenin, which was detected in the dichloromethane fraction (m/z 741.2907 [M + H]+) showed its derivative 763.2717 ([M + Na]+) and loss of water molecules 723.2800 ([M + H − H2O]+) and 705.2671 ([M + H − 2H2O]+)  . Since ce moiety  17,18].,18.8) is9, m/z 743.3055 [M + H]+) and conicaol A , which belong to the dichloromethane fraction (m/z 151. Diarctigenin (9) showed molecular ions m/z 743.3055 ([M + H]+), 765.2867 ([M + Na]+), 725.2947 ([M + H − H2O]+), and 707.2844 ([M + H − 2H2O]+ (e spectra of conicaol A were obtained as m/z 743.3060 ([M + H]+), 765.2891 ([M + Na]+), 725.2958 ([M + H − H2O]+), and 711.2811  and 9 (at 100 µM) showed significant cytotoxicity, whereas the other compounds did not show cytotoxicity at the highest dose of each compound. Based on the cell viability, the alleviation of NO production by the compounds in LPS-stimulated macrophages was estimated at non-cytotoxic concentrations. Of all compounds, compounds 3, 6, 7, 8, and 10 notably inhibited LPS-mediated production of NO in a dose-dependent manner. Among them, dibenzylbutyrolactone lignans, we selected two compounds which have slightly showed more inhibitory activity and have a different part of molecules. Compounds 8, which have 7, 8 unsaturated double-bond, and compound 10, which have C-O-C bond linkage in dimer structure, represent anti-inflammatory potential among lignan subclass from the methylenechloride fraction of F. viridissima roots. To verify that the reduced production of NO by compounds 8 and 10 was due to the transcriptional regulation of iNOS, an enzyme responsible for the production of NO, the iNOS protein expression level was measured by immunoblotting. Corresponding to the NO inhibition by compounds 8 and 10, the LPS-induced iNOS expression was notably reduced by these compounds .2Cl2 fraction of F. viridissima as described previously [All tested compounds were isolated from CHeviously . All iso® BEH C18 column . The mobile phase was composed of 0.1% (w/v) formic acid in water (solvent A) and acetonitrile (solvent B) in the following gradients; 0 min (A:B 90:10), 8 min (A:B 40: 60), 12 min (A:B 0:100), 15 min (A:B 0:100), 15.1 min (A:B 90:10), and 18 min (A:B 90:10). Injections were carried out with an autosampler (4 °C), the injection volume was 1 μL, and the column temperature was 40 °C. The flow rate was 0.3 mL/min for a total run time of 18 min. The ESI parameters were as follows: negative ion mode, positive ion mode; cone gas flow rate, off; cone voltage, 40 V; source temperature, 120 °C; desolvation gas flow, 600.0 L/h; and capillary voltage, 3.0 kV. The collision gas was argon, and the nebulizer and auxiliary gas was high purity nitrogen gas. Leucine enkephalin  was used as the lock mass. The collision energy was set to 20 to 40 eV.The analysis of the dichloromethane subfractions was performed on a Waters Xevo G2 Q-TOF mass spectrometer , which was equipped with an electrospray ionization interface with Waters ACQUITY UHPLC system . The UHPLC-MS data were obtained by MassLynx 4.1 software . The separation of the compounds was carried out on ACQUITY UHPLC2. RAW 264.7 macrophage, a mouse monocyte cell line, was obtained from ATCC . Cells were incubated in Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal bovine serum  and antibiotics , at 37 °C in a humidified air incubator with 5% CO4 cells/well) were seeded into a 96-well plate. After overnight incubation, cell culture medium was removed, various concentrations of compound diluents were applied to the cells, and then incubated for 24 h. Same volume of DMSO was treated to compound-untreated group to exclude the effect of DMSO on cell viability. After incubation, the cells were treated with EZ-Cytox solution  for additional 1 h at 37 °C. Cell viability was determined by measuring the absorbance of the resulting formazan product at 450 nm using a VersaMax Microplate Reader .RAW 264.7 cells (6.0 × 102−), which is one of two primary, stable and nonvolatile breakdown products of NO, reflects the quantity of NO in the supernatant [4 cells/well) were seeded into a 96-well plate and incubated overnight for cell adhesion. After adhesion, compounds were applied to the cells with indicated concentrations, LPS (1 µg/mL) was subsequently treated to the cells, and cells were then incubated for 24 h. After 100 µL cultured media was transferred to a new 96-well plate, 100 µL Griess reagent , and 0.1% N-(1-naphthyl) ethylenediamine in distilled water) was added to each well [Nitrite assay is based on the fact that measurement of nitrite (NOernatant . RAW 264ach well . Sodium 8 and 10 (5 and 25 µM) following LPS (1 µg/mL) stimulation for 24 h; subsequently, the cells were washed with ice-cold PBS. Cells were lysed in lysis buffer containing 0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid, 1% glycerol, 10 mM NaF, 1 Na3VO4, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and were centrifuged at 15,814 × g for 30 min at 4 °C. Supernatants were used for immunoblotting analyses. Same amounts of cell lysates were separated on 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to a nitrocellulose membrane  using wet blotting method. The membrane was blocked for 1 h with 5% nonfat-dried milk in Tris-buffered saline with 0.05% Tween-20 (TBST) buffer, followed by overnight incubation at 4 °C with 1:1000 dilution of primary antibodies. Each membrane was further incubated for 2 h with secondary peroxidase-conjugated goat immunoglobulin G . The target proteins were detected using an enhanced chemiluminescence (ECL) solution. Protein levels were quantified by scanning the immunoblots and analyzing them using LabWorks software .RAW 264.7 cells were treated with compounds t-test, performed by Prism 3.0 , and * p < 0.05 was considered statistically significant.Cell viability assay, nitrite assay, and immunoblotting were repeated three times. Each result was represented as mean ± standard deviation (SD). Differences between experimental conditions were assessed by Student’s


Muscarinic receptor antagonists  synergistically enhanced the relaxant effects of β2-adrenergic receptor agonists  in guinea pig trachealis. This crosstalk was inhibited by iberitoxin, a large-conductance Ca2+-activated K+ (KCa) channel inhibitor, whereas it was increased by verapamil, a L-type voltage-dependent Ca2+ (VDC) channel inhibitor; additionally, it was enhanced after tissues were incubated with pertussis or cholera toxin. This synergism converges in the G proteins /KCa channel/VDC channel linkages. Muscarinic receptor antagonists competitively suppressed, whereas, β2-adrenergic receptor agonists noncompetitively suppressed muscarinic contraction. In concentration-inhibition curves for β2-adrenergic receptor agonists with muscarinic receptor antagonists, EC50 was markedly decreased, and maximal inhibition was markedly increased. Hence, muscarinic receptor antagonists do not bind to allosteric sites on muscarinic receptors. β2-Adrenergic receptor agonists bind to allosteric sites on these receptors; their intrinsic efficacy is attenuated by allosteric modulation . Muscarinic receptor antagonists enhance affinity and efficacy of β2-adrenergic action via allosteric sites in β2-adrenergic receptors (synergism). In conclusion, KCa channels and allosterism may be novel targets of bronchodilator therapy for diseases such as asthma and COPD.To advance the development of bronchodilators for asthma and chronic obstructive pulmonary disease (COPD), this study was designed to investigate the mechanism of functional antagonism between β When (n = 8) A, and va) n = 8, B. Under n = 10, n = 8, p < 0.01, p < 0.05) and 19.0 ± 4.3% [95% CI: 15.40–22.60] (p < 0.01), respectively  in the presence of verapamil (1 μM) , respectively  in the presence of IbTX (30 nM)  to increase Gs activity for six hours, the inhibitory effects of this combination of these two agents were markedly improved to 66.6 ± 9.7% [95% CI: 56.42–76.78]  and 70.8 ± 8.5% [95% CI: 61.88–79.72] , respectively  in the presence of IbTX (30 nM); in contrast, this was increased to a 56.4 ± 7.3 percent inhibition  in the presence of verapamil (1 μM)  and 65.6 ± 6.6% [95% CI: 58.67–72.53] , respectively (340/F380 (an indicator of concentration of intracellular Ca2+) was decreased by 46.5 ± 5.2% [95% CI: 38.23–54.77] , on the other hand, IbTX with verapamil augmented the values of F340/F380 to 38.5 ± 5.9% [95% CI: 27.26–49.74]  was combined with tiotropium (1 nM), MCh (10 μM)-induced contraction was attenuated by 33.7 ± 5.3% [95% CI: 29.91–37.49] n = 10, A. In thel (1 μM) B. In con (30 nM) C. Moreovectively A. Similay n = 8, D. This el (1 μM) D. Pre-exectively D. When p50 and the maximal effect of these curves for these agents under each experimental condition are summarized in 50 was 0.6 ± 0.3 μM [95% CI: 0.26–0.68] (n = 6), and maximal response was observed at 100 μM (50 increased to 2.8 ± 0.9 [95% CI: 1.86–3.75] (n = 6), 3.9 ± 1.2 [95% CI: 2.64–5.16] (n = 6), 2.3 ± 0.7 μM [95% CI: 1.57–3.04] (n = 6), respectively (not significant), and the maximal contraction was not diminished under this experimental condition  were cumulatively applied to the tissues pre-contracted with MCh (1 and 10 μM), these agents inhibited MCh-induced contraction in a concentration-dependent manner, and complete inhibition was observed under these experimental conditions . When tht 100 μM B. In theondition B.50 and the maximal inhibition are summarized in When procaterol and salbutamol were cumulatively applied to the tissues pre-contracted with MCh (1 and 10 μM), the maximal inhibition was observed for each experimental condition; the inhibitory effects of these agents were attenuated in a concentration-dependent manner at higher concentrations than the concentrations that produce the maximal effects A,B. Simin = 10) [95% CI: 2.22–14.18], 6.8 ± 1.9% (n = 10) [95% CI: 1.39–12.20] and 6.2 ± 1.4% [95% CI: 2.22–10.18] (n = 10), respectively, for this extended period. Although these agents were slow to act, the time-dependent effects were modest under this experimental condition. Procaterol and salbutamol were cumulatively applied to the tissues pre-contracted with MCh (10 μM) with tiotropium (1 nM). In the presence of tiotropium (1 nM), concentration-inhibition curve for procaterol against MCh (10 μM)-induced contraction were significantly dissociated from the curves for procaterol in the absence of tiotropium. In the curves for procaterol with tiotropium, the values of EC50 were significantly decreased to 1.6 ± 0.4 nM [95% CI: 1.27–1.91]  were exposed to the tissues pre-contracted with MCh (10 μM) for 120 min, the maximal inhibition of tiotropium was 8.2 ± 2.1% -induced contraction, and complete relaxation (100% inhibition) was observed for each experimental condition. The maximal effects of these agents were not attenuated at higher concentrations than those concentrations than produce the maximal effects in the absence and presence of tiotropium, unlike β2-adrenerigic receptor agonists. In the concentration-inhibition curves for forskolin and db-cAMP, EC50 was not significantly reduced in the present of tiotropium (1 nM)  A,B, whic50 were 50.8 ± 15.4 nM [95% CI: 34.64–66.97] , and the value of the maximal inhibition were 80.3 ± 7.8% [95% CI: 72.11–88.49]  reduced responsiveness to β2-adrenergic receptor agonists ; and (2) enhanced responsiveness to the combination of β2-adrenergic receptor agonists with muscarinic receptor antagonists (synergism).This study used physiological methods to demonstrate that G proteins/K2-adrenergic receptor agonists  with a muscarinic receptor antagonist (tiotropium) synergistically enhanced the relaxant effects against muscarinic contraction  and muscarinic receptor antagonists  ,4350 wer2-adreneric receptor agonists against muscarinic contraction, affinity and efficacy modulation of these agonists were examined using concentration-inhibition curves for these agonists on a muscarinic receptor agonist. Isoproterenol completely antagonized muscarinic contraction, and the complete inhibition induced by isoproterenol was not attenuated . At higher concentrations than those that produce the maximal relaxation, procaterol, salbutamol and formoterol caused a concentration-dependent contraction, unlike isoproterenol, suggesting that the primary effects  of these agents via orthosteric sites are reduced by efficacy modulation via binding to allosteric sites    [4,11,442-adreneric receptor agonists and muscarinic receptor antagonists, affinity and efficacy modulation of β2-adreneric receptor agonists with muscarinic receptor antagonists were examined using concentration-inhibition curves for these agonists on a muscarinic receptor agonist. Allosteric modulation occurs in the interaction mediated by ligands related to GPCRs [2-adrenergic receptor agonists with muscarinic receptor antagonists may synergistically inhibit muscarinic contraction via allosterism (affinity and efficacy modulation). In the concentration-inhibition curves for procaterol, salbutamol and formoterol with muscarinic receptor antagonists , the values of EC50 were markedly decreased; the maximal effects of these β2-adrenergic receptor agonists were markedly augmented in each experimental condition -induced contraction with 1 nM of atropine, tiotropium and glycopyrronium was roughly equivalent to MCh (3 μM)-induced contraction , which are unrelated to β2-adrenergic receptors, did not cause synergistic effects in the presence of tiotropium  was added to increase the solubility of Fura-2/AM. The intensities of excitation fluorescence at 340 nm and 380 nm were measured after background subtraction by exposure of the mucosal side to excitation light. The ratio of F340 to F380 (F340/F380) was used as a relative indicator of intracellular Ca2+ concentration [3 (5.9 mM), CaCl2 (2.4 mM), MgCl2 (1.2 mM), and glucose (11.8 mM), bubbled with a mixture of 99% O2 and 1% CO2 (pH 7.4). After equilibrating the preparation in normal bathing solution, the experiments were started, and the temperature of the organ bath was maintained at 37 °C. The resting tone was abolished by the addition to indomethacin (1 μM) throughout the experiments. All animal procedures in this study were approved by the Animal Care and Use Committee, Kindai University Faculty of Medicine .The methods were similar to those described previously ,47,48. Mntration ,7,8. StrThe synergistic effects of the combination of procaterol or salbutamol with tiotropium were evaluated using the Bliss independence (BI) theory. This model assumes that two or more agents act independently, with different modes and sites of action , and is expressed by the following equation: E = E(x) + E(y) − E(x) × E(y) where E is the fractional effect, and x and y are the doses of two compounds in a combination experiment. If the combined experimental value is higher than the expected value, the interaction is synergistic. If it is lower, the interaction is antagonistic ,49,50.2 receptor) Gi, the inhibitory G protein of adenylyl cyclase, via adenosine diphosphate (ADP) ribosylation of the α-subunit of Gi protein, the strips of tracheal smooth muscle were incubated with 1 μg/mL petussis toxin (PTX) for 6 h and then the PTX was washed out. To examine the effects of activated Gs, the stimulatory G protein of adenylyl cyclase coupled to β2-adrenergic receptors, via ADP ribosylation of the α-subunit of Gs protein, the tissues were incubated with 2 μg/mL cholera toxin (CTX) for 6 h and then the CTX was washed out. The involvement of KCa channels and VDC channels was examined by application of iberiotoxin (IbTX), a potent selective inhibitor of KCa channels, and verapamil, a selective inhibitor of VDC channels. To examine allosteric effects  in the relaxant action of β2-adrenergic receptor agonists on muscarinic contraction were cumulatively applied to the tissues pre-contracted by methacholine . Moreover, to examine allosteric effects (affinity and efficacy modulation) in the relaxant action of the combination of β2-adrenergic receptor agonists with muscarinic receptors antagonists against muscarinic contraction, procaterol and salbutamol were cumulatively applied to the tissues pre-contracted by MCh (10 μM) in the absence and presence of muscarinic receptor antagonists (1 nM). The concentration-inhibition curves for β2-adrenergic receptor agonists were analyzed for each experimental condition. Time-matched control tissues were treated similarly to the test tissues, but exposed continuously to the normal bathing solution (sham incubation) instead of agents. Procaterol, salbutamol, and formoterol were used as β2-adrenergic receptor agonists. Atropine, tiotropium, and glycopyrronium were used as muscarinic receptor antagonists. Forskolin (a direct activator of adenylyl cyclase) and N6-dibutylyl cyclic AMP  were used as cAMP-related agents independent of β2-adrenergic receptors.Concentration-response curves were plotted by the cumulative addition of agents at 10 min for each concentration. To examine the effect of inhibited . IbTX was obtained from Peptide Institute . Tiotropium, verapamil, PTX, CTX, forskolin, db-cAMP and indomethacin were obtained from Sigma .50) of contraction were determined from the linear portion of each concentration-response curve. Statistical significance was assessed by unpaired Student’s t-test, one-way analysis of variance and the Dunnett test. A probability below 0.05 (p < 0.05) was considered to be significant.All data are expressed as the mean ± standard deviation (SD) with 95% confidence interval (CI). The response to an agent is described as a percentage of the maximal response. The effect of relaxant agents on MCh-induced contraction is expressed as percent inhibition by taking the control response to MCh (1 or 10 μM) for each experimental condition as 100%. The response to MCh was described as the percent contraction by taking the maximal contraction under the control condition as 100%. Values of the concentration of agents that produced a 50% response (EC2-adrenergic receptor agonists with muscarinic receptor antagonists causes a synergistic inhibition against muscarinic contraction of airway smooth muscle. The G proteins/KCa channel/VDC channel linkage is a key molecule for this synergism, and allosteric GPCRs modulation induced by crosstalk between these two receptors plays a fundamental role in this phenomenon (2-adrenergic action is not so different, not only between guinea pig trachea [Ca channels are densely distributed on the airway smooth muscle in various mammals including humans; the electrical characteristics of these channels are no different between these species [2-adrenergic receptor agonist and a muscarinic antagonist causes greater bronchodilation than monotherapy in asthma and COPD [Ca channels/VDC channels coupling may also be responsible for β2-adrenergic desensitization [Combining of βenomenon . Observa trachea ,11,24 an trachea , but als trachea  and huma trachea . KCa cha species ,52. Thesand COPD ,27,28,29tization  and airwtization . Hence, tization .