T and PWU20T were both from soil in Germany, PWU5T was isolated from soil in India and PWU37T was obtained from sheep faeces collected on the Island of Crete. Cells of each were observed to be Gram-negative, strictly aerobic, rod shaped, and to grow optimally between 28 and 34 °C, between pH 7.0 and 8.0 and without the addition of NaCl. The strains were found to be catalase and oxidase-negative and able to grow on most mono- and disaccharides, a few polysaccharides and organic acids. Their predominant menaquinone was identified as MK-7. Their major fatty acids were identified as C16:1ω7c (PWU4T and PWU20T) and C16:1ω5c (PWU5T and PWU37T). The DNA G + C contents of strains PWU4T, PWU20T, PWU5T and PWU37T were determined to be 50.2 mol%, 51.6 mol %, 39.8 mol% and 53.8 mol%, respectively. The 16S rRNA gene sequence analysis revealed that the close relatives Ohtaekwangia koreensis 3B-2T and Ohtaekwangia kribbensis 10AOT share less than 93.8% sequence similarity. The strains were classified in two groups, where PWU4T and PWU20T share 93.0% sequence similarity, and PWU5T and PWU37T share 97.5% sequence similarity. However, the members of each group were concluded to represent different species based on the low average nucleotide identity (ANI) of their genomes, 69.7% and 83.8%, respectively. We propose that the four strains represent four novel species of two new genera in the family Cytophagaceae. The type species of the novel genus Chryseosolibacter is Chryseosolibacter histidini gen. nov., sp. nov. with the type strain PWU4T (= DSM 111594T = NCCB 100798T), whilst strain PWU20T (= DSM 111597T = NCCB 100800T) is the type strain of a second species, Chryseosolibacter indicus sp. nov. The type species of the novel genus Dawidia is Dawidia cretensis gen. nov., sp. nov. with the type strain PWU5T (= DSM 111596T = NCCB 100799T), whilst strain PWU37T (= DSM 111595T = NCCB 100801T) is the type stain of a second species, Dawidia soli sp. nov.Four novel strains were isolated: PWU4 Cytophagaceae was originally introduced in 1940 by Stanier  were identified. The aim of the present study was to explore the taxonomic status of the four bacteria as novel species by using a polyphasic approach.Since the ‘golden age’ of antibiotic discovery, members of the phylum T, PWU20T and PWU37T were isolated from soil samples collected in May 1990 at Braunschweig, Germany ; in May 1989 at Lucknow, Uttar Pradesh, India ; in September 1991 at Braunschweig ; strain PWU5T was isolated from sheep faeces with plant residues collected in July 1988 at Crete Island (35.2463N 25.09705E). The strains were isolated using a dilution method on agar plates following the protocol of Reichenbach  web server . After 5 days incubation at room temperature, cells were harvested from E broth medium and the pellet was further processed through DNA extraction following the manufacturer’s protocol. The 16S rRNA genes was amplified using the bacterial universal primer set 27F and 1492R following the protocol of Mohr et al. . After thttp://ggdc.dsmz.de/distcalc2.php , while strains PWU5T and PWU37T grow at 21–34 °C (optimum at 28–34 °C). Moreover, strain PWU4T grows at pH 5.5–8.0 (optimum at pH 7), strain PWU5T grows at pH 6.5–8.5 (optimum at pH 7), strain PWU20T grows at pH 6.5–9.0 (optimum at pH 7) and strain PWU37T grows at pH 5.0–9.5 (optimum at pH 7.4–8.0). The reference strains Ohtaekwangia koreensis (3B-2T) and Ohtaekwangia kribbensis (10AOT) , respectively. Reference strains such as members of the genus Ohtaekwangia, which were isolated from the marine environment, tolerate a concentration up to 0.2% (w/v) only. Members of the closely related genus Chryseotaela, tolerate up to 1.0% of NaCl (w/v)   or facultatively anaerobic. No growth was observed for any the strains under anaerobic conditions. Catalase and oxidase activities along with flexirubin-type pigments for all the strains were negative, whereas the reference strains are positive for these characteristics  , C16:1ω5c (43.8%), iso C15:0 (43.6%) and C16:1ω5c (38.5%), respectively. In contrast to strain PWU4T where C16:1ω7c was found to be the major fatty acid, this fatty acid was much less abundant in strain PWU37T (9.0%). Moreover, C16:1ω5c was found to be the major fatty acid in strains PWU5T and PWU37T but not in strains PWU4T and PWU20T. Albers and Siebers , O. kribbensis 10AOT (92.0%) and Chryseolinea soli KIS68-18T (91.0%), whereas the current closest relatives of strain PWU5T are O. koreensis 3B-2T (93.6% 16S rRNA gene sequence similarity), O. kribbensis 10AOT (93.1%), and Chryseolinea serpens RYGT (92.3%). The current closest relatives of strain PWU20T are O. kribbensis 10AOT (92.5% 16S rRNA gene sequence similarity), O. koreensis 3B-2T (92.0%), and C. soli KIS68-18T (90.6%). The current closest relatives of strain PWU37T are O. kribbensis 10AOT (93.7% 16S rRNA gene sequence similarity), O. koreensis 3B-2T (93.2%) and C. soli KIS68-18T (92.0%). Notably, all of these values are below the 94.5% 16S rRNA threshold suggested by Yarza et al.  revealed values of 13% to 50% and confirmed that all of strains represented distinct new species. Strains PWU4T, PWU5T, PWU20T, and PWU37T shared ANI values of 69.2%, 69.9%, 69.5% and 69.7% with O. koreensis 3B-2T, respectively . The major identified polar lipid is phosphatidylethanolamine. Member of the family Cytophagaceae. The type species is Chryseosolum histidini.Gram-stain negative, rod-shaped, asporogenous, non-motile, mesophilic, heterophilic and aerobic bacteria. Catalase and oxidase negative. Growth is observed on D-gluconic acid and methyl pyruvate. The major cellular fatty acids are Chryseosolibacter histidini .In addition to the characteristics listed in the genus description, the species has the following characteristics: cells are 2.56–6.67 µm long and appear as single cells. Colonies are irregular and yellow on E agar plates after 5 days of cultivation. Growth occurs between 21 and 40 °C (optimum 30 °C), between pH 5.5 and 8.0 (optimum pH 7) and NaCl tolerance 0.4% (w/v). Positive for phosphatase alkaline, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, phosphatase acid, naphthol-AS-B1-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamidase, α-mannosidase, α-fucosidase, urease, esterase and alcaline phosphatase. Weak activities of lipase (C14) and β-glucoronidase. No growth is observed on dextrin, D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose, stachyose, D-raffinose, α-D-lactose, D-melibiose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, α-D-glucose, D-mannose, L-fucose, D-arabitol, glycerol, gelatin, glycyl-L-proline, L-alanine, L-arginine, L-serine, pectin, D-galacturonic acid, L-galactonic acid lactone D-gluconic acid, glucuronic acid, glucuornicamide, mucic acid, D-lactic acid methyl ester, citric acid, D-malic acid, L-malic acid, bromo-succinic acid, tween 40, γ-amino-butryric acid, α-keto-butyric-acid, acetoacetic acid, and acetic acid. The DNA G + C content of the type strain is 50.2 mol%.T (= DSM 111594T = NCCB 100798T) was isolated from a soil sample collected in May 1990 at Braunschweig, Germany (52.22090N 10.50902E). The 16S rRNA gene and whole-genome sequences of PWU4T have been deposited in GenBank/EMBL/DDBJ under accession numbers MW182516 and JAHESF000000000, respectively.The type strain PWU4Chryseosolum indicus .4, L-alanine, L-arginine, L-histidine, L-galactonic acid lactone D-gluconic acid, p-hydroxy-phenylacetic acid, L-malic acid, bromo-succinic acid, tween 40, α-keto-butyric-acid, propionic acid, and acetic acid. The DNA G + C content of the type strain is 39.8 mol %.In addition to the characteristics listed in the genus description, the species has the following characteristics: cells are 2.32–6.41 µm long and appear as single cells. Colonies are irregular and yellow on E agar plates after 5 days of cultivation. Growth occurs between 21 and 40 °C (optimum 28 °C), between pH 6.5 and 9.0 (optimum pH 7) and NaCl tolerance below 0.8% (w/v). Positive for phosphatase alkaline, leucine arylamidase, valine arylamidase, cystine arylamidase, chymotrypsin, phosphatase acid, naphthol-AS-B1-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucoronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamidase, α-mannosidase, urease, esterase and alcaline phosphatase. Weak activities of esterase (C4), esterase lipase (C8), lipase (C14), trypsin and no activities for α-fucosidase. No growth is observed on dextrin, D-maltose, D-cellobiose, gentiobiose, N-acetyl-D-glucosamine, N-acetyl-β-D-galactosamine, D-fructose, D-frucose, L-rhamnose, inosine, D-sorbtiol, D-mannitol, D-arabitol, myo-inositol, D-fructose-6-POT (= DSM 111597T = NCCB 100800T) was isolated from a soil sample collected in May 1989 at Lucknow, Uttar Pradesh, India (26.8684N 80.90979E). The 16S rRNA gene and whole-genome sequences of strain PWU20T have been deposited in GenBank/EMBL/DDBJ under accession numbers MW182517 and JAHESD000000000, respectively.The type strain PWU20Dawidia iso-C15:0 and C16:1ω5c. The major respiratory quinone is menaquinone-7 (MK-7). The major identified polar lipids is phosphatidylethanolamine (PE). Member of the family Cytophagaceae. The type species is Dawidia cretensis.Gram-stain negative, rod-shaped, asporogenous, non-motile, mesophilic, heterophilic and aerobic bacteria. Catalase and oxidase-negative. Growth is observed on D-cellobiose, L-fucose, D-arabitol, L-malic acid, and α-keto-butyric-acid. The major cellular fatty acids are Dawidia cretensis .In addition to the characteristics listed in the genus description, the species has the following characteristics: cells are 4.37–7.62 µm long and appear as single cells. Colonies are irregular and yellow on E agar after 5 days of cultivation. Growth occurs between 21 and 34 °C (optimum 28 °C), between pH 6.5 and 8.5 (optimum pH 7) and NaCl tolerance below 0.6% (w/v). Positive for phosphatase alkaline, leucine arylamidase, valine arylamidase, cystine arylamidase, phosphatase acid, naphthol-AS-B1-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamidase, esterase and alkaline phosphatase. Weak activities of esterase (C4), esterase lipase (C8), lipase (C14) and negative for trypsin, chymotrypsin, β-glucoronidase, α-mannosidase, α-fucosidase and urease activities. No growth is observed on dextrin, D-turanose, α-D-lactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-arginine, L-histidine, L-pyroglutamic acid, D-gluconic Acid, glucuronic acid, glucuornicamide, mucic acid, quinic acid, D-saccharic acid, methyl pyruvate, citric acid, γ-amino-butryric acid, and β-hydroxy-D,L-butyric acid. The DNA G + C content of the type strain is 51.6 mol %.T (= DSM 111596T = NCCB 100799T) was isolated from sheep faeces with plant residues collected in July 1988 on the Island of Crete (35.2463N 25.09705E). The 16S rRNA gene and whole-genome sequences of strain PWU5T have been deposited in GenBank/EMBL/DDBJ under accession numbers MW182518 and JAHESE000000000, respectively.The type strain PWU5Dawidia soli .4, D-aspartic acid, D-serine, gelatin, L-alanine, L-arginine, L-aspartic Acid, L-histidine, pectin, L-galactonic acid lactone D-gluconic acid, D-gluconic acid, glucuornicamide, mucic acid, p-hydroxy-phenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, L-lactic acid, citric acid, α-keto-glutaric acid, D-malic acid, bromo-succinic acid, tween 40, α-hydroxy-butyric acid, β-hydroxy-D,L-butyric acid, acetoacetic acid, propionic acid, acetic acid and formic acid. The DNA G + C content of the type strain is 53.8 mol %.In addition to the characteristics listed in the genus description, the species has the following characteristics: cells are 3.1–6.43 µm long and appear as single cells. Colonies are irregular and yellow on E agar plate after 5 days of cultivation. Growth occurs between 21 and 34 °C (optimum 34 °C), between pH 5.0 and 9.5 (optimum pH 7.4 until 8.0) and NaCl tolerance below 1.0% (w/v). Positive for phosphatase alkaline, leucine arylamidase, valine arylamidase, cystine arylamidase, phosphatase acid, naphthol-AS-B1-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamidase, α-fucosidase, esterase and alkaline phosphatase. Weak activities of esterase (C4), esterase lipase (C8), lipase (C14) and no activities for trypsin, chymotrypsin, β-glucoronidase, α-mannosidase, and urease. No growth is observed on dextrin, D-maltose, gentiobiose, D-turanose, D-raffinose, α-D-lactose, D-melibiose, D-salicin, N-acetyl-D-glucosamine, N-acetyl-β-D-galactosamine, N-acetyl-D-galactosamine, α-D-glucose, D-fructose, D-sorbitol, myo-inositol, D-glucose-6-PO4, D-fructose-6-POT (= DSM 111595T = NCCB 100801T) was isolated from a soil sample collected in September 1991 at Braunschweig (52.21501N 10.53329E). The 16S rRNA gene and whole-genome sequences of strain PWU37T have been deposited in GenBank/EMBL/DDBJ under accession numbers MW182519 and JAHESC000000000, respectively.The type strain PWU37


Ligustrum robustum have been applied as Ku-Ding-Cha, a functional tea to clear heat, remove toxins, and treat obesity and diabetes, in Southwest China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of eight new monoterpenoid glycosides (1–8) and three known monoterpenoid glycosides (9–11). Compounds 1–11 were tested for the inhibitory activities on fatty acid synthase (FAS), α-glucosidase, α-amylase, and the antioxidant effects. Compound 2 showed stronger FAS inhibitory activity (IC50: 2.36 ± 0.10 μM) than the positive control orlistat (IC50: 4.46 ± 0.13 μM), while compounds 1, 2, 5 and 11 displayed more potent ABTS radical scavenging activity (IC50: 6.91 ± 0.10~9.41 ± 0.22 μM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM). This study provided a theoretical basis for the leaves of L. robustum as a functional tea to treat obesity.The leaves of  Ligustrum (Oleaceae) and the genus Ilex (Aquifoliaceae) 20D-91.9 ; UV (MeOH) λmax: (log ε) 214 (4.2), 244 (4.1), 331 (4.4) nm; IR (film) νmax: 3375, 2923, 1694, 1630, 1601, 1515, 1446, 1376, 1261, 1025, 928, 836, 811 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 647.2679 [M + Na]+ .Compound 2: white amorphous powder. [α]23D-29.9 ; UV (MeOH) λmax (log ε): 209 (3.9), 230 (3.9), 314 (4.4) nm; IR (film) νmax: 3375, 2927, 1689, 1632, 1604, 1515, 1445, 1263, 1169, 1037, 832 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 631.2728 [M + Na]+ .Compound 3: white amorphous powder. [α]25D-11.0 ; UV (MeOH) λmax (log ε): 208 (3.9), 230 (3.9), 316 (4.4) nm; IR (film) νmax: 3370, 2926, 2855, 1696, 1605, 1514, 1448, 1262, 1169, 1036, 833 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 647.2680 [M + Na]+ .Compound 4: white amorphous powder. [α]23D-20.9 ; UV (MeOH) λmax (log ε): 208 (3.9), 229 (3.9), 315 (4.4) nm; IR (film) νmax: 3369, 2924, 2854, 1695, 1632, 1604, 1515, 1448, 1262, 1170, 833 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 665.2784 [M + Na]+ .Compound 5: white amorphous powder. [α]23D-29.3 ; UV (MeOH) λmax (log ε): 209 (3.9), 228 (3.9), 315 (4.4) nm; IR (film) νmax: 3375, 2926, 1694, 1632, 1605, 1515, 1377, 1262, 1169, 1038, 833 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 661.2833 [M + Na]+ .Compound 6: white amorphous powder. [α]23D-71.0 ; UV (MeOH) λmax (log ε): 209 (3.9), 230 (3.9), 313 (4.4) nm; IR (film) νmax: 3410, 2973, 1696, 1604, 1515, 1381, 1260, 1168, 1046, 834 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 777.3312 [M + Na]+ .Compound 7: white amorphous powder. [α]23D-71.0 ; UV (MeOH) λmax (log ε): 208 (3.9), 230 (3.9), 316 (4.4) nm; IR (film) νmax: 3410, 2973, 1696, 1604, 1515, 1381, 1260, 1168, 1046, 834 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 777.3312 [M + Na]+ .Compound 8: white amorphous powder. [α]23D-17.8 ; UV (MeOH) λmax (log ε): 208 (3.9), 230 (3.9), 316 (4.4) nm; IR (film) νmax: 3391, 2925, 1697, 1632, 1605, 1515, 1446, 1264, 1169, 1041, 834 cm−1; 1H NMR  data, see 13C NMR  data, see /zm 661.2831 [M + Na]+ .Compound 1–8 (2 mg) in MeOH (0.1 mL) were added to 1 M H2SO4 aqueous solution (2 mL) and heated in 95 °C water bath for 6 h, respectively. The hydrolyzed solution was neutralized with 1 M Ba(OH)2, filtered and concentrated to a small amount. The monosaccharides in the concentrated solution were identified by TLC with authentic samples, developing with EtOAc-MeOH-HOAc-H2O . The fR values of d-glucose, d-mannose and l-rhamnose were 0.43, 0.46 and 0.73, respectively.Compounds 1 or 2 (20 mg) was hydrolyzed with cellulase (30 mg) in HOAc-NaOAc buffer solution  at 37 °C for 12 h. The hydrolyzed product was extracted with Et2O and purified on silica gel column (eluting with CH2Cl2), to give (R)-linalool and (S)-linalool (4:6) confirmed by [α]27D +3.5  or +2.8 .Compound α-glucosidase and α-amylase, and the DPPH and ABTS radical scavenging effects of compounds 1–11 were evaluated according to the methods described in the literature  at a different position in 2. The acid hydrolysis experiment of 2 gave d-glucose and l-rhamnose identified by TLC. The HMBC experiment of 2 (δH 4.39 (H-1′ of glucosyl) and δC 81.5 (C-3 of aglycone), between δH 5.17 (H-1″ of rhamnosyl) and δC 84.4 (C-3′ of glucosyl), and between δH 4.30, 4.45 (H-6′ of glucosyl) and δC 169.0 (carbonyl of coumaroyl). Additionally, the enzymatic hydrolysis experiment of 2 gave (R)-linalool and (S)-linalool (4:6). The 1H and 13C NMR signals of 2 were assigned by 1H-1H COSY, HSQC and HMBC experiments (2 was confirmed as a mixture (R:S = 4:6) of 3(R)- and 3(S)-linaloyl-(3-O-α-l-rhamnopyranosyl)-(6-O-trans-p-coumaroyl)-O-β-d-glucopyranoside, which is a new monoterpenoid glycoside and named ligurobustoside T1.Compound ata of 2  were siment of 2  showed teriments . Thus, c3 was analyzed as C31H44O13 by HRESIMS . The 1H and 13C NMR data of 3 (2 except for some data of the aglycone. The HSQC experiment of 3 displayed the correlations between δH 4.78 (H-8a of aglycone), 4.88 (H-8b of aglycone) and δC 111.4 (C-8 of aglycone), meaning that the C-6 double bond in 2 was replaced by the C-7 double bond in 3. The 1H-1H COSY experiment of 3 (δH 1.22 (H-4 of aglycone), 3.95 (H-6 of aglycone) and δH 1.60 (H-5 of aglycone), meaning that a hydroxyl was linked at C-6 in 3. Thus, the aglycone of 3 was 3,7-dimethyl-octa-1,7-diene-3,6-diol. The acid hydrolysis experiment of 3 gave d-glucose and l-rhamnose identified by TLC. The HMBC experiment of 3 (δH 4.38 (H-1′ of glucosyl) and δC 81.4 (C-3 of aglycone), between δH 5.17 (H-1″ of rhamnosyl) and δC 84.4 (C-3′ of glucosyl), and between δH 4.30, 4.45 (H-6′ of glucosyl) and δC 169.0 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 3 were assigned by 1H-1H COSY, HSQC and HMBC experiments -(3-O-α-l-rhamnopyranosyl)-(6-O-trans-p-coumaroyl)-O-β-d-glucopyranoside. It is a novel monoterpenoid glycoside named ligurobustoside T2.Compound ata of 3  are simient of 3  displayeent of 3  displayeeriments . Therefo4 was analyzed as C31H46O14 by HRESIMS . The NMR spectra of 4 showed two stereoisomers 4a and 4b (2:1). The 1H NMR spectrum of 4a ; (2) a trans double bond at δH 6.34, 7.64 ; (3) a monosubstituted double bond at δH 5.19 , 5.24  and 5.92 ; (4) two anomeric protons at δH 4.41 , 5.18 ; (5) a methenyl at δH 3.21 ; (6) two methylene groups at δH 1.32–1.90 ; (7) four methyl groups at δH 1.11, 1.14, 1.36 , 1.25 . The 13C NMR spectrum of 4a -(3-O-α-l-rhamnopyranosyl)-(4-O-trans-p-coumaroyl)-O-β-d-glucopyranoside (lipedoside B-VI) -(4-O-trans-p-coumaroyl)-O-β-d-glucopyranoside, which is a new monoterpenoid glycoside and named ligurobustoside T6.Compound ata of 6  are simient of 6  showed teriments . Thus, c7 was determined as C37H54O16 by HRESIMS . The 1H and 13C NMR data of 7 ) in 6 was replaced by the cis-p-coumaroyl ) in 7. The acid hydrolysis experiment of 7 yielded d-glucose and l-rhamnose identified by TLC. The HMBC experiment of 7 (δH 4.41 (H-1′ of glucosyl) and δC 81.6 (C-3 of aglycone), between δH 5.29 (H-1″ of inner rhamnosyl) and δC 79.8 (C-3′ of glucosyl), between δH 5.13 (H-1′′′ of outer rhamnosyl) and δC 80.6 (C-4″ of inner rhamnosyl), and between δH 4.86 (H-4′ of glucosyl) and δC 166.9 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 7 were assigned by 1H-1H COSY, HSQC and HMBC experiments -α-l-rhamnopyranosyl]-(4-O-cis-p-coumaroyl)-O-β-d-glucopyranoside. It is a new monoterpenoid glycoside, named ligurobustoside T7.Compound ata of 7  are relaent of 7  showed teriments . Thus, c8 was analyzed as C32H46O13 by HRESIMS . The NMR spectra of 8 exhibited two stereoisomers 8a and 8b (2:1). The 1H NMR spectrum of 8a ; (2) two trans double bonds at δH 6.35, 7.64 , 5.44 , 5.55 ; (3) an olefinic proton at δH 5.41 ; (4) two anomeric protons at δH 4.31 , 5.17 ; (5) two methylene groups at δH 4.27 , 2.76 ; (6) four methyl groups at δH 1.23, 1.23, 1.65 , 1.24 ; and (7) a methoxy at δH 3.12 . The 13C NMR spectrum of 8a -1--(3-O-α-l-rhamnopyranosyl)-(4-O-trans-p-coumaroyl)-O-β-d-glucopyranoside (ligurobustoside I) [trans-p-coumaroyl was linked at different positions, and there was another methyl in 8a. The HMBC experiment of 8a (δH 3.12 (OCH3) and δC 76.4 (C-7 of aglycone). The NOEDS experiment of 8a (δH 5.41 (H-2 of aglycone) and δH 2.76 (H-4 of aglycone). Therefore, the aglycone of 8a was -7-methoxy- 3,7-dimethyl-octa-2,5-dien-1-ol. The acid hydrolysis experiment of 8 gave d-glucose and l-rhamnose identified by TLC. The HMBC experiment of 8a (δH 4.31 (H-1′ of glucosyl) and δC 66.3 (C-1 of aglycone), between δH 5.17 (H-1″ of rhamnosyl) and δC 84.0 (C-3′ of glucosyl), and between δH 4.35, 4.50 (H-6′ of glucosyl) and δC 169.1 (carbonyl of coumaroyl). The 1H and 13C NMR signals of 8 were assigned by 1H-1H COSY, HSQC and HMBC experiments -1--(3-O-α-l-rhamnopyranosyl)-(6-O- trans-p-coumaroyl)-O-β-d-glucopyranoside.Compound um of 8a  displayeum of 8a  revealednt of 8a  showed tnt of 8a  displayent of 8a  displayeeriments . Consequ8b ) in 8b. The HMBC experiment of 8b (δH 4.27 (H-1′ of glucosyl) and δC 66.3 (C-1 of aglycone), between δH 5.16 (H-1″ of rhamnosyl) and δC 84.0 (C-3′ of glucosyl), and between δH 4.31, 4.48 (H-6′ of glucosyl) and δC 168.1 (carbonyl of coumaroyl). So, 8b was identified as -1--(3-O-α-l-rhamnopyranosyl)-(6-O-cis-p-coumaroyl)-O-β-d-glucopyranoside. In conclusion, compound 8 is a mixture of novel monoterpenoid glycosides 8a and 8b, named ligurobustoside T8–9.The NMR data of 8b  are simint of 8b  displaye9–11 (NMR data see 9a) and H (9b), ligurobustoside C (10), ligurobustosides K (11a) and L (11b), respectively, by direct comparison with published spectral data  [Compounds 13C NMR) ,9.1–11 from the leaves of L. robustum were tested for the inhibitory activities on FAS, α-glucosidase, α-amylase, and the antioxidant effects. The results of bioactivity assays are shown in 2 revealed stronger FAS inhibitory activity (IC50: 2.36 ± 0.10 μM) than the positive control orlistat (IC50: 4.46 ± 0.13 μM); compound 2 showed weaker α-glucosidase inhibitory effect than the positive control acarbose; compounds 2–6, 8, 9 and 11 displayed weaker α-amylase inhibitory effect than the positive control acarbose; compounds 1, 2, 5 and 11 exhibited more potent ABTS radical scavenging activity (IC50: 6.91 ± 0.10~9.41 ± 0.22 μM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM), while compound 1 displayed weaker DPPH radical scavenging activity (IC50: 19.74 ± 0.23 μM) than L-(+)-ascorbic acid (IC50: 13.66 ± 0.13 μM).Compounds 2, 6 and 10 with strong FAS inhibitory activity might be a part of the constituents with anti-obesity activity in L. robustum. In addition, the results suggested that the FAS inhibitory activity would reduce or disappear when the monoterpene unit of glycoside was substituted with hydroxyl, or the trans-p-coumaroyl of glycoside was replaced by other groups.Because FAS is a potential therapeutic target for anti-obesity drugs ,18,19, cL. robustum resulted in the separation of eleven monoterpenoid glycosides (1–11), including eight new compounds (1–8) identified with spectroscopic method , and physical and chemical methods. The biological study showed that compound 2 revealed stronger FAS inhibitory activity (IC50: 2.36 ± 0.10 μM) than the positive control orlistat (IC50: 4.46 ± 0.13 μM); compounds 1, 2, 5 and 11 displayed more potent ABTS radical scavenging activity (IC50: 6.91 ± 0.10~9.41 ± 0.22 μM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 μM); compound 2 revealed also moderate α-glucosidase and α-amylase inhibitory activities. This study provided a theoretical basis for the leaves of L. robustum as a functional tea to treat obesity.In summary, the phytochemical research on the leaves of


Cimicifuga yunnanensis. The new structures were elucidated by analysis of spectroscopic data. Compounds 3–5, 7–9, and 11, without obvious cytotoxicity at 50 μM, were evaluated for inhibiting the mRNA expressions of atherosclerosis-related factors of CD147 , matrix metalloproteinase 2 (MMP-2) and MMP-9 in phorbol-12-myristate-13-acetate (PMA) induced Human monocytic THP-1 cells by using a quantitative real-time PCR method (q-PCR). Among them, aglycones 7 and 8 showed potent activities, whereas all tested glycosides were inactive. Compounds 7 and 8 suppressed the mRNA expression of CD147 in a dose-dependent manner, with an IC50 value of 3.38 ± 0.27 μM and 8.25 ± 0.33 μM, respectively. Besides, 7 dose-related down-regulated the mRNA expression of MMP-2, and MMP-9, having an IC50 value of 6.32 ± 0.31 μM and 11.57 ± 0.23 μM, respectively. Meanwhile, 8 at 10 μM reduced the mRNA expression of MMP-2 and MMP-9 by 35% and 25%, respectively. Significantly, the migration ability of the induced THP-1 cells was potently and dose-dependently inhibited by 7, with an IC50 value of 5.87 ± 0.27 μM.Eleven new 9,19-cycloartane triterpenes  and one undescribed lanostane-type aglycone (10) were identified from the aerial parts of  Cimicifuga yunnanensis. CTs aglycones 7 and 8 potently down-regulated the mRNA expression of CD147, MMP-2, and MMP-9.Eleven new 9,19-cycloartane triterpenes (CTs)  and one undescribed lanostane-type aglycone (10) were identified from  Otherwise, a life-threatening acute coronary syndrome, including acute myocardial infarction (AMI) and unstable angina pectoris (UA) would happen to them.4–6 CD147, extracellular matrix metalloproteinase inducer, and metalloproteinases (MMPs), such as MMP-2 and MMP-9 (a novel marker of AMI), are overexpressed in advanced atherosclerotic plaques by monocytes/macrophages and have been shown to contribute to AMI and UA through degradation of the extracellular matrix.7–10 Of note, CD147 stimulates macrophages to produce MMP-2 and MMP-9 in a paracrine or autocrine way.4,11 Therefore, to suppress the expression of CD147 and MMPs represents a promising strategy for anti-atherosclerosis.Cardiovascular diseases (CVDs) are leading causes of death globally, by which more than 17.0 million people die every year.Cimicifuga genus  are famous herb medicines in Europe, the United States, and East Asia.12,13 These herbs mainly contain 9,19-cycloartane triterpenes (CTs) with diverse bioactivities, such as cytotoxicity,14,15 anti-angiogenic,16 anti-inflammatory,17,18 and neuro-protective.19,20 Recently, we identified two CTs, yunnanterpene G (YG) and 12β-hydroxycimiacerol (HC), with anti-atherosclerosis potentials by potently suppressing the mRNA expressions of CD147 and MMPs.21,22 As a part of our successive program to explore bioactive CTs from Cimicifuga spp, nine unreported CTs glycosides  and two new aglycones (7 and 8), together with one undescribed lanostane-type triterpene (10) were identified from the aerial parts of C. yunnanensis . While, 8 has comparable activity as YG at this concentration. Moreover, 7 dose-dependently down-regulated the mRNA expression of MMP-2, and MMP-9 and suppressed the migration ability of the induced THP-1 cells, having an IC50 value of 6.32 ± 0.31 μM, 11.57 ± 0.23 μM, and 5.87 ± 0.27 μM, respectively. Conversely, all tested glycosides  were inactive at 10 μM. Described herein are the isolation, structure elucidation, and biological activities of compounds 1–12.Plants of nanensis , an indi2.2.1.37H54O10 for compounds 1 and 2 were determined by the HRESIMS . The IR spectra showed absorptions for OH (3443 cm−1 for 1 and 3441 for 2), and carbonyl (1733 cm−1 both for 1 and 2) groups. In the 1H NMR  and 1.07 , a secondary methyl group at δH 0.95 , six tertiary methyl groups at δH 0.95–1.47, and an anomeric proton at δH 4.79  were observed. The 13C spectrum of 1 (δC 171.1 (s), two olefinic carbons at δC 114.5  and 148.2 , and six oxygenated carbon atoms at δC 88.3 , 77.1 , 73.5 , 63.6 , 63.7 , and 69.2 , respectively. These data suggested that 1 was a highly oxygen-bearing CTs glycoside with a seven-ring skeleton.The same molecular formula Ce 1H NMR  spectrumrum of 1  showed t1H–1H COSY (2CHCHCH(CH3)CH2– , together with the diagnostic ketal signal at δC 106.4 , as well as the pair of geminal signals for CH2-26 at δH 3.91 and 3.97 , indicated that 1 was a acteol-type CTs.12,23 HMBC couplings from H-16 (δH 4.63), H-22 (δH 1.65 and 2.19) to C-23 (δC 106.4), H-22 (δH 1.65 and 2.19) to C-24 (δC 63.6), and H-26 (δH 3.91 and 3.97) and H-24 (δH 3.73) to C-23 (δC 109.5) and C-25 (δC 83.4), further supported this deduction. The location of sugar unit at C-3 was inferred from HMBC correlation between the anomeric proton at δH 4.85  and the methine signal at δC 88.5 (C-3). In addition, the sugar was determined as l-arabinose by comparing its TLC and specific rotation with a standard after acid hydrolysis. Structurally, 1 resembles to 26-deoxyactein,23 with main differences as that an acetoxy group substituted at C-12 and the presence of a double bond at C-7 and C-8. These elucidations were confirmed by HMBC association from H-12 (δH 5.23) to the ester carbonyl group (δC 171.1), and the 1H–1H COSY correlation of the olefinic proton resonance (δH 5.15) and H-6 (δH 1.54 and 1.82).The –1H COSY  spin sys3-28 , and H-20 with CH3-18  were observed, which helped to establish the relative configuration of the core structure of 1. Moreover, the characteristic ROESY correlations of H-21/H-24 and H-24/CH3-27 further decided the configuration of ring F and G, as well as the ternary epoxy ring and ring G as shown -en-acteol-3-O-α-l-arabinopyranoside and 7(8)-en-23-epi-acteol-3-O-α-l-arabinopyranoside, respectively.In the ROESY spectrum , cross-pas shown . Intensixyactein .23 Final37H58O12 based on the HRTOF-ESIMS at m/z 717.3823 [M + Na + H2O]+ . The 1H NMR spectrum  and 0.58 , and an anomeric proton at δH 4.79 . The 13C NMR spectrum (δC 171.1 (s), and an α,β-unsaturated ketone unit at δC 207.3 , 148.6  and 151.0 . Aforementioned data indicated that 3 was a CTs glycoside. The sugar unite was determined as l-arabinose by the same way as that of 1. The NMR data of aglycone part of 3 resembled that of 24-O-acetyl-16(17)-en-hydroshengmanol-3-O-β-d-xylopyranoside,20 except that the acetoxy group was changed to C-12 and the OH-16 was replaced by a carbonyl group. HMBC correlations of H-12 (δH 5.74) with the ester carbonyl group (δC 170.8), and CH3-28 (δH 1.31) with the carbonyl carbon (δC 207.3) further confirmed these elucidations. ROESY cross-peaks of H-3/H5, H-12/CH3-28, and H-20/CH3-18 in 3 suggested the α-orientation of H-3, H-12, and CH3-21. The β-orientation of H-24 was deduced by the ROESY correlation of H-24/H-20. In addition, identical to isodahurinyl-type molecules, H-24 of 3 was a singlet in 1H NMR spectrum, suggesting S configuration of C-24 .15 Therefore, the structure of 3 was determined as 12β-acetoxy-16(17)-en-isodahurinyl-3-O-α-l-arabinopyranoside.Compound 3, white powder, had molecular formula Cspectrum  showed cspectrum  indicate37H58O10 based on the HREIMS at m/z 662.4058 [M]+ . The NMR spectroscopic data for 4 resembled those of -12β-acetoxy-16,23-epoxy-24,25-dihydroxy-3β-(β-d-xylopyranosyloxy)-9,19-cyclolanost-22(23)-ene (AC),24 with major differences in sugar unit. In addition, the sugar moiety was attached to C-3 and determined as l-arabinose by the same way as that of 1. A α-orientation of H-3, H-12, H-16, H-17, and CH3-21 were determined by ROESY couplings of H-3 with H-5, H-12, H-16, and H-17 with CH3-28, and H-20 with H-17, respectively. Whereas, a β-orientation of H-8 was deduced by the correlation of H-8/CH3-18. In addition, identical to that of AC -12β-acetoxy-16,23-epoxy-24,25-dihydroxy-3β-(β-d-xylopyranosylo-xy)-9,19-cyclolanost-22(23)-ene, the characteristic ROESY association of CH3-18/CH3-26 was observed in 4, indicating it shares the same configuration at C-24 as R in AC . Thus, the structure of 4 was determined as 12β-acetoxy-22(23)-en-15-deoxy-isodahurinyl-3-O-α-l-arabinopyranoside.Compound 4 possessed the molecular formula of C37H58O11, given by the HREIMS . The IR spectrum showed the presence of hydroxyl (3431 cm−1), carbonyl (1730 cm−1) and olefinic (1629 cm−1) groups. The NMR data of aglycone part for 5 (O-α-d-xylopyranoside.25 The main differences were that a methine (C-22) at δC 86.6 was absent, instead, there's another methylene (δC 42.1), and the upfield shifts of C-23, C-24, and C-25 by 3.1 ppm, 5.4 ppm and 8.6 ppm, respectively. These changes could be explained as that, in 5, a methylene replaced a methine at C-22, and two hydroxy groups instead of the ternary epoxy ring at C-23 and C-24. These deductions were further confirmed by the HMBC coupling of H-20 (δH 4.81)/C-22 (δC 42.1). The sugar unit was connected to C-3 and identified as l-arabinose using same approaches as those of 1. In addition, the orientations of core structure and the configuration of C-24 of 5 were determined on the basis of the ROESY associations as those of 4. Therefore, the structure of 5 was determined as 12β-acetoxy-23,24-dihydroxy-7(8)-en-15-deoxy-isodahurinyl-3-O-α-l-arabinopyranoside.Compound 5 was purified as white powder, with the molecular formula Crt for 5  and 2 wem/z 715.3666 [M + Na]+ , the molecular formula of 6 was determined as C37H56O12. 1H NMR resonances due to a downfield shifted cyclopropane methylene at δH 0.57  and 1.07 , an olefinic proton at δH 6.73 , an acetyl methyl group at δH 2.24, a secondary methyl signal at δH 1.11 , six singlet methyl groups at δH 1.00–1.59, as well as an anomeric proton at δH 4.76  were observed (l-arabinose by using same approaches as those of 1. Comparison of NMR data of 6 and hydroxyshengmanol-7(8)-en-15-one-3-O-β-d-xylopyranoside26 revealed the aglycone part of the two compounds were identical, except for an acetoxy group substituted at C-12 in 6, which further supported by the HMBC correlation of H-12 (δH 5.49) and the ester carbonyl group (δC 170.7). A α-orientation of the substituents at C-3, and C-12 were determined by ROESY correlations of H-3/H-5 and H-12/CH3-28. Whereas, correlations of H-20/CH3-18 and H-23/H-20 indicated the β-orientation of H-23. The stereochemistry of C-24 was elucidated as S by comparison of coupling constant of H-24 with known compounds .15,26 The molecular formula C30H44O7 of 7 was deduce from its HRTOF-ESIMS at m/z 539.2988 [M + Na]+ . The spectroscopic features of 7 were identical to 6 except for a carbonyl group and a hydroxy group at C-3 and C-12, respectively. HMBC correlation of CH3-29 (δH 1.11) with the carbonyl carbon (δC 214.6) and the upfield shift of C-12 by 4.8 ppm further confirmed these elucidations. Same orientations of H-8, H-12, H-17, and H-23, as well as the configuration of C-24 between 7 and 6 were determined on the basis of the ROESY associations and comparison of coupling constant of H-24 with known compounds. Thus, the structure of 6 and 7 were determined as 12β-acetoxy-7(8)-en-15-one-hydroxyshengmanol-3-O-α-l-arabinopyranoside and 12β-hydroxy-7(8)-en-3,15-dione-hydroxyshengmanol, respectively.On the basis of the HRTOF-ESIMS peak at observed , indicat30H48O5, was established by HREIMS , indicating 7 degrees of unsaturation. The 30 carbon signals of 8 were similar to the aglycone resonances of actaeaepoxide-3-O-β-d-xylopyranoside.25 The main differences were that no double bond at C-7 and C-8, and the absence of an acetoxy group at C-12 in 8. Moreover, 1H–1H COSY correlations of H-6  with H-7 , H-11  with H-12  confirmed these deductions. The orientations of H-16, H-17, and H-22 were assigned as α by analysis of ROESY spectrum. Besides, the characteristic ROESY correlation of H-22/H-24 further suggested the configuration between ring E and the ternary epoxy ring as shown . In the 1H NMR spectrum  and 2.01 , an anomeric proton at δH 4.82 , an olefinic hydrogen atom at δH 5.21 , four tertiary methyl groups at δH 1.12–1.60, and a secondary methyl signal at δH 0.90 , were observed. The 1H–1H COSY spectrum indicated that 9 had part structure of –CHCH(CH3)CH2– . Aforementioned data together with HMBC associations of H-22  with the carbonyl carbon (δC 211.2) and the oxygenated carbon at δC 82.3 , and H-17 (δH 2.25) with the oxygenated carbon at δC 81.9  and C-24, exhibited that 9 was a foetidonol-type CTs glycoside. The sugar unit was connected to C-3 and identified as l-arabinose using same ways as those of 1. Compound 9 had a similar structure as that of foetidonol-3-O-β-d-xylopyranoside,27 except for a double bond at C-7 and C-8, and a hydroxy group at C-11. The 1H–1H COSY coupling of H-6 and the olefinic proton at δH 5.21, and the HMBC correlation of H-11 (δH 4.60) and C-9, as well as the molecule weight further confirmed these elucidations. Therefore, the structure of 9 was determined as 11β-hydroxy-7(8)-en-foetidonol-3-O-α-l-arabinopyranoside.The molecular formula of compound 9 was determined as Cspectrum , signalsm/z 488.3508 [M]+  for the molecular formula of C30H48NO5. The 1D NMR spectroscopic data . These data indicated that 10 possessed one more tertiary methyl group in the skeleton than a usual CTs. In 13C NMR spectrum, a pair of tetrasubstituted olefinic carbons at δC 134.9  and 136.0 , and the characteristic ketal signal for C-16 of cimigenol-type CTs at δC 112.7 (s) were observed. HMBC associations of H-7/C-8 , H-11/C-9 , H-1/CH3-19 , and CH3-19/C-9 and C-10, located the double bond at C-8 and C-9, and the CH3-19 at C-10, respectively. The rest of NMR resonances of 10 were identical to those of cimigenol.28 Therefore, the structure of 10 was determined as 19β-methyl-8(9)-en-cimigenol.The HREIMS of 10 exhibited a molecular ion at pic data  of 10 shm/z 657.3613 [M + Na]+  in the positive ion HRTOF-ESIMS, corresponding to the molecular formula C35H54NaO10, which is 2 Da less than that of 15,16-seco-14-formyl-16-oxo-hydroshengmanol-3-O-α-l-arabinopyranoside.14 When its spectroscopic data -en-hydroshengmanol-3-O-α-l-arabinopyranoside.Compound 11 gave a pseudo-molecular ion at pic data  and 2 we37H54O10 from its HREIMS at m/z 658.3707 [M]+ . The spectroscopic features of 12 resembled to those of 15,23-O-diacetyl-7(8)-en-shengmanol-3-O-α-l-arabinopyranoside29 except for the substituent group at C-23. For 12, the oxygenated methine of C-23  was absent, showing instead a carbonyl carbon at δC 205.5. This difference was due to a carbonyl group at C-23 in 12, which confirmed by the HMBC correlations of H-22 and H-24 with carbonyl signal at δC 205.5. The sugar unit and the relative configuration of 12 were elucidated by the same ways as those of aforementioned compounds. Therefore, the structure of 12 was determined as 15α-acetoxy-23-oxo-7(8)-en-cimicidanol-3-O-α-l-arabinopyranoside.Compound 12 was assigned a molecular formula C2.2.11 Cultured by adding 100 nM PMA for 24 h, the cells became flat and amoeboid in shape, and adhered to the dish bottom  with q-PCR 2−ΔΔCt method. Compounds 3–5, 7–9 and 11, and Yunnanterpene G (YG), the previously identified active compound (positive control), were firstly tested at 10 μM in the differentiated THP-1 cells for 24 hours. The aglycones (7 and 8) noticeably suppressed the mRNA expression of CD147 (P < 0.001). Conversely, the tested glycosides, 3–5, 7 and 11, were inactive. Of note, 7 is more potent than YG (P < 0.05), while, 8 showed same level of activity as YG. Dose–response studies further revealed that 7 and 8 down-regulated the mRNA expression with an IC50 value of 3.38 ± 0.27 μM and 8.25 ± 0.33 μM, respectively  and 25% (P < 0.01), respectively. In addition, the mRNA expression of MMP-2, and MMP-9 were significantly and dose-dependently down-regulated by 7, having an IC50 value of 6.32 ± 0.31 μM and 11.57 ± 0.23 μM, respectively.ectively . Importa2.5.4,5 Because of compound 7 showed significant inhibition on the mRNA expression of CD147 and MMPs, the regulatory effect of this molecule on the migration of PMA-induced THP-1 cells was further studied by scratch wound assay. As a result, 7 (after 24 h incubation) potently and dose-related decreased the number of migrated cells with an IC50 value of 5.87 ± 0.27 μM  from C. foetida, with notable inhibitions on CD147 and MMPs mRNA expression, were identified. Successive investigations on the aerial parts of C. yunnanensis led to characterize eleven new CTs, including nine glycosides  and two aglycones (7 and 8), along with one undescribed lanostane-type triterpene (10). Compounds 7 and 8, two aglycones, showed noticeable inhibitory effects on the mRNA expression of CD147 and MMPs, as well as migration ability of the induced THP-1 cells. By contrast, all tested glycosides were inactive. It is worth noting that 7 is the most potent molecule among these four active CTs. Given the critical roles of CD147 and MMPs in stabilizing atherosclerotic plaques, 7 may have a promising effect in retarding the development of the vulnerability of the plaque, deserve to conduct more sophisticated animal studies in future.Natural products (NPs) are important resources of active molecules for modern drug development.In summary, our studies show that CTs, specially aglycones, are potential resources of anti-atherosclerosis bioactive agents and deserve further extensive exploration.4.4.1.254, Qingdao Marine Chemical, Inc.). Bruker DRX-500 and Avance III-600 MHz spectrometers  were used to record 1D and 2D NMR data with solvent signal as internal reference. ESIMS, HREIMS and HRESIMS were obtained from a Shimadzu LCMS-IT-TOF MS , a Waters AutoSpec Premier P776 MS  or an Agilent G6230 TOF MS . Shimadzu IR-450 instrument was used to evaluate infrared spectra with KBr pellets. A JASCO P-1020 digital polarimeter was applied to test optical rotations, using MeOH as solvent. Quantitative-PCR (q-PCR) was conducted on ProFlex™ PCR system .Column chromatography (CC) were conducted with Silica gel  and Lichroprep RP-18 . Waters 2695 liquid chromatography system was applied to run Semipreparative HPLC with a YMC-Pack 10 mm × 250 mm column (Pro C18 RS). Thin-layer chromatography was carried out on precoated TLC plates  were collected from Litang county, Garze Tibetan autonomous prefecture, Sichuan, China, in October 2013. A voucher specimen (No. 20131007) has been deposited at Department of Respiratory Medicine, The Third People's Hospital of Kunming, Yunnan, 650041, P. R. China.The aerial parts of 4.2.2Human monocytic THP-1 cells were bought from Kunming Cell Bank, Kunming Institute of Zoology, Chinese Academy of Sciences. Penicillin/streptomycin was added to all reagents used for cell culture. RevertAid™ First Strand cDNA Synthesis Kit, DMEM/F12 medium, FBS , and FITC-labeled anti-human CD68 and CD11b antibodies were purchased from Thermo Fisher . Restriction enzymes, Taq polymerase and PrimeScript RT reagent Kit were obtained from Takara Bio (Japan). Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma Chemical Co .4.3.Cimicifuga yunnanensis (4.7 kg) was extracted at room temperature by MeOH . The extract (489.7 g) was obtained after evaporation of MeOH under vacuum at 50 °C. The extract was divided by silica gel CC  eluted with CHCl3–MeOH  to yield fractions A (52.6 g), B (67.4 g), C (55.7 g), D (29.2 g) and E (23.3 g). Sub-fractions (B.1–B.7) were further obtained by silica CC , eluted with CHCl3–Me2CO from 40 : 1 gradient to 5 : 1. Compounds 7 (3.2 mg), 8 (2.4 mg) and 10 (2.2 mg) were purified from fraction B.4 (7.9 g) by RP-18 CC  eluting with MeOH–H2O from 60 : 40 to 100 : 0 and semipreparative HPLC . Subsequently, further silica CC  on fraction C, eluting with CHCl3–Me2CO from 20 : 1 gradient to 1 : 1, gave six sub-fractions (C.1–C.6). Another five sub-fractions (C.3.1–C.3.5) were obtained by RP-18 CC , eluting with MeOH–H2O from 50 : 50 to 100 : 0. Fraction C.3.3 (7.7 g) gave compounds 1 (2.7 mg), 2 (3.2 mg), 3 (2.1 mg), 4 (2.2 mg), 11 (2.1 mg), and 12 (3.1 mg) by RP-18 CC  eluting with MeOH–H2O from 60 : 40 to 75 : 25 and semipreparative HPLC . Compound 5 (2.5 mg), 6 (2.3 mg), and 9 (3.7 mg) were purified from fraction C.3.4 (6.9 g) by successively silica gel CC , and semipreparative HPLC .The aerial parts of 4.3.1α]19D = −97.3 ; IR (KBr): νmax 3443, 2963, 2876, 1733, 1632, 1453, 1378, 1251, 1084, 1029, 987 cm−1; ESIMS (positive) m/z 681 [M + Na]+; HREIMS (positive) m/z 658.3703 [M]+, calcd for C37H54O10, 658.3717; 1H NMR  and 13C NMR  data see White powder; [4.3.2α]19D = −39.1 ; IR (KBr): νmax 3441, 2962, 2874, 1733, 1630, 1455, 1382, 1247, 1070, 1028 cm−1; ESIMS (positive) m/z 681 [M + Na]+; HREIMS (positive) m/z 658.3694 [M]+, calcd for C37H54O10, 658.3717; 1H NMR  and 13C NMR  data see White powder; [4.3.3α]20D = −10.9 ; IR (KBr) νmax: 3424, 2931, 2872, 1706, 1642, 1455, 1381, 1137, 946 cm−1; ESIMS (positive) m/z 717 [M + Na + H2O]+; HRTOF-ESIMS (positive) m/z 717.3823[M + Na + H2O]+, calcd for C37H58NaO12, 717.3826; 1H NMR  and 13C-DEPT  data see White powder; [4.3.4α]19D = −28.4 ; IR (KBr): νmax 3440, 2932, 2870, 1734, 1628, 1452, 1383, 1244, 1072, 993 cm−1; ESIMS (positive) m/z 685 [M + Na]+; HREIMS (positive) m/z 662.4058 [M]+, calcd for C37H58O10, 662.4030; 1H NMR  and 13C NMR  data see White powder; [4.3.5α]19D = −29.3 ; IR (KBr): νmax 3431, 2964, 2876, 1730, 1629, 1451, 1383, 1249, 1069, 1025, 987 cm−1; ESIMS (positive) m/z 701 [M + Na]+; HREIMS (positive) m/z 678.3990 [M]+, calcd for C37H58O11, 678.3979; 1H NMR  and 13C NMR  data see White powder; [4.3.6α]19D = −18.82 ; IR (KBr) νmax: 3423, 2928, 2873, 1736, 1629, 1458, 1384, 1129, 945 cm−1; ESIMS (positive) m/z 715 [M + Na]+; HRTOF-ESIMS (positive) m/z 715.3666 [M + Na]+, calcd for C37H56NaO12, 715.3670; 1H NMR  and 13C-DEPT  see White powder; [4.3.7α]19D = −50.78 ; IR (KBr) νmax: 3441, 2925, 2854, 1753, 1631, 1454, 1384, 1141, 997 cm−1; ESIMS (positive) m/z 539 [M + Na]+; HRTOF-ESIMS (positive) m/z 539.2988 [M + Na]+, calcd for C30H44NaO7, 539.2985; 1H NMR  and 13C-DEPT  see White powder; [4.3.8α]24D = −304.0 ; IR (KBr): νmax 3424, 2925, 2873, 1450, 1384, 1068, 1037, 1008 cm−1; ESIMS (positive) m/z 511 [M + Na]+; HREIMS (positive) m/z 488.3499 [M]+, calcd for C30H48NO5, 488.3502; 1H NMR  and 13C NMR  see White powder; [4.3.9α]19D = −40.37 ; IR (KBr) νmax: 3420, 2928, 2855, 1721, 1632, 1451, 1384, 1139, 985 cm−1; ESIMS (positive) m/z 599 [M + Na]+; HRTOF-ESIMS (positive) m/z 599.3188 [M + Na]+, calcd for C32H48NaO9 599.3196; 1H NMR  and 13C-DEPT  see White powder; [4.3.10α]19D = −38.4 ; IR (KBr): νmax 3432, 2932, 2870, 1627, 1455, 1375, 1229, 1076, 1028, 973 cm−1; ESIMS (positive) m/z 511 [M + Na]+; HREIMS (positive) m/z 488.3508 [M]+, calcd for C30H48NO5, 488.3502; 1H NMR  and 13C NMR  see White powder; [4.3.11α]24D = −20.22 ; IR (KBr) νmax: 3424, 2968, 2877, 1715, 1634, 1454, 1382, 1141, 995 cm−1; ESIMS (positive) m/z 657 [M + Na]+; HRTOF-ESIMS (positive) m/z 657.3613 [M + Na]+, calcd for C35H54NaO10 657.3615; 1H NMR  and 13C-DEPT  see White powder; [4.3.12α]19D = −46.4 ; IR (KBr): νmax 3452, 2967, 2882, 1749, 1454, 1380, 1233, 1063, 1030, 991 cm−1; ESIMS (positive) m/z 681 [M + Na]+; HREIMS (positive) m/z 658.3707 [M]+, calcd for C37H54O10, 658.3717; 1H NMR  and 13C NMR  data see White powder; [4.4.2. Cells (5 × 105 to 106 per mL) were subjected to differentiation by 100 nM PMA for 24 h with DMEM/F12 serum medium. After incubation, the adherent cells were washed with DMEM/F12. Nonattached cells were removed by aspiration.Human monocytic THP-1 cells were cultured at 37 °C by DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO4.5.5) were washed 3 times by phosphate-buffered saline (PBS) and then respectively treated with fluorescein isothiocyanate (FITC)-conjugated anti-CD68 antibody, and FITC-conjugated anti-CD11b antibody, for 20 minutes. Cells were washed by PBS and then tested on a PARTEC CyFlow® Cube flow cytometer. Data were processed by the CytExpert software.The expression of CD68 and CD11b on surface of the differentiated THP-1 cells were tested by suing Flow cytometry. In a dark condition, cells , and incubated at 37 °C with 5% CO2 for 24 h. Then, 0.1 mL of DMEM/F12 with different concentrations of compounds 1–12  were added to instead of the original medium, and cultured for another 48 h. Cell viability was evaluated by MTT assay: each well was added with 20 μL of MTT to a final concentration of 0.5 g L−1 for 4 h before using 150 μL DMSO to solubilize the reactive dye. Each well was recorded by a Bio-Rad microplate reader to absorbance value of 570 nm. All the experiments were repeated in triplicate.The differentiated THP-1 cells were seeded onto gelatinized 96-well culture plates (5 × 104.7.5 cells per mL) following the manufacturer's instructions. cDNA was synthesized from the isolated total RNA based on the instruction of the PrimeScript RT reagent Kit. Briefly, PrimeScript RT Enzyme Mix 1 (0.5 μL), 5 × PrimeScriptTM Buffer (2 μL), oligo dt Primer (0.5 μL) and Random 6 mers (0.5 μL) were gently mixed with 1 μg RNA from each sample to a reaction volume of 10 μL with RNase free water, then incubated for 15 minutes at 37 °C to activate the reverse transcriptase enzyme. Finally, the reaction was stopped by 85 °C for 5 seconds.RevertAid™ First Strand cDNA Synthesis Kit was applied to extract total RNA of differentiated THP-1 cells treated with compounds 3–5, 9 and 11 (10 μM), along with 7 and 8  for 24 h . All the experiments were carried out in triplicate.After reverse transcription, cDNA was used to carry out real-time quantitative RT-PCR on ProFlex™ PCR system by SYBR Premix Ex Taq (Takara). The final volume of TR-PCR reaction is 25 μL, containing 12.5 μL SYBR green master mix, 1 μL cDNA, 0.5 μL each forward and reverse primer, and 10.5 μL nuclease-free water. For information of primers see Table S1.4.8.The differentiated THP-1 cells were seeded and grown into full confluence in 6 well plates. 2% FBS DMEM/F12 media was used to inactivated cell proliferation for 12 h, then wounded with pipette tips. Fresh DMEM/F12 medium with or without 1, 3, 10, 25, and 50 μM of 7 was added to the scratched monolayers. Images were took after 24 hours using a Nickon inverted microscope . The migration cell number of PMA-induced THP-1 cell group was defined as control. All the experiments were performed in triplicate.4.9.t-test. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001 are considered significant.Data are presented as mean ± SD. Statistical analysis of data was performed with Student's CD147Extracellular matrix metalloproteinase inducer, EMMPRINMMP-2Matrix metalloproteinase 2MMP-9Matrix metalloproteinase 9PMAPhorbol-12-myristate-13-acetateq-PCRQuantitative real-time PCR methodCVDsCardiovascular diseasesAMIAcute myocardial infarctionUAUnstable angina pectorisCTs9,19-Cycloartane triterpenesYGYunnanterpeneHC12β-HydroxycimiacerolNPsNatural productsNi-Hong Lu: investigation; methodology; writing-original draft. Jie Li and Yong-Rui Yang: formal analysis; writing-original draft. Hong-Lu Liu: data curation. Ying-Rong Du: supervision; writing – review & editing.The authors declare no competing financial interest.RA-011-D1RA07828C-s001


O-acetyl-2-fluoro-l-fucose (2F-Fuc) is a potent inhibitor of the plant cell elongation. After feeding plant seedlings with 2F-Fuc, this monosaccharide derivative is deacetylated and converted by the endogenous metabolic machinery into the corresponding nucleotide-sugar, which then efficiently inhibits Golgi-localized fucosyltransferases. Among plant cell wall polymers, defects in the fucosylation of the pectic rhamnogalacturonan-II cause a decrease in RG-II dimerization, which in turn induce the arrest of the cell elongation. In order to perform the inhibition of the cell elongation process in a spatio-temporal manner, we synthesized a caged 3,4-di-O-acetyl-1-hydroxy-2-fluoro-l-fucose (1-OH-2F-Fuc) derivative carrying a photolabile ortho-nitrobenzyl alcohol function at the anomeric position: 3,4-di-O-acetyl-1-ortho-nitrobenzyl-2-fluoro-l-fucose (2F-Fuc-NB). The photorelease of the trapped 1-OH-2F-Fuc was performed under a 365 nm LED illumination. We demonstrated that the in planta elimination by photoexcitation of the photolabile group releases free 2F-Fuc in plant cells, which in turn inhibits in a dose-dependent manner and, reversibly, the root cell elongation.Glycan metabolic engineering is a powerful tool for studying the glycosylation in living plant cells. The use of modified monosaccharides such as deoxy or fluorine-containing glycosides has been reported as a powerful pharmacological approach for studying the carbohydrate metabolism. 1,3,4-tri- Glycan metabolic engineering takes advantage of the cellular metabolism and associated enzymes to modify the structures of glycans in living organisms . One of O-acetyl-2-fluoro-l-fucose (2F-Fuc) was demonstrated to inhibit the fucosylation of cell wall polysaccharides and the protein N-glycosylation at micromolar concentrations in plant root seedlings. 2F-Fuc is able to inhibit all plant fucosyltransferases +: [C17H20FKNO8] calc m/z: 424.0810, found 424.0818.HMRS  δ: 8.12 , 7.92 , 7.68 , 7.46 , 5.29 , 5.21 , 5.12 , 5.01 , 4.70 , 4.63 , 3.87 , 2.18 , 2.06 , 1.24 . 13C-NMR  δ: 170.6, 170.3 (2 COCH3); 147.1 (C-ar), 134.2 (CH-ar); 128.7 (CH-ar); 128.4 (CH-ar); 125.0 (CH-ar); 100.4 ; 88.2 ; 71.6 ; 71.0 ; 69.6 (C-5); 66.8 (CH2-ph); 20.9, 20.7 (2 COCH3); 16.0 (C-6).19F (1H uncoupled)-NMR  δ: −207.08. α anomer: 1H-NMR  δ: 8.11 , 7.87 , 7.69 , 7.48 , 5.50 , 5.36 , 5.35 , 5.19 , 5.12 , 4.84 , 4.22 , 2.17 , 2.06 , 1.15 . 13C-NMR  δ: 170.6, 170.2 (2 COCH3); 147.1 (C-ar), 134.00 (CH-ar); 128.6 (CH-ar); 128.3 (CH-ar); 124.9 (CH-ar); 96.8 ; 85.6 ; 71.7 ; 68.8 ; 68.0 (CH2-ph); 65.3 (C-5); 20.8, 20.7 (2 COCH3); 15.8 (C-6). 19F (1H uncoupled)-NMR  δ: −208.63.β anomer: O-acetyl-1-hydroxy-2-fluoro-l-fucose (1-OH-2F-Fuc).Synthesis of 3,4-di-3 in methanol  was added at 0 °C to a solution of 1,3,4-tri-O-acetyl-2-fluoro-l-fucose  in dry THF . The reaction mixture was stirred at 0 °C under an argon atmosphere for 5 h. The reaction mixture was concentrated under vacuum. The residue was purified by flash chromatography over silica gel  to give a mixture of anomers  of the expected product  as a white solid. 1H, 19F and 13C NMR data of this compound were consistent with the literature data [NHure data  and also−): [M-H]−: [C10H14FO6] calc m/z: 249.0774, found 249.0775.HMRS  was added in an NMR tube, followed by the internal reference hexafluoroisopropanol (2 mM). This solution was analyzed by proton-uncoupled 19F and 1H NMR (t = 0), then irradiated at 365 nm and monitored over 10 min by 19F and 1H NMR analysis.A solution of 2F-Fuc-NB and/or 1-OH-2F-Fuc in a mixture of DArabidopsis thaliana Col-0 seeds were surface sterilized in 70% (v/v) ethanol for 5 min then in 30% (v/v) sodium hypochlorite for 2 min. Seeds were then washed 6 times in sterile water. Sterilized seeds were deposited on Murashige and Skoog (MS 2.2 g/L) mineral medium containing 0.8% plant agar. The plates were placed vertically at 4 °C for 48 h before being transferred in a growth chamber under a 16 h light/8 h dark cycle at 25 and 22 °C, respectively. Light equipment consisted of RGB LED light covering the visible spectra with the exception of UV light  0%, Blue (400–500 nm) 12%, Green (500–600 nm) 16%, Red (600–700 nm) 57% and Far-red (700–800 nm) 16% (manufacturer’s data)).For treatments with 2F-Fuc and 2F-Fuc-NB, molecules were dissolved in DMSO with a maximum DMSO concentration for the highest concentration of treatment (10 µM) corresponding to 0.25% of DMSO. Control conditions were performed in 0.25% DMSO. 2F-Fuc or 2F-Fuc-NB or DMSO (negative control) was added to the medium in a tube and homogenized by shaking before pouring the medium in the Petri dishes. Plants were grown for 7 days or gently transferred onto a treatment-free medium after 3 days and then placed for 4 more days in the growth chamber. Transfer of seedlings was achieved with plastic tweezers in order to avoid any damage.After 3 days of growth on 2F-Fuc-NB supplemented medium, the Petri dish was exposed at room temperature (20 °C) to a 365 nm LED light for 10 min. Alternatively, after 3 days of growth on 2F-Fuc-NB supplemented medium, seedlings were transferred in a Petri dish containing control (treatment-free) medium. The Petri dish was then exposed to a 365 nm LED light for 10 min. The photolysis was always performed in the same manner, i.e., the bottom of the Petri dish faced the 365 nm LED. The Petri dish was then placed back in the growth chamber.Seedlings were observed with an inverted microscope (Leica DMI 6000B), and images were acquired with the Leica DFC 450C camera. After 7 days of growth, Petri dishes containing seedlings were scanned with a high-resolution scanner (HP Expression 11000 XL), and roots were measured using Image J software . Sixty rp-value < 0.05, ** p-value < 0.01).Significant differences were determined by one-way ANOVA followed by Tukey’s HSD posthoc multiple comparison test. Data are marked by different letters when significantly different with respect to the control conditions (*


More importantly, all the active inhibitors are toxic to the breast cancer cell line and non-cytotoxic for the normal breast cell line. These compounds would be a suitable choice to investigate in the in vivo models to check their efficacy against these particular targets. These newly identified human carbonic anhydrase inhibitors have potential to be considered as therapeutic leads for the treatment of CA-related diseases, especially for breast and lung tumors and glaucoma. Furthermore, lead optimization and preclinical and clinical investigations of these compounds are necessary to develop potential drug entities for the treatment of cancer.Here, we report the eco-friendly synthesis and antitumor potential of the imidazole hybrids of pyrimidine. The results showed that all the compounds possess excellent inhibition of tumors, promoting enzymes hCA-IX and hCA-II. Furthermore, the selectivity index showed that compounds 1–11) that specifically target cytosolic carbonic anhydrase (CAs) isoenzymes, including human CA-II and human CA-IX . A highly eco-friendly aqueous approach was used for the formation of a carbon–carbon bond by reacting aromatic nitro group substitution of nitroimidazoles with carbon nucleophiles. The in vitro results indicate that this new class of compounds (1–11) includes significant inhibitors of hCA IX with IC50 values in the range of 9.6 ± 0.2–32.2 ± 1.0 µM, while hCA II showed IC50 values in range of 11.6 ± 0.2–31.1 ± 1.3 µM. Compound 2 (IC50 = 12.3 ± 0.1 µM) showed selective inhibition for hCA-II while 7, 8, and 10 (IC50 = 9.6–32.2 µM) were selective for hCA-IX. The mechanism of action was investigated through in vitro kinetics studies that revealed that compounds 7, 3, 11, 10, 4, and 9 for CA-IX and 1, 2, and 11 for CA-II are competitive inhibitors with dissociation constant (Ki) in the range of 7.32–17.02 µM. Furthermore, the in situ cytotoxicity of these compounds was investigated in the human breast cancer cell line MDA-MB-231 and compared with the normal human breast cell line, MCF-10A. Compound 5 showed excellent anticancer/cytotoxic activity in MDA-MB-231 with no toxicity to the normal breast cells. In addition, in silico molecular docking was employed to predict the binding mechanism of active compounds with their targets. This in silico observation aligned with our experimental results. Our findings signify that imidazole-based hybrids could be a useful choice to design anticancer agents for breast and lung tumors, or antiglaucoma compounds, by specific inhibition of carbonic anhydrases.Imidazole-based pyrimidine hybrids are considered a remarkable class of compounds in pharmaceutical chemistry. Here, we report the anticancer bioactivities of eleven imidazole-based pyrimidine hybrids ( Human carbonic anhydrases hCAs: EC 4.2.1.1) are zinc-containing enzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons. Zinc plays an essential part of the reaction by acting as a Lewis acid to ionize the water molecule. To date, fourteen diverse isoforms  of CAs have been identified with diverse molecular features (quaternary structure), organ and tissue distribution, subcellular localization, catalytic properties, and the ability to interact with different classes of inhibitors ,4..2.1.1 arMammalian CAs regulate various physiological functions including pH control, bicarbonate metabolism, and cellular pressure . The abnKnown α-CA inhibitors include various anions, phenol, hydroxyurea, carboxylates, organic phosphates, and phosphonates ,18,19. Tm/z. 1H-NMR and 13C-NMR spectra were recorded in DMSO-d6 by using Bruker 600 MHz and 150 MHz spectrophotometers, respectively. Splitting patterns are presented as follows; s, singlet; d, doublet; dd, double doublets; t, triplet; and m, multiplet. Chemical shifts are described in δ (ppm) and coupling constants are calculated in Hz. The progress in all the reactions was observed via TLC, which was performed on 2.0 × 5.0 cm aluminum sheets pre-coated with silica gel 60F254 to a thickness of 0.25 mm (Merck). The chromatograms were visualized by UV light (254–366 nm) or iodine vapors. The aforementioned compounds were successfully synthesized and characterized.All the reagents and compounds used in the synthesis were received directly from the suppliers and used without further purification. Methanol, pure ethanol, and other solvents were also acquired in sufficient purity from various sources and employed in the reaction medium without purification. All the targeted compounds were resynthesized in Anton Paar Microwave Synthesis Reactor. The melting points were taken on Stuart melting point apparatus SMP10. IR spectra (KBr) were recorded on a Bruker FT-IR IFS48 spectrophotometer. EI mass spectra were attained using HR-ESIMS: Agilent Technologies, 6530. Exact mass and data are presented as A G10 vial of the Anton-Paar microwave synthesizer containing 6 mL of distilled water was charged with 1 mmol of active methylene(s) and 1 mmol of corresponding nitroimidazole(s). The reactions were irradiated under 90 °C for 5–10 min. A colored precipitate appeared as the resulting product. The resulting crude solids were filtered and washed with water, followed by methanol. Some of the compounds were purified through column chromatography using DCM: MeOH (9.5:0.5 to 3:1) solvents system over silica. After washing and purification, the solid materials were dried in a vacuum drying oven . The str3-hydroxy-2-(1-(2-hydroxyethyl)-2-methyl-1H-imidazol-5-yl) cyclohex-2-en-1-one (1)−1): V(C=O) = 1684, V(C=C) = 1600, V(C=N) = 1515, V(C-O) = 1276; 1H NMR : δ 14.89 , 7.56 , 4.95 , 4.01 , 3.64 , 2.48 , 2.28 , 1.82 ; HRMS (ESI+): calculated for C12H16N2O3 (M + H+) 237.27, found 237.12.Yield: 35% (70 mg) as light brown solid; purification: chromatography: DCM: MeOH (9.5:0.5 to 3:1) over silica; m.p = 186–190 °C; FTIR (cm2-(5-(2-hydroxy-6-oxocyclohex-1-en-1-yl)-2-methyl-1H-imidazol-1-yl) ethyl benzoate (2)−1): V(C=O) = 1713, V(C=C) = 1588, V(C-O) = 1266; 1H NMR : δ 7.89 , 7.63 , 7.50 , 6.60 , 4.33 , 4.11 , 2.44 , 2.15 , 1.76 ; HRMS (ESI+): calculated for C19H20N2O4 (M + H+) 341.38, found 341.14.Yield: 24% (65 mg) as greenish-yellow solid; purification: filtration, washed with water and methanol; m.p. = 150–154 °C; FTIR (cm6-hydroxy-5-(2-methyl-1H-imidazol-5-yl) pyrimidine-2,4-dione (3)−1): V(C=O) = 1687, V(C=O) = 1637, V(C=C) = 1580, V(C-O) = 1244; HRMS (ESI+): calculated for C8H8N4O3 (M + H+) 209.18, found 209.04; 1H NMR : δ 13.15 (s O121H), 9.66 , 7.33 , 2.52 .Yield: total 78% (161 mg) as pink solid; purification: filtration, washed with water and methanol; m.p. = 215 °C decomposed; FTIR (cm6-hydroxy-5-(2-methyl-1H-imidazol-5-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (4)−1): V(C=O) = 1644, V(C=C) = 1590, V(C=N) = 1522, V(C-O) = 1272; 1H NMR : δ 13.33 , 13.09 , 11.09 , 7.43 , 2.53 ; HRMS (ESI+) calculated for C8H8N4O2S (M + H+) 225.25, found 225.02.Yield: total 71% (166 mg) as grey solid; purification: filtration, washed with water and methanol; m.p. = not determined (above 300 °C); FTIR -2-methyl-1H-imidazol-1-yl) ethyl benzoate (5)−1): V(C=O) = 1714, V(C=C) = 1566, V(C=N) = 1515, V(C-O) = 1269; 1H NMR : δ 13.64 , 9.44 , 7.89 , 7.65 , 7.52 , 7.15 , 4.45 , 4.42 , 2.64 ; HRMS (ESI+): calculated for C17H16N4O5 (M + H+) 357.34, found 357.11.Yield: 69% (249 mg) as white powder; purification: DCM: MeOH (9.5:0.5 to 3:1) over silica; m.p. = 275–280 °C; FTIR pyrimidine-2,4-dione (6)−1): V(C=O) = 1633, V(C=C) = 1592, V(C=N) = 1555, V(C-O) = 1253; 1H NMR : δ 13.23 , 7.44 , 3.31 , 2.53 ; HRMS (ESI+): calculated for C10H12N4O3 (M + H+) 237.24, found 237.04.Yield: total 75% (185 mg) pink solid product; purification: DCM: MeOH (9.5:0.5 to 3:1) over silica; m.p. = not determined (above 300 °C); FTIR -2-methyl-1H-imidazol-1-yl) ethyl benzoate (7)−1): V(C=O) = 1706, V(C=O) 1665 = V(C=C) = 1635, V(C=N) = 1526, V(C-O) = 1275; 1H NMR : δ 13.76 , 10.85 , 7.89 , 7.64 , 7.53 , 7.20 , 4.44 , 4.43 , 2.64 . HRMS (ESI+): calculated for C17H16N4O4S (M + H+) 373.41, found 373.09.Yield: 69% (240 mg) dark brown product; purification: filtration, washed with water and methanol; m.p = 225–230 °C; FTIR (cm6-hydroxy-5-(1-(2-hydroxyethyl)-2-methyl-1H-imidazol-5-yl) pyrimidine-2,4 -dione (8)−1): V(C=O) = 1684, V(C=C) = 1626, V(C=N) = 1547, V(C-O) = 1277; 1H NMR : δ 9.37 , 7.04 , 5.05 , 3.95 , 3.55 , 2.56 ; HRMS (ESI+): calculated for C10H12N4O4 (M + H+) 253.23, found 253.09.Yield: 78% (176 mg) as pale yellow powder; purification: filtration, washed with water and methanol; m.p = not determined (above 300 °C); FTIR (cm6-hydroxy-5-(1-(2-hydroxyethyl)-2-methyl-1H-imidazol-5-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (9)−1): V(C=O) = 1645, V(C=C) = 1584, V(C=N) = 1520, V(C-O) = 1278; 1H NMR : δ 13.07 , 11.10 , 7.54 , 5.07 , 4.09 , 3.67 , 2.58 ; HRMS (ESI+): calculated for C10H12N4O3S (M + H+) 269.29, found 269.09.Yield: 76% (193 mg) as pale yellow powder; purification: filtration, washed with water and methanol; m.p. = not determined (above 300 °C); FTIR (cm6-hydroxy-5-(1-(2-hydroxyethyl)-2-methyl-1H-imidazol-5-yl)-1,3-dimethylpyrimidine-2,4-dione (10)−1): V(C=O) = 1707, V(C=C) = 1626, V(C=N) = 1562, V(C-O) = 1261; 1H NMR : δ 13.48 , 7.11 , 4.98 , 3.93 , 3.51 , 3.07 , 2.60 ; HRMS (ESI+): calculated for C12H16N4O4 (M + H+) 281.28, found 281.10.Yield: 70% (185 mg) as light pink crystalline solid; purification: filtration, washed with water and methanol; m.p. = 163–165 °C; FTIR -2-methyl-1H-imidazol-1-yl) ethyl benzoate (11)−1): V(C=O) = 1724, V(C=O) = 1679, V(C=C) = 1575, V(C-O) = 1258; 1H NMR : δ 13.66 , 7.86 , 7.64 , 7.49 , 7.16 , 4.42 , 4.41 , 3.03 , 2.65 ; HRMS (ESI+): calculated for C19H20N4O5 (M + H+) 385.39, found 385.15.Yield: 78% (273 mg) as light pink crystalline solid; filtration, washed with water and methanol; m.p. = 180–185 °C; FTIR . All kinetic graphs were plotted by GraFit program . Different parameters and their values, such as the values of intercepts, slopes, correlation coefficients, and their standard error means (SEMs), were calculated by the nonlinear regression analysis using the same program (GraFit program).In vitro inhibition of human CA  was performed in 96-well plate. Each well of the 96-well plate comprised 20 µL of the test compound (0.5 mM), 20 µL of the purified enzyme (0.1 mg/mL), 140 µL of the assay buffer , and 20 µL of the substrate , making a final volume of 200 µL. The assay procedure started with the incubation of the test compound, desired enzyme, and 140 µL of buffer for 15 min. Soon after the incubation, the substrate was added to each well and the plate was placed in the microplate readers  for 30 min at 25 °C, and the absorbance was recorded at one-minute intervals . All the1–11) was determined by MTT -2, 5-diphenyl tetrazolium bromide) assay using an aggressive breast cancer cell line, MDA-MB-231. This cell line is ER, PR, and E-cadherin negative, and it lacks the growth factor receptor HER2. It represents a good model of triple-negative breast cancer and is commonly used for in vitro and in vivo studies  assay was used to determine the decrease in cancer cell viability induced by the synthesized hybrids. For MDA-MB-231, the IC50 values, percent inhibition, and viability of compounds (1–11) are given in 5 showed more inhibitory potency in the MDA-MB-231 cell line with an IC50 value of 53.65 μM, followed by compounds 8 (55.65 μM), 9 (58.623 μM), 10 (60.93 μM), 2 (62.848 μM), 11 (63.096 μM), 7 (64.009 μM), 4 (75.65 μM), 3 (76.07 μM), 6 (79.43 μM), and 1 (96.01 μM). To determine whether the cytotoxic effects of the compounds were selective for malignant cells as compared to non-malignant cells, the non-tumorigenic (MCF-10A) cells were exposed to the compounds at varying concentrations  in a similar manner as the cancer cells. The results  of imidazole–pyrimidine hybrids ( results  depict t3, 4, 7, and 9–11 were identified as excellent inhibitors of hCA-IX, while compounds 1, 2, and 11 showed good inhibitory activity for hCA-II. The selectivity index showed that compounds 7, 8, and 10 are selective inhibitors of hCA-IX, while compound 2 is a selective inhibitor of hCA-II. More importantly, all the active inhibitors are non-cytotoxic for the normal breast cell line, while compound 5 is more toxic to the breast cancer cell line. These newly identified human carbonic anhydrase inhibitors have potential to be considered as therapeutic leads for the treatment of CA-related diseases such as cancer and glaucoma upon further optimization.Green chemistry is an eco-friendly approach for the synthesis of new hybrids for different beneficial purposes. We report here, for the first time, the synthesis of the imidazole hybrids of pyrimidine through a green chemistry approach. Our synthesized hybrids were then evaluated against human carbonic anhydrase isozymes. In the in vitro analysis, compounds


Monopterus albus) with six isonitrogenous and isoenergetic experimental diets of basic feed supplemented with different levels of methionine . This study built upon previous research findings that showed dietary methionine restriction  inhibited hepatic fatty acid metabolism and intestinal fatty acid transportation, but both are improved by dietary supplementation with a suitable level of methionine . Hence, M0 and M8 were selected to investigate how methionine regulates the gut microbiota and lipidomics of M. albus. Compared with M0, values for gut bacterial Sobs, Shannon, ACE, and Chao1 indices of M8 were remarkably increased (p < 0.05), with Fusobacteria, Firmicutes, and Proteobacteria the dominant phyla and Cetobacterium, Plesiomonas, and Bacillus the main genera in the community under the M0 vs. M8 treatments. However, compared with M0, the proportion of phyla consisting of Fusobacteria decreased in M8, as did the Cetobacterium and Lactococcus at the genus level; conversely, the proportions corresponding to Firmicutes, Proteobacteria, and Chioroflexi phyla increased in M8, as did the Clostridium and Streptococcus genera. Many edges appeared in the circus and networks, demonstrating the interspecies interactions among different operational taxonomic units (OTUs). In addition, various OTUs within the same phylum were clustered within one module. Cooperative interactions were predominant in the two networks, while competitive interactions were prevalent in their submodules. Gut microbiota mainly played roles in nutrition  transport and metabolism under the M0 vs. M8 treatments. The PLS-DA scores indicated a significant difference in the main lipidomic components between the M0 and M8 treatment groups. Namely, the TG(26:0/16:0/17:0), TG(28:0/16:0/16:0), TG(26:0/16:0/16:0), and TG(30:0/16:0/16:0)—among others—comprising the gut content were reduced under the M8 treatment (p < 0.001). The genus Clostridium was positively correlated with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(18:0/18:1/20:3), and other compounds, yet negatively correlated with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), and TG(16:0/16:0/24:0), among others as well. According to the lipidomics analysis, the predicted KEGG pathways mainly included lipid and glycan biosynthesis and metabolism, and digestive, sensory, and immune systems. In conclusion, methionine restriction disturbed the microbial community balance and induced microbial dysfunctions, whereas methionine supplementation improved the homeostasis of gut microbiota and lipid metabolism of the rice eel.An 8-week feeding trial was conducted using the rice field eel ( The gut cavity is home to a diverse and abundant microbiome, which has a pivotal role in maintaining the host’s physiological homeostasis . Gut micvia multiple direct and indirect biological mechanisms (Barchydanio rerio var.), finding that its gut microbiota could stimulate the uptake of fatty acids and formation of lipid droplets by the intestinal epithelium and liver, with the sclerenchyma enhancing absorption capacity of fatty acids by host’s intestinal cells, thereby promoting an increase in the number of lipid droplets. Meanwhile, the size of these lipid droplets increased as bacterial abundance increased, which the authors demonstrated was primarily regulated by golden rod bacteria (Chrysobacterium hispanicum) and Pseudomonas sp. (P. adaceae). Later, Gut microbiota can affect their host’s lipid metabolism chanisms ; hence, chanisms . Semova M. albus) is subtropical freshwater benthic fish with considerable economic value, which is why it is widely raised in central and south China  to use lipidomics to reveal how gut microbiota affect the host’s lipid metabolism and absorption. This study demonstrates the alteration of the microbiome and lipid-related products in response to methionine restriction, and thus may provide a new perspective on the theory of lipid metabolism.Here, following our previous study , soy proA basic diet , as prepared in previous studies , was supThe proximate analysis  of the experimental feed treatments was determined based on our previous paper . Amino aM. albus were purchased from Changde, China. Similar-sized M. albus (25.08 ± 0.31 g) were reared in 18 float cages (2.0 m × 1.5 m × 1.5 m), three per dietary treatment (triplicates), with 60 fish per cage. More details about the experiment can be found in our recent paper  {No. 094}. All experimental fish were anesthetized with eugenol  to reduce their suffering.M. albus. After the 8-week feeding trial, from each cage the gut contents of five fish were sampled, and these five samples under each experimental group were used for the sequencing analysis.Our previous research showed that a methionine-deficient diet  inhibited hepatic fatty acid metabolism  and inte™ DNA Isolation Kit . High-throughput sequencing was performed on the Illumina MiSeq platform, in which all sequences were classified into operational taxonomic units (OTUs) at a minimum 97% similarity by the QIIME  software pipeline, after first removing any low-quality scores  with the FASTX-Toolkit . For full details about the bioinformatics analysis and molecular ecological network construction, please refer to our previous study . Finally, the qualitative and quantitative results of each sample were obtained via its mass spectrum data analysis. LC–MS platform used was the UHPLC-Q Exactive system , with differences considered significant at p < 0.05.Our data were analyzed on an online cloud platform of Majorbio .p < 0.01, p < 0.01, p < 0.05, p < 0.05, respectively; Both the Coverage and Simpson were similar between the M0 (0g/kg) and M8 (0g/kg) treatment groups. Compared with M0, gut bacterial Sobs, Shannon, ACE, and Chao1 for M8 were significantly increased ; while TG(30:1/18:1/18:1) and PE(11:0/20:2) of gut content in M8 (Met5) were remarkably decreased (p < 0.05), TG(6:0/12:4/ 20:2), TG(8:0/11:3/23:1), TG(8:0/10:1/22:0), TG(16:1e/11:4/ 12:2), TG(6:0/12:0/20:4), TG(6:0/6:0/24:1), DG(16:1e/18:3), DG(18:3e/20:5), WE(3:0/19:1), WE(3:0/16:1), WE(3:0/18:2), Cer(d19:2/20:0 + O), WE(3:0/24:2), TG(30:0/18:0/18:1), TG(26:0/16:0/17:0), TG(28:0/16:0/16:0), TG(26:0/16:0/16:0), TG(20:0e/18:0/20:0), TG(18:0e/18:0/19:0), TG(30:0/16:0/16:0), TG(28:0/16:0/17:0), TG(30:0/16:0/18:0), TG(30:0/16:0/17:0), TG(30:0/18:0/22:0) and TG(30:0/16:0/20:0) of gut content in M8 (Met5) were remarkably decreased , BisMePG(17:1/22:6) and PE(10:0/11:3) of gut content in M8 were remarkably increased (Acetobacterium had significant positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(16:0/16:1/17:1), TG(15:0/14:0/16:1), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), and TG, but significant negative correlations with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:0/18:0/18:1), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), TG(18:0/18:0/18:3), and TG. While g__Rhodoblastus was positively correlated with TG, it was negatively correlated with TG. The g__Rhodoplanes had positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/ 18:1), TG(16:0/16:1/17:1), TG(15:0/14:0/16:1), TG(19:1/18:1/18:2), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), TG(15:0/14:0/18:2), and TG, but negative correlations with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:0/18:0/18:1), TG (30:1/16:0/18:0), TG(29:0/18:0/18:1), TG(18:0/18:0/18:3), and TG. For g__Rhodopseudomonas, it was positively correlated with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(16:0/16:1/17:1), TG(15:0/14:0/16:1), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), and TG(15:0/14:0/18:2), TG though negatively correlated with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:0/18:0/18:1), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), TG(18:0/18:0/18:3), and TG. Significant negative correlation with TG(15:0/16:1/18:3), TG(16:1/13:0/14:0), TG(16:0/17:0/18:1), and TG (18:0/18:1/20:3) were found for g__norank_f__Barnesiellaceae (p < 0.05). By contrast, g__norank_f__Caldilineaceae had positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), DG(16:1/18:1), TG(16:0/16:1/17:1), TG(16:0/16:0/17:1), TG(15:0/14:0/16:1), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), and TG, for which negative correlations with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:0/18:0/18:1), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), TG(18:0/18:0/18:3), TG(19:0/18:1/18:2) were also present (p < 0.05). The g__norank_f__Rhizobiales_Incertae_Sedis had a positive correlation with TG, as did g__norank_f__norank_o__Chloroplast with TG(18:1/18:1/ 22:5), TG(16:0/17:0/18:1), TG (18:0/18:1/20:3), and TG along with a negative correlation with TG(18:0/ 18:0/18:3) and TG. For g__norank_f__norank_o__PeM15, it had positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG (18:0/18:1/20:3), and TG but negative correlations with TG(16:0/17:0/24:0), TG(30:1/16:0/18:0), TG(18:0/18:0/18:3), and TG. The g__norank_f__norank_o__RBG-13-54-9 was positively correlated with both TG(18:1/18:1/ 22:5) and TG and negatively correlated with TG. Positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(16:0/16:1/17:1), TG(16:1/14:0/17:1), and TG, as well as negative correlation with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), TG(18:0/18:0/18:3), and TG(19:0/18:1/18:2) were found for g__norank_f__norank_o__ Saccharimonadales (p < 0.05). With respect to g__unclassified_c__Gammaproteobacteria, it had positive and negative correlation, respectively, with TG(18:1/18:1/22:5) and TG. Both g__unclassified_f__Chloroflexaceae and g__unclassified_f__Chromatiaceae had positive correlations with TG, while the latter was negatively correlated with TG. The g__unclassified_f__ Rhodobacteraceae was positively correlated with TG(18:1/18:1/22:5) and TG while negatively correlated with TG. For g__ unclassified_f__Xanthobacteraceae, it had a positive correlation with the following: TG(18:1/18:1/22:5), DG(16:1/18:1), TG(16:0/16:1/17:1), TG(16:0/16:0/17:1), TG(15:0/14:0/16:1), TG(16:0/14:0/14:0), and TG(16:1/14:0/17:1), TG. However, it had a negative one with these: TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), and TG(18:0/18:0/18:3), and TG. The g__unclassified_k__norank_d__Bacteria was negatively correlated with TG(15:0/16:1/18:3) and TG, as was g__unclassified_o__ Bacteroidales with TG(15:0/16:1/18:3), TG(16:1/13:0/14:0), TG(16:0/ 17:0/18:1), and TG . For g__unclassified_o__Micrococcales, it had positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(16:0/16:1/17:1), TG(15:0/14:0/16:1), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), and TG in addition to negative correlation with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), and TG. The g__unclassified_o__Rhizobiales was positively correlated with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(16:0/ 16:1/17:1), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), and TG, but negatively correlated with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:0/18:0/18:1), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), TG(18:0/18:0/18:3), and TG. A positive correlation between g__Chloronema and TG(18:1/18:1/22:5) was found (p < 0.05). The g__Christensenellaceae_R-7_group had positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(16:0/16:1/17:1), and TG and negative ones with TG(16:0/17:0/24:0), TG(18:0/18:0/18:3), and TG. Many correlations were significant for g__Clostridium_sensu_stricto_1, these being positive vis-à-vis DG(18:1/22:5), TG(15:0/20:5/22:1), TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG (18:0/18:1/20:3), DG(16:1/18:1), DG(18:1/14:0), DG(18:3e/16:1), TG(19:1/16:0/18:0), TG(18:0/17:1/20:0), TG(16:0/16:1/17:1), DG(15:0/18:1), TG(16:0/16:0/17:1), TG(18:0/16:0/17:1), TG(15:0/14:0/16:1), TG(19:1/18:1/18:2), TG(16:1/14:0/17:1), and TG; and negative vis-à-vis TG(18:0/ 17:0/20:0), TG(16:0/17:0/24:0), TG(30:0/18:0/18:1), TG(30:1/16:0/ 18:0), TG(29:0/18:0/18:1), TG(16:0/16:0/24:0), TG(26:0/16:0/17:0), TG(26:0/16:0/16:0), TG(28:0/16:0/16:0), TG(30:0/16:0/16:0), TG(28:0/16:0/17:0), TG(18:0/18:0/18:3), TG(19:0/18:1/18:2), WE (3:0/22:2), and WE. By contrast, g__Epulopiscium only had a negative correlation with TG. For g__Leptolyngbya_ANT.L52.2, there were positive correlations with TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), DG(16:1/18:1), TG(16:0/16:1/17:1), TG(16:0/16:0/17:1), TG(15:0/14:0/16:1), TG(16:0/14:0/14:0), TG(16:1/14:0/17:1), and TG and negative correlations with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(30:1/16:0/18:0), TG(29:0/18:0/18:1), TG (18:0/18:0/18:3), TG. Finally, g__ Macellibacteroides was negatively correlated with TG(18:1/18:1/ 22:5), TG(16:0/17:0/18:1), DG(16:1/18:1), and DG was chosen as the main reference lipid. We found that TG(20:0e/14:0/16:0), TG(16:0/17:0/22:0), WE(3:0/18:1), WE(3:0/18:2), TG(18:0/18:0/18:3), TG(19:0/18:1/ 18:2), TG(30:0/18:0/18:1), TG(30:1/16:0/18:0), TG(29:0/18:0/ 18:1), WE(3:0/24:2), TG(16:0/16:0/24:0), TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(26:0/16:0/17:0), TG(30:0/16:0/16:0), TG(28:0/16:0/17:0), WE(3:0/22:2), TG(26:0/16:0/16:0), TG(28:0/ 16:0/16:0), Hex1Cer(d18:1/18:0 + 2O), TG(16:0/6:0/11:3), and TG(16:0/6:0/9:0), TG(11:0/10:1/14:3) all had significant positive correlations with TG(18:1/18:1/22:5). Conversely, it was negatively correlated with TG(16:0/16:0/16:0), DG(18:1/22:5), DG (18:1/20:3), TG(15:0/20:5/22:1), TG(16:0/17:0/18:1), TG(18:0/ 18:1/20:3), TG(15:0/16:1/18:3), TG(16:1/13:0/14:0), TG(15:0/ 16:0/16:1), DG(15:0/18:1), DG(18:3e/16:1), DG(18:1/14:0), DG (16:1/18:1), TG(19:1/16:0/18:0), TG(16:0/16:1/17:1), TG(16:0/ 16:0/17:1), TG(18:0/16:0/17:1), TG(16:1/14:0/17:1), TG(15:0/ 14:0/18:2), TG(18:0/17:1/20:0), TG(19:1/18:1/18:2), TG(15:0/ 14:0/16:1), TG(16:0/14:0/16:0), TG(15:0/16:0/16:0), TG(15:0/ 16:0/18:3), and TG and M8 (8 g/kg) treatment groups to characterize community succession of gut microbiota and how it regulates lipid mechanisms.Our previous studies indicated that a methionine-deficient diet not only impairs muscle fiber growth and the development and differentiation of formance  and induM. albus . To bettM. albus. Our results are similar to another study done on mice, where a suitable methionine supplementation improved the intestinal barrier functioning, inflammatory response, and lipid metabolism by regulating their gut microbiota .Here, compared with M0, the gut bacterial Sobs, Shannon, ACE, and Chao1 in the M8 group were significantly increased. This phenomenon indicated that restricting methionine intake lowered the diversity of gut microbiota and rendered the community more alike; hence, a suitable dietary level of methionine could promote the balance of gut microbiota of crobiota . In the M. albus . Nonethebacteria  whose prbacteria , corrobobacteria . Cetobacacterium , being tacterium , 2020, frmicutes . This phM. albus. Evidently, the dominant phyla in M0 were Firmicutes, Proteobacteria, Actinobacteria, Chloroflexi, and Cyanobacteria; the dominant phyla in M8 were Firmicutes, Proteobacteria, Actinobacteria, Chloroflexi, and Cyanobacteria. This pattern of dominance was also similar between the networks of the two dietary methionine treatment groups. Our study indicates that methionine restriction induced gut microbiota dysbiosis, such that the predicted ecological network within microbial community was starkly affected by methionine-deficient diet, and interact among different gut microbial community , TG(18:1/18:1/22:5), TG(16:0/17:0/18:1), TG(18:0/18:1/20:3), etc., and negative correlations with TG(18:0/17:0/20:0), TG(16:0/17:0/24:0), TG(16:0/16:0/24:0), etc. In addition, enriched KEGG pathways for metabolism mainly included those for lipid and glycan biosynthesis and metabolism, and those for digestive, sensory, and immune systems. Therefore, methionine restriction may affect lipid metabolism by mediating the succession of the gut microbiota community. Methionine restriction also inhibited the hepatic lipid deposition of M. albus, and chiefly downregulated hepatic fatty acid synthesis, especially for unsaturated fatty acids , the latter chosen from among the main lipids to investigate correlations with gut bacterial taxa and other lipids. Many kinds of saturated fatty acids, such as TG(16:0/17:0/22:0), TG(16:0/16:0/24:0), and TG(18:0/17:0/20:0), have significant positive correlations with TG(18:1/18:1/22:5), whereas most unsaturated fatty acids, such as  have significant negative correlations with TG(18:1/18:1/22:5). The mechanisms linking fatty acids metabolism and succession of gut microbiota community are highly complex and deserve further systematic study.Plotting the PCA and PLS-DA scores lets one visually display the classification of samples, such that the greater separation degree of the two groups, the more significant are their differences . Here, tM. albus . We infeM. albus, which reduced the average connectivity and number of connectors while also decreasing competitive interactions within the ecological network; however, the composition of gut microbiota went unchanged. In stark contrast, methionine supplementation clearly improved the homeostasis of gut microbiota and lipid metabolism.In conclusion, a methionine restriction diet disturbed the balance of gut microbiota by deteriorating the submodules of https://github.com/mineraltsai/manuscript_FM.The original contributions presented in the study are publicly available. This data can be found at: Our study was approved by Committee of Laboratory Animal Management and Animal Welfare of Hunan Agricultural University  No. 094.YaH was in charge of methodology, data curation, and writing—original draft. MC was in charge of data curation and software. WC was in charge of formal analysis and writing—review and editing. YiH was in charge of funding acquisition, writing—review and editing, and validation. All authors contributed to the article and approved the submitted version.This study was financially supported by National Natural Science Foundation of China (grant no. 32172986).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Quercetin is one of the most powerful bioactive dietary flavonoids. The in vivo biological study of quercetin is extremely difficult due to its very low solubility. However, diorganotin complexes of quercetin are more useful when contrasted with quercetin due to increased solubility. In the present study, quercetin, substituted biguanide synthesized in the form of Schiff base and its di-alkyl/aryl tin (IV) complexes were obtained by condensing Schiff base with respective di-alkyl/aryl tin (IV) dichloride. Advanced analytical techniques were used for structural elucidation. The results of biological screening against Gram-positive/Gram-negative bacteria and fungi showed that these diorganotin (IV) derivatives act as potent antimicrobial agents. The in silico investigation with dihydropteroate (DHPS) disclosed a large ligand–receptor interaction and revealed a strong relationship between the natural exercises and computational molecular docking results. QuerceAlthough quercetin has a powerful pharmacological potential, its low solubility makes it difficult to act as biocidal agent ,16. ConsOn the other hand, organotin complexes are the most diverse segment of chemical compounds with applications ranging from material science and catalytic activities to therapeutic agents for various diseases due to their wide variety of interesting structural possibilities . OrganotThe chemical structures of various reported organometallic complexes of flavonoids support the magnificent radical scavenging ability. Valentina Uivarosi et al. found that the metal-flavonoid complexes have enhanced the physiochemical and biological properties of flavonoids . In anot2L) with the Sn atom was verified by evaluating the IR spectra of ligand and complexes (1–4) (−1 confirming v(O-H) stretching vibration of coordinated water molecules and -OH groups of the ligand. The absorption band obtained in Schiff base ligand (H2L) at 1657 cm−1 was found to be absorbed at lower values by 20–30 cm−1 and observed at 1627–1637 cm−1 in the spectra of diorganotin (IV) complexes , [Et2SnL], [Bu2SnL], and [Ph2SnL] through displaying peaks at m/z = 819.05, 845.49, 901.49, and 941.46.LCMS technique has been utilized for the determination of mass, and thus the molecular weight of the compounds. The spectra of the ligand contained a peak at m/z = 669.57, which has been attributed to [M + H] ion accompanied by the nitrogen rule and, currently, in full settlement with the proposed molecular mass. Mass spectrometry of the complexes showed the formation of  calculated for [C36H31N5O12Sn]: 845.10; found: 845.49; Anal calc. for [C36H31N5O12Sn]; C 51.21; H 3.70; N 8.29; found: C 51.73; H 4.30; N 8.59: FT-IR data ; 3382 v(OH)phenolic; 1605 v(C=N) aromatic; 1622 v(C=N)aliphatic; 1297 v(C-O-C); 428 v(Sn-C); 520 v(Sn-O); 467 v(Sn-N) 1 HNMR : 12.21(5-OH), 8.21(7-OH), 7.4(2’-H), 7.1(6’-H), 6.6(5’-H), 6.2(8-H), 6.2(6-H), 2.6(NH), O.88-0.90(Et-H): 13 CNMR : 162.5(C=N), 160.4(C-7), 157.2(C-5), 149.2(C-3’), 157.2(C-4’), 149.2(C-9), 145.8(C-2), 133.9(C-3), 133.1(C-1’), 121.8(C-5’), 119.8(C-2’), 115.6(C-6’), 100.6(C-6), 99.4(C-10), 94.7(C-8), 7.5(Me-C); 119 Sn NMR : −190.68. UV-Vis; λ max (DMSO 10-3), 414 and 262 nm.Yield: 76%, light orange solid; mp: 178–181 °C; m/z [M+] calculated for [C40H39N5O12Sn]: 901.16; found: 901.49; Anal calc. for [C40H39N5O12Sn]; C 53.35; H 4.37; N 7.78; found: C 54.73; H 4.48; N 7.59: FT-IR data ; 3384 v(OH)phenolic; 1607 v(C=N) aromatic; 1628 v(C=N)aliphatic; 1299 v(C-O-C); 482 v(Sn-C); 516 v(Sn-O); 460 v(Sn-N) 1 HNMR : 12.20(5-OH), 8.20(7-OH), 7.3(2’-H), 7.3(6’-H), 6.8(5’-H), 6.8(8-H), 6.6(6-H), 2.5(NH), O.9-2(Bu-H): 13 CNMR : 162.5(C=N), 160.4(C-7), 157.4(C-5), 149.0(C-3’), 157.4(C-4’), 149.0(C-9), 146.1(C-2), 134.1(C-3), 133.9(C-1’), 121.7(C-5’), 119.9(C-2’), 115.8(C-6’), 100.7(C-6), 99.9(C-10), 94.5(C-8), 33(Bu-C); 119 Sn NMR : −213.47. UV-Vis; λ max (DMSO 10-3), 420 and 262 nm.Yield: 64%, dark orange solid; mp: 168–171 °C; m/z [M+] calculated for [C44H31N5O12Sn]: 941.10; found: 941.46; Anal calc. for [C44H31N5O12Sn]; C 56.19; H 3.32; N 7.45; found: C 56.73; H 3.68; N 7.65: FT-IR data ; 3394 v(OH)phenolic; 1612 v(C=N) aromatic; 1624 v(C=N)aliphatic; 1299 v(C-O-C); 463 v(Sn-C); 519 v(Sn-O); 473 v(Sn-N); 1 HNMR : 12.19(5-OH), 8.20(7-OH), 7.2(2’-H), 7.2(6’-H), 6.7(5’-H), 6.9(8-H), 6.8(6-H), 2.5(NH), 7.29–7.36(Ph-H): 13 CNMR : 163.5(C=N), 161.4(C-7), 157.8(C-5), 149.7(C-3’), 157.4(C-4’), 149.7(C-9), 146.2(C-2), 132.2(C-3), 133.7(C-1’), 121.9(C-5’), 119.6(C-2’), 115.6(C-6’), 100.5(C-6), 99.4(C-10), 94.9(C-8), 147.9–124.1(Ph-C); 119 Sn NMR : −314. UV-Vis; λ max (DMSO 10-3), 430 and 262 nm.Yield: 69%, orange solid; mp: 176–180 °C; m/z [M+] calculated for [CStaphylococcus aureus (MTCC 9760), Bacillus subtillis (MTCC 1133), and Gram-negative strains, i.e., Escherichia coli (MTCC 589), Pseudomonas aeruginosa (MTCC 9048). The antifungal activity was carried out against fungal species, such as Scopulariopsis canadensis (MTCC 567) and Aspergillus niger (MTCC 9933). Ciprofloxacin and fluconazole were used as standard antimicrobials to compare the activities of tested compounds while 2% DMSO was used as a blank. The solutions were prepared by dissolving 5 mg of each compound in 5 mL of 2% DMSO as solvent. Then, 1 mL of the prepared solution was diluted with 9 mL of DMSO for the concentration of the solution to reach a value of up to 100 ppm [The in vitro antimicrobial screening of the newly designed ligand and its corresponding diorganotin (IV) derivatives were evaluated against different strains of microorganisms by determining the minimum inhibitory concentration (MIC). This activity has been evaluated using serial dilution and agar well diffusion technique . The ant 100 ppm ,70.E. coli 24 kDa DNA gyrase domain (PDB ID: 1KZN), and UDP-N-acetylglucosamine pyrophosphorylase (6TN3) were obtained from the protein data bank and prepared for molecular docking using AutoDock Tool 1.5.6 [E. coli 24 kDa DNA gyrase domain, the binding coordinates were assigned as center_x = 18.84; center_y = 24.05; center_z = 36.31. For UDP-N-acetylglucosamine pyrophosphorylase, the binding coordinates were assigned as center_x = 22.81; center_y = 60.59; center_z = −4.47. The search space was set up at 30 × 30 × 30 Å for all docking studies. Twenty possible binding conformations for each ligand were generated by AutoDock Vina suite software using genetic algorithm (GA-LS) searches [The crystal structures of dihydropteroate synthase (PDB ID: 1AD4), ol 1.5.6 . Chain Asearches ,73. Selesearches .The back bone of the chemical structure of complexes is the presence of bi-flavonoid moiety, i.e., the presence of four benzene rings with two pyranone rings. The eight flavonoid hydroxyl groups present at 11, 12, 16, and 18th and 32, 33, 37, and 39th positions of each complex are responsible for anti-oxidative properties. Four >C=N bonds present at 6, 27, 43, and 46th positions provide excellent biocidal properties, whereas the double bonds present between the 4th and 5th positions and 25th and 26th positions of bi-flavonoid moiety are responsible for effective inhibitory activities . Further4 was the most effective agent as an antimicrobial along with the substantial interactions with DHPS (1AD4) receptor protein.The current research involved the synthesis of novel, Schiff base ligand using quercetin and biguanide as initial materials. In the subsequent step, diorganotin (IV) complexes of the designed Schiff base ligand were synthesized. The spectroscopic records confirmed that the oxygen atom of the 3-OH group and imine nitrogen atoms are coordination sites for central tin atom, which result in the formation of penta-coordinated complexes. Antimicrobial screening displayed that the diorganotin (IV) complexes have greater inhibition potential than the Schiff base ligand, which may be due to the increase in lipophilicity and permeability through the cell membrane following the formation of complexes. The excessive lipid solubility of organotin compounds ensures better cell penetration and interaction with intercellular sites. Furthermore, the molecular docking proved that the complex formed in the case of