Brucea javanica (B. javanica) seeds, also known as “Melada pahit” in Indo-Malay region are traditionally used to treat diabetes. The objective of this study was to determine antidiabetic, antioxidant and anti-inflammatory effects of B. javanica seeds on nicotinamide (NA)-streptozotocin (STZ) induced type 2 diabetic (T2D) rats and to analyze its chemical composition that correlate with their pharmacological activities.B. javanica seeds was fractionated with n-hexane, chloroform and ethyl acetate. An active fraction was selected after screening for its ability to inhibit α-glucosidase and glycogen phosphorylase α (GP-α). Isolation and characterization were carried out by using column chromatography, NMR and LCMS/MS. All isolates were assayed for inhibition of GP-α and α-glucosidase. Antidiabetic effect of active fraction was further evaluated in T2D rat model. Blood glucose and body weight were measured weekly. Serum insulin, lipid profile, renal function, liver glycogen and biomarkers of oxidative stress and inflammation were analyzed after 4-week treatment and compared with standard drug glibenclamide.A hydroethanolic extract of 50 = 45.08 μM) was 10-fold higher than that of caffeine (IC50 = 457.34 μM), and α-glucosidase inhibitory activity (IC50 = 26.41 μM) was 5.5-fold higher than that of acarbose (IC50 = 145.83 μM), respectively. Compounds (4), (6), and (7) inhibited GP-α activity in a concentration-dependent manner with IC50 values of 357.88, 297.37, and 214.38 μM, and their inhibitory effect was higher than that of caffeine. These compounds exhibited weak potency on α-glucosidase compared with acarbose. Compounds (1), (2), and (3) showed no inhibition on both GP-α and α-glucosidase. In vivo study showed that EAF treatment significantly reduced blood glucose level, increased insulin and glycogen contents, decreased markers of oxidative stress and inflammation, and lipid levels in T2D rats compared with untreated group.Ethyl acetate fraction (EAF) exerted good inhibitory potential for α-glucosidase and GP-α compared with other fractions. Chromatographic isolation of the EAF led to the identification of seven compounds: vanillic acid (1), bruceine D (2), bruceine E (3), parahydroxybenzoic acid (4), luteolin (5), protocatechuic acid (6), and gallic acid (7). Among them, Compound (5) was identified as the most potent inhibitor of GP-α and α-glucosidase and its GP-α inhibitory activity (ICB. javanica seeds.The EAF has potential therapeutic value for the treatment of T2D via acting as GP-α and α-glucosidase inhibitors by improving hepatic glucose and carbohydrate metabolism, suppressing oxidative stress, and preventing inflammation in T2D rats. According to the results, the efficacy of EAF could be due to the presence of luteolin along with synergistic effect of multiple compounds such as parahydroxybenzoic acid, protocatechuic acid, and gallic acid in  Type 2 diabetes (T2D) is a metabolic dysfunction characterized by hyperglycemia resulting from insulin resistant and β-cell dysfunction. T2D affected 382 million people globally in 2013, and it is estimated to rise up to 592 million by 2035 , α-glucoSeveral studies reported that oxidative stress has a direct link to the pathogens of diabetes that lead to insulin resistant, β-cell dysfunction, and impaired glucose tolerance in hyperglycemic subjects , 12. It B. javanica seeds, exhibited blood glucose lowering effect in both nondiabetic mice and STZ-induced diabetic rats at lower dose (1 mg/kg b.w.) during 0-8 h screening  × 100 [The fractions from hibitors  and werehibitors . Brieflyl] × 100 .n-hexane and DCM (90:10–0:100) followed by DCM and MeOH (100:0–70:30) as mobile phase. All fractions were concentrated, monitored by TLC, and visualized under UV light. Total 91 fractions were collected and the fractions with similar Rf values were pooled together to afford twelve fractions (F1-F12). F1 appeared as white crystals which showed a single spot on TLC and yield compound 1 (12 mg). F7 and F9 identified as light yellow powder to yield compound 2 (45 mg) and 3 (56 mg). F3 was subjected to Sephadex LH20 column, eluted with DCM and MeOH (65:35 – 55:45) to get 40 fractions. Fractions 29–35 were combined and dried to get compound 4 (199 mg). Sephadex LH20 column of F5, eluted with DCM and MeOH (65:35 – 45:55), yielded 35 fractions. Fractions 22–27 were combined to get compound 5 (9 mg). F6 was fractionated in the same fashion as above to afford 36 fractions. Fraction 27 showed a single spot on TLC and dried to yield compound 6 (53.8 mg). Sephadex LH20 column of F8 using DCM and MeOH (60:40 – 20:80) yielded 50 fractions. Upon drying, fraction 20 gave rise to crystals showing single spot on TLC and designated as compound 7 (7.6 mg).The EAF (7.6 g) was loaded onto a column packed with silica gel . Gradient elution was performed using 1H, 13C & DEPT) and 2D-NMR  spectra were recorded in deuterated pyridine (C5D5N) and deuterated methanol (CD3OD) using Bruker Avance III 400 NMR spectrometer. Chemical shifts (δ) were expressed in ppm and coupling constants (J) were given in Hz.1D- . The mobile phase  was eluted at a flow rate of 0.5 mL/min. The elution was gradient . The total run time was 30 min, and the injection volume was 10 μL.α-glucosidase inhibition activity of isolated compounds were determined according to the method described in section 2.3 and GP-α inhibition activity was measured according to our previously reported method .Sprague Dawley (SD) rats of both sexes were obtained from Animal Experimental Unit of the University of Malaya where they were bred and housed according to Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (USA). The animal protocol performed in this study was approved by the Institutional Animal Care and Use Committee at the University of Malaya. Ethic No: ISB/23/05/2013/AA (R).n = 6, 3 male and 3 female in each group) and were housed in cages under standard laboratory conditions with a12-h dark–light cycle and humidity-controlled environment with a room temperature of 22 ± 3 °C and relative humidity of 65 ± 5%. The rats were allowed access to Laboratory Rodent Chow and drinking water ad libitum and were received human care according to the guidelines.Total 30 SD rats  were dived into 5 groups  and diabetes was induced by single intraperitoneal (i.p) injection of STZ (60 mg/kg b.w.) freshly prepared in 0.1 M citrate buffer (pH 4.5) 15 min after i.p injection of NA (100 mg/kg b.w.) dissolved in normal saline. Diabetes was confirmed 3 weeks after NA-STZ induction by measuring tail vein blood glucose levels using glucose meter . The rats having blood glucose levels higher than 11 mmol/L were considered as diabetic and selected for study .n = 6; 3 M/3 F) as following. Group I: non-diabetic control (NDC) and Group II: diabetic control (DC) consisted of rats were allowed to free access of water; Group III and IV were treated orally with EAF (25 and 50 mg/kg/day b.w.) diluted in distilled water, Group V was treated orally with Glibenclamide (10 mg/kg/day b.w.) and served as standard drug. All groups except group 1 are diabetic. The selected doses (25 and 50 mg/kg/day b.w) were based on prior acute oral toxicity study − . 1H NMR : δ 3.91 , 4.90 , 6.85 , 7.57 , 7.57 . 13C NMR : δ 55.0 (C-8), 112.4 (C-2), 114.4 (C-5), 121.7 (C-1), 123.9 (C-6), 147.3 (C-3), 151.3 (C-4), 168.8 (C-7).Compound 1 was isolated as colorless needle. Its molecular formula of C20H26O9 on the base of its HRESIMS ion peak at m/z 411.3018 [M + H]+ . See Table 1H NMR  and 13C NMR .Compound 2 was isolated as white amorphous powder. Its molecular formula was determined to be C20H28O9 as determined by HRESIMS ion peak at m/z 435.3028 [M + Na]+ . See Table 1H NMR  and 13C NMR .Compound 3, a white, amorphous powder, gave a molecular of C7H6O3 as determined by HRESIMS ion peak at m/z 137.00 [M-H]− . 1H NMR : δ 7.90 , 6.83 . 13C NMR : δ 114.6 , 121.3 (C-1), 131.6 , 162.0 (C-4), 168.7 (C-7).Compound 4, a white, amorphous powder, gave a molecular of C15H10O6 on the base of its HRESIMS ion peak at m/z 285.10 [M-H]− . 1H NMR : δ 6.23 , 6.46 , 6.56 , 6.92 , 7.39 , 7.40 . 13C NMR : δ 93.6 (C-8), 98.7 (C-6), 102.5 (C-3), 103.9 (C-10), 112.8 (C-2′), 115.4 (C-5′), 118.9 (C-6′), 122.3 (C-1′), 145.6 (C-3′), 149.6 (C-4′), 158.0 (C-5), 161.8 (C-9), 164.4 (C-2), 165.0 (C-7), 182.5 (C-4).Compound 5 was isolated as a yellow, amorphous powder. Its molecular formula was determined to be C7H6O4 as determined by HRESIMS ion peak at m/z 153.00 [M-H]− . 1H NMR : δ 6.82 , 7.45 .7.46 , 13C NMR : δ 114.4 (C-5), 116.3 (C-2), 121.7 (C-1), 122.5 (C-6), 144.7 (C-), 150.1 .Compound 6, a white, amorphous powder, gave a molecular of C7H6O5 was determined by HRESIMS ion peak at m/z 169.07 [M-H]− . 1H NMR : δ 8.08 , 13C NMR : δ 110.3 , 122.7 (C-1), 140.3 (C-4), 147.4 , 169.4 (C-7).Compound 7 was isolated as colorless needle. Its molecular formula of CB. javanica seeds were tested for α-glucosidase and GP-α inhibition activity in vitro and results are summarized in Table 50 = 45.08 μM) was 10 times more potent than that of standard GP-α inhibitor caffeine (IC50 = 457.34 μM), and its α-glucosidase inhibitory activity (IC50 = 26.41 μM) was 5.5 times more potent than that of acarbose (IC50 = 145.83 μM). Compounds 4, 6, and 7 are phenolic derivatives of benzoic acid. Interestingly, it can be observed that the increased number of hydroxyl substituent on the skeleton of benzoic acid significantly increased potency of those compounds against GP-α and α-glucosidase. Compound 1, which is a direct structural analogue of para-hydroxybenzoic acid (4) without the methoxy group, showed little to no inhibition (22.91%) of GP-α at 12 mM, and did not inhibit α-glucosidase at the concentrations tested up to 6 mM, suggested that the methoxy functionality in compound 1 was influential on its biological activity. Compounds 2 and 3 exhibited very weak inhibitory activity against both GP-α (19.88% and 32.74%) at 5 mM and α-glucosidase (14.74% and 16.14%) at 2.5 mM.The isolated compounds from B. javanica seeds was evaluated on T2D rat model. As Table p < 0.05) elevated in DC group compared to non-diabetic control group. Treatment with EAF (25 and 50 mg/kg b.w.) and GLI (10 mg/kg b.w.) in T2D rats, once a day for 28 days, caused significant reductions in FBG levels compared to initial day. The reduction of FBG levels for EAF (25 and 50 mg/kg b.w.) and GLI (10 mg/kg b.w.) treated T2D rats were 29.78, 44.77, and 45.44%, respectively, compared to initial day of the respective groups. There was no significant difference for FBG levels in NDC rats throughout the experimental period.Antihyperglycemic activity of EA fraction from B. javanica seeds and GLI on body weight in experimental rats was summarized in Table The effect of EAF from p <0.05) reduced to 30.26%, 32.46%, and 29.97% after treated with EAF (25 and 50 mg/kg) and GLI when compared to the values at 30 min  in the diabetic control group compared to non-diabetic control group. Treatment with EAF at 50 mg/kg dose exhibited significant (p < 0.05) increase of serum insulin by 23.7% compared to diabetic control group. Treatment with EAF at 25 mg/kg dose and GLI increased serum insulin levels by 10% and 16.5% respectively, and no significant (p > 0.05) changes in the serum insulin levels were observed in these groups compared to diabetic control group. Finally, EAF treatment at 50 mg/kg dose exhibited slight higher activity than that of glibenclamide. In addition, HOMA-IR levels in EAF treated groups were significantly improved by 53.0% and 57.1% compared to the diabetic control group  in DC rats, and it was restored near to normal levels (NDC) after treated with EAF (50 mg/kg). Treatment with EAF at the dose of 25 mg/kg increased HDL level, but unable to normalize it (p > 0.05) when compared with NDC group. By comparison, GLI improved serum lipid profiles in diabetic rats, and there was no significant (p > 0.05) changes in serum TG, TC, HDL, and LDL levels when compared with NDC group.As shown in Table p < 0.05) in DC group compared to the NDC group. Treatment with EAF (50 mg/kg) and GLI significantly reversed these alterations and there were no significant difference in the levels of liver enzymes and urea when compared with NDC group. Treatment with EAF (25 mg/kg) did not reduce ALT level to normal when compared with NDC group. The creatinine levels were not significantly (p > 0.05) altered in STZ (DC), EAF, and as well as GLI treated groups in comparison with NDC group.As Table p < 0.05) reduction of serum MDA levels by 42.94% and 51.56% respectively as compared to the DC group, and the values of MDA in these groups were reversed to nearly normal as compared to the NDC group (p > 0.05). The GSH level was significantly decreased in the serum of DC group compared to NDC group  showed significant increase in serum MDA level compared to NDC group Fig. . Treatmeoup Fig. . Signifip < 0.05) reduced and restored near to normal level in diabetic rats treated with EAF (25 and 50 mg/kg), indicating that EAF effectively reduced the levels of proinflammatory cytokines in NA-STZ induced T2D ratsThe serum pro-inflammatory cytokine  levels were significantly increased in DC rats compared with NDC Table . Howeverp < 0.05) in NA-STZ treated rats (DC) and almost 60% of glycogen was lost when compared with NDC. Treatment with EAF and GLI increased hepatic glycogen accumulation, exhibiting values of glycogen similar to those of NDC group (Table Glycogen levels in the liver were exhausted significantly (up Table .Brucea javanica (L.) Merr is a well-known medicinal plant originated from tropical Asia. It is extensively used for the treatment of various diseases including diabetes in traditional medicine system among indigenous people in Malaya peninsula [B. javanica seeds exhibited significant concentration-dependent inhibition on both GP-α and α-glucosidase enzymes activity in vitro compared to n-hexane, chloroform, and water fractions indicating that compounds in EAF could be involved in activities of carbohydrate and glycogen metabolism. Hence, based on in vitro assay, EAF was selected for further study.eninsula . HoweverIn this study, T2D was developed in SD rats after NA-STZ treatment, as characterized by hyperglycemia compared to untreated rats (NDC). Treatment with EAF to diabetic rats significantly reduced blood glucose levels, increased glucose tolerance during OGTT, and increased insulin levels compared to untreated rats (DC). These results indicated that EAF exert antihyperglycemic effect by inhibiting GP-α and α-glucosidase activity to enhance glycogen synthesis and slowdown digestion of carbohydrates, thereby improve regulation of glucose in diabetic condition.The body weight lowering effect is often found to be associated with diabetic conditions. It was observed that during the experimental period, body weight of EAF treated rats were slightly decreased compared to initial date. To evaluate the decrease in body weight either caused by diabetes or EAF, markers for liver  and kidney (urea and creatinine) toxicity were measured. In T2D rats, serum ALP, ALT, AST, and urea contents were significantly elevated after NA-STZ treatment, but significantly decreased in EAF treated rats without alteration of creatinine levels, indicating that EAF did not cause any damage to the liver and kidney when treated up to 50 mg/kg b.w. Hyperlipidemia is one of the major factors linked with hyperglycemia due to insulin deficiency during diabetes and correlated with carbohydrate metabolism. Elevation of cholesterol and triglyceride levels in T2D demonstrates abnormal lipoprotein metabolism associated with hyperglycemia , 26. As KB pathway [As shown in Table  pathway , 28. The pathway . TherefoIt has been reported that hyperglycemia and/or reduced antioxidant capacity of the body in diabetic condition result in elevation of ROS and RNS and consequently increased oxidative stress , 31. To The ability of liver to store glycogen is impaired due to lack of insulin or insulin resistance. It is often linked with enhanced activity of glycogen phosphorylase to improve glycolysis, ultimately, causes hyperglycemia , 33. To B. javanica seeds and all of the isolated compounds were tested for GP-α and α-glucosidase inhibition activity. As shown in table para-hydroxybenzoic acid increased hepatic glycogen content [B. javanica seeds.In normal condition, starch was broken down to glucose by α-glucosidase after meals and utilized by cells as an energy source . Glucose content , protoca content , gallic  content , 40, pre content , prevent content  in diabepara-hydroxybenzoic acid, protocatechuic acid, and gallic acid existed in B. javanica seeds acting on several processes. However, possible synergistic effects of these compounds are yet to be evaluated in vivo.The EAF showed antihyperglycemic and antioxidant potential, and reduced the levels of pro-inflammatory cytokines in T2D rats. Our results indicated that the effects of EAF in T2D rats may be due to the presence of luteolin acted as potential α-glucosidase and GP-α inhibitors. Furthermore, it could also be a result of synergistic effect of


The interplanar angle between the ring systems is 40.71 (3)°. In the crystal, molecules are connected by N—H⋯O=C and N—H⋯Nthiazole hydrogen bonds, forming ribbons parallel to the b axis.In the title compound, the hydrazide N atom bonded to the C=O group is planar, whereas that bonded to the SO 16H15N3O3S2, the hydrazide N atom bonded to the C=O group is planar, whereas that bonded to the SO2 group is pyramidally coordinated. The interplanar angle between the ring systems is 40.71 (3)°. Molecules are connected into ribbons parallel to the b axis by two classical hydrogen bonds N—H⋯O=C and N—H⋯Nthiazole.In the title compound, C The structure of (1) was determined on the basis of its spectroscopic data and elemental analysis . In order to establish the structure of the product unambiguously, its crystal structure was determined.Benzothiazoles are versatile heterocyclic compounds with potential pharmaceutical applications  in the solid state , forming layers parallel to (100).Molecules are connected by two pairs of classical hydrogen bonds across inversion centres, to form ribbons parallel to the s Table 2. A C—H⋯Oet al., 20162—NH—NH—C(=O)—C gave six hits: EYOZIB, KUKYOG, XOVFEV, XOZDOG, YOTKAU and ZIVVUX.A search of the Cambridge Database  in pyridine (10 ml) was added gradually to a stirred solution of 2-(benzo[d]thiazol-2-yl)acetohydrazide  in pyridine (10 ml) at 273 K. The reaction mixture was then stirred at room temperature for 3 h (TLC control). After the reaction was completed, the mixture was poured into ice-water with continuous stirring and neutralized with 1 N HCl solution to pH 7. The precipitate thus formed was filtered off, washed with water and recrystallized from ethanol to give colourless crystals . IR : ν 3427 (NH), 3164 (Ar CH), 2929, 2858 , 1692 (C=O); 1H NMR : δ 2.26 , 3.95 , 7.15 , 7.44 , 7.52 , 7.62 , 7.96 , 8.07 , 9.95 , 10.52 .A solution of aromatic = 0.95, C—Hmethylene 0.99 Å) with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 3Despite the slightly larger ellipsoid of the benzothiazol sulfur atom S1, there is no evidence for significant mixing (disorder) of the sites N3/S1.10.1107/S2056989017008738/hg5489sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017008738/hg5489Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017008738/hg5489Isup3.cmlSupporting information file. DOI: 1555516CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


III center coordinated by one chloride ligand, four N atoms and a bridging oxo ligand in a distorted octahedral geometry. The bridging oxo ligand is connected to a second FeIII atom with three coordinating chloride ligands.The bimetallic title compound contains an Fe III atom in the solvated title compound, [Fe2Cl4O(C26H28N4)]·CHCl3, adopts a distorted six-coordinate octahedral geometry. It is coordinated by one chloride ligand, four N atoms from the -N,N′-bis[(quinolin-2-yl)methyl]cyclohexane-1,2-diamine ligand, and a bridging oxido ligand attached to the second FeIII atom, which is also bonded to three chloride ions. A very weak intramolecular N—H⋯Cl hydrogen bond occurs. In the crystal, the coordination complexes stack in columns, and a grouping of six such columns create channels, which are populated by disordered chloroform solvent molecules. Although the Fe—Cl bond lengths for the two metal atoms are comparable to the mean Fe—Cl bond lengths as derived from the Cambridge Structural Database, the Fe—O bond lengths are notably shorter. The solvent chloroform molecule exhibits ‘flip’ disorder of the C—H moiety in a 0.544 (3):0.456 (3) ratio. The only directional interaction noted is a weak C—H⋯Cl hydrogen bond.The first Fe A chloride ion and a bridging oxido ligand, which connects the two metal cations, complete the axial coordination. The distortions from the ideal octahedral geometry occur both in the equatorial and the axial positions. The equatorial angles vary widely from 74.96 (9)°, as in the case of the N1—Fe1—N2 angle, to 133.98 (9)° for the untethered N1—Fe1—N4 angle. The axial ligands exhibit a bent conformation with a Cl1—Fe1—O1 angle of 166.06 (7)°. In contrast, the second Fe metal cation, Fe2, exhibits a near ideal tetrahedral coordination geometry composed of one O atom and three Cl atoms. As expected based on the difference in the saturation of the coordination sphere, the Fe—Cl and the Fe—O distances for Fe1 are longer than that for Fe2. The single Fe—Cl distance for Fe1 is 2.3560 (8) Å, whereas the average Fe2—Cl distance of 2.232 (9) Å is more than 0.1 Å shorter, a statistically significant variation. Similarly, the Fe—O distances for Fe1 and Fe2 are also statistically significantly different at 1.808 (2) Å and 1.756 (2) Å, respectively. The bond lengths in the title compound are comparable to the mean Fe—Cl distances from the CSD for Fe complexes in an octahedral coordination [2.33 (7) Å] and a tetrahedral coordination [2.23 (3) Å]. In contrast, the Fe—O distances for both the octahedral and tetrahedral configurations in the title compound are shorter than the mean distances from CSD . A very weak intramolecular N3—H3⋯Cl4 hydrogen bond (Table 21) is present only as the M (left-handed) conformer.There is one coordination complex and one molecule of chloroform solvent in the asymmetric unit. The coordination complex features two Fe(III) metal cations. One of the metal cations, Fe1, assumes a distorted octahedral coordination Table 1. The tetd Table 2 occurs. a-, b-, and c-axis directions. As there are no additional symmetry elements present, the resulting space group, P212121, is chiral. The absolute structure was unequivocally established, as evidenced by a Hooft y parameter of 0.003 (6), using anomalous dispersion. Apart from a weak C—H⋯Cl bond from the chloroform molecule to one of the chloride ions bonded to Fe2 -N,N′-bis[(quinolin-2-yl)methyl]cyclohexane-1,2-diamine ligand. The packing is illustrated in Fig. 2The molecules in the crystal structure are related by twofold screw axes running along the 2 Table 2, the molet al., 2016R,2R)-N,N′-bis[(quinolin-2-yl)methyl]cyclohexane-1,2-diamine ligand motif. Of the five, only one structure showed coordination to iron  and the Cl1—Fe—N angles show a narrow distribution from 92–94°, except for Cl1—Fe—N1, which is 84°.In our survey of the Cambridge Structural Database -N,N′-bis[(quinolin-2-yl)methyl]cyclohexane-1,2-diamine (R-QMC): In a 50 mL round-bottom flask -1,2-cyclohexanediamine  and 2-quinolinecarboxaldehyde  were refluxed in ethanol (10 mL) for 3 h. A yellow precipitate formed that was isolated by filtration, washed twice with ethanol, and dried in vacuo producing the unreduced form of the ligand (QMC), . 1H NMR : δ 1.24 , 1.56 , 1.97 , 3.62 , 7.48 , 7.65 , 7.74 , 8.03 , 8.06 , and 8.52 . The reduced form of the ligand : δ 1.24 , 1.56 , 1.97 , 3.62 , 4.22 , 7.55 , 7.61–7.73 , 7.81 , 8.06 , 8.08 .R,2R)-N,N′-bis[(quinolin-2-yl)methyl]cyclohexane-1,2-diamine}chloridoiron(III))-μ-oxido-[trichloridoferrate(III)] chloroform monosolvateSynthesis of  was dissolved in 50/50 mixture of dichloromethane and ethanol (20 mL) in a 50 mL round-bottom flask. Iron(II) chloride  was added to the flask to give a brown-colored solution. The reaction was allowed to mix for 6 h under gentle heat producing a brown-colored precipitate. The precipitate was filtered and washed twice with cold solvent then dried under vacuum for 30 minutes producing a brown powder . Brown prisms of (1) suitable for X-ray analysis were obtained by slow solvent diffusion of diethyl ether into a concentrated complex solution made in chloroform.R,2R)-N,N′-bis[(quinolin-2-yl)methyl]cyclohexane-1,2-diamine]chloridoiron(III)}-μ-oxido-[trichloridoferrate(III)], there is one molecule of chloroform solvent in the asymmetric unit. The solvent molecule exhibits extensive positional disorder over three positions. Initially, the disorder was modeled with chloroform molecule in an idealized geometry, where the 1,2 and the 1,3 bond lengths were constrained. As the refinement converged, the geometry constraints were lifted. The chlorine atoms Cl6 and Cl7 were modeled over two positions, with the major component contributing 54.4 (3)%. The carbon atom C27 required modeling over three positions with the major component contribution of 54.4 (3)% and the two minor components contributing 24.1 (4)% and 21.5 (4)%. The C–Cl distances for all of the disorder components were restrained to be similar. In addition, Cl6A–Cl7A and Cl7A–Cl5 were restrained to be similar. The absolute structure was unequivocally determined by anomalous dispersion.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017007952/hb7682sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017007952/hb7682Isup2.hklStructure factors: contains datablock(s) I. DOI: 1552964CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Chelate bond lengths are consistent with an unreduced ligand bound to the  2(C20H17BrN2)]·0.5CH2Cl2, comprises a bidentate iminopyridine ligand and two Cl atoms bound to a zinc2+ cation in a distorted tetrahedral arrangement. The chelate bond lengths are consistent with localized C=N double bonds and a C—C single bond, as expected for an unreduced ligand bound to a closed-shell transition metal cation. Apart from weak nonclassical C—H⋯Cl hydrogen bonds between the complex molecules and the disordered solvent molecules (occupancy = 0.5), no further significant intermolecular interactions are observed.The solvated title compound, [ZnCl To this end, the preparation of the titular zinc(II) complex featuring the unreduced ligand was undertaken. Inclusion of a bromine functionality in the remaining ortho position of the pyridine ring allows for the introduction of an additional donor arm that differs from the imine fragment . The Zn2+ cation lies 0.3855 (3) Å above the plane defined by the chelate (N2/C7/C8/N15), in a distorted tetrahedral arrangement  of the ligand are consistent with the depiction as localized C=N double bonds, and as a C—C single bond. A comparison of the observed bond lengths with the average bond lengths for neutral and doubly-reduced α-iminopyridine  are observed between neighbouring molecules. Notably, neither dimerization nor stoichiometric binding of solvent to the metal cation is observed for this complex, in contrast to some base metal complexes of similar ligands  in diethyl ether (200 ml) at 195 K, was added n-BuLi  dropwise over 5 min. The solution was stirred at 195 K for 1 h, after which benzonitrile  was added dropwise over 5 min. The resultant solution was allowed to warm to room temperature, at which point the yellow solution turned dark red. After 1 h, cold aqueous 3 M HCl (250 ml) was added to the solution causing the dark-red solution to turn yellow, and the organic phase was removed. To the aqueous layer, 3 M NaOH (250 ml) was added, and the mixture was extracted with diethyl ether (3 × 100 ml). The organic fractions were combined, dried over MgSO4, and concentrated under reduced pressure. The product (2) was recrystallized from ethanol, yielding a light-yellow crystalline solid .Following the method of Kobayashi and co-workers , 2,6-dimethylaniline , ∼30 mg of p-toluenesulfonic acid catalyst, and toluene (300 ml) were fitted with a Dean–Stark apparatus, and brought to reflux for 6 d. The mixture was washed with a saturated aqueous solution of NaHCO3, dried over MgSO4, and concentrated under reduced pressure. The resultant brown (crude) product was purified by column chromatography (silica 50–70 ml) with a 4:1 (v/v) hexanes–ethyl acetate mixture as eluant (RF = 0.62) to yield 3 as a bright-yellow solid .Following the method of Meneghetti  al. 1999, a round3  solids were added to a Schlenk flask fitted with a magnetic stirrer bar, and the flask was flushed with argon. Anhydrous tetrahydrofuran (10 ml) was added to the flask, and the solution was allowed to stir for 16 h. The solvent and other volatiles were removed in vacuo, and the residue was rinsed with dry pentane to yield 23-ZnCl as a yellow solid . Single crystals suitable for X-ray diffraction were obtained by diffusion of diethyl ether into a saturated solution of 23-ZnCl in CH2Cl2. 1H NMR  δ 8.01 , 7.95 , 7.60 , 7.49 , 7.39 , 7.21 , 7.01–6.92 , 5.30 , 2.30 . 13C{1H} NMR : δ 169.2 (C=N), 150.3 (aryl ipso-C), 144.5, 142.4, 142.2 (aryl p-CH), 133.9 (aryl m-CH), 131.8 (phenyl p-CH), 130.4 (phenyl ipso-C), 128.9 (aryl o-C), 128.8 (phenyl m-CH), 128.6 (pyridine CH), 127.8 (phenyl o-CH), 127.0 (aryl m-CH and pyridine CH), 19.2 (CH3); m.p. 529–537 K. Analysis calculated (%) for C20.5H18N2BrCl3Zn: C 45.26, H 3.33, N 5.15; found: C 45.19, H 3.40, N 5.06.Anhydrous zinc(II) chloride  and Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017007812/wm5393sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017007812/wm5393Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017007812/wm5393sup4.pdfNMR spectrum. DOI: 10.1107/S2056989017007812/wm5393sup5.pdfNMR spectrum. DOI: 1552501CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Fh1 in pancreatic β cells (Fh1βKO mice) appear normal for 6–8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1βKO mice led to dysregulated metabolism in β cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking   •Fh1 loss in β cells causes progressive Hif1α-independent diabetes•Fh1 loss in β cells impairs ATP generation, electrical activity, and GSIS•Elevated fumarate is a feature of diabetic murine and human islets•“Normoglycemia” restores GSIS in Fh1βKO islets Adam et al. have shown that progressive diabetes develops if fumarate hydratase is deleted in mouse pancreatic β cells. Such β cells exhibit elevated fumarate and protein succination and show progressively reduced ATP production and insulin secretion. The depleted insulin response to glucose recovers when diabetic islets are cultured in reduced glucose. The resulting rise in cytoplasmic Ca2+ initiates exocytosis of insulin granules. In addition to this “triggering” effect, glucose amplifies insulin secretion at a stage subsequent to Ca2+ influx. Several intracellular factors might mediate this amplifying effect, including ATP, NADPH, and glutamate  catalyzes the hydration of fumarate to malate. FH is also a tumor suppressor, mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC) . Loss ofThe high levels of fumarate that accumulate in FH-deficient cells cause post-translational modification of cysteine residues in proteins to form S-(2-succino)-cysteine (2SC), a process known as succination . This inVhl), an integral component of the HIF1α degradation pathway, in β cells leads to HIF1α stabilization, a switch from oxidative to glycolytic metabolism and consequent glucose intolerance . These mice had normal glucose tolerance for the first 6–8 weeks of life, and their β cells had essentially normal properties, including GSIS, despite the lack of a key Krebs-cycle enzyme. However, Fh1βKO mice subsequently developed rapidly progressing diabetes, culminating in severe glucose intolerance, reduced islet insulin content, and almost complete loss of GSIS.We explored the role of FH in insulin secretion using a mouse model in which Fh1 was deleted specifically in pancreatic β cells  by intercrossing an Fh1 conditional knockout mouse  and prolyl hydroxylase dehydrogenase 3 (Phd3/Egln3) was increased in Fh1βKO islets (Deletion of -β cells K. ExpresO islets K.Blood glucose levels were measured in free-fed CTL and Fh1βKO littermates from 6 to 20 weeks. While young (6–8 weeks of age) Fh1βKO mice were nearly normoglycemic, they subsequently (at >9 weeks of age) developed severe hyperglycemia (>20 mM) L. PlasmaHif1αfl/fl mice (Fh1 and Hif1α (Fh1Hif1αβKO mice). Deletion of Hif1α, confirmed by loss of HIF1α staining in all nuclei of Fh1Hif1αβKO islets  . The glucose intolerance of Fh1βKO mice was unaltered in the Fh1βNrf2DKO mice (cyt) (Fh1βKO+FHcyt)  . The rolunt GSIS . To dete of Nrf2  to deletO+FHcyt)  fully reTo explore the cause of glucose intolerance in Fh1βKO mice, we examined insulin secretion from the perfused pancreas. Glucose elevation from 1 mM to 6 mM transiently stimulated insulin release ∼10-fold in stage II CTL mice but not in Fh1βKO littermates A. NevertThese differences in insulin release correlated with a reduction in insulin content. In islets from stage II mice, insulin content and insulin granule density were reduced by ∼50% in Fh1βKO islets C–2E. PanFh1 deletion disrupts the Krebs cycle, we analyzed glucose utilization  and oxidation   . In bothabolism) A. Glucosabolism) B, suggesabolism) .Figure 3cyt in mice , we measured 16 key amino acids in islets at both 1 mM and 20 mM glucose. Glucose elevation increased levels of 15 amino acids in CTL islets but had little effect in stage II Fh1βKO islets (ATP-dependent) pathway (+ (to test the amplifying effect of glucose). Increasing glucose from 2 mM to 20 mM amplified insulin secretion by 600% in CTL islets but only by 150% in Fh1βKO islets  and glucose (m0 to m+6) isotopomers in islets from stage II Fh1βKO and CTL mice incubated in 1 mM or 20 mM glucose with either i and pHi in stages I and III Fh1βKO β cells and CTL cells. Changes in [Ca2+]i were similar in stage I CTL and Fh1βKO β cells , the responses to glucose and glyceraldehyde were similar  consistently increased [Ca2+]i. Basal pHi was lower in stage III Fh1βKO β cells than either CTL or stage I Fh1βKO β cells  must occur distal to FH. Labeling experiments in mouse embryonic fibroblasts (MEFs) lacking Fh1 show that fumarate exits the mitochondria to the cytoplasm but is then metabolized to aspartate via the urea cycle, which re-enters the Krebs cycle . We speculate that a small elevation of plasma glucose induces a vicious cycle of impaired insulin secretion and hyperglycemia. In part, this arises because, as blood glucose increases, more glucose enters metabolism, causing even greater stress on the β cell with increased fumarate levels. It is possible that metabolism of cytosolic fumarate and anaplerosis eventually become unable to maintain the Krebs cycle and that, once this occurs, rapid deterioration of both GSIS and glucose tolerance occurs.ATP-channel closure, and suppression of electrical activity and [Ca2+]i.Our results indicate that Fh1βKO mice eventually develop diabetes because glucose is no longer able to stimulate insulin secretion. This is because of a failure of mitochondrial metabolism that culminates in impaired ATP production, defective K+ across the inner mitochondrial membrane, the electron transport chain produces the alkalinization of the mitochondrial matrix (4H4O4) is an acid with two hydroxyl groups with pKa of 3.0 and 4.4; thus, loss of FH activity can accordingly be expected to result in acidification not just of the cytoplasm  but also of the mitochondrial matrix.Why does ATP production fail? Pumping Hl matrix  and membi than β cells from stage III mice. At higher plasma glucose levels, mitochondrial glucose metabolism will accelerate due to the increased substrate, leading to greater fumarate generation, acidification of both cytoplasmic and mitochondrial matrices, and a progressive impairment of ATP production. Also, acidification may influence the activity of other enzymes involved in β cell metabolism with optimal activity at alkaline pH 23HerrCre recombinase, Rip2-Cre+/−), Fh1fl/fltm1PjpHif1αfl/flRip2-Cre+/−, Fh1tm1Pjpfl/flRip2-Cre+/−Gt(ROSA)26Sortm1(CAG-FH)Pjp;Tg(Cdh16-cre)91Igr, Fh1tm1PjpGt(ROSA)26Sortm1(CAG-FH∗)PjpTg(Cdh16-cre)91Igr, and Fh1tm1Pjpfl/flRip2-Cre+/−Nrf2−/− mice  and littermate controls (designated CTL). The constitutive Nrf2 KO mouse was generated from an embryonic stem cell (ESC) clone obtained from Riken, Japan. Genotyping was performed by Transnetyx, but primer details and PCR conditions can be obtained from J.A. NMRI and C57BL/6J mice (designated wild-type) purchased commercially were used in a few cases.Animal experiments were conducted in accordance with the UK Animals Scientific Procedures Act (1986) and University of Oxford local ethical guidelines. We used male and female adult mice of the following strains: Mouse tissues were fixed in 10% neutral-buffered formalin, dehydrated, and processed for paraffin wax embedding and sectioning (3 μm). H&E sections were generated for all samples. IHC was carried out using the EnVision Kit (Dako) as per the manufacturer’s protocol, with the following antibodies: FH (Autogen Bioclear), HIF1α (Cayman), insulin , glucagon , and 2SC .Blood glucose levels were determined with an Accuchek Aviva meter after 16 hr of fasting and at time points following intraperitoneal injection of 2 g glucose per kilogram of body weight. Animals were culled at the end of the test, and pancreata were processed as described earlier.Insulin secretion was measured by in situ pancreas perfusion or in static incubations of isolated islets . Insulin4 plaque-forming units [PFUs] per islet) delivering Perceval, a recombinant sensor of ATP/ADP  for a further 50 min at room temperature and imaged using an open chamber at 34°C. The bath solution containing various stimuli was perifused continuously at the rate of 200 μL/min. The ratiometric dye SNARF-5F was excited at 543 nm, and emission was collected at 650 nm and 600 nm. Fluo-4 was excited at 488 nm and imaged at 530 nm. Images were acquired at the frequency of 0.03 Hz. The pH calibration of each trace was performed using the high K+-nigericin technique. Valinomycin (5 μM) was added to the extracellular solution to abolish the K+ gradient (Simultaneous time-lapse imaging of [Cagradient .http://fiji.sc/Fiji). The numerical time series data were analyzed using the IgorPro package (Wavemetrics). Statistical significance of the differences between paired or unpaired samples was tested using Friedman or Kruskall-Wallis tests, respectively, with Nemenyi post hoc analysis, as implemented in the R package  using open-source FIJI software .All data are given as mean ± SEM unless indicated that they are mean ± SD. Other than for image analysis, as indicated earlier, statistical significance was determined with significance set at <0.05, using either ANOVA with Tukey’s multiple comparison or Student’s t test (where indicated). Statistical significance was determined using GraphPad Prism v.6.0d  via the Human Tissue Laboratory at Lund University Diabetes Centre. Islets were hand picked, and their quality was assessed prior to research use. Experiments in Oxford were performed using islets from donors  with the following parameters: age, 47.8 years ± 6.7 years; BMI, 28.4 ± 5.8. In Lund, the characteristics of ND  and diabetic  islet donors were as follows: age (years), 61.24 ± 10.45 (CTL) and 60.43 ± 7.72 (T2D), p = 0.82; sex, expressed as male/female, 14/7 (CTL) and 2/5 (T2D), p = 0.44; BMI, 27.11 ± 3.06 (CTL) and 28.9 ± 5.35 (T2D), p = 0.42; and HbA1c, 5.85 ± 0.45 (CTL) and 6.41 ± 0.56, p = 0.04. Data are given as mean ± SD; groups were compared using the Student’s t test or using two-tailed Fisher’s exact test.Pancreatic islets were isolated from deceased donors under ethical approval obtained from the local human research ethics committees in both Oxford and Lund. All donors gave informed research consent. Islets were obtained from the Diabetes Research & Wellness Foundation Human Islet Isolation Facility, OCDEM, University of Oxford, and the Nordic Center for Clinical Islet Transplantation (J.A., P.J.P., and P.R. designed the study. J.A., R.R., M.V.C., N.T., A.H., A.I.T., Q.Z., E.R., N.J.G.R., R.M.d.R., G.O., H.W.D., P.S., K.S., K.K., K.I., B.M.K., J.T.-R., H.M., A.C., T.S., and P.J.P. collected and analyzed the data. A.L., A.S., C.W.P., N.F., and P.R. analyzed data and contributed expertise. P.J.P., M.V.C., A.S., A.I.T., and J.A. made the final figures. J.A., A.S., P.J.P., P.R., and F.M.A. wrote the manuscript, which was critically edited by all authors.


The title compound crystallized with two conformationally very similar independent molecules (A and B) in the asymmetric unit. In the crystal, the individual molecules are linked by pairs of N—H⋯O hydrogen bonds, forming A–A and B–B inversion dimers, with  17H13NO2, crystallizes with two conformationally very similar independent molecules (A and B) in the asymmetric unit. In the crystal, the individual molecules are linked by pairs of N—H⋯O hydrogen bonds forming A–A and B–B inversion dimers, with R22(10) rings. They stack alternately up the a-axis direction and are linked by C—H⋯π interactions, forming sheets parallel to the ab plane.The title compound, C The cyclohexene rings of the tetrahydrocarbazole moieties have half-chair conformations in both molecules. The mean plane of the tetrahydrocarbazole moiety  is inclined to the furan ring by 12.89 (14)° in molecule A, and 12.09 (14)° in molecule B.The title compound, crystallizes with two independent molecules -2-furylmethylene-2,3,4,9-tetrahydro-1H-carbazol-1-one skeleton gave four hits. These include (E)-2-[(furan-2-yl)methylidene]-7-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one -2-(furan-2-ylmethylidene)-8-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one -6-chloro-2-(furan-2-ylmethylidene)-2,3,4,9-tetrahydro-1H-carbazol-1-one , and 0.99 Å (methylene H), and refined as riding with Uiso(H) = 1.2Ueq(C). Reflections 002 and 100 were obstructed by the beam stop and omitted from the refinement.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017017972/su5413sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989017017972/su5413Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017017972/su5413Isup3.cdxSupporting information file. DOI: 1811751CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-(4-methoxyphenyl) caffeamide (K36E) on melanogenesis was investigated.The derivative of caffeamide exhibits antioxidant and antityrosinase activity. The activity and mechanism of B16F0 cells were treated with various concentrations of K36E; the melanin contents and related signal transduction were studied. Western blotting assay was applied to determine the protein expression, and spectrophotometry was performed to identify the tyrosinase activity and melanin content.Our results indicated that K36E reduced α-melanocyte-stimulating hormone (α-MSH)-induced melanin content and tyrosinase activity in B16F0 cells. In addition, K36E inhibited the expression of phospho-cyclic adenosine monophosphate (cAMP)-response element-binding protein, microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). K36E activated the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase 3 beta (GSK3β), leading to the inhibition of MITF transcription activity. K36E attenuated α-MSH induced cAMP pathways, contributing to hypopigmentation.p-CREB, p-AKT, p-GSK3β, tyrosinase, and TRP-1, and activated the transcription factor, MITF. K36E may have the potential to be developed as a skin whitening agent.K36E regulated melanin synthesis through reducing the expression of downstream proteins including  Tyrosinase undergoes maturation and activation through multiple mechanisms, including copper binding, glycosylation, and phosphorylation, resulting in melanin synthesis [Melanin plays a pivotal role in preventing photodamage and photocarcinogenesis of the skin; however, abnormal accumulation of melanin induces hyperpigmentation disorders such as age spots and melasma , 2. Melaynthesis .N-(4-methoxyphenyl) caffeamide , DL-dithiothreitol, H-89 dihydrochloride hydrate, phenylmethanesulfonyl fluoride, and L-tyrosine were purchased from Sigma-Aldrich Chemical Co. . Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and trypsin- ethylenediaminetetraacetic acid (EDTA) were purchased from the GIBCO Invitrogen Corporation . An antibody recognising MITF was obtained from Abcam . Antibodies recognising p-CREB and CREB were purchased from Cell Signalling Technology, Inc. . Antibodies recognising phospho-AKT, AKT, and phospho-glycogen synthase kinase 3 beta (p-GSK3β) were obtained from GeneTex, Inc. . Antibodies recognising actin, GSK3β, TRP-1, and tyrosinase were obtained from Santa Cruz Biotechnology, Inc. .K36E was synthesized and identified by Professor Yueh-Hsiung Kuo with a purity of 99.9% . α-MSH wThe activity of mushroom tyrosinase was spectrophotometrically determined with minor modifications to the previously described procedure , 21–23. 2.B16F0 cells were purchased from the Bioresource Collection and Research Centre in Taiwan and cultured in DMEM supplemented with 10% FBS and 100 units/mL of penicillin and streptomycin at 37 °C in 5% COCell growth experiments were performed using a 3--2, 5-diphenyltetrazolium bromide (MTT) assay with minor modifications to the previously described procedure , 24, 25.4 cells per well and incubated overnight. The cells were exposed to a medium containing α-MSH and K36E for 48 h. Arbutin (1 mM) was the positive control. NaOH (2 N) was added to each well to lyse the cells, which were then centrifuged. The melanin content in the supernatant was measured at 405 nm by using an ELISA reader .The melanin content in B16F0 cells was measured by using a method modified from previous studies , 8, 23. The tyrosinase activity of B16F0 cells after K36E treatment was measured with slight modification on the method described in previous studies , 26, 27.The rate of tyrosinase activity was calculated using the following equation:p-AKT (1:5000), CREB (1:1000), p-CREB (1:1000), GSK3β (1:500), p-GSK3β (1:500), MITF (1:1000), TRP-1 (1:500), and tyrosinase (1:200). The PVDF membranes were incubated with the corresponding conjugated anti-immunoglobulin G horseradish peroxidase . Immunoreactive proteins were detected using an Enhanced Chemiluminescence Plus kit , and signal strengths were quantified using a densitometric program (MultiGauge V2.2). The results of western blot assays represented at least three individual experiments.Western blot analysis was used to demonstrate the effects of K36E on the expression of melanogenesis-related proteins in B16F0 cells as previously described , 28, 29.t-test or ANOVA as well as Scheffe’s test through SPSS software (version 12.0). P values <0.05 indicated significance.Values were expressed as the mean ± standard deviation from the results of at least three individual experiments. Differences in the effects of various treatments were compared using the Student’s K36E at 1000 μM significantly reduced the mushroom tyrosinase activity. The mushroom tyrosinase inhibitory effect at 500, 750 and 1000 μM was 6.8% ± 1.6%, 14.0% ± 7.1% and 36.8% ± 1.1%, respectively. In addition, the inhibition rate of 2 mM arbutin on mushroom tyrosinase activity was 65.3% ± 2.5%.2O2 was 48.9% ± 7.5% after 48 h treatment. The cell viability was acceptable for developing a material for cosmetics. According to International Organization for Standardization (ISO) 10993–5:2009 , cell viability higher than 80% is considered as noncytotoxicity. The results indicated that treatment with 0.5 to 2.5 μM K36E for 48 h had no cytotoxic effect on the B16F0 cells.Cell viability after treatment with 1, 1.5, 2, 2.5, 4, 5, 10, 25, and 50 μM K36E was 92.7% ± 2.0%, 91.7% ± 2.1%, 90.9% ± 2.2%, 87.8% ± 4.2%, 72.8% ± 1.0%, 68.5% ± 2.4%, 54.6% ± 1.6%, 38.3% ± 0.8%, and 28.4% ± 2.3%, respectively. Hydrogen peroxide was the positive control, and the cell viability of 0.1 μM HFigure K36E significantly inhibited tyrosinase activity in B16F0 cells after treatment for 48 h Fig. . The levTo examine whether the inhibition of melanogenesis by K36E was related to the expression levels of melanogenesis-related proteins, including tyrosinase and TRP-1, B16F0 cells were incubated with α-MSH (0.5 μM) and various concentrations of K36E (1–2.5 μM) for 48 h. Although tyrosinase expression exhibited a 2.7-fold increase compared with that in the control after treatment with α-MSH, K36E significantly suppressed tyrosinase expression in a dose-dependent manner  after 1 h and 1.5-fold increase after 2 h; the level of p-GSK3β also displayed a 1.2-fold increase after 1 h compared with that in the control, suggesting that K36E suppressed melanogenesis in B16F0 cells by activating the AKT and GSK3β signalling pathways, resulting in the inhibition of MITF expression and transcription activity and, thus, inhibiting the expression of the tyrosinase gene.As shown in Fig. Tyrosinase and its activity play a major role in controlling melanogenesis –33. Agenp-AKT and p-GSK3β, possibly reducing MITF transcription to suppress tyrosinase gene expression. Previous studies have reported that the activation of AKT inhibited melanogenesis in melanocytes [MITF is the most critical transcription factor that regulates melanogenesis by inducing the expression of melanogenic genes , 40. MITanocytes , 44. ThuUV exposure stimulates the secretion of α-MSH in keratinocytes. α-MSH binds to MC1R in melanocytes, resulting in cAMP production and PKA activation . The sigK36E reduced MITF expression by inhibiting CREB phosphorylation. Additionally, K36E inhibited MITF expression by upregulating the phosphorylation of AKT and GSK3β, which subsequently inhibited the expression of tyrosinase and TRP-1 and thereby reduced melanin biosynthesis. Normal melanocytes and in vivo studies may be applied for further investigation into the effect of K36E on melanogenesis. In conclusion, K36E may be a candidate for the regulation of melanogenesis and it is likely to have various applications in skin whitening products in the future.


However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20 mg L−1) and its 21 derivatives. Anti-Staphylococcus aureus assay reveals that the antibacterial activity of three intermediates is even stronger than that of helvolic acid. Notably, we observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis. These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics.Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P The biosynthetic pathway of fusidane-type antibiotics, such as helvolic acid, is largely undiscovered. Here, the authors investigate the biosynthesis of helvolic acid, thereby determining the sequence of the enzymatic reactions involved in the process and the intermediates formed. Ginsenosides and ganoderic acids are drug candidates3, and ginsenoside Rg3 has been clinically approved to treat cancers in China. However, extremely low amounts of these medicinal constituents in natural hosts have significantly hampered their applications5. Combinatorial biosynthesis is a feasible option to improve the yields of target compounds and expand chemical diversity, which however requires a thorough understanding of their biosynthesis. To date, only a few triterpenoids such as ginsenosides6 and betulinic acid7 have been demonstrated.Triterpenoids possess a variety of pharmacological activities and serve as an invaluable source of pharmaceuticalsZ,24-dien-3β-ol (2) with unique tetracyclic skeleton, which is formed by enzymatic cyclization of (3S)-2,3-oxidosqualene8. Thus, after the formation of the tetracyclic C-20 protosteryl tertiary intermediate cation9, the cyclization reaction is terminated by elimination of H-17 to form a double-bond between C-17 and C-20 without the backbone rearrangement. Helvolic acid (1)10, fusidic acid11, and cephalosporin P112 are the most representative fusidane-type antibiotics 15. Along with the advancement of global antimicrobial resistance, fusidane-type antibiotics have drawn renewed attentions because they have no cross-resistance to commonly used antibiotics16.Fusidane-type antibiotics, a group of fungi-derived triterpenes, originate from protosta-17(20)ics Fig. , all of 17. Exploring fusidane-type antibiotics biosynthesis could offer an opportunity to yield structurally novel derivatives, but the genetic and molecular basis for the biosynthesis of fusidane-type antibiotics remained obscure until 2009 when helvolic acid putative biosynthetic gene cluster consisting of nine genes , functioned to convert (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3β-ol (2), which underwent dehydrogenation to form 3-keto (3) by HelC , or oxidation to generate 4β-carboxylic acid (4) by HelB1 18. Nevertheless, it is still not clear whether the proposed nine-gene cluster is enough for helvolic acid biosynthesis, and if so, what the functions and reaction order of the six other genes are, and whether biosynthetic intermediates with more potent antibacterial activity than the end product helvolic acid exist.The remarkable bioactivity and little cross-resistance of fusidane-type antibiotics have inspired many attempts to search for more active congeners. Great efforts have been focused on modifications of fusidane skeleton by means of chemical derivatization and biotransformationA. oryzae NSAR120, and observe the production of helvolic acid (~20 mg L−1), confirming that the putative gene cluster is sufficient for helvolic acid biosynthesis. Then, we determine the function of each gene and reaction sequences by reconstitution of the nine genes one by one in A. oryzae NSAR1, which enables us to isolate 21 derivatives and find three intermediates with more potent activity than helvolic acid. Remarkably, we observe an unusual C-4 demethylation process that is distinct from the sterol biosynthesis. These findings have provided a basis for constructing fusidane-type derivatives using biosynthetic approaches.In this work, we first introduce all the nine genes into the heterologous expression system A. fumigatus Af293 as the hel cluster and renamed all nine genes . As a result, an additional peak (1) (~20 mg L−1) was detected Z,24-dien-3β-ol (2) and its 3-keto derivative 3 when co-expressing helA and helC in A. oryzae NSAR1  analysis, an additional peak at m/z 410 was observed in the mycelia extract of AO3, but not in the parent strain A. oryzae NSAR1 , HelB1 P450), and HelC (SDR) have been studied through reconstitution in otosta-170Z,24-dieAR1 Fig. . HRESIMS50, and H. 5 Fig. , which i. 5 Fig. 21, 22.Fi5 was the biosynthetic intermediate of helvolic acid, among the remaining six genes, only the P450 enzymes HelB2-4 and the dehydrogenase HelE might be engaged in the next modification. Thus we individually introduced helB2, helB3, helB4, and helE into the strain AO3 to generate transformants AO4–AO7. HPLC analyses of these strains showed that only introduction of helB2 (AO4) gave two additional peaks 6 and 7  is responsible for hydroxylation of the C-16β position  led to the appearance of additional peaks in the HPLC profile  accounts for the acetylation of C-16β hydroxyl group  was responsible for converting C-20 methyl group to carboxylic acid  might function after HelB4. We then introduced helB3 and helE into the strain AO14, respectively. The helB3 containing transformant strain AO17 yielded 17   into AO14 also resulted in the production of 19  and HelE  acts on the C-6 hydroxyl   Fig. .A. oryzae NSAR1 revealed that HelB1 and HelC were responsible for the removal of the C-4β methyl group, which is distinct from the sterol C-4 demethylation process  and purified  analysis , C-3 sterol dehydrogenase (3βHSD), and 3-keto reductase (3SR)7, 9, and 14 by HelC raised a doubt that the biosynthetic routes including HelB2-mediated conversion of 5 to 7, HelD2-mediated conversion of 7 to 9, and HelB4-mediated conversion of 9 to 14 may not exist. To test this hypothesis, feeding experiments for HelB2, HelB4 and HelD2 were carried out. The active expression of HelB2 in AO24, HelB4 in AO25, and HelD2 in AO26 were confirmed by feeding them with substrates 4, 8, and 6, respectively  and its 21 derivatives via reconstitution in A. oryzae, which provided an opportunity for structure-activity relationship analysis. We tested their inhibitory effects against Staphylococcus aureus 209P by the 2-fold dilution method  of 0.5 μg mL−1. The structure-activity relationship analysis revealed that both C-20 carboxyl group and 3-keto were important for the antibacterial activity, which is consistent with previous findings13. In addition, we firstly found that acetylation of the C-6 hydroxyl group was detrimental to the activity, as 17 and 22 exhibited higher activity than their corresponding acetylated products 20 and helvolic acid, respectively. These facts suggested that the end products are not always to be the most active molecules, and using biosynthetic approaches to access intermediates would be an effective way to obtain bioactive analogs.In the course of our work, we obtained helvolic acid ~20 mg L0 mg L−1 S)−2,3-oxidosqualene  by HelA, HelB1-mediated and HelB2-mediated oxidation at C-4 and C-16, HelD2-dependent acetylation of 16-OH, oxidation of C-21 by HelB4, and HelC-dependent oxidative decarboxylation yield the fusidane skeleton 14, which is further modified in three additional steps mediated by HelB3, HelD1, and HelE to give helvolic acid (1)  and its 21 derivatives via reconstitution in A. oryzae NSAR1. We investigated the antibacterial activity of all these compounds and found that the C-20 carboxyl group and 3-keto are essential for their antimicrobial activity against S. aureus, which is consistent with the previous report13. It is worth noting that three intermediates 17, 19 and 22 exhibit more potent antibacterial activity than the end product helvolic acid. Based on the structural difference between 17 (MIC: 1 μg mL−1) and 20 (MIC: 16 μg mL−1), and 22 (MIC: 0.5 μg mL−1) and helvolic acid (MIC: 2 μg mL−1), we could conclude that the free C-6 hydroxyl group is important for the antibacterial activity and further acetylation of 6β-OH is deleterious to the activity. These results also give a good example that in some cases, not all the biosynthetic genes in the cluster are designed to increase the biological activities of secondary metabolites, which is also shown in the cylindrocyclophane biosynthesis31. Our results suggested that the biosynthetic intermediates and shunt products might be an invaluable source of drug discovery. However, natural products obtained via the conventional method are usually the end products biosynthesized in the hosts32, and access to the biosynthetic intermediates is always difficult. Therefore, biosynthetic approaches, especially heterologous expression, could allow the isolation of as many intermediates as possible33, which will be an effective way to obtain more active derivatives.In this study, we obtained helvolic acid  was purchased from Oceanpak Alexative Chemical Co., Ltd. . Petroleum ether, ethyl acetate (EtOAc), and acetone were analytical-grade from Fine Chemical Co., Ltd. . Formic acid was purchased from Kemiou Chemical Reagent Co., Ltd. .Acetonitrile . Plasmid purification kits and agarose gel DNA extraction kits were purchased from Sangon Biotech Co., Ltd. . PCR was carried out using a Mastercycler nexus gradient  with KOD -Plus- polymerase . In-Fusion® HD Cloning Kit and T4 DNA polymerase were purchased from Takara Bio Inc. . Other DNA modification reagents were purchased from Thermo Fisher Scientific Inc. .−1 and stayed at 230 °C for 2 min. It then continued with a second ramp by rising at a rate of 10 °C min−1 to 310 °C and held for 10 min. Helium was used as the carrier gas.GC–MS analysis was performed on Thermo Finnigan Trace GC Ultra equipped with a HP-5MS column  and coupled with Thermo Finnigan Trace DSQ. The temperature of the ionization chamber was 300 °C and the electron impact ionization voltage was 70 eV. The oven temperature began at 75 °C and held for 5 min, then increased to 230 °C at a rate of 20 °C min18-MS-II column . Elution was subjected to a linear gradient .HPLC and LC–MS were carried out on an Ultimate 3000 HPLC system (Dionex) and an amaZon SL ion trap mass spectrometer (Bruker) using electrospray ionization with a Cosmosil 5C3: δH 7.26/δC 77.0; Pyridine-d5: δH 7.21/δC 123.5) as internal standards.UV data, IR data and optical rotations were respectively measured on the JASCO V-550 UV/vis spectrometer, JASCO FT/IR-480 plus spectrometer, and JASCO P1020 digital polarimeter from JASCO International Co., Ltd., Tokyo, Japan. The HRESIMS data were obtained on a Micromass Q-TOF mass spectrometer . 1D and 2D NMR spectra were obtained with Bruker AV 400 and Bruker AV 600 spectrometers  using the solvent signals (CDClThe semi-preparative HPLC was performed on an Ultimate 3000 HPLC system (Dionex) using a YMC-Pack ODS-A column . Medium-pressure liquid chromatography (MPLC) was equipped with a dual-pump gradient system, a UV preparative detector, and a Dr. Flash II fraction collector system . Column chromatography (CC) was performed with silica gel  and ODS .Aspergillus oryzae NSAR1 20 served as the host for heterologous expression of genes. Transformants of A. oryzae NSAR1 were grown in 10 mL DPY medium  for 1–2 days at 28 °C and 150 rpm. The cells were then transferred into Czapek-Dox (CD) medium  to induce the expression of heterologous genes under the amyB promoter.E. coli DH5α in LB medium supplemented with appropriate antibiotics, and E. coli BL21(DE3) was used for expression of HelC.Standard gene engineering experiments were performed using Aspergillus fumigatus Af293 genomic DNA with the primers listed in Supplementary Table helA gene was ligated into the SmaI-linearized pTAex3 vector using the In-Fusion® HD Cloning Kit to yield pTAex3-helA. The helB1 was cloned into the KpnI site of the pUSA vector to yield pUSA-helB1. The other seven genes were ligated into the pTAex3 vector digested with EcoRI or (and) KpnI to create pTAex3-helB2, pTAex3-helB3, pTAex3-helB4, pTAex3-helC, pTAex3-helD1, pTAex3-helD2, pTAex3-helE. pUSA-helC was constructed by ligation of the fragment amplified from pTAex3-helC using HelC-EcoRI-F/HelC-EcoRI-R to the SmaI-digested pUSA vector.All the nine genes in the helvolic acid gene cluster were amplified from helB1-helC was constructed by ligation of the helC-containing fragment generated from the BamHI-digestion of pTAex3-helC to the BamHI-linearized pUSA-helB1. pAdeA-helB2, pAdeA-helB3, pAdeA-helB4, pAdeA-helE, pPTRI-helB3, and pPTRI-helE were constructed by the insertion of the fragments amplified from pTAex3-helB2, pTAex3-helB3, pTAex3-helB4, pTAex3-helE with the primers pTAex3-Pamy-F/pTAex3-Tamy-R into the XbaI-digested pAdeA or SmaI-digested pPTRI. For construction of plasmids harboring two or three genes, DNA fragments including the amyB promoter and terminator were amplified from pTAex3-based plasmids with the corresponding primers, and then ligated with the XbaI-digested pAdeA or HindIII-digested pPTRI using the In-Fusion® HD Cloning Kit. All of the expression plasmids are listed in Supplementary Table pUSA-A. oryzae NSAR1 was inoculated into the 10 mL DPY medium and cultivated for two days at 28 °C and 150 rpm. Then the cells were transferred into 100 mL DPY medium and continued growing for one day at 28 °C and 150 rpm. Mycelia were collected by filtration, and cell walls were digested using 1% Yatalase (Takara) in 0.6 M (NH4)2SO4, 50 mM maleic acid, pH 5.5 at 30 °C for 3 h. After removing residues by filtration, protoplasts were centrifuged at 1500 rpm for 10 min and washed with Solution 2 , and then adjusted to 2 × 107 cells mL−1 in Solution 2. Mixtures of 200 μL protoplasts solution and <15 μL plasmids (10 μg) were incubated on ice for 30 min, and subsequently 1.35 mL Solution 3  was added to the aliquot. After 20 min incubation at the room temperature, 10 mL Solution 2 was added, and then the mixture was subjected to centrifugation at 1500 rpm for 10 min. The precipitates were suspended in 200 μL Solution 2 and spread on the lower selective medium 2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, 2% glucose and 1.2 M sorbitol as well as 0.15% methionine, 0.1% arginine, 0.01% adenine, and 0.1 μg mL−1 pyrithiamine hydrobromide if necessary, pH 5.5) with 1.5% agar, and then covered with the selective upper medium containing 0.8% agar. The plates were incubated at 30 °C for five to seven days. All of the transformants used in the work are listed in Supplementary Table The spore suspension of To analyze the metabolites in different transformants, the seed broth cultured in 10 mL DPY medium for 2 days were inoculated into 100 mL CD medium in a 500 mL flask at 28 °C for 5–6 days on a rotary shaker at 150 rpm. Then, mycelia were collected by filtration and extracted with acetone at the room temperature, and if necessary, the culture medium was extracted with EtOAc. The extract concentrated under reduced pressure was resuspended for analysis. For isolation of metabolites, extract was subjected to the column chromatography and further purification by semi-preparative HPLC (Supplementary Methods).helC using SDR-NdeI-F/SDR-HindIII-R was cloned into the expression vector pET28a at the NdeI and HindIII sites in frame with an N-terminal His6-tag sequence. E. coli BL21(DE3) harboring pET28a-helC was inoculated into 10 mL LB medium with 50 mg L−1 kanamycin and cultured at 37 °C and 200 rpm overnight, and then 1.5 mL seed broth was transferred into the 500 mL flask with 150 mL LB medium and 50 mg L−1 kanamycin, and grew at 37 °C and 200 rpm until OD600 reached up to 0.5–0.7. The gene expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18 °C for 12–16 h. Cells were harvested by centrifugation at 4 °C and 5000 × g for 10 min. The pellet was washed with 50 mM Tris-HCl (pH 8.0) and resuspended in lysis buffer , followed by sonication on ice for 10 min. After centrifugation at 4 °C and 10,000 ×  g for 10 min, the supernatant was further filtered through a 0.45 μm filter to remove cell debris and other particulate materials. The filtrate was subjected to a Ni SepharoseTM 6 Fast Flow  affinity chromatography column equilibrated with binding buffer . After elution with wash solution  to remove host proteins, the bound HelC was eluted with elution solution . The fraction containing HelC was concentrated and buffer-exchanged using the Amicon centrifugal filter Ultracel®-10K . The purified HelC was flash frozen in liquid nitrogen and stored at −80 °C for further use. In the course of expression and purification, HelC was confirmed using SDS-PAGE. Protein concentration was quantified according to the Bradford method via PierceTM BCA Protein Assay Kit  and using bovine serum albumin as the standard protein.The fragment coding HelC amplified from pTAex3-In vitro enzymatic reaction for HelC was carried out in a reaction mixture  containing 100 mM Tris-HCl (pH 8.0), 5 mM NAD trihydrate, 2.35–7.00 μM HelC, and 0.5 mM substrate. Following incubation at 30 °C overnight, the mixture was extracted with 200 μL EtOAc. After evaporation of solvents, the residue was resuspended in methanol for HPLC analysis.A. oryzae NSAR1 transformant strain only containing helB1, helB2, helB4, or helD2 was cultured in 10 mL DPY medium for 1–2 days, and then was inoculated into 50 mL CD medium for expression of exogenous genes. After cultivation for 1 day, the culture was supplemented with 1.0 mg of substrate dissolved in 20 μL DMSO, and was further incubated for 5 days. Mycelia were harvested and extracted with acetone. After removal of solvents under reduced pressure, samples were dissolved in methanol for LC–MS analysis.The Staphylococcus aureus 209 P was evaluated using the 2-fold dilution assay36. The bacterial strain was inoculated on nutrient agar  at 37 °C for 1–2 days, then cells were collected with normal saline and adjusted to a concentration of 107–109 mL−1 as seed broth. Compounds were dissolved into DMSO and adjusted to 50 mg L−1. A total of 100 μL seed broth was added to 200 mL nutrient solution, and then 200 μL of mixtures was transferred into the first test well of each line in the 96-well while 100 μL was added in the other wells in the same line. 0.5 μL sample solution was added into the first well with the concentration of 128 mg L−1, then 100 μL was transferred to the second well until the twelfth well with the final concentration of 0.06 mg L−1. Tobramycin was used as the positive control and DMSO as the negative control. The 96-well microtiter plates were placed at 37 °C for 24 h. The MIC was defined based on the minimal concentration in which no bacteria were observed.In vitro antibacterial activity against Gram-positive strain 1: A white powder; HRESIMS (positive) m/z 591.2932 [M + Na]+ , see Supplementary Fig. β,16β-diacetyloxy-29-norprotosta-1,17(20)Z,24-trien-3,7-dione-21-oic acid (helvolic acid37)  m/z 425.3793 [M + H]+ , see Supplementary Fig. Z,24-dien-3-one18  m/z 457.3665 [M + H]+ , see Supplementary Fig. β-hydroxy-protosta-17(20)Z,24-dien-29-oic acid , but are slightly revised  m/z 411.3600 [M + H]+ , see Supplementary Fig. Z,24-dien-3-one , the detailed NMR assignments of which are not available prior to the present study ; UV (CH3OH) λmax (log ε) 206 (4.05), 252 (3.33); IR (KBr) νmax 3429, 2947, 2872, 1697, 1621, 1455, 1375, 1027 cm−1; HRESIMS (positive) m/z 473.3622 [M + H]+ , see supplementary Fig. 6 is identified as 3β,16β-dihydroxy-protosta-17(20)E,24-dien-29-oic acid (Supplementary Note Compound ), 252 3.; IR ; UV (CH3OH) λmax (log ε) 205 (3.67), 248 (3.18); IR (KBr) νmax 3421, 2941, 2872, 1705, 1624, 1463, 1375, 1046 cm−1; HRESIMS (positive) m/z 427.3572 [M + H]+ , see Supplementary Fig. 7 is identified as 16β-hydroxy-29-norprotosta-17(20)E,24-dien-3-one ; UV (CH3OH) λmax (log ε) 206 (3.85), 253 (2.79); IR (KBr) νmax 3445, 2944, 2875, 1729, 1705, 1449, 1378, 1239, 1027 cm−1; HRESIMS (positive) m/z 537.3557 [M + Na]+ , see Supplementary Fig. 8 is identified as 16β-acetyloxy-3β-hydroxy-protosta-17(20)E,24-dien-29-oic acid ; UV (CH3OH) λmax (log ε) 207 (3.65), 248 (3.05); IR (KBr) νmax 2950, 2869, 1708, 1454, 1373, 1245 cm−1; HRESIMS (positive) m/z 491.3472 [M + Na]+ , see Supplementary Fig. 9 is identified as 16β-acetyloxy-29-norprotosta-17(20)E,24-dien-3-one ; UV (CH3OH) λmax (log ε) 205 (3.77), 246 (2.97); IR (KBr) νmax 3429, 2965, 2938, 2851, 1688, 1466, 1449, 1375, 1098, 1027 cm−1; HRESIMS (positive) m/z 473.3615 [M + H]+ , see Supplementary Fig. 10 is identified as 3β,20-dihydroxy-protosta-16,24-dien-29-oic acid with the absolute configuration of C-20 unsolved (Supplementary Note Compound ε) 205 3.7, 246 ; UV (CH3OH) λmax (log ε) 205 (3.91), 249 (3.05); IR (KBr) νmax 3450, 2965, 2938, 2872, 1697, 1469, 1449, 1375, 1098, 1027 cm−1; HRESIMS (positive) m/z 473.3611 [M + H]+ , see Supplementary Fig. 11 is identified as 3β,20-dihydroxy-protosta-16,24-dien-29-oic acid with the absolute configuration of C-20 unsolved ; UV (CH3OH) λmax (log ε) 205 (3.84), 246 (2.78); IR (KBr) νmax 3459, 2962, 2941, 2869, 1702, 1452, 1375, 1106 cm−1; HRESIMS (positive) m/z 427.3569 [M + H]+ , see Supplementary Fig. 12 is identified as 20-hydroxy-29-norprotosta-16,24-dien-3-one with the absolute configuration of C-20 unsolved ; UV (CH3OH) λmax (log ε) 206 (3.87), 245 (2.48); IR (KBr) νmax 3442, 2960, 2936, 2872, 1702, 1455, 1381, 1109 cm−1; HRESIMS (positive) m/z 427.3568 [M + H]+ , see Supplementary Fig. 13 is identified as 20-hydroxy-29-norprotosta-16,24-dien-3-one with the absolute configuration of C-20 unsolved ; UV (CH3OH) λmax (log ε) 206 (3.54), 225 (3.45); IR (KBr) νmax 2957, 2944, 2872, 1729, 1708, 1455, 1373, 1253 cm−1; HRESIMS (positive) m/z 521.3244 [M + Na]+ , see Supplementary Fig. 14 is identified as 16β-acetyloxy-29-norprotosta-17(20)Z,24-dien-3-one-21-oic acid ; UV (CH3OH) λmax (log ε) 206 (3.56), 222 (3.51); IR (KBr) νmax 3439, 2971, 2944, 2869, 1729, 1708, 1449, 1375, 1250, 1033 cm−1; HRESIMS (positive) m/z 523.3408 [M + Na]+ , see Supplementary Fig. 15 is identified as 16β-acetyloxy-3β-hydroxy-29-norprotosta-17(20)Z,24-dien-21-oic acid ; UV (CH3OH) λmax (log ε) 206 (3.82), 226 (3.74); IR (KBr) νmax 3456, 2968, 2953, 2869, 1729, 1466, 1375, 1267, 1024 cm−1; HRESIMS (positive) m/z 545.3474 [M + H]+ , see Supplementary Fig. 16 is identified as 16β-acetyloxy-3β-hydroxy-protosta-17(20)Z,24-dien-21,29-dioic acid  m/z 551.2987 [M + Na]+ , see Supplementary Fig. β-acetyloxy-6β-hydroxy-29-norprotosta-17(20)Z,24-dien-3,7-dione-21-oic acid (CAS: 1379525–35–5), the detailed NMR assignments of which are not available prior to the present study ; UV (CH3OH) λmax (log ε) 207 (3.86), 225 (3.75); IR (KBr) νmax 3432, 2953, 2938, 2869, 1708, 1621, 1387, 1355, 1267, 1027 cm−1; HRESIMS (positive) m/z 553.3139 [M + Na]+ , see Supplementary Fig. 18 is identified as 16β-acetyloxy-3β,6β-dihydroxy-29-norprotosta-17(20)Z,24-dien-7-one-21-oic acid  m/z 497.3238 [M + H]+ , see Supplementary Fig. β-acetyloxy-29-norprotosta-1,17(20)Z,24-trien-3-one-21-oic acid , the detailed NMR assignments of which are not available prior to the present study  m/z 593.3081 [M + Na]+ , see Supplementary Fig. β,16β-diacetyloxy-29-norprotosta-17(20)Z,24-dien-3,7-dione-21-oic acid  ; UV (CH3OH) λmax (log ε) 206 (3.71), 223 (3.60); IR (KBr) νmax 3450, 2977, 2944, 2872, 1747, 1440, 1375, 1259, 1027 cm−1; HRESIMS (positive) m/z 573.3428 [M + H]+ , see Supplementary Fig. 21 is identified as 6β,16β-diacetyloxy-3β-hydroxy-29-norprotosta-17(20)Z,24-dien-7-one-21-oic acid  m/z 527.3009 [M + H]+ , see Supplementary Fig. β-acetyloxy-6β-hydroxy-29-norprotosta-1,17(20)Z,24-trien-3,7-dione-21-oic acid (helvolinic acid37) (Supplementary Note Compound All relevant data are available within the article and its Supplementary Information and from the corresponding authors on request.Supplementary InformationPeer Review File


Many studies report that, under some circumstances, amending soil with biochar can improve field capacity and plant-available water. However, little is known about the mechanisms that control these improvements, making it challenging to predict when biochar will improve soil water properties. To develop a conceptual model explaining biochar’s effects on soil hydrologic processes, we conducted a series of well constrained laboratory experiments using a sand matrix to test the effects of biochar particle size and porosity on soil water retention curves. We showed that biochar particle size affects soil water storage through changing pore space between particles (interpores) and by adding pores that are part of the biochar (intrapores). We used these experimental results to better understand how biochar intrapores and biochar particle shape control the observed changes in water retention when capillary pressure is the main component of soil water potential. We propose that biochar’s intrapores increase water content of biochar-sand mixtures when soils are drier. When biochar-sand mixtures are wetter, biochar particles’ elongated shape disrupts the packing of grains in the sandy matrix, increasing the volume between grains (interpores) available for water storage. These results imply that biochars with a high intraporosity and irregular shapes will most effectively increase water storage in coarse soils. Biochar is charcoal made for the purpose of soil amendment . AmendinWater movement and storage in soils are crucial for nutrient delivery and plant productivity. Biochar has the potential to alter soil hydrology and to drive shifts in the amount of water stored in soils. To understand how biochar amendment may influence water delivery to plants, we must understand how biochar affects soil hydrologic processes. However, while many studies report effects of specific biochars on specific soil water properties , 10, theθfc) and at permanent wilting point (θpwp), and the amount of plant available water (θpaw) of soil change with biochar amendment is an efficient way to quantify how biochar affects soil water conditions and plant growth. We use the water retention curve, which defines equilibrium water content (θ) at a given soil water potential (ψ), to extract the key parameters of water content at saturation (θs), θfc (water content at ψ = -33kPa), θpwp (water content at ψ = -1500kPa), and θpaw (= θfc - θpwp)  calculated from sample mass with water , dry sample mass , sample volume (V = 2.5 x 10−4 m3) and water density (ρw = 1000 kg/m3).For Hyprop measurements, each sample was poured without intentional compaction into a stainless-steel cylinder V/ρw, m3/m3]. Based on a suite of experiments, it is difficult to prepare several samples with same ψ. Therefore, we used the WP4C data to estimate one θpwp (without error bars) for our biochar-sand mixtures and sand.For WP4C measurements we added de-aired, purified water to sand and biochar-sand mixtures to make samples with water content from 0.000 mθfc and WP4C measurements we can determine θpwp, which then allows us to calculate plant available water (θpaw = θfc.- θpwp).With Hyprop measurements we can determine ρb =Md/V) of each sample (sand or biochar-sand mixture) (Md) and total sample volume (V = 2.5 x 10−4 m3), which is the volume of the stainless-steel cylinder for the Hyprop.We calculated the dry bulk density (mixture)  using meϕT, volume fraction of intrapores plus interpores) (Ms (kg) is mass of sand, Mb (kg) is mass of biochar, ρss (kg/m3) is skeletal density of sand, and ρsb (kg/m3) is skeletal density of biochar   of the b biochar .Soil water potential is composed of pressure potential, gravitational potential, osmotic potential, and perhaps by other potential terms , althougTo properly describe the water retention characteristics of our biochar-sand mixtures with interpores and intrapores, we used a bimodal van Genuchten model (VGbi)  to fit tθr is the residual water  and θs is the saturated water content (k is the number of “pore systems” (i.e. interpores and interpores) that form the total pore size distribution . In our mixtures with two types of pores (interpores and intrapores), we assume that k is equal to 2, and wi is the weighting factors of each sub soil water retention curve for each pore system where 0 < wi < 1 and Ʃwi = 1. Where αi (>0) is the inverse of the air entry pressure and ni (>1) is a measure of the pore-size distribution the for each sub soil water retention curve, respectively. With these constraints, we simplify In  content , k is thThe fitted parameters and goodness of fit were determined using the MATLAB Curve Fitting toolbox .D50) and aspect ratio (AR) which is D50 divided by median of the maximum distance between two parallel tangential lines of a particle projection  . To quanojection . We usedθs, θfc, ρb, and ϕT between replicated measurements. We then performed statistical comparisons of θs, θfc, ρb, and ϕT between different biochar-sand mixtures and sand by the one-way analysis of variance (ANOVA), followed by Tukey-Kramer’s post hoc test if differences were deemed significant at a p-value less than 0.05. We also computed Pearson correlation coefficients (R) to assess the relationships between θs, θfc, θpwp, θpaw, and ρb as well as the relationships between θs, θfc, θpwp, θpaw, and ϕT.We used Levene’s test to confirm equality of variances of Biochar grain size played an important role in the water retention of sand-biochar mixtures. Field capacity, permanent wilting point, and plant available water of sand-biochar mixtures all increased with biochar particle size.θs) of fine (0.39 ± 0.03 m3/m3) and coarse (0.37 ± 0.04 m3/m3) biochar-sand mixtures were statistically the same (p>0.05) as that for sand (0.34 ± 0.02 m3/m3); however, the water content at saturation of the medium biochar-sand mixtures (0.41 ± 0.01 m3/m3) was 21% higher (p<0.01) than that for sand , the field capacity of medium (0.042 ± 0.002 m3/m3) and coarse (0.050 ± 0.005 m3/m3) biochar-sand mixtures increased by 68% (p<0.01) and 100% (p<0.01), respectively (3/m3) increased by 12% relative to sand but was not statistically significant (p = 0.25). Similarly, permanent wilting point increased from 0.005 m3/m3 for sand to 0.007 m3/m3 (40% increase), 0.010 m3/m3 (100% increase), and 0.010 m3/m3 (100% increase) for fine, medium, and coarse biochar-sand mixtures, respectively . These increases in θfc and θpwp resulted in increases in θpaw for medium and coarse biochar-sand mixtures. Sand had θpaw of 0.018 ± 0.005 m3/m3, whereas θpaw was 0.021 ± 0.001 m3/m3 for fine biochar-sand mixtures . Plant available water was higher for medium and coarse biochar-sand mixtures: 0.032 ± 0.002 m3/m3 (78% increase) and 0.040 ± 0.005 m3/m3 (122% increase) (Compared to the ectively . Field cncrease) .p = 0.55, 0.22, and 0.08, respectively). Total porosity increased 9.3% (p<0.05) for all biochar-sand mixtures  and coarse sand-sand mixture (0.37 ± 0.01 m3/m3) were the same to that of sand alone (0.34 ± 0.02 m3/m3) within error. The water retention curves of fine sand-sand mixtures and coarse sand-sand mixtures continued to overlap with that of the sand sample for all ψ <-1.8kPa , the bulk density of fine sand-sand mixture (1600 ± 00 kg/m3) and coarse sand-sand mixture (1580 ± 30 kg/m3) were increased (p<0.05) by 5.2% and 4.0%, respectively. Correspondingly, the total porosity of the fine sand-sand mixture (0.40 ± 0.0 m3/m3) and coarse sand-sand mixture (0.43 ± 0.01 m3/m3) were 7.0% and 4.7% lower (p<0.05) than that of sand (0.41 ± 0.01 m3/m3).Compared with sand (bulk density = 1520 ± 20 kg/mOur results suggest that the pores inside biochar (intrapores) and the pores created between biochar particles and soil particles (interpores) play fundamentally different roles in soil water retention when capillarity pressure is the main component of soil water potential. Intrapores control water retention at lower soil water potential values causing an increase in field capacity, permanent wilting point, and plant available water for medium and coarse biochar-sand mixtures. However, interpores control water retention at higher soil water potential values for fine biochar-sand mixtures.Pc) was the major component of soil water potential.To understand how pore type and size act to control soil water retention in biochar-sand mixtures, we used a simple calculation to estimate pore diameters that correlate with our observed increases in water retention. We assumed that capillary pressure , θc (°) is contact angle between the water-air interface and biochar/sand surface and d (m) is the pore diameter. Contact angle describes the hydrophilicity or hydrophobicity of a solid surface [θc of biochar resulting in uncertainties of biochar’s θc. While acknowledging these uncertainties, we assumed θc = 55° for biochar, which is the minimum reported contact angle of fresh biochar in existing studies using direct and indirect methods [θ >90°), then water entry pressure is positive [Here  surface . The con surface , 28, liq surface , particl surface , and sur surface . This le methods –29, 31. positive . In thisd) <0.01 mm [ψ < -16.5 kPa when d is less than 0.01 mm. This suggests that the pores smaller than 0.01 mm control water retention of our biochar-sand mixtures when ψ is less than -16.5 kPa. Given the small size of these pores, we assume this represents intrapores (pores inside biochar particles). We did not use θc = 0° for biochar because there is unlikely that this biochar is fully hydrophillic. However, if θc = 0° for biochar, then ψ is equal to -28.8 kPa which is close to -16.5 kPa considering ψ spans several orders of magnitude.Most biochar intrapores have diameters  is larger than 0.1 mm if we assume d is 40% of particle diameter (>0.251 mm) for packed spheres [θc≈0°) because previous studies showed that biochar only causes a small degree of change in contact angle in soil [ψ will be greater than -2.88 kPa when interpore size >0.1 mm. Therefore, interpores would be more likely control water retention curves at higher ψ.For a mono-dispersed sand (0.251–0.853 mm), the interpore size  of the parent biochar  (ϕT) increased, the amount of water held at a number of pressures  increased for sand amended with medium and coarse biochars. We found positive relationships between θi and ϕT, θfc and ϕT, θpwp and ϕT, θpaw and ϕT  , θpwp (at ψ = -1500kPa), and θpaw (= θfc- θpwp) (The high intraporosity (1-ρe/ρs)  suggestsnd 0.75) . Based oc- θpwp)  caused bc- θpwp) .θfc, θpwp, and θpaw decreased as biochar particle size decreased  is less than -16.5 kPa. The water content held by medium and coarse biochars (m3 water/m3 biochar) at -16.5 kPa from measured water retention curves (θb) is showed in The water content of biochars determined from water retention curves were lower than the water content that parent biochar can store. In section 4.1 we documented that intrapores control water retention of our biochar-sand mixtures when soil water potential  is the difference in the water content at ψ = -16.5 kPa between the biochar-sand mixture and the sand, or water content increased by biochar at ψ = -16.5 kPa; and fvb  is volume fraction of biochar which can calculated from Mb (kg) is the biochar mass; ρeb  is the sample volume of the mixture. Therefore, by combing Eqs Where θdiff = 0.016 m3/m3 and Mb = 7.5 x 10−3 kg result in a θb of 0.30 m3 water/m3 biochar. Similarly, for coarse biochar, θdiff = 0.027 m3/m3 and Mb = 7.5 x 10−3 kg result in a θb of 0.52 m3 water/m3 biochar. Therefore, the amount of water (0.52 m3 water/m3 biochar) held by coarse biochar intrapores at ψ = -16.5 kPa is slightly less than the maximum water (0.6 m3 water/m3 biochar) that can be stored by parent biochar intrapores. However, water held by medium biochar intrapore at ψ = -16.5 kPa is half of the water content that can be stored by parent biochar intrapores. We interpret this to mean that the decrease in water stored in medium biochar is due to destruction of intrapores caused by grinding biochar into smaller particles.For medium biochar, ϕT  in plant-available water after mixing coarser biochar with sand at a laboratory timescale. Over the timescale of field application, biochar particle size, intraporosity, and hydrophobicity might change, likely altering soil water retention. For instance, biochar particle size can be reduced by natural forces such as freeze and thaw cycles , plant r% in planψ and by increasing interporosity due to elongated biochar particle shape increasing interpores space at higher ψ when capillarity pressure is the main component of soil water potential. This suggests that to increase plant-available water (θpaw) in sandy soils, biochar with high intraporosity and an irregular shape will be most effective. Various production factors  may be useful to produce biochar with varying porosity. Further studies are needed to address how long biochar intraporosity, particle size, and particle shape will last after field application.In this study, we used a simple sand-biochar system to develop a mechanistic understanding of how biochar’s internal pores (intrapores) and the pores between biochar and sand particles (interpores) affect soil water retention. In our experiments the addition of biochar to sand increased initial water content and field capacity. Our controlled particle size and porosity conditions allowed the development of conceptual models that connect biochar properties to soil water benefits. We propose that the increase of water retention of sandy soils by biochar addition is caused by biochar intraporosity at lower S1 FigPhotomicrograph of (a) fine biochar (<0.251 mm), (b) fine sand (<0.251 mm), (c) medium biochar (0.251–0.853 mm), (d) sand (0.251–0.853 mm), (e) coarse biochar (0.853–2.00 mm), (f) coarse sand (0.853–2.00 mm) and (g) parent biochar (2.00–2.30 mm).(TIF)Click here for additional data file.S2 FigD) is the shortest chord of a particle projection (results close to screening/sieving).Particle diameter ((TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


The synthesis of a new asymmetrically substituted hemilactide is reported and its structural analysis, including X-ray crystallographic data, is reported. 9H10O5, the hemilactide heterocycle exhibits a twist-boat conformation. The bulkier propynyloxymethyl group is in an axial position with a gauche conformation for the CH2–O–CH2–C segment. In the crystal, molecules are linked by pairs of C—H⋯O hydrogen bonds, forming inversion dimers. The dimers are linked by further C—H⋯O contacts, forming a three-dimensional structure.The number of known asymmetrically substituted hemilactides, important precursors for obtaining regular derivatives of polylactide polymers, is still limited and structural characterization of most of them is incomplete. In the title racemic 1,4-dioxane-2,5-dione derivative, C In general, the bond lengths and angles are in normal ranges for organic carbohydrates. The hemilactide heterocycle exhibits a twisted boat conformation, where atoms C1, C2 and O1 are in one plane and atoms C1, C3, C4 and O2 are in another plane; the planes are inclined at a dihedral angle of 27.9 (2)°. The values of the observed puckering parameters [θ = 84.8 (3)° and φ = 308.2 (3)°] deviate slightly from those corresponding to an ideal boat conformation (θ = 90° and φ = 300°). Two stereocentres represented by the C1 and C3 atoms have opposite chirality, i.e.R,S , the substituents at which adopt a trans configuration with respect to the ring, by minimizing repulsive interactions. The bulkier propynyloxymethyl group is located above the ring, i.e. in the axial position with a gauche conformation for the C6—O5—C7—C8 segment, at a dihedral angle of 71.3 (2)°. A similar conformation has been observed in meso-3,6-dipropargyloxymethyl-1,4-dioxane-2,5-dione  returned 25 entries, including different lactide stereoisomers  via a three-step pathway  followed by the etherification with propargyl alcohol to 2-hydroxy-1-(propynyloxymethyl)propanoic acid (4) and the final double esterification of 4 with bromopropyonyl bromide. The final purification of 1 was performed by auto-flash-chromatography on silica, using chloroform as eluent to give, after evaporation under reduced pressure, a white crystalline solid .The desired product Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017008581/ff2148sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017008581/ff2148Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017008581/ff2148sup3.pdfDetails related to the synthesis, purification and characterization techniques used in the study. DOI: Click here for additional data file.10.1107/S2056989017008581/ff2148Isup4.cmlSupporting information file. DOI: 1555000CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: