In this study, modified maifanite (MMF) prepared by acidification, alkalization, salinization, calcination and combined modifications, respectively, were first applied to treat sediment P. The morphology and microstructure of MMF samples were characterized by X-ray fluorescence (XRF), Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM) and Brunauer-Emmett-Teller (BET). Various adsorption parameters were tested, such as dosage of maifanite, time, operation pH and temperature. The adsorption mechanisms were also investigated and discussed. Results showed that CMMF-H2.5-400  exhibited the highest P adsorption capacity. Thus, it was selected as the in-situ adsorbent material to control the internal P loading. Under the optimal conditions of dynamic experiments, the adsorption rates of TP, IP, OP, Fe/Al-P and Ca-P by CMMF-H2.5-400 were 37.22%, 44.41%, 25.54%, 26.09% and 60.34%, respectively. The adsorption mechanisms analysis revealed that the adsorption of P onto CMMF-H2.5-400 mainly by ligand exchange. Results of this work indicated that the modification treatment could improve the adsorption capacity of maifanite, and CMMF-H2.5-400 could be further applied to eutrophication treatment.Sediment phosphorus (P) removal is crucial for the control of eutrophication, and the  Phosphorus (P) is an essential element that can trigger severe eutrophication4. Many recent reports have shown that both nitrogen (N) and P, which are the key limiting nutrients in eutrophication play a dominant role in aquatic ecosystem6. Accordingly, P limitation occurs in spring, while N becomes the limiting factor in summer and fall7. The change in the dual limitation paradigm is because the P concentration in water bodies can be sustained by the release of P from sediments, whereas loss of N through denitrification often occur in warm season8. The release of sediment P, also known as internal P loading, has been recognized that the supply of sediment P to the growth of algae has two phases, which is dominated by the cycling of Iron (Fe)-bound P (Fe-P) and algal degradation. The internal loading of P from sediments plays a primary role in seasonal N limitation for harmful algal bloom (HAB), which in turn can drive the release of sediment P9. Fe is a redox-sensitive element and its redox cycle play a vital role in controlling the P mobility in sediments10. Some recent studies revealed that the formation of anoxic condition at the sediment-water interface during HAB events (HABs), causes the reductive dissolution of Fe (III) oxyhydroxides and the release of Fe-P12. Furthermore, the algal degradation appears during HABs, resulting in the release of P from the degraded algal cells13. In addition, previous studies found that the distribution of soluble reactive P (SRP) and labile P stratified into two layers, called static layer and active layer. In static layer, the concentration of soluble reactive phosphate and labile P remained at a low level, which is the key to sustain the immobilization effectiveness14. The active layer not only retains P liberated from the underlying old sediment, but also draws P out of the overlying fresh sediment, thus reducing P release from the sediment16. More recently, the use of high-resolution dialysis (HR-Peeper) and diffusive gradient in thin films (DGT) techniques for investigating the P mobilization processes in sediments had achieved great progress18.Eutrophication has become a worldwide water quality issue19. Reducing internal P loading in sediments has been proposed as an essential technique for eutrophication control21.The sediment plays a great role in the overall P dynamics of water bodies-it may either act as a sink to adsorb P or an internal source to release P on the base of concentration gradientsex-situ or in-situ techniques. Sediment dredging, which could reduce P from sediment permanently, has been widely applied to control sediment P in lakes23. Nevertheless, dredging may destroy the lake ecosystems and enhance the risk in contaminants resuspension, nor are they cost-effective. Therefore, in-situ techniques for reducing the sediment internal P loading have received increasing attention in recent years.The techniques for reducing the release of sediment P are either in-situ techniques have been applied to reduce internal P loading in sediment, including in-situ capping25, hypolimnetic aeration27, phytoremediation29, and in-situ physicochemical adsorption31, etc. Among these, in-situ physicochemical adsorption turns out to be an efficient technique for controlling eutrophication in lakes. The selection of an effective in-situ adsorbent materials, which should be environmentally friendly, geographically available, and economically feasible, is critical for the in-situ physicochemical adsorption technology. Some natural or modified clay minerals selected as P adsorbent materials have been applied to reduce the sediment internal P loading.Many 33. Maifanite has great surface area and strong adsorption capacity due to the vesicular pores and sponge shaped morphological structure35. It could adsorb P via chemisorption due to the aluminum (Al) and iron (Fe), etc. in the maifanite36. The market price of raw maifanite (RMF) is about 300–400 yuan (¥)/t. Due to its low cost and high capability, maifanite has been applied to soil improvement35, which contains amino acid and some trace elements, and could be further combined with ecological remediation. The adsorption capacity of RMF material is usually limited, attributed to the presence of impurity and the permanent negative charges due to isomorphous substitution effect37. Thus, there is a need to modify RMF to enhance its confined capacity of adsorption.Maifanite is a kind of natural clay minerals with an eco-friendly characteristic, which is abundant in Chinain-situ physicochemical adsorbent materials to adsorb sediment P in all fractions. This study investigated the adsorptive effects of sediment P in all fractions with MMF for the first time. The specific objectives of the study were to explore the optimal modification methods of maifanite applied as adsorbent material and investigate the adsorption effects and mechanisms of MMF on sediment P in West Lake, Hangzhou, China.Here, RMF granules were modified by acidification, alkalization, salinization, calcination and combined modifications, respectively. Modified maifanite (MMF) granules were prepared and tested as promising −1, 1022.29 mg·kg−1, 630.02 mg·kg−1, 494.45 mg·kg−1 and 499.79 mg·kg−1, respectively. This illustrated the internal P loading, which was mainly due to the quantities of Fe/Al-P and Ca-P, in West lake was volatile and at the risk of P release. Additionally, the values of CEC and pH of lake sediments were 52.4 meq/100 g and 7.39, respectively.The quantities of TP, IP, OP, Fe/Al-P and Ca-P in sediment from West Lake were 1652.31 mg·kg−1, 318.35 mg·kg−1, 308.29 mg·kg−1, 311.644 mg·kg−1 and 304.94 mg·kg−1, respectively. The corresponding adsorption rates were 17.24%, 19.27%, 18.66%, 18.86%, and 18.46%, respectively. Based on these results, MMF-H2.5, MMF-OH3.0 and MMF-La5.0 samples were further calcined at 400 °C to obtain the combined MMF samples, named as CMMF-H2.5-400, CMMF-OH3.0–400 and CMMF-La5.0–400, respectively. The adsorption quantities of sediment TP by CMMF-H2.5-400, CMMF-OH3.0–400 and CMMF-La5.0–400 were 331.75 mg·kg−1, 314.99 mg·kg−1, and 321.7 mg·kg−1, respectively. The corresponding adsorption rates were 20.08%, 19.06% and 19.45, respectively. CMMF-H2.5-400 exhibited the highest adsorption capacity. Thus, CMMF-H2.5-400 was selected as the optimal adsorption material in the following experiments.Compared with the RMF, MMF samples exhibited higher adsorption capacities Fig. . The mod2, Al2O3, Na2O, CaO and Fe2O3. The modification treatment resulted in an obvious increase in the content of SiO2 and a significant decrease in MgO and P2O5 of MMF samples. Typically, SiO2 and Al2O3 were the major compositions of these maifanite samples. Compared to the RMF, the content of Na2O in MMF-OH3.0 was increased. It indicated Na ion from 3.0 mol/L NaOH was intercalated in the layers of the RMF after the NaOH modification. This was also confirmed by XRD analyses.The XRF results Table  showed t39. In the spectrum of RMF and MMF samples  and Margarite (CaAl2(Si2Al2)O10(OH)2). Additionally, it is obvious that the d001 value increased after the modification treatment , who reported that the main mineral diffraction peak position of maifanite did not change after calcining at 500 °C45. On the other hand, some peaks were disappeared and weakened a little after the modification treatment, which might be caused by the decrease of impurity after the modification treatment. This was consistent with BET and FTIR results.XRD analyses of various maifanite samples were carried out to discuss the crystal structure and identity. XRD patterns Fig.  revealedrns Fig.  of maifa+, Na+, La3+, respectively, on the MMF-H2.5, MMF-OH3.0, MMF-La5.0, and CMMF-H2.5-400 structures48. Calcination led to microporous, followed by the removal of surface water, bound water and water of hydration of CMMF-400 , the total pore volume (Vt), the volume of micropores (Vmikro), the external surface  and the average pore size (Dp) before and after the modification were given in Table BET and an obvious decrease in the Dp. It could provide more active sites for the adsorption reaction, and made the surface more available for the sediment P. These findings supported the SEM results. The pHPZC of sorbents depended on various factors, including crystallinity nature, Si/Al ration, operation temperature and sorption capacity of the electrolytes, contents of impurity and degree of H+ and OH− ions adsorption, etc. Thus, it might vary from adsorbent to adsorbent. Chutia et al.52 revealed that pHPZC had a significant impact in As(V) adsorption by natural zeolites. That’s because the adsorption of multivalent cation occurred effectively at a pH below pHPZC. Compared to the RMF, the pHPZC values of MMF-H2.5, MMF-OH3.0, MMF-La5.0, CMMF-400 and CMMF-H2.5-400 were increased in different degrees, respectively. On the other hand, The CEC of MMF samples was found to be higher than RMF, and the CEC of CMMF-H2.5-400 was the maximum. The above information revealed that modification treatments could improve the sediment P adsorption capacity of maifanite.Generally, the BET equation has been applied to measure and compare the specific surface areas of a variety of porous materials31. Figure 54. From Fig. 55. The highest adsorption quantities for RMF and CMMF-H2.5-400 were found at 12 g and 2 g, respectively. The adsorption amounts of TP, IP, OP, Fe/Al-P and Ca-P by CMMF-H2.5-400 (2 g dosage) were 444.01 mg·kg−1, 343.44 mg·kg−1, 100.56 mg·kg−1, 195.87 mg·kg−1 and 131.53 mg·kg−1, respectively. The corresponding adsorption rates were 26.87%, 33.60%, 15.96%, 39.61% and 26.32%, respectively. The adsorption amounts of TP, IP, OP, Fe/Al-P and Ca-P by RMF (12 g dosage) were 360.23 mg·kg−1, 289.83 mg·kg−1, 70.41 mg·kg−1, 185.31 mg·kg−1 and 100.53 mg·kg−1, respectively. The corresponding adsorption rates were 21.80%, 28.35%, 11.18%, 37.48% and 20.12%, respectively. Based on the above results, optimal dosages were fixed as 2 g and 12 g, respectively for CMMF-H2.5-400 and RMF and pursued in further investigations.The amount of the sorbent dosage plays a vital role in sediment P adsorption. Experiments were carried out with various dosages of RMF and CMMF-H2.5-400, respectively at 20 ± 2 °C, pH 7.0 ± 0.2 and shaken at 200 rpm for 12 h to investigate the effects of maifanite dosage. Figure To investigate the effects of stirring time on P adsorption by maifanite, the experiments were carried out with 2 g CMMF-H2.5-400 or 12 g RMF at 20 ± 2 °C, pH 7.0 ± 0.2 and shaken at 200 rpm. Figure 56. To investigate the influence of extremely high or low pH value on adsorption of sediment P, the experiments were carried out at different pH conditions ranged from 2.0 to 12.0. Figure opt) 2.0 and decreased with the increasing pH value. The adsorption of TP by RMF increased with the increasing pH (2.0–6.0), then decreased when pH exceeded 6.0 (P < 0.05). The adsorption amounts of TP, IP, OP, Fe/Al-P and Ca-P by CMMF-H2.5-400 at pHopt 2.0 were 604.86 mg·kg−1, 440.62 mg·kg−1, 164.23 mg·kg−1, 126.67 mg·kg−1 and 290.70 mg·kg−1, respectively. The corresponding adsorption rates were 36.61%, 43.10%, 26.07%, 25.62% and 58.16%, respectively. The adsorption amounts of TP, IP, OP, Fe/Al-P and Ca-P by RMF at pHopt 6.0 were 514.38 mg·kg−1, 373.60 mg·kg−1, 140.78 mg·kg−1, 188.83 mg·kg−1 and 161.69 mg·kg−1, respectively. The corresponding adsorption rates were 31.13%, 36.55%, 22.35%, 38.19% and 32.35%, respectively. When the pH value increased from 2.0 to 12.0, the adsorption quantity of Ca-P by CMMF-H2.5-400 and RMF, respectively, decreased immensely (P < 0.05) while the adsorption amount of Fe/Al-P was the highest in alkaline conditions (P < 0.05). These results were agreement with the previous reports57. The effects of P adsorption in various pH were due to a series of mechanisms, including chemical interaction, ligand exchange, electrostatic attraction/repulsion and coagulation/precipitation58.The operation pH is a key factor that controls the P adsorption from sediments60. Shallow lakes are usually isothermal, and the sediment is susceptible to temperature variations61. Thus, the temperature may have a larger influence on adsorption P in sediment from shallow lakes. The results were depicted in Fig. 62. The adsorption amounts of TP, IP, OP, Fe/Al-P and Ca-P by CMMF-H2.5-400 at 20 °C were 614.91 mg·kg−1, 454.03 mg·kg−1, 160.88 mg·kg−1, 129.01 mg·kg−1 and 301.59 mg·kg−1, respectively. The corresponding adsorption rates were 37.22%, 44.41%, 25.54%, 26.09% and 60.34%, respectively. On the other hand, the adsorption amounts of TP, IP, OP, Fe/Al-P and Ca-P by RMF at 20 °C were 521.08 mg·kg−1, 390.36 mg·kg−1, 130.72 mg·kg−1, 194.69 mg·kg−1 and 175.93 mg·kg−1, respectively. The corresponding adsorption rates were 31.54%, 38.18%, 20.75%, 39.38% and 35.20%, respectively. Additionally, the adsorption quantities of P in all fractions decreased in various degrees above 20 °C. The results could be explained that the process of desorption as well as the Brownian movement were enhanced due to the high temperature60.Operation temperature is a significant factor influencing the P adsorption in sediment, which could remarkably improve p releasein-situ treatment, static experiments were carried out with time from 0 d to 30 d. Figure −1, 433.92 mg·kg−1, 134.07 mg·kg−1, 341.30 mg·kg−1 and 85.45 mg·kg−1, respectively. The corresponding adsorption rates were 34.38%, 42.45%, 21.28%, 69.03% and 17.10%, respectively. On the other hand, the adsorption quantities of TP, IP, OP, Fe/Al-P and Ca-P by RMF at 20 d were 457.41 mg·kg−1, 296.53 mg·kg−1, 147.48 mg·kg−1, 224.01 mg·kg−1 and 68.70 mg·kg−1, respectively. The corresponding adsorption rates were 27.68%, 29.01%, 23.41%, 45.31% and 13.75%, respectively. Figure 31.To simulate the P adsorption under 2, Fe2O3, MgO, K2O, TiO2 and P2O5 were increased respectively. However, the contents of Al2O3, CaO, Na2O and P2O5 were decreased respectively. Figure −1, which referred to the O-H bending vibration with Fe (III), Al (III) species present on the surface of CMMF-H2.5-400 after adsorption51. It indicated that Fe (III), Al (III) species were intercalated into interlayers of CMMF-H2.5-400 by adsorption process. Additionally, the peaks of CMMF-H2.5-400 at 3432 cm−1, 1634 cm−1 and 1093 cm−1 were shifted to 3413 cm−1, 1627 cm−1 and 1085 cm−1, respectively. For the RMF, a new adsorption peak emerged at 3568 cm−1, which were attributed to broad OH-stretching in RMF after adsorption. On the other hand, the peaks of RMF at 3430 cm−1 and 776 cm−1 were shifted to 3420 cm−1 and 778 cm−1, respectively. Comparing the Fig. 63. The d001 values of CMMF-H2.5-400 and RMF were 3.73 nm and 3.07 nm, respectively, before adsorption . Table BET. These findings supported the SEM results.Ion-exchange occurred and altered the elements content of CMMF-H2.5-400 and RMF, respectively, during the adsorption process Table . The conon Table 64,65. Fu68. Surface hydroxyl groups are protonated in the ligand exchange process at low pH. That’s because, compared to the hydroxyl groups, -OH2+ is easier to displace from the metal binding sites43. Therefore, it was likely that the adsorption of phosphate onto CMMF-H2.5-400 mainly using ligand exchange. This result was in agreement with our previous study, which reported that MBG adsorbed phosphorus mainly by anionic coordination exchange adsorption31. On the CMMF-H2.5-400 samples, phosphate replaced the hydroxyl groups, which were then released into the solution. The adsorption of phosphate could be speculated to take place as follows: the phosphate in the sediment was first transferred to the sites on the adsorbent; then, chemical complexation/ion exchange occurred at the active sites70.Phosphate is adsorbed onto clay minerals via electrostatic, ligand exchange, and Lewis acid-base interaction63. Meanwhile, the hydroxyl and hydration base could be swop out31.Comparing the SEM images of the CMMF-H2.5-400 before and after adsorption process, we observed an aggregated morphology and some large flakes in CMMF-H2.5-400 samples after adsorption process. Additionally, the size of the intra-particle voids was decreased due to a stacking structure formed by some thin lamellas  is located on the western side of Hangzhou City, China. West Lake, which has been listed in the World Heritage Site in 2011, is a typical eutrophic lake with an area of 6.5 km4+-N and NO2-N of overlying water above sediments in the lake was 0.06 ± 0.01 mg/L, 3.57 ± 0.03 mg/L, 0.15 ± 0.02 mg/L and 0.12 ± 0.01 mg/L, respectively. The pH values of the interstitial water and overlying water were 8.07 and 7.9, respectively.The sampling site is located in a severe eutrophic region  in Xiaonan Lake, one sub lake of West Lake. The surface lake sediments, at a depth of 0–10 cm, were collected by a Peterson grab sampler (model HNM1-2) on May 14th, 2017. The sediment samples were then stored in plastic bags. After transportation to the laboratory, the sediment samples were air-dried. The content of TP, TN, NHRMF  granules were purchased from Kangyuan maifanite factory in mengyin country, Shandong Province, China. Firstly, the RMF granules were soaked in deionized water for 10 h and then washed with deionized water until the pH value reached 7.0. After that, the samples were dried at 105 °C for 24 h. Finally, the samples were cooled to room temperature for further studies, which were used as RMF. To improve the sediment P adsorption capacity of RMF, acidification, alkalization, salinization, calcination and combined modifications were used to modify the RMF granules, respectively. The preparation of various MMF samples was listed in Table To explore the optimal modification method of RMF, 5 g sediment samples and 8 g various MMF samples were added to Erlenmeyer flasks, containing 250 mL 0.02 mol/L KCl solution with pH 7.0 ± 0.2, then shaken at 200 rpm for 12 h in a thermostatic shaker  at 20 ± 2 °C. After the adsorption process, the mixture was filtered through a steel screen (60 mesh) to remove maifanite granules. The sediment was then dried in an oven at 55 °C for 24 h. Each treatment consisted of three replicates.Dynamic adsorption experiments were carried out in thermostatic bottle shakers. Typically, 250 mL KCl solution (0.02 mol/L) was poured into Erlenmeyer flasks, and then 5 g sediment samples and different dosages  of maifanite samples  were added into the Erlenmeyer flasks. After that, they were shaken at 200 rpm for certain hours  at different pH (2.0–12.0). The dynamic adsorption experiments were carried out at temperatures of 5 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, and 60 °C. Two drops of 0.1% chloroform were added in each Erlenmeyer flask to inhibit bacterial activity.Static adsorption experiments were carried out in Erlenmeyer flasks with pH 7.0 ± 0.2 in the dark condition for 0–30 d at 20 ± 2 °C. A certain quantity of maifanite  and 5 g sediment samples were added to Erlenmeyer flasks. Two drops of 0.1% chloroform were added in each Erlenmeyer flask to inhibit bacterial activity.72 were used for determining P fractions. The P fractions can be characterized as TP , IP (inorganic P), OP (organic P), Fe/Al-P  oxides extracted by NaOH) and Ca-P  extracted by HCl). The presence of metals in sediment can mediate the transport of P. Each P fraction concentration was measured directly using the ammonium molybdate spectrophotometric method with an UV-visible spectrophotometer at 700 nm . The cation exchange capacity (CEC) of the sediment samples was analyzed using NH4Cl-NH4OAc method. Sediment pH was measured in 1:10 (w/v) solid/water suspensions by a PHS-3C digital pH meter .The Standards and Measurements and Testing (SMT) protocol−1. The mineralogical and chemical compositions of the lake RMF and MMF samples were determined by X-ray diffractometer  operating with CuKα radiation  over the range (2 Theta) of 5–70°. The microstructure of RMF and MMF samples was characterized by scanning electron microscope . The CEC of maifanite samples, which was defined by the ability of maifanite granules to adsorb the cations, was analyzed using the ammonium acetate method. The specific surface areas were calculated by nitrogen adsorption using the Brunauer-Emmett-Teller (BET) equation on an analyzer . The t-plot method was applied to gain the volume of micropores and the surface of mesopores together with the external surface. The total pore volume was derived from the nitrogen volume adsorbed at the relative pressure p/p0 → 1 73. The batch equilibrium techniques were applied to estimate the points of zero charge (pHpzc) of maifanite samples52.The chemical compositions of RMF and MMF samples were determined by X-ray fluorescence . Fourier transform infrared (FTIR) spectra of the RMF and MMF samples were measured by a FTIR spectrometer  in the wavenumber range of 400–4000 cmAll the chemicals and reagents used were analytical grade. All glassware and sample bottles were presoaked before use in diluted HCl solution for at least 12 h followed by washing with deionized water and drying in oven. Deionized water was used for preparing solutions.A) were calculated using the following equations:q is the adsorption quantity of P per unit weight of maifanite samples (mg·kg−1), and q0 and qe (mg·kg−1) are the initial and final P quantity, respectively.The amount of P adsorbed on the maifanite granules and the adsorbed efficiency  was used to plot various figures. All statistical analyses were estimated using SPSS 18.0 . Analyses of the variance  were applied to test the significant differences between the dependent variables (the adsorption quantity of P) and independent variables (the corresponding adsorption parameter). The difference was considered statistically significant when the significance level was smaller than 0.05.


The Sm doping level varied between 1 mol % to 8 mol %. Processing parameters, such as sintering temperature and annealing temperature, were optimized to obtain high-quality samples. Based on their dielectric properties, the optimum sintering and annealing temperatures were found to be 300 °C and 825 °C, respectively. Leakage-free square-shaped ferroelectric hysteresis loops were observed in all samples. The remnant polarization was maximized in the 5 mol %-doped sample (~35 μC/cm2). Furthermore, remnant magnetization was increased after the Sm doping and the 8 mol%-doped sample possessed the largest remnant magnetization of 0.007 emu/g. Our results demonstrated how the modified solid-state-reaction method proved to be an effective method for preparing high-quality BiFeO3 ceramics, as well as how the Sm dopant can efficiently improve ferroelectric and magnetic properties.Sm-doped BiFeO The material shows great potential in applications of information storage, spintronics, and sensors [2 along the [001]hex direction [In the past decade, considerable attention has been devoted to the study of multiferroics BiFeO sensors . Moreove sensors . BFO has sensors ,2,3. Theirection . Howeverirection ,5. Recenirection . In this0.88Sm0.12FeO3) and found that the synthesis methods considerably affects the crystal structures as well as the ferroelectric properties [0.9Sm0.1FeO3) using a sol-gel routine and found that the sample was too leaky to obtain reasonable ferroelectric hysteresis loop [1-xSmxFeO3, x = 0.05, 0.075, 0.1, 0.125, 0.20, 0.25) using a conventional solid-state method and found that the rhombohedral phase favors  the piezoelectric properties [Walker et al. used conventional solid-state and mechano-chemical activation-assisted solid-state synthesis methods to prepare Sm-doped BFO ceramics  or non-magnetically-active ions  [3+ site. Meanwhile, the antiferromagnetic Néel temperature will be decreased by the doping [Another issue concerning the application of BFO material is the zero remnant magnetization (Mr) as a result of the antiferromagnetic spin ordering. This is because for the magnetoferroelectric (ME) applications, the Mr is a must for the ME coupling to occur ,13,14,15 Al, Zn) ,20,21,22e doping . Herein,3+ possesses smaller ionic radius compared to La3+. Kan et al. found that a smaller ionic radius at the Bi-site of the rare-earth doped BFO will induce antiferroelectric-like double-hysteresis P–E loops [It was previously found that the La-doped BFO undergoes structural transformation from rhombohedral to triclinic or orthorhombic around 10 mol % ,11. Sm3+–E loops . Moreove–E loops . Tao et –E loops . Karimi –E loops . This wa–E loops . 1−xSmxFeO3 (x = 0.15–0.18) ceramics and verified the existence of three different phases: ferroelectric R3c, antiferroelectric Pnam, and paraelectric Pnma [R3c phase. Additionally, in this doping range the electrical properties of the doped BFO ceramics, for example the piezoelectric coefficients, were better than those heavily doped [Arnold et al. reviewed the structural phase transitions in rare-earth doped BFO materials and found that average Bi-site ionic radii are the primary driving forces for phase transitions, and that synthesis methods considerably account for the controversy in the literature . Yang etric Pnma . Howeverric Pnma . Based oly doped . The comTherefore, in current study the doping level was set to be 1 mol %, 3 mol %, 5 mol %, and 8 mol %  in order to rule out the lattice structure effects. There were two effects expected for the Sm dopants: first, that it would substitute the bismuth ions; second, that it may partially destroy the antiferromagnetic cycloidal spiral structure which may result in produce remnant magnetization ,30. Our 2O3 and Fe2O3 (purity: 99.9%) were weighed and ball-milled  in ethanol alcohol for 24 h. The mixture was calcined  at 650 °C for 5 h in air followed by furnace cooling. The calcined powders were ground and ball-milled again for 48 h together with the additive of Sm2O3 (purity: 99.99%). The amount of Sm2O3 was 1 mol %, 3 mol %, 5 mol % and 8 mol % relative to the BFO raw powders. Then, polyvinyl alcohol (PVA) was added into the dried powders as a binder. The powders were pressed  into disks with a diameter of 10mm. After burning the binder at 650 °C for 2 h, the samples were sintered. The sintering temperature varied between 815–845 °C, with an interval of 5 °C. During the sintering process, we adopted a much higher heating/cooling rate  than the conventional method (~2 °C/min), as shown in Stoichiometric amounts of high purity BiX-ray diffractometer  with a Cu Kα source and field-emission scanning electron microscope  were used to study the crystal structures and microstructures, respectively. The Raman scattering spectroscopy  were taken at room temperature with backscattering geometry. For electrical measurements, the samples were thinned to 0.3~0.4 mm plates and silver electrodes (2 mm in diameter) were fired on both sides The dielectric properties were measured on an HP4294A impedance analyzer . The ferroelectric hysteresis loops were taken on a modified Sawyer-Tower circuit  and the measurement frequency was set to 100 Hz. A sinusoidal waveform  was used for the applied electric field. No pre-poling was applied. A vibrating sample magnetometer  was used to characterize the magnetic properties.2O3 was around 830 °C [2O3. d 830 °C , we attr2Fe4O9 and Bi25FeO39 [The liquid phase bismuth oxide facilitated the BFO phase formation and produced a dense microstructure. However, increasing the sintering temperature led to the bismuth loss and bismuth vacancies, as well as oxygen vacancies ,5,6. Suci25FeO39 . Furtheri25FeO39 . In currIn order to further improve the dielectric property, the 825 °C-sintered samples were annealed in air at different temperatures ranging from 10–500 °C with an interval of 100 °C. The dielectric constant and dielectric loss after annealing are shown in Based upon the dielectric measurements results, the optimum processing conditions can be summarized as below: sintering at 825 °C for 30 min followed by annealing at 300 °C for 5 h. The calcination temperature did not show prominent effects on the phase formation as well as properties of BFO ceramics.The frequency dependence of dielectric constants and losses of samples prepared under the optimized conditions are shown in 0.9Sm0.1FeO3) were around 125 and 0.40, respectively [1−xSmxFeO3, x = 0.05, 0.075), the dielectric constant and dielectric loss are around 60 and 0.01, respectively [0.85Sm0.15FeO3), the dielectric constant and dielectric loss are 150 and 0.01, respectively [The dielectric properties of the samples prepared by this method were better than those of the Sm-doped BFO ceramics prepared by normal solid-state method or sol-gel method , and comparable to those of the samples prepared by spark plasma sintering method. The dielectric constant and dielectric loss, at 1kHz, for the sol-gel derived Sm-doped BFO ceramics (Biectively . Furtherectively .The SEM plane-views of the 825 °C-sintered samples are shown in 25FeO39 (indicated by arrows in the figure) was observed. No traces of Sm2O3, Fe2O3, or SmFeO3 were observed. This suggests that Sm3+ has been dissolved into the lattices of BFO to form a normal rhombohedral structure. The diffraction peaks shift to higher 2θ values as the Sm dopant level was increased. For example, the major peak (110) shifts from 31.84° for Sm1 sample to 31.95° for Sm8 sample, as shown in 3+ (0.958 Å) is smaller than that of Bi3+ (1.030 Å). In order to further confirm this, we did the Rietveld refinement using the MAUD software [R3c space group) was used for the fitting [WP(%) = 25.93. The large values of Rwp comes from the signals of the secondary phase (Bi25FeO39), as indicated in R3c). The refinement results for other Sm-doped samples exhibit very similar behavior. The calculated lattice constant-a/c were obtained from the Rietveld refinement, which are 5.5829 Å/13.8568 Å, 5.5820 Å/13.8571 Å, 5.5818 Å/13.8517 Å, and 5.5806 Å/13.8443 Å for the Sm1, Sm3, Sm5, and Sm8 samples, respectively, as plotted in 3, 387.5 Å3, 387.9 Å3, and 385.5 Å3 for the Sm1, Sm3, Sm5, and Sm8 sample, respectively. The refinement results confirmed that the Sm dopant leads to the decrease of the lattice constants (a and c) as well as the unit cell volume.The XRD θ–2θ diffraction patterns of these samples were shown in software  and ICSD fitting ,9. The r−1, 170 cm−1, and 219 cm−1, denoted as A1-1, A1-2, and A1-3 in −1/170.1 cm−1/218.6 cm−1 to 141.4 cm−1/173.2 cm−1/229.9 cm−1 for Sm1 and Sm8 samples, respectively. This frequency shift confirms that the Sm substitutes for Bi in the BFO samples: since the Sm element (atomic weight: 150.36) is lighter than Bi (atomic weight: 208.98), Sm doping will decrease the effective mass and increase the frequency of the normal modes  [The Raman scattering spectra are shown in onstant) .r) was increased from 10.5 μC/cm2 for Sm1 sample to 16.1 μC/cm2 for Sm3 sample, and maximized at 28.0 μC/cm2 in the Sm5 sample, then slightly decreased to 26.3 μC/cm2 as the doping level further increased to 8 mol %. The coercive fields (Ec) also exhibit such trend, changing from 69.0 kV/cm, 80.0 kV/cm, 97.3 kV/cm to 95.6 kV/cm for Sm1, Sm3, Sm5, and Sm8, respectively. The inset in r and Ec. Moreover, the Pr can be further increased up to 35.0 μC/cm2 (Ec = 98.0 kV/cm) in the Sm5 sample as the sintering temperature increased to 835 °C. The ferroelectric polarization vs. electric field (P–E) hysteresis loops of the 825 °C-sintered samples are shown in In the Sm1–Sm5 samples, the hysteresis loops under lower electric field (<120 kV/cm) showed resemblance of antiferroelectric (AFE) double-hysteresis loops  will lead to frozen defects and suppress the double-hysteresis behavior. Meanwhile, the second step  will lead to the migration/redistribution of the defects and cause a domain-wall pinning, favoring the double-hysteresis behavior. Therefore, these two competing mechanisms will account for the observed hysteresis shown in Based upon the above discussions, it can state that the Sm dopants may suppress the migration/redistribution of the defects, and hence the absence of the double hysteresis loops in the Sm8 sample. This hypothesis can be partially supported by the dielectric constants/losses shown in m) of 18 kOe. The remnant magnetization (Mr) was increased from 0.002 emu/g to 0.007 emu/g, as the Sm doping level increased from 1% to 8%, as shown in the inset of c) of the Sm8 sample is 0.8 kOe. The observation of the remnant magnetization in the doped BFO is believed to be due to the collapse the space modulated antiferromagnetic spin structure [3+ ions) with a long period of 620 ± 20 Å [3+–O–Fe3+ bond will bend due to the structure distortion, and, the antiferromagnetic ordering is then corrupted. It was shown that the remnant magnetization was increased up to 1emu/g in the doped BFO ceramics with a seriously-distorted rhombohedral structure [3+ ion is magnetically-active, and it can also be a possible source of the remnant magnetization. The remnant magnetization and polarization is both necessary for ME applications. Bearing the considerably improved ferroelectric and magnetic properties as discussed above, the Sm-doped BFO material shows promise for the ME applications.tructure ,20,21,220 ± 20 Å  and the tructure . Besides2). The remnant magnetization appears after Sm doping and the Sm8 shows the largest Mr of 0.007 emu/g with a coercive field of 0.8 kOe. It was found that Sm-dopant can improve the dielectric, ferroelectric, and magnetic properties of BFO ceramics.Sm-doped BFO ceramics with low doping levels  were obtained using a modified conventional solid-state-reaction method. The preparation procedure was modified with a fast heating (30 °C/min) and cooling process (100 °C/min). The sintered samples were annealed in air for 5 h at temperatures from 100 °C to 500 °C. Based upon their dielectric properties, the optimum sintering and annealing temperatures were found to be 825 °C and 300 °C, respectively. The Sm1 sample exhibits the lowest dielectric loss <0.01 at frequencies from 1 kHz to 3 MHz. Leakage-free and saturated ferroelectric P–E loops were observed in all samples. The remnant polarization was maximized in the 835 °C-sintered Sm5 sample (~35 μC/cm


Terminalia chebula, native to Southeast Asia, is a popular medicinal plant in Ayurveda. It has been previously reported to have strong antioxidant and anti-inflammatory efficacy. In this study, we aimed to investigate if fruit extract from T. chebula might protect neuronal cells against ischemia and related diseases by reduction of oxidative damage and inflammation in rat pheochromocytoma cells (PC12) using in vitro oxygen-glucose deprivation followed by reoxygenation (OGD-R) ischemia and hydrogen peroxide (H2O2) induced cell death. Cell survival was evaluated by a 2--2,5-diphenyltetrazolium bromide (MTT) assay. Free radical scavenging, lipid peroxidation and nitric oxide inhibition were measured by diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA) and Griess reagent, respectively. We found that T. chebula extract: (1) increases the survival of cells subjected to OGD-R by 68%, and H2O2 by 91.4%; (2) scavenges the DPPH free radical by 96% and decreases malondialdehyde (MDA) levels from 237.0 ± 15.2% to 93.7 ± 2.2%; (3) reduces NO production and death rate of microglia cells stimulated by lipopolysaccharide (LPS). These results suggest that T. chebula extract has the potential as a natural herbal medicine, to protect the cells from ischemic damage and the possible mechanism might be the inhibition of oxidative and inflammatory processes. Natural antioxidants, from medicinal or edible plants, have recently received much attention as promising agents for reducing the risk of oxidative stress-induced neurological diseases ,2. ReactTerminalia chebula Retz. (Combretaceae), commonly known as chebulic myrobalan, is a popular medicinal plant in the Ayurvedic system of medicine. The main compounds from T. chebula, such as gallic acid, ellagic acid, chebulanin, chebulinic acid, rutin casuarinin, and quercetin, were reported to possess antioxidant effects [T. chebula has been used to treat kidney and urinary disorders, nervous disorders, colic pain, chronic cough, sore throat, asthma, etc. It is also used as laxative, antitussive, diuretic, digestive, antidiabetic, and as a cardiotonic remedy [T. chebula has been reported to exhibit anticancer [T. chebula is also reported to possess strong anti-inflammatory activity [ effects ,8. Tradic remedy ,10. T. cticancer , antidiaticancer , antimutticancer , antibacticancer  and cardticancer . T. chebactivity .T. chebula extract may have protective effects on neuronal cell death against ischemia and related diseases. In this study, we aimed to investigate the protective effect of T. chebula extract against OGD-R and hydrogen peroxide (H2O2) induced cell injury on the rat pheochromocytoma cell line (PC12 cells). The reason for choosing PC12 cells is that, they display phenotypic characteristics of adrenal chromaffin cells and sympathetic neurons and therefore, PC12 cells are a well-known model for neuronal differentiation [in vitro ischemia model [T. chebula extract. H2O2 induced cell death; an extensively used in vitro oxidative stress model [T. chebula extract in PC12 cells. Furthermore, the activation of the primary microglia is one of the major reasons of cellular inflammation in ischemic condition and lipopolysaccharide (LPS) activated BV2 cells are reported as the suitable alternatives for primary microglia in the culture or animal experiments [T. chebula extract on LPS activated microglia cells (BV2 cell). We also determined the diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, lipid peroxidation and nitric oxide (NO) inhibition activity of T. chebula extract.Based on the information regarding antioxidant and anti-inflammatory effects, we hypothesized that fruit of ntiation ,18. OGD-ia model ,20, was ss model ,22, was eriments . TherefoT. chebula extract is shown in T. chebula extract were gallic acid and ellagic acid.The 3D HPLC spectrum of T. chebula extract showed dose dependent radical scavenging activities with a maximum of 96% inhibition against DPPH radical at 100 μg/mL. The effective concentration of T. chebula extract to scavenge 50% of DPPH free radical (EC50) was found as 5.5 ± 1.1 μg/mL. Ascorbic acid (EC50 of 4.7 ± 0.01 μg/mL) was used as a positive control. T. chebula extract.T. chebula extract was measured by an MTT assay. T. chebula extract (at 0.1–10 μg/mL) didn’t show any cytotoxic effects on PC12 cells . To examine whether T. chebula extract protects against OGD-R induced cell death, PC12 cells were incubated without glucose solution in the hypoxia chamber for 4 h followed by 24 h of reoxygenation with glucose solution in normal incubator. Different concentrations of T. chebula extract were treated 30 min before and during 4 h OGD. Cell viability in OGD group was 50.4 ± 1.5% whereas cell viability in T. chebula extract treated cells  were 62.4 ± 2.2% (p < 0.05), 68.0 ± 2.0% (p < 0.01), and 54.8 ± 1.2% (p < 0.05) respectively, as compared to the control group (set 100%). Baicalein at 0.27 μg/mL (1 μM) treated cells showed 74.0 ± 2.8% (p < 0.001) cell viability against OGD-R . Cell viability in the H2O2 (200 μM) treated group was 70.4 ± 1.5% and that in T. chebula extract treated cells  was 80.6 ± 3.6% (p < 0.05), 91.4 ± 3.6% (p < 0.005), and 86.9 ± 4.4% (p < 0.01) respectively, as compared to the control group (100%) (p < 0.05) cell viability against H2O2. Previous study [T. chebula at 0.5 μM showed 100.8 ± 9.2% (p < 0.01) cell viability.To determine whether p (100%) A. BaicalT. chebula extract can inhibit the formation of MDA induced by H2O2. The cells treated with H2O2 significantly increased MDA levels as compared with control cells (set 100%). Pretreatment of T. chebula extract at 0.1 μg/mL and 1 μg/mL significantly reduced MDA level from 237.0 ± 15.2% in the H2O2 treated group to 115.8 ± 5.8% (p < 0.001) and 93.7 ± 2.2% (p < 0.001) respectively , was found as 68.7 ± 2.0% whereas T. chebula extract  treated cells showed viability of 76.8 ± 4.9%, 78.2 ± 2.2% (p < 0.05), and 87.1 ± 4.3% (p < 0.01) respectively (p < 0.001) in LPS treated cells, and 16.6 ± 0.7 μM, 10.0 ± 0.7 μM (p < 0.001) and 7.2 ± 0.9 μM (p < 0.001) in T. chebula extract  treated cells, respectively (p < 0.001) cell viability and 8.7 ± 1.2 μM (p < 0.001) NO release in BV2 cells.In order to elucidate the effect of ectively A. The amectively B. CurcumT. chebula significantly reduced PC12 cell death induced by 4 h of OGD followed by 24 h of reoxygenation and 24 h exposure of H2O2. T. chebula extract showed the strong scavenging activity against DPPH free radicals and markedly reduced the H2O2-induced MDA production. T. chebula extract also reduced LPS induced cell death and inhibited the production of NO by BV2 cells in a dose dependent manner.In the present study, fruit extract of etc. [As OGD-R mimics tissue ischemia, it results in the production of ROS, release from glutamate toxicity, and inflammatory cytokines ,26. Geneetc. . Once thetc. . Among setc. ,31,32.T. chebula extract significantly protected the PC12 cells against OGD-R. The exposure of PC12 cells to 4 h of OGD followed by 24 h of re-oxygenation resulted decrease in cell viability as compared to control (non-OGD) cells. Incubation of PC12 cells with T. chebula extract, 30 min before and during OGD period, led to significant increase in cell viability. It is reported that, OGD mediates the oxidative stress in the cells by activating the different oxidative cascades such as activation of lipoxygenase, IκB kinase, nuclear factor-κB, and OGD-R is the major source of ROS in ischemia [T. chebula extract markedly scavenged the nitrogen-centered free radical DPPH (by 96%) in a dose dependent manner, similar to that of ascorbic acid, so the potential nature of antioxidant activity of T. chebula extract might be the key factor to sequester the ROS, that are generated in the ischemic cascades, like OGD-R.ischemia ,34. In oT. chebula extract (70%) against H2O2 induced PC12 cell death, was found to be significantly higher than that of the positive control, baicalein (46.3%). H2O2 is not a free radical and has a limited reactivity; however, it is the major precursor of the highly reactive hydroxyl radical. Previous studies have shown a close association between H2O2 and neurodegenerative disease, and it has been suggested that H2O2 levels are increased during pathological conditions, such as ischemia [2O2, readily damage biological molecules that can eventually lead to apoptotic or necrotic cell death [T. chebula extract after H2O2 induced cell death was higher in less concentration of extract than that in higher concentration .The maximum protective effect of ischemia ,36. ROS,ll death . In our ntration A. The metrations ,39. ThisT. chebula extract markedly scavenged the nitrogen-centered free radical DPPH in a dose dependent manner. From HPLC analysis, it was observed that main phenolic compounds in T. chebula are gallic acid and ellagic acid, which are well-known natural antioxidants [T. chebula is mostly related to the hydroxyl group in gallic acid and ellagic acid.oxidants . Plant poxidants . The DPP2O2 treatment was significantly inhibited by T. chebula. H2O2 is believed to be the major precursor for highly reactive free radicals, expressed indirectly by the increase in MDA levels, which is one of the most important organic expressions of oxidative stress in various neuronal diseases including ischemia [T. chebula against ischemia and H2O2 induced cell death.Our results showed that MDA production during Hischemia . Oxidatiischemia . Previouischemia ,44. TherT. chebula extract inhibited the accumulation of NO by 76.2%, at 10 μg/mL. LPS activated BV2 cells release NO in the culture medium which is recognized as a mediator and regulator in pathological reactions, especially in acute inflammatory responses [T. chebula extract significantly inhibited NO release by LPS activated BV2 cells, elucidating that it can inhibit the activation of microglia. Therefore, anti-inflammatory effect through the inhibition of NO release could be another possible protective mechanism of T. chebula against ischemia.esponses . In addiesponses ,47,48. Tesponses , a potenesponses . Activatesponses . T. chebT. chebula and its active compounds gallic acid and ellagic acids are previously reported to inhibit cytochrome p450 enzyme and protect against mitochondrial dysfunction [T. chebula treatment significantly increased the level of Cytochrome C oxidase and other mitochondrial enzymes. Gallic acid down-regulates the protein expression and activity of caspase-3, an essential effector molecule in the course of programmed cell death [T. chebula is also reported to inhibit inducible nitric oxide synthesis by decreasing iNOS protein and iNOS mRNA levels suggesting the possible intercellular mechanism of T. chebula extracts and its active constituents [T. chebula extract against OGD-R induced PC12 cell death and the inhibition of activated microglia must be due to the antioxidant and anti-inflammatory potential of T. chebula extract and its polyphenols.Besides these, function ,53. T. cll death . T. chebtituents . Taken tT. chebula were collected from Pokhara, Nepal after proper identification by the experts. A voucher specimen (HP203) was deposited in the Herbarium of the Kyung Hee University, College of Oriental Medicine, Seoul, South Korea. Dulbecco’s modified Eagle’s Medium (DMEM), LPS, Escherichia coli (0127:138), 3--2,5-diphenyltetrazolium bromide (MTT), 2,2'-, DPPH, gallic acid, ellagic acid, thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), dimethyl sulfoxide (DMSO) and Griess reagent were all purchased from Sigma Chemical Co. , Fetal Bovine Serum (FBS), penicillin and streptomycin were obtained from Gibco BRL . All reagents and solvents used were of HPLC grade, unless specified.The fruits of T. chebula fruit powder (100 g) was extracted under reflux with 70% methanol (1000 mL). The extract was evaporated with a rotary evaporator under reduced pressure to remove the organic solvent and then lyophilized until dryness. A stock solution of extract was prepared in DMSO and deionized water, respectively. Final DMSO concentration did not exceed 0.1% (v/v) in all treatments.T. chebula was analyzed by the HPLC with a 600 pump  using 250 mm × 4 mm HypersilTM Gold C18 column , at a flow rate of 1 mL/min under the following conditions: solvent A (0.5% v/v H3PO4) and solvent B (CH3CN); a linear gradient from 5% to 50% v/v of solvent B in A for 60 min. The isolated compounds were monitored with photodiode array detector . Content of major compounds was calculated by comparing with the standard compounds.Methanol extract of T. chebula extracts were determined by the colorimetric assay as described previously [384, CA, USA) at 725 nm. Gallic acid (10–500 mg/L) was used for calculation, and the results were expressed as mg of gallic acid equivalents (GCE) per gram of extract.Free radical scavenging activity was measured spectrophotometrically by DPPH free radical scavenging assay  using aseviously  with som2. PC12 cells were seeded at a density of 1.5 × 103 cells/well whereas BV2 cells were seeded at a density of 5 × 104 cells/well and cultured at 37 °C in DMEM is supplemented with 10% heat-inactivated FBS, penicillin (1 × 105 U/L) and streptomycin (100 mg/L) in a 5% CO2 incubator.PC12 cells and BV2 cells were obtained from the Medical Department, Kyung Hee University, Seoul, Korea, and maintained at 37 °C in a humidified atmosphere containing 5% COT. chebula extract  and incubated for 28 h. For OGD injury, PC12 cells were first washed with phosphate buffer saline  then with glucose-free DMEM. The cultures were then placed in fresh glucose free DMEM and kept in a hypoxic chamber  containing 95% N2 and 5% CO2 for 4 h. At the end of the exposure period, glucose solution was added, and the cells were incubated (5% CO2 at 37 °C) for an additional 24 h. Samples of different concentrations were treated 30 min before and during OGD exposure. The control group was treated with the same amounts of serum free glucose DMEM and incubated. Baicalein, a potent antioxidative and neuroprotective compound [Oxygen-glucose deprivation followed by re-oxygenation experiments were carried out 24 h after seeding the cells. For the measurement of cytotoxic effects, PC12 cells were treated with compound  was used2O2 treatment, PC12 cells were washed with FBS free DMEM. Dilution of H2O2 was made from a 30% stock solution into DMEM just prior to each experiment and 200 μM solution added to the cells. The culture plates were incubated for an additional 24 h. Samples of different concentrations were treated 2 h before and during H2O2 exposure. Control group (without H2O2) was incubated under the same conditions.For H2O2 (200 μM) in the presence or absence of T. chebula extract (0.1 and 1 μg/mL), cultures were washed with ice-cold PBS, pooled in 0.1 mol/L PBS-5% Triton X-100 buffered solution, and incubated for about 1 h at 37 °C. Trichloroacetic acid  was added to 250 μL of cellular lysate and centrifuged . Aliquots (450 μL) of supernatant were mixed with equal volume of 0.5% (w/v) TBA. The mixture was boiled at 100 °C for 30 min. After cooling MDA formation was measured at 520 nm and the results were expressed as a percentage of the control group.Malondialdehyde (MDA), the most abundant lipid peroxidation product from PC12 cells, was measured using the TBA colorimetric assay. Briefly, 24 h after the treatment of the cells with HT. chebula extract, BV2 cells were incubated with/without 1 μg/mL of LPS, in the absence or presence of different sample solutions for 24 h. For the measurement of NO produced by activated cells, 90 μL of the supernatant from the BV2 cells was mixed with equal volume of the Griess reagent. The OD was measured at 540 nm. Curcumin, a potent anti-inflammatory compound [For LPS induced cell death and nitric oxide inhibitory assay of compound  was usedCell viability was evaluated by observing the ability of viable cells to reduce yellow colored MTT to purple colored formazan. MTT was dissolved in DMEM and added to the culture at a final concentration of 0.5 mg/mL. After an additional 2 h of incubation, the media was carefully removed and 100 µL DMSO was added to each well. The OD was measured on a plate reader at 570 nm. Results were expressed as a percentage of the control group.post hoc test using GraphPad Prism 4 . p < 0.05 is considered as statistically significant for the experimental analysis. Each experiment was performed at triplicate.All results are presented as mean ± S.E.M. Significant differences between experimental groups were determined by using one way ANOVA followed by the Tukey’s T. chebula extract showed a protective effect against OGD-R, H2O2 and LPS induced cell death. T. chebula has an antioxidative and anti-inflammatory activity which could be the possible mechanism for the protection of cells against ischemia. Our results suggest that T. chebula fruit could be the useful remedy for ischemia, oxidative stress and activated microglia-induced secondary damage, which occurs in many neurodegenerative diseases. The exact protective mechanism of T. chebula extract against OGD-R is unknown, however, it can be estimated that the protective effect of T. chebula might be due to its antioxidative and anti-inflammatory effects. Further study is necessary to evaluate the protective effect of T. chebula extract on in vivo ischemia.In conclusion,


Bandeiraea simplicifolia isolectin B4 (IB4)-binding neurons amounted to ∼61%, ∼57%, and ∼31% of DRG neurons IR for the InsR. Of the IB4-positive population only neurons expressing the InsR were responsive to insulin. In contrast, TRPV1-IR nociceptive and CGRP-IR peptidergic neurons showed increased tendency for neurite outgrowth which was further enhanced by insulin. However, the responsiveness of DRG neurons expressing the InsR was superior to populations of DRG neurons which lack this receptor. The findings also revealed that besides the expression of the InsR, inherent properties of peptidergic, but not non-peptidergic nociceptive neurons may also significantly contribute to the mechanisms of neurite outgrowth of DRG neurons. These observations suggest distinct regenerative propensity for differing populations of DRG neurons which is significantly affected through insulin receptor signaling.Apart from its pivotal role in the regulation of carbohydrate metabolism, insulin exerts important neurotrophic and neuromodulator effects on dorsal root ganglion (DRG) neurons. The neurite outgrowth-promoting effect is one of the salient features of insulin’s action on cultured DRG neurons. Although it has been established that a significant population of DRG neurons express the insulin receptor (InsR), the significance of InsR expression and the chemical phenotype of DRG neurons in relation to the neurite outgrowth-promoting effect of insulin has not been studied. Therefore, in this study by using immunohistochemical and quantitative stereological methods we evaluated the effect of insulin on neurite outgrowth of DRG neurons of different chemical phenotypes which express or lack the InsR. Insulin, at a concentration of 10 nM, significantly increased total neurite length, the length of the longest neurite and the number of branch points of cultured DRG neurons as compared to neurons cultured in control medium or in the presence of 1 μM insulin. In both the control and the insulin exposed cultures, ∼43% of neurons displayed InsR-immunoreactivity. The proportions of transient receptor potential vanilloid type 1 receptor (TRPV1)-immunoreactive (IR), calcitonin gene-related peptide (CGRP)-IR and  Insulin, besides being essential in the regulation of glucose homeostasis, is involved in several neuronal processes such as neural survival and neurite outgrowth in both the central and the peripheral nervous system . InsulinBandeiraea simplicifolia isolectin B4 (IB4)-binding non-peptidergic neurons  . These n neurons . It has  neurons .Recent findings indicated that insulin may modulate the activation of nociceptive sensory neurons through indirectly opening the TRPV1 . Immunohin vivo  were terminally anesthetized and DRGs were removed under aseptic conditions. DRGs were collected in a culture medium  containing 1 mM l-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin, and 300 mg/dL glucose  and 4% Ultroser G  without insulin. DRG neuron cultures were prepared as described previously (2 incubator. DRGs were triturated with a fire-polished Pasteur pipette. The cell suspension was spun through 21% Percoll® (Sigma-Aldrich). Following re-suspension in the supplemented culture medium, DRG cells were plated on poly-DL-ornithine (Sigma-Aldrich) coated glass coverslips and kept at 37°C in a 5% CO2 atmosphere. Insulin (Sigma-Aldrich) was administered to the culture medium at concentrations of 10 nM and 1 μM for 48 h. The concentrations of insulin applied in the present experiments are similar to those found in commonly used serum-free media  was performed.Adult male Wistar rats weighing 300–350 g  for 10 min and then washed twice in phosphate-buffered isotonic NaCl solution . In single and multiple labeling experiments, the following primary antibodies and lectins were used: mouse anti-β3-tubulin , rabbit polyclonal anti-InsRα subunit IgG , polyclonal guinea pig anti-TRPV1 IgG , polyclonal goat anti-CGRP IgG  and biotin-conjugated IB4 . Primary antibodies were dissolved in PBS containing 0.3% Triton X-100 and applied to the cultures overnight at 4°C. After rinsing in PBS, the following secondary antibodies were applied: donkey anti-rabbit IgG labeled with DL488 (1:500), donkey anti-guinea pig IgG labeled with Cy3 (1:500) and AMCA-avidin conjugates were used as secondary antibodies . After incubation for 2 h, the specimens were rinsed in PBS and covered with Prolong Gold antifade mounting medium (Invitrogen). The selectivity and specificity of the polyclonal rabbit anti-TRPV1 and anti-InsR antisera (Santa Cruz Biotechnologies) were assessed in DRG sections by the lack of staining with the TRPV1 antibody in specimens obtained from TRPV1facturer . ControlPhotomicrographs were taken with a Leica DMLB fluorescence microscope  equipped with a Retiga 2000R digital camera . In control and insulin-treated cultures, at least 30 neurons per culture were counted and analyzed. Data collection was performed blinded to the experimental conditions. Each image was analyzed using ImagePro Plus 7 image processing and analysis software  and ImageJ image analysis software . Neurons exhibiting IB4 binding and immunoreactivity for the InsR, TRPV1, and CGRP or a combination of these molecular markers were counted. To assess the effect of insulin on neurite outgrowth, first, we examined the percentages of neurite-bearing DRG neurons in the control and the insulin-treated cultures. Thereafter, by using the ImageJ (NIH) simple neurite tracer program, we measured three quantified parameters of neurons with neurites longer than the soma diameter and clearly identifiable axonal arborization: total neurite length , maximum neurite length (length of longest neurite) and neurite branch points (number of neurite branch points).post hoc Tukey’s multiple comparison test, as appropriate. A p-value of less than 0.05 was considered to be statistically significant for all tests. Statistical analysis was made by using the Dell Statistica software .Data were collected from three to nine independent experiments. All data are presented as the mean ± standard error of the mean (SEM). To test the normality of the distribution of data in each experimental group, the Shapiro–Wilk test was applied. Statistical comparisons of data were performed with Fisher’s exact probability test or a one-way ANOVA with p < 0.05). These observations are in accord with previous findings showing the lack of a significant effect of insulin on neuronal counts in DRG cultures . The difference between the cultures exposed to 10 nM and 1 μM were also significant (p < 0.05). Neurite outgrowth was examined by analyzing three quantified parameters of neurons with neurites longer than the soma diameter and clearly identifiable axonal arborization: total neurite length , maximum neurite length (length of the longest neurite), and neurite branch points (number of neurite branch points). The results showed that administration of insulin at a concentration of 10 nM for 48 h increased total neurite length by 2.08-fold, maximum neurite length by 1.88-fold and neurite branch points by 2.35-fold as compared to control cultures . Administration of insulin, at a concentration of 1 μM, increased maximum neurite length by 1.64-fold (p < 0.05), whereas no significant changes were observed in total neurite length and neurite branch points . Therefore, only the effects of insulin applied at a concentration of 10 nM were analyzed in detail in this study.Administration of insulin at concentrations of 10 nM and 1 μM significantly increased the percentages of neurite-bearing DRG neurons by 50.74 ± 2.52% and 26.86 ± 1.78%, respectively, as compared to controls . The size-frequency distribution of the InsR-IR neurons revealed that InsR-positive DRG neurons are small-medium sized neurons. The mean cross-sectional area of the InsR-IR and InsR-immunonegative DRG neurons amounted to 321.11 ± 11.2 μm2 and 526.39 ± 15.22 μm2, respectively . Of the TRPV1-, CGRP-, and IB4-positive DRG neurons 63.09 ± 2.42%, 48.13 ± 2.37%, and 49.58 ± 3.7%, respectively, showed InsR-IR . Administration of insulin at concentrations of 10 nM and 1 μM did not significantly alter the percentage distribution of InsR-, TRPV1-, and CGRP-IR, and IB4-binding DRG neurons (p < 0.05). Analysis of the TRPV1- and the CGRP-IR and the IB4-binding populations revealed that 60.92 ± 3.38%, 56.98 ± 2.81%, and 31.11 ± 3.7% of the InsR-IR neurons were TRPV1-, CGRP-, and IB4-positive, respectively . The proportions of TRPV1-IR, CGRP-IR, and IB4-binding neurons expressing the InsR were significantly different (p < 0.05). The InsR was expressed in 42.06 ± 3.39%, 51.04 ± 4.26%, and 28.29 ± 2.4% of the TRPV1-IR, CGRP-IR, and IB4-binding neurons, respectively . The percentages of neurons which colocalized the InsR with TRPV1, CGRP, and IB4 amounted to 26.5 ± 2.17%, 24.33 ± 3.25%, and 13.82 ± 2.37%, respectively .Neurochemically distinct subpopulations of cultured DRG neurons were assessed by counting DRG neurons which showed immunoreactivity for the InsR, TRPV1, CGRP, or IB4 binding, respectively. In control cultures, 43.68 ± 1.71% of DRG neurons were IR for the InsR .To reveal the neurite outgrowth-promoting effect of insulin on different subpopulations of DRG neurons, the three quantified parameters of InsR-, TRPV1-, and CGRP-IR and IB4-binding DRG neurons were analyzed in control and insulin-treated cultures. Photomicrographs in p < 0.05). Our data also demonstrated that in InsR-IR neurons, insulin significantly increased the total neurite length from 536.61 ± 20.29 μm to 1390.21 ± 27.17 μm, the maximum neurite length from 74.42 ± 7.74 μm to 174.33 ± 8.36 μm, and the neurite branch points from 22.56 ± 5.14 to 123.15 ± 4.0, respectively . Statistical analysis revealed that differences between each quantified parameter of insulin-treated neuronal populations which express or lack the InsR were significant (p < 0.05).Our data revealed that insulin significantly increased the total neurite length from 513.21 ± 25.86 μm to 853.95 ± 52.65 μm, the maximum neurite length from 66.57 ± 12.9 μm to 127.88 ± 11.21 μm, and the neurite branch points from 25.38 ± 4.32 to 65.38 ± 9.02 in the InsR-immunonegative neuronal population . Conversely, in neurons which displayed the InsR, but not the TRPV1, insulin significantly increased the total neurite length from 522.98 ± 26.44 μm to 1150.89 ± 21.01 μm, the maximum neurite length from 74.426 ± 7.22 μm to 160.36 ± 4.77 μm, and the neurite branch points from 22.56 ± 6.2 to 90.38 ± 2.99, respectively . Statistical analysis revealed that differences between each quantified parameter of insulin-treated neuronal populations which express or lack the InsR were significant (p < 0.05). In DRG neurons which showed colocalization of the InsR and TRPV1, insulin significantly increased the total neurite length from 542.08 ± 25.17 μm to 1326.97 ± 22.77 μm, the maximum neurite length from 85.84 ± 9.02 μm to 188.93 ± 5.2 μm, and the neurite branch points from 24.56 ± 4.88 to 124.05 ± 3.84, respectively . Statistical analysis revealed that differences between each quantified parameter of insulin-treated InsR-IR neuronal populations which express or lack the TRPV1 were significant (p < 0.05).Our morphological analysis also revealed that insulin significantly increased total neurite length from 539.02 ± 27.91 μm to 969.45 ± 25.62 μm, the maximum neurite length from 65.85 ± 8.01 μm to 125.59 ± 5.04 μm, and the neurite branch points from 22.91 ± 5.32 to 48.04 ± 3.65 of neurons which were IR for TRPV1, but not for the InsR . In the InsR-IR and CGRP-immunonegative DRG neurons, insulin significantly increased the total neurite length from 522.99 ± 26.59 μm to 1099.66 ± 19.77 μm, the maximum neurite length from 54.99 ± 8.01 μm to 140.67 ± 3.99 μm, and the neurite branch points from 24.97 ± 3.66 to 106.37 ± 3.01, respectively . Statistical analysis revealed that differences between each quantified parameter of insulin-treated neuronal populations which express or lack the InsR were significant (p < 0.05). In InsR- and CGRP-IR neurons insulin significantly increased all the three quantified parameters: the total neurite length from 535.62 ± 30.6 μm to 1292.86 ± 26.88 μm, the maximum neurite length from 59.67 ± 6.44 μm to 182.55 ± 5.8 μm, and the neurite branch points from 26.55 ± 5.89 to 131.55 ± 3.99 . Statistical analysis revealed that differences between each quantified parameter of insulin-treated InsR-IR neuronal populations which express, or lack CGRP-IR were significant (p < 0.05). The statistical analysis revealed that the differences between the treated populations were also significant (p < 0.05).Our data also showed that in the CGRP-IR and InsR-immunonegative DRG neuronal population, insulin significantly increased the total neurite length from 565.47 ± 3.94 μm to 984.33 ± 24.03 μm, the maximum neurite length from 57.4 ± 9.06 μm to 110.81 ± 4.54 μm, and the neurite branch points from 21.47 ± 4.73 to 60.5 ± 4.99, respectively . In contrast, in IB4-positive neurons displaying the InsR, insulin treatment significantly increased total neurite length from 543.56 ± 30.96 μm to 1210.81 ± 20.08 μm, the maximum neurite length from 70.3 ± 7.99 μm to 156.81 ± 10.1 μm, and neurite branch points from 28.67 ± 7.99 to 119.21 ± 4.7, respectively .Analysis of the insulin responsiveness of the IB4-positive DRG neurons revealed that neurons which lack the InsR failed to show changes in any of the quantified parameters examined . However, in the course of their development, expression of TrkA is downregulated and non-peptidergic, IB4-binding neurons lose their NGF-sensitivity and become sensitive to GDNF . Severalvo e.g., . This isvo e.g., .in vitro and in vivo findings by showing that InsR-IR DRG neurons are small-medium sized (The expression of the InsR has been demonstrated in a relatively high proportion of DRG neurons in both rats and mice ,b. The pum sized ,b.The results demonstrate that populations of DRG neurons which express the InsR show an increased propensity for neurite outgrowth as compared to populations which lack this receptor. This is especially evident in the IB4-binding population of neurons; only those neurons which express the InsR exhibit an increase in neurite outgrowth upon insulin exposure. The exact mechanism of insulin-induced increased neurite outgrowth is not clearly established, but the role of the InsR has been implicated . It has per se appear to promote neurite outgrowth. Signaling mechanisms involved in the mediation of the neurite outgrowth-promoting effect of insulin may share common pathways contributing to the expression and sensitization of the TRPV1 by NGF (However, other specific traits, such as the expression of TRPV1 and/or CGRP also significantly modulate neurite outgrowth. Hence, although the results suggest that the expression of the InsR enhances neurite outgrowth in all populations of DRG neurons examined, the expression of TRPV1 and/or CGRP 1 by NGF . It has 1 by NGF . Hence, 1 by NGF . CGRP-IR1 by NGF . It can 1 by NGF . Taken t1 by NGF . HoweverImportantly, it has also been reported that similar to NGF application, insulin treatment facilitates the formation of collateral sprouting of small diameter neurons . The preThe present experiments confirm and extend previous findings on the neurite outgrowth-promoting effect of insulin and the expression of the InsR in cultured adult rat DRG neurons. The most important new observation of this study is that neurons which express the InsR exhibit a significantly higher propensity for the neurite outgrowth-promoting effect of insulin as compared to neurons which lack this particular receptor. Further, the data indicate that peptidergic CGRP-IR and TRPV1-IR nociceptive neurons expressing the InsR may be identified as the major subpopulations of DRG neurons which exhibit insulin-induced increased neurite outgrowth. The significance of the InsR in promoting neurite outgrowth is further supported by the observation that insulin increased neurite outgrowth only in a subpopulation of IB4-positive neurons which express the InsR. These findings suggest distinct regenerative propensity of differing populations of DRG neurons which is significantly affected through InsR signaling. In addition, the different responsiveness of separate chemotypes of primary sensory neurons to insulin may interfere with the development of pathologies associated with diabetes mellitus.BL, PS, and GJ were responsible for the study concept and design. BL, LP, ID, and PS were responsible for the data collection and analysis. BL, GJ, IN, and PS were interpreted the data, revised for intellectual content. All authors were involved in manuscript editing and have approved the final version for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E.coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts’ activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E.coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM -dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature.(2 Biocatalysts may represent a green and sustainable alternative to chemical catalysts. They can be used under mild conditions and are biodegradable . Due to anti-specific hydrogenations of activated alkenes [Escherichia coli (E. coli) cells [The asymmetric reduction of C=C double bonds is a widely employed reaction, since up to two stereogenic centers can be generated . In this alkenes . Thus, t alkenes . However alkenes ,7,8. Thii) cells , makes ti) cells , which fi) cells .Nostoc sp. PCC7120 (NostocER1) [Enzyme engineering was applied to reduce the strong NADPH-dependency of OYEs. In a previous work, we were able to influence the cosubstrate preference of the cyanobacterial ene reductase 1 from stocER1)  with a hR)-carvone to -dihydrocarvone was carried out. Dihydrocarvone is used in the synthesis of different molecules of biological interest, e.g., tetraoxane derivatives as compounds with antimalarial activity [R)-carvone, especially when compared to Baker’s yeast [E. coli biocatalysts overexpressing optimized NostocER1 mutants were applied.In this study, these optimized enzymes should be applied in a biotransformation of industrial relevance. Therefore, the reduction of -activity . Due to ’s yeast ,19, recoE. coli BL21(DE3) cells were constructed . This was followed by an enzyme production phase (Phase III), which was induced by 200 µM isopropyl β-d-1-thiogalactopyranoside (IPTG) after 23.1 h. The exponential feeding protocol was adjusted to a reduced specific growth rate of µset = 0.04 h−1 at an initial temperature of 20 °C. With this strategy, four parallel fed-batch processes were conducted to achieve identical biocatalyst production conditions. Thereby, a comparable course and maximum of the biocatalyst concentrations for each variant could be accomplished (−1 was consumed after 2.5 h (Phase I) and it was possible to control the specific growth rates between 0.139 ± 0.001 h−1 and 0.155 ± 0.006 h−1 by exponential glucose feeding (Phase II). During the enzyme production phase (Phase III), the specific growth rates were successfully maintained between 0.035 ± 0.001 h−1 and 0.038 ± 0.001 h−1 by exponential glucose feeding. Since the additional energy required for protein production can influence the biomass yield coefficient, an increased substrate supply was set at the beginning of this phase. Since no change of the coefficient was observable, glucose was overfed and accumulated up to 2.4 g L−1. Thus, to enhance the growth metabolism, the temperature was increased from 20 to 23 °C after 30.5 h of process time. Hereby, glucose concentrations could be kept below 0.2 g L−1.A protocol adjusted from Wilms et al. was established for the production of the whole-cell biocatalysts . Studies acetate . Controlmplished . The ini−1 was achieved after 48.3 h with the E. coli strain overexpressing the wild type enzymes (NostocER1-FDH). The constructs with the two NostocER1-variants Loop 1,2a-FDH and Loop 1,5-FDH showed comparable maximal CDW concentrations after 48.3 h . The lowest final CDW concentration of 50.6 ± 1.6 g L−1 was observed with the NostocER1-variant Loop 1,5,2a-FDH. This was, however, due to an oxygen limitation because of a blocked waste-gas filter of the stirred-tank bioreactor between 12.3 h and 16.3 h of process time.A maximal cell dry weight (CDW) concentration of 53.5 ± 1.6 g LE. coli cells were mechanically disrupted and the enzymatic activities were determined in the supernatant. Since the four applied NostocER1-variants possess different activities with NADH [500) of the respective purified NostocER1-variant . In order to ensure the same for the cosubstrate regeneration activity, the supernatant activities of the FDHs were determined and compared to one of the cells possessing the wild type enzymes. Since all constructs hold the gene encoding for the same FDH, a direct comparison of the FDH activities was possible -carvone (1) to form -dihydrocarvone (2). Intracellular NADH is consumed by the NostocER1 wild type or the three optimized enzyme variants. The formed NAD+ is recycled inside the whole-cell biocatalysts via the formate dehydrogenase (FDH) from Mycobacterium vaccae by oxidation of formate (3) to carbon dioxide (4). Since 1 is poorly soluble in aqueous media and shows antimicrobial activity, the Amberlite® XAD4 resin was added for in situ substrate feeding and product removal (SFPR). This resin resulted in beneficial results compared to other SFPR strategies, i.e., different organic solvents or ionic liquids [The recombinant  liquids . A schemFirst, this reaction setup was applied in stirred small-scale batch biotransformations to evaluate technical replicability. Second, the evaluation of preparative biotransformations with all biocatalysts was performed on a 0.7 L scale in stirred-tank bioreactors.R)-carvone were conducted on a 15 mL scale in triplicates. The E. coli BL21 (DE3) cells possessing optimized NostocER1-variants  were applied in three parallel batch biotransformations per construct -dihydrocarvone formation rate of 2.2 ± 0.2 mmol h−1 gCDW−1. The rates of the Loop 1,5-FDH construct (b) (1.4 ± 0.1 mmol h−1 gCDW−1) and the Loop 1,5,2a-FDH construct (c) (1.9 ± 0.1 mmol h−1 gCDW−1) were reduced by 38.0 ± 5.3% and 14.9 ± 1.6%, respectively.To compare the biocatalysts’ activity, the initial product formation rates were determined per biocatalyst amount. Loop 1,2a-FDH construct (a) revealed the highest rate with an initial -carvone to -dihydrocarvone within 8 h of a reaction time  showed a product concentration of 97.9 ± 1.5 mM after 2 h process time, the other three E. coli strains ranged between 187.0 ± 1.4 mM  and 126.6 ± 0.8 mM . The same differences were observed with respect to the initial -dihydrocarvone formation rates per applied biomass . The application of the Loop 1,2a-FDH construct increased the product formation rate by a factor of 2.1 to 2.8 ± 0.1 mmol h−1 gCDW−1. The other two biocatalysts also showed an increased product formation rate. The Loop 1,5-FDH construct resulted in an increase by a factor of 1.3 to 1.8 ± 0.1 mmol h−1 gCDW−1 and the Loop 1,5,2a-FDH construct in an increase by a factor of 1.7 to 2.3 ± 0.2 mmol h−1 gCDW−1. A comparable stereospecificity of all biocatalysts could be observed, which results in similar diastereomeric excesses (de) of the product -dihydrocarvone compared to the side product -dihydrocarvone. The whole-cell biocatalysts with NostocER1-FDH and the Loop 1,2a-FDH revealed the highest stereospecificity after ≥96% conversion with a de of 95.4%. The corresponding de of Loop 1,5-FDH (93.6%) and Loop 1,5,2a-FDH (94.3%) were slightly lower.Parallel batch processes were performed with three recombinant ion time a. Thereb biomass b. E. colR)-carvone was successfully reduced to -dihydrocarvone using recombinant E. coli whole-cell biocatalysts overexpressing nostocER1 variants and fdh genes. The application of bacterial whole-cell biocatalysts for this reduction reaction was already demonstrated to be beneficial in comparison to other microorganisms, since conversions of ≥96% were reached whereas the undesired reduction of carbonyl bonds by endogenous carbonyl reductases was not observed [In this study,  in combination with a NADP+-accepting mutant of formate dehydrogenase (FDH3M) from Mycobacterium vaccae resulted in highly active E. coli whole-cell biocatalysts [R)-carvone were converted to 290.4 mM -dihydrocarvone in a batch process on a liter-scale after 9 h by applying a biocatalyst concentration of 36 gCDW L−1. A space-time yield of 32.3 mmol L−1 h−1 was achieved with an initial -dihydrocarvone formation rate of 1.3 mmol h−1 gCDW−1. In this study, the NostocER1 wild type was overexpressed in combination with the NAD+-preferring FDH wild type. Thereby, E. coli biocatalysts could be provided showing a similar activity compared to literature. On a 0.7 L scale 285.4 mM -dihydrocarvone were formed in a batch process within 8 h with an initial product formation rate of 1.3 ± 0.1 mmol h−1 gCDW−1, which results in a space-time yield of 35.7 mmol L−1 h−1. Thus, within this process time, a yield of 95.1% was achieved based on 210 mmol applied substrate (R)-carvone. However, further improved biocatalysts were generated by exchanging the NostocER1 wild type with different NADH-accepting NostocER1-variants -dihydrocarvone formation rates. However, no further improved space-time yields were observed with these two variants in comparison to the NostocER1 wild type biocatalysts.In a previous work, the overexpression of the NADPH-preferring cyanobacterial ene reductase from atalysts . 300 mM variants . The impterature . This waR)-carvone has already been studied using many different microorganisms, including bacteria [Hanseniaspora guilliermondii [Saccharomyces cerevisiae and the addition of trehalose to the reaction mixture, the substrate loading could be increased to 16.6 mM and a conversion of 74% was reached [The bio-reduction of -carvone. In this context, Pseudomonas putida was used to fully convert 6.9 mM of the substrate with a high stereospecificity within 84 h [E. coli cells overexpressing the ene reductase NostocER1 and a FDH for cosubstrate regeneration. The high effectiveness of this biocatalyst was demonstrated in a biphasic system with the adsorbent resin XAD4, which results in the, by far, highest product formation rate and space-time yield for the bio-reduction of (R)-carvone in literature [R)-carvone within 6 h of process time. The product formation rate was increased by a factor of up to 2.1 and the space-time yield by a factor of 1.8. Thereby, the Loop 1,2a NostocER1-variant, i.e., the variant with the highest kcat with NADH of the three applied NostocER1-variants  concentration in E. coli, which is up to 20-fold higher compared to NADP(H) in exponentially growing cells [cat), which increases the importance of this parameter.Besides yeasts, bacteria proved to be beneficial for the selective reduction of -carvone, but also demonstrate that cosubstrate engineering of an NADPH-dependent oxidoreductase is useful, even if whole-cell biocatalysts are applied.Another important aspect of this study is that we could not only improve the bioreduction of (R)-carvone (98%), dihydrocarvone , and 20% ) and Amberlite® XAD4 were obtained from Sigma Aldrich . Media components, Kanamycin sulfate, and NAD(H) were purchased from Carl Roth . Enzymes for DNA manipulation were bought from New England Biolabs  and primers were synthesized by Eurofins Genomics . E. coli DH5α  was used for cloning and E. coli BL21 (DE3)  for protein expression and biotransformation.(fdh gene from Mycobacterium vaccae (GenBank BAB69476.1) were amplified using the primers listed in ® High-Fidelity DNA Polymerase  was applied according to the manufacturer’s protocol. For nostocER1 and all variants of this gene, the restriction sites NcoI and BamHI were used. Additionally, fdh NdeI and KpnI was applied.Standard procedures were used for polymerase chain reaction, ligation, transformation, and plasmid and cell preparation as described by Mülhart . The conl-scale was performed in four-fold parallel lab-scale stirred-tank reactors with an initial volume of 0.3 L . Temperature, dissolved oxygen concentration (DO), pH, and gas flow rate were monitored during the production of the whole-cell biocatalysts. The temperature was initially set to 37 °C for the batch phase, which was reduced to 30 °C for the fed-batch phase and was further reduced to 20–23 °C after induction for the enzyme production phase. The pH was controlled to pH 7.0 by 12.5% (v/v) ammonia and 1 M phosphoric acid addition. Solved oxygen availability was achieved in the reactors by the supply of sterile air with a sparger at the bottom and dispersion of the gas bubbles with two six-blade Rushton turbines mounted on the axes at a distance of 25 mm. The DO concentration was controlled to a set point of 20% air saturation by varying the stirrer speed between 500 and 1600 rpm. The air flow was set to 60 L h−1 representing an air flow of at least 3.33 volume per volume of the vessel capacity per minute (vvm). DO sensor calibration was performed prior to inoculation and 100% air saturation was achieved by stripping with air at 37 °C. Afterward, the reactors were stripped with nitrogen gas until 0% air saturation was achieved.Biocatalyst production in terrific broth (TB) medium containing 12 g L−1 peptone from casein, 24 g L−1 yeast extract, 5.1 g L−1 glycerol, 2.1 g L−1 KH2PO4, 12.6 g L−1 K2HPO4, and 34 mg L−1 kanamycin. The pH was adjusted to pH 7.2 and 4 mL of the TB medium were inoculated either with a single cell colony or cells from a cryo-stock in a 13 mL single use cultivation tube . Seed cultures and biocatalyst production in stirred-tank bioreactors were carried out in defined medium, according to Wilms et al. [−1 were grown overnight at 37 °C in shaking flasks without baffles . Cells from the seed cultures were harvested by centrifugation  and the required amount of the cell pellet was resuspended in fresh medium, according to Wilms et al. Afterward the re-suspended cells were used for inoculation of the stirred-tank bioreactors by sterile single-use syringes through a cannula.Pre-cultures were produced with s et al. . Seed cu−1. Kanamycin was used as a selection marker at a concentration of 34 mg L−1. For fed-batch cultivations, a growth rate of µset = 0.15 h−1 was set by using a biomass yield coefficient of 0.4 gCDW gS−1 and, adding a feeding solution with 100 g L−1 glucose, 19.8 g L−1 (NH4)2HPO4 and 34 mg L−1 kanamycin, according to Sun et al. [set = 0.04 h−1 and a feeding solution with 500 g L−1 glucose, 99 g L−1 (NH4)2HPO4, and 34 mg L−1 kanamycin was used. Furthermore, 0.5 g L−1 MgSO4 · 7 H2O and 3 mL L−1 trace element solution [−1 thiamin was supplemented to the reaction medium. Cells were transferred to centrifugation tubes after cell cultivation and were stored for less than 24 h at 4 °C until further use for preparative biotransformations. The cells for the analysis of the technical replicability were mixed with 12.5% (w/v) glycerol and stored at −60 °C.Glucose served as a substrate for the initial batch phase at a concentration of 1 g Ln et al. . Temperasolution  were addE. coli cell suspension was mixed with 0.5 g glass beads  and disrupted for 5 min and 25 s−1 in a Mixer Mill MM 400 . Debris was separated by centrifugation  and the activity in the supernatant was determined photometrically by oxidation (ER) or reduction (FDH) of 500 µM NAD(H) at 340 nm. Assays were conducted in sodium phosphate buffer  at 30 °C using F96 microwell plates  and a Multiscan™ FC Photometer . Reactions were initiated by the addition of 10 mM maleimide (ER) or 250 mM sodium formate (FDH) as a substrate. Reaction rates were determined by automated linear regression using MATLAB R2015b .Enzyme activities were determined in cell lysates. Therefore, 1 mL R)-carvone were carried out on two different scales: 15 mL for the evaluation of technical replicability and 0.7 L for the preparative biotransformation in the stirred-tank bioreactors. All biotransformations were performed with 300 mM (R)-carvone and 450 mM sodium formate in 300 mM sodium phosphate buffer (pH 7.0). Amberlite® XAD4 with a wet mass ratio of 3:1 to (R)-carvone was used for in situ substrate feeding and product removal. (R)-carvone and adsorbent resin  were incubated for at least 2 h in buffer (50% (v/v) of the total reaction volume) prior to the biotransformations. Reactions were initiated by the addition of the whole-cell biocatalysts resuspended in 300 mM sodium phosphate buffer and the samples were incubated at 25 °C.Biotransformations of . Mixing was achieved with x-shaped magnetic stir bars  using a multiple stirrer plate Variomag Poly 15  at 900 rpm. Sampling was done with a volume of 300 µL each.On the 15 mL scale, batch biotransformations were performed in glass vials with rolled rim equipped with snap caps . Furthermore, 100 µM NAD+ were added to the reaction mixture. Mixing and dispersion of the gas phase was achieved with two six-bladed Rushton turbines mounted on the axes at a distance of 25 mm and a speed of 700 rpm. The pH was controlled to pH 7.0 by automated addition of 1 M phosphoric acid. Sampling was done with a volume of 1 mL each.On the 0.7 L scale, batch biotransformations were performed in parallel stirred-tank reactors (DASGIPv/v), EtOAc was added to the aqueous-adsorbent resin samples and extraction was performed for 15 min at 25 s−1 in a Mixer Mill MM 400  for enhanced mass transfer. The phases were separated by centrifugation  and 100 µL of the organic phase was dissolved in 300 µL EtOAc and 100 µL (R)-limonene (36 mM in EtOAc) as an internal standard.Prior to the analytical procedure, the samples had to be extracted using EtOAc. Therefore, between 33% and 50% -limonene 7.5 min, -DHC 17.4 min, -DHC 18.8 min, and (R)-carvone 20.2 min. Compound concentrations were determined using a standard from 0.1 mM to 30 mM of the reference compounds. The application of a biphasic system led to high fluctuations of the determined concentrations. Therefore, substrates and products were summarized and normalized to the applied total concentration of 300 mM. Diastereomeric excesses (de) were determined by comparison of the -dihydrocarvone concentration with the -dihydrocarvone concentration. Corresponding standard deviations of three technical replicates were between 0.01% and 0.04%. Initial -dihydrocarvone formation rates were determined by linear regression until a product concentration of 150 mM was achieved.Analytics were performed using a gas chromatograph GC-2010 Plus  equipped with a flame ionization detector (FID) and a chiral Lipodex-E column . The injection volume was set to 1 µL and the split ratio was 10. The injector temperature was set to 200 °C and the detector temperature was set to 250 °C. All runs were performed with the same oven temperature program, i.e., 60 °C for 3 min followed by 3 °C min


The main outcome was to determine the degree of change in HbA1c, while secondary outcomes included body weight, blood pressure, and blood lipids. The volunteers lost significant weight (85.7 ± 3.2 kg to 76.7 ± 2.8 kg) and lowered systolic (134.0 ± 1.6 to 123.3 ± 1.1 mmHg) and diastolic (89.9 ± 1.3 to 82.6 ± 1.0 mmHg) blood pressure. HbA1c dropped to 5.6%. Most blood lipids were significantly altered, including HDL cholesterol (43.1 ± 4.4 to 52.3 ± 3.3 mg/dl), triglycerides (177.0 ± 19.8 to 92.1 ± 8.7 mg/dl), and the TG : HDL ratio (4.7 ± 0.8 to 1.9 ± 0.2). LDL cholesterol was not significantly different. AST and ALT, plasma markers of liver health, were reported for eight patients and revealed no significant changes. These findings indicate that a short-term intervention emphasizing protein and fat at the expense of dietary carbohydrate functionally reversed the diabetes diagnosis, as defined by HbA1c. Furthermore, the intervention lowered body weight and blood pressure, while eliciting favorable changes in blood lipids.Because low-carbohydrate diets are effective strategies to improve insulin resistance, the hallmark of type 2 diabetes, the purpose of reporting these clinical cases was to reveal the meaningful changes observed in 90 days of low-carbohydrate (LC) ketogenic dietary intervention in female type 2 diabetics aged 18-45. Eleven women (BMI 36.3 kg/m Insulin resistance is the most common health disorder worldwide , affectiα-glucosidase inhibitors) or increase renal glucose excretion . However, while varyingly effective in reducing blood glucose and insulin and improving insulin resistance, side effects such as osmotic diarrhea [Because of the prevalence and pathologies arising from insulin resistance, numerous pharmacological therapies are commonly utilized to improve insulin sensitivity. Several of these therapies seek to either inhibit intestinal glucose absorption  were at or above 0.5 mmol/l  with significance set at a p value of ≤0.05.Subject data were analyzed via a two-tailed paired 2. All eleven subjects lost significant weight, with an average postintervention weight of 76.7 ± 3.2 kg (p < 0.0001), further reflected in a reduction in BMI to 32.7 ± 1.5 kg/m2  and 82.6 ± 1.0 mmHg  are relevant indicators of long-term glycemic control and, therefore, indicative of diabetes severity. As glucose levels are consistently elevated, glucose can nonenzymatically bind hemoglobin on erythrocytes. Traditionally, the diabetic range for HbA1c is 6.5%, and diabetic strategies are primarily focused on getting HbA1c close to or below that cut-off. All subjects were diagnosed with diabetes, with an average HbA1c of 8.9 ± 0.4%. Within 90 days of a LCHF intervention, HbA1c dropped to 5.6 ± 0.3% (p < 0.005), while HDL cholesterol increased (p < 0.005). The TG : HDL ratio decreased significantly from 4.6 ± 0.8 to 1.6 ± 0.2 (p < 0.005). Importantly, the TG : HDL ratio is a telling predictor of heart disease, with a much stronger correlation to heart disease than LDL cholesterol [Blood tests often measure the prototypical blood lipids of interest, which include LDL cholesterol, HDL cholesterol, and triglycerides. Each of these has been classically considered to alter the risk of heart disease to varying degrees. Insofar as heart disease is common among type 2 diabetes , the conlesterol .Various liver disorders are associated with insulin resistance and type 2 diabetes; as such, plasma levels of AST and ALT, considered a reflection of liver health, were determined in eight of the patients Figures . DespiteThe purpose of this manuscript is to report the efficacy of a short-term low-carbohydrate (LC) dietary intervention to improve glycemic control in a group of recently diagnosed subjects with type 2 diabetes. Most importantly, we found that a low-carbohydrate ketogenic diet, wherein carbohydrates constituted roughly 5% of calories, elicited a remarkable reduction in HbA1c from a diabetic level (8.9%) to the standard (5.6%) in only 90 days. Furthermore, the intervention resulted in significant improvements across other cardiometabolic markers, including body weight, BMI, blood pressure, and the triglyceride (TG) : HDL ratio.Standards of Medical Care in Diabetes, stated, “research indicates that low-carbohydrate eating plans may result in improved glycemia and have the potential to reduce antihyperglycemic medications for individuals with type 2 diabetes” [Meaningful limitations exist in this study that temper overreaching with regard to the findings. Firstly, the sample size of the report is very small. Secondly, the intervention involved self-selecting patients in a clinical setting, where the subjects were self-motivated and informed of the intervention at the diagnosis of type 2 diabetes. These two concerns are somewhat mitigated when considered in light of evidence from longer and much larger trials, wherein carbohydrate restriction yields similar, albeit less dramatic, outcomes. Very recently, Hallberg et al.  found reiabetes” .Despite our efforts to scrutinize insulin resistance, the lack of plasma insulin levels in this study prevents any definitive conclusions with regard to insulin resistance. Future studies from our group will rectify this deficiency in order to more thoroughly explore the effects of our LC intervention on insulin sensitivity. Nevertheless, the lipid changes observed in the subjects in this pilot study reflect an insulin-sensitizing effect, insofar as the TG : HDL ratio, which is considered a surrogate for insulin resistance , 19, wasBecause of the relevance of HbA1c in disease outcomes related to diabetes, such as neuropathy , nephropWe submit these data as modest, yet undeniable, evidence that a dietary intervention that restricts carbohydrate and emphasizes unrestricted consumption of protein and fat elicits a favorable metabolic state, including the dramatic reduction in HbA1c. Moreover, such an intervention, unlike various insulin secretagogues , 25, has