Eucalyptus robusta, Eurya nitida, Melastoma sanguineum, Melaleuca leucadendron, Lagerstroemia indica, Caryota mitis, Lagerstroemia speciosa and Gordonia axillaris possessed the highest antioxidant capacities and total phenolic contents among those tested, and could be potential rich sources of natural antioxidants and functional foods. The results obtained are very helpful for the full utilization of these wild fruits.In order to identify wild fruits possessing high nutraceutical potential, the antioxidant activities of 56 wild fruits from South China were systematically evaluated. The fat-soluble components were extracted with tetrahydrofuran, and the water-soluble ones were extracted with a 50:3.7:46.3 (v/v) methanol-acetic acid-water mixture. The antioxidant capacities of the extracts were evaluated using the ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays, and their total phenolic contents were measured by the Folin–Ciocalteu method. Most of these wild fruits were analyzed for the first time for their antioxidant activities. Generally, these fruits had high antioxidant capacities and total phenolic contents. A significant correlation between the FRAP value and the TEAC value suggested that antioxidant components in these wild fruits were capable of reducing oxidants and scavenging free radicals. A high correlation between antioxidant capacity and total phenolic content indicated that phenolic compounds could be the main contributors to the measured antioxidant activity. The results showed that fruits of  It has been proven that free radicals play an important role in many diseases, such as cardiovascular diseases, cancer, neurodegenerative diseases, diabetes and ageing ,2,3. AntWild fruits are receiving increasing interest from researchers because of their medicinal properties, nutritional value, vitamin and mineral contents. Concerning their medicinal properties, the most commonly studied benefit is their antioxidant effects ,27,28,29The aim of this study was to systematically evaluate the antioxidant activities and total phenolic contents of 56 wild fruits from South China, to screen out wild fruits with high antioxidant capacity and total phenolic content, and to investigate the relationship of antioxidant capacity and total phenolic content. The results from this study should prove helpful for the full utilization of these wild fruits.Fifty-six wild fruit samples were collected from different regions of South China, and were authenticated by a botanist. The scientific names of the plants and sampling location of the wild fruit are shown in The antioxidants in wild fruits are either water-soluble or fat-soluble. When determining total antioxidant activities of wild fruits, both water-soluble and fat-soluble components should be considered. In this study, the fat-soluble components of wild fruits were extracted with tetrahydrofuran, and their water-soluble components were extracted with a mixture of methanol-acetate acid-water . The ferMelastoma sanguineum [143 ± 2.00 µmol Fe(II)/g], Eucalyptus robusta [125 ± 2.49 µmol Fe(II)/g], Lagerstroemia indica [118 ± 1.49 µmol Fe(II)/g], Melastoma candidum [95.2 ± 5.89 µmol Fe(II)/g], Rosa laevigata [86.9 ± 2.18 µmol Fe(II)/g], Eurya nitida [83.3 ± 2.63 µmol Fe(II)/g], Viburnum fordiae [75.7 ± 2.06 µmol Fe(II)/g] and Gordonia axillaris [74.4 ± 3.25 µmol Fe(II)/g], had the highest antioxidant capacities, while Areca triandra had the lowest antioxidant capacity among the 56 wild fruits at 0.67 ± 0.04 μmol Fe(II)/g wet weight. For the fat-soluble fractions, the FRAP values varied from 0.61 ± 0.04 to 377 ± 3.92 μmol Fe(II)/g wet weight with a 618-fold difference, and eight wild fruits, Eucalyptus robusta [377 ± 3.92 µmol Fe(II)/g], Eurya nitida [345 ± 9.39 µmol Fe(II)/g)], Lagerstroemia speciosa [182 ± 7.89 µmol Fe(II)/g], Melaleuca leucadendron [146 ± 5.38 µmol Fe(II)/g], Melastoma sanguineum [145 ± 8.44 µmol Fe(II)/g)], Caryota mitis [138 ± 2.50 µmol Fe(II)/g], Lagerstroemia indica [137 ± 4.26 µmol Fe(II)/g] and Diplospora dubia [117 ± 6.56 µmol Fe(II)/g], had the highest antioxidant capacities, while Areca triandra again had the lowest antioxidant capacity among the 56 tested wild fruits with a value of 0.61 ± 0.04 μmol Fe(II)/g wet weight. For the combined value of water-soluble and fat-soluble fractions, the FRAP values varied from 1.28 ± 0.08 to 502 ± 6.41 μmol Fe(II)/g wet weight with a 392-fold difference, and eight wild fruits, Eucalyptus robusta [502 ± 6.41 µmol Fe(II)/g], Eurya nitida [428 ± 12.0 µmol Fe(II)/g], Melastoma sanguineum [288 ± 10.4 µmol Fe(II)/g], Lagerstroemia indica [254 ± 5.75 µmol Fe(II)/g], Lagerstroemia speciosa [225 ± 9.68 µmol Fe(II)/g], Melaleuca leucadendron [214 ± 8.86 µmol Fe(II)/g], Gordonia axillaris [180 ± 11.2 µmol Fe(II)/g] and Caryota mitis [174 ± 4.00 µmol Fe(II)/g], had the highest antioxidant capacities.As seen from the data in the table, the FRAP values varied from 0.67 ± 0.04 to 143 ± 2.00 μmol Fe(II)/g wet weight with a 213-fold difference for the water-soluble fractions, and eight wild fruits, namely •+ radical, and can measure antioxidant capacities of hydrophilic and lipophilic compounds in the same sample , Melaleuca leucadendron [138 ± 4.71 μmol Trolox/g], Melastoma sanguineum [130 ± 8.77 μmol Trolox/g], Melastoma candidum [93.8 ± 4.47 μmol Trolox/g], Viburnum fordiae [88.0 ± 2.32 μmol Trolox/g], Gordonia axillaris [79.4 ± 0.40 μmol Trolox/g], Lagerstroemia indica [73.2 ± 0.81 μmol Trolox/g] and Rosa laevigata [67.7 ± 0.32 μmol Trolox/g], had the highest free radical scavenging capacities, while in this assay Asparagus cochinchinensis with 0.37 ± 0.01 μmol Trolox/g wet weight showed the lowest free radical scavenging capacity among the tested wild fruits. For the fat-soluble fractions, the TEAC values varied from 2.47 ± 0.12 to 957 ± 5.79 μmol Trolox/g wet weight with a 387–fold difference, and eight wild fruits, Eucalyptus robusta [957 ± 5.79 μmol Trolox/g], Eurya nitida [422 ± 11.6 μmol Trolox/g], Caryota mitis [361 ± 10.4 μmol Trolox/g], Melaleuca leucadendron [323 ± 2.17 μmol Trolox/g], Melastoma sanguineum [275 ± 12.7 μmol Trolox/g], Gordonia axillaris [265 ± 11.7 μmol Trolox/g], Lagerstroemia speciosa [264 ± 16.8 μmol Trolox/g] and Lagerstroemia indica [119 ± 1.12 μmol Trolox/g], had the highest free radical scavenging capacities, whereas Aralia armata showed the lowest free radical scavenging capacity with 2.47 ± 0.12 μmol Trolox/g wet weight. For the combined value of the water-soluble and fat-soluble fractions, the TEAC values varied from 3.38 ± 0.15 to 1,140 ± 7.93 μmol Trolox/g wet weight with a 337–fold difference, and eight wild fruits, Eucalyptus robusta , Eurya nitida [478 ± 14.1 μmol Trolox/g], Melaleuca leucadendron [461 ± 6.88 μmol Trolox/g], Melastoma sanguineum [404 ± 21.5 μmol Trolox/g], Caryota mitis [389 ± 10.8 μmol Trolox/g], Gordonia axillaris [344 ± 12.1 μmol Trolox/g], Lagerstroemia speciosa [322 ± 17.4 μmol Trolox/g] and Lagerstroemia indica [192 ± 1.93 μmol Trolox/g], had the highest free radical scavenging capacities.As seen from Eucalyptus robusta, Eurya nitida, Melastoma sanguineum, Melaleuca leucadendron, Lagerstroemia indica, Caryota mitis, Lagerstroemia speciosa and Gordonia axillaris, had the highest antioxidant activities among the 56 tested wild fruits based on the consideration of the combined results obtained by FRAP and TEAC assays. These wild fruits exhibited quite high antioxidant activities when compared with some vegetables and common edible fruits , Melastoma sanguineum [9.68 ± 0.10 mg GAE/g], Melaleuca leucadendron [9.28 ± 0.37 mg GAE/g], Melastoma candidum [8.56 ± 0.30 mg GAE/g], Gordonia axillaris [7.88 ± 0.44 mg GAE/g], Diplospora dubia [7.49 ± 0.42 mg GAE/g], Rosa laevigata [7.45 ± 0.28 mg GAE/g] and Eurya nitida [6.27 ± 0.36 mg GAE/g] showed the highest total phenolic contents, while Areca triandra had the lowest total phenolic content with 0.06 ± 0.01 mg GAE/g wet weight. For the fat-soluble fractions, the total phenolic contents varied from 0.38 ± 0.02 to 40.7 ± 2.49 mg GAE/g wet weight with a 107–fold difference, and eight wild fruits, Eucalyptus robusta [40.7 ± 2.49 mg GAE/g], Eurya nitida [28.8 ± 0.60 mg GAE/g], Caryota mitis [17.5 ± 0.71 mg GAE/g], Gordonia axillaris [16.7 ± 0.64 mg GAE/g], Melaleuca leucadendron [16.4 ± 1.22 mg GAE/g], Lagerstroemia speciosa [14.9 ± 0.32 mg GAE/g], Viburnum sempervirens [13.9 ± 0.12 mg GAE/g] and Melastoma sanguineum [13.6 ± 0.39 mg GAE/g], showed the highest total phenolic contents. Meanwhile, Diospyros kaki had the lowest total phenolic content with 0.38 ± 0.02 mg GAE/g wet weight. For the combined value of the water-soluble and fat-soluble fractions, the total phenolic contents varied from 0.49 ± 0.04 to 54.8 ± 3.05 mg GAE/g wet weight with a 112–fold difference, and eight wild fruits, Eucalyptus robusta [54.8 ± 3.05 mg GAE/g], Eurya nitida [35.0 ± 0.96 mg GAE/g], Melaleuca leucadendron [25.6 ± 1.59 mg GAE/g], Gordonia axillaris [24.6 ± 1.08 mg GAE/g], Melastoma sanguineum [23.3 ± 0.49 mg GAE/g], Caryota mitis [21.4 ± 0.85 mg GAE/g], Lagerstroemia speciosa [20.1 ± 0.35 mg GAE/g] and Viburnum sempervirens [18.5 ± 0.40 mg GAE/g], showed the highest total phenolic contents. The total phenolic contents of these eight wild fruits were very high when compared with other vegetables and common edible fruits reported in the literature . As seen from 6.27 ± 0. mg GAE/g13.6 ± 0. mg GAE/gterature . R2 = 0.8916 and 0.8793 for the results from the FRAP and TEAC assays, respectively). In addition, the results from the water-soluble fractions showed a positive linear correlation between the antioxidant capacity and total phenolic content . Furthermore, the results from the fat-soluble fractions also showed a positive linear correlation between the antioxidant capacity and total phenolic content . These results indicated that the phenolic compounds could be the main contributor of the antioxidant activities of these wild fruits. This result was in agreement with many previous studies [The correlation between total antioxidant capacities and the total phenolic content of the 56 tested wild fruits is shown in  studies ,27.P = 0.010 < 0.05) and total phenolic contents (P = 0.005 < 0.05), but no significant difference in FRAP values (P = 0.954 > 0.05). The fat-soluble fractions possessed stronger scavenging free radical activity and higher phenolic content than the water-soluble fractions. In addition, comparison of FRAP value, TEAC value and total phenolic content of the wild fruits among different sampling locations: Guangdong province, Hong Kong and Jiangxi province, showed that there was no significant difference in FRAP values (P = 0.257 > 0.05), TEAC values (P = 0.200 > 0.05) and total phenolic contents (P = 0.226 > 0.05). Furthermore, three wild fruits, Gordonia axillaris, Rhodomyrtus tomentosa and Caryota mitis, were simultaneously collected both in Guangdong province and Hong Kong. Although their FRAP value, TEAC value and total phenolic content were different (data not shown), there was no significant difference in the three parameters. Although these wild fruits grow in different regions of South China, three regions all belong to the sub-tropical climate.Comparison of FRAP value, TEAC value and total phenolic content between water-soluble fraction and fat-soluble fraction showed that there was a significant difference in TEAC values (s-triazine (TPTZ), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2'-azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS) and gallic acid were purchased from Sigma–Aldrich . Iron (III) chloride hexahydrate, iron (II) sulfate heptahydrate, acetic acid, sodium acetate, potassium persulphate, tetrahydrofuran and sodium carbonate were obtained from Tianjing Chemical Factory . Ethanol, methanol and hydrochloric acid were obtained from Kelong Chemical Factory . All chemicals used in the experiments were of analytical grade, and deionized water was used throughout. Fifty-six wild fruit samples were collected from various locations in South China, and are authenticated by the botanist Dr. Xin-Sheng Qin from the College of Forestry, South China Agricultural University. The scientific names of the plants and sampling sites of the wild fruit are shown in Folin–Ciocalteu’s phenol reagent, 2,4,6-tri(2-pyridyl)-The wild fruits were washed with deionized water to remove dirt on their peels, and were given an airing at room temperature. Then, the wild fruits were ground into fine particles/slurry with a special grinder for herbal medicines. A weighed amount (about 1.00 g) of these particles was extracted with tetrahydrofuran (10 mL) at room temperature for 30 min in a shaking water bath according to the literature [4 solution, and the results were expressed as µmol Fe(II)/g wet weight of wild fruit.The FRAP assay was carried out according to the procedure described in the literature with minor modifications ,42. Brie•+ stock solution was prepared from 7 mM ABTS and 2.45 mM potassium persulfate in a volume ratio of 1:1, and then incubated in the dark for 16 h at room temperature and used within 2 days. The ABTS•+ working solution was prepared by diluting the stock solution with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm. All samples were diluted approximately to provide 20–80% inhibition of the blank absorbance. One hundred microliters of the diluted sample was mixed with ABTS•+ working solution (3.8 mL) and after 6 min of incubation at room temperature, the absorbance of the reaction mixture was measured at 734 nm, and the percent of inhibition of absorbance at 734 nm was calculated. Trolox was used as a reference standard, and the results were expressed as µmol Trolox/g wet weight of wild fruit.The TEAC assay was carried out according to the method established in the literature with slight modifications . BrieflyTotal phenolic contents were determined according to the literature ,37. Briep < 0.05.All the experiments were performed in triplicate, and the results were expressed as mean ± SD (standard deviation). Statistical analysis was performed using SPSS 13.0 and Excel 2003. The difference was considered significant at Eucalyptus robusta, Eurya nitida, Melastoma sanguineum, Melaleuca leucadendron, Lagerstroemia indica, Caryota mitis, Lagerstroemia speciosa and Gordonia axillaris possessed the highest antioxidant capacities and total phenolic contents among the wild fruits tested. These fruits could be potential rich resources of natural antioxidants, and could be developed into functional foods or drug for the prevention and treatment of diseases caused by oxidative stress. In the future, the specific components with high antioxidant capacities in these wild fruits should be isolated and identified, and explored for their health effects against oxidative stress.The antioxidant activities and total phenolic contents of 56 wild fruits collected from South China were evaluated. Generally, these wild fruits had high antioxidant capacities and total phenolic contents. A significant correlation between the FRAP value and the TEAC value suggested that antioxidant components in these wild fruits were capable of reducing oxidants and scavenging free radicals. A high correlation between antioxidant capacity and total phenolic content indicated that phenolic compounds could be the main contributors to the antioxidant activity of these wild fruits. The fruits of


Molecules of the title compound are linked into a complex three-dimensional network by a combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds. 15H15IN4O, the central piperazine ring adopts an almost perfect chair conformation with the pyrimidine substituent in an equatorial site. The planar amide unit makes a dihedral angle of 80.44 (7)° with the phenyl ring. A combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds links the molecules into a complex three-dimensional network structure, augmented by a π–π stacking interaction and an I⋯N halogen bond, all involving different pairs of inversion-related molecules. Comparisons are made with the structures of a number of related compounds.In 1-(2-iodobenzoyl)-4-(pyrimidin-2-yl)piperazine, C The chaon Fig. 3 links thon Fig. 4. The comx, y, z) and , which are components of the hydrogen-bonded chain along [001], are strictly parallel with an interplanar spacing of 3.4295 (10) Å and a ring-centroid separation of 3.4924 (6) Å, thus giving rise to a π–π stacking interaction  Å and C12—I12⋯N41i 174.83 (7)° , which can be regarded as a halogen bond -4-(pyrimidin-2-yl)piperazine-1-carboxamide (III) crystallizes with Z′ = 2 in space group P21/c  type as originally reported. However, the original report overlooked the presence of C—H⋯O hydrogen bonds which, in combination with the N—H⋯O hydrogen bond within the selected asymmetric unit, generates a second chain, this time running parallel to the [010] direction -4-methylpiperidine-1-carboxamide (IV), the assembly was reported  chains built from N—H⋯O hydrogen bonds. However, the presence in (IV) of a C—H⋯O hydrogen bond was overlooked, and the two hydrogen bonds together generate a complex sheet structure lying parallel to (100) on Fig. 6, so that0) Fig. 7. FinallyN,N-dimethylformamide (5 ml) and the resulting mixture stirred for 20 mins at 273 K. A solution of 1-(2-pyrimidyl)piperazine  in N,N-dimethylformamide (5 ml) was then added and stirring was continued overnight at ambient temperature. The reaction was confirmed to be complete using thin-layer chromatography, and the mixture was then quenched with water (10 ml) and extracted with ethyl acetate (20 ml). The organic layer was separated and washed successively with an aqueous hydrochloric acid solution (1 mol dm−3), a saturated solution of sodium hydrogencarbonate and then with brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature and in the presence of air, of a solution in methanol; m. p. 450–452 K.1-(2-Pyrimidyl)piperazine was purchased from Sigma–Aldrich. For the synthesis of compound (I)2), and with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901801811X/wm5479sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901801811X/wm5479Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901801811X/wm5479Isup3.cmlSupporting information file. DOI: 1886570CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This new 2,6-bis(imino)pyridine derivative with terminal 4-chlorophenyl rings crystallizes with two independent molecules in the asymmetric unit. 21H17Cl2N3, contains two crystallographically independent molecules (A and B). Both molecules have E configurations for both imine double bonds with regard to the aryl and pyridine groups. The conformations of the two molecules differ with the 4-chlorophenyl rings being inclined to the central pyridine ring by 77.64 (6) and 86.18 (6)° in molecule A, and 80.02 (5) and 43.41 (6)° in molecule B. In the crystal, molecules are linked by a number of C—H⋯π interactions, forming layers parallel to the bc plane.The asymmetric unit of the title compound, C Metal cA and B), illustrated in Fig. 1E-configurations for both imine double bonds with regard to the aryl and pyridine groups. The C=N bond lengths of the imine groups are in a narrow range, 1.2675 (15) to 1.2808 (14) Å  are inclined to the central pyridine ring (N2/C9–C13) by 77.64 (6) and 86.18 (6)°, respectively. In molecule B, the dihedral angles between the 4-chlorophenyl rings (C22–C27 and C37–C42) and the central pyridine ring (N5/C30–C34) are 80.02 (5) and 43.41 (6)°, respectively. The terminal ring (C37–C42) in molecule B adopts a significantly different conformation from the other benzene rings, as shown in Fig. 2Mercury ethan-1-imine) gave 25 hits. The crystal structure of the 2,6-bis[1-(phenylimino)ethyl]pyridine molecule was reported in 2001  was reported earlier in 1975  and 1.274 (5) Å, respectively, while the benzene rings are inclined to the central pyridine ring by 60.2 (2) and 55.2 (2)°, respectively. While the conformation of molecule A conforms to the overall limits, that of molecule B does not, with the terminal ring (C37–C42) being inclined to the pyridine ring by only 43.41 (6)°.A search of the Cambridge Structural Database  derivatives have also been reported, viz. 2,6-bis[1-ethyl]pyridine  and 88.0 (3)° in EMEJIP and 81.13 (6) and 74.22 (7)° in EYACUD. In the crystals of these two compounds, as in the crystal of the title compound, the H⋯N and H⋯Br/Cl intermolecular distances all exceed the sum of their van der Waals radii.The crystal structures of two 2,6-dihalogeno  in 20 ml of toluene was added 20 mg of p-toluenesulfonic acid  was added to this solid mass followed by the addition of hexane (ca 10 ml). The solution was then kept in the deep-freezer at 253 K. The title compound was obtained as a yellow solid in 31% yield . A very dilute solution of the compound was prepared in a 1:1 mixture of ethanol and hexane. On slow evaporation of the solvents at room temperature, pale-yellow crystals were obtained over a period of two weeks.To a solution of 2,6-diacetylpyridine  and p-chloroaniline  in 5 mL of absolute ethanol was added three drops of acetic acid. The reaction mixture was refluxed for 24 h, cooled to room temperature and then approximately 15 mL of hexane were added. The mixture was heated on a water bath and filtered hot using filter paper. The solution was kept in a deep freezer at 253 K. The title compound was obtained as a yellow solid in 26% yield .An alternate method for the synthesis is as follows: To a solution of 2,6-diacetylpyridine  and Spectroscopic data: IR : 3072 (w), 1638 (s), 1567 (w), 1482 (s), 1450 (w), 1362 (s), 1322 (w), 1297 (m), 1216 (s), 1171 (w), 1148 (w), 1119 (m), 1091 (m), 1010 (w), 994 (w), 955 (w), 842 (s), 787 (s), 743 (w), 723 (m), 672 (m), 635 (w), 597 (m), 532 (w), 517 (m); 1H NMR : 2.40 , 6.79 , 7.35 , 7.88 , 8.32 ; 13C NMR : 16.6, 121.0, 122.9, 129.4, 129.5, 137.3, 150.0, 155.6, 168.3.Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018017966/su5463sup1.cifCrystal structure: contains datablock(s) Global, I. DOI: 10.1107/S2056989018017966/su5463Isup2.hklStructure factors: contains datablock(s) I. DOI: 1886124CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-isochromen-1-one and 8-amino-3,4-diethyl-6-methyl-1H-isochromen-1-one, are described. The intermolecular contacts in the crystals were analysed using Hirshfeld surface analysis and two-dimensional fingerprint plots.The crystal structures of two new isocoumarin derivatives, 8-amino-6-methyl-3,4-diphenyl-1 H-isochromen-1-one, C22H17NO2, (I), and 8-amino-3,4-diethyl-6-methyl-1H-isochromen-1-one, C14H17NO2, (II), are new isocoumarin derivatives in which the isochromene ring systems are planar. Compound II crystallizes with two independent molecules (A and B) in the asymmetric unit. In I, the two phenyl rings are inclined to each other by 56.41 (7)° and to the mean plane of the 1H-isochromene ring system by 67.64 (6) and 44.92 (6)°. In both compounds, there is an intramolecular N—H⋯O hydrogen bond present forming an S(6) ring motif. In the crystal of I, molecules are linked by N—H⋯π interactions, forming chains along the b-axis direction. A C—H⋯π interaction links the chains to form layers parallel to (100). The layers are then linked by a second C—H⋯π interaction, forming a three-dimensional structure. In the crystal of II, the two independent molecules (A and B) are linked by N—H⋯O hydrogen bonds, forming –A–B–A–B– chains along the [101] direction. The chains are linked into ribbons by C—H⋯π interactions involving inversion-related A molecules. The latter are linked by offset π–π interactions [intercentroid distances vary from 3.506 (1) to 3.870 (2) Å], forming a three-dimensional structure.The title compounds, 8-amino-6-methyl-3,4-diphenyl-1 The isocoumarin nucleus is a rich structural pattern in natural products  of compound II are illustrated in Fig. 2H-isochromen-1-one moiety substituted by two ethyl groups, an amino group and a methyl group. The bond lengths and angles in the two independent molecules agree with each other within experimental error. The normal probability plot analyses  for both bond lengths and angles show that the differences between the two symmetry-independent molecules are of a statistical nature. For both compounds, the bond lengths and angles are close to those observed for a similar structure  ring motif: see Table 1The molecular structure and conformation of compound I is illustrated in Fig. 1H-isochromen-1-one (O1/C1–C9) ring system by 67.64 (6) and 44.92 (6)°, respectively. The 1H-isochromen-1-one moiety is planar (r.m.s. deviation = 0.021 Å) and atom O2 deviates from the mean plane by 0.041 (1) Å. In II, the 1H-isochromen-1-one ring system in each molecule (A and B) is also planar  and atoms O2A and O2B deviate from their respective mean planes by 0.052 (2) and 0.014 (2) Å, respectively.In I, the phenyl rings (C11–C16 and C17–C22) are inclined to each other by 56.41 (7)° and to the mean plane of the 1b-axis direction  Å  and Cg3⋯Cg4iv = 3.870 (2) Å ; symmetry codes: (i) −x, −y, −z; (iv) −x, −y + z − Cg1, Cg2, Cg3 and Cg4 are centroids of the (O1A/C1A–C4A/C9A), (C1A/C5A–C9A), (O1B/C1B–C4B/C9B) and (C1B/C5B–C9B) rings, respectively].In the crystal of II, the two independent molecules are linked by N—H⋯O hydrogen bonds involving the amino H atom of molecule s Table 2. The ribs Table 2: intercet al., 2007CrystalExplorer17  through white to blue . The dnorm surface was mapped over an arbitrary colour scale of −0.125 (red) to 1.528 (blue) for compound I and −0.178 (red) to 1.537 (blue) for compound II. The red spots on the surface indicate the intermolecular contacts involved in hydrogen bonding.The Hirshfeld surfaces of I and II mapped over b), O⋯H/H⋯O at 14.0% , C⋯H/H⋯C contacts at 15.4%  and H⋯N/N⋯H at 1.4%  followed by the C⋯C contacts at 2% . For II, they reveal a similar trend, with the principal intermolecular contacts being H⋯H at 61.7% , O⋯H/H⋯O at 15.6% , C⋯H/H⋯C contacts at 14.6% , and C⋯C contacts at 5.1%  followed by the H⋯N/N⋯H at 2.2% . In both compounds, the H⋯H intermolecular contacts predominate, followed by O⋯H/H⋯O contacts. However, the C⋯C contacts are significantly different: 2% cf. 5.1% for I and II, respectively.The fingerprint plots are given in Figs. 10% Fig. 10c, C⋯H/H% Fig. 10d and H⋯% Fig. 10e follow% Fig. 10f. For I% Fig. 11b, O⋯H/H% Fig. 11c, C⋯H/H% Fig. 11d, and C% Fig. 11e follow% Fig. 11f. In boet al., 2016H-isochromen-1-ones gave only one hit, viz. 8-amino-3,4-bis(4-methoxyphenyl)-1H-isochromen-1-one  ring motif as in compound (I)A search of the Cambridge Structural Database -dione (1.0 equiv), diphenylacetylene (1.2 equiv), [RhCp*Cl2]2 (3.0 mol %), Cu(OAc) (1.0 equiv) and dimethylformamide (5 ml). The flask was sealed using a Teflon-coated screw cap and the reaction was continuously heated at 383 K for 24 h. The mixture was then cooled to ambient temperature, diluted with 25 ml of ethyl acetate, filtered through a celite pad, and washed with 40–60 ml of ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the residue was purified by column chromatography using silica gel which led to the desired product, compound I.Compound II: An oven-dried round-bottom 25 ml flask with a magnetic stirrer bar was charged with 7-methyl-2H-benzo[d]oxazine-2,4(1H)-dione (1.0 equiv), hex-3-yne (1.2 equiv), [RhCp*Cl2]2 (3.0 mol %), Cu(OAc) (1.0 equiv) and dimethylformamide (5 ml). The flask was sealed using a Teflon-coated screw cap and the reaction was continuously heated at 383 K for 24 h. The mixture was then cooled to ambient temperature, diluted with 25 ml of ethyl acetate, then filtered through a celite pad and washed with 40–60 ml of ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the residue was purified by column chromatography using silica gel, which led to the desired product, viz. compound II.Colourless block-like crystals of compounds I and II were obtained by slow evaporation of solutions in ethanol.Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms. The crystal of compound II diffracted extremely weakly beyond 20° in θ and the data set was restricted to a maximum θ angle of 23.8°.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019009435/su5498sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989019009435/su5498Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019009435/su5498IIsup5.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019009435/su5498Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019009435/su5498IIsup5.cmlSupporting information file. DOI: 1937678, 1937677CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


C. elegans. Unlike lethal heat shock, mild heat activates a unique transcriptional program mimicking pathogen defense responses, characterized by the enhanced expression of innate immune and detoxification genes. Surprisingly, the expression of defense response genes persists long after heat exposure, conferring enhanced stress resistance even in aged animals. Further studies identify the histone acetyltransferase CBP-1 and the chromatin remodeling SWI/SNF complex as epigenetic modulators of the long-lasting defense responses. Histone acetylation is elevated by heat stress and maintained into agedness thereafter. Accordingly, histone acetylation levels were increased on the promoters of defense genes. Moreover, disruption of epigenetic memory abrogates the longevity response to early hormetic heat stress, indicating that long-lasting defense responses are crucial for the survival of aged animals. Together, our findings provide mechanistic insights into how temperature stress experienced in early life provides animals with lifetime health benefits.Early exposure to some mild stresses can slow down the aging process and extend lifespan, raising the question of how early life stress might impact the somatic health of aged animals. Here, we reveal that early life heat experience triggers the establishment of epigenetic memory in soma, which promotes long-lasting stress responses and longevity in  C. elegans, we reveal a mechanism of how early life heat stress impacts the health of aged animals and promotes longevity. We find that early exposure to mild heat activates basal innate immune and detoxification responses, which are surprisingly maintained long after temperature drop, even into agedness, and therefore contribute to lifespan extension. Remarkably, the long-lasting defense responses require epigenetic memory established by histone acetyltransferase CBP-1 and chromatin remodeling complex SWI/SNF. As a previous study has reported high temperature-induced transgenerational epigenetic effects in germline that involve histone methylation, our findings suggest that temperature may trigger distinct epigenetic changes in soma and germline, conferring adaptations in both the parental generation and their offspring.Organism aging is a deleterious process characterized by the progressive decline of cellular and tissue functions in the late life stage. However, the rate of aging is often determined by an organism’s historic experiences that occur in early life. For example, some mild stresses experienced in early life can extend animal lifespan, implying that early stresses might impact the health of aged animals, the mechanisms of which remain largely unknown. Here, by using the model organism  C. elegans show that mitochondrial stress induced during development can promote longevity via modulation of histone methylation , AU78[T24B8.5p::gfp::unc-54-3' UTR], AY101[F35E12.5p::gfp], CL2166[gst-4p::gfp], TJ375[hsp-16.2p::gfp], DA465[eat-2(ad465)], CB4037[glp-1(e2141)], DR1572[daf-2(e1368)].i OP50-1  and mainE. coli OP50-1 bacteria were cultured overnight at 37°C in LB, after which 150 μl of bacterial culture was seeded on 60 mm NGM plates. For the dead OP50-1 experiment, E. coli was killed by UV irradiation , heat  or kanamycin (400 μg/mL). The death of bacteria was confirmed by streaking bacteria onto LB plates and verifying the lack of colony formation after 37°C culture for 48 hours. For the RNAi experiment, HT115 bacteria containing specific dsRNA-expression plasmids (Ahringer library) [P. aeruginosa strain PA14 was cultured overnight at 37°C in LB and seeded on slow killing (SK) plates, then incubated for 24 hours at 37°C followed by another 24 hours at 25°C.library)  were cullibrary) . The patlibrary) . P. aeruFor the pathogen resistance assay, day 1 adult worms, unless otherwise indicated, were transferred to SK plates and incubated at 25°C for survival analysis. For TBHP (Sigma) resistance, day 1 adult worms, unless otherwise indicated, were transferred to NGM plates supplemented with 12.6 mM TBHP and incubated at 20°C for survival analysis. For heat shock resistance, worms at the indicated stages were transferred to NGM plates and incubated at 35°C for survival analysis.cbp-1 RNAi treatment, the bacterial culture of the cbp-1 RNAi strain was diluted at a ratio of 1:20 with the vector control.RNAi was induced at room temperature for 24 hours after seeding. Then, L1 worms were added to RNAi plates to knockdown the indicated genes. For snb-1 was used to normalize samples. Primer sequences are listed in qRT-PCR was performed as previously described . BrieflyDay 1 adult worms, unless otherwise indicated, were collected, washed in M9 buffer and then paralyzed with 1 mM levamisole. Fluorescence microscopic images were taken after mounting on slides. The expression levels of GFP were scored as low, medium and high. Briefly, dim green signals in the anterior or posterior of the intestine, green signals throughout the intestine and bright signals throughout the intestine are categorized as low, medium and high expression, respectively.Day 1 adult worms, unless otherwise indicated, were collected and sonicated in RIPA buffer  containing 1 mM DTT and proteinase inhibitor (Sigma) before boiling and loading. Antibodies against acetyl-histone H4 (Millipore), phospho-p38 (Promega) and tubulin (Sigma) were used.E. coli strains. For experiments with developmental exposure to 25°C, worms were kept at 25°C from the egg to L4 stage and then shifted to 15°C thereafter. Worms were transferred every day during the reproductive period. Worms that died of vulva burst, bagging, or crawling off the plates were censored.Lifespan assays were performed as previously described  with modTo compare the effects of temperatures on gene expression, 20°C-cultured L4 parent worms were moved to 15°C and 25°C separately, and allowed to mature further and lay eggs, which hatched and developed into middle-to-late L4 stages at these temperatures. The 15°C-cultured parent worms were allowed to lay eggs about 40 hours earlier than the 25°C-cultured ones, making the two groups developmentally equivalent across temperatures. Total RNA from 15°C and 25°C-cultivated L4 stage worms was extracted using TRIzol reagent and used to generate sequencing libraries using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina. The quality and quantity of the libraries were determined using an Agilent Bioanalyzer 2100  and Qubit 3.0 , respectively. The libraries were sequenced at a paired-end 150 bp read length on an Illumina HiseqX Ten. RNA-seq reads were aligned to the reference genome WBcel235 using Tophat v.2.0.6 . The difChIP was performed as previously described with some modifications . Synchropost-hoc test was used for Data were presented as mean ± SEM. Survival data were analyzed by using log-rank (Mantel-Cox) test. The levels of fluorescence micrographs were analyzed by using Chi-square and Fisher’s exact tests. Benjamini-Hochberg adjusted p values were used for Figs S1 FigT24B8.5 mRNA levels at 15°C and 25°C measured by qPCR. n = 3 for each group. Data are presented as mean ± SEM. ***p < 0.001 via Student’s t test. (B) Expression of F35E12.5p::GFP upon 25°C cultivation. (C-E) The expression of T24B8.5p::GFP was induced at 25°C in worms fed with dead E. coli OP50 killed by UV irradiation (C), heat (D), and kanamycin (E). (F) Pathogen resistance of worms precultivated with UV-killed OP50 at 15°C and 25°C. ***p < 0.001 versus 15°C controls via log-rank test. Scale bar = 100 μm.(A) The endogenous (TIF)Click here for additional data file.S2 Figpmk-1 mutation on defense gene expression at 25°C. n = 3 for each group. Data are presented as mean ± SEM. ***p < 0.001 versus WT controls via two-way ANOVA followed by Bonferroni post-hoc test. (B-C) Lifespan of WT and pmk-1 mutants fed on dead bacteria at 25°C (B) and 15°C (C). ***p < 0.001 versus WT controls via log-rank test.(A) Effects of (TIF)Click here for additional data file.S3 FigF35E12.5p::GFP (A) and gst-4p::GFP (B) was examined on day 1, day 4, and day 7. Arrows indicate GFP signals in the posterior of worms. (C) Worms were treated as indicated in S3A and S3B Fig. The mRNA levels of T24B8.5, F49F1.7 and cyp-37B1 were examined by qPCR on day 1, day 4, and day 7. n = 3 for each group. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001 versus 15°C controls via Student’s t test. (D) Persistent resistance to TBHP of worms precultivated at 25°C until day 1 adults. *p < 0.05, **p < 0.01, ***p < 0.001 versus 15°C controls via log-rank test. (E) Persistent mRNA expression of hsp-16.2 in worms exposed to 35°C for one hour on day 1. n = 3 for each group. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001 versus 20°C controls via Student’s t test. (F) Persistent resistance to heat shock stress of worms exposed to 35°C for one hour on day 1. **p < 0.01, ***p < 0.001 versus 20°C controls via log-rank test. Scale bar = 100 μm.(A-B) Worms were precultivated at 25°C until day 1 adults and transferred to 15°C thereafter. The expression of (TIF)Click here for additional data file.S4 Figcbp-1 RNAi on the mRNA expression of cbp-1. n = 3 for each group. Data are presented as mean ± SEM. ***p < 0.001 via Student’s t test. (B) The effects of cbp-1 RNAi on the mRNA expression of T24B8.5, F49F1.7 and C32H11.4 were examined by qPCR at 15°C and 25°C. n = 3 for each group. Data are presented as mean ± SEM. ***p < 0.001 versus control RNAi via two-way ANOVA followed by Bonferroni post-hoc test. (C) Effects of RNAi targeting genes regulating histone acetylation on T24B8.5p::GFP expression at 25°C. HATs: histone acetyltransferases. Number of biological replicates (n): control (95), cbp-1 RNAi (78), K03D10.3 RNAi (105), T02C12.3 RNAi (85), ZK856.9 RNAi (73), trr-1 RNAi (64), ZK1127.3 RNAi (102), mrg-1 RNAi (105). ***p < 0.001 versus control RNAi via Chi-square and Fisher’s exact tests. (D) Effects of cbp-1 RNAi on gst-4p::GFP expression at 25°C. (E) Effects of cbp-1 RNAi on TBHP resistance at 15°C and 25°C. ***p < 0.001 versus 15°C RNAi via log-rank test. (F-H) Effects of cbp-1 RNAi on stress gene induction in eat-2 (F), daf-2 (G) and glp-1 (H) mutants. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus control RNAi via Student’s t test. (I) mRNA levels of T24B8.5 measured as indicated in (A) Effects of 20-fold diluted (TIF)Click here for additional data file.S5 Figcbp-1 RNAi on acetylated H4 levels at 15°C and 25°C. (B) Effects of pmk-1 mutation on acetylated H4 levels at 15°C and 25°C. (C) Schematic of promoter regions amplified by ChIP-qPCR. (D) Acetylated H4 levels on the promoters of acdh-4 and pde-4 detected by ChIP-qPCR. n = 3 for each group. Data are presented as mean ± SEM. (E) The expression levels of acdh-4 and pde-4 at 15°C and 25°C examined by RNA-seq analysis. n = 3 for each group. Data are presented as mean ± SEM and ns indicates Benjamini-Hochberg adjusted p > 0.05.(A) Effects of (TIF)Click here for additional data file.S6 Figswsn-1 RNAi on gst-4p::GFP expression at 25°C. (B) The SWI/SNF complex components in C. elegans. Asterisks indicate corresponding RNAi clones in C. elegans Ahringer RNAi collection. (C) TBHP resistance of swsn-1 RNAi worms precultivated at 15°C and 25°C. ***p < 0.001 versus 15°C RNAi via log-rank test. Scale bar = 100 μm.(A) Effects of (TIF)Click here for additional data file.S1 TableGenes with a fold change of > 2 (either up or down) and FDR of < 0.01 were listed as 25°C-regulated genes.(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


The N—H group is involved in the formation of an intramolecular hydrogen bond.In the title compound, C 20H18NO3P, the C=O and P=O groups of the carbacylamidophosphate (CAPh) fragments are located in a synclinal position relative to each other and are pre-organized for bidentate chelate coordination of metal ions. The N—H group is involved in the formation of an intramolecular hydrogen bond. In the crystal, molecules do not form strong intermolecular interactions but the molecules are linked via weak C—H⋯π interactions, forming chains along [001].In the title compound, C The phosphorus atom environment has a distorted tetrahedral configuration. The C1–C6 phenyl ring is almost coplanar with the P=O bond [the C6—C1—P1—O1 torsion angle is −5.7 (2)°] while the C7–C12 phenyl ring is rotated significantly relatively to the P=O bond as defined by the C8—C7—P1—O1 torsion angle of −72.7 (2)°.The conjugation between the carbamide group and the anisole substituent is broken, as evidenced by the value of the C13—C14 bond length of 1.496 (3) Å, which is comparable to the mean value for non-conjugated Cet al., 1990et al.,1997et al., 2004abet al., 2014i.e. chains, dimers and more seldom monomers. These motifs are realized through existence of hydrogen bonds with participation of the –N—H group. In crystal of (I)c-axis direction -2-methoxybenzamide (I)o-methoxybenzamide  and triethylamine  in 20 ml of dioxane was added chlorodiphenylphosphine  under an inert atmosphere. The mixture was stirred under reflux for 60 min and evaporated to dryness to give a pasty residue, which was mixed with 20 ml of acetone and then a solution of 0.01 mol of H2O2 in 5 ml of acetone was added dropwise under vigorous stirring at 273 K. The brownish solution was evaporated and the residue was mixed with 50 ml of 10% aqueous HOAc. The solid precipitate was filtered, washed with cold water (2 × 20 ml) and recrystallized from i-PrOH [2.8 g (80%)]. Single crystals suitable for X-ray diffraction were grown from dilute i-PrOH solution by slow evaporation after one week.To a solution of −1): 3271m [ν(NH)], 3059w, 3011w, 2985w, 2949w, 2924w, 2843w, 1671vs [ν(CO)], 1601m, 1486w, 1461vs (Amide-II), 1436s, 1294m, 1242m, 1225vs [ν(PO)], 1181m, 1160w, 1125s, 1109m, 1045w, 1012m, 868w [(PN)], 840m, 786m, 767m, 754s, 726m, 702m, 668w, 634w, 592w, 543m, 524s, 512m, 487m, 442w. 1H NMR (DMSO-d6): C—H 3.95 , 7.07 , 7.22 , 7.58 , 7.67 , 7.88 , N—H 9.90  ppm. UV–Vis abs. : 240, 295.M.p. 431–434 K. IR  = 1.2Ueq or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901900762X/lh5904sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901900762X/lh5904Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901900762X/lh5904Isup3.cmlSupporting information file. DOI: 1918613CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


When all isolates were tested for their AMPK activation activities, seven compounds (1 and 3–8) were significantly activated AMPK phosphorylation in mouse C2C12 skeletal muscle cell lines. Since G. longipes contained a significant amount of active compound 1 (over 2.08% per dried raw plant), it suggested the potential of this plant to be developed as a functional food or botanical drug that enhances muscle proliferation by activating AMPK signaling pathways.The aging population is growing rapidly around the world and there is also an increase in sarcopenia, which is characterized by decreased muscle mass, strength and function in the elderly population. AMP-activated protein kinase (AMPK) is an essential sensor and regulator of glucose, lipid and energy metabolism throughout the body. Previous studies have shown that AMPK pathway activation by regular exercise and appropriate dietary control have beneficial effects on skeletal muscle. In the process of searching for new AMPK activators from medicinal plants, we isolated and characterized eight new 12,23-dione dammarane triterpenoids ( Skeletal muscle decreases approximately 3–8% per decade after 30 years of age, and muscle loss rapidly increases by about 25–30% after 60 years of age2. This loss of muscle mass, strength and function is the key cause of many diseases in elderly people, and the increased risk of injury and falls due to sarcopenia can also lead to continuous functional dependence and disability. The decreased muscle mass is also associated with a continuous increase in fat mass and consequently a change in body composition, and this in turn is associated with a higher incidence of insulin resistance in the elderly people4. Therefore, an increase of muscle mass in the elderly population is a promising approach to alleviate many of the symptoms of geriatric diseases5.One of the most distinctive effects of aging is the involuntary loss of muscle mass, strength and function, which is termed sarcopenia6. Conversely, increased physical inactivity and obesity due to a sedentary lifestyle are associated with attenuated muscle mass and reduced AMP-activated protein kinase (AMPK) activity8. AMPK plays an important role in the regulation of cellular energy homeostasis, and the activation of AMPK promotes glucose uptake, fatty acid oxidation, mitochondrial biogenesis and insulin sensitivity. In eukaryotes, there are three subunits of the AMPK, including the catalytic α-subunit (α1 and α2), regulatory β-subunit (β1 and β2) and γ-subunit 10. The regenerative process in skeletal muscle tissue generally involves a coordinated sequence of the activation of satellite cells, which proliferate to produce a population of myogenic progenitors (termed myoblasts)12. Especially, the AMPKα1 isoform has an important role in enhancing Warburg-like glycolysis through non-canonical sonic hedgehog (Shh) signaling in myogenic cells during muscle regeneration. AMPKα1 knock-out in satellite cells reduces their activation and also inhibit the proliferation and differentiation of myogenic progenitor cells7. Taken together, the activation of AMPK  is a key mediator linking obesity and impaired muscle regeneration, providing an effective drug target to facilitate muscle regeneration in obese and elderly populations9.Recent studies suggested that muscle loss can be prevented by exercise in both young and older peoplesGynostemma longipes, commonly called “That diep dom”, a Vietnamese traditional herb used widely by the local community for tonic, treatment of diabetes and health strengthening. A fraction from this plant was then found to show potential activity in accelerating the muscle proliferation in the myoblast C2C12 cell model. The main ingredients of G. longipes are known as dammarane triterpenes14 similar to other species of Gynostemma genus16. Dammarane triterpenes have been also reported to play a significant role in the development of natural drugs for the treatment of different metabolic diseases such as diabetes mellitus, metabolic syndrome, aging and neurodegenerative diseases17. In this study, bioassay-guided fractionation and isolation of G. longipes were performed, and resulted in the purification of nine 12,23-dione dammarane triterpenes (1–9). All isolates were evaluated for their enhancement effects on muscle proliferation through activation of the AMPK pathway using the C2C12 myoblast cell model.Our ethnopharmacological investigation of medicinal plants with AMPK activation activity resulted in the selection of G. longipes was successfully identified using the DNA barcoding marker techniques with 4 DNA sequences. Result in Fig. The authentication of medicinal plants based on genomic tools is an efficient and accurate method in the past decades. In this study, G. longipes afforded eight new dammarane triterpenes (1–3 and 5–9), along with one known compound gypentonoside A (4)  Fig. .Figure 21 was obtained as a white crystal with c 0.1, MeOH). The molecular formula of compound 1 was deduced as C48H78O17 from the [M − H]− high resolution electrospray ionization mass spectrometry (HRESIMS) ion peak observed at m/z 925.5157   peak at 254 nm suggested the existence of an α,β-unsaturated carbonyl group. The IR broad peak at 3404 cm–1 appeared in companion with the peak at 1035 cm–1 indicated the presence of a hydroxyl group. The proton nuclear magnetic resonance (1H NMR) spectrum demonstrated oxygenated methine proton at δH 3.25  and eight methyl singlets at δH 2.15, 1.70, 1.50, 1.17, 1.12, 1.11, 0.84, and 0.76 (each 3 H)  spectrum revealed two ketone carbons (δC 211.7 and 201.7) and one oxygenated quaternary carbons (δC 74.0)  correlations from H-17 (δH 2.77), H-21 (δH 1.50) and H-22 (δH 2.90 and 2.72) to C-20 (δC 74.0). One ketone group was located at C-12 from the HMBC experiment showing cross-peaks from H-11 (δH 2.24 and 2.22), H-13 , and H-17 (δH 2.32) to C-12 (δC 211.7)  and C-24/25 (CH = C) by HMBC cross peaks from H-22 , H-24 (δH 6.28) to C-23 (δC 201.7), from H-22 , H-26 (δH 2.15), and H-27 (δH 1.71) to C-24 (δC 126.4)  and H-5 (δH 0.66) demonstrated an α orientation for H-3. Both H-17 and Me-21 were also determined as α orientation based on the coupling constant of H-13 (J = 9.6 Hz) and NOESY correlations from H-17 (δH 2.77) to H-21 (δH 1.50) and H-30 (δH 0.84) -dihydroxydammar-24-en-12,23-dione. The diagnostic NMR data suggested the existence of three sugar units in compound 1. Acid hydrolysis of 1 with HCl afforded d-glucopyranose and l-rhamnopyranose, based on liquid chromatography analysis following treatment with l-cysteine methyl ester hydrochloride and benzyl isothiocyanate derivatization. The coupling patterns of anomeric protons indicated β configuration for the glucopyranosyl  unit and α configuration for the two rhamnopyranosyl  units. The HPLC-MS experiment at the positive mode showed fragment ions at 909 [M + H − H2O (18)]+, 763 [M + H − H2O – rha (146)]+, 617 [M + H − H2O – rha – rha (146)]+, 455 [M + H − H2O – 2 × rha – glu (162)]+ corresponding to the presence of a sugar chain consisting of 2 rhamnoses and 1 glucose. The linkage patterns of three sugar units were analyzed based on the HMBC experiment  and C-3 (δC 88.5), H-1″ (δH 5.92) and C-2′ (δC 78.3), H-1′′′ (δH 5.70) and C-3′ (δC 87.5). Therefore, compound 1 was elucidated as 3β,20(S)-dihydroxydammar-24-en-12,23-dione-3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→3)]-β-d-glucopyranoside.Compound .4) Fig. . The nuc84) Fig. . Therefoent Fig.  which al2 was obtained as a white and amorphous powder. Its molecular formula of C54H88O22 was evident by the negative HRESIMS [M − H]− ion peak at m/z 1087.5682  . The 1H, 13C NMR, and heteronuclear single quantum correlation (HSQC) spectroscopic data of 2  and the addition of 6 carbon resonances at C-1ʹʹʹʹ (δC 98.8), C-2ʹʹʹʹ (δC 73.0), C-3ʹʹʹʹ (δC 72.9), C-4ʹʹʹʹ (δC 74.0), C-5ʹʹʹʹ (δC 78.2), and C-6ʹʹʹʹ (δC 63.3). When compared with 1, a downfield shift of C-20  and an upfield shift of C-21  were observed, which supported the attachment of the glucopyranosyl unit to C-20. Further HMBC analysis also constructed a link between H-1ʹʹʹʹ (δH 5.16) and C-20 -dihydroxydammar-24-en-12,23-dione-3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→3)]-β-d-glucopyranosyl-20-O-β-d-glucopyranoside.Compound 2 Tables –S13 were-20 Fig. . Accordi3 was obtained as a white and amorphous powder. The negative HRESIMS showed the deprotonated molecular ion peak at m/z 967.5276 [M − H]−  indicating that the molecular formula is C50H80O18  and a methyl singlet . When compared to 1, a downfield shift of C-4ʹʹ  and an upfield shift of C-5ʹʹ  demonstrated the substitution of an acetyl moiety to C-4ʹʹ. The HMBC spectrum showed a cross peak from H-4ʹʹ (δH 5.79) to acetyl carbonyl (δC 171.9) confirming the linkage -dihydroxydammar-24-en-12,23-dione-3-O-α-l-rhamnopyranosyl-(1→2)-[(4-O-acetyl)-α-l-rhamnopyranosyl-(1→3)]-β-d-glucopyranoside.Compound 3 Tables –S19 alsoage Fig. . Accordi5 was obtained as a white and amorphous powder. The negative HRESIMS showed a deprotonated molecular ion peak at m/z 779.4597 [M − H]−  indicating the molecular formula to be C42H68O13 ]+, 617 [M + H − H2O − rha (146)]+, 455 [M + H − H2O – rha − glc (162)]+ corresponding to the existence of a oligosaccharide chain [-glc-rha]. The 1H, 13C, HSQC and HMBC NMR spectroscopic data of 5 . Accordingly, compound 5 was determined as 3β,20(S)-dihydroxydammar-24-en-12,23-dione-3-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranoside.Compound 5 Tables –S26 coul6 was obtained as a white and amorphous powder. The negative HRESIMS showed a deprotonated molecular ion peak at m/z 471.3393 [M − H]− , representing the molecular formula C30H48O4  to H-5 (δH 0.80) and H-28 (δH 0.77) suggested OH-3 to be β-oriented, which was also supported by the coupling pattern of H-3  -dihydroxydammar-24-en-12,23-dione.Compound a Tables –S31 show7 was obtained as a white and amorphous powder. The negative HRESIMS showed a deprotonated molecular ion peak at m/z 471.3452 [M + H]+ , which represents the molecular formula C30H46O4  and the disappearance of H-3 resonance, together with the deshielded chemical shift of C-2  and C-4  suggested ketone oxidation of OH-3. Thus, 7 was identified as 20(S)-hydroxydammar-24-en-3,12,23-trione.Compound 7 Tables −S36 show8 was obtained as an white, amorphous powder, and its molecular formula of C48H80O18 was evident on the basis of a negative [M − H]− HRESIMS ion peak at m/z 943.5374  , C-25  and a downfield shift of C-23  were observed, which supported the attachment a hydroxyl group at C-25. Further HMBC analysis also established the connection between Me-26, Me-27 , and C-25 (δC 70.0) ,25-trihydroxydammaran-12,23-dione 3-O-α-l-rhamnopyranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→3)]-β-d-glucopyranoside.Compound 8 Tables −S40 show.0) Fig. . Thus, c9 was obtained as a white and amorphous powder. The molecular formula of compound 9 was deduced as C48H76O16 from the [M − H]− HRESIMS ion peak observed at m/z 907.5066 . The HPLC-MS experiment at positive mode also demonstrated the presence of a glycoside chain identical with compound 1. The 1H, 13C, and HSQC NMR spectroscopic data of 9 , and downfield shifts of C-20  and C-22  were observed, suggesting the dehydration of this compound at C-20. The HMBC spectrum exhibited cross peaks from H-21 and H-17 to C-20 and C-22, respectively -[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside.Compound 9 Tables −S45 showely Fig. , further1 as main ingredient was the highest as 130.01 mg/g in 70% EtOH extract. This was equivalent to 20.75 mg/g of the raw material of G. longipes.Extraction yields by using different solvents 100% MeOH, water, 30% EtOH, 50% EtOH, 70% EtOH and EtOH were 11.5%, 16.5%, 18.5%, 17.5%, 14%, and 7.5%, respectively. As a result of the quantitative analysis, the content of compound 19. Recently, several studies have demonstrated that decreased proliferation of myoblasts and cytotoxicity can reduce the number of muscle fibers22. Therefore, mouse C2C12 cells, also known as myoblastic cells, were chosen in this research because they are a valid model to study muscle cell proliferation20. In a search for new bioactive natural products with promising activity for muscle proliferation, we found that the 95% EtOH eluted fraction on SP70 resin of the total extract could potentially be a ‘hit’ using an in vitro screening method. This fraction stimulated an increase in proliferation of C2C12 myoblast cell about 20–30% compared to the negative control during 24 and 48 hours of treatment using 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) and 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) methods , which play an important role in the proliferation and differentiation of myoblasts to form or repair muscle fibersods Fig. . The proods Fig. . As AMPKner Fig. , and S53G. longipes resulted in the isolation of nine compounds (1–9) and all of the isolates were evaluated for their enhancing effect on muscle proliferation in C2C12 myoblast cells. After 48 hours of treatment, cell viability was assessed using the MTT and WST-1 methods. Seven compounds (1 and 3–8) increased cell viability by about 20–30% , it was selected to examine the effects on the proliferation of two cancer cell lines, MCF-7 and MDA-MB-231 breast cancer cells. As shown in Supplementary Fig. G. longipes (10 μg/mL) or compound 1 (10 μM) for 2 days. These results suggested that the active fraction and major compound of G. longipes stimulate muscle cell proliferation selectively and do not induce the outgrowth of harmful cancer cell lines.Bioassay-guided isolation from 30% Fig. . Notably24. In order to evaluate the potential activities of compounds from G. longipes, nine isolates (1–9) were measured the stimulatory effects on the p-AMPK and p-ACC expressions using mouse C2C12 myotubes. Results in Supplementary Figs 5, 6, and 7 increased the phosphorylation of AMPK and ACC proteins strongly, while compounds 1, 3, 4, and 8 exhibited less efficient. Therefore, potential candidates  were further confirmed the activation effect on p-AMPK in C2C12 myoblasts. These compounds clearly increased the phosphorylation of the target protein compared to Aicar as a positive control  < 1 (3 sugar units) < 5 (2 sugar units) < 6 and 7 (no sugar). It was also noteworthy that with the same number of sugar units, the attachment of a sugar unit to C-20 remarkably reduced the activity (2 < 4). Furthermore, the hydration of olefinic bond at C-20 and C-24 slightly increased the effect in the following order: 9 (two double bonds) < 1 (one double bond) < 8 (no double bond). The change of hydroxy (6) and ketone substitutions (7) at C-3 did not cause any difference in activity.Considering the structures and activities of all nine isolates, the core structure of the dammarane skeleton might contribute mainly to the AMPK activation effect. Further examination of structure-activity relationships (SAR) revealed that compounds with a longer saccharide side chain at C-3 showed lower AMPK inhibition effects: p-AMPK were analyzed using the mouse C2C12 myoblast model. Interestingly, co-treatment of compound C with the active fraction or compound 1 decreased the percentage of cell viability after 48 hours of incubation, compared to the number of C2C12 cells exposed to the fraction or compound 1 alone  or the SP70-EtOH 95% fraction (20 μg/mL) and incubated for 8 hours. Next, the cells were incubated for 2 hours with or without BrdU. The BrdU incorporation into the nucleus was assessed using a fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody and DAPI staining. As shown in Fig. G. longipes or compound 1 compared to DMSO treatment alone. We also evaluated the BrdU-labeled cells using a flow cytometer for BrdU and PI staining. When C2C12 cells were incubated with or without BrdU treatment, the fluorescence intensity was significantly increased from 33.46 ± 0.58% to 55.41 ± 0.88%. In order to confirm the concentration dependence in the cells, compound 1 was treated for 8 hours at different concentrations  and then incubated with BrdU for 2 hours. The BrdU positive cell population increased in a concentration-dependent manner to 65.22 ± 3.43%, 59.90 ± 0.84% and 56.00 ± 0.81%, respectively  can be used to label the DNA of cells in the S-phase. This method is useful for quantifying the degree of DNA synthesis during cell proliferationely Fig. .Figure 61 alone amino]-2-deoxy-d-glucose (2-NBDG), to monitor intracellular glucose uptake. As illustrated in Fig. 1 as well as insulin (as a positive control) increased glucose uptake in mouse C2C12 myoblasts.Recent studies suggested the importance of AMPKG. longipes on the mitochondrial ATP synthesis was examined by treatment mouse C2C12 myoblasts with the active fraction and compound 1 in approximately 70 minutes. The ATP levels of the active fraction and compound 1 increased drastically from 30 to 50 minutes comparison with the vehicle group  staining method29. C2C12 myoblasts were exposed to compound 1 (20 μM) for 12 and 24 hours. The cell cycle distributions at different phases were significantly changed, compared to the vehicle group. After 12 hours of incubation with compound 1, the cells with 2 N DNA content (G0/G1 phases) decreased remarkably from 72.07 ± 0.63% to 65.09 ± 2.49%, while the cells with >2 N DNA content (S or G2/M phases) increased about 8% compared with negative control  activated significantly AMPK phosphorylation in mouse C2C12 cell lines. The result of AMPK activation by compound 1 could stimulate glucose uptake into the cells and increase mitochondrial ATP synthesis. The effect of dammarane triterpenes on DNA synthesis during cell proliferation also showed that compound 1 increased the BrdU positive cell population in a dose-dependent manner. Analysis of cell cycle using flow cytometry displayed clearly that compound 1 reduced the 2 N DNA content (G0/G1 phases) while the ratio of G2/M phases was significantly increased. These results suggested strongly that compound 1 enhances significantly the proliferation of C2C12 myoblast cells  in G. longipes suggested that it may be a promising candidate for development of functional food or botanical drug. These results also indicated that new dammarane-type compounds are promising candidates for muscle proliferation via activation of AMPK signaling pathways and could be further studied and developed as therapeutics for geriatric diseases.In this study, we reported the findings of eight new 12,23-dione dammarane triterpenes from lls Fig. . Further18 (particle size: 40–63 μm) were purchased from Merck  and Sephadex LH-20 from Sigma-Aldrich  were used for column chromatography (CC). Silica gel 60 F254 and RP-18 F254 TLC plates were obtained from Merck . A Gilson HPLC purification system, equipped with an Optima Pak C18 column , was used at a flow rate of 2 mL/min and UV detection at 205 and 254 nm.Optical rotations were measured in a JASCO P-2000 polarimeter . IR data were recorded on a Nicolet 6700 FT-IR spectrometer . NMR data were analyzed using an AVANCE 500 MHz spectrometer  or a JNM-ECA 600 MHz spectrometer . HRESIMS values were analyzed using an Agilent Technologies 6130 Quadrupole LC/MS spectrometer equipped with an Agilent Technologies 1260 Infinity LC system  and an INNO C18 column . Silica gel (particle size: 63−200 μm) and RP-CG. longipes C. Y. Wu based on the morphological characteristics by Prof. Tran Van On, Head of Department of Botany, Hanoi University of Pharmacy, Hanoi, Vietnam. A voucher specimen was deposited in the Medicinal Herbarium of Hanoi Univeristy of Pharmacy (HNIP) with the accession number HNIP.18500/16 , 99.6% (rbcL gene), 99.8% (rpl20-rps12 intergenic spacer region) and 99.9% (matK gene). This additional genetic information supported morphological authentication of Gynostemma longipes  with some modifications. PCR amplification and sequencing were performed by Macrogen Inc.  using 4 pairs of primers: ITS1-F (5′-TCCGTAGGTGAACCTGCGG-3′), ITS4-R (5′- TCCTCCGCTTATTGATATGC-3′) for ITS1–5.8S-ITS2 region; matK8-F (5′-AAATCCTTCGCTCCTGGGTG-3′), matK710-R (5′-GAATCGATCCAGGCCGTCTT-3′) for partial maturase K gene, rbcL134-F (5′-GGACAACTGTGTGGACCGAT-3′), rbcL590-R (5′-AAACGGTCTCTCCAACGCAT-3′) for partial ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene and rp143-F (5′-ACCTTCCCGACCACGATTTC-3′), rp686-R (5′-CGGAACAACCCCGTTCACTA-3′) for rpl20-rps12 intergenic spacer region. All nucleotide sequence data were recorded in the GenBank sequence database, National Center for Biotechnology Information (NCBI), with the accession numbers KX619478, KX619479, KX619480 and KX619481 respectively. DNA sequencing data were analyzed using the Geneious DNA sequencing analysis software . DNA sequences were compared to published sequences available from Genbank using the Basic Local Alignment Search Tool (BLAST). All analysis sequences were matched with G. longipes (1.5 kg) was powdered, sonicated with 70% EtOH (3 × 3 L), filtered and the solvent was evaporated in vacuo. The crude extract obtained (262 g) was suspended in water, absorbed onto Sephabeads SP70 resin and washed with water, 50% EtOH, 95% EtOH, 100% EtOH and acetone in a sequential elution process. The 95% EtOH fraction (75 g) was subjected to silica gel column chromatography  using a gradient of EtOAc/MeOH/H2O (from 20/1/0.1 to 4/1/0.1) to give 9 sub-fractions (C1-C9) based on the thin-layer chromatography profile. Compound 1 (50.5 g) was obtained by direct crystallization from sub-fraction C7. Sub-fraction 8 was chromatographed on C18- reversed phase silica gel (RP-C18) column chromatography eluting with MeOH/H2O  and HPLC , flow rate 2 mL/min) to yield compounds 2 (12.5 mg) and 3 (21.3 mg). Sub-fraction C9 was chromatographed on RP-C18 column chromatography [MeOH/H2O ] to yield 8 subfractions C9–1 to C9-8. Fractions C9-3 by preparative HPLC , flow rate 2 mL/min) gave compounds 4 (20.4 mg) and 5 (10.5 mg). Fraction C9-4 followed by HPLC , flow rate 2 ml/min) led to the isolation of compounds 6 (22.2 mg) and 7 (17.1 mg). The 100% EtOH fraction was applied sequentially to Sephadex LH-20 using 100% MeOH and HPLC , flow rate 2 mL/min) to yield compounds 8 (18.5 mg) and 9 (16.0 mg).The aerial part of c 0.1, MeOH); UV (MeOH) λmax (log ε) 244 nm (3.8); IR (KBr) vmax 3404, 2973, 2938, 1697, 1616, 1073, 1055, 1010 cm–1; 1H and 13C NMR data, Tables m/z 925.5157 [M−H]− .white crystal; mentclass2pt{minimc 0.1, MeOH); UV (MeOH); λmax (log ε) 244 nm (3.8); IR (KBr) vmax 3400, 2970, 2938, 1698, 1617, 1069, 1053, 1008 cm–1; 1H and 13C NMR data, Tables m/z 1087.5682 [M−H]− .white, amorphous powder; mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 244 nm (3.8); IR (KBr) vmax 3402, 2971, 2938, 1702, 1616, 1060, 1054, 1010 cm–1; 1H and 13C NMR data, Tables m/z 967.5276 [M−H]− .white, amorphous powder; mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 244 nm (3.7); IR (KBr) vmax 3403, 2972, 2940, 1702, 1616, 1060, 1054, 1010 cm–1; 1H and 13C NMR data, Tables m/z 779.4597 [M−H]− .white, amorphous powder; mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 244 nm (3.9); IR (KBr) vmax 3403, 2972, 2940, 1702, 1616, 1060, 1010 cm–1; 1H and 13C NMR data, Tables m/z 471.3393 [M−H]− .white, amorphous powder; mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 244 nm (3.8); IR (KBr) vmax 3403, 2972, 2940, 1702, 1616, 1060, 1054, 1010 cm–1; 1H and 13C NMR data, Tables m/z 471.3452 [M + H]+ .white, amorphous powder, mentclass2pt{minimc 0.1, MeOH); IR (KBr) vmax 3403, 2972, 2940, 1702, 1616, 1060, 1054, 1010 cm–1; 1H and 13C NMR data, Tables m/z 943.5374 [M-H]− .white, amorphous powder, mentclass2pt{minimc 0.1, MeOH); UV (MeOH) λmax (log ε) 275 nm (3.6); IR (KBr) vmax 3403, 2972, 2940, 1702, 1616, 1060, 1054, 1010 cm–1; 1H and 13C NMR data: Tables m/z 907.5066 [M−H]− .White amorphous powder, mentclass2pt{minim1 (20 mg) was hydrolyzed with 1 N HCl  at 90 °C for 1 hours. The solution was neutralized by NaOH 10%, dried and suspended in H2O and partitioned with EtOAc. The residual H2O layer was concentrated and dissolved in pyridine (1.0 mL) and 5.0 mg of l-cysteine methyl ester hydrochloride was added. The mixture was kept for 1 hours at 60 °C, and then 4.4 μL of benzylisothiocyanate was added. This solution was filtered through a 0.2-µm Whatman hydrophilic membrane filter into an HPLC sample vials immediately before LC-MS analysis. Analysis was performed using an Agilent 1200 HPLC system  with INNO C18  at a column temperature of 30 °C. Chromatographic condition was carried out using a mobile phase of 27% MeCN/H2O isocratic elution at a flow rate 0.6 mL/min in 60 minutes. The sugar derivatives showed the retention time at 21.3 and 16.8 minutes, and were identical to the trimethylsilyl-l-cysteine derivatives of authentic d-glucose and l-rhamnopyranose, respectively.Compounds in vacuo and lyophilized respectively, the extraction yield of each extract was calculated by the percentage of each extract in 1 g of raw material. For preparation of stock solutions, plant extracts were dissolved in extracted solvents at concentrations of 1 mg/mL. This solution was filtered through a 0.2-µm Whatman hydrophilic membrane filter into an HPLC sample vials immediately before LC-MS analysis. Analysis and validation were performed using an Agilent 1200 HPLC system  with INNO C18  at a column temperature of 30 °C. All chromatographic separations were carried out using a mobile phase of 10% to 100% MeCN/H2O gradient elution at a flow rate 0.6 mL/min , where y and x correspond to the peak area and concentration, respectively. The relative amount of compound in each of the extracts (mg/g of the extract) were calculated using this equation.The plant extracts were prepared using different extraction solvents with water, 30% EtOH, 50% EtOH, 70% EtOH, 100% EtOH and MeOH, respectively. After the extracts were dried 2. The cell proliferation assay was carried out using the -2,5-diphenyl-2H-tetrazolium bromide (MTT)  or the WST-1 cell proliferation reagent . Briefly, C2C12 cells were seeded onto a 96-well plate at 4,000 cells/well and incubated for 8 hours. The cells were then treated with different concentrations of compounds or fraction dissolved in low-serum medium containing 5% FBS. After 24 hours or 48 hours of incubation, 2 mg/mL MTT solution (20 μL) was added to each well and incubated for 4 hours. Formazan was dissolved in 100 μL of DMSO and then absorbance was measured with an Absorbance Microplate Reader  at 550 nm. In addition, 10 μL of the WST-1 cell proliferation reagent was added to each well to evaluate cell viability, and incubated for 2 hours, and absorbance was measured at 440 nm. For the cell counting assay, experiments were performed using 36 mm cell culture dishes. After 48 hours of incubation with the tested compound, cells were harvested and evaluated with a CountessTM automated cell counter . For measuring the cell viability of cancer cell lines, MCF-7 and MDA-MB 231 cells were seeded onto 96-well plate at 4,000 cells/well and incubated for 8 hours. Next, cells were cultured in DMEM containing various compounds. After 2 days of incubation, cell viability was evaluated by the above procedure.C2C12 cell lines  were maintained in growth medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , and cells were incubated at 37 °C and 5% COµm, Immobilon-P, USA). Membranes were then incubated with primary antibodies for p-AMPKα Thr172, AMPKα, p-ACC Ser79, ACC  or mouse monoclonal actin . After incubation with secondary antibodies, membranes were detected using an enhanced chemiluminescence Western blot detection kit .C2C12 myoblasts were seeded into 6-well plates and incubated until a confluence of 70–80%. To prepare C2C12 myotubes, cells were incubated with DMEM supplemented with 2% horse serum . The differentiation medium was changed every 2 days until myotubes were formed. For Western blot analysis, myotubes or myoblasts were incubated with various concentrations of compounds or fractions for 30–60 minutes. The cells were washed with cold PBS and lysed using the lysis buffer  and centrifuged at 12,000 rpm for 20 minutes. The protein concentrations were determined using a protein assay kit . Aliquots of lysates were electrophoresed on 8% or 12% SDS-polyacrylamide gels and then transferred electronically to polyvinylidene fluoride (PVDF) membranes  was added to the culture cells followed by incubation at 37 °C for 2 hours. The cells were then washed three times with PBS and fixed with 4% formaldehyde in PBS for 15 minutes at room temperature. After washing three times with PBS, the cells were permeabilized with Triton X-100 buffer  for 20 minutes at room temperature. Next, the cells were incubated on ice for 20 minutes with 2 N HCl solution. After removing the acid solution, the cells were incubated with phosphate/citric acid buffer (pH 7.4) for 10 minutes at room temperature. Cells were washed three times with Triton X-100 permeabilization buffer and incubated overnight with anti-BrdU monoclonal antibody  diluted 1:50 in PBS (pH 7.4). Cells were continuously incubated with FITC-conjugated goat anti-mouse lgG  for 1 hour and then stained with 500 nM DAPI solution  for 10 minutes at room temperature. The slides were fixed with a mounting medium for fluorescence detection  and images were observed using fluorescence microscopy .C2C12 myoblasts were grown on sterile glass coverslips and the cells were treated with test samples. After incubation for 8 hours, 50 µM BrdU labeling solution was added to the culture medium and incubated for 2 hours. After removing BrdU medium, the cells were washed with PBS and harvested by trypsin solution. Cells were permeabilized using precooled 70% ethanol solution and further incubated at 4 °C for 1 hour. After centrifugation at 1000 rpm for 2 minutes, the pellet cells were continually added to a 2 N HCl solution containing 0.5% Triton X-100 reagent and incubated at room temperature for 30 minutes. Cells were then washed with PBS containing 1% BSA  and resuspended in PBS containing 0.5% Tween 20 and 1% BSA. Cells were incubated with anti-BrdU monoclonal antibody  for 4 hours at room temperature. Cells were resuspended in PBS containing 20 µg/mL propidium iodide (PI)  and kept in the dark for 30 minutes. Then, double-stained cells were analyzed using a flow cytometer  and at least 10,000 cells were counted for each analysis.For flow cytometry with anti-BrdU antibody staining, C2C12 myoblasts were maintained in 36-mm culture dishes. After treatment with different concentrations of the test compounds, the cells were incubated for 8 hours. Next, 50 µg/mL of PI in PBS solution and incubated at room temperature for 30 minutes. Cellular DNA content was analyzed using a flow cytometer . At least 10,000 cells were counted for each assay. At each phase of the cell cycle, cell distribution was displayed using a histogram.C2C12 myoblasts were maintained in 36-mm culture dishes and cells were treated with the test compounds. After 12 or 24 hours incubation, cells were harvested using trypsinization and washed twice with pre-cooled PBS. Cells were then fixed with pre-cooled 70% ethanol solution and incubated overnight at 4 °C. After washing with PBS, cells were stained sequentially with 20 4 cells/well using glucose-free medium containing 10% FBS. After 1 day of incubation, cells were treated with test samples or insulin (as a positive control) in the presence or absence of 2-NBDG . The cells were continually incubated for 1 hour at 37 °C and they were then washed with cold PBS. The fluorescence intensity was measured with excitation/emission at 450/535 nm using a fluorescence microplate reader .C2C12 myoblasts were seeded into black 96-well plates at 10µL). The cell lysates and ATP standard dilutions (20 µL) were transferred to white 96-well plates and then luciferase mixture (80 µL) was added to each well. ATP levels were measured using a luminescence microplate reader .Intracellular ATP production was evaluated using ATP bioluminescence determination assay kit . C2C12 myoblasts were grown on 24-well plates overnight and the cells were then exposed to test samples from 0 to 70 minutes. After that, the cells were lysed using lysis buffer  followed by Duncan’s or Tukey’s post hoc test as appropriate, conducting in SPSS Statistics 23 . Statistical significance was accepted at *Supporting information


R,S)-(4-nitrophenyl)-3-[(4-nitrobenzylidene)amino]-5(S)-(propan-2-yl)imidazolidin-4-one was derived from an initial reaction of 2(S)-amino-3-methyl-1-oxobutanehydrazine at its hydrazine moiety to provide a 4-nitrobenzylidine derivative, followed by a cyclization reaction with another molecule of 4-nitrobenzaldehyde to form the chiral five-membered imidazolidin-4-one ring.A 1:1 epimeric mixture of 2-(4-nitrophenyl)-5(S)-(propan-2-yl)imidazolidin-4-one, C19H19N5O5, was isolated from a reaction mixture of 2(S)-amino-3-methyl-1-oxobutanehydrazine and 4-nitrobenzaldehyde in ethanol. The product was derived from an initial reaction of 2(S)-amino-3-methyl-1-oxobutanehydrazine at its hydrazine group to provide a 4-nitrobenzylidene derivative, followed by a cyclization reaction with another molecule of 4-nitrobenzaldehyde to form the chiral five-membered imidazolidin-4-one ring. The formation of the five-membered imidazolidin-4-one ring occurred with retention of the configuration at the 5-position, but with racemization at the 2-position. In the crystal, N—H⋯O(nitro) hydrogen bonds, weak C—H⋯O(carbonyl) and C—H⋯O(nitro) hydrogen bonds, as well as C—H⋯π, N—H⋯π and π–π interactions, are present. These combine to generate a three-dimensional array. Hirshfeld surface analysis and PIXEL calculations are also reported.A 1:1 epimeric mixture of 3-[(4-nitrobenzylidene)amino]-2( In MolA, the configurations at atoms C12 and C14 are S. In MolB, the configurations at atoms C12 and C14 are R and S, respectively .The title compound, ly Fig. 1. The asyMolA and MolB, respectively. In the case of MolA, the Cremer & Pople puckering parameters  of 0.287 (2)Å and φ(2) of 54.7 (5)° for reference bond N11—C12; for MolB, Q(2) is 0.103 (3)Å and φ(2) is 230.3 (15)° for reference bond N21—C22. In MolA, the dihedral angles between the mean planes of the imidazoline ring and the benzene ring (pivot atom C121) is 45.83 (18)°, between the imidazoline ring and the benzene ring (pivot atom C131) is 28.04 (12)° and between the two benzene rings is 69.86 (11)°. In MolB, the dihedral angles between the mean planes of the imidazoline ring and the benzene ring (pivot atom C221) is 59.83 (13)°, between the imidazoline ring and the benzene ring (pivot atom C131) is 6.86 (13)° and between the two benzene rings is 66.38 (11).In both molecules, the imidazoline rings are puckered, the puckers in each case being a twist at C12—N13 and C22—N23 in c) has two nitro groups, whose O atoms can act as acceptors for hydrogen bonding, and three rings that are able to participate in π–π stacking. Fig. 1c) shows the two molecules labelled for the nitro O atom and the oxo atoms (O15 and O25), as well as the identification of ring A (benzene rings with pivot atoms C131 and C231), B (benzene rings with pivot atoms C121 and C221) and C (imidazoline rings).As seen, each of the molecules of the asymmetric unit Fig. 1c has twPLATON analysis , C—H⋯O(nitro) and C—H⋯O(oxo) hydrogen bonds, and C—H⋯π, N—O⋯π and π–π intermolecular interactions. All details of the hydrogen bonding (molecular contacts) and π–π stacking are given in Tables 1MolB and the nitro-group atoms O128/O129 in MolA , which generate chains running parallel to the , with an H⋯Cg2 distance of 2.95°, an angle at H of 128° and a C143⋯Cg2 distance of 3.638 (3)°.A ng Fig. 2. These πPLATON analysis. The Hirshfeld analysis, generated using CrystalExplorer , indicated red areas related to specific intermolecular short contacts  act as acceptors for H atoms of MolB.Briefly, the Hirshfeld surface analysis revealed that in PIXEL3.1  in the asymmetric unit and each has five O atoms that may be involved in the formation of hydrogen bonds, which are labelled in Fig. 1c). In short, each molecule has two 4-NO2-phenyl substituents, one substituent connected to the imine C atom, ring A , and the other to the imidazoline ring, ring C . In addition, there is a carbonyl O atom in heterocyclic ring C , together with a potential donor, i.e. the –NH group on the same ring.The compound crystallized with two molecules  and blue (MolB), the partner to the specific epimer in the molecular pair is coloured in standard element colours and any other relevant molecule is coloured grey.PIXEL energy calculations, as implemented in I and II connect MolA with MolA . These pairs relate MolA⋯MolA and MolB⋯MolB in chains, as can be visualized in Figs. 4I and pairs VII differ by about 5 kJ mol−1  and this may be due to the presence of an additional C—H⋯π interaction in I that is not detected in VIII[VII?]. The similar substructures a/IIbII and a/IXbIX, are built utilizing similar C—H⋯O interactions, involving the O atom of the nitro group of ring B. Nevertheless, the total energies for those pairs also differ by about 5 kJ mol−1, this time with a higher value for pairs IX due to a higher contribution of the dispersion term.Substructures A Table 3 and subtB Table 3. There iIII. In this substructure, the nitro O atoms of ring A act as acceptors in both molecules, but they interact with different H atoms, e.g. (i) a methyl H atom to form the O138 ⋯H24D—C243 hydrogen bond in the MolA⋯MolB contact and (ii) an H atom of the imidazoline ring thereby generating an O239⋯H12—C12 hydrogen bond in the MolB⋯MolA contact  with the molecules at  (for VI) and  (for VII). Although those molecules are not connected in a classical way, the pairs make a significant contribution to the lattice stabilization energy, i.e. −32.5 and −25.9 kJ mol−1, respectively, for VI and VII. These pairs are depicted in Fig. 6VI shown in order to clarify a possible path explaining the electronic interactions, while in pair VII, the those interactions are made viamolB of the asymmetric unit.Finally, two more substructures have been identified as energetically important in the stabilization of the supramolecular structure for MolA and MolB. The FP plots show two pairs of spikes pointing south-west and ending at  that are due to O⋯H/H⋯O close contacts, the light blue in the middle is due to the H⋯H and C⋯C close contacts. The percentages for atom–atom contacts were taken from the FP plots and are given in Table 4MolB and the N⋯H and H⋯H contacts that are higher in MolA.Fig. 9et al., 20161 is the 1:1 epimeric mixture of 5-isobutyl-2-(2-nitrophenyl)-3-(phenylamino)imidazolidin-4-one -4-benzyl-1-methyl-5-oxoimidazolidin-2-yl]benzonitrile -5-benzyl-2-(4-fluorophenyl)-3-methylimidazolidin-4-one imidazolidin-1-ium chloride --4-(4-hydroxybenzyl)-5-oxoimidazolin-1-yl]acetyl}glycyl)-l-phenylalanyl-l-leucine 4′-O-ester -4-isopropyl-5-oxo-3-(3-oxobutyl)-1-(pyridin-2-yl)imidazolidin-2-yl]benzonitrile  was converted to 2(S)-amino-3-methyl-1-oxo-butanehydrazine (3) in two stages, as outlined in Scheme 13 (1 mmol) in ethanol (10 ml) was added 4-nitrobenzaldehyde (2.2 mmol). The reaction mixture was stirred for 20 h at 351 K and rotary evaporated. The residue was purified by column chromatography using a mixture of 9.7:0.3 (v/v) dichloromethane–methanol as eluent. Further purification was achieved by crystallization from ethanol. The crystal of 1 used in the structure determination was obtained by slow evaporation of an ethanol solution at room temperature.To a stirred solution of 1H NMR : δ 0.96 , 0.97 , 1.42 , 2.00 , 2.09 , 4.06 , 7.66–7.71 , 7.82–7.85 , 8.422–8.48 .M.p. 411–414 K. 13C NMR : δ 17.2,17.4,18.9, 29.9, 30.3, 61.8, 62.53, 124.0, 123.7, 128.1, 128.1, 128.3, 128.9, 140.2, 1140.1, 146.2, 146.3, 147.7, 148.2, 148.7, 171.1, 171.6. IR : ν 3015 (br), 1670, 1518, 1337.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989019013938/lh5929sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019013938/lh5929Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019013938/lh5929Isup3.cmlSupporting information file. DOI: 1944779, 1944779CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Here we assessed the effects of ambient fine particulate matter  were compared to in situ ambient air exposed mice as the study control. The concentrated PM2.5 exposed group presented increased levels of the oxidized nucleoside 8-oxo-7,8-dihydro-2′-deoxyguanosine in lung and kidney DNA and increased levels of the etheno adducts 1,N6-etheno-2′-deoxyadenosine and 1,N2-etheno-2′-deoxyguanosine in kidney and liver DNA, respectively. Apart from the genotoxic effects, the exposure to PM2.5 led to decreased levels of the epigenetic mark 5-hydroxymethylcytosine (5-hmC) in lung and liver DNA. Changes in lung, liver, and erythrocyte antioxidant enzyme activities were also observed. Decreased glutathione reductase and increased superoxide dismutase (SOD) activities were observed in the lungs, while the liver presented increased glutathione S-transferase and decreased SOD activities. An increase in SOD activity was also observed in erythrocytes. These changes are consistent with the induction of local and systemic oxidative stress.Mice exposed to concentrated ambient PM2.5 at a concentration that mimics 24-h exposure to the mean concentration found in ambient air presented, after 3 months, increased levels of DNA lesions related to the occurrence of oxidative stress in the lungs, liver, and kidney, in parallel to decreased global levels of 5-hmC in lung and liver DNA. Genetic and epigenetic alterations induced by pollutants may affect the genes committed to cell cycle control, apoptosis, and cell differentiation, increasing the chance of cancer development, which merits further investigation.Mice exposed daily to PMThe online version of this article (10.1186/s12989-018-0276-y) contains supplementary material, which is available to authorized users. Epidemiologic studies conducted mostly in the United States and Europe have shown that cardiovascular diseases, respiratory diseases, and cancer of different tissues  are empirically associated with exposure to air pollution –12. None10, and sulfur oxides) has been observed, except for ozone, even with the increase in vehicular fleet and fuel consumption pyrene, can be metabolized to reactive intermediates able to damage biomolecules, including the DNA [a]pyrene pyrene , 27. Thea]pyrene . Based oAmong the useful biomarkers for the assessment of chemical carcinogenesis pathways, DNA adducts and oxidized nucleobases or nucleosides provide information on early events triggered by exposure, such as the bioactivation of xenobiotics and oxidative stress, while DNA methylation changes depict early biological effects that may evolve to cancer . AccordiN2-etheno-2′-deoxyguanosine  in vitro presented decreased 5-hmC levels -2′-deoxyguanosine and [15N5]-2′-deoxyadenosine were provided by Cambridge Isotope Laboratories . All the other reagents were obtained from Sigma-Aldrich Co. . The catalogue numbers of the reagents are described in Additional file All the chemicals employed here were of the highest purity grade commercially available. Chromatography grade acetonitrile and methanol, isopropyl alcohol, chloroform, hydrochloric acid, ethanol, magnesium chloride, and tris(hydroxymethyl)-aminomethane were obtained from Carlo Erba Reagents . Sodium hydroxide, potassium phosphate, and ammonium acetate were acquired from Merck . DNA extraction solutions were obtained from QIAGEN . DNase I was acquired from Bio Basic Inc. . 5N5-2′-deN2-Etheno-2′-deoxyguanosine  and the isotopic standards [15N5]1,N2-εdGuo, [15N5]1,N6-εdAdo and [15N5]8-oxodGuo were prepared as described +, m/z 176 [M – 2′-deoxyribose + H]+. [15N5]1,N2-εdGuo: positive ESI-MS: m/z 297 [M + H]+, m/z 181 [M – 2′-deoxyribose + H]+. [15N5]1,N6-εdAdo: UV, λmax = 260 nm, ε = 10,300 M− 1 cm− 1 [m/z 281 [M + H]+, m/z 165 [M – 2′-deoxyribose + H]+. [15N5]8-oxodGuo: UV, λmax = 294 nm, ε = 9700 M− 1 cm− 1 [m/z 289 [M + H]+, m/z 173 [M – 2′-deoxyribose + H]+.1,escribed , 70. The 1 cm− 1 , pH 7.0; 1 cm− 1 , pH 7.0; 1 cm− 1  in water2O, λmax 277 nm, ε = 8500 M− 1 cm− 1 [m/z 242 [M + H]+, m/z 126 [M – 2′-deoxyribose + H]+. The standard was used for quantification of 5-mC and 5-hmC, but the retention time of 5-hmC was defined using a sample of calf thymus DNA presenting a main peak with m/z 258 [M + H]+ → m/z 142 [M - 2′-deoxyribose + H]+.5-methyl-2′-deoxycytidine (5-mC) standard was obtained by HPLC purification from a solution of commercial dCyd (1 mg/mL). Its spectral properties are as follows: UV in H 1 cm− 1 ; positiv⍛ 1310/09). The animals were submitted to a controlled exposure system in a Harvard Ambient Fine-Particle Concentrator designed to concentrate PM2.5 up to 30 times. It works separating particles from gases by particle inertia using virtual impaction 1,N6-εdAdo and [15N5]1,N2-εdGuo (250 fmol/μL), and 6 μL (15 units) of deoxyribonuclease I  were added. The samples were incubated at 37 °C, 90 rpm for 1 h. Then, 6 μL (0.006 units) of phosphodiesterase I from Crotalus atrox  and 7.5 μL (15 units) of alkaline phosphatase from bovine intestinal mucosa  were added, incubating again at 37 °C, 90 rpm for 1 h. Finally, samples were centrifuged at 14,000 g for 10 min. Aliquots of 10 μL were withdrawn for quantification of deoxynucleosides  by HPLC/PDA. The residual volume of sample was submitted to solid phase extraction, as described below.For analyses of etheno adducts in liver, aliquots containing 150 μg of DNA were transferred to a final volume of 200 μL of deionized water. 7.5 μL of 200 mM Tris/MgClAnalyses of etheno adducts in kidney and lung were performed using 100 μg of DNA, maintaining the correct proportions of enzymes and the other reagents.15N5]8-oxodGuo in the injection volume.The same procedure was used for quantification of 8-oxodGuo in liver, lung and kidney, using 80 μg of DNA and 1000 fmol of the internal standard [15N5]1,N6-εdAdo were also hydrolyzed for analyses of 5-mC and 5-hmC, using the same procedure adjusted to a final volume of 60 μL. The hydrolyzed samples were, then, added to 140 μL of acetonitrile, vortexed for 20 s, centrifuged at 9300 g for 10 min, and aliquots of 20 μL were injected into the HPLC-ESI-MS/MS system described below.Samples containing 12 μg DNA and 3000 fmol of + → m/z 160 [M - 2′-deoxyribose + H]+ and m/z 281 [M + H]+ → m/z 165 [M - 2′-deoxyribose + H]+ for detection of 1,N6-εdAdo and respective internal standard [15N5]1,N6-εdAdo; m/z 292 [M + H]+ → m/z 176 [M - 2′-deoxyribose + H]+ and m/z 297 [M + H]+ → m/z 181 [M - 2′-deoxyribose + H]+ for detection of 1,N2-εdGuo and respective internal standard [15N5]1,N2-εdGuo; m/z 284 [M + H]+ → m/z 168 [M - 2′-deoxyribose + H]+ and m/z 289 [M + H]+ → m/z 173 [M - 2′-deoxyribose + H]+ for detection of 8-oxodGuo and respective internal standard [15N5]8-oxodGuo; m/z 242 [M + H]+ → m/z 126 [M - 2′-deoxyribose + H]+ for detection of 5-mC; m/z 258 [M + H]+ → m/z 142 [M - 2′-deoxyribose + H]+ for detection of 5-hmC; m/z 228 [M + H]+ → m/z 112 [M - 2′-deoxyribose + H]+ for detection of dCyd.Analyses were carried out with multiple reaction monitoring (MRM) by using the following fragmentations: 15N5]8-oxodGuo (1000 fmol); 1 to 40 fmol of 1,N6-εdAdo and 1,N2-εdGuo, with fixed amounts of [15N5]1,N6-εdAdo and [15N5]1,N2-εdGuo (200 fmol); and 150 to 1500 pmol of dCyd, 5 to 500 fmol of 5-mC for quantification of 5-hmC, and 5 to 80 pmol of 5-mC for quantification of 5-mC, with a fixed amount of [15N5]1,N6-εdAdo (300 fmol). Data were acquired and processed using Analyst software 1.4 (Applied Biosystems/MDS Sciex). The molar fractions 8-oxodGuo/dGuo, 1,N6-εdAdo/dAdo, 1,N2-εdGuo/dGuo, 5-mC/(5-mC + 5-hmC + dCyd), 5-hmC/(5-mC + 5-hmC + dCyd) present in each DNA sample were determined. The following chromatography conditions were used for the analyses. All solvents used were previously filtered and degassed.The calibration curves were constructed at the intervals of 367 to 5875 fmol of 8-oxodGuo, with a fixed amount of 8-oxodGuo (1000 fmol) to 100 μg of calf thymus DNA and carrying out the enzymatic hydrolysis and analysis. Samples were processed in quadruplicate in two different days. The limit of quantitation (LOQ) was estimated from the lowest amount of 8-oxodGuo injected on column, with S/N = 10.Method accuracy and precision were determined by adding varying amounts of 8-oxodGuo  and a fixed amount of 1,N6-εdAdo and [15N5]1,N2-εdGuo (200 fmol in the injection volume) were added to 100 μg of calf thymus DNA and the analyses were carried out. Samples were processed in quadruplicate in two different days for method accuracy and precision assessment. Recovery was calculated by adding the internal standards [15N5]1,N6-εdAdo and [15N5]1,N2-εdGuo (200 fmol) to 100 μg of calf thymus DNA before and after solid phase extraction, carrying out the analyses as described above. The DNA used for recovery calculation was contaminated with 7.5 fmol of 1,N6-εdAdo and 20 fmol of 1,N2-εdGuo in the beginning of the process. The limit of detection (LOD) was estimated from the lowest amount of adducts added to the calf thymus DNA sample.Varying amounts of 1,3OH  at a flow rate of 1 mL/min and 30 °C. The PDA detector was set at 260 nm. Calibration curves were constructed at intervals of 0.05–1 nmol for dGuo and dAdo.The quantification of normal 2′-deoxynucleosides was carried out with a Shimadzu  HPLC system equipped with two LC-20AT pumps, a photo diode array detector (PDA-20AV), an auto-injector (Proeminence SIL-20 AC), and a column oven (CTO-10AS/VP) controlled by a CBM-20A communication module and the software LC-Solution. Elution system was as follows: a 250 mm × 4.6 mm i.d., 5 μm, Luna C18(2) column  attached to a C18(2) guard column , eluted with a gradient of 0.1% formic acid and CH2.5) were compared using t test for 8-oxodGuo, 1,N2-εdGuo, 1,N6-εdAdo, 5-mC and antioxidant enzymes activities, or Mann-Whitney test for 5-hmC. Results were considered statistically significant when P value was less than 0.05. Normality of the samples was checked by the Kolmogorov-Smirnov test and homogeneity of variances with the Levene test. The statistical analyses were conducted using GraphPad Prism version 6 for Windows .Data were expressed as average ± SEM or average ± SD, as indicated in the text and tables. Means between the two groups , is presented in Additional file For 8-oxodGuo quantification, the system illustrated in Additional file N6-εdAdo and 1,N2-εdGuo   were 0.3 fmol for 1,N6-εdAdo and 1 fmol for 1,N2-εdGuo.The calibration curves presented in Additional file o) Table . Intra-d2.5 from a busy traffic crossroad in São Paulo City presented altered antioxidant system activity in lung, liver, and erythrocytes, concomitantly to increased levels of lung and kidney 8-oxodGuo, kidney 1,N6-εdAdo, and liver 1,N2-εdGuo, as well as decreased levels of lung and liver 5-hmC.A complex mixture of gases, vapors and particles of varying size and chemical composition is found in urban air, resultant from diverse polluting human activities , 17. To 2.5 (682 ± 532 μg/m3) for 1 h daily for 3 months (from infancy to adulthood) were compared to a control group exposed to ambient air, and both groups were treated inside the chambers of a Harvard Ambient Fine-Particle Concentrator located near the crossroad. The total intake dose (ID) of 70.2 μg (1.08 μg per day of exposure) was estimated according to Alexander et al. . If mice were exposed to 20 μg/m3 PM2.5 for 24 h, then the estimated intake dose of particles would be 0.864 μg/day (56.16 μg after 65 days). Thus, we can assume that the adopted exposure protocol mimics a 24 h exposure to concentrations of ambient fine particulate matter found in São Paulo City.In the present study, mice exposed to ambient air enriched in PMr et al. , with thr et al.  pyrene in vitro and in vivo, respectively. It was evident the broad effect of benzo[a]pyrene modulating the DNA methylation in both systems . A total of 153 genes with altered promoter methylation (hypermethylation or hypomethylation) were found in the exposed murine skin, 45 of them (29%) occurred in the exposed human cells. In contrast, 10,447 genes with altered promoter methylation were detected in the exposed human cells [Jiang and coworkers  assessedan cells . Delattean cells  providedan cells . StudiesLINE1 and Alu repeat elements have been used as surrogate assays for quantifying total 5-mC in the majority of the studies on associations between ambient PM exposure and global DNA methylation alterations , 66, 67.2, PM10, PM2.5 and O3 was associated with decreased total 5-mC levels in human blood DNA; Janssen and coworkers [2.5 exposure; and Nys and coworkers [2.5 and PM10 and decreased 5-mC and 5-hmC total levels in human buccal cell DNA. So far, to the best of our knowledge, this study is the first to quantify 5-mC and 5-hmC by HPLC-ESI-MS/MS in different tissues  of mice selectively exposed to ambient PM2.5. Our observations encourage further investigation of the effects of PM2.5 exposure on the profiles of the genomic methylation and hydroxymethylation of target tissues to obtain a better understanding of the deregulated molecular pathways that may culminate in disease  development [Using HPLC based methods, De Prins and coworkers  found thoworkers  observedoworkers  were theelopment .2.5 at a concentration that mimics 24-h exposure to the mean concentration found in ambient air, presented altered antioxidant system activity in lung, liver, and erythrocytes, increased levels of DNA lesions related to oxidative stress in lung, liver, and kidney, and decreased global levels of 5-hmC in lung and liver DNA. This is the first direct demonstration of the occurrence of these effects due to exposure to PM2.5 as it occurs in the environment. Genetic and epigenetic alterations induced by pollutants may affect the genes committed to cell cycle control, apoptosis, and cell differentiation, increasing the chance of cancer development, which merits further investigation.Mice exposed daily, for 3 months, to PMAdditional file 1:Table S1. Reagents used in the study. Table S2. Parameters used in the ESI-MS/MS equipment for detection of the lesions and epigenetic marks in DNA. Figure S1. System of two columns used for 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) analyses. A) Configuration used in the first 16 min and from 32 to 46 min of the chromatography; B) Configuration used in the interval 16 – 32 min, allowing further separation and peak narrowing in column B prior to elution to the ESI source of the mass spectrometer. Figure S2. Calibration curves obtained by HPLC-ESI-MS/MS for quantification of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), 1,N2-etheno-2′-deoxyguanosine , 1,N6-etheno-2′-deoxyadenosine , 2′-deoxycytidine (dCyd), 5-methyl-2′-deoxycytidine (5-mC), and 5-hydroxymethyl-2′-deoxycytidine . (DOCX 738 kb)