Dimocarpuslongan Lour. seeds. Its structure was determined as 3-(2-acetyl-1H-pyrrol-1-yl)-5-(prop-2-yn-1-yl)dihydrofuran-2(3H)-one by spectroscopic methods and HRESIMS.A new natural product named longanlactone was isolated from  Dimocarpuslongan Lour. ] is an evergreen tree of the Sapindaceae family, which is widely cultivated in Southern China, India, and Southeast Asia + ; positive ESIMS m/z 232 [M+H]+, 254 [M+Na]+, 270 [M+K]+; [α]D20 -9° (C = 0.2 in acetone); UV: λmax (ε) 202 (9867), 243 (sh. 3253), 253 (3541), 286 (5924); IR (KBr) νmax: 3306, 3130, 3116, 2992, 2926, 2111, 1770, 1640, 1548, 1486, 1037, 870, 775, 760 cm−1; 1H-NMR  and 13C-NMR  see Dimocarpuslongan Lour. seeds was identified as 3-(2-acetyl-1H-pyrrol-1-yl)-5-(prop-2-yn-1-yl)dihydrofuran-2(3H)-one by its spectral data, including MS, and 1D and 2D NMR. In summary, the new compound longanlactone isolated from


Landoltia punctata led to the isolation and identification of 17 compounds, four of which were new and identified as -9,19-cycloartane-3,22,24,25-tetraol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (1), -9,19-cycloartane-3,24,25-triol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (2), 3,4'-dihydroxy-7,3'-dimethoxyflavan-5-O-β-d-glucopyranoside (3) and 3,4'-dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-d-glucopyranoside (4). Their structures were elucidated by spectroscopic, chemical, and biochemical methods. Thus, cycloartane triterpenoids were discovered in the Lemnaceae family for the first time. Compound 3 showed antioxidant capacity in the positively charged 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS+•) and superoxide anion radical scavenging assays.Chemical investigation on the constituents of  Lemna, Landoltia, Spirodela, Wolffia and Wolffiella -[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (1), -9,19-cycloartane-3,24,25-triol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (2), 3,4'-dihydroxy-7,3'-dimethoxyflavan-5-O-β-d-glucopyranoside (3) and 3,4'-dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-d-glucopyranoside (4), (5), luteolin (6), apigenin-7-O-β-glucoside (7), luteolin-7-O-β-glucoside (8) , 6,apigenin  [13], 6-apigenin  [14], β-tosterol , stigmasmasterol  [17) [163 were described.This is the first report of cycloartane-type triterpenoids from the Lemnaceae family. Here the isolation and structural elucidation of these compounds as well as bioactivities of compound 1 was obtained as an amorphous powder. Its HR-ESI-MS quasi-molecular ion peak at m/z 985.5371 ([M+Na]+) corresponded to a molecular formula of C48H82O19, which was supported by the NMR signals. 1H-NMR spectrum of 1 showed characteristic signals of a cyclopropane CH2 at δH 0.64  and 0.44 , six tertiary Me groups  and a secondary Me group at δH 0.95 (H3-21) -[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside. Consequently, the structure of 2  supported a molecular formula of C48H82O18. The NMR spectra of 2 were very similar to those of 1, except for one less oxygenated methine at δC 71.3. Detailed inspection of the HMBC spectrum led to a conclusion that 2 had one less OH on C-22 than 1, on the basis of the key HMBC correlation of 21-CH3 (δH 1.01) with C-22 (δC 23.2). The linkage of three glucoses on the triterpenoid aglycon was determined to be the same way as 1 on the basis of HMBC and 1H,1H-COSY signals. The stereochemistry of 2 was determined on the basis of NOESY cross peaks of H-8 and H-19a/18-CH3, 18-CH3 and H-12a, H-19a and 29-CH3, H-16b and 30-CH3, 21-CH3 and H-17, H-17 and 30-CH3. Acid hydrolysis of 2 gave d-glucoses, which was identified by comparing the optical rotation value with an authentic sample. Finally the structure of 2 was determined to be -9,19-cycloartane-3,24,25-triol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside.The HR-ESI-MS spectrum of 3 was obtained as colorless needles. Its molecular formula was deduced to be C23H28O11 from the quasi-molecular ion peak at m/z 479.1549 ([M–H]−) in the HR-ESI-MS spectrum, indicating 10 degrees of unsaturation. The IR spectrum revealed the existence of OH (3434 cm−1) and aromatic ring (1623 cm−1). The 1H-NMR spectrum displayed two meta-coupled protons at δH 6.49  and 6.29 , and three ABX system aromatic protons at δH 7.22 , 6.99 , and 6.87 . In addition, two methoxyl H-atoms at δH 3.82  and 3.95 , two CH at δH 4.29 (br. s) and 4.98 (br. s), and one CH2 at δH 3.09  and 3.02  were observed. Meanwhile an anomeric H-atom at δH 4.95  together with the signals at δH 3.42-3.55 indicated the presence of a β-linked glycosyl group. Enzymatic hydrolysis of 3 with β-d-glucosidase afforded d-glucose, which was identified by direct comparison with the authentic sample by TLC -[β-d-glucopyranos-yl(1→6)]-β-d-glucopyranoside (1): Amorphous powder.  −3.0 . IR (KBr) νmax 3411, 2932, 1075 cm−1. 1H and 13C-NMR: See m/z 985.5371 [M+Na]+ .-9,19-Cycloartane-3,24,25-triol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (2): Amorphous powder.  −4.0 . IR (KBr) νmax 3418, 2891, 1043 cm−1. 1H and 13C-NMR: See m/z 969.5406 [M + Na]+ .3,4'-Dihydroxy-7,3'-dimethoxyflavan-5-O-β-d-glucopyranoside (3): Colorless needles.  −42.0 . UV (MeOH) λmax (log ε) 218 (4.60), 227 (3.96), 282 (3.52) nm. IR (KBr) νmax 3434, 1623, 1080 cm−1. 1H and 13C-NMR: See m/z 479.1549 . 3,4'-Dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-d-glucopyranoside (4): Amorphous powder.  −20.0 . UV (MeOH) λmax (log ε) 226 (4.02), 280 (3.60) nm. IR (KBr) νmax 3420, 2926, 1597, 1424, 1079 cm−1. 1H and 13C-NMR: See m/z 509.1662 .+•, and superoxide anion radical scavenging models complying with the previously published literature [According to the reported , antifunterature .L. punctata. The result showed that apigenin or luteolin flavonoids are the main constituents of this species, which is in good agreement with the previous reports and our previous study results that the transcripts for key enzymes of flavonoid biosynthesis in L. punctata expressed in high abundance at the transcriptional level [L.punctata could be related to environmental stressors. Meanwhile cycloartane triterpenoids were discovered in Lemnaceae family for the first time in this study. Many cycloartane triterpenoids possessed diverse bioactivities such as anti-inflammatory, anti-tumor, anti-viral, immuno-regulatory, hypoglycemic, cardiovascular system, nervous system and hepato-protective effects [L.punctata is a new source for cycloartane triterpenoids. In this study, we identified 12 flavonoids including two new flavanol glucosides from al level ,10,34. F effects ,36, some


Among the known ones, 6, 10, and 13 were isolated from the rosmarinus genus for the first time. Their structures were elucidated by chemical and spectroscopic methods. Moreover, the effects on sodium oleate-induced triglyceride accumulation (TG) in HepG2 cells of the above-mentioned compounds and 16 other isolates (17–32) reported previously to have been obtained in the plant were analyzed. Results show that eight kinds of flavonoids  and seven kinds of other known isolates  possessed significant inhibitory effects on intracellular TG content in HepG2 cells. Among them, the activities of compounds 1 and 20 were comparable to that of orlistat, which suggested that these compounds in this plant might be involved in lipid metabolism.Sixteen flavonoids ( Rosmarinus officinalis L., is a perennial edible herb, belonging to Labiatae, commonly known as rosemary. R. officinalis extract has been reported to have antioxidant, anti-inflammatory, antidiabetic and anticancer properties , lutcuronide  c = 0.79, MeOH)}. Its molecular formula was determined to be C23H20O13 by negative-ion HRESI-TOF-MS . Acid hydrolysis of 1 yielded d-glucuronic acid, which was identified by comparing the Rf value on TLC plate with the authentic sample. The 1H- and 13C-NMR , 6.51 , 6.81 , 7.01 , 7.66 , 7.69 , 12.94 ), a β-d-glucuronyl moiety ), together with an acetyl group ; δC 20.9 (COCH3), 169.1 (COCH3)) in the structure. A downfield shift of 0.3 ppm for the C-2′′, and upfield shift by 2.0 ppm both for the C-1′′ and 3′′ compared with those of luteolin 3′-O-β-d-glucuronide (5)  to obtain 14 fractions (Fr. 9-10-2-1–9-10-2-14). Fractions 9-10-2-5 (21.5 mg) and 9-10-2-6 (14.5 mg) were combined and subjected to PHPLC ) to give homoplantaginin . Fraction 9-10-4 was separated by PHPLC ) to yield 13 fractions (Fr. 9-10-4-1–9-10-4-13). Fraction 9-10-4-12 (116.0 mg) was subjected to silica gel CC  → MeOH) and PHPLC ) to give luteolin 3′-O-β-d-glucuronide , luteolin 3′-O-(3′′-O-acetyl)-β-d-glucuronide , and luteolin 3′-O-(4′′-O-acetyl)-β-d-glucuronide . Fraction 10 (6.3 g) was isolated by PHPLC through gradient elution ) to yield 35 fractions (Fr. 10-1–10-35). Fraction 10-24 (142.2 mg) was purified by Sephadex LH-20 CC (MeOH) and PHPLC ) to yield 6-hydroxy luteolin-7-O-β-d-glucopyranoside . Fraction 10-25 (149.4 mg) was separated by PHPLC ) to yield six fractions (Fr. 10-25-1–10-25-6). Fraction 10-25-5 (20.8 mg) was further purified by Sephadex LH-20 ), and luteolin-7-O-β-d-lutinoside  was obtained. Fraction 10-26 (93.7 mg) was separated by PHPLC ) to yield luteolin-7-O-β-d-glucoside . Fraction 10-27 (756.1 mg) was subjected to Sephadex LH-20 CC (MeOH) to yield nine fractions (Fr. 10-27-1–10-27-9). Fraction 10-27-5 (114.3 mg) was isolated by PHPLC ) to give both officinoflavonoside B  and apigenin-7-O-β-d-lutinoside . Furthermore, fraction 10-27-7 (149.8 mg) was purified by PHPLC ) to yield luteolin 3′-O-β-d-glucuronide  and nepitrin . Fraction 10-28 (473.3 mg) was also subjected to Sephadex LH-20 CC (MeOH) to yield seven fractions (Fr. 10-28-1–10-28-7). Fraction 10-28-6 (222.0 mg) was prepared by PHPLC ) to give luteolin 3′-O-β-d-glucuronide . Furthermore, fraction 10-30 was isolated by Sephadex LH-20 CC (MeOH) and PHPLC ) to yield officinoflavonoside A . Fraction 10-31 (198.5 mg) was separated by PHPLC ) to give 4′,5,7-trihydroxy-3′-O-β-d-glucuronic acid-6′′-methyl ester .The EtOH fraction (36 g) was subjected to normal phase silica gel CC (CHCl3 partition (200 g) of the rosemary extract was subjected to silica gel CC  → CHCl3–MeOH–H2O  → MeOH) to yield 23 fractions (Fr. 1–23). Fraction 9 (56.3 g) was further subjected to silica gel CC (Pet. Ether (PE) → PE-EtOAc  → EtOAc) to yield 19 fractions (Fr. 9-1–9-19). Fraction 9-16 (5024.0 mg) was purified by PHPLC ), and 13 fractions (Fr. 9-16-1–9-16-13) were given. Fraction 9-16-4 (629.8 mg) was subjected to PHPLC ) to give acacetin .The CHClOfficinoflavonoside A (1): Yellow powder; [α]c = 0.79, MeOH); IR νmax (KBr) cm−1: 3209, 2927, 2861, 1736, 1656, 1607, 1505, 1435, 1356, 1301, 1255, 1167, 1081, 1039; UV λmax (MeOH) nm (log ε): 335 (4.20), 265 (4.15). 1H-NMR  and 13C-NMR  spectroscopic data, see m/z 503.0838 [M – H]− .Officinoflavonoside B (2): Yellow powder; [α]c = 0.59, MeOH); IR νmax (KBr) cm−1: 3332, 2923, 1658, 1603, 1570, 1514, 1462, 1355, 1276, 1192, 1067, 1014; UV λmax (MeOH) nm (log ε): 340 (4.17), 267 (4.11). 1H-NMR  and 13C-NMR  spectroscopic data, see m/z 6231595 [M – H]− .6′′-O-(E)-p-Coumaroylnepitrin (13): Yellow powder; The NMR data of 13 in DMSO-d6 are reported for the first time. 1H-NMR : δ 6.70 , 6.96 , 7.45 , 6.89 , 5.23 , 3.41 , 3.39 , 3.30 , 3.86 , , 4.43 , H2-6′′), 7.27 , 6.60 , 7.46 , 6.29 , 3.77 , 13.03 . 13C-NMR : δ 164.5 (C-2), 102.3 (C-3), 182.0 (C-4), 152.5 (C-5), 132.5 (C-6), 156.1 (C-7), 94.0 (C-8), 152.0 (C-9), 105.7 (C-10), 120.8 (C-1′), 113.1 (C-2′), 145.9 (C-3′), 150.5 (C-4′), 115.9 (C-5′), 119.0 (C-6′), 99.8 (C-1′′), 72.9 (C-2′′), 76.3 (C-3′′), 69.9 (C-4′′), 73.7 (C-5′′), 63.4 (C-6′′), 124.6 (C-1′′′), 115.5 , 129.9 , 159.7 (C-4′′′), 144.9 (C-7′′′), 113.4 (C-8′′′), 166.4 (C-9′′′), 60.2 (6-OCH3). Negative-ion mode m/z 623.1413 [M − H]− .6′′-O-(E)-Feruloylnepitrin (14): Yellow powder; The NMR data of 14 in DMSO-d6 are reported for the first time. 1H-NMR : δ 6.63 , 6.95 , 7.39 , 6.83 , 7.39 , 5.23 , 3.40 , 3.38 , 3.32 , 3.83 , , 4.44 , H2-6′′), 7.13 , 6.60 , 6.83 , 7.45 , 6.37 , 3.76 , 13.07 , 3.71 . 13C-NMR : 164.6 (C-2), 102.1 (C-3), 182.0 (C-4), 152.4 (C-5), 132.4 (C-6), 156.1 (C-7), 94.0 (C-8), 152.0 (C-9), 105.7 (C-10), 120.6 (C-1′), 113.0 (C-2′), 146.0 (C-3′), 150.5 (C-4′), 115.8 (C-5), 119.1 (C-6′), 99.8 (C-1′′), 73.0 (C-2′′), 76.3 (C-3′′), 69.7 (C-4′′), 73.7 (C-5′′), 63.1 (C-6′′), 125.2 (C-1′′′), 111.0 (C-2′′′), 147.7 (C-3′′′), 149.3 (C-4′′′), 115.3 (C-5′′′), 122.7 (C-6′′′), 145.2 (C-7′′′), 113.8 (C-8′′′), 166.4 (C-9′′′), 60.2 (6-OCH3), 55.4 (3′′′-OCH3). Negative-ion mode m/z 653.1514 [M − H]− .1 and 2. A solution of officinoflavonoside A  was hydrolyzed with 10% H2SO4 in 50% EtOH (2 mL) at 100 °C for 3 h. The reaction mixture was neutralized with ion exchange resin Amberlite IRA-400 (OH− form) and evaporated to dryness. The sugar was found to be glucuronide by comparison of the Rf value (Rf = 0.21) on TLC plate ) with the authentic d-glucuronide.Acid Hydrolysis of 2, 2.0 mg) in 1 M HCl (1 mL) was heated under reflux for 3 h, respectively. The reaction mixture was dealt with the similar methods as those used to officinoterpenosides A–D [l-rhamnose (i) and d-glucose (ii) from 2 presented in the aqueous was carried out by comparison of its retention time and optical rotation with that of authentic samples, tR: (i) 9.1 min , and (ii) 17.6 min .Meanwhile, a solution of officinoflavonoside B , 2.0 mg Materials. A human hepatoma HepG2 cell line was obtained from Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College . Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin were purchased from Thermo SCIENTIFIC . Fetal Bovine Serum (FBS) was purchased from PAN-Biotech GmbH . TG assay kits were purchased from Wako Pure Chemical Industries, Ltd. . Sodium oleate and orlistat were obtained from Sigma-Aldrich Corporation .Cell culture. HepG2 cells were grown in DMEM supplemented with 10% FBS and 1% antibiotics (100 unit/mL penicillin and 100 mg/mL streptomycin). Cells were maintained in an atmosphere of 95% air and 5% CO2 at 37 °C in subconfluent condition.Sodium oleate-induced TG accumulation. TG accumulation inhibitory effects were screened as previous report [R. officinalis for another 48 h. Meanwhile, an anti-obesity drug, orlistat (5 µmol/L), was used as a positive control. Cells were then rinsed with phosphate buffered saline twice and 200 µL deionized water was added per well to get cell lysate by ultrasonication. Intracellular TG contents in the lysates were finally evaluated using a GPO-POD method as the kit protocol provided. Briefly, the method is based on the enzymatic hydrolysis of triglyceride to glycerol and free fatty acids by lipoprotein lipase. The glycerol is phosphorylated by adenosine triphosphate in the presence of glycerolkinase to form glycerol-3-phosphate and adenosine diphosphate. glycerol-3-phosphate is oxidized by glycerophosphate oxidase (GPO) to form dihydroxyacetone phosphate and hydrogen peroxide. Purple quinoneimine complex is produced by the peroxidase (POD) catalyzed coupling of and 4AAP and ESPAS with hydrogen peroxide. The final absorbance was monitored at 492 nm using a microplate reader . Concentration setting of test sample and orlistat were established by pre-test results. Under the concentration, there were no treatment-related changes in cell viability (Data not shown).s report . BrieflyOil red O staining After a 48 h-treatment in 24-multiwell plates, cells were washed with phosphate-buffered saline (PBS) and fixed in 10% paraformaldehyde for 30 min at room temperature, followed by washing twice with deionized water and incubated in Oil Red O working solution  for 1 h. The unbound dye was removed by washing with 75% ethyl alcohol for once and images were immediately captured under an inverted phase contrast microscope . For quantitative analysis, stained cells were then dissolved in isopropanol and absorbance was measured at 492 nm.p < 0.05 were considered significant.Statistical analysis was performed using one-way ANOVA with the Tukey’s test for multiple comparisons. All data were expressed as mean ± S.E.M. and values of 1–16) including two new ones, officinoflavonosides A (1) and B (2), were obtained from the aerial parts of R. officinalis. Among the known ones, 6, 10, and 13 were isolated from the rosmarinus genus for the first time, and the NMR data of 13 and 14 in DMSO-d6 were reported for the first time. Their structures were elucidated by chemical and spectroscopic methods and their effects on TG accumulation were analyzed in HepG2 cells. The results showed that six kinds of flavonoids  together with the two novel ones (compounds 1 and 2) and seven kinds of other known isolates  possessed significant inhibitory activities on TG accumulation in vitro. Among them, the activities of compounds 1 and 20 were comparable to that of orlistat, which suggested that these compounds contained in the R. officinalis might be involved in the lipid metabolism.Taken together, 16 flavonoids (


Aphanamixis polystachya resulted in isolation of four new tetranortriterpenes (1–4) in addition to one protolimonoid ,14(15)-diene-3-oate (5)), five known limonoids (rohituka 3 (6), rohituka 7 (7), nymania 1 (8), rubrin G (9), prieurianin (10)) and a steroid ). Their structures were determined by spectroscopic analyses, including 2D-NMR  and high-resolution electrospray ionization mass spectrometry (HRESIMS). Cytotoxic and anti-inflammatory activities of these compounds were evaluated. Compounds 4 and 5 showed significant inhibition against superoxide generation and elastase release by human neutrophils in response to  (FMLP/CB).Phytochemical investigation of the acetone extract from the roots of  Aphanamixis polystachya  R.N. Parker (Meliaceae) is a tropical neem tree native to Asia, especially China and India. According to previous studies, the meliaceaeous plants are famous for the limonoid-producing source and the pest control function in agriculture + .Aphataiwanin B (2). Colorless amorphous solid; c 0.1, MeOH); IR (CH2Cl2) vmax 3484, 3071, 1740, 1643, 983, 902 cm−1; 1H-NMR (CDCl3) and 13C-NMR (CDCl3) spectroscopic data, see m/z 653.3681 [M + Na]+ .Aphataiwanin C (3). Colorless amorphous solid; c 0.1, MeOH); IR (CH2Cl2) vmax 3510, 2930, 1740, 1650, 972, 900 cm−1; 1H-NMR (CDCl3) and 13C-NMR (CDCl3) spectroscopic data, see m/z 671.3785 [M + Na]+ .Aphataiwanin D (4). Colorless amorphous solid; c 0.1, MeOH); IR (CH2Cl2) vmax 3491, 2932, 1741, 1642, 900 cm−1; 1H-NMR (CDCl3) and 13C-NMR (CDCl3) spectroscopic data, see m/z 671.3763 [M + Na]+ .Cytotoxicity was tested against human Hep-G2 (hepatocellular carcinoma), HEp-2 , A549 (lung carcinoma) and MCF-7 (breast adenocarcinoma) tumor cell lines. The assay procedure using MTT -2,5-diphenyltetrazolium bromide) was carried out as previously described ,19. The c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate. Genistein was used as a standard compound, which showed inhibition of 65.0 ± 5.7 and 51.6 ± 5.9 at 10 μg/mL, respectively, on superoxide anion generation and elastase release.Neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide generation and elastase release were carried out according to a procedure described previously . Superox1–4 ,14(15)-diene-3-oate (5) +) and 13C-NMR/DEPT spectra. The IR absorption bands revealed the presence of hydroxy (3479 cm−1), ester (1740 cm−1), and double bond (1645 cm−1) functionalities in 1. The 1H-NMR data of 1 , two methoxy singlets , exomethylene , an olefinic methine doublet , and five oxygen-bearing methine signals . The 13C-NMR , two olefinic methylenes , an olefinic methine (δC 119.1), three olefinic quaternary carbons , five oxymethines , four aliphatic methines , six aliphatic methylenes , two methoxyls , and seven methyls . This accounted for 6 of the 10 degrees of unsaturation, indicating that 1 is a tetracyclic triterpenoid with two acetyl and two methoxyl moieties.Compound ata of 1  exhibite 13C-NMR  and DEPT1 exhibited four proton spin systems of H-1 (δH 5.49)/H2-2  and H-5 (δH 2.44)/H2-6 /H-7 (δH 5.16); H-9 (δH 2.18)/H2-11 /H2-12 ; and a proton sequence between H-15 (δH 5.27)/H2-16 /H-17 (δH 1.92)/H-20 (δH 2.21)/H-21 (δH 4.78) and between H-20/H2-22 /H-23 (δH 4.23) /H-24 (δH 3.83). In the HMBC spectrum , C-5 (δC 44.3), C-9 (δC 34.5) and C-10 (δC 44.3), and the H2-29 had correlations with C-4 (δC 145.1), C-5 and C-28 (δC 23.0) suggesting the ring B. In addition, the HMBC correlations of H3-18 (δH 1.00) with C-13 (δC 46.3), C-12 (δC 33.5), C-14 (δC 159.2), C-17 (δC 52.9), and of H3-30 (δH 1.14) with C-7 (δC 74.7), C-8 (δC 42.5), C-9 (δC 34.5) and C-14 (δC 159.2) established rings C and D. Moreover, the HMBC correlations of both H3-26 (δH 1.78) and H2-27  with C-24 (δC 78.1) and C-25 (δC 144.9) indicated a methyl vinyl group to be attached at the oxygenated C-24. The HMBC correlations of the methoxy protons (δC 3.39) with C-21 (δC 104.6) and of H-21 (δH 4.78) with C-23 (δC 80.7) revealed the tetrahydrofuran ring with a methoxy group at C-21. The remaining methoxy (δH 3.36) group was found to be connected with C-3 (δC 172.0), and the H-1 (δH 5.49) and H-7 (δH 5.16) were connected with the acetyl carbonyl carbons  indicated that the acetyl groups were attached at C-1 and C-7, respectively. This 2D NMR spectroscopic analysis was used to identify 1 as an A-seco apo-tirucallane triterpenoid +). The similar 1H- and 13C-NMR spectroscopic data of 3 and 1 suggested that they are close analogues. However, the 1H-NMR spectrum of 3 exhibited eight methyl singlets  instead of seven in 1 and only three olefinic protons  instead of five in 1, implying that a double bond was missing in 3. In the 13C-NMR spectrum, compound 3 was found possessing an additional methyl (δC 26.5) and lacking an olefinic methylene, in comparison to 1. The additional methyl group was assigned at C-27 by observation of the HMBC correlations from H3-27 (δH 1.26) and H3-26 (δH 1.27) to C-25 (δC 73.1) and C-24 (δC 76.8). The relative configuration of 3 was determined on the basis of the NOESY experiment and comparing the J values of 3 with those of 1. The result was identical to 1, suggesting the same configuration. On the basis of above interpretations, the structure of 3 was categorized into the group of ring A-seco apotirucallol and a name aphataiwanin C was given.Compound 4 was isolated as an amorphous solid, 36H56O10 as 3, as derived from HRESIMS at m/z 671.3763 ([M + Na]+). The IR spectrum revealed that 4 contained a hydroxyl (3491 cm−1), ester (1741 cm−1), and double bond (1642 cm−1) functionalities. The 1H- and 13C-NMR spectra of 4  in 3 and the oxygenated carbon C-23 shifted upfield to δC 76.9. The 1H-1H COSY and HMBC of 4 revealed cross peaks similar to those of 3, suggesting that compound 4 was an epimer of 3. The configuration at C-21 was determined by NOESY experiment. NOESY correlations between H-21/H-22β, H-20/H-22α and H-22α/H-23 indicated that the methoxy group was α-oriented. Other NOESY correlations were the same as those of 3. Thus, the structure of compound 4 was elucidated and the name aphataiwanin D was given.Compound tra of 4  were sim5 possessed significant activity  against MCF-7 cells and mild activity against Hep-G2 and A549 cells . Simultaneously, compounds 1 and 10 also showed mild activities against Hep-G2 and HEp-2, respectively, with IC50 values of 16–17 μg/mL , lung carcinoma (A549), epithelial type 2 (HEp-2), and breast adenocarcinoma (MCF-7) cell lines in vitro. Among these compounds, 17 μg/mL . In term FMLP/CB . Only co1–5 belong to a group of protolimonoids, ring A-seco apotirucallol, while 6–10 can be classified as rings A,B-seco prieurianins. Among these secondary metabolites, liaphataiwanins A (1) and D (4), 5, and prieurianin (10) were found to have mild cytotoxicities against cancer cells and moderate to potent anti-inflammatory activities. Compound 5, which has the moiety of six-member ether ring, showed the best biological function. The chemical constituents and the evaluation of cytotoxicities against human cancer cells and anti-inflammatory activities reported herein may provide beneficial information for further phytotherapy research.In the present study, 10 tetranortriterpenoids, including four new compounds and a steroid, were isolated; compounds


Talaromyces verruculosus, a rhizosphere fungus of Stellera chamaejasme L., (−)-8-hydroxy-3-(4-hydroxypentyl)-3,4-dihydroisocoumarin (1) and (E)-3-pyrrolidin-1-yl)acrylic acid (2) were isolated and evaluated for their antimicrobial activities. Their structures were elucidated by UV, IR, MS, 1H-NMR, 13C-NMR and 2D NMR spectra. Compound 1 exhibited the significant activities in vitro against two strains of bacteria and four strains of fungi. Compound 2 gave slight activities on the fungi at 100 µg mL−1, but no activities on the bacteria. Compound 1 should be considered as a new lead or model compound to develop new isocoumarin antimicrobial agents.From the ethyl acetate extract of the culture broth of  The molecular formula indicated six degrees of unsaturation. The IR spectrum revealed the presence of an OH group , C=O groups  and CH3 groups ; The UV disclosed absorption maxima at λmax 262 nm (logε 4.01), due to the presence of a conjugated system; The 1H-NMR spectrum exhibited the presence of two methyl groups at δH 2.35  and 1.93 , two olefinic protons at δ 7.78  and 6.91 , and one methylene group at δH 3.35 . The above findings were further supported by the DEPT spectrum. The 13C-NMR spectrum displayed 10 signals, of which five were assigned to quaternary carbon atoms by DEPT. Based on the large coupling constant of the signals at δH7.78  and 6.91 , a trans configuration was established for the ∆6-double bond. In the HMBC spectrum, the correlations between H-6 and C-2, C-5, C-8 suggested that the CH=CHCO2H group was attached to a nitrogen atom, and the correlations between H-10 and C-3, C-9, C-11 and between H-11 and C-3, C-9, C-10 suggested the occurrence of one propan-2-ylidene moiety at the C-3 position. From the foregoing, it was concluded that compound 2 was (E)-3-pyrrolidin-1-yl) acrylic acid, in agreement with its X-ray crystallographic analysis result D25 −40.4 ; UV (95% EtOH): λmax (logε) = 212 (4.30), 246 (3.75), 314 (3.56) nm; IR (KBr): νmax = 3419, 3080, 2946, 1657, 1617, 1584, 1462, 1239, 1120 cm−1; 13C-NMR  δ ppm: 169.8 (C-1), 162.0 (C-8), 139.3 (C-10), 136.1 (C-6), 117.9 (C-5), 116.1 (C-7), 108.3 (C-9), 79.6 (C-3), 67.8 (C-14), 38.7 (C-13), 34.7 (C-11), 32.8 (C-4), 23.6 (C-15), 21.1 (C-12); 1H-NMR  δ ppm: 11.04 , 7.44 , 6.92 , 6.72 , 4.59-4.65 , 2.96-2.99 , 1.89–2.00 , 1.77-1.85 , 1.60–1.77 , 1.50–1.56 , 3.88 , 1.25 , 1.69 ; Main HMBC correlations: δH 2.96–2.99 (H-4) / δC 79.6 (C-3), 117.9 (C-5), 108.3 (C-9), 139.3 (C-10); δH 6.72 (H-5) / δC 32.8 (C-4), 108.3 (C-9); δH7.44 (H-6) / δC 116.1 (C-7), 139.3 (C-10); δH 6.92 (H-7) / δC 117.9 (C-5), 108.3 (C-9); δH1.89-2.00 (H-11a), 1.77-1.85 (H-11b) / δC 79.6 (C-3); δH 1.50–1.56 (H-13) / δC 23.6 (C-15); δH 1.25 (H-15) / 67.8 (C-14), 38.7 (C-13); δH1.69 (14-OH) / 67.8 (C-14), 23.6 (C-15); (+)-ESI MS m/z = 251.0 [M+H]+; (+)-ESI HR MS m/z = 251.1279 [M+H]+ . (E)-3-pyrrolidin-1-yl)acrylic acid (2). White powders, Rf = 0.54 , 0.76 , m.p. 194–195 °C; UV (95%EtOH): λmax (logε) = 262 (4.01) nm; IR (KBr): νmax = 3431, 2924, 1714, 1628, 1359, 1234 cm−1; 13C-NMR  δ ppm: 173.9 (C-8), 170.9 (C-5), 168.3 (C-2), 154.1 (C-9), 133.2 (C-6), 119.3 (C-3), 110.9 (C-7), 48.6 (C-4), 24.4 (C-11), 21.0 (C-10); 1H-NMR  δ ppm: 7.78 , 6.91 , 3.35 , 2.35 , 1.93 ; Main HMBC correlations: δH 7.78 (H-6) / δC 173.9 (C-8), 170.9 (C-5), 168.3 (C-2); δH 6.91 (H-7) / δC 133.2 (C-6); δH 2.35 (H-10) / δC 154.1 (C-9), 119.3 (C-3), 24.4 (C-11); δH 1.93 (H-11) / δC 154.1 (C-9), 119.3 (C-3), 21.0 (C-10); (+)-ESI MS m/z = 209.9 [M+H]+; (+)-ESI HR MS m/z = 210.0762 [M+H]+ .E. coli and S. aureus. Half milliliter of the 24 h cultured fresh broth at 37 °C was diluted by 50 mL of the same broth to indicate a 0.5 McFarland (McF) turbidity (ca. 1.5 × 108colony forming unit). A tested compound was dissolved in dimethylsulfoxide (DMSO) to provide a sample solution of 0.8 mg mL−1, and then double-fold serial dilutions (0.625–160 μg mL−1) were made by adding Mueller-Hinton broth. Each sample solution (100 μL) was completely mixed with the bacterial suspension (100 μL) in a well of 96-well polystyrene plate. After incubation of the plates for 24 h at 37 °C, the absorbance of each well was measured at 630 nm with a microtiter plate reader (BioRad). According to the same, were prepared and used as positive control. Five percent DMSO in the same broth served method as the tested compounds, the double-fold serial solutions of ceftriaxone sodium (0.156–2.50 μg mL−1), a broad spectrum antibiotics as negative control. All experiments were performed in triplicate. The minimal inhibitory concentration  was defined as the concentration of a compound required to reduce the absorbance to less than 50% of that of the negative control.The antibacterial activity was determined by method reported in the literature . A LB  medium slants were subcultured in Petri dishes prior to testing. Sample solution (1 mg mL−1) in acetone was completely mixed with the autoclaved PDA medium to provide the medium containing 50 µg mL−1 of sample, and poured into Petri dishes. Five percent acetone in PDA medium served as negative control. PDA medium containing 50 (or 100) µg mL−1 of thiabendazole, a broad-spectrum antifungal drug, was used as positive control. When the medium in the plates was partially solidified, a 5-mm thick and 4 mm diameter disc of fungus cut from earlier subcultured Petri dishes was placed at the centre of the semi-solid medium. The treated and control dishes were kept in an incubator at 26 (±2) °C for 72 h. The diameters (in mm) of inhibition zones were measured in three different directions. Growth inhibition rates were calculated according to the following formula and expressed as means ± SD:d0: Diameter of the fungus cut, dc: Diameter of the untreated control fungus, ds: Diameter of the sample-treated fungus. The antifungal activity e method  with sli1 and 2 of Talaromyces verruculosus isolated in the rhizosphere soil of Stellera chamaejasme L. and reported their UV, IR, MS, 1D and 2D NMR spectroscopic data for the first time. Compound 1 showed significant antimicrobial activities against four strains of fungi  at 50 µg mL−1 and two strains of bacteria (S.aureus and E. coli) with MIC values of 2.5 µg mL−1 and 5.0 µg mL−1, respectively. Compound 2 was slightly active against the fungi at 100 µg mL−1and its MIC values on the bacteria were more than 100 µg mL−1. Compound 1 should be considered as a new lead or model compound to develop new isocoumarin antimicrobial agents.In conclusion, the present study described the isolation, characterization and antimicrobial activities of two secondary metabolites


This is the first study to address the effect of point chirality toward G-quadruplex DNA stabilization using purely small organic molecules. Enantioselective behavior toward the majority of ligands was observed, particularly in the case of parallel conformations of c-KIT2 and k-RAS. Additionally, Nε-Boc-l-Lys-NDI and Nε-Boc-d-Lys-NDI discriminate between quadruplexes with parallel and hybrid topologies, which has not previously been observed with enantiomeric ligands.Four pairs of amino acid-functionalized naphthalenediimide enantiomers ( The demand for alternative approaches to the classical chemotherapy as principal treatment of cancer has led to the development of advanced techniques, and to the identification of new targets . Among t®, porphyrinoids, quinolones, alkaloids, carbazoles, acridine derivatives −, found: 723.2873. Nεd-Lys NDI:-Boc- The reaction was performed on 1 equivalent of NDA  and 2.1 equivalents of Nε-Boc-d-lysine  by using the general procedure. Yield: 554 mg, 98%; 1H NMR : δ 12.85 , 8.75 , 6.67  = 6.0 Hz, 2H), 5.53  = 9.4, 5.0 Hz, 2H), 2.85–2.79 , 2.27–2.17 , 2.10–2.01 , 1.21 ; 13C NMR : δ 171.1, 162.8, 155.9, 131.7, 126.8, 126.4, 77.6, 54.0, 36.3, 31.2, 29.8, 28.6, 23.7; FTMS-NSI: m/z calcd for C36H44N4O12: 723.2883 [C36H44N4O12-H]−, found: 723.2874.Nα-d-Lys and Nε-d-Lys NDIs) were obtained from Nα-Boc-d-Lys and Nε-Boc-d-Lys NDIs via the following general procedure: a 25-mL round-bottomed flask equipped with a stirrer bar was charged with starting material (NDI) suspended in CH2Cl2 (5 mL) and trifluoroacetic acid (5 mL) was added to the suspension. The reaction mixture was stirred at room temperature for 3 h. The solvents were removed under reduced pressure, and the residue was subsequently washed with diethyl ether (2 × 30 mL), filtered, and vacuum dried to yield a light brown solid in both cases.The deprotected derivatives . Yield: 331 mg, 85%; 1H NMR : δ 13.05 , 8.66 , 8.16 , 4.04  = 7.6 Hz, 4H), 3.92–3.84 , 1.88–1.72 , 1.72–1.60 ; 13C NMR : δ 171.5, 163.1, 158.9, 158.4, 130.9, 126.8, 126.6, 52.3, 50.2, 30.2, 27.4, 24.2, 22.4; FTMS-NSI: m/z calcd for C26H29N4O8: 525.1993 [C26H29N4O8-H]+, found: 525.1980.Nε-d-Lys NDI: The aforementioned general procedure was following using Nε-Boc-d-Lys NDI (275 mg). Yield: 176 mg, 62%; 1H NMR : δ 12.39 , 8.74 , 7.59 , 5.53  = 8.9, 5.3 Hz, 2H), 3.37  = 7.0 Hz, 4H), 2.31–2.23 , 2.08–2.00 , 1.08  = 7.0 Hz, 4H); 13C NMR : δ 171.1, 162.9, 131.7, 126.9, 126.5, 53.8, 39.1, 36.2, 28.5, 27.3, 23.4. The MS data could not be acquired because of the aggregation issue. CD and UV-vis experiments were performed on an Applied Photophysics Chirascan Circular Dichroism Spectrophotometer  equipped with a Peltier temperature controller. The following parameters were used for full spectra measurements: wavelength scanning range 220–420 nm; temperature 5.0 °C; scanning increments 1 nm; monochromator bandwidth 2.5 nm; sampling time-per-point 0.5 s; pathlength cuvette 1 mm.max) with the following parameters: temperature range 5–95 °C with 1 °C increments; temperature slope 1 °C min−1; temperature equilibration time 45 s; monochromator bandwidth 2.5 nm; sampling time-per-point 2.0 s. The VT CD data was processed using QtiPlot® , and fitted to the Boltzmann equation  studies, the ligand  was added to the DNA sequence solution previously annealed. Any samples with a maximum ellipticity <4.0 mdeg were transferred to a quartz cuvette with a longer pathlength . For VT experiments, a single-wavelength spectrum was recorded using the wavelength with the maximum ellipticity  and screened them against G4-forming sequences in telomeres (h-TELO) and the oncogene promoters c-KIT1, c-KIT2, k-RAS and BCL-2. To the best of our knowledge, this is the first study to explore the effect of point chirality in metal-free ligands and chiral recognition toward stabilizing G4 DNA sequences. The heat map table in l-NDIs (a) vs. d-NDIs (b) as G4 and dsDNA binders.We have synthesized the Nε-Boc-Lys NDI interacting with c-KIT2, where the l-enantiomer stabilizes the quadruplex sequence 16 °C more than the d-enantiomer; the latter has a mild destabilizing effect on the c-KIT2 oligonucleotide (Nα-Lys NDI enantiomers showed differential stabilization toward this quadruplex in favor of the l-enantiomer. All of the NDI ligands displayed enantiomeric differentiation in the interaction with c-KIT1, k-RAS and BCL-2 with the exception of Nα-Boc-Lys NDI and c-KIT1, and Nε-Lys NDI with both c-KIT1 and BCL-2. h-TELO does not have a strong interaction with any of the NDI ligands studied.Enantioselectivity was observed for certain ligand/quadruplex pairs adopting parallel or hybrid conformations. The biggest stabilization difference between the enantiomers was observed for cleotide . The proThe enantioselectivity observed cannot be explained by the aromatic π-π stacking of the NDI core on the terminal G-tetrads, thus implying that the sidechain interaction with the loops is the cause of this selectivity. This study paves the way toward further exploration of chiral ligands for G4 DNA with the hope of developing a new generation of G-quadruplex sequence selective anticancer drugs.


Compounds were characterized with analytical data and assayed in vitro for their antimycobacterial, antifungal, antibacterial and photosynthesis-inhibiting activity. 5-tert-Butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12) has shown the highest antimycobacterial activity against Mycobacterium tuberculosis (MIC = 6.25 µg/mL), as well as against other mycobacterial strains. The highest antifungal activity against Trichophyton mentagrophytes, the most susceptible fungal strain tested, was found for 5-chloro-N-(3-trifluoromethylbenzyl)-pyrazine-2-carboxamide . None of the studied compounds exhibited any activity against the tested bacterial strains. Except for 5-tert-butyl-6-chloro-N-benzylpyrazine-2-carboxamide  and 5-tert-butyl-6-chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide , only moderate or weak photosynthesis-inhibiting activity in spinach chloroplasts (Spinacia oleracea L.) was detected.A series of twelve amides was synthesized  Tuberculosis (TB) is considered to be one of the most frequent and widespread nowadays infectious diseases especially in developing countries. In 2010, there were about 8.8 million new cases of TB and 1.4 million deaths (including deaths from TB among HIV-positive people) [pncA gene and mutation of this gene is primarily responsible for resistance to PZA [Mycobacterium tuberculosis fatty acid synthase-I (FAS-I) in whole-cell and cell-free assays suggests that the disruption might be a consequence of the inhibition of membrane synthesis [M. tuberculosis FAS-I has been definitively confirmed by Saturation Transfer Difference NMR spectroscopy (STD-NMR), a NMR technique used to characterize ligand–protein interactions [et al. [trans-translation and its role in M. tuberculosis is multifaceted. Pyrazinamide (PZA), a nicotinamide analogue, is one of the most important first-line drugs used in TB-therapy . Along we to PZA . POA’s ie to PZA . Otherwiynthesis ,9,10. Reractions . Boshoff [et al.  reported [et al. ,9. Final [et al. . RpsA istert-butyl-N-(3-hydroxy-4-chlorophenyl)-pyrazine-2-carboxamide and 5-tert-butyl-6–chloro-N-(3-fluorophenyl)-pyrazine-2-carboxamide interacted with the D· intermediate, i.e., with the tyrosine radical which is situated at 161th position on D2 protein occurring on the donor side of PS 2 [·/D· intermediate confirmed that also some members of the PET chain between Z·/D· intermediate and plastoquninone were partially damaged in the light by these carboxamides. Chlorophyll a fluorescence quenching due to treatment of chloroplast suspension with above mentioned pyrazinamides indicated their interaction with pigment-protein complexes in PS 2 [Several pyrazine derivatives were found to possess herbicidal activity summarized in review paper . Many py of PS 2 . Due to  in PS 2 .2- bridge, namely on N-benzylpyrazine-2-carboxamides. Earlier studies have shown some interesting anti-mycobacterial activity in a series of substituted N-phenylpyrazine-2-carboxamides [N-benzylpyrazine-2-carboxamides, i.e., to study the influence of incorporated methylene moiety in the connecting bridge and to continue in the study of the substituent variability influence on the biological activity. In this study, we focused on binuclear pyrazinamide analogues containing the -CONH-CHoxamides ,19 and b1–12, is shown in tert-butyl-6-chloropyrazine-2-carboxylic [1, R2 and R3 of individual compounds 1–12 are listed in Synthesis of 5-chloropyrazine-2-carboxylic acid chloride  from 5-hrboxylic  acids wiP/Clog P values of the compounds 1–12 were calculated using the program CS ChemBioDraw Ultra version 12.0  and also measured by means of the RP-HPLC determination of capacity factors k with subsequent calculation of log k. The results are shown in Lipophilicity, one of the most important physicochemical properties of the compound, which seems to be a key factor related to the cell transmembrane transport and other biological processes, can either be determined experimentally or predicted by means of commercially available programmes. In this work Log N-(4-methoxybenzyl)pyrazine-2-carboxamide (4), while 5-tert-butyl-6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (10) was the most lipophilic compound of this series. Based on log k values, lipophilicity increased for substituents in pyrazine part in the following order: 5-chloropyrazine < 6-chloropyrazine < 5-tert-butyl-6-chloropyrazine. In the case of substituents in the benzyl part of the molecule lipophilicity increased this way: 4-OCH3 < H < 4-Cl < 4-CF3. The dependence of the calculated Clog P values on the measured log k parameters showed an approximate linearity and the corresponding correlation can be expressed by the following regression equation:The lowest lipophilicity was shown by 5-chloro-k and calculated Clog P values were observed for 5-chloro and 6-chloro group in pyrazine part. This may be caused by the used calculating program for Clog P, that does not distinguish difference between substituent’s lipophilicity in position C(5) and C(6) in pyrazine part of molecule. Consequently, it can be assumed that log k values specify lipophilicity within this series of compounds more precisely than calculated Clog P values.The differences between experimentally determined log in vitro towards Mycobacterium tuberculosis and several Mycobacteria Other Than Tuberculosis (MOTTs)  were 6.25 µg/mL. These activities were fully comparable with PZA. More importantly, 5-tert-butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12) showed significant activity against tested MOTTs, which are unsusceptible to PZA. The vast majority of compounds exhibited only modest antimycobacterial activity expressed as MIC in µg/mL, or with respect to the molecular weight of final products in µmol/L (see All synthesized compounds were assayed TTs) see . The mosol/L see . tert-butyl in the C(5) position. The obtained results provide some insights into the SAR in this miniseries. With respect to the benzyl part, the most suitable substituents are 4-methoxy and 3-trifluoromethyl groups, especially with disubstitution in the pyrazine moiety. The most significant substitutions in the pyrazine part are chlorine in the C(6) and k, see tert-butyl-6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide  displayed one of the highest activity against M. tuberculosis. On the other hand, 6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (8) with the lowest lipophilicity (log k = 0.1838) showed the same activity against M. tuberculosis as compound 10 (MIC = 6.25 µg/mL). There is no clear dependence between lipophilicity  and 6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamides (6), only weak antifungal activity was found. In comparison with fluconazole  compounds 2 (MIC = 15.62 µmol/L) and 6 (MIC = 62.5 µmol/L) exhibited moderate in vitro antifungal activity against Trichophyton mentagrophytes (TM), the most susceptible fungal strain evaluated. For the results see The evaluation of in vitro antibacterial activity [Staphylococcus aureus CCM 4516/08, Staphylococcus aureus H 5996/08–methicilin resistant, Staphylococcus epidermidis H 6966/08, Pseudomonas aeruginosa CCM 1961, Escherichia coli CCM 4517. Enterococcus sp. J 14365/08, Klebsiella pneumoniae D 11750/08 and Klebsiella pneumoniae J 14368/08–ESBL positive. None of the synthesized compounds exhibited any activity against the tested strains. All prepared compounds were tested for their activity ,24 again50 values. Compounds 4, 5, 7 and 8 were not tested due to their low solubility in tested medium. The IC50 values varied in the range from 7.4 to 1,623.0 µmol/L, see 50 = 1.9 µmol/L).All studied compounds were evaluated for their photosynthetic electron transport (PET) inhibition in spinach chloroplasts, which was reflected in the inhibition of oxygen evolution rate. The photosynthesis-inhibiting activity of the compounds has been expressed as IC9 (IC50 = 7.4 µmol/L) and 11 (IC50 = 13.4 µmol/L). In general, photosynthesis-inhibiting activity of the studied compounds is dependent on their lipophilicity  fluorescence. Fluorescence emission spectra of Chla in spinach chloroplasts treated with compound 2 are shown in a-protein complexes in the thylakoid membrane was reflected as decreased fluorescence of the pigment , 11 (triangles) and 6 (circles)  see B. Bindin·/D· intermediate on the donor side of PS 2, to chloroplasts treated with the studied compounds in which PET was inhibited at about 80‑90%, PET was restored only to 77‑88%. This indicates that the site of PET inhibition is situated not only on the donor side of PS 2 in the section between the primary electron donor of PS 2 (H2O) and Z·/D· intermediate but also in the photosynthetic transport chain from P 680 to plastoquinone QB occurring on the acceptor side of PS 2. Similar sites of action were determined previously for 5-tert-butyl-N-(3-hydroxy-4-chlorophenyl)-pyrazine-2-carboxamide and 5-tert-butyl-6–chloro-N-(3-fluorophenyl)-pyrazine-2-carboxamide [By the addition of DPC, an artificial electron donor acting in Zboxamide .254 TLC plates . Compounds were purified using an automated chromatograph CombiFlash Rf  using columns filled with Kieselgel 60, 0.040–0.063 mm ; gradient elution (hexane/ethyl-acetate), detection wavelength 260 nm, monitor wavelength 280 nm. NMR analysis was performed on Varian Mercury VX-BB 300  at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts were recorded as δ values in parts per million (ppm) and were indirectly referenced to tetramethylsilane (TMS). IR spectra were recorded in KBr blocks on Nicolet Impact 400 . Elementary analysis was performed on CE Instruments EA-1110 CHN analyser . Melting points were determined on Stuart SMP30 melting point apparatus  and are uncorrected.All organic solvents used for the synthesis were of analytical grade. All chemicals were purchased from Sigma-Aldrich . The reactions were monitored using Merck Silica 60 Ftert-butyl-6-chloropyrazine-2-carboxylic [in vacuo. The crude acyl chloride was dissolved in dry acetone (20 mL) and added dropwise to a stirred solution of the corresponding benzylamine (0.5 mmol) with triethylamine (0.5 mmol) in dry acetone (10 mL). Then, the reaction mixture was stirred at room temperature for about 1 h. The reaction was monitored using TLC with hexane/ethyl acetate 2:1 mixture as eluent. After this time, the solution was evaporated till dryness with silica gel and purified using a flash column chromatography .To a solution of 6-chloropyrazine-2-carboxylic  or 5-terrboxylic  acid (0.In the case of 5-chloropyrazine-2-carbonyl chloride  synthesiN-Benzyl-5-chloropyrazine-2-carboxamide (1). White crystalline compound. Yield: 81%; m.p. 101.9–103.1 °C; 1H-NMR (CDCl3) δ 9.21 , 8.49 , 7.98 , 7.40–7.26 , 4.67 ; 13C-NMR (CDCl3) δ 162.0, 152.1, 144.0, 142.6, 142.4, 137.5, 128.8, 127.8, 127.7, 43.5; IR (cm−1) 3384 (N-H), 1660 (C=O); Anal. Calcd. For C12H10ClN3O (247.68): 58.19% C, 4.07% H, 16.97% N; Found: 58.38% C, 4.23% H, 16.89% N.5-Chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (2). White crystalline compound. Yield: 65%; m.p. 91.3–92.7 °C; 1H-NMR (CDCl3) δ 9.20 , 8.51 , 8.09 , 7.65–7.40 , 4.72 ; 13C-NMR (CDCl3) δ 162.2, 152.3, 144.0, 142.5, 142.3, 138.6, 131.2, 131.1 , 129.3, 124.6 , 124.4 , 123.6 , 43.0; IR (cm−1) 3385 (N-H), 1665 (C=O); Anal. Calcd. For C13H9ClF3N3O (315.68): 49.46% C, 2.87% H, 13.31% N; Found: 49.51% C, 2.96% H, 13.48% N.5-Chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (3). White crystalline compound. Yield: 79%; m.p. 89.9–91.3 °C; 1H-NMR (CDCl3) δ 9.20 , 8.50 , 7.98 , 7.35–7.25 , 4.63 ; 13C-NMR (CDCl3) δ 162.1, 152.2, 144.0, 142.5, 142.4, 136.1, 133.6, 129.2, 128.9, 42.9; IR (cm−1) 3327 (N-H), 1672 (C=O); Anal. Calcd. For C12H9Cl2N3O (282.13): 51.09 % C, 3.22% H, 14.89% N; Found: 50.96% C, 3.14% H, 14.97% N.5-Chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (4). White crystalline compound. Yield: 80%; m.p. 123.2–124.0 °C; 1H-NMR (CDCl3) δ 9.19 , 8.48 , 7.90 , 7.30–7.25 , 6.99–6.62 , 4.59 , 3.80 ; 13C-NMR (CDCl3) δ 161.9, 159.2, 152.0, 144.0, 142.6, 142.4, 129.6, 129.3, 114.2, 55.3, 43.1; IR (cm−1) 3388 (N-H), 1659 (C=O); Anal. Calcd. For C13H12ClN3O2 (277.71): 56.22% C, 4.36% H, 15.13% N; Found: 56.26% C, 4.34% H, 15.01% N.N-Benzyl-6-chloropyrazine-2-carboxamide (5). White crystalline compound. Yield: 87%; m.p. 58.3–59.7 °C; 1H-NMR (DMSO-6d) δ 9.48 , 9.19 , 8.81 , 7.34–7.20 , 4.50 ; 13C-NMR (DMSO-6d) δ 163.1, 148.1, 143.8, 143.6, 143.1, 139.4, 128.4, 127.5, 127.0, 42.5; IR (cm−1) 3371 (N-H), 1670 (C=O); Anal. Calcd. For C12H10ClN3O (247.68): 58.19% C, 4.07% H, 16.97% N; Found: 58.33% C, 4.12% H, 17.08% N.6-Chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (6). White crystalline compound. Yield: 63%; m.p. 68.1–69.8 °C; 1H-NMR (CDCl3) δ 9.33 , 8.77 , 8.03 , 7.64–7.42 , 4.73 ; 13C-NMR (CDCl3) δ 161.8, 147.6, 147.5, 143.6, 142.1, 138.5, 131.2 , 130.9, 129.3, 124.6 , 124.5 , 124.0 , 43.1; IR (cm−1) 3324 (N-H), 1672 (C=O); Anal. Calcd. For C13H9ClF3N3O (315.68): 49.46% C, 2.87% H, 13.31% N; Found: 49.57% C, 2.71% H, 13.56% N.6-Chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (7). White crystalline compound. Yield: 91%; m.p. 93.2–94.5 °C; 1H-NMR (CDCl3) δ 9.32 , 8.76 , 7.97 , 7.37–7.31 , 7.28–7.20 , 4.63 ; 13C-NMR (CDCl3) δ 161.7, 147.5, 147.4, 143.7, 142.0, 135.9, 133.6, 129.2, 128.9, 42.9; IR (cm−1) 3387 (N-H), 1667 (C=O); Anal. Calcd. For C12H9Cl2N3O (282.13): 51.09% C, 3.22% H, 14.89% N; Found: 51.23% C, 3.37% H, 14.76% N.6-Chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (8). White crystalline compound. Yield: 89%; m.p. 71.4–72.6 °C; 1H-NMR (CDCl3) δ 9.32 , 8.73 , 7.88 , 7.31–7.25 , 6.91–6.85 , 4.59 , 3.80 ; 13C-NMR (CDCl3) δ 161.5, 159.2, 147.5, 147.2, 144.0, 142.0, 129.5, 129.3, 114.2, 55.3, 43.1; IR (cm−1) 3370 (N-H), 1662 (C=O); Anal. Calcd. For C13H12ClN3O2 (277.71): 56.22% C, 4.36% H, 15.13% N; Found: 56.34% C, 4.38% H, 15.03% N.N-Benzyl-5-tert-butyl-6-chloropyrazine-2-carboxamide (9). White crystalline compound. Yield: 61%; m.p. 69.7–71.3 °C; 1H-NMR (DMSO-6d) δ 9.35 , 9.04 , 7.33–7.20 , 4.48 , 1.48 ; 13C-NMR (DMSO-6d) δ 162.8, 162.0, 145.5, 142.4, 140.1, 139.3, 128.4, 127.6, 127.0, 42.6, 38.6, 28.2; IR (cm−1) 3399 (N-H), 1669 (C=O); Anal. Calcd. For C16H18ClN3O (303.79): 63.26% C, 5.97% H, 13.83% N; Found: 63.39% C, 6.13% H, 13.67% N.5-tert-Butyl-6-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (10). White crystalline compound. Yield: 59%; m.p. 74.4–75.3 °C; 1H-NMR (CDCl3) δ 9.21 , 7.98 , 7.60 , 7.58–7.45 , 4.72 , 1.53 ; 13C-NMR (CDCl3) δ 164.5, 162.3, 145.9, 140.8, 140.2, 138.8, 131.2, 131.1 , 129.3, 124.5 , 124.4 , 123.9 , 42.9, 38.9, 28.3; IR (cm−1) 3373 (N-H), 1676 (C=O); Anal. Calcd. For C17H17ClF3N3O (371.78): 54.92% C, 4.61% H, 11.30% N; Found: 54.86% C, 4.53% H, 11.35% N.5-tert-Butyl-6-chloro-N-(4-chlorobenzyl)pyrazine-2-carboxamide (11). White crystalline compound. Yield: 82%; m.p. 67.5–68.7 °C; 1H-NMR (CDCl3) δ 9.19 , 7.91 , 7.39–7.19 , 4.62 , 1.52 ; 13C-NMR (CDCl3) δ 164.4, 162.1, 140.9, 140.1, 136.2, 133.5, 131.5,129.2, 128.9, 42.7, 38.9, 28.3; IR (cm−1) 3376 (N-H), 1659 (C=O); Anal. Calcd. For C16H17Cl2N3O (338.23): 56.82% C, 5.07% H, 12.42% N; Found: 56.76% C, 5.18% H, 12.25% N.5-tert-Butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12). White crystalline compound. Yield: 84%; m.p. 71.3–72.7 °C; 1H-NMR (CHCl3) δ 9.19 , 7.84 , 7.31–7.25 , 6.91–6.85 , 4.59 , 3.80 , 1.52 ; 13C NMR (CDCl3) δ 164.1, 161.9, 159.1, 141.1, 140.2, 140.1, 129.7, 129.3, 114.1, 55.3, 42.9, 38.8, 28.2; IR (cm−1) 3324 (N-H), 1673 (C=O); Anal. Calcd. For C17H20ClN3O2 (333.81): 61.17% C, 6.04% H, 15.13% N; Found: 61.23% C, 6.17% H, 15.01% N.2O  was used as mobile phase. The total flow of the column was 1.0 mL/min, injection 20 µL, column temperature 30 °C. 210 nm as detection wavelength and 270 nm as monitor wavelength were chosen. The KI methanol solution was used for the dead time (TD) determination. Retention times (TR) of synthesized compounds were measured in minutes. The capacity factors k were calculated using Microsoft Excel according to formula k = (TR − TD)/TD, where TR is the retention time of the solute and TD denotes the dead time obtained via an unretained analyte. Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale. An Agilent Technologies 1200 SL liquid chromatography system equipped with a Diode-Array Detector SL G1315C, chromatographic pre-column ZORBAX XDB-C18 5 µm, 4 × 4 mm, Part No. 7995118-504 and column ZORBAX Eclipse XDB-C18 5 µm, 4.6 × 250 mm, Part No. 7995118-585  were used. The separation process was controlled by Agilent ChemStation, version B.04.02 extended by spectral module (Agilent Technologies Inc.). A solution of MeOH  with HP (the logarithm of the partition coefficient for n-octanol/water) and Clog P  were calculated using the program CS ChemBioDraw Ultra version 12.0 .Log M. tuberculosis H37Rv CNCTC My 331/88, M. avium CNCTC My 80/72, M. avium CNCTC My 152/73 and M. kansasii CNCTC My 235/80 . Tested compounds were dissolved in DMSO , diluted with Šula’s semisynthetic medium  and placed into microdilution panel. Tested species were added in the form of suspension in isotonic saline solution. The final concentration of DMSO did not exceed 1% (v/v), this concentration of DMSO did not affect the growth of mycobacteria. The cultures were grown in Šula’s semisynthetic medium at pH 6.0 and 37 °C. The antimycobacterial activity was determined visually after 14 days (6 days for M. kansasii) of incubation as minimally inhibition concentration , i.e., the lowest concentration of tested substance which inhibited the growth of mycobacteria.Microdilution panel method. Antimycobacterial evaluation was shielded by Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles University in Prague, Czech Republic. Four mycobacterial strains were used: Trichophyton mentagrophytes (TM), of static incubation in dark at 35 °C. Tested species: Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon asahii 1188, Aspergillus fumigates 231, Absidia corymbifera 272 and Trichophyton mentagrophytes 445 (TM).The Department of Medical and Biological Sciences at the Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Czech Republic, performed the antifungal susceptibility assays, which was carried out using microdilution broth method ,30. CompSpinacia oleracea L.) according to Masarovičová and Kráľová [et al. [2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these experiments and the samples were irradiated (~100 W/m2) from a 10 cm distance with halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples (suspension temperature 22 °C). The studied compounds were dissolved in DMSO due to their limited water solubility. The applied DMSO concentration (up to 4%) did not affect the photochemical activity in spinach chloroplasts (PET). The inhibitory efficiency of the studied compounds was expressed as the IC50 values, i.e., molar concentration of the compounds causing 50% decrease in the oxygen evolution relative to the untreated control. The comparable IC50 value for a selective herbicide 3--1,1-dimethylurea  was about 1.9 µmol/L [Chloroplasts were prepared from spinach  and aromatic amino acids in spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000  using excitation wavelength λex = 436 nm for monitoring fluorescence of Chla and λex = 275 nm for monitoring fluorescence of aromatic amino acids, excitation slit 20 nm and emission slit 10 nm. The samples were kept in the dark 2 min before measuring. The phosphate buffer used for dilution of the chloroplast suspension was the same as described above. Due to low aqueous solubility the compounds were added to a chloroplast suspension in DMSO solution. The DMSO concentration in all samples was the same as in the control (10%). The chlorophyll concentration in chloroplast suspension was 10 mg/L.The fluorescence emission spectra of chlorophyll 2- bridges was synthesized by the condensation of substituted pyrazine-2-carboxylic acid chlorides with the corresponding benzylamines. The final products were characterized by analytical data and evaluated for their in vitro antimycobacterial, antifungal, antibacterial and photosynthesis-inhibiting activity. Lipophilicity of the compounds was determined using RP-HPLC method and calculated using predicting program CS ChemBioDraw Ultra version 12.0. The obtained values were compared with each other and graphically expressed as dependence of calculated Clog P on log k. Compounds 8, 10 and 12 had in vitro antimycobacterial activity against M. tuberculosis comparable with PZA (MIC = 6.25 µg/mL). More importantly, 5-tert-butyl-6-chloro-N-(4-methoxybenzyl)pyrazine-2-carboxamide (12) was active against tested MOTTs (M. kansasii and M. avium), which are unsusceptible to PZA. No clear dependence between lipophilicity and antimycobacterial activity has been found in this series. The highest antifungal activity (MIC = 15.6 µmol/L) against susceptible strain (Trichophyton mentagrophytes) was observed for 5-chloro-N-(3-trifluoromethylbenzyl)pyrazine-2-carboxamide (2), other compounds showed only weak or none in vitro antifungal activity. None of the studied compounds exhibited any significant activity against tested bacterial strains. 5-tert-butyl-6-chloro-N-benzylpyrazine-2-carboxamide . The most active compounds in inhibition of PET in spinach chloroplasts (Spinacia oleracea L.) were 5‑ tert-butyl-6-chloro-N-benzylpyrazine-2-carboxamide  and 5-tert-butyl-6-chloro-N-(4-chlorobenzyl)-pyrazine-2-carboxamide . Based on the obtained results it can be assumed, that in this series of pyrazinamide derivatives the antimycobacterial and antifungal activities did not depend directly on compound’s lipophilicity. On the other hand, PET-inhibiting activity increased linearly with compound’s lipophilicity. A series of twelve binuclear pyrazinamide analogues containing -CONH-CH


Aquilaria sinensis collected in Hainan Province of China resulted in the isolation of a new benzenoid, named aquilarin A (1), together with two known compounds balanophonin (2) and (+)-lariciresinol (3). Their structures were elucidated by a study of their physical and spectral data. Compounds 2 and 3 exhibited cytotoxicity against SGC-7901 and SMMC-7721 cell lines.Chemical investigation of the EtOH extract of the fresh stem of  Aquilaria species in response to injury by cutting, holing, burning, or incursion of moths, microorganisms, etc., is well known as incense in the Oriental region, and has also been used as a sedative, analgesic and digestive in Traditional Medicine + ion peak at m/z 319.0789 , corresponding to the molecular formula C14H16O7. This formula can also be validated through its 1H-NMR, 13C-NMR and DEPT data. The IR spectrum displayed free hydroxyl , γ-lactone carbonyl , and aromatic ring  absorptions. The 13C-NMR spectrum of compound 1 (C δ58.9 and 68.0), two methines (C δ43.6 and 45.7), two methoxyls (C δ56.2 and 56.2), two carbonyls (C δ195.7 and 176.6), and six aromatic carbons of a symmetrical benzene ring . The 1H-NMR spectrum of 1 , two aromatic OMe at Hδ3.84 , and one phenolic OH at Hδ 5.29. The remaining oxymethylene [Hδ4.58  and Hδ4.20 ], two methines [Hδ4.58  and Hδ3.00 ], and a hydroxymethyl  were ascribed to a 9CH2O−8CH−11CH−12CH2OH fragment by 1H-1H COSY spectrum. The HMBC cross peaks (Cδ 195.7 (C-7)], and two aromatic OMe should be located at C-3 and C-5 in the symmetrical benzene ring. A γ-butyrolactone ring was deduced from the HMBC cross peaks from H-8, H-9, H-11, and H-12 to the lactone carbonyl [C δ176.6 (C-10)]. The ROESY correlations from H-8 to H-12 and H-9 to H-11 indicated the trans configuration at C-8 and C-11 -lariciresinol, respectively.Based on the comparison of the terature ,8, compo1−3 were evaluated for their cytotoxic activity against SGC-7901 and SMMC-7721 cell lines using the MTT method. Compound 2 showed cytotoxic activity against SGC-7901 cell line, and compound 3 showed cytotoxic activity against SGC-7901 and SMMC-7721 cell lines, while compound 1 was inactive (IC50 > 100 μg·mL-1) (Compounds μg·mL-1) .Melting points were obtained on a Beijing Taike X-5 stage apparatus and are uncorrected. Optical rotation was recorded using a Rudolph Autopol III polarimeter . The UV spectra were measured on a Shimadzu UV-2550 spectrometer. The IR spectra were obtained on a Nicolet 380 FT-IR instrument, as KBr pellets. The NMR spectra were recorded on a Bruker AV-400 spectrometer, using TMS as an internal standard. The HRESIMS spectra were measured with an API QSTAR Pulsar mass spectrometer. Column chromatography was performed with silica gel  and Sephadex LH-20 (Merck). TLC was preformed with silica gel GF254 .A. sinensis (Lour.) Gilg were collected in Ding’an county, Hainan province, China in November 2008, the plant was identified by Associate Professor Zheng-Fu Dai of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (No. AS20081101) was deposited.Fresh stems of A. sinensis (66.0 kg) were extracted with 95% EtOH three times (100 L × 3) at room temperature. After removal of EtOH by evaporation, the EtOH extract was suspended in water (10.0 L) and successively partitioned with petroleum ether, EtOAc, and then n-BuOH to give the corresponding Petro-extract (106.3 g), EtOAc-extract (66.0 g), and n-BuOH-extract (244.5 g), respectively. The fresh and crushed stems of 3-MeOH  to afford eight fractions (Fr.1−8). Fr.2 (25.0 g) was chromatographed on a silica gel column using a step gradient elution of Pet-Acetone  to afford eight fractions (Fr.2-1−8). Fr.2-4 (6.5 g) was subjected to column chromatography over Sephadex LH-20 using CHCl3-MeOH  as eluent to afford four fractions (Fr.2-4-1−4). Fr.2-4-1 (554.0 mg) was separated by column chromatography over Sephadex LH-20 using CHCl3-MeOH  as eluent to afford compound 1 (5.0 mg). Fr.2-4-2 (4.4 g) was submitted to column chromatography over silica gel, eluenting with gradient CHCl3-MeOH to afford compounds 2 (10.0 mg) and 3 (3.0 mg).The EtOAc fraction (66.0 g) was subjected to vacuum liquid chromatography (VLC) over silica gel, eluting with a gradient of CHClAquilarin A (1): Armorphous powder, M.p. 167−168 ºC. [α]25D = − 78.3 . UV (MeOH): λmax (log εmax): 306 (1.41), 226 (1.66), 214 (1.39) nm. IR (KBr): ν = 3,428, 2,920, 2,851, 1,766, 1,586, 1,511, 1,464, 1,380, 1,116 cm-1. HR-MS [(+)-ESI]: m/z = 319.0789 . 1H and 13C-NMR: see Balanophonin (2): Yellow oil, [α]25D = + 12.1 . ESI-MS m/z: 379 [M+Na]+. 1H-NMR : δ 9.62 , 7.41 , 7.13 , 7.03 , 6.90 , 6.89 , 6.88 , 6.59 , 5.63 , 3.97 , 3.67 , 3.92 , 3.86 . 13C-NMR : δ 151.5 (C-1), 144.8 (C-2), 112.2 (C-3), 128.1 (C-4), 118.2 (C-5), 129.1 (C-6), 153.2 (C-7), 126.3 (C-8), 193.6 (C-9), 145.9 (C-1'), 146.7 (C-2'), 108.7 (C-3'), 132.2 (C-4'), 119.4 (C-5'), 114.4 (C-6'), 88.9 (C-7'), 53.0 (C-8'), 63.9 (C-9'), 56.0 (2×OCH3).( + )-Lariciresinol (3): Armorphous powder. [α]25D = + 31.0 . UV (MeOH): λmax (log εmax): 221 (1.02), 282 (2.17) nm. ESI-MS m/z: 383 [M+Na]+. 1H-NMR : δ 6.88 , 6.87 , 6.84 , 6.81 , 6.70 , 6.69 , 4.79 , 4.06 , 3.92 , 3.89 , 3.88 , 3.79 , 3.75 , 2.92 , 2.73 , 2.55 , 2.41 . 13C-NMR : δ 134.8 (C-1), 108.3 (C-2), 146.5 (C-3), 145.1 (C-4), 114.2 (C-5), 118.8 (C-6), 82.9 (C-7), 52.6 (C-8), 61.0 (C-9), 132.3 (C-1'), 111.2 (C-2'), 146.6 (C-3'), 144.0 (C-4'), 114.4 (C-5'), 121.2 (C-6'), 33.4 (C-7'), 42.4 (C-8'), 72.9 (C-9'), 56.0 (2×OCH3).control − ODsample)/ODcontrol. The IC50 value, which is defined as the concentration of sample needed to reduce 50% of absorbance relative to the vehicle-treated control, was determined using the Bliss method. The same experiment was repeated independently three times to obtain a mean IC50 value and its standard deviation. The IC50 values are listed in The 3--2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the previously reported method . The inh1), together with two known lingnans balanophonin (2) and (+)-lariciresinol (3), were isolated from the 95% ethanol extract of the fresh stem of A. sinensis, which were different from those of Chinese eaglewood. Meanwhile, the cytotoxicity against SGC-7901 and SMMC-7721 cell lines of compounds 1− 3 was evaluated for the first time.Although much attention has been paid to the phytochemical investigation of Chinese eaglewood, little is known about the chemical constituents of the fresh healthy wood. Previous studies revealed the characteristic components of Chinese eaglewood were sesquiterpenes and chromone derivatives ,3,4,5,6.


H)α-dauca-11(12)-ene-7α,14-diol (1) and 1α,2α-epoxy-10(H)α-dauca-11(12)-ene-7α,14-diol (2) were isolated from the plateau medicinal plant Daphne aurantiaca Diels. (Thymelaeceae). Their structures were elucidated by 1D and 2D NMR spectroscopy, as well as HR-ESI-MS data.Two new daucane sesquiterpenoids 1β,2β-epoxy-10( Daphne aurantiaca Diels. (Thymelaeceae) is an evergreen shrub widely distributed in the plateau area of southwest China and used traditionally in folk medicine  in dauca-1,11-diene-7α,14-diol shifted up-field to δH 3.27  in 1, which suggested that compound 1 was generated from dauca-1,11-diene-7α,14-diol with C-1-C-2 double bond oxidized to a epoxy group, combined with the analysis of its molecular formula. The key HMBC correlations ], H-7 [δH 3.85 ], and H-14 to C-1 [δC 67.5 (s)] further clarified this hypothesis.The IR spectrum displayed the presence of hydroxyls  of 1 were elucidated by comparison of NMR data with dauca-1,11-diene-7α,14-diol and NOESY spectrum ] and H-2/H-4 [δH 1.88 ]. Moreover, the α-orientation of H-10 in compound 1 was determined by NOE of H-13 [δH 1.66 ]/H-15 [δH 0.99 ]. Thus, the structure of compound 1 was assigned as showed in H)α-dauca-11(12)-ene-7α,14-diol.The other correlations in the HMBC and -1H COSY  spectrumspectrum  and dete2 was obtained as a yellow oil and had the same molecular formula as 1 based on HR-ESI-MS m/z [M+Na]+ 275.1617 , with four degrees of unsaturation. The IR spectrum also displayed the presence of hydroxyls  and olefinic bond  absorptions. The 1H and 13C-NMR spectroscopic data of 2 was extremely similar to those of 1 except that C-2 (δC 59.4) in compound 1shifted down-field to δC 61.8 in 2 and H-2 (δH 3.27) in 1 shifted up-field to δH 3.15 in 2, which hinted compound 2 was the isomer of 1 with different configuration of C-1 and C-2 (epoxy group). This was confirmed by the NOESY experiment. The relative configurations of the stereogenic centers  of 2 were determined to be the same as those of compound 1 by NOE of H-15 (δH 0.85)/H-7 (δH 4.29) and H-15/H-13 (δH 1.73). The a-orientation of epoxy group was deduced by key NOE of H-2/H-12 (δH 4.85) and H-7/H-14 (δH 4.07 and 3.30). The correlations in the HMBC and 1H-1H COSY spectrum α-dauca-11(12)-ene-7,14-diol. Finally the anti-HIV-1 and inhibitory AChE activities of compounds 1 and 2 were evaluated. The two new compounds showed no activity against HIV-1 and AChE.Compound spectrum  further 3 as solvent); ESI-MS and HR-ESI-MS, API QSTAR Pulsar 1 spectrometer. Silica gel , RP-18  and Sephadex LH-20  were used for column chromatography. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph equipped with a Zorbax SB-C18, 9.4 mm × 25 cm column. Fractions were monitored by TLC and spots were visualized by heating after spraying with 5% H2SO4 in ethanol.Spectra were recorded on the following instruments: optical rotation, Jasco P-1020 polarimeter; UV, Shimadzu double-beam 210A spectrometer; IR, Tensor 27 spectrometer (KBr pellets); NMR; Bruker AV-400 or a DRX-500 spectrometer , identified by Prof. H. Sun and Dr. L.L. Yue , was deposited in Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.The stems of Daphne aurantiaca (4.5 kg) were extracted with 95% EtOH (15 L) under reflux three times. The extract was concentrated and suspended in water followed by successive partition with petroleum ether (3 × 4 L) and EtOAc (3 × 4 L). The EtOAc extract (300 g) was separated by silica gel column using a gradient solvent CHCl3/MeOH (9:1–3:1 10 L) to afford fractions A–C. Fraction A (40 g) was separated by silica gel column using a gradient solvent petroleum ether/EtOAc (20:1–1:2 8 L) to afford fractions A1–A4. Fr. A2 (5g) was purified over Sephadex LH-20 column (CHCl3/MeOH 1:1 3 L) to give two subfractions A2-1 and A2-2. Subfr.A2-1 (2 g) was subjected to repeated RP-18 column (MeOH/H2O 2:1 2 L) to obtain the mixture two compounds 1 and 2. The mixture (42.3 mg) was further separated by semi-preparative HPLC (MeOH/H2O 45:55 0.5 L) to yield 1 (11.7 mg) and 2 (8.5 mg).The dried and powdered stems of 1β,2β-Epoxy-10(H)α-dauca-11(12)-ene-7,14-diol (1). Yellow oil; [α]D17: +1.29 ; UV (MeOH) λmax (logε) 202 (3.54), 229 (2.66); IR (KBr) νmax 3425, 2950, 2878, 2858, 1635, 1454, 1383, 1107, 1067, 1055, 1024, 889; 1H and 13C-NMR see m/z [M+Na]+ 275 (100); HR-ESI-MS m/z [M+Na]+ 275.1628 .1α,2α-Epoxy-10(H)α-dauca-11(12)-ene-7,14-diol (2). Yellow oil; C15H24O3, [α]D18: +15.67 ; UV (MeOH) λmax (logε) 202 (3.57), 237 (2.47); IR (KBr) νmax 3419, 2949, 2930, 2855, 1636, 1451, 1382, 1024, 972, 890; 1H and 13C-NMR see m/z [M+Na]+ 275 (100); HR-ESI-MS m/z [M+Na]+ 275.1617 .50) and cytotoxicity assay against C8166 cell line (IC50) using MTT methods as described in the literature [50) was determined from the dose response curve and calculated by Reed and Muench method [The anti-HIV activity was evaluated by the inhibition assay for the cytopathic effects of HIV-1  acid , acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical . Compounds were dissolved in DMSO. The reaction mixture  containing phosphate buffer (pH 8.0), test compound (50 μM), and acetyl cholinesterase (0.02 U/mL), was incubated for 20 min (30 °C). The reaction was initiated by the addition of 20 μL of DTNB (0.625 mM) and 20 μL acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay. The hydrolysis of acetylthiocholine was monitored at 405 nm after 30 min. Tacrine (Sigma-Aldrich 99%) was used as positive control with final concentration of 0.333 μM, and DMSO was used as negative control with final concentration of 0.1%. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compounds).Acetylcholinesterase inhibitory activity was assayed by the spectrophotometric method developed by Ellman  with sliDaphne aurantiaca led to the isolation of two new dacane sesquiterpenoids named 1β,2β-epoxy-10(H)α-dauca-11(12)-ene-7α,14-diol (1) and 1α,2α-epoxy-10(H)α-dauca-11(12)-ene-7α,14-diol (2), which were isomers. The evaluation on anti-HIV-1 and inhibitory AChE activities of two new dacane sesquiterpenoids showed these two isolates exhibited no activity against HIV-1 and AChE.The phytochemical investigation of


Panax notoginseng. Chromatographies and spectral analysis methods were combined to isolate and identify PNS. Moreover, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. As a result, eleven new dammarane-type triterpenoid saponins, notoginsenosides NL-A1–NL-A4 (1–4), NL-B1–NL-B3 (5–7), NL-C1–NL-C3 (8–10), and NL-D (11) were isolated, and their structures were identified by using various spectrometric techniques and chemical reactions. Among them, compounds 4 and 11 were characterized by the malonyl substitution at 3-position. The 3-malonyl substituted dammarane-type terpennoids were first obtained from natural products. In addition, compounds 1, 2, 5, 6, and 8–10 were found to play an important role in suppressing NO levels at 50 μM, without cytotoxicity. All inhibitory activities were found to be dose-dependent.Inflammation is a very common and important pathological process that can cause many diseases. The discovery of anti-inflammatory drugs and the treatment of inflammation are particularly essential. Dammarane-type triterpenoid saponins (PNS) were demonstrated to show anti-inflammatory effects in the leaves of  Panax notoginseng (Burk.) F. H. Chen is generally known as San qi in Chinese. As recorded in Chinese medical book “Compendium of Material Medica”, its root was traditionally used in Asia for the treatment of trauma, body pain, inflammation, and cardiovascular diseases since ancient times , and one α-l-arabinofuranosyl [δ 5.66 ]. Its 13C-NMR spectrum showed forty-seven signals. After subtracting the seventeen carbon resonances that belonged to the sugar units, the remaining thirty resonances were attributable to a triterpene skeleton. In the 1H-NMR spectrum, eight signals could be assigned to methyls , two signals belonged to oxygenated methylene , and the signals for one trans-olefin  indicated that 1 was a dammarane-type triterpene saponin derivative. In order to solve the problem of overlapping for the three glycosyl groups, HSQC-TOCSY experiment was performed. In the HSQC-TOCSY spectrum, correlations were found between the following proton and carbon pairs: δH 4.95 (H-1′) and δC 71.8 (C-4′), 75.7 (C-2′), 78.7 (C-3′), 107.1 (C-1′); δH 4.42, 4.62 (H2-6′) and δC 63.0 (C-6′), 71.8 (C-4′), 78.4 (C-5′); δH 5.18 (H-1″) and δC 71.9 (C-4″), 75.1 (C-2″), 78.8 (C-3″), 98.2 (C-1″); δH 4.13, 4.66 (H2-6″) and δC 68.3 (C-6″), 71.9 (C-4′), 76.4 (C-5″); δH 5.66 (H-1‴) and δC 83.3 (C-2‴), 110.0 (C-1‴); δH 4.87 (H-2‴) and δC 78.9 (C-3‴), 83.3 (C-2‴), 85.9 (C-4‴), 110.0 (C-1‴); δH 4.21, 4.31 (H2-6‴) and δC 62.7 (C-5‴), 78.9 (C-3‴), 83.3 (C-2‴), 85.9 (C-4‴). In conjunction with the HSQC spectrum, the spectroscopic data of the above-mentioned three glycosyls were assigned. According to the proton and proton correlations observed in its 1H–1H COSY spectrum  to δC 88.8 (C-3); δH 5.18 (H-1″) to δC 83.2 (C-20); δH 5.66 (H-1‴) to δC 68.3 (C-6″) displayed in its HMBC spectrum. The resonance of C-25 were shifted downfield by about 11 ppm, as compared with notoginsenoside Fh5 with 3β,12β,20(S),25-tetrahydroxydammar-23-ene as aglycone -20-O-[β-d-xylopyranosyl(1→6)]-β-d-glucopyranoside  in 2. As compared to compound 1, one more β-d-glucopyranosyl [δ 5.50 ] and one more β-d-xylopyranosyl [δ 5.39 ] presented in 3, which was clarified by its ESI-Q-Orbitrap MS determination . Meanwhile, as compared to 1, δC-1′ and δC-2′ of 3 were shifted upfield and downfield by , respectively, which suggested C-2′ in 3 might be substituted by the glycosyl group. The cross peaks from δH 4.98 (H-1′) to δC 88.8 (C-3); δH 5.20 (H-1″) to δC 83.2 (C-20); δH 5.00 (H-1‴) to δC 69.9 (C-6″) were observed in 2; while the long range correlations from δH 4.92 (H-1′) to δC 89.0 (C-3); δH 5.50 (H-1″) to δC 82.9 (C-2′); δH 5.39 (H-1‴) to δC 84.6 (C-2″); δH 5.17 (H-1⁗) to δC 83.2 (C-20); δH 5.64 (H-1′′′′′) to δC 68.4 (C-6⁗) were found  was a white powder. Its molecular formula was determined to be C44H72O17, on the basis of the ESI-Q-Orbitrap MS  and NMR data ] and one α-l-arabinopyranosyl [δ 5.02 ]. Forty-four signals were observed in its 13C-NMR spectrum. Except for the signals that belonged to the above-mentioned aglycone and glycosyl groups, the other three signals could be assigned to two ester carbonyls  together with one methylene [δC 43.8 (C-2′)]. Combining with the chemical shift [δ 3.83 ] of methylene proton, the presence of malonyl was deduced, which was confirmed by the cross peaks from δH 3.83 (H2-2′) to δC 168.3 (C-1′), 169.9 (C-3′). Moreover, the covalent connectivities of above-mentioned moieties were established by the long-range correlations from δH 4.81  to δC 168.3 (C-1′); δH 5.20 (H-1″) to δC 83.3 (C-20); δH 5.02 (H-1‴) to δC 69.0 (C-6″)  from the NOESY correlations beween δH 4.01 (H-12) and δH 1.38 (H-9); δH 0.78 (H-5) and δH 1.38 (H-9), 4.81 (H-3) and vicinal coupling constants of H-3 with H2-2 . Then, its aglycone was also confirmed to be 3β,12β,20S-trihydroxy-25-hydroperoxydammar-23-ene. On the basis of the above-mentioned evidence, the structure of notoginsenoside NL-A4 (4) was determined.Notoginsenoside NL-ANMR data  analysis0 (C-6″) . The NMR4 (4) was a white powder. Its molecular formula was determined to be C44H72O17, on the basis of the ESI-Q-Orbitrap MS  and NMR data ] and one α-l-arabinopyranosyl [δ 5.02 ]. Forty-four signals were observed in its 13C-NMR spectrum. Except for the signals that belonged to the above-mentioned aglycone and glycosyl groups, the other three signals could be assigned to two ester carbonyls  together with one methylene [δC 43.8 (C-2′)]. Combining with the chemical shift [δ 3.83 ] of methylene proton, the presence of malonyl was deduced, which was confirmed by the cross peaks from δH 3.83 (H2-2′) to δC 168.3 (C-1′), 169.9 (C-3′). Moreover, the covalent connectivities of above-mentioned moieties were established by the long-range correlations from δH 4.81  to δC 168.3 (C-1′); δH 5.20 (H-1″) to δC 83.3 (C-20); δH 5.02 (H-1‴) to δC 69.0 (C-6″)  from the NOESY correlations beween δH 4.01 (H-12) and δH 1.38 (H-9); δH 0.78 (H-5) and δH 1.38 (H-9), 4.81 (H-3) and vicinal coupling constants of H-3 with H2-2 . Then, its aglycone was also confirmed to be 3β,12β,20S-trihydroxy-25-hydroperoxydammar-23-ene. On the basis of the above-mentioned evidence, the structure of notoginsenoside NL-A4 (4) was determined.Notoginsenoside NL-ANMR data  analysis0 (C-6″) . The NMR1 (5) was obtained as a white powder with positive optical rotation , 6.09 , 6.23 ] and C-23–27 , as compared to that of compound 1. The ESI-Q-Orbitrap MS analysis result indicated its molecular formula was C47H80O18 , an oxygen atom disappeared when comparing it with 1, suggesting that C-25 of 5 was substituted by the hydroxyl group. The cross peaks from δH 1.57 (H3-26) to δC 30.6 (C-27), 70.0 (C-25), 142.2 (C-24); δH 1.56 (H3-27) to δC 30.7 (C-26), 70.0 (C-25), 142.2 (C-24), clarified the above-mentioned description. The NMR data of its aglycone were likable to those of the known compound, 3-O-β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-3β,12β,20(S),25-tetrahydroxydammar-23-ene-20-O-β-d-glucopyranoside-(1→6)-β-d-glucopyranoside .The molecular formula of both notoginsenosides NL-B 13C-NMR , 1H–1H Cperiment . Those rspectrum  denoted 1 (8), white powder, exhibited a molecular ion peak [M − H]− at m/z 929.51184  in ESI-Q-Orbitrap MS spectrum, which matched to the molecular formula, C47H78O18, confirmed by the NMR spectral data (−1), α,β-unsaturated ketone carbonyl (1669 cm−1), olefin (1637 cm−1), and an ether function (1077 cm−1). Its 1H- and 13C-NMR spectra showed that the signals belonged to two β-d-glucopyranosyl , and one α-l-arabinofuranosyl [δ5.71 ]. Its 13C-NMR spectrum displayed forty-seven carbons including thirty for aglycone and seventeen for sugar moieties. The 1H and 13C-NMR spectra denoted that 8 was a dammarane-type triterpene saponin derivative. Compared to 1, the chemical shifts of proton and carbon for the side chain changed significantly, as follows—one disubstituted terminal olefin signals  appeared, while the signals of one methyl and one trans-olefinic bond disappeared in its 1H-NMR spectrum; meanwhile, an additional terminal olefin  and additional carbonyl group [δC202.6 (C-24)] signals were displayed in its 13C-NMR spectrum, but one methyl, one trans-olefinic bond, as well as oxygenated quaternary carbon signals disappeared. The 1H–1H COSY experiment on 8 indicated the presence of partial structures written in bold lines, as shown in H 5.78, 6.37 (H2-26) to δC 17.8 (C-27), 144.4 (C-25), 202.6 (C-24); δH 1.85 (H3-27) to δC 125.5 (C-26), 144.4 (C-25), 202.6 (C-24), then the conjunction position of carbonyl and terminal olefin were clarified. Since the chemical shifts of its aglycone were identical to those of ginsenoside III  appeared, and one α-l-arabinofuranosyl disappeared in 9. Finally, the β-d-xylopyranosyl was clarified to have connected with 6-position of β-d-glucopyranosyl substituted at C-20 by cross peak from δH 4.91 (H-1‴) to δC 70.1 (C-6″), showed in HMBC (Notoginsenoside NL-C 13C-NMR  spectra  in HMBC .3(10) was measured to be C53H88O23  on an ESI-Q-Orbitrap MS spectrometer. Compared with 9, one more group data of β-d-glucopyranosyl [δ 5.39 ] was presented in 10 (C 75.8 (C-2′), 107.0 (C-1′) for 9; δC 83.5 (C-2′), 105.1 (C-1′) for 10], respectively, which suggested that C-2′ was displaced by the glycosyl group. The long-range correlation from δH 5.39 (H-1″) to δC 83.5 (C-2′) displayed in its HMBC experiment confirmed the above-mentioned β-d-glucopyranosyl linked with C-2′.The molecular formula of notoginsenoside NL-Ced in 10 . Moreove11) was a white powder with positive optical rotation . The IR absorptions at 3382, 1717, 1649, and 1075 cm−1 indicated the presence of OH, CO, olefin, and an ether group. The 1H and 13C-NMR -β-d-glucopyranosyl  and malonyl  as 4. Forty-four signals were observed in its 13C-NMR spectrum. Except for the signals belonging to the above-mentioned moieties, the other 30 indicated that 11 was a triterpene saponin. Its 1H-NMR spectrum showed eight methyl signals at δ0.93, 0.97, 1.05, 1.35, 1.62, 1.63, 1.66, and 1.76 , three methines bearing an oxygen function at δ4.19 , 4.32 , and 4.88 , one trisubstituted olefin signal at δ 5.32 . In its HMBC experiment, long-range correlations were observed from the following proton to carbon pairs: H-3 to C-1′; H3-18 to C-7−9, C-14; H3-19 to C-1, C-5, C-9, C-10;H3-21 to C-17, C-20, C-22; H3-26 to C-24, C-25, C-27; H3-27 to C-24−26;H3-28 to C-3−5, C-29; H3-29 to C-3−5, C-28; and H3-30 to C-8, C-13−15; H-1″ to C-20. Then, its planar structure was elucidated, which was a protopanaxatriol type saponin. The chemical shifts for proton and carbon of the B–D ring and the side chain were matched closely with those of ginsenoside U , and 12 fractions (Fr. 1–Fr. 12) were yielded. Fraction 6 (6.2 g) was further subjected to silica gel CC [CH2Cl2-MeOH-H2O  → MeOH] to give 13 fractions (Fr. 6-1–Fr. 6-13). Fraction 6-5 (650.0 mg) was isolated by pHPLC [CH3CN-1% HAc ] to gain notoginsenoside NL-A4 . Fraction 6-10 (800.0 mg) was purified by pPHPLC [CH3CN-1% HAc ], and notoginsenosides NL-A1 , NL-C1 , as well as NL-D  were given. Fraction 7 (30.0 g) was separated by MCI gel CC [MeOH-H2O ] to provide 12 fractions (Fr. 7-1–Fr. 7-12). Fraction 7-5 (480.0 mg) was further isolated by pHPLC [CH3CN-1% HAc ], and notoginsenoside NL-B1  was yielded. Fraction 7-6 (800.0 mg) was subjected to pHPLC [MeOH-1% HAc ] to give seven fractions (Fr. 7-6-1–Fr. 7-6-7). Fraction 7-6-3 (141.5 mg) was purified by pHPLC [CH3CN-1% HAc ] to gain notoginsenoside NL-C2 . Fraction 7-6-4 (179.5 mg) was isolated by pHPLC [CH3CN-1% HAc ] to produce notoginsenoside NL-A2 . Fraction 8 (25.0 g) was subjected to MCI gel CC [MeOH-H2O ], as a result, 11 fractions (Fr. 8-1–Fr. 8-11) were gained. Fraction 8-6 (1.6 g) was separated by pHPLC [MeOH-1% Hac ] to yield 13 fractions (Fr. 8-6-1–Fr. 8-6-13). Fraction 8-6-6 (135.3 mg) was further purified by pHPLC [CH3CN-1% HAc ] to obtain notoginsenosides NL-A3  and NL-B2 . Fraction 8-6-9 (223.8 mg) was isolated by pHPLC [CH3CN-1% HAc ] to gain notoginsenoside NL-C3 . Fraction 9 (15.0 g) was subjected to MCI CC [MeOH-H2O ], and 10 fractions (Fr. 9-1–Fr. 9-10) were given. Fraction 9-4 (500.0 mg) was separated by pHPLC [CH3CN-1% HAc ] to yield 11 fractions (Fr. 9-4-1–Fr. 9-4-10). Fraction 9-4-10 (10.1 mg) was further purified by pHPLC [MeOH-1% HAc ], and notoginsenoside NL-B3  was gained.The 95% EtOH eluate (150.0 g) was separated by silica gel CC − .2Notoginsenoside NL-A (2): White powder; νmax 3418, 2968, 2865, 1647, 1455, 1395, 1052, 1033, 1014 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 947.51996 [M − H]− .3Notoginsenoside NL-A (3): White powder; max 3368, 2939, 2877, 1635, 1453, 1386, 1074, 1042 cm−1; 1H-NMR and 13C-NMR  spectroscopic data—see m/z 1241.61584 [M − H]− .4Notoginsenoside NL-A (4): White powder; νmax 3379, 2946, 2877, 1718, 1646, 1456, 1384, 1080, 1041, 1010 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 871.46875 [M − H]− .1Notoginsenoside NL-B (5): White powder; νmax 3379, 2944, 2877, 1649, 1458, 1387, 1076, 1035, 1018 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 931.52838 [M − H]− .2Notoginsenoside NL-B (6): White powder; νmax 3364, 2938, 2877, 1647, 1456, 1384, 1073, 1042, 893 cm−1; 1H-NMR and 13C-NMR  spectroscopic data—see m/z 1225.62134 [M − H]− .3Notoginsenoside NL-B (7): White powder; νmax 3360, 2927, 2877, 1630, 1455, 1382, 1073, 1041, 894 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 1225.61841 [M − H]− .1Notoginsenoside NL-C (8): White powder; λmax (MeOH) nm (log ε) 218 (3.57); IR (KBr) νmax 3387, 2941, 2877, 1669, 1632, 1456, 1384, 1077, 1039 cm−1; 1H-NMR and 13C-NMR  spectroscopic data—see m/z 929.51184 [M − H]− .2Notoginsenoside NL-C (9): White powder; λmax (MeOH) nm (log ε) 219 (3.63); IR (KBr) νmax 3415, 2943, 2877, 1671, 1639, 1458, 1386, 1078, 1038, 894 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 929.51221 [M − H]− .3Notoginsenoside NL-C (10): White powder; λmax (MeOH) nm (log ε) 218 (3.69); IR (KBr) νmax 3391, 2942, 2881, 1663, 1645, 1453, 1385, 1164, 1077, 1040, 895 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 1091.56445 [M − H]− .Notoginsenoside NL-D (11): White powder; νmax 3382, 2953, 2878, 1717, 1649, 1456, 1387, 1312, 1075, 1048, 1008 cm−1; 1H-NMR  and 13C-NMR  spectroscopic data—see m/z 855.47388 [M − H]− .1–11: Compounds 1–11 were hydrolyzed by acid and an aqueous layer was obtained by using the method reported in ; a mobile phase, CH3CN-H2O ; and a flow rate of 0.7 mL/min. Then, d-xylose , l-arabinose , and d-glucose (from 1–11) and were confirmed by comparison of the retention times with the authentic samples .Acid Hydrolysis of orted in —their so1–11, as well as statistical analyses were conducted by using the method reported previously [The MTT and nitrite levels measurements of eviously .P. notoginseng. They were chemically classified as the derivatives of 20(S)-protopanaxatriol (PPT) and 20(S)-protopanaxadiol (PPD). In the present study, eleven new dammarane-type triterpenoid saponins were obtained and identified from the leaves extract of P. notoginseng. Among them, 1–10 were PPD-type saponins. It was suggested that PPD derivatives were the major PNS in P. notoginseng leaves, which was in accordance with the results found by other researchers [P. notoginseng roots [P. notoginseng leaves would be a possible replacement of the roots used in clinics.It is well-known that PNS are the major bioactive saponins in earchers ,7,8,9. Ang roots , we coul1–10 were dammarane-type with oxygenated C-17 side chains different to protopanaxadiol. Compounds 1–4, 5–7, and 8–10 featured 23-ene25-hydroperoxyl, 23-ene-25-hydroxyl, and α,β-unsaturated ketone, respectively. Among them, 23-ene-25-hydroperoxyl-substituted protopanaxadiol saponins were found in P. notoginseng leaves, first though they were found in P. notoginseng flowers [4 and 11 were characterized by the malonyl substitution at 3-position. The 3-malonyl substituted dammarane-type terpennoids are reported here for the first time. These results enrich the chemical investigations of P. notoginseng leaves.From a structural point of view, the aglycones of compounds  flowers , roots [ flowers , and rhi flowers . Additio1 (1), NL-A2 (2), NL-B1 (5), NL-B2 (6), NL-C1–NL-C3 (8–10) at noncytotoxic concentration (50 μM) decreased the NO production significantly, which indicated that they have an anti-inflammatory activity. Moreover, the inhibitory effects of all of them were found to be dose-dependent. Further investigations on their anti-inflammatory mechanisms would be beneficial.Inflammation is widespread in the clinical pathology and closely associated to the progress of many diseases. NO is implicated in a variety of inflammatory conditions, which indicates the agents block NO production might be beneficial for the treatment of inflammatory responses. In this study, pretreatment of notoginsenosides NL-A


The structures of the isolates were confirmed by spectroscopic analysis and they showed no inhibitory effects on the in vitro growth of microbial organisms  at less than 1.0 mg/mL.Two new monoterpenes,  Illicium (Illiciaceae) genus consists of aromatic evergreen trees that are distributed primarily in southwestern China and the southeast of America + , suggests the presence of four degrees of unsaturation. The IR spectrum showed a strong absorption band due to hydroxyl  groups. The 1H-NMR spectrum of 1 showed the presence of one methyl group at δH 1.71 ; one anomeric proton signal of β-glucopyranosyl moiety at δH 4.40 , indicating the existence of a β-d-linkage sugar moiety in compound 1; one exocyclic methylene protons at δH 4.77 and 5.27 (each s) -CH2(3)-CH(4)-CH2(5)-CH2(6) in its structure. The planar structural skeleton of 1 was further established on the basis of HMBC spectral data , 8-dien-2-O-β-d-glucoside (1).Compound (each s) . The 13C signals . Analysi–1H COSY  and HMBCral data  in whichspectrum . The relspectrum  in which2 was isolated as a white powder. The HRESIMS of 2 showed a pseudo-molecular ion at m/z 207.0999 [M + Na]+ , consistent with the molecular formula C10H16O3. The IR spectrum suggested the presence of hydroxyl  and carbonyl  groups. The 13C-NMR and DEPT spectra displayed 10 carbon signals, including two methyl, four methylene, one methine and three quaternary carbon signals /δc 33.4, δH 1.48 /δc 19.5 and the characteristic signals of a γ-lactone moiety at δH 2.37–2.42 , 2.43–2.49  and δC 86.2 , 33.3 , 176.6  -CH2(6)-CH(1)-CH2(8) as shown with bold line in 2 was further established on the basis of its HMBC spectrum  synthesized by Wolinsky in 1966 [2 and 3 were likely stereoisomers of each other. The correlation between CH3-7 and CH3-10 observed in the NOESY spectrum -8-p-menthen-1,2-diol (4), according to the reference [3 came from 3-isopropenyl-6-oxoheptanoic acid which is the oxidation product of compound 4.Compound  signals . 1H- and . The 1H–spectrum , in whicspectrum , in addi3–7 were identified as trans-2,4-dihydroxy-2,4-dimethyl-cis-1-acetic acid γ-lactone (3) [R, 2R, 4R)-8-p-menthen-1,2-diol [trans-sobrerol (5) [S, 2S, 4R)-p-menthane-1,2,8-triol (6) [S, 2S, 4R, 8R)-p-menthane-1,2,9-triol (7) [Compounds tone (3) ,  ,  , respectEscherichia coli, Staphyloccocus aureus and Bacillus subtilis) in vitro. A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) [The isolates were preliminarily evaluated against the test organisms (on (MIC) , howeverOptical rotations were determined on a Perkin-Elmer model 341 and Polar 3001. IR (KBr) spectra were recorded on a PE-1710 FT-IR spectrometer. 1D and 2D NMR spectra were recorded on Bruker DPX-400 NMR with TMS as internal standard. HR-ESI-MS spectra were run on a Waters-Q-Tof MS instrument. Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Meigao Chemical Company . MCI gel (10 μm) was purchased from Merck Chemicals Ltd. . Sephadex LH-20 (20–150 μm) was purchased from Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden.Illicium lanceolatum were collected in the Tianmu Mountains, Jiangsu Province, China, in September 2011, and identified by Associate Professor Dr. Mengqi Liu from the School of Pharmaceutical Science, Henan University of Traditional Chinese Medicine. A voucher specimen (2011-09-01) was deposited in the School of Pharmaceutical Science, Zhengzhou University.The fruits of I. lanceolatum (5.0 kg) were extracted with 95% EtOH (20 L) under reflux for three times, 2 h each time. The extract was concentrated under reduced pressure to give a residue. The residue was dissolved in H2O and then extracted successively with CHCl3 (each 2 L) and n-BuOH (each 2 L). The CHCl3 fraction (90 g) was subjected to silica gel chromatography  and eluted with a CHCl3/MeOH  gradient to afford eight fractions (Frs.1–8). Fr.3 (15 g) was further separated to obtain six sub-fractions (Frs.3a–f). Fr.3b (1.0 g) was subjected to silica gel column  with an eluent of petroleum ether/Me2CO (80:20) to yield compounds 3 (10 mg) and 4 (30 mg). Fr.3c (2.0 g) was chromatographed through a silica gel column  eluted with petroleum ether/EtOAc  to yield compounds 2 (20 mg) and 5 (35 mg). Fr.5 (8.0 g) was subjected to silica gel chromatography  eluted with a CHCl3/Me2CO  gradient to afford six fractions (Frs.5a–f). Sub-fractions (5a–f) were respectively chromatographed through MCI (MeOH/Water: 10:90–70:30), Sephadex LH-20 (MeOH), and then further separated by silica gel chromatography with petroleum ether/Me2CO (70:30) and CHCl3/Me2CO (80:20) to furnish compounds 1 (15 mg), 6 (5 mg) and 7 (15 mg).The powdered fruits of d-glucosidep-Mentha-1(7), 8-dien-2-O-β- (1). White amorphous powder;  +4.7 ; IR (KBr) vmax 3423, 2923, 1643, 1437, 1074, 1020 cm−1; 1H- and 13C-NMR spectral data, see m/z 337.1622 .trans-2,4-Dihydroxy-2,4-dimethyl-trans-1-acetic acid γ-lactone (2). White amorphous powder;  +133.3 ; IR (KBr) vmax 3446, 2972, 2940, 1746, 1378, 1268, 1105 cm−1; 1H- and 13C-NMR spectral data, see m/z 207.0999 .trans-2,4-Dihydroxy-2,4-dimethyl-cis-1-acetic acid γ-lactone (3). White amorphous powder;  +54.5 ; IR (KBr) vmax 3473, 2924, 2852, 1735, 1382, 1269, 1100 cm−1; 1H- and 13C-NMR spectral data, see m/z 207.0994 .Illicium. Compounds 1 and 2 were found to be new monoterpenes, and the other five compounds were found for the first time in I. lanceolatum. The compounds 1–7 showed no inhibitory effects on the growth of the tested microbial organisms at less than 1.0 mg/mL in vitro.This work was part of a series of investigations on anti-microbial compounds obtained from plants of the genus


S1) and fused aryloxazinone with unsymmetrical urea fragment (S2). The data include the analyzed NMR spectra, turbo-spray mass spectra, melting points and Rf-values. 1D and 2D NMR spectra are given in the article. The efficiency of the ligands as synergists in the isolation and separation of lantanoid ions is reported in rerence. The data represent the synthesis and structural characterization of two novel polydentate ligands possessing unsymmetrical NH-urea fragment: an open-chain substituted aromatic compound with unsymmetrical urea and secondary amine units ( Specifications TableValue of the data•The 2D NMR data permit full assignment of the signals in the spectra of the novel compounds.•The ligands offer unlimited possibilities for NH-derivatization.•The data can be useful for comparison with similar structures.1The data include synthetic details and characterization by 1D and 2D NMR spectra, mass spectrometry, TLC and m.p. of novel organic polydentate ligands.22.1f-values determination. Merck Silica gel 60 (0.040–0.063 mm) was used for flash chromatography purification of the products. The melting points were determined in capillary tubes on SRS MPA100 OptiMelt  automated melting point system. The NMR spectra were recorded on a Bruker Avance II+ 600 spectrometer  in CDCl3; the chemical shifts were quoted in ppm in δ-values against tetramethylsilane (TMS) as an internal standard and the coupling constants were calculated in Hz. The spectra were processed with Topspin 2.1 program. The low-resolution mass spectra were taken on a HP 5973 Mass Selective Detector, the turbo spray spectrum on API 150EX (AB/MAS Sciex).All reagents were purchased from Aldrich, Merck and Fluka and were used without further purification. The deuterated chloroform was purchased from Deutero GmbH. Fluka silica gel (TLC-cards 60778 with fluorescent indicator 254 nm) were used for TLC chromatography and R2.2S1 and S2 methyl)-4-methylphenol , pyridine , and aniline  in toluene (20 ml) phosgene  was added and the mixture was stirred at room temperature for 24 h. The products were partitioned between toluene and 10% aq. HCl. The water layer was washed with dichloromethane (DCM). The combined organic solutions were dried over MgSOS1: 13% (238 mg); Rf 0.22 (1% MeOH/DCM); m.p. 151.9–152.1 °C; 1H NMR 2.231 , 3.821 , 4.015 , 4.362 , 4.643 , 6.800 , 6.843 , 6.979 , 7.241 , 7.268–7.328 , 7.356 , 7.398 , 7.435 , 8.629 ;13C NMR 20.49 (CH3), 45.21 (CH2-2), 49.20 (CH2-Ph’), 51.62 (CH2–6), 52.53 (CH2-Ph), 119.21 (o-Ph”), 121.93 (Cq-6), 122.11 (p-Ph”), 123.51 (Cq-2), 127.23 (p-Ph or p-Ph’), 127.84 (p-Ph or p-Ph’), 128.03 (o-Ph’), 128.43 (o-Ph), 128.57 (Cq-4), 128.62 (m-Ph’), 128.73 (m-Ph”), 128.84 (m-Ph), 129.16 (CH-5), 130.79 (CH-3), 137.60 (i-Ph), 138.17 (i-Ph’), 140.26 (i-Ph”), 153.32 (Cq-1), 156.42 (C=O-NH); ESI (TIS)-Q m/z 932 [2M+1]+ (28), 466 [M+1]+ (100), 359 [M-NHBn]+ (56), 347 [M-M-CHNHBn+1]+ (24), 240 [M-BnNCONHPh]+ (74), 226 [M-CH2N(Bn)CONHPh]+ (20), 133 [BnNCO]+ (41).Ligand S2: 6% (120 mg); Rf 0.31 (1% MeOH/DCM); m.p. 173.7–174.4 °C; 1H NMR 2.258 , 4.279 , 4.595 , 4.652 , 4.672 , 6.749 , 6.927 , 6.995 , 7.015 , 7.255 , 7.282–7.362 , 7.406 ;13C NMR NMR 20.80 (CH3), 45.33 (CH2–8), 46.56 (CH2-2), 50.43 (CH2-Ph’), 52.53 (CH2-Ph), 117.07 (Cq-4a), 119.90 (o-Ph”), 122.82 (p-Ph”), 124.10 (Cq-8), 125.33 (CH-5), 127.55 (p-Ph or p-Ph’), 127.59 (p-Ph or p-Ph’), 128.15 (o-Ph or o-Ph’), 128.24 (o-Ph or o-Ph’), 128.76 (m-Ph”), 128.84 (m-Ph or m-Ph’), 128.89 (m-Ph or m-Ph’), 129.30 (CH-7), 134.06 (Cq-6), 135.19 (i-Ph), 137.35 (i-Ph’), 139.28 (i-Ph”), 145.41 (Cq-8a), 150.50 (Cq-2), 155.97 (C=O-NH); ESI (TIS)-Q m/z 983 [2M+1]+ (231), 492 [M+1]+ (100), 399 [M-NHPh]+ (9), 373 [M-CONPh+1]+ (42), 266 [M-BnNCONHPh]+ (30), 133 [BnNCO]+ (25).Ligand 2.3See