Their structures were determined using HR-ESI-MS data, 1D and 2D NMR spectra, 13C NMR calculations, and electronic circular dichroism (ECD) calculations. Heilaohuacids A and B (1 and 2) contain a 3,4-seco ring A and unprecedented migration of Me-18 from C-13 to C-17 or C-14 to C-18. This type of lanostane triterpenoid derivatives was rarely reported so far. More importantly, all compounds against inflammatory cytokines IL-6 and TNF-α levels on LPS-induced RAW 264.7 macrophages were evaluated, and compounds 4 and 31 significantly inhibited the release level of IL-6 with IC50 values of 8.15 and 9.86 μM, respectively. Meanwhile, compounds 17, 18, and 31 significantly inhibited proliferation of rheumatoid arthritis-fibroblastoid synovial (RA-FLS) cells in vitro with IC50 values of 7.52, 8.85, and 7.97 μM, respectively.One new 3,4- Kadsura and Schisandra. This family of medicinal plants are enriched with lanostane, cycloartane, and schinortriterpenoid (SNT) triterpenoids , gastric and duodenal ulcers, etc.  Craib. in the same genus Kadsura displayed good anti-rheumatoid arthritis, anti-inflammatory, and analgesic effects , one new 3,4-seco-17,14-friedo-lanostane triterpenoid heilaohuacid B (2), five new 3,4-seco-lanostane triterpenoids heilaohuacids C-D (3–4) and heilaohumethylesters A-C (7–9), one new 3,4-seco-cycloartane triterpenoid heilaohuacid E (5), and one new intact-lanostane triterpenoid heilaohuacid F (6) (10–31), were isolated from heilaohu  , togetheheilaohu . Their s1 was derived as a white amorphous powder, and the molecular formula C30H44O4, with 9 degrees of unsaturation, was deduced from the HR-ESI-MS at 491.3143 [M + Na]+  and its 13C NMR data. The 1H NMR data showed typical resonances for five tertiary methyls , one double methyl , and five olefinic protons . The 13C NMR data with the aid of DEPT and HSQC spectra revealed the presence of six methyls, nine methylenes, six methines (three tri-substituted double bond), and nine quaternary carbons (two carboxyl groups). Detailed analyses of 1H NMR, 13C NMR, DEPT, and HSQC spectra enabled all proton resonances of 1 to be attributed to their respective carbons . The HMBC cross-peaks  matched the experimental data better than 1-2 (R2 = 0.9928), which indicated that H3-21 has an a-orientation. To further elucidate its absolute configuration, the electronic circular dichroism (ECD) spectrum of 1 was recorded in MeOH, and it showed a good agreement with the calculated ECD spectrum of the  model  model , which s2) had the same molecular formula of C30H44O4 as compound 1, based on the HR-ESI-MS at m/z 467.3105 [M-H]− . Its 1H NMR spectrum displayed signals characteristic of five olefinic protons at δH 6.92 , 6.21 , 5.39 , 4.95 , and 4.75 . The 1H NMR spectrum also displayed one methyl doublet at δH 0.84  and five methyl groups at δH 1.85 (H3-27), 1.78 (H3-29), 1.00 (H3-18), 0.86 (H3-19), and 0.65 (H3-30). The 13C NMR and DEPT spectra data  matched the experimental data better than 2-2 (R2 = 0.9952), which indicated that H3-21 has an a-orientation. The absolute configuration of 2 was determined by ECD data. The experimental ECD spectrum of 2 exhibited a negative cotton effect around 250 nm, which was consistent with the calculated ECD data of the  model (2) was elucidated as a 5S, 9S, 10R, 13S, 17S, and 20R absolute configuration.The relative configuration of elations  of H3-19R) model . Thus, h3 had a molecular formula of C30H46O4, requiring eight indices of hydrogen deficiency by analyzing the HR-ESI-MS at m/z 469.3352 [M-H]− . Comprehensive analysis of the 1D and 2D NMR data revealed it to be the derivative of seco-coccinic acid K  and the absence of a methoxy group at C-31 in 3, which were confirmed by the HMBC correlations of H-11 with C-12, C-9, and C-13, H3-18 with C-12, and H2-1 and H2-2 with C-3. The relative configuration of H-8 was determined to be β-oriented, through the NOE correlations of H3-18 with H-8. The 5, 8, 10S, 13R, 14S, 17R, and 20R absolute configuration of 3 was determined by comparing the experimental and calculated ECD spectra . Comprehensive analyses of its NMR data  shifted from ring C to ring B, a double bond at C-24/C-25 was present, but a ketone group was absent at C-23 in 4, as supported using the HMBC spectral analyses. The relative configuration was confirmed by ROESY spectral analyses. Based on the NOE correlation of H3-19 with H-9, the H-9 was classified as β-oriented. Thus, the structure of 4 was assigned as shown in Compound NMR data , 3 sugge5 was C30H48O3, as determined by the HR-ESI-MS ion at m/z 455.3532 [M-H]− , suggesting 7° of unsaturation. The 1H and 13C NMR spectra showed typical resonances for 3,4-seco ring A δH 4.75, 4.83 , 1.68  and δC 179.4 (C-3), 149.4 (C-4), 111.6 (C-28), and a pair of methylene doublets at δH 0.42 (J = 3.8 Hz) and 0.74 (J = 3.8 Hz), characteristics of the C-19 protons and carbon of the cyclopropane ring, suggesting that six was a 3,4-seco-cycloartane triterpenoid (5 was very similar to nigranoic acid (25)  and H2-24 (δH 2.26) with C-23 (δC 211.4) supported the ketone group locate at C-23. In a ROESY experiment, H-8 (δH 1.58) showed correlations with H3-18 (δH 1.01), which indicated that H-8 was β-oriented. Hence, compound 5 was elucidated as shown and named heilaohuacid E.The molecular formula of compound erpenoid . Analysierpenoid , 3 reveacid (25) . The obv6 was obtained as a white amorphous powder and its molecular formula was deduced to be C30H46O3 based on the HR-ESI-MS showing molecular ion at m/z 453.3364 [M-H]−  with 8° of unsaturation. The 1H NMR data , six methyl singlet protons at δH 1.25 (H3-18), 0.67 (H3-19), 1.84 (H3-27), 1.07 (H3-28), 1.08 (H3-29), and 0.75 (H3-30), and two olefinic protons at δH 5.31  and 6.91 . Analyses of the 13C NMR and DEPT data , and eight quaternary carbons (two carboxyl group). These evidences indicated that compound 6 was an intact lanostane-type triterpenoid, whose 1H and 13C NMR spectroscopic data were very similar to those of coccinic acid  was assigned a molecular formula of C31H50O4 based on the HR-ESI-MS spectra and NMR data analysis   was located at C-31, confirmed by the HMBC correlations of H3-31 with the carbonyl (δC 175.1) at C-3. The similar chemical shifts, coupling constants, and NOE correlations with 16 determined the relative configurations of 7. Therefore, the structure of 7 was elucidated as shown.Heilaohumethylester A  . The pre8 was deduced to have the molecular formula of C31H52O4 from the molecular ion at m/z 511.3734 [M + Na]+  in the HR-ESI-MS data. The NMR data of 8 were highly similar to those of 7; HMBC spectral analysis showed that the obvious differences were absence of a ketone group at C-23 and a hydroxyl group was present at C-24 in 8. Comparison of the NMR data of 8 with those of a pair of 24-epimers (S)-24,25-dihydroxytirucall-7-en-3-one  and (24R)-24,25-dihydroxytirucall-7-en-3-one . This indicated that the NMR data of 8  were almost similar to the corresponding 24(S)-24,25-dihydroxytirucall-7-en-3-one, suggesting that the C-24 stereochemistry should be assigned as 24S in 8. According to analysis of the NOE effect, and the ROESY cross-peaks of H3-19 with H-9, the H-9 was classified as β-oriented. Finally, the structure of 8 was identified, and named heilaohumethylester B accordingly.Compound -epimers  with the9 was obtained as a white amorphous powder, and its molecular formula was found to be C31H48O3 deduced from HR-ESI-MS, indicating quasi-molecular ion peak at m/z 491.3499 [M + Na]+ . The 1H and 13C NMR data  (11) (E)-3,4-seco-9βH-lanosta-4 (28),7,24-triene-3,26-dioic acid (12) (Z)-3,4-seco-tirucalla-4(28),7,24-triene-3,26-dioic acid (13)  (19 and 20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31)  , abiesat10) (11) , 24(E)-3cid (12) , (24Z)-3cid (13) , seco-co (14-18) , kadsura and 20) , micrano20) (21) , schigla21) (22) , manwuwe22) (23) , 3-monom23) (24) , nigrano24) (25) , abiesat25) (26) , changna26) (27) , schigla27) (28) , schisan28) (29) , kadsula29) (30) , and sch30) (31) , by comp1–31) were evaluated using the MTT method, and methotrexate was used as the positive control (IC50 4.10 µM). The results  released by LPS-induced RAW 264.7 cells. The inflammatory activity of the isolated compounds was determined using ELISA kits, with methotrexate as positive control. The results  column. Extract fractions were analyzed using TLC, and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 in Vanillin solution. Petroleum ether (PE), hexane, ethyl acetate (EtOAc), ethanol, n-butanol (n-BuOH), methanol (MeOH), and dichloromethane (CH2Cl2) were purchased from Shanghai Titan Scientific Co. Ltd. Acetonitrile and methanol (HPLC grade) were purchased from Merck KGaA, 64,271 Darmstadt, Germany.Optical rotations of compounds were determined by a Rudolph Research Analytical Autopol Ⅲ automatic polarimeter. UV analysis of compounds was performed on a Shimadzu 2450 UV-vis spectrometer. An Applied Photophysics Chirascan plus CD spectrometer was used to determine ECD spectrum. An Agilent Technologies Cary 630 FTIR spectrometer was used to determine IR spectra of compounds. K. coccinea were collected from Huaihua, Hunan Province, People’s Republic of China, in July 2015. Plant material was identified by one of the co-authors (WW). A voucher specimen  was deposited at TCM and Ethnomedicine Innovation and Development International Laboratory, Innovative Materia Medica Research Institute, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan, People’s Republic of China.The dried roots of K. coccinea (100 kg) were extracted twice with 80% ethanol for 2 h each time under reflux and filtered. All extract solvents were evaporated under vacuum to obtain crude EtOH extract (3 kg). Half of the whole ethanol extract (1.5 kg) was suspended in H2O and successively partitioned with PE, CH2Cl2, EtOAc, and n-BuOH to give a PE-soluble fraction (182 g), CH2Cl2-soluble fraction (545 g), EtOAc-soluble fraction (330 g), n-BuOH-soluble portion (173 g), and H2O layer. The CH2Cl2-soluble fraction (545 g) was subjected to silica gel column chromatography (CC) eluted with hexane-EtOAc (80:1-0:1) to afford twelve fractions (C1–C12). Fractions C2 (13.1 g), C4 (45.2 g), and C6 (35.6 g) were separated by silica gel CC with cyclohexane-EtOAc (80:1-0:1) to yield 17 (12.0 g) and 13 (600 mg), respectively. Fraction C3 (29.5 g) was chromatographed on a silica gel column eluted with cyclohexane-EtOAc (200:1-0:1) gradients further to give 13 fractions (C3-1–13) and 12 (300 mg). Subfraction C3-5 (6 g) was separated on silica gel CC with hexane-EtOAc (100:1-20:1) to give 18 (2.0 g). Subfraction C3-7 (50 mg) was purified using semipreparative HPLC (99% MeOH in H2O) to yield compound 19 . Fraction C5 (49.5 g) was chromatographed on a silica gel column eluted with cyclohexane-CH2Cl2-EtOAc (80:1:0-200:1-0:5:1) gradients to give 12 further fractions (C5-1∼12). Subfraction C5-8 (5.0 g) was purified by using repeated silica gel CC eluted with hexane-EtOAc (40:1-0:1) and then by semipreparative HPLC with the mobile phase (93% MeOH/H2O) to obtain 30  and 8 . Subfraction C5-9 (5.0 g) was further purified by using silica gel CC with hexane- EtOAc (100:1-0:1) to obtain 14 further fractions (C5-9-1–14), 10 (15 mg) was isolated from subfraction C5-9-12 by using silica gel CC eluted with hexane-CHCl3 (20:1-0:1), and subfraction C5-9-11 (128 mg) was purified by using semipreparative HPLC with the mobile phase (72% MeOH/H2O) to yield 14  and 29 . Subfraction C5-10 (5.3 g) was chromatographed on a silica gel CC and eluted with hexane-EtOAc (40:1-0:1) gradients to give 15 (1.0 g). C5-9-12 (229.2 mg) was purified by semipreparative HPLC with the mobile phase (91% MeOH/H2O) obtained 22 , 11 , 7 , and 24 . Fraction C8 (15.5 g) was chromatographed on a silica gel column while eluted with PE-acetone (40:1-0:1) gradients to obtain 12 fractions (C8-1–12), subfraction C8-8 (15.5 g) was subjected to silica gel CC eluted with PE-acetone (40:1-0:1) to afford twelve subfractions (C8-8-1–12), 20  was isolated from the subfraction C8-8-9 (52.5 mg) by semipreparative HPLC with the mobile phase (93% MeOH/H2O), and 28  was isolated from the subfraction C8-8-8 (30.0 mg) by semipreparative HPLC with the mobile phase (90% MeOH/H2O). Fraction C9 (53.9 g) was chromatographed on a silica gel column eluted with PE-EtOAc (10:1-0:1) gradients to give 16 fractions (C9-1–16), subfraction C9-8 (5.3 g) was further subjected to silica gel CC with hexane-acetone (40:1-0:1) as mobile phase to obtain 17 further fractions (C9-8-1–17), and 1  and 26  were isolated from the subfraction C9-8-16 (120 mg) by semipreparative HPLC with the mobile phase . Compound 2  was isolated by semipreparative HPLC with the mobile phase (90% MeOH/H2O) from subfraction C9-8-15 (50 mg), subfraction C9-8-13 (68.7 mg) was purified by semipreparative HPLC with the mobile phase (70% MeOH/H2O) to obtain 4 , subfraction C9-8-14 (135 mg) was purified by semipreparative HPLC with the mobile phase  to obtain 25 , and 27 , subfraction C9-8-15 (181.7 mg) was separated on C18 column eluted with 30%–100% MeOH-H2O to yield 21 (4 mg). Subfraction C9-10 (8.0 g) was subjected to silica gel CC eluted with hexane-acetone (40:1-0:1) gradients to 16 (1.0 g). Subfraction C9-12 (3.6 g) was repeatedly purified by silica gel CC with CHCl3-EtOAc (40:1-0:1) to obtain 12 further fractions (C9-12-1–12), and subfraction C9-12-2 (89.8 mg) was purified by semipreparative HPLC with the mobile phase (76% MeOH) to yield 31 . C9-12–5 (22.9 mg) was purified by semipreparative HPLC with the mobile phase (90% MeOH/H2O) to yield 23  and 3 . C9-12–6 (50.0 mg) was purified by semipreparative HPLC with the mobile phase (92% MeOH/H2O) to yield 6  and 5 .The dried roots of 9 (18.2 mg).The EtOAc-soluble fraction (330 g) was applied to silica gel column chromatography, eluted with PE-EtOAc (100:0-0:100), to give twelve fractions (Fr. E1–E12). Fr. E5 (90 g) was separated by silica gel column chromatography using Hexane-EtOAc (100%-0) to afford eight subfractions (Fr. E5-1–8). Fr. E5-3 was purified by semipreparative HPLC  to obtain compound c 0.01 CHCl3); UV (CHCl3) λmax (log ε) 219 (4.2) nm, 248 (1.8) nm; IR (KBr) νmax 2,926, 2,853, 1,718, 1,696, 1,656, 1,561, 1,457, 1,372, 1,260, 962, and 900 cm−1; 1H- and 13C NMR data, see m/z 491.3143 [M + Na]+ .White amorphous powder [α]25 D - 11 (c 0.02 MeOH); UV (MeOH) λmax (log ε) 212 (6.7) nm, 252 (6.52) nm; IR (KBr) νmax 3,525, 2,972, 2,866, 1,791, 1,696, 1,652, 1,569, 1,472, 1,394, 1,056, 1,032, and 746 cm−1; 1H- and 13C NMR data, see m/z 467.3105 [M-H]− .White amorphous powder [α]25 D - 39 (c 0.07 MeOH); UV (MeOH) λmax (log ε) 204 (3.5) nm, 250 (2.8) nm; IR (KBr) νmax 3,414, 1,699, 1,652, 1,457, 1,372, 1,260, 962, 900, and 668 cm−1; 1H- and 13C NMR data, see m/z 469.3352 [M-H]− .White amorphous powder [α]25 D - 67 (g ε) 204 .5 nm, 25c 0.04 MeOH); UV (MeOH) λmax (log ε) 206 (6.4) nm, 245 (6.3) nm; IR (KBr) νmax 3,628, 2,950, 1,771, 1,558, 1,436, 1,374, 1,260, 1,032, 667, and 646 cm−1; 1H- and 13C NMR data, see m/z 453.3406 [M-H]− .White amorphous powder [α]25 D - 38 (c 0.02 MeOH); UV (MeOH) λmax (log ε) 204 (6.3) nm; IR (KBr) νmax 3,401, 3,223, 2,972, 2,856, 1,749, 1,699, 1,558, 1,460, 1,246, 1,056, and 643 cm−1; 1H- and 13C NMR data, see m/z 455.3532 [M-H]− .White amorphous powder [α]25 D + 19 (ε) 204 6. nm; IR (c 0.06 MeOH); UV (MeOH) λmax (log ε) 204 (3.6) nm, 240 (2.8) nm; IR (KBr) νmax 2,944, 2,899, 1,715, 1,652, 1,448, 1,402, 1,372, 1,280, 959, and 900 cm−1; 1H- and 13C NMR data, see m/z 453.3364 [M-H]− .White amorphous powder [α]25 D - 83 (c 0.02 MeOH); UV (MeOH) λmax (log ε) 205 (6.6) nm; IR (KBr) νmax 3,369, 2,946, 2,833, 1,699, 1,635, 1,506, 1,456, 1,372, 1,035, and 667 cm−1; 1H- and 13C NMR data, see m/z 509.3561 [M + Na]+ .White amorphous powder [α]25 D - 72 (c 0.03 MeOH); UV (MeOH) λmax (log ε) 205 (6.5) nm; IR (KBr) νmax 3,518, 3,199, 2,963, 1,718, 1,653, 1,558, 1,260, 1,014, 804, and 650 cm−1; 1H- and 13C NMR data, see m/z 511.3734 [M + Na]+ .White amorphous powder [α]25 D - 88 c 0.0 MeOH; UVc 0.05 CHCl3); UV (CHCl3) λmax (log ε) 240 (3.4) nm; IR (KBr) νmax 2,929, 2,856, 1,757, 1,704, 1,558, 1,460, 1,246, and 1,024 cm−1; 1H- and 13C NMR data, see m/z 491.3499 [M + Na]+ .White amorphous powder [α]25 D - 55 . RAW264.7 cell line was purchased from Fuheng Biological Technology Co., Ltd. . Human RA-FLS and RAW264.7 cells were cultured in DMEM/F12 with 10% FBS and DMEM with 10% FBS in 5% CO1–31) on release of inflammatory cytokines (IL-6 and TNF-α) in the supernatants on LPS-induced RAW264.7 cells were determined using ELISA kits  following the manufacturer’s instructions. Methotrexate was used as a positive control.Inhibition effects of all compounds  was then added to each well and incubated for 4 h. The supernatants were retrieved, and 100 μl of DMSO was added to each well and mixed by shaking for 5 min. Optical density values at 490 nm were measured using a microplate reader.K. coccinea, as a Tujia ethnomedicine, have been used to the treat rheumatoid arthritis for a long time in China. The present study has reported that nine new triterpenoids (1–9), along with 22 known analogues (10–31), were isolated from the roots of K. coccinea. Heilaohuacids A and B (1 and 2) contain a 3,4-seco ring A and unprecedented migration of Me-18 from C-13 to C-17 or C-14 to C-18; their relative and absolute configurations were determined by 13C NMR calculations and ECD data analysis. To the best of our knowledge, this type of lanostane triterpenoid derivative was rarely reported so far, which enriched the structural types of lanostane triterpenoids in K. coccinea. Additionally, compounds 4, 17, 18, 29, and 31 showed good anti-RA and/or anti-inflammatory activities. These findings suggest that lanostane triterpenoids from K. coccinea might serve as therapeutic agents for RA treatment.The roots of


Aspergillus flavus strains, providing useful information for inter- and intraspecific analyses.Nuclear genome sequences incompletely characterize the genomic content and thus the genetic diversity of fungal species. Here, we present the complete mitochondrial genome sequences of nine  Aspergillus contains over 300 fungal species of varied industrial, agricultural, and medical relevance  sequences are available  was used as a reference for the seed database (-s) for each assembly.Genomic DNA was previously isolated and sequenced using an Illumina HiSeq 2500 paired-end 2 × 250-bp platform as described by Drott et al. . Paired- Archive , extract Archive . The rea Archive . Mitogen Archive , with SP Archive  as the iA. flavus strains against the corresponding circularized mitogenome, and SAMtools was used to identify variants. The read mapping was also visualized and the variants identified using the Integrative Genomics Viewer (IGV) v2.9.4 (A. flavus (GenBank accession number NC_026920.1), Aspergillus oryzae (NC_008282.1), A. oryzae 3.042 (NC_018100.1), Aspergillus parasiticus (NC_041445.1), and A. fumigatus (NC_017016.1) serving as the references. The gene names were manually revised, and genes with low coverage and sequence similarity (<50%) were discarded. The annotation was finalized following inspection using Geneious Prime v2021.1  v2.9.4 . The mit) v2.9.4 . GeSeq v) v2.9.4 , a rapidatus NC_0016.1 serAspergillus mitogenome sequences , MZ714576.1 (PA-C-1-1-1), MZ714577.1 (NC-E-3-2), MZ714578.1 (TX-A-2-1-1), MZ714579.1 (TX-A-13-1-1), MZ714580.1 (TX-A-20-1-1), MZ714581.1 (TX-A-1-1-1), MZ714582.1 (TX-B-1-1-1), and MZ714583.1 (TX-B-2-1-1). The SRA accession numbers for the whole-genome sequencing data used for mitogenome assembly are as follows: SRR12001149 (FL-B-1-1-1), SRR12001150 (PA-C-1-1-1), SRR12001141 (NC-E-3-2), SRR12001147 (TX-A-2-1-1), SRR12001145 (TX-A-13-1-1), SRR12001144 (TX-A-20-1-1), SRR12001148 (TX-A-1-1-1), SRR12001143 (TX-B-1-1-1), and SRR12001142 (TX-B-2-1-1).The


C-glucoside flavone that exhibits a wide range of pharmaceutical activities. However, the poor solubility of vitexin limits its applications. To resolve this limitation, two glycoside hydrolases (GHs) and four glycosyltransferases (GTs) were assayed for glycosylation activity toward vitexin. The results showed that BtGT_16345 from the Bacillus thuringiensis GA A07 strain possessed the highest glycosylation activity, catalyzing the conversion of vitexin into new compounds, vitexin-4′-O-β-glucoside (1) and vitexin-5-O-β-glucoside (2), which showed greater aqueous solubility than vitexin. To our knowledge, this is the first report of vitexin glycosylation. Based on the multiple bioactivities of vitexin, the two highly soluble vitexin derivatives might have high potential for pharmacological usage in the future.Vitexin is a  C-glucoside, C-glycosylated flavone and is, thus, largely resistant to molecular transformation. Vitexin is found in various plants, such as mung bean, chasteberry, buckwheat, hawthorn, passion flowers, pearl millet, and bamboo. These agricultural plants have also been used in traditional medicine for 3000 years − ion peak at m/z: 593.3 in the electrospray ionization mass (ESI-MS) spectrum, corresponding to the molecular formula C27H30O15 and the vitexin glucoside. Furthermore, the mass–mass analysis of the [M − H]− ion peak at m/z: 593.3 showed another [M − H]− ion peak at m/z: 431.3 corresponding to vitexin (molecular weight of 432 Da) (1) and (2) were vitexin glucosides, and NMR spectroscopic analysis further defined their structures.The chemical structures of compounds ( 432 Da) . The res1H- and 13C-NMR spectra, including distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra, were obtained, and the 1H- and 13C-NMR signal assignments were conducted accordingly. The 1H-NMR spectrum of compound (1) exhibited a flavonoid pattern with four aromatic signals: an AA′BB′ system at δ H 6.16  and δ H 8.14 , and two additional aromatic signals at δ H 6.29  and δ H 6.90 . The carbon atoms of the aforementioned protons were found to resonate at 128.7 ppm (C-2′/C-6′), 116.6 ppm (C-3′ /C-5′), 103.5 ppm (C-3), and 98.2 ppm (C-6), respectively. The 1H-NMR signals reflected the C-glucosyl (δ H: 3.24 (H-5′′), 3.27 (H-3′′), 3.41 (H-4′′), 3.56 (H-6′′a), 3.78 (H-6′′b), 3.81 (H-2′′), 4.69 (H-1′′)) and O-glucosyl (δ H: 3.19 (H-4′′′), 3.28 (H-2′′′), 3.31 (H-3′′′), 3.37 (H-5′′′), 3.48 (H-6′′′a), 3.69 (H-6′′′b), 5.03 (H-1′′′)) of compound (1).The 1H- and 13C-NMR sugar signals were assigned to C-glucosyl and O-glucosyl moieties in 1D- and 2D-NMR experiments. The glycosidic linkage of the C-glucosyl unit on the flavonoid A-ring was confirmed, using HMBC and NOESY correlations occurring between H1′′/C7, C8, C9, and the NOESY correlation between H1′′/H2′′. The grafting of the O-glucosyl unit onto the flavonoid C-ring was revealed by the presence of HMBC correlations between H1′′′/C4′ and the NOESY correlation between H1′′′/H3′ (5′). The doublet signal at δH H-1′′  and H-1′′′ 5.03  with the corresponding carbon atom at C-1′′ (73.4 ppm) and C-1′′′ (100.0 ppm) was assigned to the anomeric proton and indicated a C-β-configuration and an O-β-configuration, respectively. Hence, compound (1) was assigned as vitexin-4′-O-β-glucoside (1). The NMR spectroscopic data are shown in Finally, characteristic 1H- and 13C-NMR data, compound (2) was structurally similar to vitexin-4′-O-β-glucoside (1), described above. The aromatic signals at δ H 6.61  and δH 6.88  with their corresponding carbons resonating at 98.5 ppm (C-6) and 102.6 ppm (C-3) were identified. The signals were attributed to two glucose moieties at the C-glucosyl (δ H: 3.29 (H-5′′), 3.39 (H-3′′), 3.40 (H-4′′), 3.52 (H-6′′a), 3.72 (H-2′′), 3.78 (H-6′′b), 4.87 (H-1′′)) and O-glucosyl (δ H: 3.21 (H-4′′′), 3.31 (H-3′′′), 3.35 (H-2′′′), 3.42 (H-5′′′), 3.49 (H-6′′′a), 3.71 (H-6′′′b), 4.96 (H-1′′′)) of compound (2). The anomeric signal of the C-β-glucoside and O-β-glucoside moieties resonated as a doublet at δH 4.87  and δH 4.96  with the HSQC corresponding carbon atom at 73.3 ppm (C-1′′) and 101.2 ppm (C-1′′′), respectively. An ether linkage between the H-1′′ of glucose and the C-8 (4.87/107.2 ppm), H-1′′′, and C-5 (4.96/161.2 ppm) of compound (2) was proven, using the HMBC and NOESY (H-1′′′/H6) spectra. The structure of compound (2) was thus confirmed to be vitexin-5-O-β-glucoside (2). The NMR spectroscopic data are shown in Bacillus GTs.According to O-β-glucoside and vitexin-5-O-β-glucoside was higher than that of vitexin.The overall aim of this work is to improve the aqueous solubility of vitexin through glycosylation to enable its clinical application. Given that the solubility of flavonoids is affected by the pH of the solvent, the aqueous solubility of vitexin and its two glycosylated derivatives was determined at different pH . It was C-glycosidic flavonoids  are also more resistant than O-glycosidic flavonoids to acidic and enzymatic hydrolysis and are thus more stable during oral absorption -, 431.3, 311.3; 1H-NMR : δ H 3.19 , 3.24 , 3.27 , 3.28 , 3.31 , 3.37 , 3.41 , 3.48 , 3.56 , 3.69 , 3.78 , 3.81 , 4.69 , 5.03 , 6.16 , 6.29 , 6.90 , 8.14 ; 13C-NMR : δ C 60.5 (C-6′′′), 61.2 (C-6′′),69.5 (C-4′′′), 70.4 (C-4′′), 70.9 (C-2′′), 73.2 (C-2′′′), 73.4 (C-1′′), 76.5 (C-3′′′), 77.1 (C-5′′′), 78.6 (C-3′′), 81,9 (C-5′′), 98.2 (C-6), 100.0 (C-1′′′), 103.5 (C-3), 104.1 (C-10), 104.7 (C-8), 116.6 , 124.5 (C-1′), 128.7 , 156.0 (C-9), 160.3 (C-4′), 160.4 (C-5), 162.7 (C-7), 163.3 (C-2), 182.2 (C-4).Vitexin-5-O-β-glucoside (2): yellow powder; ESI/MS m/z: 593.3 [M − H]-, 431.3, 311.1; 1H-NMR : δ H 3.21 , 3.29 , 3.31 , 3.35 , 3.39 , 3.40 , 3.42 , 3.49 , 3.52 , 3.71 , 3.72 , 3.78 , 4.87 , 4.96 , 6.61 , 6.88 , 6.89 , 8.07 ; 13C-NMR : δ C 60.6 (C-6′′′), 61.3 (C-6′′), 69.4 (C-4′′′), 70.6 (C-4′′), 71.6 (C-2′′), 73.3 (C-1′′), 73.4 (C-2′′′), 75.8 (C-3′′′), 77.1 (C-5′′′), 78.5 (C-3′′), 81,9 (C-5′′), 98.5 (C-6), 101.2 (C-1′′′), 102.6 (C-3), 105.7 (C-10), 107.2 (C-8), 115.9 , 121.2 (C-1′), 129.2 , 154.9 (C-9), 160.8 (C-7), 161.2 (C-5), 161.6 (C-4′), 164.5 (C-2), 182.3 (C-4).2O, PB at pH 6, or pH 8, for 1 h at 25 °C. The mixture was centrifuged at 10,000× g for 30 min at 25 °C. The supernatant was filtered through a 0.2 µm nylon membrane, mixed with an equal volume of methanol, and analyzed by HPLC. The concentration of the tested compounds was determined on the basis of their peak areas, using calibration curves prepared by HPLC analyses of authentic samples.The aqueous solubility of vitexin and its glucoside derivatives was examined as follows. Each compound was vortexed in double-deionized HO-β-glucoside (1) and vitexin-5-O-β-glucoside (2). These new compounds were found to have higher aqueous solubility than vitexin. Based on the multiple bioactivities of vitexin, the two new vitexin derivatives have high potential for pharmacological usage in the future.To our knowledge, this is the first report of vitexin glycosylation and the two produced vitexin glucosides: vitexin-4′-


Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A–C (1–3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey’s method.Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and  The identification of valuable agents is currently more urgent than ever. The rising drug resistance among disease-causing pathogens is only one reason to drive natural product research forward . Agents Bacillus brevis + determined by high-resolution electrospray ionization mass spectrometry (HRESIMS) , eight methine protons , four methylenes  and seven methyl groups . Analysis of HSQC and HMBC spectra (no 13C spectra were acquired) additionally revealed ten quaternary carbons (seven carbonyls and three double bond carbons). The presence of several NH groups and carbonyls and the typical proton/carbon chemical shifts of amino acid α-CH groups  suggest a peptide-like structure consisting of at least three natural amino acids, proline, alanine and isoleucine (H 6.17 and methyl group at δH 1.70 in COSY spectrum and their HMBC correlations to the quaternary carbons C-10 (δC 130.7) and C-13 (δC 163.5) indicate an α,β-unsaturated amino acid, called dehydrobutyrine (Dhb), of which two additional units were recognized in the NMR spectra. Dehydrobutyrine can be present in the Z or E configuration; however, it is often found as the Z isomer. ROESY correlations indicate the expected Z-configuration for all three dhbs  could be recognized by its characteristic chemical shifts . Careful interpretation of the HMBC correlations led to the peptide sequence Lac-allo-Ile-Dhb-Dhb-Ala-Dhb-Pro-OH (Scabimycin A (HRESIMS) . The mololeucine . The croree dhbs . Furtherree dhbs  lead to b-Pro-OH .allo-isoleucine, alanine and proline were found to have a L-configuration  was isolated as a pale-yellow solid with UV absorbance at 220 nm and a molecular ion peak with m/z 536.3069 [M + H]+ determined by HRESIMS  to C-2 (δC 170.8) and the N-HMBC correlation of H-1 to 2-NH (δN 125.0). It was found that the number and type of amino acids in scabimycin B were slightly different from those found in the amino acid sequence of scabimycin A  was isolated as a yellow, oily compound with a molecular ion peak at m/z 423.2227 [M + H]+ determined by HRESIMS . However, none of the scabimycins inhibited the protease activity.Streptomyces acidiscabies Lu19992 attracted our attention due to extensive production of three putatively unknown metabolites. These compounds were isolated and characterized via NMR spectroscopy resulting in three novel short linear peptides that harbor the rather unusual amino acid dhb. This amino acid arises from dehydration of threonine, which occurs either through action of a dehydratase enzyme or via the elimination of phosphorylated residues  and coupled to a PDA detector operating at 200–600 nm. The coupled LTQ Orbitrap mass spectrometer  was operated at m/z 200–2000. Data were analyzed with Thermo Xcalibur Version 3.0.63 software. Dereplication was performed by comparing the exact masses with the Dictionary of Natural Products database version 10.0 .The S. acidiscabies strain was precultivated in a 100 mL flask filled with 10 mL of TSB (tryptic soy broth 30 g/L) at 28 °C and 180 rpm on a rotary shaker for 24 h. For the main culture, 50 mL of DNPM medium  21 g/L, pH 6.8) was inoculated in a 500 mL flask with 1 mL of preculture and cultivated for 5 days at 28 °C and 180 rpm on a rotary shaker. After cultivation, the biomass and culture liquid were extracted separately with a mixture of acetone/methanol (1:1) and butanol, respectively. The solvent was evaporated until the residue was dry, and then the residue was dissolved in 300 µL MeOH. High-resolution LC-MS data were collected on a Dionex Ultimate 3000 UHPLC system  with stationary phase Acquity UPLC BEH C18, 100 mm × 2.1 mm, 1.7 µm column  using a linear gradient from 5 % [B] (acetonitrile + 0.1 % formic acid) against [A] (ddHS. acidiscabies was cultivated in 10 L of DNPM (100 × 500 mL flasks with 100 mL medium) as described above. After cultivation, the culture liquid was separated from the biomass and extracted twice with butanol. The solvent was removed under reduced pressure, and the residue was dissolved in methanol and centrifuged. The pellet was discarded, and the supernatant was dried, resulting in 16 g of raw material. The crude extract was dissolved in 20 mL methanol and fractionated by flash chromatography  using a Chromabond® Flash RS 330 C18 ec 360 g column . Two runs (10 mL of extract for each) were performed using Milli-Q® (MQ)  -H2O [A] and methanol [B] as eluents. A linear gradient from 30–80% [B] over 5 column volumes (CV) was applied. Fractions were tested on LC-MS, a Dionex Ultimate 3000 UPLC system using Acquity BEH C18, 50 × 2.1 mm, 1.7 µm dp column  and mobile phase: ddH2O + 0.1% formic acid [A]/acetonitrile + 0.1% formic acid [B], 5–95% [B] over 9 min, at flow rate 0.6 mL/min, coupled to amaZON SL speed mass spectrometer  with ESI source and mass range m/z 200–2000. Fractions containing the targeted compounds were pooled and further purified with size exclusion chromatography using Sephadex® LH 20  as the stationary phase  and methanol as the eluent. Fractions were collected every 15 min with a speed of 1–2 drops per second. Every third fraction was analyzed by LC-MS . The fractions containing scabimycins were further purified by semipreparative high-performance liquid chromatography (HPLC) using the following equipment: Agilent 1100 and 1260 Series HPLC  equipped with a Nucleodur C18 HTEC column  and a DAD detector operating at 200–600 nm. A linear gradient used solvent [A] MQ-H2O + 0.1% formic acid against solvent [B] acetonitrile + 0.1 % formic acid starting from 35% [B] and increasing to 45% [B] over 20 min with a flow rate of 4.5 mL/min at 45 °C.For isolation and purification of the targeted compounds, d6) at 298 K on a Bruker Avance III 700 or 500 MHz spectrometer, both equipped with a 5 mm TXI cryoprobe. The NMR shifts were relative to the position of the residual solvent signal of DMSO-d6 at δ 2.50 1H, or to the solvent itself at δ 39.5 (DMSO-d6) for 13C measurements. NMR data were analysed using Topspin, version 3.5 pl7  and Spectrus Processor 2018.2.3 .NMR spectra were acquired in deuterated dimethyl sulfoxide (DMSO-1). Lac-l-allo-Ile-Dhb-Dhb-l-Ala-Dhb-l-Pro-OH. Yellow oil; 5.0 mg; max 219 nm; δH : 9.75 , 9.20 , 9.06 , 7.66 , 7.63 , 6.47 , 6.17 , 5.52 , 4.36 , 4.33 , 4.18 , 3.98 , 3.60–3.40 , 2.14–1.77 , 1.84–1.72 , 1.83 , 1.70 , 2 × 1.64 , 1.46–1.06 , 1.30 , 0.89 , 0.84 ; δC : 174.5 (C-3), 173.2 (C-29), 2 × 170.9 , 165.3 (C-24), 163.6 (C-17), 163.5 (C-13), 131.4 (C-21), 130.7 (C-10), 129.9 (C-14), 129.1 (C-15), 124.8 (C-11), 120.2 (C-22), 66.9 (C-2), 58.5 (C-28), 55.9 (C-4), 48.5 (C-25), 48.1 (C-18), 36.8 (C-5), 28.8 (C-27), 24.8 (C-26), 23.9 (C-7), 20.6 (C-1), 17.0 (C-19), 15.2 (C-6), 2 × 12.4 , 11.8 (C-23), 10.9 (C-8); HRESIMS m/z 621.3229 [M + H]+  .Scabimycin A (2). Ac-l-allo-Ile-Dhb-Dhb-Dhb-l-Leu-OH. Yellow oil; 1.8 mg; max 220 nm; δH : 9.63 , 9.00 , 8.67 , 8.42 , 7.42 , 6.49 , 6.43 , 6.32 , 4.18 , 4.06 , 1.88 , 1.77 , 1.69 , 1.68 , 1.62 , 1.61 , 1.60–1.48 ; 1.55–1.19 , 0.92 , 0.86 , 0.84 , 0.81 ; δC : 173.6 (C-26), 171.3 (C-8), 170.8 (C-2), 163.7 (C-12), 163.0 (C-20), 162.6 (C-16), 130.3 (C-13), 2 × 129.8 (C-17/C-9), 129.4 (C-18), 128.3 (C-10), 127.5 (C-14), 58.2 (C-3), 50.5 (C-21), 39.8 (C-22); 34.8 (C-4), 24.5 (C-6), 23.7 (C-23), 22.6 (C-24), 22.0 (C-1), 20.9 (C-25), 15.1 (C-5), 3 × 12.6 (C-11/C-15/C-19), 10.7 (C-7); HRESIMS m/z 536.3069 [M + H]+  .Scabimycin B (3). Ac-l-allo-Ile-Dhb-Dhb-Dhb-OH. Yellow oil; 2.0 mg; H : 9.61 , 8.78 , 8.44 , 8.23 , 6.49 , 6.48 , 6.36 , 4.06 , 1.86 , 1.73 , 1.69 , 2 × 1.63 , 1.52–1.18 , 0.91 , 0.85 ; δC : 171.7 (C-8), 170.4 (C-2), 163.2 (C-12), 2 × 162.4 (C-20/C-16), 130.7 (C-9), 2 × 130.1 (C-18/C-14), 2 × 129.2 (C-13/C-17), 127.6 (C-10), 58.4 (C-3), 35.5 (C-4), 25.1 (C-6), 22.0 (C-1), 15.4 (C-5), 2 × 13.8 (C-15/C-19), 13.1 (C-11), 11.1 (C-7); HRESIMS m/z 423.2227 [M + H]+  .Scabimycin C (1 (200 µg) and 2 (250 µg) were treated with 100 µL of a 6 M HCl solution and heated at 110 °C for 45 min. Subsequently, the solvent was removed completely under a nitrogen stream at the same temperature within 20 min. The residues were dissolved in 110 µL H2O, and 50 µL was transferred into two new tubes for each sample. To each tube, 20 µL of 1 M NaHCO3 was added to reach pH 9. For derivatization, the samples were treated with 20 µL of 1% Marfey’s reagent  in acetone. Similarly, 20 µL of 1 M NaHCO3 and 20 µL of a 1% solution of L-FDLA were added to 50 µL of an aqueous solution (50 mmol) of each present amino acid . All samples were then incubated for 1.5 h at 40 °C and 700 rpm. To stop the reaction, 10 µL of 2 M HCl solution was added. Afterwards, 300 µL acetonitrile was added, and the samples were subjected to LC-HRMS measurements. Samples were run on an Acquity BEH C18, 100 × 2.1 mm, 1.7 µm dp column  using eluent [A] ddH2O + 0.1% formic acid and [B] acetonitrile + 0.1% formic acid. The following gradient was applied: 5–10% [B] (0–2 min), 10–25% [B] (2–15 min), 25–50% [B] (15–22 min), 50–95% [B] (22–24 min), 95 -95% [B] (24–26 min), and 95–5% [B] (26–27 min). The column oven temperature was set to 45 °C, and a mass range from m/z 100–1000 was acquired.For determination of absolute configuration using advanced Marfey’s method, samples


Withania somnifera and a number of Physalis species grown under environmentally controlled aeroponic conditions suggested that this technique is a convenient, reproducible, and superior method for their production and structural diversification. Investigation of aeroponically grown Physalis coztomatl afforded 29 withanolides compared to a total of 13 obtained previously from the wild-crafted plant and included 12 new withanolides, physacoztolides I−M (9–13), 15α-acetoxy-28-hydroxyphysachenolide C (14), 28-oxophysachenolide C (15), and 28-hydroxyphysachenolide C (16), 5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (17), 15α-acetoxy-5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (18), 28-hydroxy-5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (19), physachenolide A-5-methyl ether (20), and 17 known withanolides 3–5, 8, and 21–33. The structures of 9–20 were elucidated by the analysis of their spectroscopic data and the known withanolides 3–5, 8, and 21–33 were identified by comparison of their spectroscopic data with those reported. Evaluation against a panel of prostate cancer  and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells revealed that 8, 13, 15, and 17–19 had potent and selective activity for prostate cancer cell lines. Facile conversion of the 5,6-chlorohydrin 17 to its 5,6-epoxide 8 in cell culture medium used for the bioassay suggested that the cytotoxic activities observed for 17–19 may be due to in situ formation of their corresponding 5β,6β-epoxides, 8, 27, and 28.Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. Our recent studies on withanolides obtained from plants of Solanaceae including  The 13C NMR spectrum of 9 , three methyls , eight olefinic carbons , two ester carbonyls (δC 165.9 and 171.4), and a conjugated ketone carbonyl (δC 204.4). The absence of a signal due to an oxygenatied carbon around 87 ppm in the 13C NMR spectrum indicated that C-17 is non-oxygenated /C-17(δC 151.1), H3-21/C-20 (δC 74.4), and H3-21/C-22 (δC 80.8). The NMR chemical shifts  of one of the methyl group suggested that it was attached to an olefinic carbon. This was confirmed to be C-27 methyl group by the HMBC correlations of H3-27 [δH 1.85 (s)]/C-26 (δC 165.9), H3-27/C-25 (δC 123.4), and H3-27/C-24 (δC 148.2). The absence of a signal due to C-28 methyl group and the presence of an oxygenated CH2 group suggested the possible attachment of an O-glucosyl moiety to C-28 [3-19 [δH 1.22 (s)]/C-1 (δC 204.4), H3-27 [δH 1.85 (s)]/C-26 (δC 165.9), H3-27/C-25 (δC 123.4), and H3-27/C-24 (δC 148.2) -18-acetoxy-28β-d-O-glucopyranosyl-14α,20β-dihydroxy-1-oxo-witha-2,5,16,24-tetraenolide (9).Compounds rum of 9  showed trum of 9  displayeions see  of H3-218.2) see  preclude6 nm see , suggest10) was determined to be C36H48O12 from its HRESIMS and NMR data. The 1H NMR data of 10  and 9 (C36H48O13) indicated that 10 may be a deoxygenated analogue of 9. The assignment of the 13C NMR spectrum . This was confirmed by the up-field chemical shifts  of carbons located β to C-14 in 10 when compared with those of 9 -18-acetoxy-28β-d-O-glucopyranosyl-20β-hydroxy-1-oxo-witha-2,5,16,24-tetraenolide (10).The molecular formula of physacostolide J  were consistent with the molecular formula, C30H40O9. The 1H NMR data of 11 , 5.84 , and 6.78 ], suggesting the presence of 2,3-en-1-one and 5,6-double bond moieties similar to physachenolide D (3)  suggested that two of the methyl groups of the withanolide skeleton are substituted. This was confirmed by the presence of two oxygenated methylene signals . The two low-field oxygenated methines [δH 4.23  and 4.35 ] in the 1H NMR spectrum of 11 which coupled with each other suggested that C-23 is oxygenated. The 13C NMR spectrum of 11 (11 are not oxygenated. The absence of any oxygen-bearing substituents at C-17 was confirmed by the HMBC correlation of H3-21 [δH 1.43 (s)]/C-17 (δC 49.3) ] established the configuration of the side chain at C-17 to be β [3-21/H-17α and H3-21/H-12β (see 11 was apparent from the HMBC correlations of H3-28 [δH 2.06 (s)]/C-23 (δC 66.9) and H2-27 [δH 4.34 (s)]/C-26 (δC 164.6) (see J = 7.8 Hz) was consistent with 23β-hydroxyphysacoztolide E-type sub-structure , two oxygenated methylenes [δH 4.89 /3.70  and 3.83 /3.64 ], three olefinic protons , and two oxygenated methines . The coupling between the two oxygenated methines suggested possible hydroxylation at C-23  of the δ-lactone further confirmed that ring E is saturated. The presence of HMBC correlation of H3-27 [δH 1.28 ]/C-26 (δC 177.6)  between H-23 and H-24 was identical to that of 24,25-dihydro-23β,28-dihydroxywithanolide G  in its 1H NMR spectrum established the configuration of the side chain at C-17 as β  and from their 13C NMR signals for C-5 and C-6 . Their ECD spectra , the major withanolide of this plant. These NMR data, together with its molecular formula, indicated that 13 contained one oxygen atom more than that of physachenolide C (8). Comparison of the 1H and 13C NMR data  and 4.24 ; δC 61.9] in 13. The presence of CH2OH-18 in 13 was confirmed by the HMBC correlations of H3-27 [δH 1.88 (s)]/C-26 (δC 169.1) and H2-28/C-23 (δC 30.3) -18-acetoxy-5β,6β-epoxy-14α,17β,20β,28-tetrahydroxy-1-oxo-witha-2,24-dienolide].Based on its HRESIMS and NMR data, withanolide NMR data  suggesteNMR data  of 13 wiose of 8  revealed0.3) see . The ECD6 nm see , suggest of C-22 ,36. Thus14 was established as C32H42O12 by its HRESIMS and NMR data. Careful analysis of 1H NMR and 13C NMR spectra of 14 (13) or an oxygenated analogue of 15α-acetoxyphysachenolide C (27)  (14).The molecular formula of ra of 14  suggestee C (27) . Comparise of 27 , and the9.0) see . The pos9.0) see  in its E22 of 14 ,36. Thus15 was determined to be C30H38O10 based on its HRESIMS and NMR data, suggesting twelve degrees of unsaturation. The analysis of the 1H NMR and 13C NMR spectra (15 were similar to those of 28-hydroxyphysachenolide C (13), except for those of ring E. Comparison of the molecular formula of 15 (C30H38O10) with that of 13 (C30H40O10) indicated that 15 contains two protons fewer than 13. The presence of a low-field proton signal at 10.30 ppm in the 1H NMR spectrum and a signal at 190.2 ppm in the 13C NMR spectrum of 15 suggested that the CH2OH group attached to the ring E of 13 has undergone oxidation to a CHO group in 15. The HMBC correlations of H3-27 [δH 2.35 (s)]/C-26 (δC 165.6) and H-28 [δH 10.30 (s)]/C-23 (δC 24.5) -18-acetoxy-5β,6β-epoxy-14α,17β,20β-trihydroxy-1,28-dioxo-witha-2,24-dienolide].The molecular formula of withanolide  spectra  with the4.5) see  located 4.5) see  in its E of C-22 . The str16 was determined to have the molecular formula C30H40O10. It was suspected to be a glucoside from its molecular formula, C34H46O11, and the presence of a signal due to an anomeric proton at δH 4.23  and the typical 13C NMR signals  of the glucose moiety and was named physacoztolide M. The 1H NMR spectrum of 16 , four olefinic protons , an oxygenated methine proton [δH 4.37 ], and protons on an oxygenated methylene group [δH 4.42 (br s)]. The olefinic region of the 1H NMR spectrum of 16 was found to similar to that of physacoztolide I (9) (see above). The absence of a singlet methyl signal around 2.0 ppm in 16 suggested that it lacked the acetyl group present in 9. These data suggested 16 has a similar skeleton as that of 9 and contained three double bonds at 2(3), 5(6), and 16(17) positions, and the AcOCH2 at C-13 in 9 was replaced by a CH3 group in 16. The assignment of the 13C NMR spectrum of 16 (13C NMR data with those of 9 further confirmed that the AcO group at C-18 of 9 is replaced with a proton in 16. The presence of the double bonds at 2(3) and 5(6), and 16(17) in 16 was further confirmed by the HMBC correlations of H3-19/C-5, H3-19/C-1, H-4/C-2, H-4/C-6, and H3-18/C-17, H3-21/C-17, H-16/C-20, respectively (see δC 67.7) located the O-glycosyl moiety at C-28. The presence of the d-glucose moiety in 16 was further confirmed by the acid hydrolysis and the positive [α]D obtained for the resulting sugar. The ECD spectrum of 16 showed a positive Cotton effect at 256 nm -28β-d-O-glucopyranosy-14α,20β-dihydroxy-1-oxo-witha-2,5,16,24-tetraenolide (16).Based on its HRESIMS and NMR data, withanolide um of 16  displayeum of 16  with theum of 16  and HMBCum of 16  and compvely see . The lon6 nm see  establis of C-22 ,36. Ther1H and 13C NMR spectra  and 75.0 (C-6) for 17, 79.0 (C-5) and 74.6 (C-6) for 18, and 82.3 (C-5), and 75.6 (C-6) for 19] suggested that these are chlorohydrins containing 5-chloro-6-hydroxy substituents similar to withanolide C , protons of two oxygenated methines , and an oxygenated methylene [δH 4.43 ]. The 13C NMR spectrum of 17 (21)  are consistent with those of withanolide C [δC 80.4 (C-5) and 74.7 (C-6)] [δC 80.9 (C-5) and 74.5 (C-6)] [17. The NOESY correlations of H-7β [δH 2.54 (m)]/H-6 [δH 4.06 (br s)] and H-7α [δH 1.52 (m)]/H-6 in 17  as in physachenolide A (21) .The HRESIMS data together with their  spectra  indicatenolide C . The 13Ce A (21)  suggestium of 17  displayeum of 17  assignedum of 17  and HMBCum of 17  data ind 17 (21) , but wit7 (C-6)]  and phys5 (C-6)] , suggestn 17 see  togethere A (21) . The ECD6 nm see  establis of C-22 ,36, and of Cl-5) , respect18 was determined to be C32H43ClO11 by the analysis of its HRESIMS and NMR data. The 1H NMR spectrum of 18 , but exhibited an additional oxygenated methine signal [δH 5.20 ], two acetyl methyl signals [δH 2.08 (s) and 2.09 (s)] besides the typical signals for H-2, H-3, H-22, H2-18, and the methyl groups. These data together with the difference in molecular formulae between 18 (C32H43ClO11) and 17 (C30H41ClO9) suggested that 18 may be an acetoxylated analogue of 17. The 13C NMR spectrum of 18  − δ(9) = −3.5 ppm, γ-effect] and down-field shift of C-16  placed the acetoxy substituent at C-15. The orientation of this OAc group was determined to be α by the large coupling constant for H-15 [δH 5.20 ] in its 1H NMR spectrum, which is identical with that of 15α-acetoxyphysachenolide D (4) [β [δH 2.61 (m)]/H-6 [δH 3.97 (br s)] and H-7α [δH 1.54 (m)]/H-6 -15α,18-diacetoxy-5α-chloro-6β,14α,17β,20β-tetrahydroxy-1-oxo-witha-2,24-dienolide].The molecular formula of compound um of 18  resembleum of 18 , assigneum of 18  and HMBCum of 18 , also sude D (4) . The NOE/H-6 see , togethe 335 see  establisings A/B . On the 19 was deduced to be C30H41ClO10 from its HRESIMS and NMR data. The 1H NMR spectrum of 19 , 6.75 ], two oxygenated methylenes , two oxygenated methines , an acetate [2.12 (s)], and three singlet methyls . The 13C NMR spectrum of 19 , except for the signal assigned to C-28 which appeared at 62.0 ppm compared to that of 17 at 20.7 ppm. The above NMR data together with the difference in molecular formulae of 19 (C30H41ClO10) and 17 (C30H41ClO9) suggested that 19 could be a C-28 hydroxylated analogue of 17. The presence of OH-28 was confirmed by the HMBC correlations of H3-27 [δH 1.89 (s)]/C-26 [δC 169.2], H2-28/C-25 [δC 122.5], and H2-28/C-23 [δC 30.3]  indicated the orientation of OH-6 to be β similar to those of 17 and 18. The ECD spectrum of 19 exhibited a positive Cotton effect at 255 nm and a negative Cotton effect at 336 nm -18-acetoxy-5α-chloro-6β,14α,17β,20β,28-pentahydroxy-1-oxo-witha-2,24-dienolide] (19).The molecular formula of compound um of 19  displayeum of 19  assignedum of 19  and HMBCum of 19  closely 0.3] see . The sma6 nm see  establisings A/B ,36. Base20 was determined to have the molecular formula, C31H44O10, based on its HRESIMS and NMR data. Its 1H NMR spectrum , 6.48 ], two oxygenated methines , an oxygenated methylene , an acetyl [δH 2.03 (s)], and four singlet methyls . A signal due to an OCH3 group [δH 2.93 (s)] rare in withanolides was also encountered, indicating that 20 is a methoxylated withanolide. Comparison of the 13C NMR data of 20 (21) .Withanolide spectrum  displayeta of 20  with tho 20 (21)  revealed/C-5 see . The ori5 nm see  establis CH3O-5) . The str21) , 5α-methoxy-6β-hydroxy analogue [physachenolide A-5-methyl ether (19)] and 5α,6β-dihydroxy analogue [physachenolide A (21)] ] see , all of β-hydroxywithanolides, including physachenolide C (8), were capable of selectively inhibiting the proliferation of prostate cancer cells at nanoMolar concentrations without affecting many other cancer cells and normal human fibroblast cells c 0.34, MeOH); UV (MeOH) λmax (log ε) 224 (4.09) nm; ECD (MeOH) λmax (Δε) 338 (−2.36), 256 (4.25); 1H and 13C NMR data, see m/z 711.2986 [M + Na]+ .Physacoztolide I (10): amorphous, colorless solid; [α]c 0.25, MeOH); UV (MeOH) λmax (log ε) 224 (4.21) nm; ECD (MeOH) λmax (Δε) 338 (−2.92), 257 (4.74); 1H and 13C NMR data, see m/z 695.3037 [M + Na]+ .Physacoztolide J (11): amorphous, colorless solid; [α]c 0.25, MeOH); UV (MeOH) λmax (log ε) 224 (4.21) nm; ECD (MeOH) λmax (Δε) 338 (−2.92), 256 (2.38); 1H and 13C NMR data, see m/z 567.2539 [M + Na]+ .Physacoztolide K (12): amorphous, colorless solid; [α]c 0.10, MeOH); UV (MeOH) λmax (log ε) 223 (3.77) nm; ECD (MeOH) λmax (Δε) 338 (−2.37), 256 (1.37); 1H and 13C NMR data, see m/z 569.2727 [M + Na]+ .Physacoztolide L (13): amorphous, colorless solid; [α]c 0.30, MeOH); UV (MeOH) λmax (log ε) 224 (4.09) nm; ECD (MeOH) λmax (Δε) 342 (1.29), 258 (2.59); 1H and 13C NMR data, see m/z 583.2520 [M + Na]+ .28-Hydroxyphysachenolide C (14): amorphous, colorless solid; [α]c 0.27, MeOH); UV (MeOH) λmax (log ε) 224 (4.16) nm; ECD (MeOH) λmax (Δε) 341 (1.25), 258 (2.86); 1H and 13C NMR data, see m/z 641.2579 [M + Na]+ .15α-Acetoxy-28-hydroxyphysachenolide C (15): amorphous, colorless solid; [α]c 0.34, MeOH); UV (MeOH) λmax (log ε) 225 (4.13) nm; ECD (MeOH) λmax (Δε) 341 (1.37), 258 (3.06); 1H and 13C NMR data, see m/z 581.2355 [M + Na]+ .28-Oxophysachenolide C (16): amorphous, colorless solid; [α]c 0.25, MeOH); UV (MeOH) λmax (log ε) 225 (4.11) nm; ECD (MeOH) λmax (Δε) 340 (−2.71), 256 (5.01); 1H and 13C NMR data, see m/z 653.2937 [M + Na]+ .Physacoztolide M : amorphous, colorless solid; [α]c 0.34, MeOH); UV (MeOH) λmax (log ε) 227 (4.10) nm; ECD (MeOH) λmax (Δε) 336 (−1.24), 252 (3.82); 1H and 13C NMR data, see m/z 603.2349 [M + Na]+ .5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (18): amorphous, colorless solid; [α]c 0.17, MeOH); UV (MeOH) λmax (log ε) 227 (4.05) nm; ECD (MeOH) λmax (Δε) 335 (−1.18), 252 (3.41); 1H and 13C NMR data, see m/z 661.2437 [M + Na]+ .15α-Acetoxy-5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (19): amorphous, colorless solid; [α]c 0.10, MeOH); UV (MeOH) λmax (log ε) 227 (4.05) nm; ECD (MeOH) λmax (Δε) 335 (−1.33), 255 (2.82); 1H and 13C NMR data, see m/z 619.2292 [M +Na]+ .28-Hydroxy-520): amorphous, colorless solid; [α]c 0.45, MeOH); UV (MeOH) λmax (log ε) 225 (4.00) nm; ECD (MeOH) λmax (Δε) 335 (−0.60), 257 (1.70); 1H and 13C NMR data, see m/z 599.2836 [M + Na]+ .Physachenolide A-5-methyl ether  in MeOH (0.5 mL) was added 2N HCl solution (0.5 mL). The mixture was heated at 100 °C. After 1 h (TLC control), the reaction mixtures were concentrated and the residues thus obtained were chromatographed over a column of silica gel (0.5 g) using CHCl3/MeOH (8:2) as the eluent. Fractions containing the sugar were collected based on their TLC profiles, concentrated, dissolved in water for qualitative measurement of [α]D.To a solution of each glycoside  was added to each well, and continued to incubate for 3–4 h at 37 °C. The media were removed and 100 μL/well of DMSO was added before data acquisition using a microplate reader at 570 nm.A tetrazolium dye-based colorometric (MTT) assay was used for evaluating cytotoxicity of the compounds against cancer cell lines, LNCaP (androgen-sensitive prostate adenocarcinoma), PC-3 (androgen-insensitive prostate adenocarcinoma), DU-145 (androgen-insensitive prostate adenocarcinoma), VCaP (androgen-sensitive metastatic prostate cancer), and ACHN , and normal human lung fibroblast cells, WI-38. The cells were plated at 1000–4000 cells/well (depending on the cell growth rate) in 96-well flat-bottomed microplates. After incubation at 37 °C for 24 h in an atmosphere of 5% COα-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (17) (0.2 mg) in DMSO (1.0 μL) was added to the RPMI medium (1.0 mL) used for the cytotoxicity assays with LNCaP and ACHN cells . The solution was kept at 37 °C in a 5% CO2 incubator, and 100.0 μL samples were withdrawn for HPLC analysis at 0 min, 5 min, 2 h, 8 h, and 24 h. The HPLC analysis was carried out on an Agilent HP 1100 HPLC system with a Phenomenex Spherisorb 5 μ ODS (2) 80A, 250 mm × 4.6 mm HPLC column . The product formed was identified as physachenolide C (8) by its retention time and the peak enhancement method.A solution 5Physalis coztomatl afforded 29 withanolides including 12 new withanolides (9–20), and 17 known withanolides . Evaluation of these withanolides against a panel of prostate cancer  and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells suggested that 8, 13, 15, and 17–19 had potent and selective activity for prostate cancer cell lines. This work also resulted in the discovery that the potent cytotoxic activity of withanolide 5,6-chlorohydrins may be due to their facile conversion into the corresponding 5β,6β-epoxides in the cell culture medium used for the bioassay.Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. The work reported here further supports our previous findings that the application of the aeroponic technique for cultivation of plants of Solanaceae is a convenient, reproducible, and superior method for production and structural diversification of withanolides. Investigation of aeroponically grown


Diaporthe foeniculina SCBG-15, led to the isolation of eight new cyclohexanone derivatives, foeniculins A–H (1–8) and three new phenolic acid derivatives, foeniculins I–K (9–11). Their structures were extensively established on the basis of 1H and 13C NMR spectra together with COSY, HSQC, HMBC, and NOESY experiments. The absolute configurations were confirmed by quantum chemical ECD calculations and single-crystal X-ray diffractions. Moreover, the in vitro cytotoxic and antibacterial activities of isolated compounds 1–11 were also evaluated.Chemical investigation of an endophytic fungus  Leptospermum brachyandrum belongs to the genus Leptospermum, it is an important member in the plant family Myrtaceae , and three phenolic acid derivatives, foeniculins I–K (9–11). All of the novel compounds 1–11 possessed polymethylated skeleton . HRESIMS spectra were acquired with a Thermo MAT95XP high resolution mass spectrometer . Silica gel  was used for column chromatography. TLC analysis was carried out on silica gel plate . A Hitachi Primaide  equipped with a diode array detector (DAD) using a semi-preparative YMC ODS C18 column  was used for semi-preparative HPLC separation. All solvents were analytical grade .Optical rotations were recorded using an Anton Paar MCP-500 spectropolarimeter . UV spectra were obtained by a Shimadzu UV-2600 spectrophotometer . ECD spectra were measured with an Applied Photophysis Chirascan. IR data were measured on a Shimadzu IR Affinity-1 spectrometer . 1D and D. foeniculina SCBG-15 was isolated from the plant of L. brachyandrum, which was collected at South China Botanical Garden (SCBG), Chinese Academy of Sciences, China, in September 2016. The strain was identified by sequence analysis of rDNA ITS  region. The sequence of the ITS region of the D. foeniculina has been submitted to GenBank (Accession No. MN788609). The strain is preserved at the Laboratory of Natural Product Medicinal Chemistry, SCBG.The endophytic fungal strain D. foeniculina was fermented on an autoclaved rice solid medium  for 30 days at 28°C. After cultivation, the mycelia and rice solid medium were extracted with EtOAc for three times, and the crude extract (50 g) was obtained. The crude extract was subjected to silica gel using gradient elution with petroleum ether-EtOAc as eluent  and CH2Cl2-MeOH . Then, they were combined by TLC analysis to afford six main fractions (Fr.1-Fr.6).The fungus 18 silica gel, eluting with MeOH-H2O  to give six subfractions (Fr.5-1 to Fr.5-6). Fr.5-2 (1.94 g) was separated by Sephadex LH-20 column chromatography and eluted with CHCl3-MeOH  to afford six subfractions (Fr.5-2-1 to Fr.5-2-6). Fr.5-2-2 (1.23 g) was isolated by column chromatography on silica gel and eluted with n-hexane-EtOAc gradient  to obtain six subfractions (Fr.5-2-2-1 to Fr.5-2-2-6). Fr.5-2-2-5 (127.4 mg) was further purified by the semi-preparative HPLC system with CH3CN-H2O (10:90) as eluent to afford compounds 1 , 2 , and 3 . Fr.5-2-1 (311.4 mg) was isolated by column chromatography on silica gel and eluted with n-hexane-EtOAc gradient  to get three subfractions (Fr.5-2-1-1 to Fr.5-2-1-3). Fr.5-2-1-1 (208.8 mg) was subjected to semi-preparative HPLC with CH3CN-H2O  to give seven subfractions (Fr.5-2-1-1-1 to Fr.5-2-1-1-7). Fr.5-2-1-1-6 (28.3 mg) was purified by semi-preparative HPLC and washed with CH3CN-H2O  to afford compound 4 . Fr.5-2-1-1-1 (23.1 mg) was purified by semi-preparative HPLC equipped with a chiral column and washed with isopropanol-hexane (30:70) to afford compounds 5 , 6 , 7 , and 8 . Fr.5-2-1-1-2 (40.2 mg) was purified by semi-preparative HPLC and washed with MeOH-H2O  to afford compound 10 .Fr.5 (7.22 g) was applied to column chromatography over RP-Cn-hexane-EtOAc gradient  to get four subfractions (Fr.4-1 to Fr.4-4). Fr.4-2 (197.1 mg) was separated by Sephadex LH-20 column chromatography and eluted with CHCl3-MeOH  to afford four subfractions (Fr.4-2-1 to Fr.4-2-4). Fr.4-2-4 (13.8 mg) was further purified by the semi-preparative HPLC system and eluted with MeOH-H2O (70:30) to give compound 9 .Fr.4 (2.26 g) was isolated by column chromatography on silica gel and eluted with 2O . Fr.6-1 (2.79 g) was loaded onto Sephadex LH-20 column chromatography and eluted with CHCl3-MeOH  to give four subfractions (Fr.6-1-1 to Fr.6-1-4). Fr.6-1-2 (643.0 mg) was isolated by column chromatography on silica gel and eluted with CH2Cl2-MeOH  to get seven subfractions (Fr.6-1-2-1 to Fr.6-1-2-7). Fr.6-1-2-4 (311.0 mg) was separated by semi-preparative HPLC with CH3CN-H2O  and then repeatedly purified by semi-preparative HPLC with CH3CN-H2O  to afford compound 11 .Fr.6 (19.0 g) was separated into four subfractions (Fr.6-1 to Fr.6-4) on ODS column chromatography with MeOH-Hα)20D–12.4 ; UV (MeOH): λmax (log ε): 259 (2.77), 202 (2.41) nm; IR (KBr): 3,381, 2,996, 2,905, 2,837, 1,616, 1,559, 1,456, 1,385, 1,308, 1,229, 1,206, 1,098, 1,024, 695, 758, 733, 667, 596, 556 cm−1; HRESIMS: m/z 227.1274 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin A (1): colorless needle crystals (α)20D + 8.2 ; UV (MeOH): λmax (log ε): 267 (3.28) nm; IR (KBr): 3,370, 2,976, 2,932, 2,884, 1,717, 1,614, 1,381, 1,337, 1,242, 1,217, 1,146, 1,105, 1,026, 978, 874, 773, 739, 689, 667, 596 cm−1; HRESIMS: m/z 227.1275 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin B 2): colorless needle crystals; m. p. 120–121°C (: colorleα)20D–33.4 ; UV (MeOH): λmax (log ε): 263 (3.10) nm; IR (KBr): 3,377, 2,974, 2,926, 1,616, 1,456, 1,386, 1,333, 1,289, 1,252, 1,209, 1,141, 1,103, 1,068, 1,011, 976, 914, 883, 760, 692 cm−1; HRESIMS: m/z 227.1276 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin C (3): white solid (α)20D + 11.6 ; UV (MeOH): λmax (log ε): 269 (2.54) nm; IR (KBr): 3,329, 2,947, 2,835, 1,651, 1,456, 1,410, 1,115, 1,017, 667, 608, 546 cm−1; HRESIMS: m/z 255.1598 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin D (4): white solid (α)20D–11.6 ; UV (MeOH): λmax (log ε): 275 (2.98) nm; IR (KBr): 3,356, 1,653, 1,616, 667, 600, 552 cm−1; HRESIMS: m/z 211.1329 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin E (5): colorless needle crystals (α)20D + 12.0 ; UV (MeOH); λmax (log ε): 268 (3.34), 202 (2.23) nm; HRESIMS: m/z 233.1146 (M + Na)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin F (6): white solid 20D–13.7 ; UV (MeOH): λmax (log ε): 275 (3.08) nm; IR (KBr): 3,337, 1,636, 669, 600, 554 cm−1; HRESIMS: m/z 211.1339 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin G (7): white solid (α)20D + 10.8 ; UV (MeOH): λmax (log ε): 274 (3.23) nm; IR (KBr): 3,360, 1,636, 667, 600, 557 cm−1; HRESIMS: m/z 211.1339 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin H (8): white solid (λmax (log ε): 328 (3.02), 279 (3.33), 217 (3.47) nm; IR (KBr): 3,379, 2,976, 2,922, 2,851, 1,676, 1,608, 1,458, 1,329, 1,292, 1,220, 1,151, 1,092, 1,024, 947, 768 cm−1; HRESIMS: m/z 207.1013 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin I (9): colorless needle crystals; m. p. 108–109°C; UV (MeOH): α)20D–11.4 ; UV (MeOH): λmax (log ε): 270 (2.80) nm; IR (KBr): 3,312, 2,976, 2,930, 2,899, 1,717, 1,668, 1,607, 1,456, 1,400, 1,344, 1,271, 1,246, 1,180, 1,130, 1,032, 945, 856, 764, 669 cm−1; HRESIMS: m/z 211.1333 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin J (10): colorless oil (λmax (log ε): 305 (3.00) nm; IR (KBr): 3,358, 2,974, 2,920, 1,649, 1,626, 1,560, 1,479, 1,445, 1,362, 1,305, 1,290, 1,169, 1,113, 1,028, 953, 829 cm−1; HRESIMS: m/z 207.1025 (M + H)+ . 1H (500 MHz) and 13C (125 MHz) NMR data, see Foeniculin K (11): green needle crystals; UV (MeOH): viawww.ccdc.cam.au.ck/conts/retrieving.html.)The single-crystal X-ray diffraction data were collected at 100 K for 1, 2, 5, and 9 on Agilent Xcalibur Nova single-crystal diffractometer using CuKα radiation. Crystallographic data for 1, 2, 5, and 9 reported in this paper have been deposited in the Cambridge Crystallographic Data Centre. . Copies of these data can be obtained free of charge in vitro cytotoxic activities of compounds 1–11 were assayed against three human tumor cell lines SF-268, MCF-7, HePG-2, and normal cell line LX-2 with adriamycin as positive control. Assays were performed by the SRB method (The B method .Staphylococcus aureus (CMCC 26003) and Escherichia coli (ATCC 8739). Assays were performed by the published microdilution method for the estimation of minimum inhibitory concentration (MIC) values  values . Vancomy12H18O4 was established on the basis of (+)-HRESIMS m/z 227.1274 (M + H)+ , implying four degrees of hydrogen deficiency. The IR spectrum of 1 logically revealed the presence of carbonyl and free hydroxyl functional groups through the characteristic resonance absorptions at 1,616 and 3,381 cm−1, respectively. The 1H NMR data , 3.29 , 3.26 ] and three methyl moieties . The 13C NMR data (δC 204.1).Compound 1 was isolated as needle crystals. Its molecular formula of CNMR data  of 1 exhNMR data  combined1H-1H COSY spectrum (3-11/H-6/H2-5 suggested the presence of fragment a (C-11/C-6/C-5). The HMBC correlations from H3-10 to C-5 (δC 33.1), C-6 (δC 41.5), and C-7 (δC 204.1), H-11 to C-7, C-8 (δC 115.9), and C-8a (δC 171.0), H2-5 to C-7 and C-8a coupled with the fragment a were significantly suggested the existence of a cyclohexanone ring (ring A) with a carbonyl group located at C-7 position as well as two methyls attached at C-6 and C-8 positions, respectively. In addition, the obvious HMBC correlations from H-2 to C-4 (δC 76.0) and C-8a, H-9 to C-2 (δC 79.1) and C-3 (δC 76.3) together with the 1H-1H COSY spin system b (C-4a/C-4/C-3/C-2/C-9) confirmed the presence of the pyran ring B. Therefore, the planar structure of 1 was established as shown in In the spectrum , the cro3-11 confirmed these protons were co-facial, and assigned as α-oriented. Then, the NOE correlation between H-5 and H3-9 indicated that the methyl group at C-9 was β-oriented + in its HRESIMS spectrum, requiring four degrees of unsaturation. The 1D NMR data (1H-1H COSY cross peaks of H-2/H-3/H-4 as well as the predominant HMBC correlations from H3-9 to C-2 and C-3 as well as carbon shit of C-3 (δC 39.0).Compound 2 was obtained as needle crystals. Its molecular formula was established as CNMR data  and 2 of3-11 with H-5α indicated that these protons should be co-facial, and they were tentatively assigned as α-oriented. Moreover, H-5β exhibited a conclusive NOESY correlation with H-4, which further correlated with H-2, thus strongly suggesting that they should be located as β-oriented , H-3 , and H-4 . Comparing the 13C NMR spectra of 2 and 3, the signals attributed to the methylene C-3  and quaternary carbon C-8  indicated that they should be a pair of diastereoisomers, which showed a little structural difference on the ring B. Interestingly, the partial relative configuration of 3 was determined by NOESY experiment + , implying four degrees of hydrogen deficiency. After a careful inspection of the NMR spectra of 4 with those of 2, it could be readily disclosed that they showed very close similarity in most NMR profiles. The major difference between them was the hydroxyl group at C-4a in 2 replaced by a hydroxyethyl one in 4, which could be substantiated by its chemical shifts  in conjunct with the HMBC correlation from H2-1′ to C-4a and the 1H-1H COSY fragment H2-1'/H3-2'. Interestingly, compound 4 showed an ECD spectrum almost consistent with that of 2 , 2.98 ] and three methyl groups . The 13C NMR spectrum combined with HSQC data of 5 resolved 12 carbon resonances, and they were attributable to three methyls, two methylenes, four methines, and three quaternary carbons including a carbonyl group (δC 193.4).Compound 5 was obtained as colorless needle-like crystals. The HRESIMS of compound 5 showed a positive molecular ion peak at e 1H NMR  data of 1H-1H COSY spectrum  and b (C-2/C-3/C-9). Based on the fragment a, the HMBC correlations from H-8 to C-6, C-7, and C-4a, H3-11 to C-5, C-6, and C-7, H3-10 to C-7, C-8, and C-8a suggested the existence of a cyclohexene ring A, which possessed a hydroxyl group located at C-7 and two methyls attached at C-6 and C-8, respectively. Furthermore, the obvious HMBC correlations from H-2 to C-4, H-9 to C-2 and C-3, H-3 to C-4a as well as the 1H-1H COSY fragment b confirmed the presence of the pyran ring B. The NOESY correlations from H-7 to H-5α, H3-10, and H3-11 assigned these protons as β-orientation -7-hydroxy-2,6,8-trimethyl-2,3,5,6,7,8-hexahydro-4H-chromen-4-one and given the trivial name foeniculin E.In the spectrum , the croentation . A singlm/z 233.1146  of 6, 211.1339 (M + H)+  of 7, and m/z 211.1339 (M + H)+  of 8 indicated that compounds 7 and 8 should share the same molecular formula with C12H18O3 as that of 6. Careful comparison of the 1H and 13C NMR spectra of 6–8 -7-hydroxy-2,6,8-trimethyl-2,3,5,6,7,8-hexahydro-4H-chromen-4-one and given the trivial name foeniculin G.Compound 7 shared the same planar structure as those of 5 and 6. In its NOESY spectrum, the key NOESY correlations between H-6/HD curves  revealedα-orientation and the hydroxyl group at C-7 should be β-orientation. This deduction was consistent with the analysis of the ECD calculations -7-hydroxy-2,6,8-trimethyl-2,3,5,6,7,8-hexahydro-4H-chromen-4-one and given the trivial name foeniculin H.Compound 8 also shared very close similarity in the NMR data to those of 6. These subtle differences indicated that the methyl group at C-6 adopted an ulations . Thus, t12H14O3 was deduced from the HRESIMS peak at m/z 207.1013 (M + H)+ , which clearly suggested the presence of six indices of unsaturation. 1H NMR data of 9 , an oxymethine , a methylene , and an olefinic methine . The 13C NMR data , a ketocarbonyl (δC 200.6), three methyls , and one oxymethine (δC 87.0). The 1H-1H COSY revealed one spin-spin system (C-8/C-9/C-10). The HMBC correlations of H-5 to C-1, C-3, C-7, and C-12, H3-12 to C-3, C-4, and C-5, as well as H3-11 to C-1, C-2, and C-3 established a 3-hydroxy-2,4-dimethylph-2-en-1-one core scaffold for ring A (S absolute configuration of 9 was assigned by the X-ray diffraction (S)-8-ethyl-3-hydroxy-2,4-dimethylbenzofuran-3(2H)-one and given the trivial name foeniculin I.Compound 9 was isolated as colorless needle crystals, and the molecular formula of Cata of 9  revealedNMR data  and the r ring A . The HMBfraction . Finally12H18O3 from the (M + H)+ ion at m/z 211.1333 in HRESIMS data . The molecular unsaturation together with the 1H and 13C NMR data . The partially relative configuration of 10 was established by analyses of NOESY correlations. The key NOESY correlations between H-3/H3-11 and H-3/H3-12 strongly suggested that these two methys should be in the same orientation -10 showed a negative Cotton effect at 270 nm, which well matched with that of the experimental result  was found to have a molecular formula of Cin vitro cytotoxic activity against the tumor cell lines SF-268, MCF-7, HePG-2, and normal cell line LX-2. As a result, compound 11 exhibited mild cytotoxicity against the tumor cell line with IC50 values of 27.73, 42.54, and 25.12 µM. Compounds 1–10 were inactive to the tested tumor cell lines even at a concentration of 100 µM. The antimicrobial activity of compounds 1–11 was also evaluated against the bacteria Escherichia coli and S. aureus. However, all of them were found to be devoid of significant activity.The isolated compounds 1–11 were tested Diaporthe foeniculina SCBG-15 resulted in the isolation and structural elucidation of eleven new compounds foeniculins. The structures including absolute configurations were determined by extensive physicochemical and spectroscopic analysis, as well as ECD calculation and X-ray diffraction crystallography. All the novel compounds 1–11 possessed polymethylated skeleton. Compound 11 exhibited cytotoxic activity against the tumor cell lines SF-268, MCF-7, HePG-2 with IC50 values of 27.73, 42.54, 25.12 µM, which might serve as a promising antitumor lead compound for the drug discovery.A phytochemical investigation on the


In the Antibodies, reagents and flow cytometers subsection of the Materials and methods, there is an error in the first sentence of the first paragraph. The correct sentence is: Fluorescein isothiocyanate (FITC)-conjugated anti-Ki67 (SolA15), Rat IgG2a kappa isotype control (eBR2a), PerCP-Cy5.5-conjugated anti-CD19 (1D3), NK1.1 (PK136), CD3ε (145-2C11), CD25 (PC61.5), Allophycocyanin (APC)-conjugated anti-FcεR1α (MAR-1), CCR9 (CW-1.2), Mouse IgG2a kappa isotype control (eBM2a), Alexa Fluor 700-conjugated anti-CD45.2 (104), CD11c (N418), eFluor 450-conjugated anti-CD3ε (145-2C11), CD4 (RM4-5), CD19 (1D3), CD11b (M1/70), TCRγδ (GL3), CD11c (N418), NK1.1 (PK136), TCRβ (H57-597), Gr1 (RB6-8C5), Ter119 (TER-119), Phycoerythrin (PE)-conjugated anti-CD127 (A7R34) and anti-GATA3 (TWAJ), PE-Cyanine7 conjugated anti-CD127 (A7R34) were purchased from Thermo Fisher Scientific .In the In vitro stimulation subsection of the Materials and methods, there is an error in the first sentence of the first paragraph. The correct sentence is: ILC2s were sorted from naïve female and male lungs after ILC2 enrichment and 1000 cells were cultured in 200 μL RPMI-1640 media containing 10% FBS, P/S, 50 μM 2ME, 5 ng/mL TSLP and IL-33.In


C-β-d-arabinopyranoside-2″-O-(4‴-acetoxy)-glucoside (6) and three known flavone C-glycosides—5,7,3′,4′-tetrahydroxyflavone-6-C-xyloside-8-C-β-d-glucoside (lucenin-1) (7), 5,7,3′-trihydroxyflavone-6-C-glucoside-8-C-β-d-glucoside (vicenin-2) (8), and 5,7,4′-trihydroxy-3′-methoxyflavone-6-C-β-d-glucopyranoside-8-C-α-arabinopyranoside (chrysoeriol-6-C-β-d-glucopyranoside-8-C-α-arabinopyranoside) (9)—were isolated from aerial parts of Scleranthus perennis L. (Caryophyllaceae). Their structures were determined through the use of comprehensive spectroscopic and spectrometric methods, and a method for the quantification of the major constituents of S. perennis and S. annuus L. was developed. Furthermore, the anti-collagenase and antioxidant activities of all isolated compounds obtained from extracts and fractions from both Scleranthus species were evaluated. The highest percentage of collagenase inhibition (at 400 µg/mL) was distinguished for methanolic extracts  and ethyl acetate fractions  from S. annuus and S. perennis. Compounds 6–9 displayed moderate inhibitory activity, with IC50 values ranging from 39.59–73.86 µM.A new 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8- Scleranthus perennis L. and Scleranthus annuus L., belonging to the Caryophyllaceae family, are endemic species to Europe, North Africa, and Western Asia , 475 [M + H-hex-Ac], 343 [M + H-hex-Ac-pent], and 313 [M + H-hex-Ac-pent-OCH3] -glucoside and named scleranthoside D  . The arant-OCH3] —suggestehoside D .7 was obtained as a yellow amorphous powder. Based on the HRESIMS ion peak at m/z 581 [M + H]+, the molecular formula of C26H28O17 was determined. Based on UV spectra, 8 was assigned to the flavone derivatives and showed a bathochromic shift of band I in the presence of NaOAc/H3BO3 (29 nm), indicating an ortho-dihydroxyl group in the B-ring (luteolin derivative) + in positive mode by HRESIMS, corresponding to the molecular formula C27H30O15. According to UV spectra, 8 is different from 7 by a lack of hydroxylation at C4′. This conclusion was supported by observations of a similar value of band I appearing with NaOAc/H3BO3 (347 nm) and in MeOH (345 nm) .,22.8, a 9, isolated as a yellow amorphous powder, showed an [M + H]+ ion at m/z 595 in its HRESIMS spectrum, corresponding to the molecular formula C27H30O15. The UV spectra are typical of flavone derivatives with ortho-hydroxylation (bathochromic shift of band I in the presence of NaOAc/H3BO3) D −29.0 ; HPLC rt, 100.03 min; HRESIMS m/z = 679.205 [M + H]+ ; UV λmax nm: 263, 287; +NaOMe: 252, 304; +AlCl3: 249, 318; +NaOAc: 260, 301; +H3BO3: 263, 297); NMR spectral data , 1H NMR  H-C2′: δ 7.62 , H-C6′: δ 7.50 , H-C5′: δ 6.95 , H-C3: δ 6.58 , H-C6: δ 6.23 , H-C1″: δ 5.16 , H-C1‴: δ 4.31 , H-C4″: δ 4.18 , H-C4″: δ 4.09 , CH3: δ 4.03 , H-C2″: δ 3.90 , H-C3″: δ 3.78 , H-C6‴: δ 3.19 , H-C5‴: δ 3.17 , H-C3‴: δ 3.04 , H-C4‴: δ 2.95 , H-C2‴: δ 2.93 , OAc: δ 1.97 , OAc: δ 2.20 ; 13C NMR  C4: δ 182.74, Ac: δ 171.47, δ 170.72, C2: δ 164.83, C7: δ 163.11, C5: δ 161.39, C9: δ 155.59, C4′: δ 150.46, C3′: δ 148.06, C1′: δ 122.76, C6′: δ 120.45, C5′: δ 115.40, C2′: δ 109.73, C8: δ 104.59, C1‴: δ 104.08, C3: δ 102.87, C10: δ 102.53, C6: δ 99.43, C2″: δ 80.42, C3‴: δ 76.45, C1″: δ 74.72, C3″: δ 74.61, C5‴: δ 73.60, C2‴: δ 72.71, C5″: δ 71.38, C4‴: δ 69.20, C4″: δ 68.05, C6‴: δ 63.11, CH3: δ 55.32, Ac: δ 19.58, Ac: δ 19.32; see 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-xyloside-8-C-β-d-glucoside (lucenin-1) (7): yellow amorphous powder; HPLC rt, 17.44 min; HRESIMS m/z = 579.167 [M + H]+ ; UV λmax nm: 263, 287; +NaOMe: 252, 304; +AlCl3: 249, 318; +NaOAc: 260, 301; +H3BO3: 263, 297); NMR spectral data, see + ; UV λmax nm: 274, 345; +NaOMe: 285, 412; +AlCl3: 281, 362; +NaOAc: 283, 368; +H3BO3: 274, 247; NMR spectral data, see + ; UV λmax nm: 274, 349; +NaOMe: 285, 414; +AlCl3: 281, 360; +NaOAc: 283, 377; +H3BO3: 274, 282; NMR spectral data, see [5,7,4′-trihydroxy-3′-methoxyflavone--6-ata, see .Individual test compounds and extracts in methanol (130 µL) were added to 70 µL of a solution of DPPH . After 30 min of incubation, the absorbance was evaluated at 517 nm . The detSuppression of the production of the radical cation was measured using an antioxidant assay kit (CS0790). In brief, 10 µL of the test sample, 20 µL of myoglobin working solution, and 150 µL of ABTS working solution (containing ABTS substrate solution and 3% hydrogen peroxide) were mixed into a clear, flat-bottom 96-well plate. After 5 min of incubation, 100 µL of stop solution was added and absorbance was measured at 405 nm. The antioxidant concentration of tested samples was calculated using the equation obtained through linear regression of the Trolox standard curve.SP1–SP6, SA1–SA6, 1–9) was mixed with 190 µL of supplied reaction mix . After 1 h of incubation at 37 °C, the absorbance was measured at 594 nm. A blank solution containing 10 µL of MeOH instead of extract/compound solution was used. For further calculations, the ferrous standard curve was evaluated. Values are given as mM Ferrous equivalents. All tests were performed in triplicate.The chelating activity of the investigated substances toward ferrous ions was assessed using a ferric reducing antioxidant power assay kit. Briefly, 10 µL of each sample . Briefly, 20 µL of sample and 100 µL of reaction mix were added into separate wells in a 96-well plate. After 10 min of incubation, the absorbance endpoint was measured at 570 nm. Total antioxidant capacity was determined using a standard curve evaluated for Trolox.Clostridium histolyticum, 25 µL of Tricine buffer, and 25 µL of various levels of the extracts  or compounds (6–9) were incubated at 37 °C. After 20 min, 75 µL of FALGPA was added. Then, absorbance was measured at 335 nm. The experiment was performed in 50 mM Tris buffer. The blank was evaluated using Tricine buffer, instead of sample, and the positive control was epigallocatechin gallate (EGCG). All measurements were conducted in triplicate and the IC50 for each sample was calculated. The percentage of collagenase inhibitory activity (CoInh) was calculated as follows:The potential anti-collagenase activity was assessed as previously described . In shorAll results obtained in the anti-collagenase test are represented as the mean ± standard deviation (SD) from at least three independent replicates. Statistical analysis was carried out using the GraphPad Prism 9 software . Statistical differences and linear regression parameters for the standard curve were determined using ANOVA with corroboration of statistical significance. Calculations for regression parameters were conducted using the MS Excel 2019 software .6–9) isolated from S. perennis in this paper are newly found C-glycosylated flavones in Scleranthus plants. So far, flavone C-glycosides have only been reported in S. uncinatus Schur. [7), vicenin-2 (8), and chrysoeriol-6-C-β-d-glucopyranoside-8-C-α-arabinopyranoside (9) were previously isolated from Caryophyllaceae plants  [Scleranthus sp., as well as that of the Caryophyllaceae family. As phytochemical studies of both S. perennis and S. annuus have not been reported so far, we also performed qualitative and quantitative elucidation of all bioassay-guided extracts and fractions. All tested samples exhibited moderate anti-collagenase activity, and so, further biological models and the application of acylated C-diglycosyled flavones should be studied.Plant metabolomics may deliver large data sets, which allow for a better understanding of the cellular processes occurring in plant organisms. The growing interest and development of metabolomics research in plants provide important indicators for the chemotaxonomic description of whole plant families. The identified compounds (s Schur. . It coulnis sp.) . Further


A wide variety of 2,4-, 4-, and 2-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles were synthesized for SAR study via two different pathways. Overall, 4-halogenated 5-aryl-1-(4-methylsulfonylphenyl)imidazoles, regardless of the species of halogen, exhibited very strong inhibitory activities of PGE2 production. Among them, 4-chloro-5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazole , and 4-chloro-5-(4-chlorophenyl)-1-(4-methylsulfonylphenyl)imidazole  showed the best results.A series of halogenated 1,5-diarylimidazole compounds were synthesized and their inhibitory effects on LPS-induced PGE Cyclooxygenase (COX), is one of the key proinflammatory enzymes which catalyzes the conversion of arachidonic acid to prostaglandins (PGs). Cyclooxygenase exists in two isoforms, COX-1 and COX-2. Among them, COX-2 is inducible and known as a major isoform found in inflammatory lesions + 329.11-(4-methylsulfonylphenyl)-5-(4-methoxyphenyl)imidazole (11). Yellow solid, 87%; 1H-NMR  δ 8.02 , 7.78 , 7.30 , 7.05 , 3.11 .5-(4-chorophenyl)-1-(4-methylsulfonylphenyl)imidazole (To the solution of intermediates (1 or 11 (0.5 mmol) in CHCl3 (4 mL) was added NCS/NBS/NIS (0.75 mmol). The mixture was refluxed for 5 h, extracted with DCM, washed with aqueous NaHSO3 and brine, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography to give 4-halo and 2,4-dihalo imidazoles. Different from chlorination, 2-bromo imidazole products were separated in some bromination reactions. While iodination with NIS in acetonitrile (CH3CN) afforded 2-, 4- and 2,4-iodo imidazole products. NCS/NBS/NIS are abbreviated name of n-chlorosuccinimide, n-chlorosuccinimide, and n-chlorosuccinimide, respectively.3). White solid, 41%; 1H-NMR  δ 7.95 , 7.65 , 7.32 , 7.11 , 6.88 , 3.82 , 3.09 ; 13C-NMR  δ 159.9, 140.6, 135.0, 129.1, 129.0, 127.1, 125.7, 118.7, 114.4, 55.3, 44.4; HRMS (EI) m/z Calcd for C17H15ClN2O3S [M] + 362.0492 Found 362.0493.4-chloro-1-(4-methylsulfonylphenyl)-5-(4-methoxyphenyl)imidazole (13). Yellow solid, 20%; 1H-NMR  δ 8.00 , 7.67 , 7.32 , 7.13 , 3.10 ; 13C-NMR  δ140.8, 140.1, 135.9, 135.2, 131.0, 129.3, 129.2, 125.8, 124.9, 55.1, 49.2, 44.4; HRMS (EI) m/z Calcd for C16H12Cl2N2O2S [M] + 365.9997 Found 365.9994.4-chloro-5-(4-chorophenyl)-1-(4-methylsulfonylphenyl)imidazole (16). Yellow solid, 50%; 1H-NMR  δ 7.98 , 7.72 , 7.33 , 7.16 , 3.10 ; 13C-NMR  δ 140.5, 140.3, 137.1, 135.0, 131.2, 129.2, 129.2, 128.6, 125.8, 125.7, 117.6, 44.4; HRMS (EI) m/z Calcd for C16H12BrClN2O2S [M] + 409.9491 Found 409.9490.4-bromo-5-(4-chorophenyl)-1-(4-methylsulfonylphenyl)imidazole  in tetrahydrofuran (THF) 3 mL of lithium bis(trimethylsilyl)amide  was added dropwise at −20 °C. The mixture was stirred for 0.5 h, then solution of NCS or NBS (1.6 mmol) in THF (3 mL) was added. The reaction mixture was stirred for 0.5 h at −20 °C and 6 h at room temperature. Saturated aqueous NH4Cl was added to the mixture and extracted with ethyl acetate. The organic layer was washed with aqueous NaHSO3 and brine, dried over magnesium sulfate, and concentrated under vacuum. Following oxidation of crude 2-halogenated 5-aryl-1-(4-methylthiophenyl)imidazole with 3-chloroperbenzoic acid followed by silica gel column chromatography yielded pure 2-halogenated 5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazoles.7). White solid, 12%; 1H-NMR  δ 7.96 , 7.64 , 7.32 , 7.12 , 6.88 , 3.82 , 3.09 ; 13C-NMR  δ 159.9, 140.7, 140.1, 136.4, 131.3, 129.6, 129.0, 125.7, 119.3, 116.9, 114.3, 55.3, 44.4; HRMS (EI) m/z Calcd for C17H15BrN2O3S [M] + 405.9987 Found 405.9988.2-bromo-5-(4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazole (To the solution of 5-aryl-1-(4-methylthiophenyl)imidazoles  for 1 h and then activated with LPS (100 ng/mL) for the indicated time. SAMPLE were dissolved in DMSO and added to the culture media in serial dilution .RAW 264.7 cells were purchased from the Korea Cell Line Bank  and maintained in DMEM medium containing 10% FBS, streptomycin sulfate, penicillin, HEPES, and sodium bicarbonate in a 5% CO5 cells/mL with 10% fetal bovine serum cell culture medium, and 1 mL of cell suspension were added to each hole of a 24-well plate for 24 h in a 37 °C incubator. Cytotoxicity was determined by MTT assay after treating various concentrations of SAMPLE  for another 24 h after overnight incubation. The formed formazan crystals in the cells were dissolved by DMSO, followed by measurement at 540 nm.RAW 264.7 cells, 2 × 105 cells/mL) were seeded onto a 24-well plate and then incubated with/without LPS (100 ng/mL) in the presence or absence of SAMPLE  for 24 h. Nitrite levels of cellular supernatants were measured using the Griess reaction and estimated to reflect the concentration of NO. The absorbance was measured at the wavelength of 540 nm using the microplate reader. In all experiments, fresh culture media were used as blanks. The levels of nitrite in the samples were determined using the standard curve of sodium nitrite. PGE2 concentration in the medium was measured using an ELISA kit for PGE2 .RAW 264.7 cells  or Dup-697 (100 nM) were used.Samples were evaluated for its potency and selectivity of inhibition in vitro using COX Inhibitor Screening Assay . Recombinant COX-1 (ovine) or COX-2 (human) proteins were pre-incubated with Compound 3 for 10 min at 37 °C. The reaction was started by the addition of 100 µM arachidonic acid and allowed to proceed for 2 min. The reaction was terminated by the addition of an HCl solution containing SnClp < 0.05 compared with the control group, and * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the LPS-stimulated group.Results are expressed as the mean ± SD of triplicate experiments with similar patterns. Statistically significant values were compared using ANOVA and Dunnett’s post hoc test, and P values of less than 0.05 were considered statistically significant. #