Geometries of the mono-, di-, tri-, and tetra-ionic thymine  complexes were optimized at the M06-2X/6-311++G level. The IR and UV-Vis spectra, QTAIM, and NBO analysis for the most stable T-4H2O-Br−-1, T-3H2O-[Cnmim]+-Br−-1, T-2H2O-[Cnmim]+-2Br−-1, and T-1H2O-2[Cnmim]+-2Br−-1 hydrates were presented in great detail. The results show that the order of the arrangement stability of thymine with the cations (T-[Cnmim]+) by PCM is stacking > perpendicular > coplanar, and with the anion (T-Br−) is front > top. The stability order for the different microhydrates is following T-5H2O-1 < T-4H2O-Br−-1 < T-3H2O-[Cnmim]+-Br−-1 < T-2H2O-[Cnmim]+-2Br−-1 < T-1H2O-2[Cnmim]+-2Br−-1. A good linear relationship between binding EB values and the increasing number (x + y) of ions has been found, which indicates that the cooperativity of interactions for the H-bonding and π-π+ stacking is varying incrementally in the growing ionic clusters. The stacking model between thymine and [Cnmim]+ cations is accompanied by weaker hydrogen bonds which are always much less favorable than those in T-xBr− complexes; the same trend holds when the clusters in size grow and the length of alkyl chains in the imidazolium cations increase. QTAIM and NBO analytical methods support the existence of mutually reinforcing hydrogen bonds and π-π cooperativity in the systems.The exploration of the ionic liquids’ mechanism of action on nucleobase’s structure and properties is still limited. In this work, the binding model of the 1-alkyl-3-methylimidazolium bromide ([C Vibrio fischeri Br, n = 4, 6, 8, 10, 12) and DNA using the pyrene probing fluorescence. Wang et al. [nmim]Br ILs  occupy the DNA strands as expected +-2Br− among a series of DFT methods as M06L, M06-2X, B3LYP, CAM-B3LYP, LC-ɷPBE, and ωB97XD methods.It has been found that the hybrid meta-GGA functionals M06-2X , includinmim]Br  at the M06-2X/6-311++G/PCM/water level. Based on the most stable configurations existing between thymine and the increased ion clusters, the microhydrated mono-, di-, tri-, and tetra-ionic thymine are calculated to explore the cooperativity of the NCIs established between thymine-cation, thymine-anion, and thymine-cation-anion and the effects of the intermolecular NCIs on the structures and properties of diketone thymine in aqueous solution.In this work, we have first computed and found the most preferred binding modes of thymine with [CwH2O-y[Cnmim]+-xBr−-k, where the number w of a water molecule is 5 to 1. In contrast, the ion number’s sum (x + y) varies from 0 to 4. The item k denotes the stable configuration for the given w and (x + y). Relative energies (ER), as showed in Equation (1), zero-point vibrational energy (ZPVE) corrected, is defined as the difference between the energy of the most stable structure T-wH2O-y[Cnmim]+-xBr−-1 and other forms T-wH2O-y[Cnmim]+-xBr−-k (k > 1).The optimized configurations recognized as local minima were confirmed by the absence of symmetry constraints and imaginary vibrational frequencies evaluated. To simplify the identification, the involving microhydrated ionic complexes are labeled as T-EB), Equation (2), is computed as the discrepancies between the total energy of the optimized complexes and the sum of the energies of the isolated fragments that constitute the studied clusters and is considered the basis set superposition error (BSSE) +-xBr−k systems in aqueous solution. All the quantum chemistry calculations have been performed using the Gaussian-16 suites of programs Br  are constructed and shown in − anion is expected to have stronger proton affinity and prefers to appear around the N1-H  and N3-H  of thymine. In contrast, the [Cnmim]+  cations are partial to approaching the C2=O7  and C4=O8  sites of thymine. In turn, regarding the cations, the positive charges are more evenly dispersed around the hydrogen atoms of head groups (+C(2/4/5)-H) and decrease significantly with the increasing length of the alkyl chain, resulting in possible interactions with the Br− anion and the H-bonding acceptors (HBAs) in thymine; we have also considered that the forming HBs of thymine and 1~5 water molecule(s) explicitly acted as both an H-bonding donor and an H-bonding acceptor (shown in Electronic Supplementary Information (ESI) 2O-1 hydrate will be replaced orderly by the T-4H2O-Br−, T-3H2O-[Cnmim]+-Br−, T-2H2O-[Cnmim]+-2Br−, and T-1H2O-2[Cnmim]+-Br− species (vide infra). The van der Waals distance of Br···H (3.05 Å) +Mono-cationic T-[C. In terms of the relative position of the [Cnmim]+ cations binding to thymine, the T-[Cnmim]+ systems can be partitioned into three arrangements  and +C-H···O8 . The strongest interaction was established by the planes of the imidazolium and pyrimidine ring approximately paralleling each other and accompanying the +C-H···O7 hydrogen-bonding on one end of the complexes, which is quasi-exclusively with the cations increasing alkyl chains. In these T-[Cnmim]+-1 systems, the binding energies follow the trend T-[C2mim]+-1 > T-[C4mim]+-1 < T-[C6mim]+-1 > T-[C8mim]+-1 > T-[C10mim]+-1 , which indicates that the interaction formed between the [C6mim]+ and thymine is slightly more favorable than the binding of other cations to base.ngements : stackinnmim]+-1 and T-Br−-1, the interaction of thymine with [Cnmim]+ cations presents weaker base-ion strength than that with Br− anion  are accompanied by the +C-H···O HB and include Br− anion side sandwiched between the two aromatic rings, interacting via HBs with N1-H and +C(2)-H, wherein the lengths and angles of HBs range within 2.445~2.450 Å and 131.04~131.45° (+C(6)-H···O7), 2.426~2.434 Å and 145.77~146.14° (N1-H···Br−), 2.827~2.835 Å, 118.01~118.44° (+C(2)-H···Br−). On the other hand, the C2=O7 and N1-H bonds in thymine are elongated by 0.003 Å and 0.012 Å, while the +C(6)-H and +C(2)-H bonds are instead shortened by 0.010 Å and 0.000 Å, respectively. It signifies that the thymine base exhibits a higher sensitivity to the H-bonding environment provided by the Br− anion rather than the [Cnmim]+ cations, while the π-π+ interactions still dominate in the base-cation contacts. Upon close inspection of the binding energy of thymine with each cation increasing n, the T-[C6mim]+-Br−-1 complex is presented again as a better intermolecular action in this arrangement . As expected, the strength of thymine-ions binding for the most stable T-[Cnmim]+-Br−-1 complex is strong enough to break the intermolecular interaction for the lowest energy T-2H2O-1 microhydrate.To better understand how thymine interacts with the cations and anion simultaneously, 12 kinds of structures of T-[Cnmim]+ system, as shown in nmim]+-Br−-5), where the Br− anion allows itself on top of the imidazolium cations and forms N1-H···Br− HB with thymine, and the [Cnmim]+ cations form double +C(2)-H···O7 and +C(6)-H···O7 HBs with thymine in an edge-to-face manner—adding an anion and leading to a greater stabilization T-[Cnmim]+-Br−-1, which are differing from T-[Cnmim]+-1 complexes in the position of the HB binding sites between the base and cations, occupying the “sugar edge” site [nmim]+-Br−-9), which are reminiscent of the coplanar HBs in the A-T base pair involving the “Watson-Crick” site  +-Br−-5 in the perpendicular complexes (50.46 > 49.37 < 50.54 > 49.90 > 48.95 kJ/mol), while the slight distinctiveness of T-[C8mim]+-Br−-9 in the coplanar series (46.33 > 47.55 < 48.54 < 48.96 > 47.75 kJ/mol). From the EB vales, to T-[Cnmim]+-Br−-1, 5, 9 complexes, the stable order is stacking > perpendicular > co-planar.3.1.4. Tri-ionic T-[Cnmim]+-2Br−.In common with the T-+ cations, it is mandatory for the extension to obtain the 1:3 forms T-[Cnmim]+-2Br− complexes, the most stable T-[Cnmim]+-2Br−-1 complexes for the independent arrangement are listed in di-ionic thymine systems, a little difference is the T-[C8mim]+-2Br−-1, which is the most favorable energetic complex with the altering carbon atom number n . But consistently, this trend also holds for the microhydrated T-2H2O-[C8mim]+-2Br−-1 in the following Given that the thymine nucleobase is more sensitive to the Brnmim]+-2Br− complexes. The most exciting result is that the preponderance of the stacking isomers was an absolute advantage in the three arrangements mentioned above, that is the stable sequence is stacking (S) > Perpendicular (P) > coplanar HB (H), which is shown to be entirely independent of the cationic alkyl chain length and to have the same tendencies as their magnitudes of EBSSE values order. In the most stable T-2[Cnmim]+-2Br−-1 structures, the thymine nucleobase, sandwiched between the two cationic rings piling up a π+···π···π+ stacking interaction along with two weaker +C(6)-H···O7 HBs, interacts via a single HB with each Br− anion, which also forms HBs contacts with the cations situated above or below the pyrimidine ring. Upon the intercalation of a second ionic pairwise, the base-anion, base-cation, and cation-anion interactions are enhanced obviously by the additional N3-H···Br−, π···π+ stacking along with +C(6)-H···O7 and +C(2)-H···Br− HBs, analogous to the order for interaction strengths of those in the T-[Cnmim]+-Br−-1 structures; the result of EB in absolute values again observed preferential T-2[C6mim]+-2Br−-1 form in the cations increasing n (109.47 > 107.80 < 109.91 > 109.71 > 107.61 kJ/mol).Shortly, a new dimension in studied complexes is being investigated. To further understand the strength and properties of interactions between thymine and ILs, it is natural to continue to expand the system of the action to 1:2 base-IL forms, T-2+-xBr−-1 complexes, the thymine nucleobase is geometrically and energetically more sensitive to the H-bonding environment provided by the Br− anion rather than the π-π+ stacking imparted by the imidazolium cations, with a weaker +C-H···O7 HB  hydrates.In order to further explore the effects of water molecules and the cation and anion of ionic liquid on the geometric structure, electronic structure, spectroscopy and energetics of the thymine, we constructed T-wFree-ionic T0H2O hydrates w-. The solvation-first-shell effect, including 1–5 explicit water molecules, was approximately considered, and the corresponding structures are presented in Mono-ionic T1H2O-Br− hydrates w-. In comparison, the T-0wH2O-1  hydrates are always less stable than those of the T-1wH2O-Br−-1 counterparts, which can be interpreted as a matter that the charged Br− anion replaces the neutral water molecule and interacts with thymine, resulting in a more favorable system and a better correlation between the calculated EB and increasing water molecule numbers w (correlation coefficient R2: 0.919 > 0.894). Satisfactorily, the arrangement of four water molecules is fairly consistent with the NMR experimental results +-Br−-1 should be focused on. A good linear relationship was revealed between the EB values and increasing water number 2w. The regression coefficients are up to 0.99 with the progressive hydration, irrespective of the cationic alkyl chain lengths ···O7 HBs exchanged by the N1-H···Ow and Ow-H···O7 HBs. In − anion is reflected in angles, 142.08~144.11°, and distances, 2.454~2.482 Å of N1-H···Br− in different complexes, which is weaker than that for the T-4H2O-Br−-1 (2.363 Å and 161.58°). It is also the case for N3-H···Br− HB. These results indicate that a larger separation between thymine and Br− anion is observed as a consequence of the cations participating, embodied in π···π+ centroids distances 3.306~3.327 Å. At the same time, Br− interaction is reflected in HBs, the angles in 155.31~160.64° and distances in 2.840~2.882 Å for N1-H···Br−, and the variation of N1-H bond length is about 0.011 Å. lengths a. From ttability . In the e” sites ) is replEB values for the T-3H2O-[Cnmim]+-Br−-1 complexes given a varying n reveals a similar tendency to the T-[Cnmim]+-Br−-1, that the binding intensity can be arranged orderly as follows: T-3H2O-[C2mim]+Br−-1 > T-3H2O-[C4mim]+Br−-1 < T-3H2O-[C6mim]+Br−-1 > T-3H2O-[C8mim]+Br−-1 > T-3H2O-[C10mim]+Br−-1 (126.09 > 125.90 < 127.37 > 126.52 > 126.32 kJ/mol), among which the T-3H2O-[C6mim]+Br−-1 has been discussed by the more favorable arrangement, although to a minor extent.Analysis on the absolute Tri-ionic T3H2O-[Cnmim]+-2Br− hydrates w-. Sequentially, the stacking T-[Cnmim]+-2Br−-1 complexes have participated in stepwise adding of 1~2 explicit water molecule(s) to fill in the “sugar edge” site and “Watson-Crick” site with the two anions, respectively. The hydrous T-2H2O-[Cnmim]+-2Br−-1 is worthy of being mentioned and reproduces the same binding energy order as the anhydrous T-[Cnmim]+-2Br−-1, which the T-2H2O-[C8mim]+-2Br−-1 slightly favored.4H2O-2[Cnmim]+-2Br− hydrates (w4 = 1)Tetra-ionic T-w. Finally, one additional water molecule to the “Watson-Crick” site based on the T-2[Cnmim]+-2Br−-1 reproduces the solvent effect described effectively as a hybrid model and simulated the first explicit solvation shell [2O-2[Cnmim]+-2Br−-1 illustrates a perturbation of N3-H···Ow and Ow-H···O7 HBs in T-3H2O-[Cnmim]+-Br−-1 through a replacement of N3-H···Br− and the second π-π+ stacking along with +(C6-H)···O7 HB, which causes much greater stability. Very consistently, there are no noticeable quantitative discrepancies, with the increasing alkyl chain length in cations, the stabilities for the T-1H2O-2[Cnmim]+-2Br−-1, hydrates to the T-3H2O-[Cnmim]+-Br−-1 complexes still follow the order: T-1H2O-2[C2mim]+-2Br−-1 > T-1H2O-2[C4mim]+-2Br−-1 < T-1H2O-2[C6mim]+-2Br−-1 > T-1H2O-2[C8mim]+-2Br−-1 > T-1H2O-2[C10mim]+-2Br−-1, among which the T-1H2O-2[C6mim]+-2Br−-1 is the greatest and the most favorable one (137.46 > 134.71 < 137.63 > 136.71 > 136.29 kJ/mol).on shell . The T-12O-1 < T-4H2O-Br−-1 < T-3H2O-[Cnmim]+-Br−-1 < T-2H2O-[Cnmim]+-2Br−-1 < T-1H2O-2[Cnmim]+-2Br−-1. The gradual decreasing x and y), the more stable the T-wH2O-y[Cnmim]+-xBr− microhydrates. EB values. Increasing ion number(s) could be observed and is independent of alkyl chain length, indicating a cooperative interaction between thymine and the imidazolium IL in aqueous solutions. It has been pointed out [y[Cnmim]+-xBr− complexes. Herein, the most energetically stable one of each system  is employed for analysis of the spectral features and electronic structures to explore the thorough nature of intermolecular thymine-ILs interactions.As discussed above and in data listed in  finding ,43 that nted out ,43,82,83nmim]+ , and water molecule are shown in −1, and the fingerprint region, 500–1500 cm−1, so they are not well suited to follow the thymine-ion(s) interactions. −1; the main contribution for these two peaks involves ν(N1-H)  and ν(N3-H) , successively. These values of both stretching modes are well coordinated with Singh’s theoretical results (3639 and 3596 cm−1) +-xBr−-1 when interacting with the ILs in aqueous solution, as discussed above.The infrared vibration spectrum of pure thymine, +-Br−-1 hydrates  in mixtures with water have only a little correlation with the alkyl chain. The same point also holds for the tri- and tetra-ionic species in aqueous solutions.As shown in 2O-[Cnmim]+-Br−-1 for the counterpart in an isolated state mainly involve vibrations of the four interaction sites , in 2O-[C6mim]+-Br−-1 are yielding in 3453, 3281, 1749, and 1713 cm−1, with the comparison of the T-4H2O-Br−-1, experiences a respective blue-shift, among which the first two bands are almost determined by the stretching ν(N-H) modes. It implies the formation of new NCIs between thymine and cations at the expense of the old HBs between the base and water molecule, which was pointed out in a reported work [4mim]X . This interaction which either weakens strengths or the broken bonds for the intrinsic HBs between [C4mim]+ cation and the anions causes a high-wavenumber shift upon an aqueous dilution. Closer observation of the mainly respective vibrations corresponding to the four maxima, the v7 (100%), v10 (100%), v32 (98%), and v34 (98%), absorption is weakened relative to the T-4H2O-Br−-1; this indicates the π-π+ stacking formation.Admittedly, the red-shifted changes in the maxima peak for thymine in T-3Hted work  on the Inmim]Br into the “wobble” site , equivalent to the replacement of the two molecules in T-3H2O-[Cnmim]+-Br−-1. In 2O-[C6mim]+-Br−-1, the amount of redshift from 3453 to 3400 cm−1, assigned to the complete ν(N1-H) mode (100%), and the absorption intensity enhanced from 956 to 1245 cm−1, result in a stronger intermolecular N1-H···Br− HB arises from the di- to tetra-ionic microhydrates. However, the peak assigned mainly to the ν(N3-H) mode suffers a blue-shifted wavelength due to the NCI change from the N3-H···Ow to N3-H···Br− HB. The three vibration modes within 1800~1700 cm−1 for the T-1H2O-2[C6mim]+-2Br−-1 correspond to almost the same wavenumbers as those for T-3H2O-[C6mim]+-Br−-1, but the intensities show a visible increment, which is ascribed to the increasing π-π+ stacking. Thus, upon the participation of a second ion pair, in an aqueous solution, thymine is still more sensitive to H-bonding environments with Br− anion than stacking and weaker +(C2-H)···O7 ambiances with the [Cnmim]+ cations, which lives up to the conclusion obtained by Leist et al. +-Br−-1, T-2H2O-[Cnmim]+-2Br−-1 (barring T-2H2O-[C2mim]+-2Br−-1), and T-1H2O-2[Cnmim]+-2Br−-1, the S0→S1 excitation is mainly attributed to the HOMO-2→LUMO (57%), HOMO→LUMO (52~53% for the varying cations), HOMO→LUMO (34~38% for the different cations), and HOMO-3→LUMO (74~78% for the varying cations) excitation, one after another . Thus, the participation of the second ion pair permits a stronger H-bonding and stacking environment conferred by thymine and the imidazolium of IL.It is interesting to note that in addition to the bathochromism, with the hampered hydration, the hypochromic effect has existed in the T-s et al. . They arnmim]Br  in essence. As a local descriptor, the QTAIM, more precise criteria compared with the geometric respect, is employed to describe the H-bonding and van der Waals NCIs +-Br−-1, T-2H2O-[C6mim]+-2Br−-1, and T-1H2O-2[C6mim]+-2Br−-1 hydrates are shown in −, T-[C6mim]+, and [C6mim]+-Br− fragments: electron density ρ(r) and its gradient Laplacian ∇2ρ(r), the kinetic G(r), potential V(r), and total H(r) energy densities. ∇2ρ(r) > 0 is characterized by a closed-shell interaction (including H-bonding and π-stacking), in which the ρ(r) value is typically small or, more specifically, ~0.01 a.u. or less for H-bonding interaction and ~0.001 a.u. for van der Waals contact +-Br−-1 and T-1H2O-2[C6mim]+-2Br−-1 show a greater ρ(r) value for the latter. In the case of interactions between cation-anion, some of the corresponding ρ(r) values are small enough to be distinguishd only by the rather rough geometrics. tetra-ionic T-1H2O-2[Cnmim]+-2Br−-1 hydrates than the tri-, di-, and mono-ionic species. It seems plausible that the ρBCP values have negative correlations with the corresponding H-bond distances while positive ones have the π···π+ distance of centroids.For the four hydrates, Popelier ,103. The2O-[C6mim]+-Br−-1 hydrate and seven BCPs and five CCPs in the T-1H2O-2[C6mim]+-2Br−-1 used to describe π-π+ interactions. The sum of ρBCP and ρCCP (ƩρBCP and ƩρCCP) and the sum of corresponding Laplacian (Ʃ∇2ρBCP and Ʃ∇2ρCCP) are calculated to compare the π-π+ interaction between T-3H2O-[C6mim]+-Br−-1 and T-1H2O-2[C6mim]+-2Br−-1. It transpires that the ƩρBCP and Ʃ∇2ρBCP between two aromatic π rings in tetra-ionic hydrate are greater than those in di-ionic counterpart, although the ƩρCCP and Ʃ∇2ρCCP are almost equal for the two systems . It indicates that the π-π+ interaction between thymine and the imidazolium cation is much stronger upon adding two equivalents of [C6mim]Br. EHB and the ρ(r) values at BCPs for the T-3H2O-[C6mim]+-Br−-1 (0.933), T-2H2O-[C6mim]+-2Br−-1 (0.960), and T-1H2O-2[C6mim]+-2Br−-1 (0.966) hydrates, orderly, which signifies cooperative effect between intermolecular T-Br−, T-[C6mim]+, and [C6mim]+-Br− fragments incrementally present along with the increasing ion clusters, and supports a linear dependence between EB values and increasing ionic number as discussed before.wH2O-y[Cnmim]+-xBr−-1 hydrates. The stabilization of orbital interaction (E2) is calculated charge transfer energies between the Lewis type NBO’s and non-Lewis type NBO’s, followed by Equation (5).iq is the donor orbital occupancy, iε and jε are the diagonal elements  of the donor (i) and acceptor (j). Generally, the higher the E2 values, the greater the stabilities of the targeted complexes [E2 = E2π→π+ + E2π←π+) between thymine and the imidazolium cations [Cnmim]+  is used to describe as the π-π+ interaction. According to the NPA charges in nmim]Br in aqueous solutions are listed in − (lpBr−) and 2-center antibond (BD*) N-H orbitals, which orientates unequivocally to the lpBr−→σ*N1-H for the T-4H2O-Br−-1 and T-3H2O-[Cnmim]+-Br−-1 while the lpBr−→σ*N3-H for the T-2H2O-[Cnmim]+-2Br−-1 and T-1H2O-2[Cnmim]+-2Br−-1. Of the greatest contributor in per ionic thymine hydrate, the specific value shows 74.99, 40.63~45.81 (for varying n), 68.13~69.01, and 75.28~76.33 kJ/mol E2 energy in the respective T-4H2O-Br−-1, T-3H2O-[Cnmim]+-Br−-1, T-2H2O-[Cnmim]+-2Br−-1, and T-1H2O-2[Cnmim]+-2Br−-1 hydrates. Comparison of the di-ionic hydrates, the amount of NPA charge transfer doubles in the tetra-ionic species, where the π-π+ stacking characterized by the ∑E2 values shows 37.24~39.12 kJ/mol for the varying cations and is more than that in T-3H2O-[Cnmim]+-Br−-1 showing 20.82~21.69 kJ/mol. These calculated results coincide with geometrics, energetics, spectroscopy, and AIM analysis.The natural population analysis (NPA) using NBO method was also performed to focus on the atomic charges and orbital interaction energy for the studied T-omplexes . Especiad, p) was used to calculate the three typical interaction modes of cation and two anionic interaction modes between1-alkyl-3-methylimidazolium bromide  ionic liquids to the thymine (T) in aqueous environment. At the same time, the effect of the first solvation shell was investigated. The geometric structure, energetics, spectral characteristics and electronic structure were compared and analyzed, and the hydrogen bond interaction and π-π interaction in the complxes were explored in detail.In this work, the density functional theory method M06-2X/6-311++G(2PCM Model. The relative and binding energies (ER and EB) show the same trend for the favorable arrangements of thymine with the cations (T-[Cnmim]+): stacking > perpendicular > coplanar and with the Br− anion (T-Br−): front > top. The interaction of thymine with [Cnmim]+ cations presents a weaker base-ion strength than that with Br− anion, irrespective of the growing ion clusters increasing overall stabilities or the progressive solvation.Solvation Model. Upon the addition of water-explicit molecule(s) gradually, the relative stabilities continue following: T-5H2O-1 < T-4H2O-Br−-1 < T-3H2O-[Cnmim]+-Br−-1 < T-2H2O-[Cnmim]+-2Br−-1 < T-1H2O-2[Cnmim]+-2Br−-1. A good linear-like relationship between EB values and the increasing number (x + y) of ions indicates the cooperative effect between H-bonding and π-π+ stacking. Additionally, with the increasing lengths of the cationic alkyl chain, regardless of the di- or tetra-ionic hydrates, n equals 6, which is more favored energetically, albeit slightly.mono-, di-, tri-, and tetra-ionic thymine suggest that the formation of N-H···Br− HB between thymine and the anion results in more significant redshifts and incrementing strengths (bathochromism). In contrast, π-π+ stacking interaction between thymine and the imidazolium cations leads to less critical shifts with varying alkyl chain length and decrementing strengths (hypochromism).To the isolated thymine (T), the IR and UV-Vis spectra for the microhydrated −, T-[Cnmim]+, and [Cnmim]+-Br− fragments and fits well with the high linear correlation between EB values and increasing ionic number; the E2 value of lpBr−→σ*N-H is the maximum contribution to the charge transfer energies and is well-beyond the ∑E2 values for the sum of E2π→π+ and E2π←π+.The QTAIM and NBO calculations suggest that the cooperativity effect exists between T-Br


In the original article, there was a mistake in the legend for (A−D) IL-6 IL-1β, TNF-α, and TGF-β in cell supernatant detected by ELISA (n = 3). (E) Apoptosis (Annexin V+/PI− and Annexin V+/PI+) detection by FACS in each experimental group. (F–H) WB and quantification of P-p38, p38, P-ASK1, and ASK1. β-actin was used as a loading control. (I–J) M1 (F4/80 + CD86+) and M2 (F4/80 + CD163+) macrophage proportions detected by FACS in RAW264.7 and statistical analysis (n = 3). (K) WB of iNOS P-p65 p65.  Quantification of band densities fromWB images in (k), (n = 3). (M) mRNA levels of iNOS in lung tissues detected by qPCR (n = 3). All data were presented as mean ± SEM; *p < .05, **p < .01, ***p < .001, and ****p < .0001.AKEX0011 inhibited RAW264.7 from secreting pro-inflammatory cytokines, blocked p38 MAPK signaling, and reduced silica-induced apoptosis and M1 polarization. There were eight cell groups: PBS Control (abbreviated as “Control” in the graphs), PBS + AKEX0011 (200 μg/ml) (abbreviated as “AKEX”), Silica pre, Silica pre + AKEX0011 (100 μg/ml) (abbreviated as “Si pre + AKEX L”), and Silica pre + AKEX0011 (200 μg/ml) (abbreviated as “Si pre + AKEX H”), Silica post, Silica post + AKEX0011 (100 μg/ml) (abbreviated as “Si post + AKEX L”), and Silica post + AKEX0011 (200 μg/ml) (abbreviated as “Si post + AKEX H”). The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


H)-on-3-yl]-3-(4-methylcyclohex-1-yl)-1,2,4-oxadiazole, was proposed. Its molecular and crystal structures were defined and described, whereas the biological activity was predicted with molecular docking.The synthesis of the potential antimicrobial and antiviral drug, 5-[1-benzyl-6-fluoro-7-(piperidin-1-yl)-quinolin-4-1,4-dihydroquinoline-3-carbonitrile as a starting compound that undergoes substitution with hydroxylamine and subsequent cyclization with 4-methylcyclohexane-1-carboxylic acid. It crystallizes from 2-propanol in the triclinic space group PThe title compound, C These purely synthetic compounds have been known since 1962 when the first drug from this group, nalidixic acid, was discovered. The scope of their application has changed and substantially broadened from that time since fluorine atoms were included in the 6-position of the quinoline molecule. Today, fluoroquinolones have positive pharmacokinetic properties, high oral bioavailability, a wide spectrum of action, and good tolerability -on-3-yl]-3-(4-methylcyclohex-1-yl)-1,2,4-oxadiazole.One of the ways to create fluoroquinolones with combined action is the synthesis of hybrid structures that contain several pharmacophores. The basic fluoroquinolone molecule can be structurally modified in several directions at once, which significantly expands both the synthetic possibilities and the opportunities for further research into the biological activity of new compounds -on-3-yl]-3-(4-methylcyclohex-1-yl)-1,2,4-oxadiazole and one of isopropyl alcohol  Å, θ = 178.0 (3)°, φ = 269 (6)°. Atoms N4 and C14 deviate from the mean plane of the other atoms of this ring by 0.652 (2) and −0.696 (3) Å, respectively. Atom N4 has a pyramidal configuration, the sum of bond angles centered at it is 345°. The benzyl substituent is located in the position -ac relative to the endocyclic N3–C3 bond [the C3—N3—C17—C18 torsion angle is 103.8 (3)°] and rotated around the N3–C17 bond [N3—C17—C18—C23 = −35.2 (4)°]. The cyclohexyl fragment also adopts a chair conformation as well  with C24 and C27 deviating from the mean plane through the other ring atoms by 0.590 (3) and −0.622 (4) Å, respectively. It rotates more than the piperidine moiety [the dihedral angle between the mean planes of cyclohexane and oxadiazole derivatives is 46.7 (2)°].The asymmetric unit contains a molecule of 5- should be mentioned. The molecules of the title compound are bound in pairs with π–π stacking  and the pairwise interactions are complemented by the strong O—H⋯O and weak C—H⋯O hydrogen bonds involving two isopropyl alcohol molecules . Therefore, the strongly bound tetramers  Å; H2BA⋯H2BB’, 2.28 Å, symmetry operation: −x + 2, −y, −z + 1). They indicate the existence of repulsion between these isopropyl molecules and so shed light on the reasons for the greater advantage of position A  Å,  A Fig. 3.4.i.e. regions where the specified promolecular electron density exceeds the procrystal density. It was modernized with 2D-fingerprint plots in CrystalExplorer17  emerge on the surface of the title compound  and two more regions exhibit for the isopropyl alcohol . Indeed, the biggest shortening occurs for the strongest hydrogen bonds: O1A—H1A⋯O2 or O1B—H1B⋯O2 in positions A and B, respectively. The regions where the shortening of dnorm is less pronounced are associated with the C16—H16B⋯O1A hydrogen bond and the C30⋯F1 short contact. In addition, with a renormalization of dnorm from  to , the appearance of other intermolecular contacts described above is visible . As expected, the Hirshfeld surface of the isopropanol repeats the contacts O—H⋯O and C16—H16B⋯O1A. Thereby, the appearance of the tetramers described above is also confirmed using Hirshfeld surface analysis, especially after renormalization.A Hirshfeld surface analysis  below the van der Waals radii of the corresponding atoms . Sharp peaks, whose appearance is usually associated with the formation of intermolecular interactions, are found for the C⋯H and O⋯H contacts, as well as for the ‘less significant’ C⋯C contact (3.9% of the area). They point out the hydrogen bonds and stacking interactions in a manner similar to the conventional supramolecular analysis. Similarly to it, the three contributions exceeding 5.0% of the Hirshfeld surface area are found for isopropyl alcohol: H⋯H, 78.1%; O⋯H, 15.3%, and N⋯H, 5.7%; with just one type below the van der Waals radii of the corresponding atoms: O⋯H . The contributions of intermolecular contacts do not differ significantly for positions A and B, except for the appearance of the short contact C2B⋯C2B′ . The dispersion is many times superior to the electrostatic and polarization components in it . The other observation is that the aforementioned tetramers contain the sole interaction of the title molecules in the crystal that has the electrostatic component higher than the dispersive one . It is also the strongest interaction for the isopropanol molecule . Since hydrogen bonding is a directed and thus more rigid interaction than the non-specific interactions like dispersion, the tetramers including two molecules of the title compound and two molecules of isopropyl alcohol can be distinguished as a basic structural motif . However, any short contacts or hydrogen bonds are not observable in this pair of molecules and the interactions of tetramers are completely isotropic according to the energies . At that, the influence of the short contact C30⋯F1 is negligible, because the interaction energy in the dimer including the symmetry equivalent x − 1, y, z − 1 of the title compound is very small . Since the electrostatic interactions are weak around the p-methylcyclohexyl moiety, it is possible to notice that the rotation of the corresponding group is affected mostly by the dispersive interactions among the intermolecular ones. The isopropanol exhibits strong hydrogen bonding within the tetramers, but its molecules endure just minor interactions with the neighbors not connected by hydrogen bonds (Table 2B (the short contact C2B⋯C2B′) significantly affects the intermolecular interactions in the corresponding pairs of isopropanol molecules . This leads to a loss in the total interaction energy from −23.4 to −20.6 kcal mol−1 in position B, making it less energetically favorable for the isopropyl alcohol because of the steric repulsion (lower dispersive interaction) of the methyl moieties.The interactions in the stacking-bonded dimer of the title compound turned out to be two times stronger than any other interaction of an individual molecule in the crystal structure  were utilized for this purpose. The first one is the capsid of the Hepatitis B virus . Hence, for the docking procedure, we can use any of the above-mentioned chains. We used chain A in the actual calculations.The crystal structure of the HBV capsid Y132A contains 157 amino acid residues; the molecular weight is 109.09 kDa. Methyl 4-(2-bromo-4-fluorophenyl)-6-(morpholin-4-ylmethyl)-2-pyrimidine-5-carboxylate was used as a reference ligand. For 5E0I there are six protein chains designated as N-[(5-Methylisoxazol-3-yl)carbonyl]alanyl-l-valyl-N1--4-(benzyloxy)-4-oxo-1-{[(3R)-2-oxopyrrolidin-3-yl]methyl}but-2-enyl)-l-leucinamide was used as a reference ligand.The crystal structure of the COVID-19 main protease contains 306 amino acid residues; the molecular weight is 34.51 kDa. These target macromolecules had previously been utilized for similar research, and therefore we proceeded with them both to obtain the docking results, and determine whether we could enhance the antiviral properties that were observed for some fluoroquinolones that had been hybridized with heterocycles.Jmol , LogS (by ESOL) and topological polar surface area (TPSA)  are presented in the left part of this figure. It can be seen that the obtained lengths are quite large, but fall within the typical range for hydrogen bonds (2.5–4.0 Å).An analysis of the geometric location in the active sites of the selected targets showed that the formation of complexes is facilitated by hydrogen bonds (shown with dotted red arrows) and hydrophobic  intermolecular interactions (designated in yellow) Fig. 8. The D⋯Ain vitro research of both the antimicrobial and antiviral activity.Therefore, the investigated compound is promising for further 7.The starting reagents are commercially available and, as well as solvents, were purchased from Sigma Aldrich and were used without further purification.1 in the reaction scheme), 1-benzyl-6-fluoro-4-oxo-7-(piperidin-1-yl)-1,4-dihydroquinoline-3-carbonitrile, was synthesized via N-alkylation of 6,7-difluoroquinolin-4-one-3-nitrile, followed by amination of the resultant intermediates with piperidine according to the procedures described in Spiridonova et al.  and N,N′-carbonyldiimidazole (1.1 mmol) were dissolved in N,N-dimethylformamide and stirred at 373 K for 20 minutes. After that hydroxylamine (1.1 mmol) was added and heating was maintained for 6 h. The mixture was cooled to room temperature, then water was added, and the obtained precipitate was filtered and recrystallized from an isopropanol–DMF mixture. Then 1 mmol of the obtained compound (2), N,N′-carbonyldiimidazole (1.1 mmol) (compound 3) and 4-methylcyclohexane-1-carboxylic acid (1 mmol) (compound 4) were dissolved in N,N-dimethylformamide (80 mL). The mixture was stirred at 333 K for 1 h. The mixture was then cooled to room temperature and the obtained precipitate was filtered, washed with ethanol and recrystallized from an ethanol–DMF mixture.Compound Further crystallization by slow evaporation of a solution in isopropanol was carried out to provide single block-like colorless crystals suitable for X-ray diffraction analysis (m.p. 476–477 K).8.1H NMR and 101 MHz for 13C NMR. For the NMR spectra, DMSO-d6 was used as a solvent. Chemical shift values are referenced to residual protons (δ 2.49 ppm) and carbons (δ 39.6 ppm) of the solvent as an internal standard.The NMR spectra were recorded on a Varian MR-400 spectrometer with standard pulse sequences operating at 400 MHz for max = 254 nm), API-150EX mass-spectrometer and using a Zorbax SB-C18 column, Phenomenex (100 × 4 mm) Rapid Resolution HT Cartridge 4.6×30mm, 1.8-Micron. Elution started with a 0.1 M solution of HCOOH in water and ended with a 0.1 M solution of HCOOH in acetonitrile using a linear gradient at a flow rate of 0.15 ml min−1 and an analysis cycle time of 25 min.LC/MS spectra were recorded on an ELSD Alltech 3300 liquid chromatograph equipped with a UV detector  δ 9.08 , 8.06 , 7.35–7.23 , 6.31 , 5.43 , 3.27–3.20 , 3.13 , cycloaliphatics: , 0.91 . 13C NMR  δ 181.35, 181.33, 178.80, 165.60, 151.45, 149.00, 146.21, 144.87, 144.75, 138.83, 138.80, 137.36, 129.16, 128.13, 127.52, 122.73, 122.66, 113.97, 113.75, 104.96, 104.91, 104.27, 54.03, 50.90, 50.86, 33.49, 33.36, 30.48, 27.72, 25.90, 24.07, 20.97.Characteristics of title molecule: LC/MS: [MH]9.et al., 2016H)-on-3-yl]-3-(4-methylcyclohex-1-yl)-1,2,4-oxadiazole. The compound with the closest structure is N-benzyl-1-butyl-6-fluoro-4-oxo-7-(piperidin-1-yl)-1,4-dihydroquinoline-3-carboxamide . During the refinement, the distances between the atoms of the disordered isopropyl alcohol were restrained to the following values: 1.524 Å for bonds C1A—C2A, C1B—C2B, C1A—C3A, C1B—C3B with an estimated standard deviation of 0.015 Å . They were subject to a ‘rigid bond’ restraint as well with an estimated standard deviation of 0.0025 Å2.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989023001305/dx2050sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023001305/dx2050Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989023001305/dx2050Isup3.cmlSupporting information file. DOI: 2241448CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Objective: Accumulated evidence highlights the biological significance of oxidative stress in tumorigenicity and progression of colorectal cancer (CRC). Our study aimed to establish a reliable oxidative stress-related signature to predict patients’ clinical outcomes and therapeutic responses.Methods: Transcriptome profiles and clinical features of CRC patients were retrospectively analyzed from public datasets. LASSO analysis was used to construct an oxidative stress-related signature to predict overall survival, disease-free survival, disease-specific survival, and progression-free survival. Additionally, antitumor immunity, drug sensitivity, signaling pathways, and molecular subtypes were analyzed between different risk subsets through TIP, CIBERSORT, oncoPredict, etc. approaches. The genes in the signature were experimentally verified in the human colorectal mucosal cell line (FHC) along with CRC cell lines (SW-480 and HCT-116) through RT-qPCR or Western blot.Results: An oxidative stress-related signature was established, composed of ACOX1, CPT2, NAT2, NRG1, PPARGC1A, CDKN2A, CRYAB, NGFR, and UCN. The signature displayed an excellent capacity for survival prediction and was linked to worse clinicopathological features. Moreover, the signature correlated with antitumor immunity, drug sensitivity, and CRC-related pathways. Among molecular subtypes, the CSC subtype had the highest risk score. Experiments demonstrated that CDKN2A and UCN were up-regulated and ACOX1, CPT2, NAT2, NRG1, PPARGC1A, CRYAB, and NGFR were down-regulated in CRC than normal cells. In H2O2-induced CRC cells, their expression was notably altered.Conclusion: Altogether, our findings constructed an oxidative stress-related signature that can predict survival outcomes and therapeutic response in CRC patients, thus potentially assisting prognosis prediction and adjuvant therapy decisions. Approxivia the Genomic Data Commons (GDC). The raw counts were standardized to count-per-million (CPM) using the edgeR package (Transcriptome profiling (RNA-seq) of colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) was performed, and normal tissue samples were extracted from The Cancer Genome Atlas (TCGA)  package . The thr package , GSE3958 package , and GSE package  were acqp ≤ 0.05 utilizing the edgeR package. Adjusted p was calculated through the Bonferroni and Hochberg method. In total, 1,399 oxidative stress-related genes were extracted from the GeneCards according to relevance score≥7 , a prognosis gene signature was developed with the glmnet package , disease-free survival (DFS), disease-specific survival (DSS), and progression-free survival (PFS) based on the clinical data. Uni- and multivariate Cox regression models were established on the gene signature, and clinical parameters and OS with the survival package. Through the survival-ROC package, receiver operator characteristic curves (ROCs) were drawn, followed by the area under the curve (AUC) value.p < 0.05 as the filtering condition.Immune cell infiltrations were estimated across CRC tissues through Cell Type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), a deconvolution approach proposed by via the TIP approach   algorithm , signifin 2.6.0) .www.cancerRxgene.org) (Drug Sensitivity data were acquired from the Genomics of Drug Sensitivity in Cancer (GDSC) database (ene.org) . IC50 vaene.org) .GSEA was carried out through the Java platform . Gene se2. To induce oxidative stress, the cells were administrated H2O2 in the medium, which was changed daily.Human colorectal mucosal cell lines (FHC) and CRC cell lines (SW-480 and HCT-116) were maintained in DMEM with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37°C humidified incubator with 5% COACOX1, 5ʹ-TAACTTCCTCACTCGAAGCCA-3ʹ (forward), 5ʹ-AGTTCCATGACCCATCTCTGTC-3ʹ (reverse); CDKN2A, 5ʹ-GATCCAGGTGGGTAGAAGGTC-3ʹ (forward), 5ʹ-CCCCTGCAAACTTCGTCCT-3ʹ (reverse); CPT2, 5ʹ-CATACAAGCTACATTTCGGGACC-3ʹ (forward), 5ʹ-AGCCCGGAGTGTCTTCAGAA-3ʹ (reverse); CRYAB, 5ʹ-CCTGAGTCCCTTCTACCTTCG-3ʹ (forward), 5ʹ-CACATCTCCCAACACCTTAACTT-3ʹ (reverse); NAT2, 5ʹ-ACCTGGACCAAATCAGGAGAG-3ʹ (forward), 5ʹ-TGTTCGAGGTTCAAGCGTAAAT-3ʹ (reverse); NGFR, 5ʹ-CCTACGGCTACTACCAGGATG-3ʹ (forward), 5ʹ-CACACGGTGTTCTGCTTGT-3ʹ (reverse); NRG1, 5ʹ-CGGTGTCCATGCCTTCCAT-3ʹ (forward), 5ʹ-GTGTCACGAGAAGTAGAGGTCT-3ʹ (reverse); PPARGC1A, 5ʹ-TCTGAGTCTGTATGGAGTGACAT-3ʹ (forward), 5ʹ-CCAAGTCGTTCACATCTAGTTCA-3ʹ (reverse); UCN, 5ʹ-CAACCCTTCTCTGTCCATTGAC-3ʹ (forward), 5ʹ-CGAGTCGAATATGATGCGGTTC-3ʹ (reverse); and GAPDH, 5ʹ-ACAACTTTGGTATCGTGGAAGG-3ʹ (forward), 5ʹ-GCCATCACGCCACAGTTTC-3ʹ (reverse). With GAPDH as an internal control, the relative expression was quantified using the 2−ΔΔCt method.RNA extraction was performed using the TRIzol reagent  and DNase I, followed by reverse transcription into complementary DNAs (cDNAs) utilizing the Superscript Reverse Transcriptase Kit . RT-qPCR was implemented with the Super SYBR Green Kit  using the ABI7300 RT-qPCR system . The primer pairs included ACOX1 , CDKN2A , CPT2 , CRYAB , NAT2 , NGFR , NRG1 , PPARGC1A , UCN , or GAPDH  at 4°C. The next day, the membrane was incubated with horseradish peroxidase-linked secondary antibodies at room temperature for 1 h. Protein bands were developed using the ECL reagent , and gray values were quantified via ImageJ software.Protein was extracted from cells using RIPA lysis buffer, and protein concentration was assessed using the Bradford protein assay kit . Protein samples were subjected to 8 or 12% SDS-PAGE gels and transferred onto PVDF membranes, followed by incubation with the primary antibody of p < 0.05 was set as statistical difference.Statistical analysis was generated through R 3.6.1. Statistical difference between groups was computed with unpaired Student’s t-test, Wilcoxon test, Kruskal–Wallis test, or one-way analysis of variance. Two-tailed versus 51 normal tissues (ACOX1 expression + 0.0280830167034478 * CDKN2A expression + (-0.163084055105811) * CPT2 expression + 0.0548399857226341 * CRYAB expression + (-0.0107247779354099) * NAT2 expression + 0.0267977941327448 * NGFR expression + (-0.160185818265943) * NRG1 expression + (-0.00515077740891848) * PPARGC1A expression + 0.10199017424903 * UCN expression. For CRC prognosis, ACOX1, CPT2, NAT2, NRG1, and PPARGC1A were protective factors, and CDKN2A, CRYAB, NGFR, and UCN were risk factors  were randomly allocated into the training set (N = 298) and testing set (N = 299) at 1:1 ratio. TCGA patients (NRF2 and KEAP1). Compared with normal tissues, up-regulated KEAP1 and down-regulated NRF2 were found in CRC tissues at the transcriptional level  exhibited down-regulation in the high-risk subset  were down-regulated in the high-risk subset  along with CRC cell lines (SW-480 and HCT-116) through RT-qPCR or Western blot. al cells . Next, wRC cells , indicatACOX1, CPT2, NAT2, NRG1, PPARGC1A, CDKN2A, CRYAB, NGFR, and UCN to predict CRC patients’ clinical outcomes with the LASSO approach.Oxidative stress-related signatures have been established in acute myeloid leukemia , melanomSENP7 can sense oxidative stress to maintain metabolic fitness and antitumor effects of CD8+ T cells  compared with human colorectal mucosal cells (FHC). In H2O2-induced CRC cells, their expression was remarkably altered. Butyrate-induced colonocyte differentiation determines CDKN2A as a prognostic biomarker of CRC recurrence , thereby facilitating precision oncology of CRC.In summary, this study proposed an oxidative stress-related signature composed of


Acacia hydaspica possesses varied pharmacological attributes. We aimed to examine the antimicrobial potential and isolate the active antimicrobial metabolites. The plant extract was fractionated and the antimicrobial activity of the crude extract, fractions and compounds was tested by agar well diffusion and agar tube dilution and broth dilution methods. Bacterial strains selected for bioactivity testing were Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii while selected strains from kingdom fungi were Candida albicans, Cryptococcus neoformans, Fusarium solani and Aspergillus. The active compounds were isolated from Acacia hydaspica by bioassay-guided fractionation and identified by nuclear magnetic resonance and spectroscopic techniques. S. aureus cell surface proteins, Autolysins (Atl), Clumping factor A (ClfA), and Fibronectin Binding Proteins (FnBP), were molecularly docked with Catechin 3-O-gallate (CG) and Methyl gallate (MG) and binding energy and molecular interactions between the proteins and compounds were analyzed. Ethyl acetate (AHE) and Butanol (AHB) fractions of A. hydaspica were the most active fractions against tested microbial strains. Therefore, both were subjected to bioassay-directed fractionation which led to the isolation of one pure active antimicrobial AHE and one active pure compound from AHB fraction besides active enriched isolates. Methyl-gallate (MG) and catechin-3-gallate (CG) are active compounds extracted from AHE and AHB fractions respectively. In antibacterial testing MG significantly inhibited the growth of E. coli (MIC50 = 21.5 µg/ml), B. subtilus (MIC50 = 23 µg/ml) and S. aureus (MIC50 = 39.1 µg/ml) while moderate to low activity was noticed against other tested bacterial strains. Antifungal testing reveals that MG showed potent antifungal activity against F. solani (MIC50 = 33.9 µg/ml) and A. niger (MIC50 = 41.5 µg/ml) while lower antifungal activity was seen in other tested strains. AHB fractions and pure compound (CG) showed specific antibacterial activity against S. aureus only (MIC50 = 10.1 µg/ml) while compound and enriched fractions showed moderate to no activity against other bacterial and fungal strains respectively. Molecular docking analysis revealed that CG interacted more strongly with the cell surface proteins than MG. Among these proteins, CG made a stronger complex with ClfA (binding affinity − 9.7) with nine hydrophobic interactions and five hydrogen bonds. Methyl gallate (MG) and catechin 3-O-gallate (CG) are the major antimicrobial compound from A. hydaspica that inhibit the growth of specific microbes. The occurrence of MG and CG endorse the traditional antimicrobial applicability of A. hydaspica, and it can be a legitimate alternative to control specific microbial infections.The online version contains supplementary material available at 10.1186/s13568-022-01501-y. Scientific research on raw or crude herbs containing several chemical constituents has attracted attention towards the isolation and synthesis of pure phytochemicals and the chemical preparation of specific structural analogues. There is no doubt that several of our conventional modern-day medicines either directly or indirectly resulted from natural products. World Health Organization (WHO) declared that traditional medicines are harmless cures for ailments of both microbial and non-microbial origins WHO . SeveralAcacia genus have also been accounted to be useful in conventional drugs to cure several microbial infections and an array of related diseases including malaria, sore throat  and toothache (bark)  to obtain a fine powder of plant sample and then this plant material was utilized for solvent extraction. The powder (3 kg) plant sample was soaked in 6 l of crude methanol (95%) solution for 72 h at 25 °C and extracted thrice. Filteration was done using Whatman filtrating paper No. 1 and the filtrate was evaporated at 40 °C using a rotary vacuum evaporator  and the crude methanol extract (AHM) was obtained.n-hexane, chloroform, ethyl acetate and n-butanol to obtain their soluble fractions. The filtrate was concentrated using a rotary evaporator. The scheme of fractionation is summarized in our previous publications , chloroform (AHC), ethyl acetate (AHE), n-butanol (AHB) and remaining water (aqueous) fraction (AHA) were weighed to determine the resultant mass and quantified in terms of percentage of dried plant sample. Fractions were stored at 4 °C until used for experimentation.Fractional separation of crude methanol extract was done by solvent–solvent extraction. Briefly crude methanol extract (12 g) was dissolved in distilled water (500 ml) in a separating funnel of 1000 ml capacity and successively partitioned with The initial antibacterial and anti-fungal screening of crude extract AHM and its derived fractions  were carried out using the agar well diffusion method and agar tube dilution technique respectively  and Micrococcus luteus (ATCC 10240), and four Gram-negative E.g. Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 15224), Klebsiellapneumoniae (MTCC 618), Pseudomonas aeroginosa (ATCC 27853). Bacteria used for inoculum preparation were grown in a nutrient broth medium (MERCK). Antifungal activity of A. hydaspica methanol extract/fractions was analyzed against 4 fungal strains i.e. Aspergillus flavus (ATCC 0064), Aspergillus fumigatus (ATCC 66), Aspergillus niger (ATCC 0198), Fusarium solani (ATCC 0300). Fungal strains were maintained on Sabouraud Dextrose Agar (MARK) slants. Bacterial culture turbidity was checked using McFarland 0.5 of Mc Farland standard turbidity scale corresponding to about 1.5 × 106 colony forming unit (CFU) per ml The microbial suspensions were standardized by adjusting the optical density to 0.1 for bacteria and 0.09 for yeast at 600 nm . These dilutions correspond to about 2.5 × 105 spores/ml for yeast and 106 CFU/ml for bacteria.The antibacterial activity of extract/fractions was checked against 6 bacterial strains, two Gram-positive e.g., Serial dilutions were made for all crude fractions by using DMSO at a concentration of 32, 16, 8.0, 4.0 and 2.0, 1.0, 0.5, and 0.25 mg/ml.2 g of nutrient agar (NA) was mixed with 100 ml distilled water (pH 7.0) to make a nutrient agar medium and then autoclaved. NA medium was cooled up to 45 °C and poured into pre-labelled Petri plates (14 cm). Then 75 ml of nutrient broth media (NB) was dispensed in each plate and left for solidification. Ten wells (8 mm) per plate were prepared 8 mm by using a sterile borer. Swabbing of each test bacterial strain was done or a bacterial lawn was made on the plates with the help of a sterile cotton swab to make sure equal growth over the entire surface area. 100 μl of each dilution of extract/fractions was added into the respective well, cefixime (1 mg/ml) as a positive control and DMSO was used as a vehicle (negative) control in each plate. For each bacterial strain plates were made in triplicate. After sample loading plates were incubated for 24 h at 37 °C. Next to incubation, the spans of the zone of growth inhibition were measured in millimeters (mm) with the help of a Vernier caliper, and the antibacterial activity was assessed thrice.A quantity of 12 mg/ml stock solutions of extract/fractions was made and 67 µl of the sample was used from the initial stock. Terbinafine (12 mg/ml in DMSO) was used as positive control while DMSO was used as a negative control.Media for fungal growth was prepared by dissolving 6.5 g/100 ml in distilled water (pH 5.6). 4 ml of Sabouraud dextrose agar (SDA) was dispensed into screw-capped test tubes and autoclaved for 20 min at 121 °C. After sterilization, the tubes in the autoclave were allowed to 50 °C. 67 μl of test samples from the initial stock solution was added into 4 ml of non-solidified sterilized SDA media to get 200 μg/ml concentration of extract in the media. After the addition of test samples, the tubes were placed at room temperature at a slanting position for solidification. The tubes were inoculated after solidification with a 4 mm diameter piece of fungus extracted from a 7 days old fungal culture. Positive and negative reference sample tubes were inoculated similarly. Next, the sample tubes were kept at 28 °C for 7 days. After that, the antifungal activity was examined by quantifying the linear fungal growth in mm. The growth impediment in the test sample was calculated by comparing the growth in the vehicle control tube. The percentage of growth obstruction was calculated by the following equationAfter the initial antimicrobial screening of crude extract/fractions, AHE and AHB were selected based on their efficacy against microbial strains and lower MIC values for further screening and isolation of bioactive antimicrobial compounds.Detail fractionation procedure for ethyl acetate fraction is reported already in our previous investigations , E. faecalis (ATCC 51299), B. subtilis (ATCC 6633), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), K. pneumoniae (ATCC 13883), A. baumannii (ATCC 19606) while selected strains from kingdom fungi were C. albicans (ATCC 10231), C. neoformans (ATCC 66031), F. solani (ATCC 0300) and A. niger (ATCC 0198).Antimicrobial screening of fractions obtained at each isolation step was performed by broth dilution protocol based on the NCCLS  procedures for antimicrobial vulnerability testing, 14th informational Suppl, NCCLS; M100-S14: 2002, Wayne, PA  of an antimicrobial agent that inhibits the growth of microorganisms (showing no turbidity). Initial screening of antimicrobial activity was performed using 200 µg/ml concentration of each fraction, serial dilutions were made for determining the MIC of active isolates.Cryptococcus neoformans requires a longer incubation typically 24–48 h). The plates were analyzed by recording the optical density (OD) at 600 nm. Data were analyzed as follows:Bacteria and fungi were grown overnight in 15 ml tubes with shaking (225RPM) at the optimal growth temperature for each organism. The turbidity of microbial cultures was adjusted by adding appropriate culture media depending on the test organism under testing, to obtain turbidity equivalent to the 0.5 McFarland test scale using a spectrophotometer at 600 nm OD  value of media blanks from each Abs before calculating the % GI.VLC–AHE fractions with potent antimicrobial activity were mixed and subjected to flash chromatography for further bioactivity-targeted purification of active compounds.® Normal-phase Silica Flash Column, 69-2203-340, TELEDYNE ISCO). The spectrum was monitored at all wavelengths (200–780 nm) with a total run time of 89.2 min. The pattern of step gradient was obtained by holding the mobile phase to the same gradient of the solvent mixture at which the peak starts to appear until the whole peak becomes eluted. 146 fractions collected with FC were pooled into 12 fractions based on their similar TLC (95:5 methanol:chloroform) pattern (observed under UV detection at 254 and 365 nm and staining with Vanillin-HCL reagent) and chromatogram spectral peak appearance. These 12 fractions were submitted for bioactivity testing. Out of these 12 fractions (ISCO-AHE/F1-F12), the fractions which showed promising antimicrobial potential against various tested strains were selected. The MIC50 of the active fraction was calculated and the sample was subjected to NMR and mass spectroscopic analysis studies for identification of metabolite. Analytical HPLC indicated the purity of the compound.Flash chromatography was done on Teledyne ISCO Combi-flash RF-200 automated system. VLC-AHE/F4-F6 fractions (4 g) were combined using methanol and mixed in neutral acid wash standard super cell NF and dried down completely by a rotary evaporator. The dried sample was loaded on Redisep Column (RediSep2O:MeOH), starting with 100% dH2O and ends with 100% DCM. Fractions were collected at each polarity gradient to yield 10 fractions (VLC-AHB/F1-F10). Fractions were dried using a rotary evaporator. Similar to AHE fractions, these fractions were tested for antimicrobial activity, and the active fraction VLC-AHB/F1 was afterwards refined by Sephadex LH 20-column chromatography.15 g of AHB was fractionated by VLC over reverse phase C18 (LiChroprep C18) using gradient elution with polar to nonpolar solvent  was chromatographed on Sephadex LH-20 (3 cm × 120 cm) using MeOH as eluent. The volume of each fraction collected was set to 500 ml and 20 fractions were collected. Fractions were then combined based on a similar TLC (95:5 methanol:chloroform) pattern  and got eight fractions (Sp-AHB/F1-F8). These eight fractions were submitted for bioactivity screening and fraction “Sp-AHB/F4” was shown to be the most potent antimicrobial fraction. 2O  and mobile phase B: acetonitrile. Method: 0–5 min; 15% B in 85% A (isocratic run), 5–25 min; up to 70% B in 30% A, 25–27 min; up to 100% B, 27.1–32 min; 15% B in 85% A (isocratic run). The flow rate was 3 ml/min and the injection volume was 50 μl. The spectrum was monitored at λ 220, 254, 280 and 330 nm. The Fractions were pooled based on chromatogram peaks into 10 fractions (RP-HPLC-AHB/F1-F10) eluted at different retention times with different patterns of UV absorption. These 10 fractions collected by Semi-prep RP-HPLC were tested again for antibacterial activities. RP-HPLC-AHB/F5 showed the most promising antimicrobial potential. Analytical HPLC indicated the purity of the compound. NMR and mass spectroscopic techniques were utilized to identify the compound structure.Semi-prep RP-HPLC was conducted on Agilent HPLC 1260 series using Grace Vision HT C18 column . The fractions were eluted using mobile phase A: H2O  and mobile phase B was acetonitrile. A gradient of time 0–5 min (isocratic run) for 85% A in 15% B, 50–25 min for 15 to 100% B, followed by an isocratic 100% B till 30 min of the run. The flow rate was 1 ml/min and the injection volume was 20 µl. All the test mixtures were scrutinized at 220, 254, 280, 330, and 360 nm wavelengths. Each time column was revamped for 10 min before the subsequent run. The whole chromatographic tasks were accomplished at ambient temperature.A chromatography experiment was run to validate the purity of compounds by employing HPLC–DAD equipped with a Grace Vision HT C18 column . Stock solutions of compounds were made in methanol at 0.5 mg/ml of concentration. Briefly, mobile phase A was H1H and 13C-NMR) for each compound was acquired on a Varian 600 MHz CDD NMR machine with 1H and 13C frequencies of 599.664 and 150.785 MHz, respectively at 25 °C. Spectra of active compounds were obtained in Methanol-d4 and DMSO-d6. Broad-spectrum 1D and 2D Fourier transform methods were exploited as crucial to achieve unambiguous signal assignments. Additionally, 2D shift-correlation analysis , 1H-coupled 13C spectra and selective 1H-decoupling were employed extensively to determine long-range JCH coupling constants and allocate all quaternary carbons unequivocally (DEPT). The DEPT study of resolution improved spectra  was executed on Varian NMR and ACD/NMR processor (Academic Edition software). 1H and 13C chemical shifts were described in ppm comparative to DMSO-d6 (δ 2.5 and δ 39.5 for 1H and 13C respectively), CD3OD  or inner Me4Si standard . The spectral information of isolated compounds is coordinated with published data.NMR Spectrum  (Agilent system).S. aureus compared to other strains so we have selected this bacterial specie for docking studies. Several cell surface proteins are present in S. aureus, among which three proteins: Autolysins (Atl), Clumping factor |A (ClfA), and Fibronectin binding protein (FnBP) play important roles in its virulence by promoting its division, colonization, and surface adhesion, respectively , ClfA (ID: AF-Q53653), and FnBP (ID: AF-A0A0H2XKG3) were obtained from AlphaFold database . Data were analyzed by One-way ANOVA followed by Tukey’s comparison test using Graph pad prism 9 software.A. hydaspica were collected and subjected to bioassay-guided extraction and fractionation as described in the “The aerial parts  of A. hydaspica and its consequent fractions were checked out for antibacterial potency using Gram + ive and Gram −ive bacteria. The antibacterial activity of extract/fractions is calculated by measuring the zone of inhibition in mm and minimum inhibitory concentration (MIC) in mg/ml , while the rest of the fractions did not inhibit the growth of E. coli. Similarly, maximum inhibitory activity was exhibited by AHE against B. subtilus and S. aureus. AHE showed 25 ± 0.5 mm and 23 ± 1.0 mm zone of inhibition against B. subtilus and S. aureus respectively, followed by AHB (20 ± 0.5 mm against B. subtilus and 22 ± 1.5 against S. aureus). AHM showed moderate activity against B. subtilus (18 ± 1.5 mm) and the least (10 ± 0.5 mm) inhibitory activity against S. aureus. Bacillus subtilus and S. aureus strains showed resistance to AHH, AHC and AHA fractions as indicated by no zone of inhibition. Bordetella bronchiseptica and M. luteus showed resistance to all of the tested extract/fractions except AHE, which showed a moderate (20 ± 1.5 mm) effect against B. bronchiseptica and a low (8 ± 1.0 mm) effect against M. luteus. P. aeroginosa strain showed susceptibility from non to high. AHC showed no effect, while AHH, AHB and AHA showed low , AHM showed moderate (19 ± 0.5 mm) and AHE showed slightly high (17 ± 0.5 mm) zone of inhibition. AHM (10 ± 1.0 mm) showed the least antimicrobial activity and AHE exhibited moderate (16 ± 0.5 mm) against K. pneumoniae while the remaining fractions showed no activity. MIC values recorded for A. hydaspica extract/fractions ranged from 0.5 to 8.0 mg/ml. The lowest MIC was recorded for AHE against B. subtilus, S. aureus (Gram +ive bacteria) and E. coli (Gram −ive bacteria), besides AHB also showed the lowest MIC against S. aureus.Crude methanol extract of ml Table . The actA. hydaspica, crude extract and its consequent fractions were executed against four different fungal strains. The activity was calculated as percentage growth inhibition relative to the control group. Table A. hydaspica. AHM and AHE were shown to be active against all tested fungal strains with variable potency. AHM showed good inhibitory activity against F. solani (65 ± 1.5%) and A. flavus (70 ± 1.0%) while moderate antifungal activity against A. niger (55 ± 2.0%) and A. fumigatus (50 ± 0.5%). Significant growth inhibition was recorded in F. solani (85 ± 1.5%) and A. niger (75 ± 2.0%) strains when treated with AHE, while A. flavus (60 ± 0.5%) and A. fumigatus (55 ± 1.0%) showed moderate growth inhibition upon treatment with AHE. Treatment with AHB results in moderate growth inhibition of F. solani (60 ± 1.5%) while non-significant growth inhibition was noticed against other fungal strains. AHH, AHC and AHA exhibited slight antifungal activity against all tested strains.In vitro antifungal potency of n-butanol (AHB) fractions for further fractionation of active constituents. The isolated pure active compounds/compound with antimicrobial activity were further tested against respective microbial strains to get MIC50 values.Initial antimicrobial screening led to the selection of the ethyl-acetate (AHE) and Fractionation of AHE was done by VLC and eluted fractions were combined into 12 fractions (VLC-AHE/F1-F12) based on TLC. Antimicrobial activity was checked for all VLC fractions (VLC-AHE/F1-F12) against seven bacterial, two yeast and two fungal strains using a micro-broth dilution assay. The initial antimicrobial screening was performed with a 200 µg/ml dose of eluted fractions. MIC was calculated only for active fractions. VLC-AHE/F4-F6 eluted with a 7:3 to 5:5 ratio of DCM:MeOH solvent showed comparatively high antimicrobial activity against tested strains in comparison to other fractions. Table Flash system using DCM as solvent: A and MeOH as solvent: B. For the initial 5 min an initial isocratic run with 0% A was performed, then 5.3% B for the next 3.2 min, 7.4% B from 10–28 min and 10.3% B from 29–44 min. Then a linear gradient was selected for the run on CombiFlash systems as it provided adequate resolution for most compounds. This gradient was useful for purifying natural products where the desired compound was not known. Additional file Flash systems. 146 fractions collected fractions were pooled into 12 fractions (ISCO-AHE/F1-F12) according to their similar TLC pattern and spectral peaks.The VLC-AHE/F4-F6 fractions (4 g) were combined and loaded on the Combi50 was calculated for that fraction only (Table E. coli) and two Gram +ive (B. subtilus and S. aureus) bacteria. Table 50 values of ISCO-AHE/F3 against susceptible strains ranged from 21.5–39.0 µg/ml. ISCO-AHE/F5 is moderately active against E. coli (MIC = 151.2 µg/ml) and ISCO-AHE/F7 showed good antibacterial activity only against B. subtilus (MIC = 59.3 µg/ml).The results of the antimicrobial activity of isolated ISCO fractions are summarized in Table 2O:MeOH in varying proportions. AHB is initially partitioned into 10 fractions (VLC-AHB/F1-F10) based on similar TLC patterns. The antimicrobial activity of VLC fractions (VLC-AHB/F1-F10) was evaluated at 200 µg/ml dose. Results indicate that only VLC-AHB/F1 showed good (75%) growth inhibitory activity against S. aureus, while other microbial strains showed resistance. Therefore, the active fraction VLC-AHB/F1 was further purified.By using C18 on vacuum liquid chromatographic (VLC) separation column using mixtures of dHS. aureus to get the active component. 100 µg/ml dose of each fraction was used for initial screening against S. aureus. Results showed that 4 fractions (Sp-AHB/F3-F6) possess antibacterial activity against S. aureus. Sp-AHB/F4 showed the lowest MIC50 (concentration inhibiting 50% growth of bacteria) of 17.1 µg/ml  based on similar TLC behavior. Eight fractions obtained from Sephadex separation (Sp-AHB/F1-F8) were tested only against HPLC examination was performed to check the purity of the Sp-AHB/F4 fraction. HPLC chromatogram of Sp-AHB/F4 showed that it is an enriched fraction that needed further bioassay-guided purification  eluted at different retention times with different patterns of UV absorption. The Additional file S aureus.Sp-AHB/F4 was further refined by semi-preparative high-performance liquid chromatography (Semi-prep RP-HPLC) using the C18 column. The fractions were eluted using mobile phase A: HS. aureus was carried out using a micro-broth dilution assay. 100 µg/ml dose of each fraction was used for initial screening against S. aureus  value of RP-HPLC-AHB/F5 was shown to be 10.1 µg/ml . ESI–MS (−) m/z 183.0534 [M−H].White needle crystals. , δ3.79 , δ 7.11 ;13C NMR  δ 51.0 (OCH3), δ 108.90 , δ 120.91 (C-1), δ 137.76 (C-4), δ 145.12 , δ 166.27 (C=O).13C NMR. 8 Carbons and 5 protons attached to carbon were observed in the 13C NMR and 1H NMR spectra. To determine the position and number of hydroxyl groups, the NMR solvent was shifted to DMSO-d6 as hydroxyl was not seen with Acetone-d6. 1H NMR  clearly reveal the presence two hydroxyls at δ9.44 and one hydroxyl at δ9.11. Close examination of the 1H NMR and 13C NMR spectrum showed a symmetrical molecule with two aromatic protons, δ 7.11 , three hydroxyls, two hydroxyls at δ C 145.12 , and one hydroxyl at δ C 137.76 (C-4), a methyl δ3.79  and an ester carbonyl δ 166.27 (C=O). 1H NMR and 13C NMR spectra are included in Additional file 8H8O5 showed a molecular peak at m/z 183.0534 [M−H] in its negative–ion ESI–MS spectrum.The molecular formula was determined from the MS and A. hydaspica led to the isolation of one active pure antimicrobial compound from AHB beside the active enriched fractions. The HPLC, 1H NMR, 13 C NMR and MS indicated that the bioactive fraction RP-HPLC-AHB/F5 is a pure compound and the compound was identified as Catechin 3-O-gallate. The detailed 1H NMR and 13C NMR data are included in Additional file Bioassay-directed fractionation of 2O) C22 H 18 O10. ESI–MS (−) m/z 441.0988 [M−H].Golden brown powder, : δ 7.045 , δ 5.86 , δ 5.95 , δ 4.56 , δ 3.95 , δ 2.84 , δ 2.5 .13C NMR (Methanol-d4-150.79 MHz): δ 27.07 , δ 66.96 , δ 81.53 , δ 100.66, δ 105.57 , δ 105. 57 , δ 109.18 , δ 113.77, δ 114.84 , δ 119.201 , δ 130.48 , δ 139.07 , δ 145.24 , δ 150.34 , δ 155.38, δ 156.16 , δ165.70 .1H NMR and 13C NMR of the compound were similar to the assignment of catechin 3-O-gallate signals of those reported in previous literature  signal at m/z 442.088 [M−H]. 1H NMR and 13C NMR spectra are included in Additional file The A hydaspica respectively.The purity of active fractions was determined based on the absorption spectrum obtained by analytical RP-HPLC analysis. Absorption spectra of the purified active constituent from AHE and AHB were achieved at 210–600 nm by using a spectrophotometer coupled with a UV-diode array detector (HPLC–DAD). Isolated active components AHE [ISCO-AHE/F3 and AHB [RP-HPLC-AHB/F5] showed single-peak chromatograms. The spectra of the peaks presented an indication of the family of polyphenolic compounds. Figure Acacia hydaspica ethyl acetate extract (AHE) yields a 37.5 mg/g dry sample of methyl gallate whereas a 29 mg/g dry sample of Catechin-3-O-gallate was obtained from AHB.O-gallate made stronger molecular interaction with Atl, ClfA, and FnBP in comparison to methyl gallate  fractions of A. hydaspica methanol extract. Hence AHE and AHB fractions showing lower MIC values and significant microbial growth inhibitory potential were selected for bioassay-guided isolation of lead antimicrobial compounds. Extract/fraction showed specificity in activity against different bacterial strains. The ethyl acetate fraction is the most active fraction against both Gram +ive and Gram −ive bacterial strains and showed lower MIC values. Current findings are in line with the outcomes of Guta et al.  and a et al. , illustra et al.  study inon Cowan .A. hydaspica has potent activity against A. fumigatus strain and A. solani while moderate activity against A. niger and A. flavus. Among the tested fractions, AHE seems to be best potent fraction against all tested strains, inhibit significantly the growth of F. solani and A. niger; whereas moderately inhibits the growth of A. flavus and A. fumigatus. AHB showed inhibitory potential only against F. solani and moderately inhibit its growth while non-significant inhibition was observed against other strains. The varied potency of various fractions of A. hydaspica against tested fungal strains confers a diversity of phytochemicals in different fractions that results in variable/specific potency of activity. Among all tested samples from A. hydaspica ethyl acetate fraction presented the uppermost antifungal activity against all tested strains. Physiologically active flavonoid constituents in AHE and AHM might be responsible for the antifungal activity. Studies point out that flavonoids signify novel leads, and forthcoming research may permit the advancement of pharmacologically suitable antimicrobial mediators or classes of agents  , B. subtilus (MIC50 = 23 µg/ml) and S. aureus (MIC50 = 39.1 µg/ml) while moderate to low activity was noticed against other tested bacterial strains. A study by Mashram, Mahesh and Satish reported the anti-microbial activity of A. nilotica against S. aureus, B. subtilis and E. coli. The leaf and bark extracts showed zone of inhibition ranging from 7.5–16 and 8–15.5 mm respectively and the E. coli strain seems to be more susceptible  and A. niger (MIC50 = 41.5 µg/ml) while lower antifungal activity was seen in other tested strains. AHB fractions and pure compound (CG) showed specific antibacterial activity against S. aureus only while compound and enriched fractions showed moderate to no activity against other bacterial and fungal strains respectively. Catechin 3-O-gallate (CG) showed higher inhibitory potential against S. aureus with MIC50 10.1 µg/ml in comparison to Methyl gallate (MG). However overall MG is a more effective antimicrobial compound isolated from A. hydaspica as it showed inhibitory potential against various strains. A study by Ahmed and coworkers (S. aureus (MIC of 50 µg/ml). It is well acquainted that green tea is a major source of catechins and investigations provide clear proof that the catechin compounds  of green tea constitute its antibacterial activity . These outcomes are noteworthy as S. aureus is among the major sources of both nosocomial and communal-acquired infections globally  as well as fungal strains. Both AHE and AHB fractions possess modest antibacterial activity against various bacterial and fungal strains, the lowest activity was recorded in AHB fractions. Bioassay-guided isolation leads to the isolation of lead antimicrobial compounds from AHE and AHB. Structures of purified compounds were elucidated by 1D and 2D NMR and mass spectrometric analysis (ESI and APCI). Details of NMR data have been published in our previous investigations of the antioxidant activity of compounds  and Methyl gallate (MG) was accessed to target Staphylococcus aureus cell surface proteins, Autolysins (Atl), Clumping factor A (ClfA), and fibronectin-binding protein (FnBP). These proteins promote the adhesion and proliferation of S. aureus and induce its pathogenicity -epicatechin-3′-O-gallate against the adhesion capacity of Porphyromonas gingivalis was evaluated through molecular docking and in vitro testing. Molecular docking analysis revealed the anti-adhesive activity of epicatechin-3-O-gallate--epicatechin-3′-O-gallate , 5–25 min; up to 50% B in 50% A, 25–30 min; upto 100% B, 30.1–35 min; 15% B in 85% A (isocratic run). Figure S4. Analytical HPLC Chromatogram of Sp-AHB/F4 at various wavelengths. Method: 0 min–5 min; 15% B in 85% A (isocratic run), 5–25 min; up to 50% B in 50% A, 25–30 min; upto 100% B, 30.1–35 min; 15% B in 85% A (isocratic run). Figure S5. Semi-prep RP-HPLC chromatograph of Sp-AHB/F4 fraction indicating the partitioning of fractions according to the spectral peaks. Method: 0 min–5 min; 15% B in 85% A (isocratic run), 5–25 min; up to 70% B in 30% A, 25–27 min; up to 100% B, 27.1–32 min; 15% B in 85% A (isocratic run). Figure S6. 1H NMR spectrum of Methyl gallate (MG). Solvent: acetone-d6, Frequency (MHz): 599.67, Nucleus: H, Temperature: 25 °C, Pulse sequence: s2pul, Acquisition time (sec): 1.704, Number of transits: 16, Original point count: 16,384, Spectrum offsets (Hz): 3598.0154, Spectrum type: Standard, sweep width (Hz):9615.4. Figure S7. 13C NMR spectrum of Methyl gallate (MG). Figure S8. 1H NMR spectrum of Catechin 3-O-gallate (CG). Solvent: methanol-d4, Frequency (MHz): 599.67, Nucleus: H, Temperature: 25 °C, Pulse sequence: s2pul, Acquisition time (sec): 1.7039, Number of transits: 32, Original point count: 16,384, Spectrum offsets (Hz): 3598.0154, Spectrum type: Standard, sweep width (Hz): 9615.38. Figure S9. 13C NMR spectrum of Catechin 3-O-gallate (CG).Additional file 2: Table S1. Antibacterial activity of VLC-AHE fractions (Percent growth inhibition). Table S2. Antifungal activity of VLC-AHE fractions (Percent Growth Inhibition). Table S3. Antibacterial activity of A. hydaspica: Isolated fractions/compounds from AHE by flash chromatography (ISCO). Table S4. Antifungal activity of A. hydaspica: Isolated fractions/compounds from AHE by flash chromatography (ISCO). Table S5. Antibacterial activity of A. hydaspica isolated fractions from AHB by Sephadex LH20 chromatography against S. aureus. Table S6. Antibacterial activity of A. hydaspica isolated fractions from AHB by Semi-prep RP-HPLC against S. aureus. Table S7.1H NMR data of antimicrobial compounds isolated from A. hydaspica (Coupling constant J in Hertz). Table S8.13C NMR data of antimicrobial compounds isolated from A. hydaspica. Table S9. Docked complexes of bacterial cell surface proteins and methyl gallate and catechin 3-O-gallate along with the vina score and cavity size.


Short contacts play a more decisive role in the molecular packing compared to van der Waals interactions.The syntheses and crystal structures are reported of 4-phenoxy-substituted phthalonitriles with methoxy groups in the  ortho- and meta-positions of the terminal benzene rings, respectively, namely, 4-(2-methoxyphenoxy)phthalonitrile and 4-(3-methoxyphenoxy)phthalonitrile, both C15H10N2O2. The https://journals.iucr.org/e/issues/2023/03/00/molecular structure was determined using the single-crystal X-ray diffraction method. It is shown that short contacts play a more decisive role in the molecular packing compared to van der Waals interactions.The syntheses and crystal structures are reported of 4-phenoxy-substituted phthalonitriles with methoxy groups in the  For both nitriles, the C13, C14, N1, N2 and O1 atoms are practically coplanar to the A ring with a maximum deviation of 12° in the case of the C13N1 fragment. The O1, O2, and C15 atoms and the B rings are essentially coplanar. The plane of the methoxy group (C15/O2) and its B-ring pivot atom (C3 or C2) is at an angle to the B-ring plane of only 2.21 (6)° (meta) or 1.43 (15)° (ortho). The torsion angles (C15—O2—C3—C2 for meta and C15—O2—C2—C1 for ortho) are 1.32 (15) and −179.1 (2)°, respectively.Both substituted nitriles crystallize as solvent-free crystals; the structures are illustrated in Figs. 13.A group of the A ring with the cyano group (C14≡N2) acceptor attached to the A ring of an adjacent molecule  is also found in this structure, which additionally supports the packing. In the case of 4-(3-methoxyphenoxy)phthalonitrile, because of the favorable spatial arrangement of two A rings of neighboring molecules, stabilization occurs largely through respective π–π interactions. The planes of the A rings of two neighboring molecules are parallel to each other, but offset . The distance between the centers of the A rings is 3.6632 (6) Å (centroid–centroid distance). These geometric characteristics imply the presence of a significant intermolecular π–π attraction phthalonitrile, stabilization of the intermolecular packing is realized mainly through the formation of hydrogen bonds between the donor C8—H8 Tables 1. The res4.et al., 2016et al., 2002ortho- and meta-positions results in only two and one hits, respectively. The ortho-isomers are a phthalonitrile dimer bridged by the o-phenoxy moiety  on silica-gel plates.Synthesis of substituted phthalonitriles: 4-nitrophthalonitrile and 2/3-methoxyphenol in a 1:1 molar ratio were placed in a flask and dissolved in DMF. Further, after complete dissolution of the reagents, 1 mol of potassium carbonate and 1/3 portion of water (in relation to DMF) were added to the mixture. The reaction mass was stirred at 353–363 K for 2.5 h, after which it was cooled to 278 K and poured into a threefold excess (by volume) of 15% aqueous NaCl solution. The precipitate was filtered off, recrystallized from 50% aqueous 2-propanol solution and then dried at 343 K. As a result, light crystals of 4-(2-methoxyphenoxy) phthalonitrile (75%) and 4-(3-methoxyphenoxy) phthalonitrile (89%) were obtained, respectively. Crystals were obtained by slow evaporation of solvent from a saturated solution of phthalonitriles in chloroform.6.Uiso(H) = 1.5Ueq(C) for CH3 groups and Uiso(H) = 1.2Ueq(C) for other groups).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989023000518/yz2027sup1.cifCrystal structure: contains datablock(s) o-C15H10N2O2, m-C15H10N2O2, global. DOI: 10.1107/S2056989023000518/yz2027o-C15H10N2O2sup3.hklStructure factors: contains datablock(s) o-C15H10N2O2. DOI: Click here for additional data file.10.1107/S2056989023000518/yz2027o-C15H10N2O2sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023000518/yz2027o-C15H10N2O2sup7.molSupporting information file. DOI: 10.1107/S2056989023000518/yz2027m-C15H10N2O2sup2.hklStructure factors: contains datablock(s) m-C15H10N2O2. DOI: Click here for additional data file.10.1107/S2056989023000518/yz2027m-C15H10N2O2sup6.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023000518/yz2027m-C15H10N2O2sup7.cmlSupporting information file. DOI: 2215782, 2215781CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Several properties and characterizations of We introduce and study a new class of generalized open sets in generalized topological spaces, called  For a GT μ on X, the pair μ will be called μ-open sets, and a subset A of μ-closed if μ-open. A space A is a subset of a space μ-closure of Aμ-closed sets containing A, and the μ-interior of Aμ-open sets contained in A.Let μ on X is called a strong GT μ is a strong GT. Strong GTs were introduced by Lugojan μ-space. The term “minimal structure” was used sometimes in literature to mean a collection μ in which the only assumption is that A GT see e.g. .μ-b-open sets and μ-β-open sets. In Section μ-regular open sets and regular μ-semi-open sets in terms of μ-preopen.In GTSs, several types of generalized open sets have been studied; the primary purpose of this article is to introduce and study a new type of generalized open sets in GTSs, called 2A of a space μ-semi-open μ-preopen μ-β-open μ-α-open μ-b-open μ-regular closed A is called μ-semi-closed  if μ-semi-open ; and A is called μ-b-closed if μ-b-open and called regular μ-semi-open if there exists a μ-regular open set U such that A subset μ-semi-open sets by σ, the class of μ-preopen sets by π, the class of μ-β-open sets by β, the class of μ-α-open sets by α, and the class of μ-b-open sets by b; we will also denote the class of regular μ-semi-open sets by rσ, the class of μ-regular open sets by r, and the class of μ-regular closed sets by For a space Proposition 2.1Letbe a space. Then(i);(ii).Proposition 2.2Let A be a subset of a space. Then(i) If A is μ-β-open, thenis μ-regular closed;(ii) If A is both μ-semi-closed and μ-β-open, then A is μ-semi-open.Proposition 2.3Letbe a space. Then(i)Each of the families σ, π, α, and β is a GT;(ii)The family b is a GT.σ, b, and β is a strong GT, while π and α need not. The next proposition tells, however, that the statements: μ is a strong GT, α is a strong GT, and π is a strong GT are equivalent.Concerning Proposition 2.4Letbe a space. Then the following are equivalent:(i)is a strong GTS;(ii)is a strong GTS;(iii)is a strong GTS.Proof(i)⇒(ii) Since α ⊂π, so (ii)⇒(iii) Since (iii)⇒(i) Since μ-regular open sets and regular μ-semi-open sets.At the end of this section, we review some decompositions of Proposition 2.5Let A be a subset of a space. Then the following are equivalent:(i) A is μ-regular open;(ii) A is μ-open and μ-semi-closed;(iii) A is μ-open and μ-β-closed;(iv) A is μ-α-open and μ-semi-closed;(v) A is μ-α-open and μ-β-closed;(vi) A is μ-preopen and μ-semi-closed.Proposition 2.6Let A be a subset of a space. Then the following are equivalent:(i) A is μ-regular open;(ii) A is μ-open and μ-b-closed;(iii) A is μ-α-open and μ-b-closed.Proposition 2.7Let A be a subset of a space. Then the following are equivalent:(i) A is regular μ-semi-open;(ii) A is μ-semi-open and μ-semi-closed;(iii) A is μ-b-open and μ-semi-closed;(iv) A is μ-β-open and μ-semi-closed;(v) A is μ-semi-open and μ-b-closed;(vi) A is μ-semi-open and μ-β-closed.3This section is mainly devoted to introducing and studying Definition 3.1A of a space μ-b-open set U such that A is called A subset μ-β-open sets.We study now the relationship between Proposition 3.2Let A be a-open subset of a space. Then A is μ-β-open.ProofA is μ-b-open set U such that U is μ-β-open as μ-regular closed, and therefore,A is μ-β-open. □Since Corollary 3.3Let A be a subset of a space. If A is-open, thenis μ-regular closed.ProofFollows from Corollary 3.4Let A be a subset of a space. If A is both μ-semi-closed and-open, then A is μ-semi-open.ProofFollows from The following two examples show that the converse of Example 3.5μ is a GT on X and A is μ-β-open but not -open.Let Example 3.6A is μ-β-open but not -open.Let A of a topological space β-open b-open A stand respectively for the closure of A in X and the interior of A in X.Recall that a subset μ-b-open sets and Next, we study the relationship between Lemma 3.7A be a subset of X. Then A is β-open if and only if there exists a preopen set P such that ProofNecessity. Suppose that A is β-open, that is U is open and D dense. Now,U is open and D is dense, it follows by Proposition 2.1 of P is preopen.Sufficiency. Assume that there exists a preopen set P such that P is preopen, A is β-open. □Proposition 3.8Letbe a topological space and A be a subset of X. Then A is-open if and only if A is μ-β-open.ProofA is μ-β-open. Then by μ-preopen set P such that P is μ-preopen, P is μ-b-open. Thus, A is The necessity follows from Remark 3.9μ-b-open set is μ-β-open is-open;(ii)is-closed;(iii)if and only if;(iv)if and only if.Proof(i)(ii) Follows from (i) since(iii) If (iv) If Corollary 3.16Letbe a space and A be a subset of X. Then A is-closed if and only if.ProofCorollary 3.17Letbe a space and A be a subset of X. Then A is-closed if and only if.ProofNecessity. Since A is Sufficiency. Since A is Proposition 3.18Letbe a space and A be a subset of X. Then A is-open if and only if there exists a-open set B such that.ProofNecessity. Take Sufficiency. Since B be μ-b-open set U such that A is Corollary 3.19Letbe a space and A be a subset of X. Then A is-closed if and only if there exists a-closed set B such that.ProofNecessity. Take Sufficiency. Since A is 4μ-regular open sets and regular μ-semi-open sets using The primary purpose of this section is to obtain some decompositions of μ-regular open sets using The following proposition provides decompositions of Proposition 4.1Let A be a subset of a space. Then the following are equivalent:(i) A is μ-regular open;(ii) A is μ-open and-closed;(iii) A is μ-α-open and-closed.ProofFollows from Corollary 4.2Let A be a subset of a space. Then the following are equivalent:(i) A is μ-regular closed;(ii) A is μ-closed and-open;(iii) A is μ-α-closed and-open.μ-semi-open sets using The next proposition provides decompositions of regular Proposition 4.3Let A be a subset of a space. Then the following are equivalent:(i) A is regular μ-semi-open;(ii) A is-open and μ-semi-closed;(iii) A is μ-semi-open and-closed.ProofFollows from We will now give several characterizations of spaces in which Proposition 4.4For a space, the following are equivalent:(i);(ii);(iii);(iv);(v);(vi);(vii);(viii).Corollary 4.5For a space, the following are equivalent:(i);(ii);(iii);(iv);(v);(vi);(vii);(viii);(ix).ProofFollows from At the end of this section, it might be convenient to raise the following four questions.Question 1Characterize spaces Question 2Characterize spaces Question 3Characterize spaces Question 4Characterize spaces Conclusion In a GTS b consisting of all μ-b-open sets and the class β consisting of all μ-β-open sets. Decompositions, of μ-regular open sets as well as regular μ-semi-open sets using π is the class of all μ-preopen sets, was also reachable; meanwhile, characterizing spaces, in which Mohammad Shawqi Sarsak: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.