However, photocatalytical activation has rarely been investigated. We herein present results on the light-driven CO2 reduction using either Ir(dFppy)3  or [Cu(xant)(bcp)]+,  as photosensitizer in combination with TEA (triethylamine) as sacrificial electron donor. The 3d metal catalysts have either dptacn  or dpdatcn  as ligand framework and Fe3+, Co3+ or Ni2+ as central metal ion. It turned out that the choice of ligand, metal center and solvent composition influences the selectivity for product formation, which means that the gaseous reduction products can be solely CO or H2 or a mixture of both. The ratio between these two products can be controlled by the right choice of reaction conditions. With using Cu as photosensitizer, we could introduce an intermolecular system that is based solely on 3d metal compounds being able to reduce CO2.Complexes based on nitrogen and sulfur containing ligands involving 3d metal centers are known for the electrocatalytic reduction of CO Beside l groups . Light-aeduction . Photoch2 reduction, due to the less negative NiII/I redox potential compared to Ni complexes with just N-donor atoms  was the first of its kind to be investigated in the electrocatalytic CO2 activation. Although better catalytic properties than the [Ni(cyclam)]2+ congener were observed , it was revealed that the complex seems to be instable after prolonged electrolysis time  complexes – when having π-donor atoms such as S – are more likely to show activity towards COor atoms . Interessis time .2N2 ligand sphere and two noncoordinating pyridine substituents can bind to a Lewis-acidic metal ion. The heterodinuclear Ni-Mg complex was successfully applied in photocatalytic CO2 to CO transformation using [Ru (bpy)3]2+  as photosensitizer (Ir ([Ir(dFppy)3], dFppy = 2-pyridine) and Cu ([Cu(xant)(bcp)]PF6, xant = xantphos, bcp = bathocuproine).Kojima et al. reported about a heterodinuclear nickel magnesium complex, in which the Ni ion resides in a Snsitizer . Furthernsitizer . Herein LN3 and 1-thia-4,7-di(picolyl)-diazacyclononane LN2S. We followed published procedures with slight adjustments . Briefly, LN3 was prepared beginning with the tosylation of diethylenetriamine and glycol  sulfide was synthesized via sodium ethoxide driven SN2 reaction of cysteamine hydrochloride and 2-chloroethylamine hydrochloride -1,4,7-triazacyclononane d glycol . The nexs3-tacn) . Partials3-tacn) , which wreaction . To obtac. H2SO4 . LN2Schloride , followe 2 . Work-up of the reaction mixture was done under air atmosphere leading to oxidation of the iron and cobalt compounds. Purification of the crude product was obtained via washing with CH2Cl2 to remove excess of ligand and washing with toluene (Fe(OTf)2 and Co(OTf)2) or THF (Ni(OTf)2) to remove unreacted metal salt. The desired complexes were obtained as solids, which were redissolved in a small amount of MeCN and precipitated using Et2O. Since LN3 was synthesized as hydrochloride salt (stemming from column chromatographic purification using dichloromethane), Fe-LN3 and Co-LN3 were obtained as complexes with one chloride ligand and two uncoordinated triflate counter anions, which could be verified by mass spectrometry and X-ray diffractometry. Ni-LN3 was isolated as a chloride free complex by precipitating AgCl using Ag(OTf). The complexes M-LN2S were prepared in the same fashion giving yields in the range of 44–50%. All complexation reactions should be done soon after preparation of the proligands, due to sensitivity against oxidation of the compounds LN3 and LN2S.For both compounds, m/z 516.0974 (Fe-LN3), 519.0975 (Co-LN3), 518.1018 (Ni-LN3), as well as 533.0584 (Fe-LN2S), 536.0568 (Co-LN2S) and 535.0590 (Ni-LN2S) for the corresponding [M + OTf]+ fragments  and UV/vis measurements. The UV/vis studies in DMF solution (Fe-LN3) or 502 nm (Fe-LN2S). The Ni complex Ni-LN3 only has an additional absorption maximum at 308 nm and hence is colorless. As the Ni-LN2S absorbs in a broader area from 500 to 300 nm it is isolated as light brown solid. The most striking difference is revealed for the Co complexes where d-d transitions are observed at 362 nm (ε = 320 dm3/mol*cm) for Co-LN3 and 490 nm (ε = 390 dm3/mol*cm) for Co-LN2S leading to a light red/pink color.Electrochemical and spectroscopic properties were determined solution  reveal aFe-LN3 and Fe-LN2S a reversible redox event is observed at a half-wave potential of −0.09 and 0.20 V (referenced to Fc/Fc+), respectively, representing the FeII/FeIII redox couple  than the sulfur free analogue. This result can be attributed to the electron donating property of the sulfur atom, and a similar trend is also observed for the related cobalt complexes. It is important to note, though, that for Fe-LN2S an additional reduction process occurs at −0.34 V, which we tentatively relate to the sulfur donor atom. A similar behavior is observed for Co-LN2S that shows and additional reduction process at −0.71 V, which is absent for Co-LN3.Interestingly, Co-LN3 a reversible redox event at −0.59 V is indicative for the CoII/CoIII redox couple. This event is accompanied by two irreversible reduction processes at −2.17 V and −2.59 V resulting in the formation of CoI and finally the Co0 state – most likely being a CoI(LN3−) species. In accordance with the observations for Co-LN3, Co-LN2S shows one reversible reduction at −0.35 V and three irreversible reduction events at more negative potentials at −1.93 V, −2.43 V and −2.88 V that are shifted to higher voltages when compared to N3Co-L. An assignment of these redox processes is not straightforward and currently not possible. The Ni complexes Ni-LN3 and Ni-LN2S do not show any redox processes in the investigated solvent window.In case of Co-LN3 and Fe-LN3 were obtained by slow vapor diffusion of Et2O to a high concentrated MeCN solution. Both complexes are isostructural  are observed, which is in line with Co3+ being smaller than Fe3+. The Co-Cl bond distance, on the other hand, is almost identical to the Fe-Cl bond with ∼2.23 Å ][CIO4]3•H2O was published earlier ](PF6) and [FeLN3(NCS)](PF6), which were obtained via reaction under inert conditions and, hence, possess an oxidation state for Fe of +2  as sacrificial electron donor. In earlier studies we successfully applied the following conditions  and 5 Vol% TEA as sacrificial electron donor (SR). The catalysis mixture is placed in front of a 200 W Hg-lamp equipped with a 400 nm low-wavelength cut-off filter to exclude UV light. Ir was selected because of its ability to still absorb solar photons in the region of 400–460 nm nditions : 5 × 10−0–460 nm  and its 0–460 nm   decomposition products are not responsible for the formation of the gaseous products.It can be observed, that not only the ligand but also the metal centre do have an impact on both the formed products and the catalytic activity ; Table 3mediates . When a  species . On a siCo-LN3. Similar TONs were obtained indicating that Co colloids did not form. In case of Co-LN2S the ratio of CO:H2 changes to about 2:1  in comparison to no H2 formation for Co-LN3. In case of Fe-LN2S the CO:H2 ratio stays very similar to the one of Co-LN2S, but the activity drops by about one third, and about half in comparison to Fe-LN3. For the Ni complexes the electrochemical data predicted no (photo)catalytic activity, because they do not show a reduction process in the solvent window investigated. This could be confirmed for Ni-LN3, whereas Ni-LN2S shows a moderate TONH2, which again is probably induced by the sulfur atom in the ligand system.Further evidence for molecular catalysis could be obtained by adding mercury to a catalysis solution with Co-LN2S catalytic mixture in different concentrations. Thereby a shift to higher TONH2 is observable, while TONCO is decreasing . However, there is almost no further increase in CO evolution for Cu indicating that most of the photosensitizer is decomposed already after a few hours of reaction time. The initial better performance of Cuvs.Ir is tentatively assigned to the more suitable excited state oxidation potential of Cu , which is roughly a factor of seven  (4 s−1/5·105 s−1 = 0.11) will lead to electron transfer from Ir to N3Co-L under the catalytically relevant conditions with 5·10-5 M N3Co-L.Luminescence quenching experiments were performed to obtain some insight into the reaction mechanism. The excited state of the (Ir) photosensitizer can be quenched oxidatively by the catalyst Co-LN3 ; Table 1of seven  below thut 2 μs)  this imp2 reduction in acetonitrile with [CoII(tpa)Cl]Cl (tpa = tris(2-pyridylmethyl)amine) and using Ir(ppy)3 (ppy = 2-phenylpyridine anion) as photocatalyst. Stern–Volmer analysis revealed quenching rate constants for the photosensitizer of 5.29 × 109 M−1s−1 for [CoII(tpa)Cl]Cl and 4.14 × 104 M−1s−1 for TEA, respectively. It was further noted that reductive quenching of excited [Ir(ppy)3] is not feasible from thermodynamic consideration (E1/2 [(Ir(ppy)3)*/(Ir(ppy)3)−] = +0.31 V vs. SCE) and, hence, oxidative quenching of the PS is the more favourable reaction path. These findings are also in line with previous studies by some of us 3)*/(Ir(dFppy)3)−] = +0.36 V vs. Fc/Fc+)  gave significantly greater turnover numbers and led to very fast CO2 transformation.Similar bimolecular rate constants were determined by Chan and co-workers , who invme of us , in whic Fc/Fc+)  seeminglIr complex is a somewhat stronger photo-oxidant than Ir(ppy)3  one may assume that the rate constant for bimolecular quenching of photoexcited Ir by TEA is on a similar order of magnitude, roughly 4.14·104 M−1s−1 (see above). Under the catalytically relevant reaction conditions in which 0.36 M TEA is present, this leads to a pseudo-first order rate constant of 1.5·104 s−1. Co-LN3 is present at 5·10-5 M concentration under the catalytically relevant conditions, and given a rate constant of 1.1·109 M−1s−1 for bimolecular electron transfer from photoexcited Ir to Co-LN3 (see above), one obtains a pseudo-first order rate constant of 5.5·104 s−1. Thus, under the assumption that reductive excited-state quenching of Ir by TEA is not more rapid than reductive quenching of [Ir(ppy)3] despite its 0.05 V higher oxidative power (see above), oxidative quenching of Ir by Co-LN3 is a factor of 3.6 (= 5.5·104 s−1/1.5·104 s−1) faster than reductive quenching by TEA.Though the fluorinated + is reduced by TEA to reform PS, which then restarts the cycle  was dissolved in MeCN (5 ml) and M(OTf)2 (1. eq) was added. After stirring for 3 days at room temperature, the solvent was removed. The resulting oil was washed with CH2Cl2 and toluene, dissolved in MeCN and precipitated using Et2O.General procedure for the synthesis of complexes Fe-LN3 was obtained as brownish red solid .After purification m/z: calc. for [M + OTf-]+, 516.0974; found, 516.0974.ESI-MS: UV/vis: λ 268, 312, 432 nm.Co-LN3 was obtained as pink solid .After purification 1H NMR : δ 9.24 , 8.33 , 8.13 , 7.86 , 7.72 , 7.47 , 7.21 , 6.67 , 5.09 , 4.64 , 4.46 , 4.10 , 3.79 , 3.45 , 3.31 , 3.23 , 3.01 , 2.77 , 2.54 , 2.01.13C NMR : δ 164.83 (Cquart), 162.48 (Cquart), 153.65 (Cpico), 149.45 (Cpico), 141.83 (CPico), 140.83 (CPico), 126.69 (CPico), 126.61 (CPico), 125.51 (CPico), 129.94 (CPico), 69.43 , 67.27 , 63.92 (Cmacro), 62.37 (Cmacro), 61.53 (Cmacro), 60.16 (Cmacro), 54.23 (Cmacro), 52.93 (Cmacro). 19F NMR : δ -76.95 (Ftriflat).m/z: calc. for [M + OTf-]+, 519.0956; found, 519.0975.ESI-MS: UV/Vis: 268, 362, 507 nm.Ni-LN3, Fe-LN2S, Co-LN2S and Ni-LN2S. The macrocycles LN3 and LN2S were dissolved in dry MeCN (5 ml) and M(OTf)2 (1 eq.) was added. The suspension was stirred for 3 days at room temperature and the solvent was removed subsequently. The resulting oil was washed with CH2Cl2 and toluene [Fe(OTf)2 and Co(OTf)2] or THF [Ni(OTf)2], dissolved in MeCN and the remaining chloride ions were precipitated using an excess of Ag(OTf). The filtrate was evaporated and afterwards the residue was taken up in a small amount of MeCN and precipitated using Et2O.General procedure for the synthesis of Ni-LN3 was obtained as beige solid .After purification m/z: calc. for [M + OTf-]+, 518.0984; found, 518.1018.ESI-MS: UV/Vis: 268, 308 nm.Fe-LN2S was obtained as dark brown solid .After purification m/z: calc. for [M + OTf-]+, 533.0586; found, 533.0584.ESI-MS: UV/vis: 268, 490, 502 nm.Co-LN2S was obtained as red solid .After purification m/z: calc. for [M + OTf-]+, 536.0571; found, 536.0568.ESI-MS: UV/vis: 268, 490 nm.Ni-LN2S was obtained as light brown solid .After purification m/z: calc. for [M + OTf-]+, 535.0590; found, 535.0590.ESI-MS: UV/vis: 268 nm.M-LN3 and M-LN2S containing Fe, Co and Ni, which can be applied in the photocatalytic activation of CO2. Initially, an Ir photosensitizer was used and the combination of Ir and Co-LN3 showed the highest TONCO (90 after 24 h of illumination) of all investigated complexes. The ratio of the gaseous products CO and H2 can be varied by the choice of metal ion, macrocyclic ligand and solvent composition. The product selectivity can even be adjusted to almost solely formation of CO (in case of Co-LN3) or H2 (in case of Ni-LN2S). In addition, we were able to demonstrate that a Cu photosensitizer can be used for the catalytic reaction as well, making the whole system solely 3d metal based. The initially higher catalytic activity compared to Ir (TONCO of 9 vs. 3 after 2 h reaction time) can tentatively be assigned to the more positive excited state oxidation potential of Cu making the oxidative quenching process by the catalyst more efficient. On the contrary, the long-term stability of the Cu photosensitizer is clearly inferior compared to the Ir photosensitizer with a TONCO after 24 h of illumination of 13 vs. 90.We herein describe the synthesis of two series of macrocyclic complexes First mechanistic investigations confirm that an oxidative quenching mechanism is more likely than a reductive quenching process. Further experiments are on the way to confirm possible intermediates involved, such as a hydride species, and the influence of further reaction parameters on the outcome of the photocatalytic reaction, such as changing the sacrificial electron donor. The latter might also influence the quenching process and thus the overall TON and product selectivity.


The nipagin-derived component acts as the renewable surrogate of petroleum-based dimethyl terephthalate (DMT), while the eugenol-derived component acts as the cooperative property modifier of parent homopoly(ether ester) PHN1. 1,6-Hexanediol was chosen as the spacer because of its renewability, high boiling point, and short chain to enhance the glass transition temperatures (Tgs) of materials. The molecular weights and chemical structures were confirmed by gel permeation chromatograph (GPC), NMR and FTIR spectroscopies. Thermal and crystalline properties were studied by thermal gravimetric analysis (TGA), differential scanning calorimetric (DSC) and wide-angle X-ray diffraction (WXRD). The tensile assays were conducted to evaluate the mechanical properties. The results suggested that properties of this kind of poly(ether ester)s could be finely tuned by the relative content of two components for the desired applications  suitable for different scenarios from polyethylene glycol terephthalate (PET) and polybutylene terephthalate (PBT).Naturally occurring nipagin and eugenol were used as the collaborative starting materials for poly(ether ester) polymers. In this study, two series of nipagin and eugenol-derived copoly(ether ester)s, PHN1 The reality prompts us to find bio-based substitutes to petroleum-based resources from the view of sustainable development11. PET and PBT are representative examples of widely used engineering plastics, but they are mainly produced from petroleum-based dimethyl terephthalate (DMT). Due to the huge consumption, finding the renewable surrogates for DMT without sacrificing the thermal stability and mechanical strength of materials is urgent.Currently, great majority of plastic consumed globally is produced from fossil resources. This situation brings about the gradual depletion of non-renewable materials, as well as increasingly severe environmental concerns13. These two starting materials have been found widespread applications in fields of liquid crystalline material15, epoxy resin18, benzoxazine20, coating22, and Li–S battery23. Because of the multifunctionality (phenol and vinyl) and renewability of nipagin and eugenol, based on them our group has been dedicated to polyester materials, whose thermal, crystalline, mechanical and degradable properties have been comprehensively investigated25. The influence of monomer structure on final properties was also studied26. However, the comprehensive performance of obtained polyesters can still be improvable, regardless of the renewability of starting materials. Either the glass transition temperatures (Tgs) were not high enough, or the mechanical tough and strength was not perfect. Consequently, further optimizing the performance of nipagin and eugenol-based polymer materials is particularly important for the development and utilization of such materials.Nipagin and eugenol are two naturally occurring building blocks, which exist in campanulaceae plant and clove, respectively28. In contrast, eugenol-derived unit is highly asymmetrical one, which endows the polymers low crystallinity and poor mechanical strength, while their flexibility and ductility are good24. Taking advantages of the complementary features of these two building blocks through rational molecular design may give the polymeric materials synergistic effect and desired final properties.Considering the structural difference of nipagin and eugenol, the symmetrical nipagin units can endow the polymers with high crystallinity and mechanical strength, but poor flexibility and ductility32, the low boiling points of which can lead to volatilize easily during the polycondensation stage and be difficult to control the stoichiometric ratio between diester and diol precisely, and thus caused low molecular weights. Meanwhile, due to the chain length of 1,6-hexanediol is shorter than that of 1,10-decanediol, the Tgs of obtained polymers were enhanced obviously compared with our previously synthesized polyesters with 1,10-decanediol as the spacer26. The high Tg values can make polymer materials applied in a wide temperature range. Synergistic effect between these two structurally distinctive components (nipagin and eugenol) endows the materials with desired performance, which can be modulated for a broad range of applications  suitable for a variety of scenarios.In this work, three nipagin and eugenol-derived diester monomers were firstly synthesized s were synthesized by a melt polycondensation method according to the predetermined composition ratios of nipagin and eugenol-derived dimethyl ester monomers. The polymerization routes are shown in Fig. PHN1Mn above 104 were successfully obtained, and polydispersity (Ɖ = Mn/ Mw) values are found to be fixed at about 1.7 regardless of the feed ratios, which also suggest the similar reactivity of diester monomers, where Mn and Mw are the number- and weight-average molecular weights, respectively. The apparent appearance of final products is closely related with the feed ratios. That is, with the gradual increase of eugenol-based units, the physical form of final product changes from white powder for PHN1, transforms into yellow powder for PHN195%E1(2)5%, PHN190%E1(2)10%, and PHN185%E115%, finally becomes light yellow semi-solid for PHN180%E120%, PHN185%E215% and PHN180%E220%. This phenomenon indicates that eugenol-derived units are easier to be oxidized than nipagin-derived units.The high yields suggest the high reactivity of the two kinds of monomers. The consistency of nipagin and eugenol-derived units in the feed and final product demonstrates that nipagin and eugenol-based diester monomers have similar reactivity and excellent compatibility. Copoly(ether ester)s with 1H NMR s were confirmed by 1−xE1x and PHN11−xE2x exhibited comparable thermal stability with the parent PHN1. Despite the content of eugenol-derived composition reached 20%, the temperature at which 5% weight loss (T5%) decreased just about 12 °C for both PHN11−xE1x and PHN11−xE2x relative to PHN1 s. Based on the above results we conclude that the incorporation of eugenol-derived units had little influence on thermal stability of the final product. T5% values were above 363 °C for all the samples, which is comparable with that of the petroleum-based poly (PBT) (T5% = 371 °C) with appropriate molecular weights  reported by others34. The temperature for maximum degradation rate (Td) values were almost identical for both PHN1 and PHN11−xE1(2)x, whose Td values were fixed at about 410 °C and found to be insusceptible to the changes of content in eugenol-derived composition. Furthermore, PHN11−xE1x with eugenol-derived composition below or equal to 10% featured a two-step degradation mechanism. However, for PHN11−xE1x with eugenol-derived composition above 10% and the whole series of PHN11−xE2x, single-step degradation was observed. This phenomenon could be explained by the mismatch of conformation of polymer chains at such specific compositions. Conclusion could be drawn from TGA analysis was that the copoly(ether ester)s featured excellent thermal stabilities, and the incorporation of eugenol-derived component actually had good compatibility with the parent PHN1. In a word, these bio-derived materials have excellent thermal stability.Thermal stability is an important parameter of polymer materials in practical applications, and determines the long-term usability. In this study, thermal stabilities of the copoly(ether ester)s were investigated by thermogravimetric analysis (TGA). Weight-loss curves and the corresponding derivative curves were depicted in Fig. Tg), melting and crystallization temperatures , together with their corresponding melting and crystallization enthalpies  are critical factors for the shape retention under normal practical use, as well as during molding process. Here these properties were studied by differential scanning calorimetric analysis (DSC). The DSC heating traces (2nd) are shown in Fig. Other thermal properties like glass transition s suggested that these samples could not crystallize from the melt during the first cooling run at the given cooling rate of 10 °C/min. The locally ordered packing of chain segments was not favoured. Although the Tg values gradually decreased with the incorporation of eugenol-derived units, the values changed not so much and were still in the scope of practical applications. Compared with our previous reported 1,10-decanediol-based copoly(ether ester)s (Tg <  − 8 °C)26, the Tg values (> 4.5 °C) of 1,6-hexanediol-based materials in this study has obvious improvement, which is beneficial for applying in a broad range of temperature.Both PHN136. Almost the same diffraction pattern was observed for all the poly(ether ester) materials when the diffraction angles and relative intensities were considered, indicating the crystalline mode of PHN1 was maintained in the copoly(ether ester)s. The crystallinity values can be calculated as the quotient between crystalline area and total area of the diffraction traces. The copoly(ether ester)s were found to be semi-crystalline and the crystallinities (Xc) decreased with the increase of eugenol-derived composition. The discrepancy of material attribute from DSC and WXRD analysis could be ascribed to the difference of treatment method. The samples for WXRD were directly from precipitation in methanol, resulting in a certain degree of crystallization. The X-ray diffraction results confirmed that such materials still have slightly crystalline properties, rather than completely amorphous materials. The crystallinities gradually decrease with the insertion of eugenol-derived units.In order to verify and reinforce the above DSC results and further study their crystalline ability of this kind of materials, powder wide-angle X-ray diffraction analysis (WXRD) was performed. The WXRD traces were depicted in Fig. 1−xE1x and PHN11−xE2x copoly(ether ester)s were performed on dumb-bell shaped specimens (12 × 2 × 0.5 mm3), which were prepared by casting the chloroform solution (0.1 g/mL). The stress-stain curves were depicted in Fig. 1−xE1x was slightly higher than those of PHN11−xE2x when the content of eugenol-derived composition was the same. However, the elongations at break firstly decrease and then increase with the content of eugenol-derived composition increasing. For example, elongations at break decreased from 14.5% for PHN1 37 to 7.0% for PHN190%E110%, and then increased to 10.0% for PHN180%E120%. The elongation at break didn't exhibit the same trends as the Young’s modulus and tensile strength do. This may be ascribed to the asymmetrical nature of eugenol-derived composition, leading to the difficult stacking and loose entanglement of polymer chains. Consequently, Young’s modulus and tensile strength decreases with the insertion of eugenol-derived composition, while the elongation at break did not feature the same variation tendency. Although PHN1 seems to feature the best mechanical properties  than those of copolymerized products, the necessity of copolymerization of nipagin and eugenol-derived monomers is also significant. Nipagin and eugenol are two structurally different building blocks, which will have significant influence on thermal , crystalline , and mechanical properties , which can be tailored for a broad range of applications, such as rubbery and elastomers, etc. depending on the application scenarios.Mechanical properties  are primary parameters to be considered for practical applications. To further study the synergistic effect of nipagin and eugenol-based components on mechanical properties, tensile assays of PHN134, the polymer materials in this study presented inferior tensile strength and Young’s modulus. This was largely attributed to the insertion of eugenol-derived composition impeding the crystallization of copoly(ether ester)s significantly, together with tunable elongation at break. Although we initially targeted the substitutes of petroleum-based dimethyl terephthalate (DMT) and its derived PBT-like materials, the properties presented in this study were opposite. PBT are often used as food packaging materials, which require high Tg, young modulus, and strength values, as well as specific barrier properties, but the polymer materials in this work are appropriate for other application scenarios (elastomer and rubbery), thanks to their modulated mechanical properties by insertion of disparate components. From the point of renewability, this is significant to broaden the types of polymer materials and application fields from bio-based starting materials. The harmony synergistic effect of nipagin and eugenol-derived composition could modulate the properties of such polymer materials in desired application fields.When compared with the widely-used petroleum-based poly (PBT)  materials were synthesized via the melt polycondensation method. The effect of introduction of eugenol-derived units on thermal, crystalline, and mechanical properties was emphatically studied. TGA results revealed that the copoly(ether ester)s featured fairly high thermal stability despite the content of eugenol-derived composition reached 20%. DSC and WXRD data suggested the amorphous nature for both homopoly(ether ester) and copoly(ether ester)s, regardless of the content of highly asymmetrical eugenol-based composition. Young’s modulus and tensile strengths were also found a decreasing tendency with the insertion of eugenol-based composition. More importantly, elongation at break can be tunable via the different content of eugenol-based composition. The cooperative interaction of renewable nipagin and eugenol-derived building blocks provides a practical and suitable way to obtain polymeric materials with desired performance and suitable for various application scenarios.J&K Scientific, Beijing, China), and 1,6-hexanediol  were used as received. All solvents  were analytical grade and used as received without further purification. Silica-gel slices used for thin-layer chromatography (TLC) were purchased from Qingdao Haiyang Chemical Co. Ltd .Nipagin , eugenol , methyl thioglycolate , methyl chloroacetate , 2,2-dimethoxy-2-phenylacetophenone (DMPA)  and tetrabutyl titanate (TBT) , 1,4-dibromobutane  were recorded on a Bio-Rad FTS6000 spectrophotometer at 25 °C. Polymer samples were prepared by grinding the polymeric materials adequately with KBr powder, followed by compressing the mixture to form a pellet. The molecular weights and polydispersity (Ð) values of the samples were determined by gel permeation chromatography  at 35 °C. Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1.0 mL/min. The average molecular weights were calibrated against monodisperse polystyrene (PS) standards. Thermogravimetric analysis (TGA) was carried out using a NETZSCH TG209 instrument. In a typical run, polymer sample was heated from 25 to 800 °C under a nitrogen atmosphere at a rate of 10 °C/min. The temperature leading to 5% weight loss (T5%), temperature for maximum degradation rate (Tmax), and residue weight (%) at 800 °C were recorded. Differential scanning calorimetric (DSC) analysis was carried out by a Mettler-Toledo DSC Q100 calorimeter from TA Instruments. Polymer sample after drying was first heated from room temperature to 150 °C and hold at this temperature for 10 min to eliminate thermal history, then cooled to − 30 °C. Glass transition temperature (Tg) was observed from the second heating run. All runs were carried out at a rate of 10 °C min−1. Indium was used as the calibration standards for temperature. Wide X-ray diffraction (WXRD) patterns were recorded on a D/max-2500 diffractometer using CuKα radiation with a wavelength of 0.1542 nm for dried powder samples. Crystallinity (Xc) was calculated as the quotient between crystalline area and total area. Crystalline and amorphous areas in the X-ray diffraction pattern were quantified by the PeakFit v4.12 software (SeaSolve Software Inc.) with multi-functions , deduction of reductant peaks, AutoFit , AutoScan, etc.). Tensile assays were performed at a stretching rate of 50 mm/min at 25 °C on a Testometric AX Universal Strength Testing Machine.1−xE1x, and PHN11−xE2x  copoly(ether ester)s were prepared from the mixtures of N1, E1 or E2, and 1,6-hexanediol with the pre-calculated stoichiometric composition. The polymerization was performed in a 25 mL Schleck round-bottom flask equipped with a nitrogen inlet, a vacuum distillation outlet, and a magnetic stirrer bar. The polymerization scheme is illustrated in Fig. PHN1 homopoly(ether ester), PHN11H NMR : δ = 7.99–7.97 , 6.91–6.89 , 4.66 , 4.27–4.17 , 1.76–1.62 , 1.49–1.25  ppm; 13C NMR : δ = 168.02 (-OCH2-CO-), 165.68 (Ar-CO-), 161.13 (Ar–C), 131.23 (Ar–C), 123.47 (Ar–C), 113.91 (Ar–C), 64.98–64.78 , 64.34–64.23 , 28.34–28.00 , 25.45–24.98  ppm; FTIR: 2949, 1781 (C=O), 1711 (C=O), 1606, 1510, 1253, 1170, 1105, 977, 848, 770, 697 cm−1.1H NMR : δ = 7.95–7.93 {m, (1-x)∙2H; Ar–H}, 6.88–6.86 {m, (1-x)∙2H; Ar–H}, 6.72–6.68 , 6.63–6.61 , 4.63 {s, (1-x)∙2H; ArO-CH2-CO-}, 4.60 , 4.30–4.09 {m, (1-x)∙4H; -COO-CH2-}, 4.15–4.02 , 3.81 , 3.16 , 2.62–2.57 , 1.88–1.81 {q, 3J = 8.0 Hz, x∙2H; ArCH2-CH2-CH2S-}, 1.79–1.65 {m, (1-x)∙2H; ArCOOCH2-CH2-}, 1.65–1.52 {m, (1-x)∙2H + x∙4H; -COOCH2-CH2-}, 1.50–1.17 {m, (1-x)∙4H + x∙4H; -COOCH2CH2-CH2-} ppm; 13C NMR : δ = 170.48 (-SCH2-CO-), 169.18 (ArOCH2-CO-), 168.29 (ArOCH2-CO-), 166.02 (Ar-CO-), 161.38 (Ar–C), 149.54 (Ar–C), 145.59 (Ar–C), 135.74 (Ar–C), 131.51 (Ar–C), 123.82 (Ar–C), 120.23 (Ar–C), 114.63 (Ar–C), 114.17 (Ar–C), 112.60 (Ar–C), 66.68 and 65.11 (ArO-CH2-CO-), 65.38–64.50 (-COO-CH2-), 55.87 (ArO-CH3), 34.15 (-S-CH2-CO-), 33.59 (Ar-CH2-CH2CH2S-), 31.99 (-S-CH2-CH2CH2Ar), 30.50 (ArCH2-CH2-SCH2-), 28.67–28.54 , 28.40–28.27  ppm; FTIR: 2934, 2860, 1756 (C = O), 1703 (C = O), 1603, 1510, 1422, 1265, 1166, 1105, 1070, 974, 846, 765, 693 cm−1.1H NMR : δ = 7.92–7.90 {m, (1-x)∙2H; Ar–H}, 6.85–6.83 {m, (1-x)∙2H; Ar–H}, 6.73–6.71 , 6.63–6.59 , 4.59 {s, (1-x)∙2H; ArO-CH2-CO-}, 4.19–4.10 {m, (1-x)∙4H; -COO-CH2-}, 4.03–3.97 {m, (1-x)∙4H + x∙4H; ArO-CH2-CH2- and -COO-CH2-}, 3.75 , 3.13 , 2.67–2.46 , 2.02–1.88 , 1.88–1.76 {q, 3J = 8.0 Hz, x∙4H; ArCH2-CH2-CH2S-}, 1.75–1.63 {m, (1-x)∙2H; ArCOOCH2-CH2-}, 1.62–1.47 {m, (1-x)∙2H + x∙4H; -COOCH2-CH2-), 1.46–1.12 {m, (1-x)∙4H + x∙4H; -COOCH2CH2-CH2-} ppm; 13C NMR : δ = 170.29 (-SCH2-CO-), 168.09 (ArOCH2-CO-), 165.78 (Ar-CO-), 161.22 (Ar–C), 149.20 (Ar–C), 146.57 (Ar–C), 133.87 (Ar–C), 131.31 (Ar–C), 123.62 (Ar–C), 120.13 (Ar–C), 114.00 (Ar–C), 113.21 (Ar–C), 112.20 (Ar–C), 68.58 (ArO-CH2-CH2-), 64.90 (ArO-CH2-CO-), 64.42–64.31 (-COO-CH2-), 55.71 (ArO-CH3), 33.95 (-S-CH2-CO-), 33.39 (Ar-CH2-CH2CH2S-), 31.81 (-S-CH2-CH2CH2Ar), 30.42 (ArCH2-CH2-SCH2-), 28.43–28.15 , 25.84 (ArOCH2-CH2-), 25.53–25.06  ppm; FTIR: 2935, 2860, 1756 (C=O), 1708 (C=O), 1603, 1510, 1459, 1264, 1166, 1107, 1071, 976, 847, 809, 765, 693 cm−1.Supplementary Information.


Angelica sinensis, a perennial herb that produces ferulic acid and phthalides for the treatment of cardio-cerebrovascular diseases, prefers growing at an altitude of 1800–3000 m. Geographical models have predicted that high altitude, cool temperature and sunshade play determining roles in geo-authentic formation. Although the roles of altitude and light in yield and quality have been investigated, the role of temperature in regulating growth, metabolites biosynthesis and gene expression is still unclear. In this study, growth characteristics, metabolites contents and related genes expression were investigated by exposing A. sinensis to cooler (15 °C) and normal temperatures (22 °C). The results showed that plant biomass, the contents of ferulic acid and flavonoids and the expression levels of genes related to the biosynthesis of ferulic acid  and flavonoids (CHS and CHI) were enhanced at 15 °C compared to 22 °C. The contents of ligustilide and volatile oils exhibited slight increases, while polysaccharide contents decreased in response to cooler temperature. Based on gene expression levels, ferulic acid biosynthesis probably depends on the CCOAMT pathway and not the COMT pathway. It can be concluded that cool temperature enhances plant growth, ferulic acid and flavonoid accumulation but inhibits polysaccharide biosynthesis in A. sinensis. These findings authenticate that cool temperature plays a determining role in the formation of geo-authentic and also provide a strong foundation for regulating metabolites production of A. sinensis. Angelica sinensis (Oliv.) Diels, commonly named as Dang gui, Dong quai and Tang kuei, is a perennial herb that grows at an altitude of 1800–3000 m with cool, moist and partial shade conditions [A. sinensis, one of the most important herbal drugs in traditional Chinese medicine, are used for nourishing blood, regulating female menstrual disorders and relieving pains and relaxing bowels [nditions . The roog bowels . More reg bowels . The maig bowels .A. sinensis is originally native to China with a population center in Gansu province as well as cultivated in western regions, including the following: Qinghai, Sichuan and Yunnan provinces [A. sinensis grown in Gansu region have deduced that altitude, temperature, sunshine and rainfall are the most influential ecological factors for the accumulation of bioactive compounds  [ca. 2000–2900 m) have found that there is a positive correlation of altitude with the contents of ferulic acid, volatile oils , polysaccharides, flavonoids and phenolics, while moderate altitude (ca. 2500–2600 m) is favorable for root biomass [rovinces ,2,6. Minrovinces ,8. Analyilide A) ,10. Inve biomass ,16,17,18A. sinensis plants prefer moist conditions, the excessive soil water can induce fleshy roots to rot, and soil drought will result in a significant decrease in root yield as well as ferulic acid and volatile oils contents [Generally, altitude affects climate by decreasing temperatures and increasing light intensity and rainfall. Previous studies have demonstrated that reducing light intensity with 50–75% of sunshade can increase root yield and ferulic acid accumulation, meanwhile indirectly decreasing temperature and increasing moisture of air and soil ,20; on tcontents ,22. p-coumarate 3-hydroxylase (C3H) and COMT; and (2) caffeoyl-CoA 3-O-methyltransferase (CCOAMT) pathway involved in enzymes such as 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl shikimate transferase (HCT) and CCOAMT. Flavonoid biosynthesis was catalyzed by enzymes such as chalcone synthase (CHS) and chalcone isomerase (CHI) [For the biosynthesis pathways of ferulic acid, flavonoids and phthalides, previous literature has reported that the biosynthesis of ferulic acid and flavonoids belongs to the phenylpropanoid pathway . Specifise (CHI) ,24. On tse (CHI) ,25,26.A. sinensis in response to temperatures have not been examined. In this study, we probe the role of temperatures in growth, metabolites biosynthesis and genes expression related to ferulic acid, flavonoids, volatile oils and polysaccharides to identify links between cooler growing temperatures and the formation mechanism of geo-authentic medical materials of A. sinensis.To date, plant growth, bioactive metabolites accumulation and related genes expression in Fresh and dry weights (FW and DW) of whole plants were 1.19-fold and 1.51-fold greater at 15 °C than 22 °C, which largely relied on a 1.41-fold and 1.47-fold increase in FW and DW of roots A,B. AddiFerulic acid and flavonoids contents in roots were 1.90-fold and 1.42-fold greater at 15 °C than 22 °C A,B. LiguPAL1, 4CLL4, 4CLL5, 4CLL7, 4CLL9, HCT, C3H, CCOAMT, CCR1 and COMT1) and four genes related to flavonoid biosynthesis  in roots at 15 and 22 °C were quantified. For ferulic acid biosynthesis, the relative expression levels (RELs) of seven genes, PAL1, 4CLL4, 4CLL9, HCT, C3H, CCOAMT and CCR1, exhibited an upregulation of 8.05-fold, 3.65-fold, 2.74-fold, 2.50-fold, 11.48-fold, 10.82-fold and 3.60-fold, while the other three genes, 4CLL5, 4CLL7 and COMT1, exhibited a downregulation of 0.65-fold, 0.35-fold and 0.18-fold, respectively, at 15 °C compared to 22 °C  and I3′H (isoflavone 3′-hydroxylase) exhibited a downregulation of 0.95-fold and 0.40-fold, respectively, at 15 °C compared to 22 °C (The mRNA expression levels of 10 genes related to ferulic acid biosynthesis (to 22 °C . For flato 22 °C .AIMT1) and acetyl-CoA-benzylalcohol acetyltransferase (BEAT)) and three genes related to polysaccharide biosynthesis (sucrose synthase isoform 1 (SUS1), pancreatic alpha-amylase (Amy2) and granule-bound starch synthase 1 (WAXY)) in roots at 15 and 22 °C were quantified. For volatile oil biosynthesis, the RELs of the two genes, AIMT1 and BEAT, exhibited an upregulation of 1.11-fold and 1.10-fold, respectively  [The formation of geo-authentic herbs not only lays on the human society and species but also natural environmental conditions ,25. Prevunshine) ,6,10. InA. sinensis species prefers cool environmental conditions, which is accordance with previous studies that higher-altitude improves the root yield and bioactive metabolites accumulation [Sinopodophyllum hexandrum seedings at 15 °C compared to 22 °C [Panax ginseng at 20 °C/13 °C compared to 25 °C and 35 °C/25 °C [Hypericum perforatum at 15 °C compared to 22 °C [A significant increase in plant biomass was observed at cooler temperatures of 15 °C than 22 °C, which mainly resulted from the significant increase in root diameter . The incmulation ,12,14. Sto 22 °C ; hairy r°C/25 °C ; and whoto 22 °C . PAL, C4H and 4CL  and flavonoid biosynthesis   . A signi) (COMT1  indicate)  with root-tip diameter 0.4–0.5 cm (see −2·s−1). After 15 days, plantlets that contained two leaves , stem diameter (mm), root length (cm), diameter (mm), fresh weight  and dry weight  of aerial parts and roots were measured.v/v, 20 mL) and agitated at 25 °C and 120 r/min for 72 h. The homogenate was centrifuged  at 4 °C and 5000 r/min for 10 min. The extracts were increased to 25 mL with ethanol (95% v/v) for determination of ferulic acid, ligustilide, flavonoids and polysaccharides.After air-dried roots, finely powdered aliquots (0.2 g) were soaked in ethanol . The solution of acetic acid -acetonitrile (B) was the mobile phase with gradient elution: 38–90% B (0–8 min), 90–38% B (8–12 min) and 38% B (12–14 min) at a flow rate of 1.0 mL/min. Ferulic acid and ligustilide contents were evaluated on peak area comparison with a reference standard. Representative HPLC chromatograms of standard reference and samples at 15 and 22 °C were shown in Ferulic acid and ligustilide contents were determined according to the previous protocol . Briefly2-AlCl3-NaOH method [2O (2 mL) and NaNO2 ; after oscillation, and AlCl3  was added and reacted at 22 °C for 1 min; then, NaOH  was added to stop the reaction. An absorbance reader was taken at 510 nm using a spectrometer . Flavonoid content was expressed as milligram of catechin.Flavonoids content was determined using the NaNOH method ,44. Briev/v, 1 mL); after oscillation, sulfuric acid (3 mL) was added and reacted at 22 °C for 30 min. An absorbance reader was taken at 485 nm by using a spectrometer . Polysaccharides content was expressed as milligram of sucrose.Polysaccharides content was determined using the sulfuric acid-phenol protocol method ,46. Brie2O (30 mL) and extracted in a steam distillation apparatus for 8 h; after NaCl (1.0 g) added into the extracts and left standing for 10 min, ethyl acetate (30 mL) was added; following exhaustive extraction (×3), the upper portion was pooled, filtered and dried in vacuo at 35 °C to evaporate ethyl acetate. The rate of volatile oils was expressed as extract volume (mL) of roots weight (g).The extract of volatile oils was conducted using a steam distillation method . BrieflyA. sinensis in our previously published articles [Actin was used as an internal reference. The relative expression level (REL) was evaluated based on a 2−△△Ct method [Total RNA was extracted from roots using a Plant RNA Kit . The quality of the total RNA was examined using 1.0% agarose gel electrophoresis. Based on RNA sequencing and analysis of bolting and flowering of articles ,49, 19 ct method .t-test in SPSS 22.0 was performed for independent treatments with p < 0.05 as the basis for statistical differences.All measurements were performed using three biological replicates. A A. sinensis as well as their related genes expression while inhibiting polysaccharide accumulation. These findings will provide a strong foundation for regulating plant growth and bioactive metabolites production of A. sinensis. The roles of sunshade and rainfall in the geo-authentic formation will be conducted in the upcoming studies.From the above observations, cooler temperatures significantly enhance biomass accumulation, ferulic acid and flavonoid biosynthesis in


Coronavirus disease 2019 (COVID-19) causes a global pandemic and has devastating effects around the world, however, there are no specific antiviral drugs and vaccines for the constant mutation of SARS-CoV-2.In this study, we evaluted the antiviral and anti-inflammatory activities of Liushen Capsules (LS) on different novel coronavirus in vitro, studied its therapeutic effects on novel SARS-CoV-2 infected mice and observed the LS’s clinical efficacy and safety in COVID-19.The antiviral and aiti-inflammatory effects of LS on the 501Y.V2/B.1.35 and G/478K.V1/ B.1.617.2 strains were determined in vitro. A hACE2 mouse model of novel SARS-CoV-2 pneumonia was established. Survival rates, histological changes, inflammatory markers, lung virus titers and the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blotting and immumohistochemical staining in the lungs were measured. Subsequently, the disease duration, prognosis of disease, time of negative nucleic acid and the cytokines levels in serum were used to assess the efficacy of treatment with LS in patients.The results showed that LS  could significantly inhibit the replication of the two SARS-CoV-2 variants and the expression of pro-inflammatory cytokines  induced by the virus in vitro. As for the survival experiment in mice, the survival rate of virus group was 20%, while LS-treatment groups  could increase the survival rate to 60, 100 and 100%, respectively. LS  could significantly decrease the lung titers in mice and it could improve the pathological changes, inhibit the excessive inflammatory mediators  and the protein expression of p-NF-κB p65 in mice. Moreover, LS could significantly decrease SARS-CoV-2-induced activation of p-NF-κB p65, p-IκBα, and p-p38 MAPK and increase the protein expression of the IκBα. In addition, the patient got complete relief of symptoms after being treated with LS for 6 days and was proven with negative PCR test after being treated for 23 days. Finally, treatment with LS could reduce the release of inflammatory cytokines .LS effectively alleviated novel SARS-CoV-2 or variants induced pneumonia in vitro and in vivo, and improved the prognosis of COVID-19. In light of the efficacy and safety profiles, LS could be considered for the treatment of COVID-19 with a broad-spectrum antiviral and anti-inflammatory agent. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global pandemic and had devastating effects around the world. As of 26 September 2021, the ongoing pandemic of COVID-19 had resulted in 231,703,120 confirmed cases and 4,746,620 confirmed deaths worldwide. Patients with confirmed SARS-CoV-2 infection were presented with clinical spectrum from asymptomatic infection to critical illness. Chinese epidemiological data show that COVID-19 with mild or moderate disease (including patients without pneumonia and with mild pneumonia) account for 81% and the percentage of patients with severe disease and critical illness were 14% and 5%, respectively [SARS-CoV-2 viriants have caused a pandemic around the world, which have posed an increase risk on public health. At present, there are four SARS-CoV-2 variants, including GR/501Y.V1/B.1.1.7, GH/501Y.V2/B.1.351, GR/501Y.V3/P.1 and G/478K.V1/B.1.617.2, which are considered as the variants of concern (VOC) by WHO for their higher hospitalization risks, rapid spread and decrease in effectiveness of neutralizing antibodies and some vaccines [Traditional Chinese Medicine (TCM) is extensively used in the treatment of life-threatening respiratory viral infection including SARS-CoV, MERS-CoV, dengue virus, and influenza A virus (including H1N1 and H7N9). According to the transcriptional study and some preclinical experiments, the mechanism of TCM on SARS-CoV-2 mainly include targeting SARS-CoV-2, reducing pro-inflammatory cytokines, protecting multiple organs from damage and preventing lung fibrosis . During Liushen Capsules (LS), a TCM, exhibits multiple biological activities, including anti-viral, antibacterial, anti-inflammatory, anti-tumor and immunomodulatory activities, which has been used for treating viral infection, cancer, heart disease and so on. Our previous study had indicated that LS displayed inhibitory activities against influenza A/PR/8(H1N1) and markedly ameliorated lung inflammation and injury in mice caused by influenza A/PR/8(H1N1) via TLR4/NF-кB signaling pathway . In addiLS (lot: SA01004C) was provided by Suzhou Leiyunshang Medicine Pharmaceutical Co., Ltd. . In the previous study, the index components in LS were detected by high-performance liquid chromatography (HPLC). HPLC revealed that LS contained 0.12% gamabufotalin, 0.10% arenobufagin, 0.26% telocinobufagin, 0.21% desacetylcinobufotalin, 0.25% bufotalin, 0.41% cinobufotalin, 0.27% bufalin, 0.70% resibufogenin, 0.68% cinobufagin, 1.81% cholic acid, 0.27% anserine deoxycholic acid, and 0.23% deoxycholic acid .50) = 10–7/100 μL) were clinically isolated from the First Affiliated Hospital of Guangzhou Medical University. The G/478K.V1/B.1.617.2 strain (TCID50 = 10–6.5/100 μL)and the 501Y.V2/B.1.35 strain (TCID50 = 10–6/100 μL) were clinically isolated from Guangzhou City Eighth People's Hospital. The virus was propagated and adapted as previously described [50 of SARS-CoV-2 were determined using the Reed–Muench method. Virus stocks were collected and stored at − 80 °C. Pathogen-free K18-hACE2 C57BL/6 mice  2018-0008) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.African green monkey kidney epithelial (Vero E6) cells were purchased from ATCC,which were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. The novel coronavirus (SARS-CoV-2) (50% tissue culture infective dose (TCIDescribed . The TCI4 cells/well) were seeded in 96-well plates and inoculated with 100 TCID50 of the virus at 37 °C for 2 h. The inoculum was removed, and the cells were subsequently incubated with indicated concentrations of LS or the positive control remdesivir. Following 72 h of incubation, the percentage of CPE in LS-treated cells was recorded. The 50% inhibition concentration (IC50) of the virus-induced CPE by LS was calculated as described and the selectivity index (SI) was determined from the TC50 to IC50 ratio [To determine the antiviral effects of LS against the 501Y.V2/B.1.35 and G/478K.V1/B.1.617.2 strains, the CPE inhibition assay under the nontoxic concentration of LS was employed. The Vero E6 cell monolayers (5 × 1050 ratio .6 cells/well) were grown in a 12 well plate at 37 °C for 24 h. The monolayer of cells was washed with phosphate buffered saline (PBS) and infected at MOI of 1 of the 501Y.V2/B.1.35 strain or the G/478K.V1/ B.1.617.2 strain. After incubation for 2 h, the viral suspension was removed and overlaid with 0.8% agarose in medium with or without the indicated dose of LS and remdesivir for 72 h. Subsequently, the cells were fixed by 4% formalin for 30 min and stained with 1% crystal violet for 5 min. The number of plaque was counted.The plaque reduction assay was performed as previously described . Briefly5 cells/well) in 12-well plates were washed with PBS and then were inoculated with the 501Y.V2/B.1.35 strain or G/478K.V1/ B.1.617.2 strain at MOI of 1 for 2 h. The inoculums were removed after infection, and the cells were divided into six groups: normal control group (NC), virus-infected group (virus), positive control group (remdesivir), three concentrations of LS , the cells were harvested at 48 h. Total RNA from the different groups was extracted according to the specification of RNA reagent , and reverse transcription of RNAs was quantified by using the PrimeScript™ RT Master Mix kit . Then, RT-PCR was performed on cDNA samples via the SYBR Premix Ex Tap™ II . The PCR data were analyzed using the detection system . The relative amount of PCR products was calculated using the 2−ΔΔCt method as previously described [The cell monolayer . The normal group was intranasally administered with 50 μL PBS without SARS-CoV-2 and other groups were intranasally inoculated with 50 µL of viral suspension. The infected mice were administrated LS , remdesivir (50 mg/kg), or PBS daily for 5 days by gavage after infection for 2 h. The symptoms and mortality of the mice were observed for 5 days. After infection for 5 days, the lung tissues were used to determine the lung virus titers, lung inflammatory factors and histopathology. This animal experiment was approved by the Ethics Committee of Guangzhou Medical University (2021061).50) was used to determine the lung virus titers.Lung tissues of acute lung injury experiment were homogenized in 1 mL PBS. The supernatants of homogenized lung were serially tenfold diluted and inoculated in corresponding wells of Vero E6 cells. The 50% tissue culture infective dose  for 90 min at room temperature. Lung Sects. (5 μm thickness) were incubated at 4 °C with primary antibody against p-NF-κB p65  for 24 h followed by a 15 min wash with PBS. After that, the slides were incubated with a goat anti-rabbit IgG H&L (HRP) at 1:250 for 60 min at room temperature. After being incubated with 3,3’-diaminobenzidine (DAB) and hydrogen peroxide, the slides were visualized with a fluorescence microscope  and assessed by Image-Pro Plus 6.0.The total proteins of the samples were extracted from the cells with radioimmunoprecipitation assay (RIPA) buffer . The protein concentrations of the samples were detected by using the BCA kit . Then, 30 mg of the cell extract was separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then they were transferred to a polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% BSA and incubated with different primary antibodies over night at 4 °C. And then, the membranes with different primary antibodies were incubated with different secondary antibodies for 1 h. The immune complexes were immunoblotted and the immunodetection was performed by using the enhanced chemiluminescence reagents .Two hospitalized adult patients with PCR-proven SARS-CoV-2 infection in the First Affiliated Hospital of Guangzhou Medical University were included. This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University and conducted in accordance with GCP guidelines and the Declaration of Helsinki. Written informed consent was obtained from the patients or legal representatives, and the study was registered with the requisite authority .Patient A, a 54-year-old man, was included On March 17, 2020. Patient B, a 50-year-old man, was included On March 28.Patients were recruited after satisfying the criteria: (1) age ≥ 18 years; (2) axillary temperature ≥ 37.3 °C; (3) mild, normal and severe patients with positive SARS-CoV-2 test; (4) Granting of written informed consent.Patients were excluded if (1) Severe patients satisfying the following situation: I. Respiratory failure with mechanical ventilation; II. shock; III. Combined with organ failure accepting ICU monitoring and treatment; (2) Combined with other respiratory virus infections; (3) Accepted LS treatment with confirmed SARS-CoV-2 infection in a month; (4) Patients allergic to the study drug or patients with allergies; (5) Lactating or pregnant women, women with positive urine pregnancy test or disagree with contraception after participating in the trial for three months; (6) Immunosuppressed patients: malignant tumor; AIDS; having taken immune inhibitors during the last 3 months, having had an organ or bone-marrow transplant; (7) People with severe liver damage and kidney damage; (8) Patients who gave up rescue; (9) Other patients who were not suitable to participate in this study.We randomly assigned participants to receive either basic treatment or LS (0.19 g three times a day), Arbidol Hydrochloride (200 mg three times a day) and basic treatment.Patient A was intervened with basic treatment, while Patient B was treated with LS (0.19 g three times a day), Arbidol Hydrochloride (200 mg three times a day) and basic treatment.Multiple cytokines in serum such as MCP-1, MIP-1β, IL-6, IP-10, sCD40L and so on were monitored. The serum samples were measured by a Bio-Plex Pro Human Cytokine Screening Panel . Sample size was estimated based on the above primary outcome measures. A Bio-Plex Luminex 200 XYP instrument  were used to analyze the assay plate.Results were presented as means ± standard deviation (S.D.). Data were analyzed using analysis of variance (ANOVA) with SPSS ver. 19.0 . Differences between the groups were determined by one-way ANOVA followed Tukey’s honest significant difference (HSD) test. The Wilcoxon rank-sum test was used for data that did not conform to normal distribution. Count data were presented as percentage values, and comparisons between groups were performed using a χ2 test. Differences were considered significant at P < 0.05.50 of LS toward Vero E6 cells was 4.930 μg/mL. The antiviral activities of LS and remdesivir against the 501Y.V2/B.1.35 strain and G/478 K.V1/ B.1.617.2 strain were evaluated by CPE inhibition assay. As shown in Fig. 50 values of LS and remdesivir were 0.4256 μg/mL and 4.376 μM towad to 501Y.V2/B.1.35 strain . LS  could markedly reduce the expression of IL-6, IL-8, IP-10, CCL-5 and MIP-1α in a dose-dependent manner compared with the virus group   and ARDS. Under the stimulus of the 501Y.V2/B.1.35 strain infection, the expression of IL-6, IL-8, IP-10, CCL-5, MIP-1α significantly increased after infection for 48 h compared with NC group . Treatment with LS  and remdesivir (50 mg/kg) could decrease the virus titers .To evaluate the effect of LS on resisting virus in mice, the virus titers in the lung was determined. Compared with the normal group, the virus titers of the model group were markedly increased Fig. B . Treatment with LS (160 and 80 mg/kg) could effectively reduce the expression of IP-10 and IFN-α (P < 0.01 or P < 0.05) , and treatment with LS (40 mg/kg) significantly inhibited the mRNA expression of MCP-1 and IFN-γ (P < 0.01 or P < 0.05).Interferon and Chemokines can be activated by SARS-CoV-2 in COVID-19, which plays an important role in the lung pathology of COVID-19. Thus, the production of cytokines in mice were determined. Compared with the normal group, the mRNA expression of IP-10, MCP-1, IFN-α and IFN-γ in the infected mice were significantly elevated (p < 0.05), and the expression of IκBα of the virus group were significantly decreased (p < 0.05). Compared with the virus group, the protein expression of the p-NF-κB p65, p-p38 MAPK and p-IκBα were significantly reduced in the group treated with , and IκBα was significantly upregulated, while the expression of NF-κB p65 and p38 MAPK was no significant difference. Morrover, the phosphorylations of nuclear translocation of NF-κB p65 in SARS-CoV-2-infected mice was determined by immumohistochemical staining. The result also showed that there was a lower expression of p-NF-κB p65 in the group treated with  when compared with the model group (P < 0.001 or P < 0.01). The effect of LS on inhibition of p-NF-κB p65 was superior to remdesivir. These results showed that LS could reduce the expression of cytokines in the lung probably via interruption of NF-κB/MAPK signaling pathway.To further study whether the antiviral and anti-inflammation mechanisms induced by virus of LS was related to the inhibitory of NF-κB/MAPK signaling pathway activation, the expression of the key proteins related to the NF-κB/MAPK signaling pathway was examined by western blotting and immunohistochemistryFig. B, C. ThePatient A was presented with dry cough and was included On March 17, 2020. The time to conversion to nucleic acid negativity lasted for 40 days. After receiving treatment for 56 days, patient A completely relieved his symptoms and was discharged. Patient B was recruited On March 28, 2020, who was presented with dry cough as well, but dry cough was immediately disappeared after being treated for 6 days which was markedly shorter than patient A. Patient B had shorter length of hospital stay and was discharged after being included in the study for 23 days with negative nucleic acid test twice On April 19, 2020 and April 20, 2020. Specifically, treatment with LS and was associated with accelerated symptom resolution and rapid SARS-CoV-2 clearance. These results indicated that LS could effectively improve prognosis of patient in relieving symptoms and resisting virus.During treatment, the protein expression of inflammatory factors in both patients were monitored. There was a significant increase in serum inflammatory factors  of patient A. Conversely, the inflammatory factors of patient B remained stable even lower. These results indicated that treatment with LS could prevent patient from the hyperinflammatory status.2, Ca, GLU, UREA, CCr and CREA) and cardiac function  were normal , renal function  recommended by WHO have been effective in reducing COVID-19 cases, hospitalizations and deaths. However, there is a decrease in effectiveness of vaccines  and drug50 of remdesivir against the G/478 K.V1/B.1.617.2 strain (12.19 μM) was higher than the 501Y.V2/B.1.35 strain (4.376 μM) and the novel SARS-CoV-2 (0.6505 μM) [50 of LS againt the G/478 K.V1/ B.1.617.2, 501Y.V2/B.1.35 and the novel SARS-CoV-2 was 0.331 μg/mL, 0.4256 μg/mL and 0.6024 μg/m [As of 14 September 2021, SARS-CoV-2 variants of Concern Alpha, Beta, Gamma and Delta have been prevalent in 193, 142, 96 and 180 countries, respectively . To our Our previous study has indicated that LS posed anti-viral and anti-inflammatory activities against SARS-CoV-2 in vitro. Cell-based antiviral assays can quickly determine the effects of LS on SARS-CoV-2. But the results of cell-based assays can’t fully reflect the effects of agents in vivo. The animal models play a vital role in evaluating novel therapies and vaccines. Nowadays, most of the mouse models have low lethality and vary in disease severity . The Ad5In order to further confirm the anti-novel coronavirus effects of LS on COVID-19, two cases of novel Coronavirus pneumonia were observed in the next study. Negnitive PCR-proven test is the key for discharge from the hospital. A study indicated that the median time from symptom onset to viral clearance on real-time RT-PCR was 34 days . Our resIn conclusion, LS significantly inhibited virus replication of the variants and pro-inflammatory factors induced by the variants in vitro, effectively alleviated SARS-CoV-2-induced ALI in mice and had clinical advantages for COVID-19. The therapeutic effects of LS were attributed to inhibition of proliferation of virus and anti-inflammatory effects possibly via blocking the NF-кB/MAPK signaling pathway. These results provide more evidence for the treatment of LS for COVID-19 and reveal the potential mechanism of LS against SARS-CoV-2. Therefore, LS is expected to be an effective therapeutic agent for COVID-19. To provide a strong rationale for their use on COVID-19, a large-scale randomized, double-blinded, placebo controlled clinical trial is necessary to be carried out to confirm the effect of LS on COVID-19.


Aspergillus flavus KUB2, in submerged fermentation at 30°C. Of all the substrates, sugarcane bagasse was a good source for the production of cellulolytic and also hemicellulolytic enzymes. The maximum activities of endoglucanase (CMCase), total cellulase (FPase) and xylanase using sugarcane bagasse as substrate were 8%, 75% and 165%, respectively, higher than those of the commercial substrates. The time course determination of enzyme production revealed that the highest CMCase (1.27 U/ml), FPase (0.72 U/ml) and xylanase (376.81 U/ml) activities were observed at 14 days of fermentation. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) analyses confirmed the efficient structural alteration of sugarcane bagasse caused by enzymatic actions during A. flavus KUB2 cultivation. Based on the results of the hydrolytic enzyme activities, FTIR and SEM, A. flavus KUB2 is suggested as an efficient hydrolytic enzymes producer and an effective lignocellulose degrader, while sugarcane bagasse can be applied as a low-cost carbon source for the economical production of lignocellulose hydrolytic enzymes by A. flavus KUB2.Lignocellulosic wastes, rice straw, sugarcane bagasse, rice bran and sawdust, and pure commercial carboxymethyl cellulose (CMC) and xylan were used as substrates to cultivate cellulolytic fungus,  Lignocellulose is a complex structure composed of lignin (25–30%), hemicellulose (25–30%) and cellulose (35–50%) Hagen . CelluloAspergillus genus is one of the major microbial agents of decay and decomposition and is the most common source of the production of cellulolytic enzymes (Sarkar and Aikat Aspergillus species. A. fumigatus CWSF-7 isolated from soil contaminated with lignocellulosic waste was observed to produce high titre of cellulases. The maximum endoglucanase (CMCase) activity and total cellulase (FPase) activity obtained were 1.9 U/ml and 0.9 U/ml, respectively  and FPase (0.29 U/ml) activities when cultured in medium containing CMC  and FPase (0.56 IU/ml) under submerged fermentation using rice straw as substrate  when sugarcane bagasse was used as substrate. A. niger A12 produced maximum CMCase and xylanase activities of 432 U/l (0.432 U/ml) and 714 U/l (0.714 U/ml), respectively, under submerged cultivation with sugarcane bagasse as substrate  spectroscopy and scanning electron microscopy (SEM) and compared with substrate without fungal inoculation.The present study isolated fungi from soil and decaying wood and screened them for cellulolytic activity on CMC agar and CMC broth. The selected cellulolytic fungal isolate, 2.2.1.Fungal isolates were isolated from soil-containing decomposed plant materials and wood decayed samples. A 10 g of each sample was added to 90 ml of sterile deionised water and mixed for 5 min. After serial dilutions, the suspensions were spread on potato dextrose agar (PDA) supplemented with 100 µg/ml streptomycin and incubated at 30°C for 5–7 days. Further separation was carried out until pure cultures were obtained.2.2.4)2SO4, 1.0 g/l KH2PO4, 1.0 g/l KCl, 0.5 g/l MgSO4•7H2O, 0.5 g/l yeast extract, and 15.0 g/l agar). The pH was adjusted to 5 prior to sterilisation. Inoculation was carried out by placing a 5 mm plug of 5-day-old culture on the centre of the CMC agar medium and incubating at 30°C for 4 days. The growth of fungi was measured based on the diameter of the colony. A 10 ml volume of 0.5% Congo red was then added to each plate. After 15 min, the solution was discarded and the cultures were washed with 10 ml of 1 M NaCl. The observed clear hydrolytic zone around the fungal colony indicated cellulase production. The fungal isolates producing a clear hydrolytic zone were selected for further quantitative screening in liquid medium.All fungal isolates were cultured on carboxymethyl cellulose (CMC) agar. The medium was prepared as described by Irfan et al.  with sli4)2SO4, 2.0 g/l KH2PO4, 0.4 g/l CaCl2•2H2O, 0.3 g/l MgSO4•7H2O, 1.56 mg/l MnSO4•H2O, 5.0 mg/l FeSO4•7H2O, 1.4 mg/l ZnSO4•7H2O and 2.0 mg/l CoCl2)  and calmodulin (CaM) genes was performed using primer pair Bt2a and Bt2b  using the following formula  . The spectra were measured at a spectral solution of 4 cm2.8.The surface morphology of sugarcane bagasse was observed using a scanning electron microscope  with an operating voltage of 5 kV.3.3.1In total, 57 fungal isolates were isolated from the soil containing decomposed plant materials and decayed wood samples. Initial screening of the cellulase-producing fungi was carried out on agar plates using CMC as the sole carbon source and cellulase activity was detected using staining with Congo red. This qualitative determination was based on the interaction of Congo red with intact β-D-glucan in CMC, while the area with the degradation of cellulose by the enzymes resulting in clear zones or the appearance of a pale halo  and incubated under different temperature conditions . Measurements of growth colonies and hydrolysis zones were used to evaluate the cellulase activity using the enzymatic index (EI) which is a semi-quantitative, rapid, simple and practical method for selecting and comparing the potential of microbial enzyme production. Microbial isolates with high EI values were considered to have potential for hydrolytic enzyme production , 7.5 (1.11) and 8 (1.10) as shown in A. flavus KUB2. No cellulase activity by A. flavus KUB2 was detected at an incubation temperature lower than 30°C perhaps because of lower transportation of substrate across the cells at lower temperatures, resulting in a lower yield of the products  and a hemicellulolytic enzyme (xylanase).The use of low-cost substrates for cultivating microorganism provide economical enzyme production. In this study, lignocellulosic wastes, rice straw, sugarcane bagasse, rice bran and sawdust, and pure commercial CMC and xylan were used as substrates to cultivate A. flavus KUB2. Numerous studies on the production of cellulase and xylanase using various carbon sources and microorganisms have reported different yields. A. fumigatus N2, isolated from decaying wood, had the highest CMCase activity (5.6 U/ml) and xylanase activity (91.9 U/ml) when barley straw was used as substrate , while sugarcane bagasse had xylanase activity of 5.53 U/ml. A. flavus MTCC 9390 when cultivated using pure birch wood xylan and oat bran as substrates had xylanase activity levels of 27.28 U/ml and 25.54 U/ml, respectively , FPase (0.72 U/ml) and xylanase (376.81 U/ml) were observed at 14 days of incubation , 1515 cm-1 (aromatic skeleton vibrations in lignin), 1457 cm-1 (CH2 deformation stretching in lignin and xylan), 1266 cm-1 (syringyl ring breathing and C-O stretching in lignin and xylan), 1098 cm-1 , and 898 cm-1 (β-glycosidic linkage) , FPase (0.72 U/ml) and xylanase (376.81 U/ml) at 14 days of fermentation. Additionally, A. flavus KUB2 also was efficient at the degradation of lignocellulosic biomass.In this study,


Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E , were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS+ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated. Bacillus subtilis is nonpathogenic, and displays considerable genetic diversity, even among closely related strains. Its secondary metabolites have been studied for more than 50 years, and numerous studies have revealed that the secondary metabolites are characterized by antimicrobial and other biological activities. + was dominated by common ion peaks at m/z 372.1, 250.1, 233.1, and 215.1  to H-10′  and H-12′  suggested that C-11′ (δC 22.7) was connected with C-10′ (δC 57.8) and C-12′ (δC 31.1). The assignment of the methylene group at C-12′ was confirmed by the HMBC correlation from H-11′  and H-12′  to carbonyl carbon C-13′ (δC 181.5). The HMBC correlation of H-10′  and C-13′ (δC 181.5) suggested the presence of an amide bond between C-10′ and C-12′  suggested the presence of 26 carbons, including four carbonyl carbons, six aromatic carbons, two methylenes, two methines, five methyls, and seven carbons bonded to nitrogen or oxygen. These data revealed that compound 5 possessed structural similarities to the 14′, 15′-methyl-15′-hydroxypyrrolidine amicoumacin C, but differed from the molecular formula of 14′, 15′-methyl-15′-hydroxypyrrolidine amicoumacin C by the addition of –COCH3 +) were identified .The antioxidant activities of the amicoumacins were tested according to their FRAP activities and ABTSg assays A,B. AmicB. subtilis has significant metabolic capabilities and versatile biochemical mechanisms, because of its production of structurally diverse bioactive chemical structures + at m/z 329.2452, 315.2295, and 343.2604 for these three compounds.The crude substances generated using the method described above were processed using a semipreparative HPLC system  to collect fractions from the subfractions. One milliliter of crude extract solution was loaded and separated on a column  from different subfractions using an isocratic program with acetonitrile−HB. subtilis fmb60, the acidified supernatant was adjusted to pH 7.0 with NaOH. The supernatant was then extracted with the same volume of ethyl acetate. The solvent was evaporated under reduced pressure at 50 °C to dryness. The dry matter was dissolved in methanol to collect the supernatant containing the crude substances. The crude extract was subjected to Sephadex LH-20 column chromatography  for further separation. The eluent was MeOH−H2O  and the flow rate was 12 mL/h. The eluent fraction was purified by Nexera X2 HPLC  with a gradient mobile phase  to yield compounds 4–16.To isolate the amicoumacins from For liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis, an UltiMate 3000 HPLC system  equipped with a C18 column  was used. The spray voltage was set to 4.0 kV, and the heated transfer capillary temperature was set to 350 °C. High-resolution mass spectrometry analysis was performed with a Thermo Finnigan Surveyor-LCQ DECA XP Plus  equipped with an electrospray ionization (ESI) source.d4 on Avance 600, 500, and 300 MHz Bruker NMR spectrometer .The structures were identified by nuclear magnetic resonance (NMR) spectroscopy, and the samples were carried out in methanol-Lipoamide D (1): amorphous solid, UV (MeOH) λmax (log ε) 210 (2.10), 248 (0.53), 315 (0.39) nm, HR-ESI-MS m/z 329.2452 [M + H]+ ; Soluble, methanol, DMSO, CHCl3, ethyl acetate; insoluble/poorly soluble in water.Lipoamide E (2): amorphous solid, UV (MeOH) λmax (log ε) 210 (2.08), 248 (0.51), 315 (0.37) nm, HR-ESI-MS m/z 315.2295 [M + H]+ ; Soluble, methanol, DMSO, CHCl3, ethyl acetate; insoluble/poorly soluble in water.Lipoamide F (3): amorphous solid, UV (MeOH) λmax (log ε) 210 (2.10), 248 (0.55), 315 (0.40) nm, HR-ESI-MS m/z [M + H]+ 343.2604 ; Soluble, methanol, DMSO, CHCl3, ethyl acetate; insoluble/poorly soluble in water.Amicoumacin D (4): amorphous solid, [α]23D−93.2° ; UV (MeOH) λmax (log ε) 203 (4.43), 246 (3.74), 314 (3.54) nm, HR-ESI-MS m/z 421.1971 [M + H]+ ; Soluble, methanol, DMSO, CHCl3, ethyl acetate; insoluble/poorly soluble in water.Amicoumacin E (5): amorphous solid, [α]23D−83.7° ; UV (MeOH) λmax (log ε) 203 (4.42), 246 (3.70), 314 (3.44) nm, HR-ESI-MS m/z 519.2348 [M + H]+ ; soluble, methanol, DMSO, CHCl3, ethyl acetate; insoluble/poorly soluble in water.B. subtilis fmb60 was cultured in 50 mL of medium LB at 30 °C overnight. After centrifugation at 8300× g for 5 min, the cells were washed twice with ddH2O, and then resuspended in 8 mL of SET buffer . After adding 10 mg lysozyme and incubating at 37 °C for 1–2 h in a water bath with occasional inverting, 500 μL of proteinase K (20 mg/mL) and 1 mL of 10% sodium dodecyl sulfate (SDS) were added, and the mixture was incubated at 50 °C with occasional inversion for 2 h until the solution became clear. Then, 3.5 mL of 5 M NaCl was added with inverting, after which 15 mL of phenol/chloroform/isoamyl alcohol (25:24:1) was added and mixed thoroughly by inversion to create an emulsion. After centrifugation at 8300× g for 30 min, 500 μL of the aqueous phase was transferred to a new 2 mL tube using an end-cut wide-bore 1 mL tip, then 35 μL of 3 M sodium acetate (pH 7.5) and 1.2 mL of absolute ethanol were added, and gently inverted to mix. The DNA was transferred to a 1.5 mL tube using a blue tip, rinsed with 75% ethanol, dried at room temperature (RT) for approximately 30 min, and dissolved with 200 μL ddH2O. Genomic DNA was prepared.Genomic DNA was isolated from lysed cells by phenol-chloroform extraction and ethanol precipitation . B. subtE. coli were cultivated and manipulated according to standard protocols. The strains and plasmids used in this study are listed in PacI and SpeI to release the NRPS/PKS gene cluster. The linear cloning vector p15A-cm flanked with homology arms to target genes was amplified by PCR using p15A-cm-tetR-ccdB-hyg as template, and primers  in a solvent gradient, with solvents A (water and 0.1% formic acid) and B (CH3CN and 0.1% formic acid) for 25 min: 5% B from 0 to 3 min, 5% B–95% B within 15 min, followed by 4 min with 95% B and 3 min with 5% B at a flow rate of 0.3 mL/min. MS measurements were carried out using a standard ESI source.The correct transformants were subsequently fermented. The fermentation method for 6 CFU/mL. The amicoumacins were two-fold serially diluted in 96-well plates of MHB, with 50 μL in each well and concentrations ranging from 0.78 to 100 μg/mL. Then, 50 μL of bacterial suspension was added to each well, with a final inoculum density of approximately 5 × 105 CFU/mL, and the plates were incubated statically at 37 °C for 24 h [The MICs of the amicoumacins were determined using a broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . The indfor 24 h . Qualityfor 24 h . The MIC+ radical scavenging activities of the amicoumacins were measured using commercial kits . Trolox was used as a positive control.The ferric-reducing ability power (FRAP) and ABTSp < 0.05). All of the forms were made using Excel 2010.SPSS 17.0 software was used for the experimental data processing. Antibacterial and antioxidant experiments were repeated three times, and the Duncan test was used for significance analysis (1–3), amicoumacins D, E , and known metabolites (6−16), have been identified from B. subtilis fmb60. Lipoamides D–F inhibited the growth of foodborne harmful bacteria, and amicoumacins D and E have antioxidant activities. The heterologous expression results demonstrated the function of NRPS/PKS in B. subtilis fmb60, and showed that the ace gene cluster can participate in the synthesis of amicoumacins.In conclusion, five new metabolites, lipoamides D–F (


Complexes 1–7 wereused as molecular precursors to synthesize manganese sulfide (MnS).Olelyamine-capped nanocrystals have been produced via hot injection, while the doctor blading followed by thermolysisyielded thick films. Free-standing polycrystalline powders of MnSare produced by direct thermolysis of precursor powders. All thermolysistechniques produced cubic MnS, as confirmed by powder X-ray diffraction,scanning electron microscopy, energy-dispersive X-ray spectroscopy,and Raman spectroscopy. Magnetic measurements reveal that the α-MnSnanocrystals exhibit ferromagnetic behavior with a large coercivefield strength .Manganese(II) xanthatecomplexes of the form [Mn(S With particle sizes decreasing beneath 100 nm, α-MnSdisplays decreasing TN, which was attributedto a mixture of PM and AFM phases in the nanocrystals (NCs).7At room temperature, all the three bulk phases,α, β,and γ, are paramagnetic (PM) and are antiferromagnetic (AFM)below their respective Neél temperatures of 154, 100, and 80K14 Synthetic routes for the controlled synthesis of single-phase MnSNCs have also been reported.17 For example, Hyeon etal., synthesized hexagonal MnS by heating a mixture of sulfurand MnCl2 in oleylamine at 280 °C.15 Moreover, the use of Mn(II) dithiocarbamate complexes inthe production of manganese sulfide has been reported in a study thatexamined the effect of the counter anion upon the morphology and phaseof the synthesized product.18A range of synthetic methods have beenused to produce MnS nanostructuresincluding chemical bath deposition, hydrothermal, microwave, solvothermal,and sonochemical methods.23 Compared to the classic metal dithiocarbamate single-source precursors,metal xanthates thermally decompose at lower temperatures by the Chugaevelimination.24 The biproducts formed areSCO, H2S, and the corresponding alkene, which are eithergasses or volatile liquids, thus lowering the opportunity for contaminationof the metal chalcogenide phase formed. Size control over the resultingNCs produced has been observed when increasing the alkyl chain lengthof the metal xanthate, avoiding the need for additional surfactants.26Metalxanthates are a readily prepared class of precursors thatoffer a route to a wide variety of metal chalcogenide nanometric materials.O-alkylxanthato)manganese(II)complexes  stabilizedby the bidentate N-donor ligand tetramethylethylenediamine (TMEDA)are synthesized and their crystal structures determined. The complexesare used to produce MnS by three methods: (i) hot injection usingoleylamine (OLA) as a capping agent and trioctylphosphine (TOP) asthe dispersion medium, (ii) solventless thermolysis to produce free-standingMnS powders, and (iii) doctor blade for deposition of MnS films.In this work, the novel bis(2COMe)2·TMEDA](1), [Mn(S2COEt)2·TMEDA] (2), [Mn(S2COnPr)2·TMEDA] (3), [Mn(S2COnBut)2·TMEDA] (4), [Mn(S2COnPen)2·TMEDA](5), and [Mn(S2COnHex)2·TMEDA] (6). [Mn(S2COnOct)2·TMEDA] (7) was also prepared and was characterized only by X-ray diffraction(XRD) analysis, elemental analysis, and melting point. These complexeswere prepared from the reaction of a previously prepared potassiumalkylxanthate and manganese(II) acetate tetrahydrate with the subsequentaddition of TMEDA. All the complexes were soluble in common organicsolvents such as chloroform, tetrahydrofuran (THF), and toluene. Thecomplexes were stored at −20 °C to prevent decomposition.The structures of the complexes are shown in 2, 3, 4, 6, and 7 adopt monoclinic crystal systemswith space groups C2/c, P21/c, P21/c, I2/a, and I2/a, respectively, while 1 is orthorhombic Pbca and 5 is triclinic P1̅.We report here the synthesis and single-crystalstructures of sixnovel manganese xanthates: , is shown in Table S2. The shorter Mn–S and the longest Mn–S bond lengthsinvolving the xanthate ligands were 2.5645 and 2.6750 Å, respectively,as listed in Table S1, and were in goodagreement with those reported for other analogous 1:1 adducts of Mn-dithiocarbonato(xanthate) complexes.27The molecularstructure of 6, and thus, the N–Mn–N angle was the shortestangle compared with other complexes.The symmetricmode of coordination of the xanthate ligands wasreflected in the near equivalence of the associated C–S bonddistances. Within each of the xanthate ligands, the shorter Mn–Sbond had the S atom approximately trans to a N atom, and the two Satoms forming the longer Mn–S bonds were approximately transto each other. Another difference between the structures was thatthe shortest Mn–N bond distance was observed in complex 2, 6, and 7, respectively. In contrast,the difference in the Mn–S bond distances in the asymmetricbinding of 1, 3, 4, and 5 displayed no trend with increase in the alkyl chain length.As the alkyl chain lengthincreased in the structures with identicalbinding of the two xanthates to the Mn, the difference in the bondingmodes of the two ligands became more obvious. The difference in theMn–S bond distances in the symmetrical binding decreased withan increase in the length of the alkyl chain. The Δ(Mn–S)= (longer Mn–S bond distance – shorter Mn–S distance)values for the remaining ligand were 0.11, 0.08, and 0.03 Å for 1, 2, 3, 4, and 5 were almost the same.However, in 6 and 7, the C–O bonddistances were 1.361 (5) and 1.341 (9) Å, respectively, whichwere longer than those of the other complexes. The data shown in Table S1 are consistent with a significant contributionof the resonance form of the xanthate anion that features a formalC=O bond and the negative charges on each of the S atoms. Inthe case of compound 2, the bidentate N-donor ligandshad the same Mn–N1 and Mn–N2 distances (2.293 (15) Å).Because of the restricted ligand bite, the angles N–Mn–Nand S–Mn–S were lower than 90° in a regular octahedron.The N–Mn–N angles averaged at approximately 79.22°(8) and S–Mn–S angles at 69.10° (15), as shownin Table S1. The molecular structures ofother novel complexes are shown in Table S1.The relatively short C–O bond distances of 1.333 (2) Åfor one ligand in complexes Figure S1, all the complexesdisplayed intermolecular hydrogen bonds through the sulfur atoms ofthe neighboring molecules (C–H···S), while inthe complex 4, the C–H···S interactionlinked a molecule with another one by the H from the TMEDA, exceptcomplex 5, wherein the (C–H···S)interaction was not observed. The distances of these interactionswere slightly shorter than the sum of the contact radii ,28 as shown in Table S3.The most distinctdifference between these compounds was how theligand frameworks and the presence of hydrogen bonds affected thecrystal packing in the extended solid state for all of these complexes.As shown in 2, 3, and 6 had two main modes of association between molecules;one of themwas the H from the adduct contact with the S from the other molecule(N–C–H···S) and the H from the alkylgroup contact with S from the other molecule (C–C–H···S),as shown in Figure S1. In contrast, 1 and 7 had one mode of association between molecules,which was the (N–C–H···S) interactionin 1 and the (C–C–H···S)interaction in complex 7. The complex 5 alsoexhibited interchelate distances between S from the molecule and Sfrom another molecule (3.491 Å).Furthermore, 1, 2, 3, and 4 showed a one-step decomposition to form MnS,with the decomposition profile shifting to lower temperature withincrease in the precursor alkyl chain length. For chain lengths ofpentyl and above, the thermogravimetric analysis (TGA) profiles displayeda two-step breakdown. In the case of 5 and 6 precursors, the mass residue obtained from the TGA profiles forthe first decomposition stage (57.5%) agrees with the theoreticalvalue calculated for the removal of one molecule of xanthate and halffrom a second (58%). All the six complexes gave the final solid residueamounts that matched with the predicted value for MnS; see Table S4.Thermal analyses of allthe complexes were conducted up to 600°C under nitrogen; see 30 It was observed that complex 2 showed partial decompositionat 200 °C, leading to poor crystallinity of the material produced;see Figure S3. 250 °C yielded a materialwith good crystallinity, as shown by the p-XRD measurements, 2 to 4, the fall in intensity with the precursor chain length was greaterin the (220) plane.MnS NCs weresynthesized using hot injection of manganese alkylxanthate in TOPand then injected into preheated OLA at 230 °C. OLA is used asa coordinating agent and also can catalyze the degradation of complexesat lower temperatures than the other methods.1, 2, 3, 4, 5, and 6, respectively; see details in Supporting Information. Kan et al. have successfullysynthesized cubic α-MnS NCs of different sizes ranging from20 to 80 nm by a colloidal synthesis route through the reaction ofMnCl2 and S[Si(CH3)3]2 in trioctylphosphineoxide.7 The calculatedlattice parameters and volume are given in Table S5.Estimated crystallitesizes using the Scherrer equation were foundto be 19.5, 17.8, 17.0, 14.9, 10.0, and 9.2 nm for MnS NCs from precursors Supporting Information Figure S4, Table S5†).The atomic percentages of the samples prepared byhot injectionwere determined by energy-dispersive X-ray (EDX) spectroscopy, showingthe composition to be approximately 50:50 of Mn and S and suggestingthat manganese sulfide was formed (1–5 have irregular shapes (6 have a more regular morphology acrossthe sample (The morphologyof the synthesized MnS NCs was observed by scanningelectron microscopy (SEM), and the images are shown in r shapes 5a–e, whie sample 5f.via hot injection from precursor 1 exhibiteda single bandat 635.89 cm–1 and remains approximately the samefor precursors 2, 3, 4, 5, and 6, which is in agreement with reportedvalues; see Figure S5 and Table S5.31Raman spectroscopy revealed that the MnS synthesized 2 [Mn(S2COEt)2·(TMEDA)] had not occurred when observedby XRD , whereas at 350 °C,good crystallinity demonstrated that complete conversion occurred.At lower temperaturesof 250 and 300 °C, the complete conversion of 1–6) generated MnS, with patterns that are in goodagreement with the cubic α-MnS phase .With heating for 1 h at 350 °C, all six precursors  are shown in Figure S9 and Table S6.For precursors articles 7a,b. Forpearance7c–f.The1 by solventless thermolysis exhibiteda band at 635.18 cm–1 which varied insignificantlywhen using precursors 2, 3, 4, 5, and 6 , as previouslyreported.31Raman spectroscopy revealed that the MnS synthesizedfrom precursor 1–6 were indexed to cubic manganese sulfide, α-MnS (ICDD# 03-065-0891); see 1, 2, 3, 4, 5, and 6, respectively (Table S7). The sizes ofthe crystallites are similar to those obtained by using the hot injectionmethod but are larger than those when using the solventless thermolysis.Crystallites that have preferred orientation have been observed inother studies of thin films grown by the chemical bath depositiontechnique.32The diffraction peaksof the thin films prepared from complexes Figures S11 and S7).All precursors resulted in thin films of cubic MnS; see 1, 2, 3, 4, 5, and 6 all exhibited bandsat approximately 635.89 cm–1  and arein agreement with that of bulk MnS.31Raman spectroscopy revealed that the MnS thin films obtained fromthe precursors 2 using solventlessthermolysis were studied. The room-temperature X-band EPR spectrumof α-MnS NCs obtained from complex 2 displays astrong signal with g = 2, characteristic of magneticNCs  with reduced interaction strength in smaller NCs. However, theseNCs were aggregates with smaller particles, which led to their hysteresisloop being closed.7 Puglisi etal. reported the magnetic properties of single-crystal octahedralα-MnS NCs of different sizes . Below 50 K,the NCs showed increase in FC magnetization and a maximum ZFC magnetizationat 25 K, which both confirmed of a transition between a superparamagnetic(SPM) and ferromagnetic (FM) type.16The magnetic propertiesof the α-MnS obtained from complex eticNCs 10. ThemeticNCs 11. The mC/(T – θ), providinga Curie–Weiss constant θ = −254 K , 0.081 kOe (20 nm), and 0.180 kOe (29nm).16 Yang et al. reportedthat at low temperature, hysteresis loops were presented in the FMregion since they displayed open loops with size-dependent Hc ranging from 0.01 kOe (14 nm) to 1.265 kOe(40 nm).3 This result is, to the best ofour knowledge, the first demonstration of a large coercive field  in α-MnS NCs. It is notedthat the magnetization of FM materials depends on the size, shape,and structure of these materials.34The negative sign of θ indicates that theα-MnS NCsobtained from 1), ethyl (2), n-propyl (3), n-butyl (4), n-pentyl (5), n-hexyl (6), and n-octyl (7)] were reported. Complexes 2, 3, 4, 6, and 7 adoptedthe monoclinic crystal system, while 1 was orthorhombicand 5 was triclinic. All the compounds displayed intermolecularhydrogen bonds through the sulfur atoms of the neighboring molecules(C–H···S), except complex 5, whereinthe (C–H···S) interaction was not observed.The distances of these interactions were slightly shorter than thesum of the contact radii . Furthermore, 4 and 5 exhibited intramolecular S–S distancesof 3.491 and 3.565 Å, respectively.The synthesis and thesingle-crystal X-ray structure of seven noveltetramethylethylenediamine manganese(II) bis complexesin which the alkyl group was methyl  in contrast to other studies of nanometric MnSreported in the literature.–1 and with a spectral resolution of 4 cm–1. Elementalanalysis (EA) and TGA were carried out by the Micro-elemental AnalysisService in the Department of Chemistry at the University of Manchester.EA was performed using a Flash 2000 Thermo Scientific elemental analyzer,and TGA data were obtained with a Mettler Toledo TGA/DSC stare systemin the range 30–600 °C at a heating rate of 10 °Cmin–1 under nitrogen flow. Powder XRD analyses werecarried out using an XPert diffractometer with a Cu Kα1 source(λ = 1.54059 Å), and the samples were scanned between 10and 80°; the applied voltage and current were 40 kV and 30 mA,respectively. SEM and EDX spectroscopy analysis were carried out usingTESCAN MIRA3 FEG-SEM. Raman spectra were measured using a Renishaw1000 Micro-Raman System equipped with a 514 nm laser. Single-crystalXRD data for all the complexes were obtained using Mo Kα orCu Kα radiation on a Rigaku FR-X diffractometer. The structureswere solved by the SHELXL  program.35 Non-hydrogen atoms were refined with anisotropic atomicdisplacement parameters. Hydrogen atoms were placed in calculatedpositions, assigned isotropic thermal parameters, and allowed to rideon their parent carbon atoms. Crystals suitable for single-crystalXRD were grown using vapor diffusion of hexane into a solution ofthe requisite metal xanthate in acetone.All chemicals were purchasedfrom Sigma Aldrich or Alfa Aesar and used as received. Melting pointswere determined with a Stuart melting point apparatus . Infrared spectra (IR) were recorded using a Nicolet iS5 ThermoScientific ATR instrument in the range 4000–400 cm25 In brief, potassiumhydroxide  was dissolved in excess methanol andstirred for 2 h at room temperature, followed by cooling to 0 °C.Carbon disulfide  was added dropwiseto the stirred methanolic solution and stirred for 1 h. An aqueoussolution of Mn(CH3COO)2·4H2O in 50 mL of water was added dropwise to the reactionmixture, which was stirred for 30 min to form a brown/yellow solution.TMEDA  was added to the solution while stirringfor 1 h to produce a brown precipitate. The brown precipitate wascollected by vacuum filtration and washed with deionized water. Thefinal product was dried in vacuo overnight and wassubsequently recrystallized from acetone. Yield: 83.5% (3.5 g). mp:138 °C. Elemental analysis: calc (%): C, 31.17; H, 5.76; S, 33.22;N, 7.27; Mn, 14.27%. Found (%): C, 30.98; H, 5.56; S, 33.22; N, 7.02;Mn, 13.94%. IR (νmax/cm–1): 2995(w), 1140–1193 (s), 1037 (s).Potassium methylxanthate was prepared accordingto the previous publications.2 was prepared as 1 except that excess ethanol was used in place of methanol. Yield:88.1% (3.7 g). mp: 137 °C. Elemental analysis: calc (%): C, 34.86;H, 6.34; S, 30.96; N, 6.78; Mn, 13.30% (12.2 g). Found (%): C, 34.94;H, 6.28; S, 31.26; N, 6.70; Mn, 13.01%. IR (νmax/cm–1): 2980 (w), 1142–1185 (s), 1032 (s).3 was preparedas 1 except using n-propanolin place of methanol. Yield: 77.1% (3.8 g). mp: 134 °C. Elementalanalysis: calc (%): C, 38.09; H, 6.86; S, 29.00; N, 6.35; Mn, 12.46%.Found (%): C, 37.88; H, 6.67; S, 29.37; N, 6.12; Mn, 12.18%. IR (νmax/cm–1): 2968 (w), 1145–1179 (s),1043 (s).4 was prepared as 1 except using n-butanolin place of methanol. Yield: 76.3% (4.1 g). mp: 85 °C. Elementalanalysis: calc (%): C, 40.93; H, 7.30; S, 27.26; N, 5.97; Mn, 11.71%.Found (%): C, 40.78; H, 7.15; S, 27.58; N, 5.61; Mn, 11.55%. IR (νmax/cm–1): 2958 (w), 1043 (s), 1142–1180(s).5 was prepared as for 1 except using n-pentanol in place of methanol. Yield: 78.3% (4.5 g). mp:65 °C. Elemental analysis: calc (%): C, 43.45; H, 7.70; S, 25.73;N, 5.63; Mn, 11.05%. Found (%): C, 43.41; H, 7.69; S, 25.98; N, 5.42;Mn, 10.86%. IR (νmax/cm–1): 2952(w), 1040 (s), 1145–1180 (s).The complex 6 was prepared as 1 except using n-hexanolin place of methanol. Yield: 82.9% (5.1 g). mp: 63 °C. Elementalanalysis: calc (%): C, 45.70; H, 8.06; S, 24.35; N, 5.33; Mn, 10.46%.Found (%): C, 45.30; H, 7.99; S, 24.32; N, 5.01; Mn, 10.20%. IR (νmax/cm–1): 2952 (w), 1038 (s), 1142–1182(s).7 was prepared as 1 except using n-octanolin place of methanol. Yield: 80.9% (5.5 g). mp: 60 °C. Elementalanalysis: calc (%): C, 49.55; H, 8.67; S, 22.00; N, 4.82; Mn, 9.45%.Found (%): C, 49.05; H, 8.42; S, 21.91; N, 4.65; Mn, 9.28%.TheMnS NCs were synthesized by dispersing manganese alkylxanthate (0.2g) in 2.0 mL of TOP and then injected into 8.0 mL of preheated oleylamine(OLA) at 230 °C with continuous stirring under a nitrogen atmosphere.The temperature was maintained at 230 °C for 30 min, after whichthe reaction mixture was allowed to cool to room temperature by removalfrom the heat source. Methanol (12.0 mL) was added to the reactionmixture, and the precipitate was collected by centrifugation.3 min–1). Thefurnace tube was subsequently heated to 350 °C, held at thistemperature for 1 h, and allowed to cool to room temperature.A totalof 0.4 g of manganese alkylxanthate was placed in a furnace tube undera stream of argon (300 cm3 min–1).In a typicaldeposition process, 0.02 g of manganese alkylxanthate was added to0.2 mL of THF to form a slurry. The as-prepared complex slurry waspasted on a cleaned glass substrate and distributed uniformly on theglass substrates using a doctor blade made up of stainless steel toform wet thin films of MnS. The films were then placed into furnacetube which was then heated to 350 °C for 1 h under a stream ofargon (300 cm