E)-1,4-bis(4-chlorophenyl)but-2-ene-1,4-dione (1) is composed of two p-chloro phenyl rings, each bonded on opposite ends to a near planar 1,4-trans enedione moiety [–C(= O)—CH=CH—(C=O)–]. (2E)-1,4-Bis(4-bromophenyl)but-2-ene-1,4-dione (2) has a similar structure to (1), but with two p-bromo phenyl rings and a less planar enedione group. In the crystal, molecules of (1) exhibit C—Cl⋯Cl type I interactions, whereas molecules of (2) present C–Br⋯Br type II interactions.The molecular structure of (2 E)-1,4-bis(4-chlorophenyl)but-2-ene-1,4-dione  is composed of two p-chlorophenyl rings, each bonded on opposite ends to a near planar 1,4-trans enedione moiety [–C(=O)—CH=CH—(C=O)–] [r.m.s. deviation = 0.003 (1) Å]. (2E)-1,4-Bis(4-bromophenyl)but-2-ene-1,4-dione  has a similar structure to (1), but with two p-bromophenyl rings and a less planar enedione group [r.m.s. deviation = 0.011 (1) Å]. Both molecules sit on a center of inversion, thus Z′ = 0.5. The dihedral angles between the ring and the enedione group are 16.61 (8) and 15.58 (11)° for (1) and (2), respectively. In the crystal, molecules of (1) exhibit C—Cl⋯Cl type I interactions, whereas molecules of (2) present C—Br⋯Br type II interactions. van der Waals-type interactions contribute to the packing of both molecules, and the packing reveals face-to-face ring stacking with similar interplanar distances of approximately 3.53 Å.The molecular structure of (2 We have synthesized these compounds in our laboratory as precursors to 4,4′-dibenzaldehyde cross-linkers. The reduction of the title compounds yields the saturated 1,4-diketones that, under Paal–Knorr reaction conditions, can undergo cyclization to produce the corresponding furans —CH=CH—(C=O)–] occurs in many natural and bioactive compounds, including steroids, antibiotics, and antitumor agents  and 0.011 (1) Å for (1) and (2), respectively]. In molecule (1), the carbonyl group is twisted slightly out of the chlorophenyl plane, as evidenced by the torsion angles C6—C1—C7—O1 [−15.6 (3)°] and C2—C1—C7—O1 [163.9 (2)°]. Molecule (2) shows a similar conformation with torsion angles of 14.5 (4) and −164.7 (3)° for the corresponding atoms of the inverted asymmetric unit . The chlorophenyl ring planes form a dihedral angle of 16.61 (8)° with respect to the enedione plane (O1–C7–C8–C8′–C7′–O1′) in (1), whereas the bromophenyl ring planes form a dihedral angle of 15.58 (11)° relative to the enedione plane in (2). Both molecules exhibit a pair of short intramolecular H⋯H contacts , possibly resulting from steric compression of the large phenyl halogen groups. A best fit of all symmetry independent atoms of both molecules  Å; symmetry code: (i) −1 + x, 1 + y, z] and between the Cl atoms and Cl atoms of adjacent molecules  contribute to the intermolecular interactions of (1)  , (2) exhibits trifurcated contacts between the O atoms and H and C atoms of adjacent molecules . Furthermore, the Br atoms form bifurcated contacts with the Br atoms of adjacent molecules   − θ2 (121.71°) = 42.87°, suggesting the electrophilic region of one Br atom approaches the nucleophilic region of the companion Br atom, unlike the Cl⋯Cl interactions  are stacked in close proximity along the vicinity of the a axis with an interplanar separation of 3.528 Å [centroid-to-centroid distance = 3.946 (1) Å]  stack along the vicinity of the a axis with an interplanar separation of 3.525 Å [centroid-to-centroid distance = 3.994 (1) Å], but in a crisscross-like pattern when viewed along the c axis   . The title compounds adopt an E configuration, similar to (3). They contain halogen atoms in the para position of the phenyl groups, unlike (3), but the rings are nearly planar as are those of (3), whose r.m.s value = 0.008 Å. The r.m.s. value, reflecting the planarity of the enedione moiety, in (1) is different to that of (3) (0.003 vs 0.0035 Å), and the value determined for (2) (0.011 Å). The dihedral angles between the ring planes of (1) and (2) are nearly identical to those of (3) [16° (average) vs 15.7 (1)°]. Unlike (1), its diastereomer (4) does not exhibit a planar enedione moiety and its near planar chlorophenyl rings (r.m.s deviation = 0.018 Å) form a dihedral angle of 77.4 (3)° with respect to each other. Superimposition of atom C1 of the E/Z diastereomers through the C7, Cl1, and O1 atoms yields an r.m.s. deviation of 0.033 Å. The remaining parts of the molecules are twisted from each other, with the planes containing the chlorophenyl group and adjoining carbonyl group of each stereoisomer forming a dihedral angle of approximately 79°.A search of the Cambridge Structural Database . At the conclusion of the reaction, the mixture was cooled to room temperature, poured into ice-cold aqueous 1 M HCl, and extracted several times with DCM. The combined organic layers were washed with 0.5 M NaOH and dried over Na2SO4, and the volatiles were removed under reduced pressure. The resulting red–brown solid was recrystallized in DCM, further purified with a series of cold DCM washes, and dried under reduced pressure, affording either compound (1)  or (2) . Slow evaporation of DCM solutions saturated with either (1) or (2) yielded single crystals suitable for X-ray diffraction.The title compounds were synthesized following a modified literature procedure . (1): 1H NMR : δ 7.51 , 7.97 , 8.00  ppm. 13C NMR : δ 129.48, 130.40, 135.06, 135.31, 140.77, 188.51 ppm. (2): 1H NMR : δ 7.67 , 7.92 , 7.96  ppm. 13C NMR : δ 129.53, 130.44, 132.45, 135.03, 135.69, 188.69 ppm.NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shifts (δ) are given in ppm and are referenced to tetramethylsilane (TMS) using the residual solvent (Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S205698901800230X/zl2724sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S205698901800230X/zl27241sup4.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S205698901800230X/zl27242sup5.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S205698901800230X/zl27241sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901800230X/zl27242sup5.cmlSupporting information file. DOI: 1822698, 1822697CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S(6) ring motifs with the sulfone O atom. In the crystals, molecules of (I) are linked by C—H—O hydrogen bonds, forming In the title indole derivatives, the sulfonyl-bound phenyl rings are almost orthogonal to the indole ring system, subtending dihedral angles of 88.33 (10) and 87.58 (16)°, respectively. The molecules of both (I) and (II) feature intramolecular C—H⋯O hydrogen bonds that generate  22H16N2O5S, (I) and C20H15NO3S2, (II), the sulfonyl-bound phenyl rings are almost orthogonal to the indole ring system, subtending dihedral angles of 88.33 (10) and 87.58 (16)°, respectively. In both compounds, the sulfonyl S atom has a distorted tetrahedral geometry [O—S—O = 119.98 (9) and N—S—C = 104.01 (8)° for compound (I) and O—S—O = 120.08 (18) and N—S—C = 104.91 (14)° for compound (II)] and the sum of the bond angles at N indicates sp2 hybridization. The molecules of both (I) and (II) feature intramolecular C—H⋯O hydrogen bonds that generate S(6) ring motifs with the sulfone O atom. In the crystals, molecules of (I) are linked by C—H—O hydrogen bonds, forming R44(18) ring motifs while molecules of (II) are linked by C—H—O and C—H—S hydrogen bonds, forming R22(12) ring motifs. Compound (II) was refined as an inversion twin.In the title indole derivatives, C The compound (2-methyl-1-phenylsulfonyl-1H-indol-3-yl)(phenyl)methanone . The compound (1-phenylsulfonyl-1H-indol-2-yl)(thiophen-2-yl)methanone A search of the Cambridge Structural Database (Groom Compound (I)4  was added slowly (5 min). To this, a solution of 1-phenylsulfonyl-2-methylindole  in dry DCM (10 ml) was added (5 min) and allowed to stir at room temperature for 48 h. After completion of the reaction (monitored by TLC), it was poured into ice–water (50 ml) containing conc. HCl (10 ml). The organic layer was separated and the aqueous layer was extracted with DCM (2 × 20 ml). The combined organic layer was washed with water (3 × 25 ml) and dried (Na2SO4). The subsequent purification of the crude product either by washing with MeOH or column chromatography  furnished the first title compound as a colourless solid ; m.p. 435–437 K.To a solution of 4-nitrobenzoyl chloride  in dry DCM (15 ml) at 273 K, SnClCompound (II)4  in dry DCM (20 ml) at 273 K, a solution of 1-phenylsulfonyl-2-methylindole  in dry DCM (10 ml) was added slowly (5 min). Then, it was stirred at room temperature for 30 min. After completion of the reaction (monitored by TLC), it was poured into ice–water (50 ml) containing conc. HCl (10 ml). The organic layer was separated and the aqueous layer was extracted with DCM (2 × 20 ml). The combined organic extract was washed with water (3 × 25 ml) and dried (Na2SO4). Evaporation of the solvent followed by trituration of the crude product with MeOH (5 ml) gave the second title compound as a colourless solid ; m.p. 379–381 K.To a solution of thiophene-2-carbonyl chloride  and SnClUiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms. Compound (II) was refined as an inversion twin (BASF 0.03).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017012804/ff2151sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S2056989017012804/ff2151Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017012804/ff2151Isup4.cmlSupporting information file. DOI: 10.1107/S2056989017012804/ff2151IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017012804/ff2151IIsup5.cmlSupporting information file. DOI: 1561045, 1561044CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Magnolia officinalis var. biloba fruits, nine phenylethanoid glycosides were isolated by solvent extraction, silica gel, and preparative high-performance liquid chromatography (HPLC). Their structures were elucidated by 1D and 2D NMR analyses, including COSY, HMQC and HMBC correlations, and HPLC analysis of sugar residue. Nine phenylethanoid glycosides, namely, magnoloside Ia (1), magnoloside Ic (2), crassifolioside (3), magnoloside Ib (4), magnoloside IIIa (5), magnoloside IVa (6), magnoloside IIa (7), magnoloside IIb (8) and magnoloside Va (9), were first isolated from the n-butanol fraction of Magnolia officinalis var. biloba fruits alcohol extract. Free radical scavenging activities of the nine phenylethanoid glycosides were assessed using the DPPH, ABTS, and superoxide anion radical scavenging assays. Simultaneously, protective effects of all compounds against free radical-induced oxidative damage were evaluated by two different kinds of mitochondrial damage model. The protective effects were assessed by mitochondrial swelling, the formations of malondialdehyde (MDA) and lipid hydroperoxide (LOOH), the activities of catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD). All phenylethanoid glycosides showed significant protective effects.To systematically study the chemical constituents in  Magnolia officinalis Rehd. et Wils.  or Magnolia officinalis Rehd. et Wils. var. biloba Rehd. et Wils. . They have been used as traditional Chinese medicine (TCM) for treating abdominal distention, nausea, vomiting, dyspepsia, cough, and asthma3512131415Magnolia bark extracts are derived from M. officinalis and M. officinalis var. biloba barks, into healthy food materials18et al. also confirmed that compressed mints and chewing gum containing bark extracts are effective against bacteria responsible for oral malodorM. officinalis and M. officinalis var. biloba M. officinalis and M. officinalis var. biloba fruits, which are nutritious reproductive organs of these plants, can be harvested every year. However, whether their fruits can be used as an alternative food resource remains unknown.In 2002, the Ministry of Health of the People’s Republic of China passed the notice to further standardize the management of health food raw materials, including M. officinalis var. biloba fruits in 201523252627282930M. officinalis var. biloba fruits have the same effects as those isolated from Orobanchaceae plants needs to be elucidated. Moreover, we have not validated whether the analogous components from their fruits can be associated with the traditional functions of magnolia bark extracts. Therefore, more studies are needed to further understand the function of phenylethanoid glycosides.We had already reported that phenylethanoid glycosides were isolated from M. officinalis var. biloba, we focused on the n-butanol fraction of the alcohol extract from M. officinalis var. biloba fruits and isolated nine phenylethanoid glycosides  by HR-ESI-MS and supported by the 13C NMR spectral data. The IR spectrum of 2 displaced the characteristic absorption bands for the hydroxyl group (3410 cm−1), conjugated carbonyl group (1687 cm−1), aromatic rings (1604 and 1516 cm−1), and glycosidic group (816 cm−1). The UV spectrum obtained the maximum absorption at 206, 291, and 330 nm.Compound 1H-NMR and 13C NMR spectra of 2 , 6.97 , 6.80  and δH 6.73 , 6.71 , 6.58 , a pair of trans-olefinic proton signals at δH 7.63 , 6.33 , and benzylic methylene proton signal at δH 2.80 , which were assigned to δC 115.30 (C-2′), 123.20 (C-6′), 116.45 (C-5′), 117.25 (C-2), 116.59 (C-5), 121.41 (C-6), 147.72 (C-7′), 114.71 (C-8′), and 36.73 (C-7), respectively, via HMQC correlation. Additionally, two anomeric proton resonances appeared at δH 4.85  and 4.90 , which correlated with signals at δC 100.47 (C-1″) and 97.91 (C-1″′) in the HMQC spectrum, respectively. The 1H-NMR spectrum also showed the presence of a methyl group at δH 1.29 , which indicated the presence of rhamnose in the compound. A series of signals in 1H-NMR  at δH 4.85 , 3.47 , 4.51 , 4.84 , 3.60 , 3.62 , and 3.79  and 13C NMR  at δC 100.47 (C-1″), 73.19 (C-2″), 66.45 (C-3″), 70.57 (C-4″), 74.53 (C-5″), and 62.37 (C-6″) indicated a rare β-allopyranosyl unit. Moreover, this unit was confirmed by comparing the NMR features of those of magnolosides A, B, C, D, and E1632M. officinalis barks or M. obovata barks, which also contained allopyranose. Acid hydrolysate of compound 2 contained monosaccharide components, which were identified as D-allose and L-rhamnose by HPLC analysis. In the HMBC relationship of 2 (δH 4.85) and C-8 (72.30) showed that the phenylethanol moiety was linked to C-1″ (All-C-1″); the cross peak between H-4″ (δH 4.84) and C-9′ (168.14) showed that the trans-caffeoyl moiety was linked to C-4″ (All-C-4″); and the long-range correlations between H-1″′ (δH 4.90) and C-2″ (73.19) (All-C-2″) showed that the rhamnose moiety was linked to C-2″ (All-C-2″).The tra of 2  exhibitehip of 2 , the cor2 was revealed as 2--ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(4-O-trans-caffeoyl)-β-D-allopyranoside.In light of all the above considerations, the structure of 3 was obtained as a canary yellow amorphous powder. The HR-ESI-MS data showed an accurate [M-H]− ion at m/z 769.2668 , in accordance with the empirical molecular formula of C35H46O19. The IR spectrum of 3 displaced characteristic absorption bands for the hydroxyl group (3402 cm−1), conjugated carbonyl group (1699 cm−1), aromatic rings (1603 and 1517 cm−1), and glycosidic group (814 cm−1). The UV spectrum obtained the maximum absorption at 206, 280, and 330 nm.Compound 1H-NMR and 13C NMR spectra of 3 , 4.96 , and 4.51  and resonated at δC 102.32 (C-1″″), 103.07 (C-1″′), and 102.46 (C-1′) in 13C NMR, respectively. They also could be supported in the HMQC spectrum. Two methyl groups at δH 1.14  and 1.29 , as well as δC 17.91 (C-6″′) and 17.69 (C-6″″), indicated that compound 3 may contain two rhamnoses. A series of signals in 1H-NMR  at δH 4.51 , 3.97 , 3.57 , 4.96 , 3.51 , 3.52 , and 3.63  and in 13C NMR  at δC 102.46 (C-1″), 82.68 (C-2″), 80.43 (C-3″), 69.83 (C-4″), 75.50 (C-5″), and 61.88 (C-6″) showed that the central sugar was β-glucose. The acid hydrolysate of compound 3 further confirmed the existence of D-glucose and L-rhamnose, which were identified by HPLC analysis. In the HMBC relationship of 3 (δH 4.51) and C-8 (δC 71.86) showed that the phenylethanol moiety was linked to C-1″ (Glc-C-1″); the cross peak between H-4″ (δH 4.96) and C-9′ (δC 167.91) showed that the trans-caffeoyl moiety was linked to C-4″ (Glc-C-4″); and the long-range correlations between H-1″′ (δH 4.96) to C-2″ (δC 82.68) (Glc-C-2″) and H-1″″ (δH 5.01) to C-3″ (δC 80.43) (Glc-C-3″) unambiguously showed that the two rhamnose moieties were linked at C-2″ and C-3″, respectively.The tra of 3  exhibitehip of 3 , the cor3 as 2--ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-[a-L-rhamnopyranosyl-(1 → 3)]-(4-O-trans-caffeoyl)-β-D-glucopyranoside, named crassifoliosideAll these results confirmed the structure of compound 8 was obtained as a light yellow amorphous powder. The negative-ion quasi-molecular ion peak in its HRESI-MS data at m/z 785.2520 [M-H]−  together with the 13C NMR spectral data suggested a molecular formula of C35H46O20. The IR spectrum of 8 displaced characteristic absorption bands for the hydroxyl group (3234 cm−1), conjugated carbonyl group (1692 cm−1), aromatic rings (1604 and 1518 cm−1), and glycosidic group (813 cm−1). The UV spectrum obtained the maximum absorption at 205, 280, and 330 nm.Compound 1H-NMR  and 13C NMR  spectra of 8 , 3.60 , 3.25 , 3.32 , 3.35 , 3.64 , and 3.86  and in 13C NMR at δC 104.92 (C-1″″), 74.41 (C-2″″), 77.78 (C-3″″), 71.48 (C-4″″), 77.90 (C-5″″), and 62.67 (C-6″″) deduced that the extra sugar group was β-glucose. Acid hydrolysis of 8 further gave D-allose, D-glucose, and L-rhamnose as sugar residues by HPLC analysis. The 7.25 ppm down-field shift of C-6″ in the 13C NMR spectrum indicated the attachment of the extra sugar group to C-6″ of the central sugar. This finding was also supported by the HMBC correlation (δH 4.30) and C-6″ (δC 69.62).Comparison of the tra of 8  with thorelation  of H-1″″8 was established as 2--ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 6)]-(4-O-trans-caffeoyl)-β-D-allopyranoside.Based on the above evidence, compound 5 and 6 were isolated as yellow amorphous powder, with the molecular formula of C29H36O14, as deduced from the [M-H]− peak at m/z 607.2011  and 607.2009  via HR-ESI-MS and supported by the 13C NMR spectral data. The 1H-NMR and 13C NMR spectra of 5 (1 (magnoloside A), except for the signals caused by the 4-hydroxyphenylethyl alcohol group {ortho-coupled A2B2-type aromatic protons  and two methylenes }, which was also supported by the literature6 (1 (magnoloside A) revealed that the caffeoyl group of magnoloside A was replaced by the trans-p-coumaroyl group {ortho-coupled A2B2-type aromatic protons  and trans-olefinic protons }, which was also supported by the literature5 and 6 -ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(3-O-trans-caffeoyl)-β-D-allopyranoside and 2--ethyl-O-a-L-rhamnopyranosyl-(1 → 2)-(3-O-trans-coumaroyl)-β-D-allopyranoside, respectively.Compounds tra of 5  were vererature6  with thoe5 and 6  also sho1), magnoloside B (7), magnoloside D (4), and magnoloside E (9) by comparing their 1H-NMR and 13C NMR data with those reported in the literatureM. officinalis var. biloba fruits for the first time.The other compounds were identified as magnoloside A  but different nomenclature. In this manuscript, we propose a reasonable rule of nomenclature in view of the rich structure type of phenylethanoid glycosides. Thus, the confusion and ambiguity caused by two research groups could be clarified. Different types of phenylethanoid glycosides are numberd with Roman numerals , and isomers with subscripts a, b, c… are distinguished. Therefore, compounds 1, 2, 4, 5, 6, 7, 8 and 9 were renamed magnoloside Ia (old name was magnoloside A32), magnoloside Ic (old names were magnoloside F31 and magnoloside M37), magnoloside Ib (old name was magnoloside D16), magnoloside IIIa (old name was magnoloside H36), magnoloside IVa (old name was magnoloside G36), magnoloside IIa (old name was magnoloside B33), magnoloside IIb (old name was magnoloside F37), magnoloside Va (old name was magnoloside E16).In the last few years, some confusing nomenclatures were found in the original articles36In vitro DPPH radical scavenging, ABTS radical scavenging, and superoxide anion radical scavenging activities of the isolated phenylethanoid glycosides are summarized in 381 and its structural analogs  showed significant DPPH radical scavenging activity with far smaller IC50 values  than the positive controls, such as VC (40.94 ± 0.78 μM) and BHT (89.94 ± 4.57 μM). Compounds 3, 7, and 8 expressed inferior activity compared with the above four compounds because their IC50 values  were near the value of VC. Moreover, Compound 5 and its structural isomer 6 showed large IC50 values . Some observations could be made according to the above results. Compounds 3, 7, and 8 possessed larger steric hindrance because they contained three sugars, whereas compounds 1, 2, 4, and 9 only contained two sugars. The increased steric hindrance effect of compounds 3, 7, and 8 prevented them from easily approaching the free radicals, so their DPPH radical scavenging capacity was relatively weak than compounds 1, 2, 4, and 9. Moreover, compared with the other seven compounds, compounds 5 and 6 belonged to phenylethanoid glycosides with two adjacent phenolic groups only in one side, so they exhibited poor activity.In the DPPH radical scavenging assay, compound 1, 2, and 4 also exhibited good ABTS radical scavenging activity, but the IC50 value (6.23 ± 0.06 μM) of compound 9 was large. This result was different from that of the DPPH radical scavenging assay, indicating that the apiose group in compound 9 produced a negative effect on the ABTS radical scavenging assay. By contrast, the ABTS radical scavenging ability of compound 3 was far better than that of compounds 7 and 8, which could be due to the glucose group in compound 3. The superoxide anion radical scavenging assay showed that the activity of compound 9 was the best, whereas the activity of compound 3 was the worst. These completely opposite results may be due to the fact that the apiose group in compound 9 enhanced the influence on the superoxide anion radical scavenging assay, whereas the glucose group in compound 3 minimized this influence. These different results obtained from three assays may be explained by the various mechanisms of these assays, suggesting that combined assay methods should be adopted in the screening and evaluation of bioactive compounds from natural materials.The ABTS radical scavenging assay demonstrated that compounds 1 > 2 > 4, 7 > 8.Our experimental results also showed that the substitution position of the caffeoyl group also influenced free radical scavenging activity. By analyzing the structure of similar compounds and experimental results, we found that the free radical scavenging activity of 3-caffeoyl substitution was optimal, followed by 4-caffeoyl substitution and 6-caffeoyl substitution. This conclusion could be supported by the order of activity as follows: 2+/H2O2 can produce reactive oxygen species (ROS), which lead to various adverse effects on the body tissues402+/H2O2-induced oxidative damage model are classical mitochondria models which are used to measure the protective effect against free radical-induced oxidative damage.Repeated ultraviolet B (UVB) exposure or constant treated with Fe520 will be reduced1–9 were investigated for their protective effects against free radical-induced oxidative damage.When mitochondria are damaged, swelling will occur; thus, the value of Ap < 0.001). The A532 value and A560 value of the MDA (0.075 ± 0.001) and LOOH (0.097 ± 0.001) were significantly increased in the model group compared with the control group (p < 0.001). However, the test compound groups reduced the MDA and LOOH level and showed significant effects (p < 0.001). In mitochondrial damage model caused by Fe2+/H2O2, compared with the control group, the model group showed a significant decrease in CAT, GR and SOD levels, together with a significant increase in the level of MDA and LOOH (p > 0.05).In mitochondrial damage model caused by UVB , the modand LOOH . Overall5 and 6). Furthermore, compounds with two sugars were best. These results were consistent with the experimental results of free radical scavenging assays. Therefore, we could speculate that the protective effects of the nine phenylethanoid glycosides against free radical-induced oxidative damage were attributed to their radical scavenging activity, which was caused by the number of two adjacent phenolic groups.Comparing the above experimental results, we found that protective effect of compounds containing two pair of two adjacent phenolic groups was optimal, followed by compounds containing one pair of two adjacent phenolic groups , Hubei Province, China, and identified by Prof. Youwei Wang . A voucher specimen (No. 14610) was deposited at the Traditional Chinese Medicine Specimen Museum, School of Pharmaceutical Science, Wuhan University, China.d4 (20140519), xylenol orange (20100826), and other analytical chemicals and reagents were purchased from Sinopharm Chemical Regent Co. Ltd. .2,2′-Azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was procured from Fluka . 2,2-Diphenyl-1-picrylhydrazyl (DPPH), high-performance liquid chromatography (HPLC)-grade methanol, and acetonitrile were purchased from Sigma–Aldrich Co. . D-Allose (D1218014), L-rhamnose (B1504021), L-cysteine methyl ester hydrochlorides (G14118068), and 2-thiobarbituric acid (C1510117) were purchased from Aladdin Industrial Corporation . Diagnostic kits for CAT, GR, and SOD were purchased from Nanjing Jiancheng Technology Co. Ltd. . Ascorbic acid (Vc) (F20060609), ammonium iron (II) sulfate hexahydrate (20120215), butylated hydroxytoluene (BHT) (20121211), D-glucose (20140724), dodecyl sodium sulfate (20130625), hydrogen peroxide 30% (20150911), iron (II) sulfate heptahydrate (20100506), methanol-1H NMR13,C NMR, H-H COSY, HMQC, and HMBC spectra) were obtained on a Bruker DPX-400 spectrometer using standard Bruker pulse programs  with solvent peak as references. HR-ESI-MS data were obtained on a Bruker Daltonic micrOTOF-Q II MS spectrometer . UV absorption spectra were recorded on a UV-2600 ultraviolet and visible spectrophotometer  from 200 nm to 900 nm. Infrared (IR) spectra were recorded on a Nicolet Nexus 470 spectrometer  using the compounds in the KBr pellet form. Preparative HPLC was carried out on a LabAllance Model 201 HPLC system  and a Kromasil C18 column preparative column .All NMR spectra  were extracted three times using 10 volumes of 70% alcohol (50 L) for 3 h at reflux. The filtrate was combined and concentrated under vacuum to yield a dried alcohol extract (804.9 g). The alcohol extract was suspended in distilled water (10 L) and then successively partitioned three times with chloroform (20 L), ethyl acetate (10 L), and n-butanol alcohol (10 L), which yielded the chloroform fraction (398.4 g), ethyl acetate fraction (180.8 g), and n-butanol fraction (63.4 g), respectively. All fractions were stored at −20 °C until further use.The dried and powdered n-butanol fraction (20 g) prepared from the previously described steps was further isolated on a preparative HPLC system. Each chromatographic run was carried out at a flow rate of 20 mL/min with a binary mobile phase consisting of methanol (A) and 0.1% formic acid (B) using a step gradient profile. The gradient started with 10% A, was varied to 35% A at 10 min, 46% A at 40 min, 48% A at 43 min, 100% A at 50 min, and 100% A isocratic for 10 min, and decreased to 10% A in 0.1 min. After re-equilibration at 10% A for 12 min, the next sample was injected. The temperature of the column oven was 25 °C, and 100 μL (300 mg/mL) was injected into the system every time. The peaks adsorbed at 315 nm were recorded. Compounds 1 (797.7 mg), 2 (226.4 mg), 3 (195.9 mg), and 4 (298.3 mg) were obtained at the retention times of 22.4, 32.6, 35.3, and 41.7 min, respectively. The subfraction that was collected at a retention time from 27 min to 29 min was further applied to the preparative HPLC system, which was eluted isocratically with 18% acetonitrile in water (containing 0.1% formic acid) at a flow rate of 20 mL/min. Compounds 5 (56.0 mg) and 6 (34.1 mg) were then purified. Simultaneously, the subfraction obtained at a retention time of 19.3 min was subjected to the preparative HPLC system, which was eluted isocratically with 26% methanol in water at a flow rate of 20 mL/min. Compounds 7 (37.7 mg) and 8 (52.7 mg) were then obtained. The subfraction obtained at a retention time of 38.4 min was further isolated over the preparative HPLC system, which was eluted isocratically with 43% methanol in water at a flow rate of 20 mL/min to yield compound 9 (35.9 mg).The 2, 3, 5, 6 and 8 was carried out according to the method described by previous studies462, 3, 5, 6 and 8 (each 4.0 mg) were hydrolyzed by heating in 4 M CF3COOH (4 mL) at 95 °C for 4 h. After cooling, the solution was extracted with CH2Cl2 (3 × 2 mL). The water layer was concentrated by reducing pressure and dried by vacuum. Anhydrous pyridine (0.5 mL) and L-cysteine methyl ester hydrochloride (2 mg) were added and reacted at 60 °C for 1 h. After cooling to room temperature, o-tolyl isothiocyanate (10 μL) was added to the mixture and further heated at 60 °C for 1 h. The reaction mixture was directly analyzed by reversed-phase HPLC using a Diamonsil C18 analytical column , which was eluted isocratically with 25% acetonitrile in water (containing 0.1% formic acid) at a flow rate of 1 mL/min. The temperature of the column oven was 25 °C, and 20 μL was injected into the system every time. The UV spectra were collected at 250 nm. Peaks of the hydrolysate of compounds 2, 3, 5, 6 and 8 were identified by comparing the retention times of authentic samples of D-allose (tR = 19.330), D-glucose (tR = 18.566), and L-rhamnose (tR = 33.411) after simultaneous treatment under the same conditions.Sugar analysis of compounds 0 − A)/A0] × 100 . VC and BHT with the same concentrations as the samples were used as positive controls.DPPH and ABTS, two relatively stable free radical compounds widely used to test free radical scavenging activity, were determined using a previously described method48in vivo. The capacity of each compound to scavenge superoxide radicals was examined by a pyrogallol auto-oxidation system2− directly, and the rate constant of this auto-oxidation reaction is dependent on the O2− concentration. If the test compound can scavenge O2−, the auto-oxidation reaction can slow down significantly. The auto-oxidation rate constant (Kb) of pyrogallic acid was calculated from the curve of A325 nm versus time. VC and BHT were used for comparative purposes. Simultaneously, the Kb value (10−4 A/s) of the blank control was recorded.The superoxide anion radical is the most common free radical generated Sprague–Dawley (SD) rats (200 ± 20 g) were purchased from the Laboratory Animal Center of Wuhan University, Wuhan, China. All the rats were housed under regulated conditions of 12 h light/12 h dark cycles, 25 ± 2 °C, and 30%–60% relative humidity. They were fed with a standard pellet diet  and unlimited water. The animals were allowed to acclimatize to the room conditions for 2 days. All experimental procedures were approved by the Institutional Animal Ethical Committee of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Wuhan University in Wuhan, China. The experimental methods were performed in accordance with “Regulations for the Administration of Affairs Concerning Experimental Animals” and “Guiding Opinions of Treating Experimental Animals with Good Ethics”, which have been formally released and implemented by Chinese Government.Mitochondria were isolated from SD rats according to the method described by previous studiesThe experiment consisted of the control group, model group, BHT group, and test sample group. Each group comprised the following set up: for the control group, 0.5 mL of mitochondrial protein + 0.1 mL of phosphate buffer ; for the model group, 0.5 mL of mitochondrial protein + 0.1 mL of phosphate buffer  + UVB; for the BHT group, 0.5 mL of mitochondrial protein + 0.1 mL of BHT  + UVB; and for the test compound group, 0.5 mL of mitochondrial protein + 0.1 mL of test sample  + UVB. The model, BHT, and test compound groups were irradiated for 4 h with a 20 W UVB lamp  from a 15 cm distance. The control group was kept at 37 °C for 4 h without irradiation.4(10 mM) + 20 μL H2O2 (65 mM); for the BHT group, 0.5 mL of mitochondrial protein + 0.5 mL of BHT  + 30 μL FeSO4(10 mM) + 20 μL H2O2 (65 mM); and for the test compound group, 0.5 mL of mitochondrial protein + 0.1 mL of test sample  + 30 μL FeSO4(10 mM) + 20 μL H2O2 (65 mM). All experiment groups were kept at 37 °C for 1 h.The experiment consisted of the control group, model group, BHT group, and test sample group. Each group comprised the following set up: for the control group, 0.5 mL of mitochondrial protein + 0.5 mL of phosphate buffer ; for the model group, 0.5 mL of mitochondrial protein + 0.5 mL of phosphate buffer  + 30 μL FeSO520 were used to evaluate the mitochondrial swelling degreeThe values of An = 3). The data were analyzed using one-way ANOVA. Statistically significant effects were analyzed, and the means were also compared using least-significant difference (LSD) test. Statistical significance was determined at p < 0.05.All experiments were carried out in triplicate, and the results were reported as the mean ± standard deviation .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Crystal structures of two benzothiazinones belonging to a class of compounds implicated in antimicrobial, antitumour, and HIV-RT inhibitory activity are reported. H-1,3-benzothiazin-4-one  (1) and 2,3-bis(4-methylphenyl)-2,3-dihydro-4H-1,3-benzothiazin-4-one  (2) are reported. Each structure is racemic: the asymmetric unit of 2 consists of two molecules. In both 1 and 2, the six-membered 1,3-thiazine ring is close to an envelope conformation with the chiral C atom forming the flap. In 1, the 2-aryl group is pseudo-equatorial, while in 2 it is pseudo-axial. In 1, the pendant aryl rings form a V shape with an inter-centroid distance of 3.938 (3) Å and an acute dihedral angle of 48.3 (2)° between them. Both CF3 groups are disordered over two orientations in 0.687 (19):0.313 (19) and 0.667 (16):0.33 (16) ratios. In each of the independent molecules of 2, the aryl rings are almost orthogonal to each other [dihedral angles = 85.50 (12) and 86.07 (11)°]. In both structures, the chiral C atom and the O atoms participate in C—H⋯O-type hydrogen bonding between symmetry-related molecules of 1 or the independent enantiomers in 2, forming chains along the c-axis direction in 1 and the b-axis direction in 2. Additionally, in 1, π–π contacts of both face-to-face and edge-to-face type, as well as π-H⋯O and π-H⋯F interactions are observed. In 2, a racemic mixture of molecules forms layers in the ac plane linked by weak π–π and C—H⋯π interactions.The syntheses and crystal structures of 2,3-bis[3-(trifluoromethyl)phenyl]-2,3-dihydro-4 N-Aryl (R1 = aryl or heteroaryl) 2,3-dihydro-4H-1,3-benzothiazin-4-ones display antitumor . Each has been synthesized using the same T3P/pyridine method that was used for the previously reported compounds.As part of our studies in this area, we have previously reported the crystal structures of a number of 2-aryl-3-phenyl- and 3-aryl-2-phenyl-2,3-dihydro-41, the 2-aryl group is pseudo-equatorial, unlike the structures that we have reported previously, while in 2 it is pseudo-axial (both independent molecules)  Å, θ = 118.2 (8)°, φ = 22.7 (9)° and in 2 (molecules A and B) Q = 0.5490 (19) and 0.5715 (17) Å; θ = 63.5 (2) and 116.31 (19)°; φ = 40.8 (2) and 223.5 (2)°, respectively. In 1, the pendant aryl rings form an approximate V shape with an acute dihedral angle of 48.3 (2)° and inter-centroid distance of 3.938 (3) Å between them. In each of the independent molecules of 2, the aryl rings are almost orthogonal to each other [dihedral angles = 85.50 (12) for the C1 molecule and 86.07 (11)° for the C23 molecule].The title compounds are shown in Figs. 11; C1 and C23 in 2) donates its H atom to the oxygen atom of a symmetry related molecule in 1, or the independent enantiomer in 2. This results in infinite chains along the c- and b-axis directions, respectively. In 1, these chains are further consolidated by π-H⋯O and π-H⋯F interactions   Å]. The structure also features interactions of edge-to-face type between the fused benzene and 2-aryl rings [Cg⋯Cg = 5.0083 (14) Å]. In 2, weak π–π [Cg⋯Cg = 4.735 (2) Å] and C—H⋯π interactions . Thiosalicylic acid  and then 2-methyltetrahydrofuran (2.3 mL) were added and the solution was stirred. Pyridine  and, finally, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) in 2-methyltetrahydrofuran  were added. The reaction was stirred at room temperature and followed by TLC. The mixture was poured into a separatory funnel with dichloromethane and distilled water. The layers were separated and the aqueous one was then extracted twice with dichloromethane. The organic layers were combined and washed with saturated sodium bicarbonate and then saturated sodium chloride. The organic layer was dried over sodium sulfate and concentrated under vacuum. The crude product was chromatographed on 30 g flash silica gel and then recrystallized.General: A two-necked 25 ml round-bottom flask was oven-dried, cooled under NH-1,3-benzothiazin-4-one (1): Recrystallized from 2-propanol solution. Yield: 0.5199 g (19%), m.p. 392–393 K. Colorless blocks of 1 were grown by slow evaporation from cyclohexane solution.2,3-Bis[3-(trifluoromethyl)phenyl]-2,3-dihydro-4H-1,3-benzothiazin-4-one 2: Recrystallized from 2-propanol solution. Yield: 0.6288 g (30%), m.p. 412–414 K. Colorless needles of 2 were grown by slow evaporation from ethanol solution.2,3-Bis(4-methylphenyl)-2,3-dihydro-43 groups of 1 are disordered over two orientations in a 0.687 (19):0.313 (19) ratio for the C15 group and a 0.667 (16):0.33 (16) ratio for the C22 group·The disorder was restrained using SIMU and DELU commands in SHELX for the twelve resulting atoms.Crystal data, data collection and structure refinement details for both compounds are summarized in Table 310.1107/S2056989018002049/hb7726sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018002049/hb77261sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989018002049/hb77261sup4.molSupporting information file. DOI: 10.1107/S2056989018002049/hb77262sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989018002049/hb77262sup5.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018002049/hb77261sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018002049/hb77262sup7.cmlSupporting information file. DOI: 1821702, 1821701CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The 1,2,3-benzotriazolium cations are linked between these polymer chains, via the water molecules, giving rise to left- and right-handed helical chains.The structure of this new halide-bridged polymer comprises polyanionic zigzag chains of formula [ [[tetrachloridobismuth(III)]-μ-chlorido] dihydrate], {(C6H6N3)2[BiCl5]·2H2O}n are reported. The structure comprises polyanionic zigzag chains of formula [(BiCl5)2−]n running along the c-axis direction. The 1,2,3-benzotriazolium cations are linked between these polymer chains, via the water molecules, giving rise to left- and right-handed helical chains. Hirshfeld surface analysis and fingerprint plots were used to decode the intermolecular interactions in the crystal network and determine the contribution of the component units for the construction of the three-dimensional architecture.The synthesis and the crystal structure of a new halide-bridged polymer, namely  As usual, this aromatic amine is protonated at the N3 atom and the C—C, N—N and C—N bond lengths vary from 1.358 (18) to 1.402 (15), 1.293 (15) to 1.308 (15) Å and 1.364 (16) to 1.370 (15) Å, respectively, which agree well with those observed in bis sulfate dihydrate )° Fig. 2 The over)° Fig. 2b. As usW) via N—H⋯O hydrogen bonds, forming (benzo-OW)n helical chains in a right- and left–handed sequence extending along the c-axis direction  and bottom right  of the related plots in Fig. 5The Hirshfeld surface . The formation of extensive π–π interactions is reflected in the relatively high ERCC of 3.94.The intermolecular interactions were further evaluated by using the enrichment ratio  to 1 mmol of a bismuth nitrate solution [Bi(NO3)3·5H2O], dissolved in 0.05 mL of a concentrated HCl aqueous solution. The resulting aqueous solution was stirred for 30 min. and kept at room temperature for crystallization. After two week of slow evaporation, colourless single crystals of (I)6H6N3]2[BiCl5]·2H2O (%): C 21.6 (21.0), H 2.66 (2.41), N 34.6 (33.8).The title compound was prepared by dropwise addition of an ethanolic solution of 1Uiso(H) = 1.2Ueq. The O—H and H⋯H separations in the water molecule were restrained using a DFIX model to be 0.90 and 1.46 Å, respectively, and refined with isoU(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017015134/hb7714sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989017015134/hb7714Isup2.hklStructure factors: contains datablock(s) I. DOI: 1580458CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R)- ethanol  is a valuable chiral intermediate for Aprepitant (Emend) and Fosaprepitant (Ivemend). Biocatalyzed asymmetric reduction is a preferred approach to synthesize highly optically active (R)-3,5-BTPE. However, the product concentration and productivity of reported (R)-3,5-BTPE synthetic processes remain unsatisfied.(Lactobacillus kefir (LkCR) was discovered by genome mining for reduction of 3,5-bis(trifluoromethyl) acetophenone  into (R)-3,5-BTPE with excellent enantioselectivity. In order to synthesize (R)-3,5-BTPE efficiently, LkCR was coexpressed with glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration in Escherichia coli BL21 (DE3) cells, and the optimal recombinant strain produced 250.3 g/L (R)-3,5-BTPE with 99.9% ee but an unsatisfied productivity of 5.21 g/(L h). Then, four different linker peptides were used for the fusion expression of LkCR and BsGDH in E. coli to regulate catalytic efficiency of the enzymes and improved NADPH-recycling efficiency. Using the best strain (E. coli/pET-BsGDH-ER/K(10 nm)-LkCR), up to 297.3 g/L (R)-3,5-BTPE with enantiopurity >99.9% ee was produced via reduction of as much as 1.2 M of substrate with a 96.7% yield and productivity of 29.7 g/(L h).A NADPH-dependent carbonyl reductase from E. coli/pET-BsGDH-ER/K(10 nm)-LkCR was developed for the bioreduction of 3,5-BTAP to (R)-3,5-BTPE, offered the best results in terms of high product concentration and productivity, demonstrating its great potential in industrial manufacturing of (R)-3,5-BTPE.Recombinant The online version of this article (doi:10.1186/s12934-016-0585-5) contains supplementary material, which is available to authorized users. R)- ethanol  is a key intermediate for the synthesis of neurokinin-1 receptor antagonists, such as Aprepitant (Emend) and Fosaprepitant (Ivemend), which are widely used in the treatment of chemotherapy-induced nausea and vomiting , was necessary to regenerate the reduced cofactor. In terms of biocatalytic efficiency, the requirement for NAD(P)+ addition is disadvantageous. To overcome the problem of cofactor regeneration and to improve the reaction process, co-expression of two or more enzymes in a single cell was demonstrated as a promising and effective approach in many known processes [Cys] as a co-solvent in this reaction system enabled the reduction to proceed smoothly and increased the production yield up to 98.7%  within 48 h, with an ee value of 99.9% and a productivity rate of 5.73 g/(L h)  are often used and can provide enzyme flexibility for catalysis domain separation .To effectively synthesize . Among these variants, E. coli/pET-BsGDH-ER/K(10 nm)-LkCR exhibited the highest productivity, with (R)-3,5-BTPE concentrations of 297.3 g/L at a 96.7% yield, an excellent ee value (>99.9%), and a high productivity rate [29.7 g/(L h)]. These results demonstrated that the developed biocatalytic process is scalable and has strong potential for the industrial-scale preparation of (R)-3,5-BTPE.The NADPH-dependent carbonyl reductase from R)-3,5-BTPE, NADPH, and NADP+ were purchased from TCI , J&K , Roche  and Roche, respectively. Other chemicals involved were analytical grade.3,5-BTAP, (http://www.ncbi.nlm.nih.gov). Genomic DNA was extracted and purified using a TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0 . The DNA fragment of the carbonyl reductase gene was amplified and double digested using BamHI and XhoI, and then inserted into the expression vector pET-28a . The resulting plasmid was transformed into E. coli BL21 (DE3) cells cultured at 20 °C in lysogeny broth medium  containing 0.5 mM kanamycin. When the optical density at 600 nm of the culture reached 0.6–0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and cultivation was continued at 20 °C for another 14 h.Carbonyl reductase genes used for screening were selected from the NCBI database (g for 10 min) at 4 °C, washed twice with 20 mM sodium phosphate buffer , and subsequently disrupted with an ultrasonic oscillator . The cell lysate was removed by centrifugation  at 4 °C, and the supernatant was loaded onto a 5 mL Ni–NTA FF column , which was equilibrated with 20 mM imidazole buffer (pH 7.4) with saline. Proteins were eluted with an increasing gradient of 20 mM to 500 mM imidazole buffer (pH 7.4) with saline at a flow rate of 1 mL/min. The fractions containing the target protein were collected and dialyzed against 20 mM PBS (pH 7.4) for desalting. The sample was concentrated and stored at 4 °C, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify LkCR expression and purification.Cells were harvested by centrifugation (8000LkCR was assayed by measuring the change in absorbance at 340 nm according to NADPH oxidation or NADP+ reduction using an ultraviolet (UV)/visible spectrophotometer . A molar extinction coefficient of 6.22 mM/cm for NADPH was used for the calculation  at a flow rate of 1.0 mL/min at 40 °C, and detected at 210 nm. Retention times of R- and S-3,5-BTPE were 8.0 and 9.2 min, respectively . 3,5-BTAP and (


In the cation of the title molecular salt, one of the non-H substituents on the piperidine ring occupies an equatorial site and the other an axial site. The ions are linked into sheets by a combination of one N—H⋯O and two C—H⋯O hydrogen bonds. tert-butylphenyl)-4-oxobutyl]piperidine, reacts with 3,5-dinitrobenzoic acid in methanol solution to give the title 1:1 salt, ebastinium 3,5-dinitrobenzoate, C32H40NO2+·C7H3N2O6−. In the cation, the disubstituted aryl ring exhibits orientational disorder over two sets of atomic sites having occupancies 0.706 (4) and 0.294 (6), with a dihedral angle of 41.2 (5)° between the two orientations: the bulky Ph2CH—O– substituent occupies an axial site on the piperidine ring. The two ions in the selected asymmetric unit are linked by a nearly linear N—H⋯O hydrogen bond and this, in combination with two C—H⋯O hydrogen bonds, links the ions into complex sheets.Ebastine, 4-(benzhydryloxy)-1-[4-(4- The methyl groups were permitted to rotate but not to tilt. For the H atom bonded to the N atom, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), giving an N—H distance of 0.99 (3) Å. Subject to these conditions, the occupancies of the two disordered components refined to 0.706 (4) and 0.294 (4). In the final analysis of variance there was a large value, 15.256, of K = [mean(Fo2)/mean(Fc2)] for the group of 867 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.005.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901701324X/su5391sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901701324X/su5391Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901701324X/su5391Isup3.cmlSupporting information file. DOI: 1574718CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


These luminescent nanorod bundles exhibit strong green emission at 549 nm upon excitations at 449 nm and 980 nm with quantum efficiencies of ~6.3% and ~1.1%, respectively. The TEM/HRTEM results confirm that these bundles are composed of several individual nanorods with diameter of ~100 nm and length in the range of 1–3 μm. Furthermore, two dimensional spatially resolved photoluminescence intensity distribution study has been carried out using confocal photoluminescence microscope throughout the nanorod bundles. This study provides a new direction for the potential use of such emerging dual mode nanorod bundles as photon sources for next generation flat panel optical display devices, bio-medical applications, luminescent security ink and enhanced energy harvesting in photovoltaic applications.A novel method for demonstration of photoluminescence intensity distribution in upconverting nanorod bundles using confocal microscopy is reported. Herein, a strategy for the synthesis of highly luminescent dual mode upconverting/downshift Y Designing one-dimensional 1D) rare earth nanomaterials by template-free strategies is an ultimate challenge of cutting edge scienceD rare ea2345678910111314151718192021232425282930113153+, Tm3+, Ho3+ and Yb3+ 34352O3 is a one of the most explored host lattice due to its exceptional optical, thermal and mechanical properties37384, NaYF4, LaPO4 etc. Various synthesis methods have been used for the growth of upconverting nanophosphor with different morphologies; like, nanoparticles, nanotubes, nanoflakes, nanorods etc40414343Recently, the upconversion nanomaterials have received huge attention due to their various potential applications331.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles by a facile hydrothermal method has been reported. The Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles emit strong green colour centred at 549 nm upon excitations with of 449 nm and 980 nm wavelengths. The structural analysis of these nanorod bundles have been carried out using X-ray diffraction (XRD). The morphological and microstructural investigations of these luminescent nanorod bundles have been performed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM)/high-resolution transmission electron microscopy (HRTEM) techniques. Further, 2D spectral distribution of PL intensity of these luminescent nanorod bundles have been investigated for the first time.In this article, synthesis of dual mode Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles were synthesized by a facile hydrothermal method. Details of the synthesis process for Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles have been described in experimental section. The structural analysis of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles (before and after sintering) was investigated using X-ray diffraction (XRD) technique. The XRD pattern of Y1.94(OH)3: Ho3+0.02/Yb3+0.04 nanorod bundles is illustrated in 1.94(OH)3:Ho3+0.02/Yb3+0.04 nanorod bundles have hexagonal structure with space group P63/m (JCPDS card no. 83–2042). The lattice parameters of nanorod bundles were calculated from observed d-values using a least-squares fitting method (using unit cell refinement software)1.94(OH)3:Ho3+0.02/Yb3+0.04 nanorod bundles are, a = b = 6.2384 ± 0.0040 Å and c = 3.5276 ± 0.0043 Å with cell volume of 118.8947 ± 0.0183 Å3, which is comparable to the standard lattice parameters, a = b = 6.2610 Å, c = 3.5440 Å & cell volume of 120.3100 Å3(JCPDS card no. 83–2042). 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles sintered at 1000 °C. The XRD results reveal that Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles have cubic structure (JCPDS card no. 43–1036). The estimated lattice parameters of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles are, a = b = c = 10.5841 ± 0.0125 Å and cell volume is 1185.6720 ± 4.2106 Å3 which matches well with standard lattice parameters of a = 10.6040 Å and cell volume of 1192.36 Å3. 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles, where Y atoms are substituted by Ho and Yb atoms in unit cell as per coordination number and ratio of Ho/Yb. Further, crystal structure and phase of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles were investigated using Raman spectroscopy. 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles exhibiting peaks at 128, 161, 193, 230, 293, 304, 332, 378, 433, 470 and 593 cm−1. The intense peak at 378 cm−1 represents cubic structure of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles471.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles. 1.94(OH)3:Ho3+0.02/Yb3+0.04 nanorod bundles. TGA graph demonstrates the total weight loss during heating is 25.99%. Moreover, TGA graph exhibits the weight loss occurred in three steps upto 900 °C. The weight loss in the first step was 2.91% which is attributed to the transformation of polymer complexed metal nitrate conversion into metal hydroxide (upto~252 °C). In the second step, a major weight loss upto 13.65% was observed which is related to the dehydration of hydroxide and formation of oxide nanorod bundles (upto~363 °C). In the final step, till 800 °C, weight loss of 9.43% was observed which is ascribed to removal of unused intercalated nitrates ionsY1.94(OH)3: Ho3+0.02/Yb3+0.04 nanorod bundles is shown in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles. The SEM image clearly shows uniform growth of nanorod bundles throughout the sample with diameter in the range of 0.4 to 0.6 μm and length from 6 to 10 μm. The magnified SEM image of nanorod bundles is shown in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles was investigated using energy dispersive X-ray analysis (EDAX). The EDAX spectrum was taken from the red mark region in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles is shown in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles was dispersed in ethanol and ultra-sonicated at 45 kHz for 45 min, prior to TEM characterization to avoid any agglomeration between nanorods. 2O3 (JCPDS card no. 43–1036). A plausible mechanism for the growth of nanorod bundles is demonstrated in The scanning electron microscopy (SEM) was used to probe the surface morphology of nanorod bundles. SEM image of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles were examined using PL and TRPL (time-resolved photoluminescence) spectroscopic techniques. 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles upon excitation with 980 nm wavelength. The PL emission spectrum of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod shows strong green emission peaking at 549 nm which corresponds to 5F4, 5S2-5I8 transition in Ho3+ ion491.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles can be explained using two processes; energy transfer upconversion (ETU) and excited-state absorption (ESA). In the energy transfer upconversion (ETU) process, the sensitizer ion (Yb3+) and activator ion (Ho3+)absorb the laser photons (pump photon) and get excited to higher energy state (metastable state). The excited sensitizer ion (Yb3+) transfer its energy to activator ion (Ho3+) at meta-stable state and relax back to ground state. This transferred energy excite meta-stable activator ion (Ho3+) to higher energy level. In excited state absorption (ESA) process, activator ions absorb two photons. The first photon excites the activator ion to meta-stable state and the second photon promotes it to higher excited state, which is responsible for upconversion. 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles. The inset in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles under excitation at 980 nm wavelength, where x = 0.327 and y = 0.667. 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles at excitation of 449 nm. The PL emission spectrum of nanorod bundles shows strong green emission at 549 nm. The inset in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles are almost equal at 549 nm. The excitation spectrum of nanorod bundles at fixed emission wavelength of 549 nm is illustrated in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles is demonstrated in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles for downshift and upconversion are ~6.3% (using xenon lamp as source of excitation) and ~1.1% (using 980 nm diode laser with power density 150 Wcm−2), respectively.The spectroscopic features of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles at emission wavelength of 549 nm upon excitation wavelength of 449 nm is demonstrated in 1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles is best fitted with double exponential function as given in equation (1)511 = 20.84 μs, τ2 = 80.45 μs, χ2 = 1.44, A1 = 35 and A2 = 65. The decay time of nanorod bundles are τ1 = 20.84 μs and τ2 = 80.45 μs.The double exponential decay components of Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles indicating the presence of at least two electronically excited species. The presence of activator Ho3+ ion and co-activator/sensitizer Yb3+ ion in Y2O3 host lattice could be the reason behind the observed double exponential decay. The average decay time of nanorod bundles is τav = 61.33 μs, which is calculated using equation  was recorded using a single photon counting technique using a microsecond xenon flash lamp as the source of excitation. The application of luminescent materials depends upon the observed lifetime. It is well established that lifetime in the range of milliseconds to microseconds are highly useful for several potential applications; such as optical display devices, bio-medical and security ink applications11415161.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles have dual mode (downshift as well as upconversion) upon excitation wavelengths of 980 nm and 449 nm. The observed lifetime results suggest that these nanorod bundles are highly useful for various applications such as advanced optical display devices1115The obtained spectroscopic results reveal that the Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles at different places. Further, PL mapping was performed at the same location from where the fluorescent image was taken , Yb3+(co-dopant) in Y2O3 (host lattice) nanorod bundles. Moreover, our finding could bring a better understanding about the PL intensity distribution in luminescent materials and provides a new direction that is extremely important for next generation photo emission based displays applications.We have successfully demonstrated a strategy for the synthesis of highly-luminescent dual mode upconverting Y2O3 (99.99%), Ho2O3 (99.99%), YbCl3:6H2O (99.99%), N-cetyl-N,N,N-trimethyl ammonium bromide  and HNO3 (A.R. grade) were purchased from Sigma-Aldrich and used without further purification.The precursors; Y1.94O3:Ho3+0.02/Yb3+0.04 nanorod bundles. The optimization activator and sensitizer of upconversion process in Y2O3 host lattice were optimized in our pervious report2O3 and Ho2O3 were dissolved in 20 ml D. I. water. Few drops of nitric acid were added to solution while stirring at 80 °C until the solution become transparent. Further, the precursors; YbCl3:6H2O and CTAB were dissolved in deionized water. All the above solutions were mixed by vigorous stirring at room temperature. Ethanol solution (1:5 ethanol: water) was added to the above mixed solution. Sodium hydroxide solution was added drop wise to the above solution till pH ~13 was achieved. Then, this solution was kept in hydrothermal vessel at 185 °C for 8 hours in box furnace. The pH is very crucial parameter for controlling the shape and size of nanorod bundles composed of individual dimensions of nanorod because these nanorod based bundles originate rapidly with closer nuclei formation as a co-nuclei effect at higher pH leads anisotropic growth during metal nitrate precursor to hydroxide formation. The obtained white precipitates was centrifuged several times with de-ionized water at 5000 rpm and then dried at 100 °C. The dried white powder was further sintered at 1000 °C for 5 hours to obtain the final product.The facile hydrothermal method was used for synthesis of dual mode Y1 radiation (λ = 1.5406 Å). The Raman spectrum was recorded using Renishaw inVia Raman spectrometer, with an excitation source of 514 nm. The thermogravimetric analysis (TGA) was carried out using a thermal analysis instrument with a heating rate of 10 °C min−1 in an air flow of 100 mL min−1. The surface morphology and energy dispersive X-ray analysis (EDAX) were examined by using field emission scanning electron microscope (FESEM) Carl ZEISS-SUPRA 40 VP equipped with EDAX facility. Transmission electron microscopy (TEM) and high-resolution transmission microscopy (HRTEM) were performed using a Tecnai G2 S-Twin with a field emission gun operating at 300 kV. The downconversion/downshift PL measurement was recorded using an Edinburg FLS900 fluorescence spectrometer equipped with 450 W xenon lamp as excitation source. The upconversion emission spectra were recorded using with an external power-controllable 980 nm diode laser (power density = 150 Wcm−2). To estimate the absolute luminescence quantum efficiency of nanorod bundles, an integrating sphere equipped with an Edinburgh spectrometer (Model FLS900) instrument has been used for measuring the integrated fraction of luminous flux and radiant flux with the standard methodThe crystal structure and composition of sample was identified by powder X-ray diffraction (XRD) using a Bruker AXS D8 Advance X-ray diffractometer, using Cu KaHow to cite this article: Kumar, P. et al. Experimental observation of spatially resolved photo-luminescence intensity distribution in dual mode upconverting nanorod bundles. Sci. Rep.7, 42515; doi: 10.1038/srep42515 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Interestingly, Vγ9Vδ2-T cells acquired Tfh-associated molecules such as CXCR5, PD1, CD40L, and ICOS during influenza virus stimulation, especially in the presence of CD4 T cells. Moreover, Vγ9Vδ2-T cells promoted CD4 T cells to secrete IL-13 and IL-21, and neutralizing IL-13 and IL-21 significantly reduced the number of CD19+IgD−CD38++ PCs. Using humanized mice, we further demonstrated that Vγ9Vδ2-T cells could synergize CD4 T cells to produce influenza virus-specific antibody. Our findings provide a greater scope for Vγ9Vδ2-T cells in adaptive immunity, especially for the Tfh development and humoral immune responses against influenza virus infection.Human Vγ9Vδ2-T cells recognize nonpeptidic antigens and exert effector functions against microorganisms and tumors, but little is known about their roles in humoral immune response against influenza virus infection. Herein, in the coculture of autologous human B cells, dendritic cells and/or naïve CD4 T cells, and Vγ9Vδ2-T cells, we demonstrated that Vγ9Vδ2-T cells could facilitate H9N2 influenza virus-specific IgG and IgM productions in a CD4 T cell-dependent manner. Vγ9Vδ2-T cells promoted the differentiation of CXCR5 B cells are required as a second stage of antigen-presenting cells (APCs)-CD4+ T cell conjugation to maintain and complete Tfh differentiation. Fully polarized Tfh cells are located within germinal center (GC) and have high expression of CXCR5 and Bcl-6  by vaccines is the most efficient strategy, because the neutralizing Abs can conjugate virus particles at the first contact of their invasion and promote clearance by host cells , 2. To gnd Bcl-6 , 9.Vγ9Vδ2-T cells constitute 1–5% of circulating T cells in human peripheral blood and they can be expanded rapidly in response to microbes that produce the metabolite pyrophosphate , 11. Alt−/− mice. Well-developed GCs are also present in the secondary lymphoid organs of TCRα−/− and TCRβ−/− mice  and their precursors play pivotal roles in humoral immune response by secreting antigen-specific immunoglobulin (Ig) . In the nd IL-21 –28. IL-2nd IL-21 , 30. Altnd IL-21 , 31. Whiin vivo. We demonstrated that Vγ9Vδ2-T cells could increase H9N2 influenza virus-specific IgG and IgM productions in a CD4 T cell-dependent manner both in vitro and in vivo. During this process, Vγ9Vδ2-T cells also acquired Tfh-associated features especially in the presence of CD4 T cells, indicating a reciprocal effect between Vγ9Vδ2-T and CD4 T cells in the differentiation of Tfh-like cells.The aim of our work is to examine the role of Vγ9Vδ2-T cells in antigen-specific antibody production, PC differentiation, as well as B cell Ig isotype switching during influenza virus stimulation, and then applied humanized mice to confirm their effects + monocytes were isolated from human peripheral blood mononuclear cells (PBMC) using the anti-CD14+ microbeads (Miltenyi Biotec). The CD14+ monocytes were cultured with GM-CSF (50 ng/ml) and IL-4 (10 ng/ml) for 5–7 days to differentiate into immature DCs. 5 × 104 immature DCs were seeded in 96-well U-bottom plate and were pulsed with UV-inactivated H9N2 influenza viruses  and LPS (100 ng/ml). Naïve CD4+ T cells were isolated from autologous PBMC using the naïve CD4+ T cells isolation kit (Miltenyi Biotec). 2.5 × 105 naïve CD4+ T cells in each well were cultured with the UV-H9N2 influenza viruses pulsed DCs in 96-well U-bottom plate for 5 days. On day 5 after naïve CD4 T cells and DCs being cocultured, human CD19+ B cells and Vγ9Vδ2-T cells were isolated from autologous PBMC using human CD19 microbeads (Miltenyi) and TCR γδ T cells isolation Kit (Miltenyi), respectively. 1 × 104 DCs/well were seeded in 96 well U-bottom plate the day before Vγ9Vδ2-T cells and B cells isolation and were treated with UV-H9N2 (MOI = 5). 5 × 104 CD4 T cells that have been cultured with UV-H9N2 pulsed DCs were added in 96 well U-bottom plate (with UV-H9N2 pulsed DCs) according to different groups. 1 × 105 B cells were added in all groups and 1 × 105 Vγ9Vδ2-T cells were added in 96 well U-bottom plate in indicated group. B cells were stimulated with UV-H9N2 at the MOI of 2. Cells were cultured in Iscove’s Modified Dulbecco’s Medium (Invitrogen), supplemented with 10% FBS, 50 µg/ml transferrin , 5 µg/ml insulin , and 15 µg/ml gentamicin (Invitrogen), 40 ng/ml human IL-4 (Peprotech). In all the coculture experiments, all cultured cells  were isolated from the same donor. The cell purity was above 95% after magnetic cell sorting.Human CD14Following Abs were used for flow staining: anti-TCR Vδ2 (B6), anti-TCR Vγ9 (B1), anti-CD14 (63D3), anti-CD4 (RPA-T4), anti-CD19 (HIB19), anti-CXCR5 (J252D4), anti-PD1 (EH12.2H7), anti-Bcl-6 (7D1), anti-CD40 (5C3), anti-CD40L –31, antiTo detect the cytokine production in supernatant, the bead-based flowcytomix assay was used according to the manual provided by the manufacturer . The concentration of the cytokines was calculated by extrapolating the mean fluorescence intensity on the respective standard curves and expressed as picogram per milliliter.4 DCs/well were seeded at the bottom of 96 well-transwell plate . 1 × 105 CD19+ B cells and 5 × 104 CD4 T cells that have been cultured with UV-H9N2 pulsed DCs were added directly to the bottom wells of the transwell plate. 1 × 105 Vγ9Vδ2-T cells were added in the upper chamber or the lower chamber in indicated groups. In some inserts, CD4 T cells were separated from B cells in the upper chamber. The B cells in each group were pulsed with UV-H9N2 influenza viruses (MOI = 2). On the day 6–8 after incubation, the differentiation of PCs and CD4+ Tfh cells were determined by FACS LSR II.1 × 10+ Tfh cells and PCs were determined by FACS LSRII.For IL-13 blocking, IL-13 neutralizing antibody  and IgG control antibody  were used. For IL-21 blocking, recombinant human IL-21R Fc chimera protein  and recombinant human IgG1 Fc  were used. After 6 days’ culture with cells in different groups, the differentiation of CD45 virus/well) at 4°C overnight. Supernatant from different groups was added to the blocked plate and incubated for 2 h at 37°C. HRP conjugated human IgG and IgM detection antibody was added to each well for 1 h at 37°C. TMB substrate solution was added to each well. Fifteen minutes later, the reaction was stopped by adding 2 M hydrochloric acid (HCL). Plates were read at 450 nm.The amounts of total human IgG and IgM Abs in the supernatant were determined by human IgG and IgM ELISA Quantitation Set (Bethyl). For virus-specific IgG and IgM Abs production, 96 well plates were coated with UV-H9N2 influenza viruses in coating buffer (5 × 1025 µl PBS was added in 96 well U-bottom plates from the second row to the last row. Supernatant or receptor destroyed enzyme-treated serum were added to the first row (50 μl/well) and serially diluted twofold. 4 HAU UV-inactivated H9N2 viruses in 25 µl volume PBS were added to each well. After 30 min incubation in 37°C, 0.05% turkey red blood cells were added to the plates and incubated for 40 min at room temperature. Then, the HAI titers were read and recorded.Samples of spleen from whole PBMC-humanized mice were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with rabbit anti-human CD4 , goat anti-human CD20 (Abcam), and mouse anti-human TCR γδ  Abs. The immune localization of CD4  and CD20 , CD4  and TCR γδ , and CD20  and TCR γδ  were stained in spleen section using Immunohistochemistry Double Staining Kit .−/−γc−/− mice were treated with liposomes  1 day before transplantation. The sublethally irradiated mice were transplanted i.p. with 30 × 106 PBMC or specific cell-depleted PBMC. The immune reconstitution of humanized mice was according to published paper in our group (3TCID50) through i.p. pathway on day 0 and 7. Serum was collected on day 21 postvaccination, and total IgG, IgM, and virus-specific Abs were determined by ELISA and HAI assay.Vγ9Vδ2-T cell-depleted PBMC, CD4 T cell-depleted PBMC, and both Vγ9Vδ2-T and CD4 T cell-depleted PBMC were obtained after depletion cells by magnetic microbeads (Miltenyi Biotec). 4- to 5-week-old male or female Rag2ur group , 32, 33.Human PBMC were collected from the buffy coats, which were obtained from the Hong Kong Red Cross approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB number: UW 07-154). All animal studies were approved and performed in compliance with the guidelines for the use of experimental animals by the Committee on the Use of Live Animals in the Teaching and Research (CULATR), the University of Hong Kong (CULATR number: 2986-13).t-tests or two-way ANOVA (GraphPad Prism 5.0), with differences considered significant as indicated in the figures: *p < 0.05; **p < 0.01; ***p < 0.001.Data were analyzed using paired Student’s To investigate the role of Vγ9Vδ2-T cells in antibody response against influenza virus, B cells were cocultured with CD4 and/or Vγ9Vδ2-T (γδ T) cells for 7–10 days in the presence of UV-H9N2-pulsed DCs, and the total and influenza virus-specific Abs in the supernatants were determined. As shown in Figures + Tfh cell differentiation during influenza virus stimulation. As shown in Figures +PD1+CD4+ Tfh cells . In the spleen of humanized mice reconstituted with whole PBMCs, we further observed the co-localization of CD20+ B cells, CD4 T cells, and Vγ9Vδ2-T cells. Thus, we believe that these APC-like Vγ9Vδ2-T cells present antigen to CD4 cells and support CD4+ Tfh cell differentiation as well as proliferation in a cell–cell contact-dependent manner.It is well accepted that Tfh cell differentiation is a multistep process including initial activation by DCs within the T cell zone, and followed by conjugations with B cells at the T-B border or within the follicle , 39. Prentiation . Vγ9Vδ2-ntiation . Here, win vitro studies have shown that human IL-6, IL-12, and IL-21 are involved in promoting the commitment of naïve CD4+ T cells into the Tfh lineage . All animal studies were approved and performed in compliance with the guidelines for the use of experimental animals by the Committee on the Use of Live Animals in the Teaching and Research (CULATR), the University of Hong Kong (CULATR number: 2986-13).WT initiated the project, designed the experiments, and wrote the manuscript. QC, AL, ML, and KW designed and performed the experiments. KW, KN, and ZX helped accomplish some of the animal experiments. KW, YL, and WT edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.