Arnebia hispidissima (Lehm.) DC. roots has led to the isolation of two unique polycyclic geranylhydroquinone-derived metabolites, arnebacene (1) and arnebidin (2), along with some known phenolic metabolites viz., arnebin-7 (3) and vanillic acid (4). The chemical identification of the new isolated compounds, including their relative stereochemistry, was achieved via spectroscopic analyses, including 2D NMR, and spectral comparison with related compounds. A biosynthetic pathway is proposed for the new compounds on the basis of their structure-relationship with previously isolated metabolites.A phytochemical investigation of the least polar organic extracts of  Arnebia hispidissima (Lehm.) DC. (Arabian primrose), which belongs to the family Boraginaceae, is widely distributed in the northern Africa area, through Egypt, to northern India octane core.rn India . To dateisolated ,4,5,6,7.isorders . Moreoveacterial ,9 and anacterial  which weA. hispidissima was powdered, sequentially extracted with light petroleum and then MeOH. The MeOH extract was further partitioned with H2O/n-hexane, H2O/CH2Cl2, and then with H2O/EtOAc to yield MHF, MCF, and MEF fractions, respectively. The light petroleum extract and MeOH-derived fractions were separately subjected to chromatographic fractionation and purification, utilizing a series of normal-phased chromatographic systems, to afford compounds 2 and 3 from the light petroleum extract, 1 from MCF, and 4 from MEF  data with those of the previously isolated compounds as arnebin-7 + and 239 [M-H2O-Me-MeOH]+ and in FAB-MS at m/z 269 [M-H2O+H]+ indicated the presence in the structure of 1 of at least two hydroxyl groups, a methyl and a methoxy group. The 13C-NMR spectrum displayed 17 carbon signals (3sp oxymethines), and six quaternary carbons (including one 3sp and two 2sp oxygenated carbons). The eight 2sp carbons resonating at δC 111.4–151.3 indicated the presence of four double bonds. Thus, the remaining four degrees of unsaturation indicated a tetracyclic skeleton for compound 1. The HMQC spectrum showed δH/δC correlations at 2.67 /33.2; 3.92 and 4.08 /77.9; 0.85 /19.5; and 3.79 /56.1 attributable to the presence of an isolated methylene (10-CH2), an isolated oxymethylene (12-CH2), a ring-junction tertiary methyl (10a-CH3) and an aromatic methoxy group (4-OCH3), respectively. The 1H-1H COSY experiment established three partial proton spin systems structure , a 1,2-disubstituted oxyethylene unit [b] , and a 1-oxyallyl moiety [c] . The linking of the above mentioned three partial structures [a–c], the isolated two methylenes, the tertiary methyl, the methoxy group with the remaining six quaternary carbons in the molecule (3J correlations to C-9a (qC), C-4a (qC), C-10a (qC) and C-8a (CH), respectively. Since the isolated methylene protons H2-10 and the oxymethine proton H-9 were found correlated to C-4a and C-10a and to C-9a and C-8a, respectively, therefore ring B should be fused with rings A and C through C-4a/C-9a and C-10a/C-8a, respectively  to C-5, C-8a, C-10, and C-10a positioned the single methyl group in the molecule at the ring juncture carbon C-10a. Furthermore, the HMBC correlations exhibited from H2-12 to C-8  and C-9  and from H-8a , to C-12 indicated a trisubstituted furan ring to be located at C-8, C-8a, and C-9. Acetylation of compound 1 afforded diacetate and triacetate derivatives 1a and 1b. The latter compound 1b displayed ester carbonyl absorptions at 1770 and 1734 cm−1 and no hydroxyl absorptions in the IR spectrum, whereas it exhibited successive ions peaks at m/z 370 [M-AcOH]+, 310 [M-2AcOH]+, and 250 [M-3AcOH]+ in the EI-MS due to elimination of three acetoxyl groups. Therefore, the three hydroxyl groups in 1 were determined. The C-1, C-5, and C-8 positions of these hydroxyl groups were suggested by detailed analysis of HMBC correlations of compound 1 (1b showed an upfield shift at C-1 (ΔδC-5.9) and downfield shifts at 5-CH (ΔδH + 1.06 and ΔδC + 0.8) and C-8 (ΔδC + 8.3), relative to those of compound 1, which further supported the locations of hydroxyl groups to be at C-1, C-5, and C-8, respectively. It is noteworthy to mention that the 2H singlet of the methylene protons at C-10 (δH 2.67) was transformed into two 1H doublets in the diacetate 1a  and in the triacetate 1b  derivatives as a result of acetylation of 5-OH in compound 1. On the basis of the above findings, the gross structure of compound 1 was thus deduced as illustrated in Compound  signals  which wetructure  of a 1,2molecule  was estaectively . The HMBmpound 1 . Moreove1 was interpreted from the NOE interactions  or 1b (δH 2.73) displayed NOE interactions with Me-11 and H-5 and designated as Hβ-10 whereas the other one (δH 2.49 in 1a or δH 2.54 in 1b) was found to be NOE correlated with H-8a. The α-orientation for H-8a and consequently for H-9 was thus confirmed. Moreover, one oxymethylene proton at C-12 in 1a (δH 3.92) or 1b (δH 4.05) exhibited significant NOE response with Me-11 of 1a or 1b, respectively, disclosing the β-configuration of the furan ring and thus 8-OH should be α-oriented. From the above findings, the structure and relative configuration of 1 was unambiguously established as -1,5,8-trihydroxy-4-methoxy-10a-methyl-8,9-oxymethylene-5,10a,8,8a,9,10-hexahydroanthracene and named arnebacene. The relative stereochemistry of compound ractions  observed2 was isolated as an orange-red amorphous powder. The molecular ion peak appearing in the EI-MS  and the NMR spectral data  pentanyl moiety. Furthermore, other detailed COSY and HMBC correlations  ,15. The  signals  attributNMR data  displayein-7 (3)  suggesteNMR data  with thorum of 2  revealedelations  along wielations  establis2 was identified as a novel heptacyclic arnebin-7 dimer. Comparison of proton chemical shifts and J values of H-12, H-11' and H-12' of 2 with those of 5 (4J long-range (J = 2.5 Hz) coupling occurred between H-2 and H-12' disclosed the cis configuration of H-2 octane core. The presence of 2,11-double bond is thought to catalyze the intramolecular rearrangement (cyclopropanation) in metabolite 5 to yield compound 2. Although they are few in number, cyclopropyl-containing natural products, in which the double bonds of their precursors involved in the cyclopropanation by a similar mechanism, have been reported. These can be exemplified by the biosynthesis of crythanthemic acid from dimethylallyl diphosphate +), 241 (12.5 [M-H2O-CH2O-Me]+), 189 . EI-MS 70 eV m/z: 304 , 286 , 268 , 239 , 188 . HRFAB-MS m/z 304.1307 .Acetylation of compound1: A solution of 1  in pyridine (0.3 mL) was mixed with Ac2O (0.2 mL), and the mixture was stirred at RT for 24 h. The mixture was diluted to 5 mL with distilled water, neutralized with sodium carbonate powder, extracted with ether (2 × 3 mL). The ether extract was then evaporated to dryness, dissolved in acetone-n-hexane (1:3) and subjected to normal phase HPLC using the same solvent system at a flow rate of 1 mL/min. Two UV-absorbing products: a diacetyl derivative 1a  and a triacetyl derivative 1b  were separately collected. 1a: A white amorphous powder. TLC . IR (neat) νmax 3350, 2950, 2830, 1770, 1734, 1481, 1369, 1240, 1201, 1084 cm−1. 1H-NMR  δH: 6.95 , 6.84 , 6.07 , 5.23 , 4.93 , 3.99 , 3.92 , 3.82 , 2.73 , 2.49 , 2.47 , 2.33 , 2.11 , 0.98 , 13C-NMR  δC: 170.5 , 170.4 , 155.0 , 144.0 , 133.0 , 127.1 , 126.0 , 125.8 , 120.5 , 110.1 , 78.8 , 77.9 , 72.7 , 71.4 , 55.6 , 50.5 , 34.0 , 33.0 , 21.1 , 20.0 . EI-MS 70 eV m/z: 388 , 370 , 328 , 295 (32.2 [M-AcOH-H2O-Me]+), 268 , 253 , 239 (12.5).1b: A white amorphous powder. TLC . IR (neat) νmax 2950, 2830, 1770, 1734 (strong), 1479, 1371, 1240, 1201, 1024 cm−1. 1H-NMR  δH: 6.95 , 6.85 , 6.61 , 6.10 , 5.05 , 4.90 , 4.40 , 4.05 , 3.83 , 2.73 , 2.54 , 2.83 , 2.30 , 2.12 , 2.10 , 0.97 , 13C-NMR δC: 170.7 , 170.5 , 155.0 , 144.0 , 130.0 , 129.0 , 128.0 , 127.1 , 120.5 , 110.2 , 87.1 , 76.0 , 72.6 , 71.1 , 56.3 , 47.1 , 34.0 , 33.0 , 22.2 , 21.1 , 18.3 . EI-MS 70 eV m/z: 370 , 310 , 295 (32.2 [M-2AcOH-Me]+), 253 (28.4), 250 , 241 (17.6), 227 (26.7). Compound2. Orange red amorphous powder. TLC . 1H and 13C-NMR data (CDCl3), see m/z: 538 , 523 , 495 , 469 , 457 , 415 (6.0), 403 , 391 (4.0), 202 , 391 (5.0), 317 (3.0), 279 (3.5), 239 (3.0), 202 , 189 , 149 (6.5), 108 .Compound3: Reddish violet amorphous powder, m.p. 94–95 °C. TLC . 1H and 13C-NMR data (CDCl3), see m/z: 272 , 254 , 229 (37.2), 216 , 204 , 189 , 108 .Compound4: White amorphous powder; m.p. 210–212 °C. TLC . 1H-NMR  δH: 7.60 , 7.51 , 6.90 , 3.87 . 13C -NMR  δC: 174.0 , 150.4 , 148.2 , 129.6 , 124.4 , 115.3 , 114.1 , 56.7 . EI-MS 70 eV m/z (intensity): 530 , 194 , 177 (36.5), 150 (15.4). FAB-MS m/z: 553 , 531 , 530 , 195 , 194 , 177 (57.1).Arnebia hispidissima (Lehm.) DC. roots resulted in the isolation two new polycyclic gernaylhydroquinone-derived metabolites: arnebacene (1) and arnebidin (2). Compound 1 was defined as -1,5,8-trihydroxy-4-methoxy-10a-methyl-8,9-oxymethylene-5,10a,8,8a,9,10-hexahydroanthracene. Compound 2 is reported herein as an heptacyclic arnebin-7 dimer with an unprecedented tricyclooctane core formed by the coupling of side chains.The phytochemical study of


Isaria fumosorosea ACCC 37814 to biotransform a panel of phenolic natural products, including flavonoids and anthraquinone, into their glycosides. Six new flavonoid (4-O-methyl)glucopyranosides are obtained and structurally characterized using high resolution mass and nuclear magnetic resonance spectroscopic techniques. These compounds further expand the structural diversity of flavonoid glycosides and may be used in biological study.Glycosylation is an efficient strategy to modulate the solubility, stability, bioavailability and bioactivity of drug-like natural products. Biological methods, such as whole-cell biocatalyst, promise a simple but highly effective approach to glycosylate biologically active small molecules with remarkable regio- and stereo-selectivity. Herein, we use the entomopathogenic filamentous fungus  O- or C-glycosidic bonds − ion at m/z 489.1422, indicating 4a was the methylglucosylated derivative of formononetin. This speculation was supported by the presence of resonance signals corresponding to a formononetin core, a d-glucose unit and a methoxy functional group in the 1H- and 13C-NMR spectra of 4a. HMBC interaction between the anomeric proton  and the oxygen-bearing aromatic carton at δC 161.3 (C-7) as well as the large coupling constant of this anomeric proton indicated that the glucose fragment was attached to C-7 of formononetin via an O-β-glucosidic linkage. The HMBC spectrum of 4a also displayed cross peak between the methoxy protons  and the oxygen-bearing sp3-carbon at δC 78.9 (C-4′′), confirming that the C-4 of the glucose moiety has a methoxy substituent (4a was elucidated as 4′-methoxyisoflavone 7-O-β-d-(4-O-methyl)glucopyranoside.2.0 mg of compound stituent . Hence, O-glycosides and flavone and flavonol O-glycosides account for the largest two groups of flavonoid glycosides − .4′,5-Dihydroxyflavanone 7-O-β-d-(4-O-methyl)glucopyranoside (1b): 1H- and 13C-NMR data see m/z 447.1294 [M − H]− .5,7-Dihydroxyflavanone 4′-O-β-d-glucopyranoside (1c): 1H- and 13C-NMR data see m/z 433.1122 [M − H]− .5,7-Dihydroxyflavanone 4′-O-β-d-(4-O-methyl)glucopyranoside (1d): 1H- and 13C-NMR data see m/z 623.1950 [M − H]− .5-Hydroxyflavanone 4′,7-di-O-β-d-(4-O-methyl)glucopyranoside (2a): 1H- and 13C-NMR data see m/z 461.1100 [M − H]− .4′,5,7-Trihydroxyflavone 3′-O-β-d-(4-O-methyl)glucopyranoside (2b): 1H- and 13C-NMR data see m/z 461.1097 [M − H]− .3′,5,7-Trihydroxyflavone 4′-O-β-d-(4-O-methyl)glucopyranoside (3a): 1H- and 13C-NMR data see m/z 475.1261 [M − H]− .5,7-Dihydroxy-4′-methoxyflavone-3′-O-β-d-(4-O-methyl)glucopyranoside (4a): 1H- and 13C-NMR data see m/z 489.1422 [M − H]− .4′-methoxyisoflavone 7-


SCSIO 41011. Their structures were determined by spectroscopic analysis and by comparison with literature data. And the absolute configurations were established according to the specific rotation or electron circular dichroism method. Antiviral evaluation results revealed that compounds 14, 15, 26, and 5-chloroisorotiorin displayed significant anti-IAV activities against three influenza A virus subtypes, including A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2), with IC50 values in the range of 2.52–39.97 μM. The preliminary structure-activity relationships (SARs) are also discussed. These findings expand the chemical and bioactive diversity of polyketides derived from the genus Diaporthe, and also provide a basis for further development and utilization of chromone, xanthone, and chloroazaphilone derivatives as source of potential anti-viral chemotherapy agents.Influenza A virus (IAV) is a severe worldwide threat to public health and economic development due to its high morbidity and mortality. Marine-derived fungi have been evidenced as a prolific source for the discovery of pharmacologically-active lead compounds. During the course of our search for novel bioactive substances from marine microorganisms, six new polyketides, including two octaketides ( However, two families of antiviral drugs are hitherto currently used to treat human IAV infections, which are NA inhibitors, like zanamivir and oseltamivir, and inhibitors of the viral M2 protein exemplified by amantadine and rimantadine , a negative sense RNA virus, is one of the main causes of human acute respiratory diseases characterized with high morbidity and mortality, posing a serious threat to public health and economic development  polarimeter. HRESIMS and ESIMS spectra data were recorded on a MaXis quadrupole-time-of-flight mass spectrometer and an amaZon SL ion trap mass spectrometer (Bruker), respectively. UV spectra were recorded on a Shimadzu UV-2600 PC spectrometer (Shimadzu). ECD spectra were performed on a Chirascan circular dichroism spectrometer (Applied Photophysics). IR spectra were measured on an IR Affinity-1 spectrometer (Shimadzu). The artificial sea salt was a commercial product (Guangzhou Haili Aquarium Technology Company).Semi-preparative HPLC was performed on a Hitachi Primaide apparatus using an ODS (octadecylsilanized silica) column . Chiral HPLC separation was performed using CHIRALPAK IC column . TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10–40 μm) and over silica gel (200–300 mesh) , and Sephadex LH-20 (Amersham Biosciences), respectively. Spots were detected on TLC  under 254 nm UV light. All solvents employed were of analytical grade . The NMR spectra were obtained on a Bruker Avance spectrometer (Bruker) operating at 500 and 700 MHz for Diaporthe sp. SCSIO 41011 had the same origination as that in our recent published paper . Compounds 23  and 25  were purified from Fr.2 (13 g) by an ODS column (MeOH/H2O: 10–100%) and followed by semiprep-HPLC with 72% (2.5 mL/min) and 50% MeCN (2.3 mL/min) elutions, respectively. Fr.3 (10 g) was purified on Sephadex LH-20 (MeOH), an ODS column (MeOH/H2O: 10–100%) and finally semiprep-HPLC  to afford compounds 17  and 24 . Fraction 4 (12 g) was subjected to Sephadex LH-20 (MeOH) to provide eight subfractions (Frs.4-1~4-8). Then ten subfractions (Frs.4-3-1~4-3-10) were obtained from Fr. 4-3 by an ODS column (MeOH/H2O: 10–100%). Compound 18 (150 mg) was isolated from Fr. 4-3-4 by recrystallization. Meanwhile, compounds 20 , 14  and 6  were isolated from Frs. 4-3-6, 4-3-7, 4-3-8 by semiprep-HPLC with 55% MeOH (2.5 mL/min), 50% MeCN (2.3 mL/min), and 68% MeCN (1.4 mL/min) elutions, respectively. Besides, compounds 27  and 26  was purified from Fr.4-4 and Fr. 4-6 by semiprep-HPLC with 80% MeOH (2.5 mL/min) and 60% MeCN (2.3 mL/min) elutions, respectively. Fraction 5 (4 g) was subjected to Sephadex LH-20 (MeOH) to provide five subfractions (Frs.5-1~5-5). Fr. 5-4 was further purified on semiprep-HPLC by 66% MeCN (2.5 mL/min) to give compounds 8 , 12 , and 7 . Seven subfractions (Frs.7-1~7-7) were obtained from Fr. 7 (4 g) by Sephadex LH-20 (MeOH). Then Fr. 7-4 was subjected to an ODS column (MeOH/H2O: 10–100%) to provide thirteen subfractions (Frs.7-4-1~7-4-13). Fr. 7-4-8 was further purified on semiprep-HPLC by 47% MeCN (2.5 mL/min) to give compounds 4 , 10 , and 2 , along with one subfraction (Fr. 7-4-8-6). Compounds 11  and 1  were purified from Fr. 7-4-8-6 by semiprep-HPLC by 56% MeOH (2.3 mL/min). Fr. 7-4-12 was subjected to semiprep-HPLC  to give compounds 16  and 15 . Similarly, Fr. 7-5 was subjected to an ODS column (MeOH/H2O: 10–100%) to provide eight subfractions (Frs.7-5-1~7-5-8). Compounds 19  and 28  were purified from Fr.7-5-5 and Fr.7-5-6 by semiprep-HPLC with 50% MeCN (2.5 mL/min) and 47% MeCN (2.5 mL/min) elutions, respectively. Fr. 7-5-7 was subjected to semiprep-HPLC  to afford compounds 22  and 21 . Fr.9 (7 g) was subjected to Sephadex LH-20 (MeOH) and followed by an ODS column (MeOH/H2O: 10–100%) to provide six subfractions (Frs.9-1-1~9-1-6). Fr. 9-1-4 was purified by semiprep-HPLC  to provide compounds 9 , 5 , 3 . Compund 13  was isolated from Fr.9-1-5 by semiprep-HPLC .The fermentation of the strain and the extraction and isolation of its extract was identical to that of our recent published paper , along with recently reported co-isolated 5-chloroisorotiorin  from experiments in triplicate. Student's Diaporthe sp. SCSIO 41011 was cultured on solid rice medium for 60 days. The EtOAc extract (150 g) of the fermentation was separated by continuously silica gel chromatography and semi-preparative HPLC chromatography to yield 28 aromatic polyketides (1–28). Their chemical structures were determined by comprehensive spectroscopic analyses or comparison with those reported data.The endophytic fungus 1 was obtained as colorless oil and had the molecular formula C19H26O7 as determined by a deprotonated ion peak at m/z 365.1607  in HRESIMS data. The 1H NMR data  and 3.65 , two aromatic protons at δH 6.26  and 6.19 , along with an array of methylene signals. The 13C NMR spectrum , six aromatic carbons , eight methylenes  and two methyls (δC 52.3 and 20.8). The aforementioned NMR data showed 1 was closely related structurally to the co-isolated 16-acetoxydothiorelone C (3)  at C-1′ in 1 instead of an ethyl group in 3, which was also verified by the HMBC correlation from H3-1′ to C-1 -acetoxydothiorelone A -acetoxydothiorelone A.Compound 13 was also isolated as colorless oil and gave a molecular formula of C18H22O5 relied on a deprotonated ion peak at m/z 317.1403  in the HRESIMS spectrum. The 1H NMR data  and 1.24 , three aromatic protons at δH 6.65 , 6.73  and 5.98 , along with several methylenes. The 13C NMR spectrum (Table C 173.6 and 181.4), six aromatic carbons (three oxygenated ones), six methylenes  and two methyls (δC 14.2 and 14.5). The above mentioned spectral characteristics were closely consistent to those of the co-isolated pestalotiopsone F (14)  in 13 rather than a methyl group (δH/C 3.66/52.2) at C-1′ in 14. These changes were also ascertained by the HMBC correlations from H3-1′ to C-1 and from H3-2′ to C-1′, as well as the 1H-1H COSY correlation of H3-2′ and H2-1′ , six aromatic carbons . The foregoing spectroscopic data showed great similarity to that of microsphaerophthalide F  located at C-6 in microsphaerophthalide F was replaced by an ethoxyl group (δH/C 4.60/65.3 at C-9 and 3.38/58.1 at C-10) anchored at C-6 in 17. This deduction was also supported by the HMBC correlations from H3-10 to C-9 and from H2-9 to C-5 and C-7. The barely measurable optical rotation value and quite weak Cotton effects in the ECD spectrum suggested 17 was racemic, which was also confirmed by the chiral HPLC analysis with two peaks (peak area ratio: 1:1) in the HPLC profile . However, the quantity of 17 was too little to perform further resolution. Hence, the structure of compound 17 was elucidated and the trivial name (±)-microsphaerophthalide H was assigned.Compound ta Table  of 17 re18 was obtained as colorless needle crystals and had the molecular formula C13H14O7, as evidenced by HRESIMS  and the NMR data. The highly similar NMR spectroscopic data of 18 to those of 17 indicated that their structures were closely related, except for the presence of an aldehyde group (δH/C 10.14/192.5) joined at C-4 in 18, rather than an ethoxyl group (δH/C 4.65/67.2 and 3.38/58.4) anchored at C-4 in 17. The absolute configuration of C-3 of 18 was mainly determined by comparison of the specific rotations with those reported data, as well as comparison between the computed and experimental ECD spectra. Among these reported 3-oxygenated phthalides, the 3S- and 3R- ones generally showed negative and positive specific rotations, respectively ] of 18 led to the deduction of 3R configuration in 18, which was also confirmed from experimental and calculated ECD spectra of 18 as shown in Figure 18 was single enantiomer. Consequently, the structure of 18 was determined as shown in Figure Compound 21 was obtained as a white solid and had the molecular formula C12H14O5, as determined from HRESIMS  and the NMR data. Detailed analyses of its NMR spectroscopic features implied that it was closely related structurally to convolvulopyrone  at C-10 in 21, indicating that 21 was a methyl derivative of convolvulopyrone. Besides, compound 21 and convolvulopyrone shared the same relative configuration of 7E due to the absence of a NOESY correlation between H-7 and H3-11. As a result, the structure of 21 was determined and given the trivial name methyl convolvulopyrone. However, compound 21 was likely obtained as an artifact formed in the process of extraction and purification using MeOH as a solvent.Compound 3)  -acetoxydothiorelone B (5)     -dothiorelone A (10) -2-(2-(7-acetoxyoctanoyl)-3,5-dihydroxyphenyl)acetate (11)              -2-acetate (11) was isolated as a naturally occurring compound for the first time. While sclerotinin A (22) was isolated as a diastereoisomeric mixture which could not be separated by RP-HPLC method . They were determined as 16-acetoxydothiorelone C , A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2), were then evaluated. Those compounds showed nearly no cytotoxicity against MDCK cells (IC50 > 200 μM). Compounds 14, 15, 26, and 5-chloroisorotiorin displayed significant anti-IAV activities against the above mentioned subtypes with IC50 values in the range of 2.52–39.97 μM  were inactive toward the three aforementioned IAV subtypes. Furthermore, pestalotiopsone F (14) and 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (26) exhibited obvious inhibition effect on A/FM-1/1/47 (H1N1), as well as A/Aichi/2/68 (H3N2), in a dose-dependent manner  exhibited selective inhibitions against the two IAV subtypes of A/FM-1/1/47 (H1N1) and A/Aichi/2/68 (H3N2) with the IC50 values of 6.74 ± 1.26 and 6.17 ± 1.46 μM, respectively. Comparing with the antiviral pestalotiopsones F (14) and B (15), the co-isolated siblings pestalotiopsones H (13) and A (16) were inactive toward the three IAV subtypes, which revealed methylation of the carboxyl group at C-1 for the pestalotiopsones with a C7 aliphatic branch located at C-11, and while ethylation of the carboxyl group at C-1 for the pestalotiopsones with a C5 aliphatic branch located at C-11, were essential for anti-IAV activities. Notably, among the three xanthones (25–27), only 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (26) demonstrated remarkable inhibitory effects against A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 H274Y (H1N1), and A/Aichi/2/68 (H3N2) with IC50 values of 4.80 ± 1.28, 9.40 ± 1.96, and 5.12 ± 1.49 μM, respectively, which indicated the hydroxyl group at C-3 probably promote the anti-IAV activities toward the three subtypes. By the way, 5-chloroisorotiorin  showed no obvious inhibition against A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 H274Y (H1N1), and A/Aichi/2/68 (H3N2).Additionally, based on comparison of the structural characteristics among these analogs, a preliminary structure-activity relationship is discussed. Pestalotiopsone F (1): colorless oil; UV (MeOH) λmax (log ε) 302 (3.75), 269 (3.89), 220 (4.16), 204 (4.27) nm; IR (film) νmax 3,356, 2,933, 2,833, 1,732, 1,714, 1,609, 1,591, 1,462, 1,261, 1,163, and 1,028 cm−1; 1H and 13C NMR data, Table m/z 365.1607 [M–H]− , 401.1368 [M+Cl]− .Dothiorelone O : colorleR)-acetoxydothiorelone A (2): colorless oil; [α]c 0.10, MeOH); UV (MeOH) λmax (log ε) 303 (3.69), 269 (3.78), 220 (4.14), 204 (4.17) nm; IR (film) νmax 3,358, 2,933, 2,858, 1,732, 1,714, 1,609, 1,558, 1,456, 1,265, 1,249, 1,159, and 1,024 cm−1; 1H and 13C NMR data, Table m/z 403.1741 [M+Na]+ , 419.1470 [M+K]+ , 783.3563 [2M+Na]+ .155R-acetox13): colorless oil; UV (MeOH) λmax (log ε) 291 (4.14), 250 (4.31), 242 (4.28), 217 (4.38) nm; IR (film) νmax 3,419, 2,927, 2,856, 1,732, 1,716, 1,645, 1,558, 1,456, 1,375, 1,274, 1,180, 1,161, and 1,028 cm−1; 1H and 13C NMR data, Table m/z 317.1403 [M–H]− , 353.1167 [M+Cl]− , 635.2860 [2M–H]− , 671.2626 [2M+Cl]− .Pestalotiopsone H 3: colorl17): a white solid; [α]c 0.06, MeOH); UV (MeOH) λmax (log ε) 307 (4.06), 249 (4.05), 220 (4.48) nm; IR (film) νmax 3,419, 2,935, 2,846, 1,747, 1,734, 1,604, 1,558, 1,448, 1,429, 1,375, 1,276, 1,201, 1,159, 1,093, and 1,074 cm−1; 1H and 13C NMR data, Table m/z 299.1123 [M+H]+ , 321.0954 [M+Na]+ , 619.2010 [2M+Na]+ .(±)-microsphaerophthalide H (18): colorless needle crystals; [α]c 0.10, MeOH); UV (MeOH) λmax (log ε) 367 (3.60), 295 (4.07), 245 (4.46), 201 (4.10) nm; ECD  λmax (Δε) 203 (−2.07), 225 (+4.62), 251 (−6.00) nm; IR (film) νmax 3,419, 2,945, 2,885, 1,770, 1,749, 1,668, 1,653, 1,558, 1,489, 1,375, 1,338, 1,286, 1,205, 1,097, and 1,058 cm−1; 1H and 13C NMR data, Table m/z 283.0809 [M+H]+ , 305.0632 [M+Na]+ .Microsphaerophthalide I (21): a white solid; UV (MeOH) λmax (log ε) 343 (4.05), 279 (3.73), 239 (4.51), 205 (4.12) nm; IR (film) νmax 3,446, 2,954, 2,927, 1,681, 1,635, 1,446, 1,355, 1,195, 1,182, 1,139, and 1,039 cm−1; 1H and 13C NMR data, Table m/z 239.0917 [M+H]+ , 261.0749 [M+Na]+ , 499.1590 [2M+Na]+ .Methyl convolvulopyrone 1: a whit1–2), one new chromone derivative (13), two new highly substituted phthalides (17–18), and one new α-pyrone derivative (21) along with 22 known congeners were isolated from a mangrove-associated fungus Diaporthe sp. SCSIO 41011, while methyl (R)-2-acetate (11) was isolated as a new natural compound and (±)-microsphaerophthalide H (17) was obtained as a racemic mixture. Amongst, pestalotiopsone F (14), pestalotiopsone B (15), 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate (26), and 5-chloroisorotiorin displayed pronounced anti-IAV activities against three IAV virus subtypes, including A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2) with IC50 values in the range of 2.52–39.97 μM. This work further enriched secondary metabolites in the genus Diaporthe and also expanded the chemical and bioactive diversity of polyketides, such as dothiorelones, cytosporones, phthalides, chromones, etc. Furthermore, our findings provide a basis for further development and utilization of pestalotiopsone, xanthone, and chloroazaphilone derivatives as source of potential anti-IAV chemotherapy agents.Twenty-eight aromatic polyketides, including two new octaketides (XiaL: designed the experiments and performed the isolation and characterization of all the compounds and wrote the manuscript; JY and FC: performed the antiviral experiment; XiuL: performed the isolation and purification of the fungal strain; CC: contributed to isolation of the compounds; XZ: designed the research work and wrote the manuscript; SL and YL: contributed in project design and manuscript preparation. All authors reviewed the manuscript and approved for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


OUPS-124D-1 attached to the marine alga Undaria pinnatifida. Dihydro-1,5-secovibralactone (4), a new vibralactone derivative, was isolated from the same fungal metabolites together with known vibralactone A (5), and 1,5-secovibralactone (6). The planar structures of these compounds have been elucidated by spectroscopic analyses using IR, HRFABMS, and NMR spectra. To determine the absolute configuration of the compounds, we used the phenylglycine methyl ester (PGME) method. These compounds exhibited less activity in the cytotoxicity assay against cancer cell lines.Sterepinic acids A–C ( OUPS-124D-1 derived from the marine alga Undaria pinnatifida. 5 was reported by Liu et al. + in HRFABMS. Its IR spectrum exhibited bands at 3330 and 1710 cm−1, that are characteristics of hydroxy and carbonyl groups, respectively. An analysis of the 1H and 13C NMR spectra of 1 (1H–13C heteronuclear multiple quantum coherence spectroscopy (HMQC), showed the presence of two olefin methyls (C-11 and C-12); four sp3-hybridized methylenes , including two oxygen-bearing sp3-methylenes (C-6 and C-7); one sp3-methine (C-2); two sp2-methines (C-3 and C-9); two quaternary sp2-carbons (C-4 and C-10); and one carbonyl group (C-1). In the 1H–1H correlation spectroscopy (COSY) analysis, correlations were observed between H-5 and H-6; H-2 and H-3; and H-2 and H-8, as shown by the bold lines in 1 as 6-hydroxy-4-(hydroxymethyl)-2-(3-methylbut-2-en-1-yl) hex-3-enoic acid. The elucidation of the absolute stereostructure of 1 is described below, together with those of 2–4.The molecular formula of sterepinic acid A  and C (3), were assigned the molecular formula of C24H38O7, with both compounds showing molecular weight almost twice as large as that of 1. While the general features of NMR spectra , except for the proton signal of the oxygen-bearing methylenes  in 2) and C-6 (δH 4.20 m) in 3). This phenomenon suggested that 2 and 3 were the dimers of 1. As expected, for the HMBC spectrum of 2  exhibited the molecular formula C12H20O4, containing two fewer hydrogen atoms, and one less oxygen atom than 1. Compared with the NMR spectra of 4  and H-5 (δH 4.68 ddd and 4.33 ddd), corresponding to H-2 and H-6 in 1, respectively, and the carbon signals of C-1 (δH 40.2), C-2 (δH 121.2), and C-7 (δH 174.3), corresponding to C-2, C-3, and C-1, respectively, in 1. The numbering of the carbon positions followed the numbering mentioned in a previous report D22 +58.0 ; IR (neat) νmax / cm−1: 3330, 1710. FABMS m/z (%): 229 , 211 (87.4%), 143 (34.2%), 69 (100%). HRFABMS m/z 229.1443 [M + Na]+ . 1H and 13C NMR data are listed in Sterepinic acids A (2): Pale yellow oil; [α]D22 +141.7 ; IR (neat) νmax / cm−1: 3362, 1730. FABMS m/z (%): 439 , 211 (93.5%), 69 (100%). HRFABMS m/z 439.2694 [M + H]+ . 1H and 13C NMR data are listed in Sterepinic acids B (3): Pale yellow oil; [α]D22 +53.5 ; IR (neat) νmax / cm−1: 3383, 1710. FABMS m/z (%): 439 , 211 (54.0%), 69 (96.1%). HRFABMS m/z 439.2694 [M + H]+ . 1H and 13C NMR data are listed in Sterepinic acids C (4): Pale yellow oil; [α]D22 +7.9 ; IR (neat) νmax / cm−1: 3396, 1736. FABMS m/z (%): 211  142 (37.7%), 69 . 1H and 13C NMR data are listed in Dihydro-1,5-secovibralactone  and (S)-PGME (0.054 mmol) in dry DMF (1 mL) was added to EDC-HCl (0.050 mmol), HOBt (0.050 mmol), and DMAP . The reaction mixture was stirred at room temperature 2 hours. Water (1.0 mL) was added to the reaction mixture, and then extracted using CH2Cl2. The organic layer was evaporated under reduced pressure, and the residue was purified by HPLC using MeOH–H2O (50:50) as the eluent to afford (S)-PGME amide 1a  as a pale yellow oil. To a solution of 1  and (R)-PGME (0.052 mmol) were treated with the same procedure to afford (R)-PGME amide 2a  as a pale yellow oil.1a: Pale yellow oil; HRFABMS m/z 376.2126 [M + H]+ . 1H NMR δ ppm (400 MHz in CDCl3): 1.61 , 2.16 , 2.25 , 2.47 , 2.60 , 3.22 , 3.68 , 3.79 , 4.07 , 4.11 , 4.99 , 5.54, , 5.60 , 7.24-7.34 .PGME amide 1b: Pale yellow oil; HRFABMS m/z 376.2126 [M + H]+ . 1H NMR δ ppm (400 MHz in CDCl3): 1.58 , 1.64 , 2.21 , 2.30 , 2.51 , 2.57 , 3.27 , 3.69 , 3.77 , 4.05 , 4.09 , 5.03 , 5.54, , 5.52 , 7.21–7.35 .PGME amide 1 (8.8 mg) was added trimethylsilyldiazomethane (10% in hexane) 2mL, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated under reduced pressure, and the residue was purified by HPLC using MeOH–H2O (60:40) as the eluent to afford methyl ester (6.5 mg) as a pale yellow oil.1: Pale yellow oil; [α]D22 −7.9 ; HRFABMS m/z 243.1597 [M + H]+ . 1H NMR δ ppm (600 MHz in CDCl3): 1.61 , 1.68 , 2.19 , 2.42 , 2.45 , 2.47 , 3.32 , 3.64 , 3.73 , 4.08 , 4.10 , 5.04 , 5.56, .Methyl ester of 2 (3.2 mg) in MeOH  was added concd H2SO4 (0.01 mL), and the reaction mixture was left at room temperature for 1 hr. The mixture was diluted with water, and extracted with CH2Cl2, and the extract was evaporated under reduced pressure, and then the residue was purified by HPLC using MeOH–H2O (60:40) as the eluent to afford methyl ester (0.8 mg) as a pale yellow oil.To a solution of 2, a solution of 3 (3.3 mg) in MeOH (0.5 mL) was treated with concd H2SO4 (0.01 mL), and purified by HPLC using MeOH–H2O (60:40) as the eluent to afford methyl ester (0.8 mg).Using the same procedure as above with 2, a solution of 4 (2.4 mg) in MeOH (0.5 mL) was treated with concd H2SO4 (0.01 mL), and purified by HPLC using MeOH–H2O (60:40) as the eluent to afford methyl ester (0.7 mg).Using the same procedure as above with 1–3) and dihydro-1,5-secovibralactone (4), have been isolated from a strain of Stereum sp. derived from marine sponge. Their absolute configurations were established by the application of the PGME method to 1 and the chemical transformation of 2–4. In this study, new carboxylic acids designated as sterepinic acids A–C (In the screening for the search of the seeds of antitumor agents, these compounds did not exhibit significant cytotoxic activity against three cancer cell lines.


Myoporum bontioides is a traditional medicinal plant in Asia with various biological activities, including anti-inflammatory and anti-bacterial characteristics. To identify the bioactive constituents from M. bontioides, a newly-identified flavone, 3,4′-dimethoxy-3′,5,7-trihydroxyflavone (compound 1), along with eight known compounds, were investigated in human MCF-7 breast cancer, SCC4 oral cancer, and THP-1 monocytic leukemia cells. Among these compounds, compound 1 exhibited the strongest antiproliferative activity with half-maximal inhibitory concentration (IC50) values ranging from 3.3 μM (MCF-7) to 8.6 μM (SCC4). Flow cytometric analysis indicated that compound 1 induced G2/M cell cycle arrest in MCF-7 cells. Mechanistic evidence suggests that the G2/M arrest could be attributable to compound 1’s modulatory effects on the phosphorylation and expression of numerous key signaling effectors, including cell division cycle 2 (CDC2), CDC25C, and p53. Notably, compound 1 downregulated the expression of histone deacetylase 2 (HDAC2) and HDAC4, leading to increased histone H3 acetylation and p21 upregulation. Together, these findings suggest the translational potential of compound 1 as a breast cancer treatment. Myoporum bontioides (Myoporaceae) is an evergreen shrub distributed throughout Taiwan, South China, and Japan ) (Jasco Corporation); Fourier transform IR (FT-IR), Shimadzu-IR Prestige-21 FTIR spectrophotometers (in cm−1) ; 1H-, 13C-NMR, and two-dimensional (2D)-NMR Spectra, Agilent Technologies DD2 600 spectrometers  , and EIMS and HREIMS, Finnigan Thermo Quest MAT-95XL mass spectrometer [m/z (rel. %)] .Chromatographic purification and spectroscopic characterization of the test agents were conducted using the following products and instruments. TLC, silica gel 60 FM. bontioides (Myoporaceae) were collected and identified by one of the co-authors, Dr. Wei-Yu Lin in Kinmen County, Taiwan in October 2011, and a voucher specimen (2011) has been deposited in the College of Medicine, China Medical University.The leaves of M. bontioides (3.1 kg) were ground, extracted with acetone at 25 °C, and concentrated under reduced pressure to afford a brown residue (90 g). This residue was fractionated using silica gel column chromatography using n-hexane:ethyl acetate (EtOAc), 19:1; n-hexane:EtOAc, 9:1; n-hexane:EtOAc, 4:1; n-hexane:EtOAc, 1:1, and n-hexane:EtOAc:methanol (MeOH), 1:1:1, to yield five fractions (A–E). Fractions B and E were further subjected to silica gel column chromatography to obtain compounds 1–9 as described below. Fraction E (1.0 g), CH2Cl2–MeOH (13:1) yielding fractions E1–E6; fraction E6 (238 mg), CH2Cl2:acetone (4:1) yielding 1 (10 mg) and 2 (5 mg). Fraction E5 (172 mg), CHCl3:acetone (5:1) yielding 3 (4 mg) and 4 (5 mg). Fraction E3 (59 mg), CHCl3:MeOH (9:1) yielding 5 (3 mg) and 6 (3 mg). Fraction E4 (49 mg), CHCl3:EtOAc (9:1) yielding 7 (6 mg). Fraction B (311 mg), n-hexane:EtOAc (9:1) providing fractions B1–B6; fraction B4 (105 mg), n-hexane–acetone (4:1) yielding 8 (12 mg). Fraction B3 (95 mg), n-hexane:EtOAc (4:1) yielding 9 (28 mg).The leaves of 3,4′-Dimethoxy-3′,5,7-trihydroxyflavone (1): yellow powder; UV (MeOH) λmax (log ε) nm: 209 (4.45), 255 (4.20), 268 (4.20), 295 (3.95), 355 (4.15); (AlCl3): 209 (4.47), 268 (4.24), 299 (3.98), 362 (4.11), 401 (4.12); (NaOAc): 219 (4.78), 276 (3.30), 321 (4.00), 374 (4.07); (NaOAc-H3BO3): 218 (4.77), 256 (4.15), 269 (4.15), 296 (3.90), 358 (4.09); (NaOMe): 212 (4.70), 272 (4.47), 305 (3.98), 389 (4.18); IR (KBr) υmax: 3452, 1655 cm−1; 1H-NMR : δ 3.88 , 3.95 , 6.25 , 6.51 , 7.12 , 7.64 , 7.66 ; 13C-NMR : δ 56.3 (OMe-4′), 60.2 (OMe-3), 94.5 (C-8), 99.4 (C-6), 105.9 (C-10), 112.1 (C-5′), 115.8 (C-2′), 121.8 (C-6′), 124.1 (C-1′), 139.5 (C-3), 147.3 (C-3′), 150.9 (C-4′), 156.5 (C-2), 157.8 (C-9), 163.2 (C-5), 165.0 (C-7), 179.5 (C-4); EIMS (70 eV) m/z (rel. int.): 330 [M]+ (100), 301 (10), 287 (31); HREIMS m/z 330.0743 . The spectra were showed in 2–9 were identified using spectroscopic methods and were compared with literature data [The structures of compounds ure data ,19,20,2115Ser p53, p53, CDK6, CDC25C, p-216Ser CDC25C, CDC2, p-15Tyr CDC2, cyclin A, cyclin B1, cyclin D1, HDAC2, HDAC4, p21 ; β-actin ; p-Ser/Thr-Pro MPM2 ; and acetyl Histone H3 . The enhanced chemiluminescence (ECL) system for detection of immunoblotted proteins was from GE Healthcare Bioscience . All other chemicals and reagents were obtained from Sigma-Aldrich  unless otherwise mentioned.All of the chemicals used were dissolved in dimethyl sulfoxide (DMSO) and were added to the culture medium at indicated concentrations to a final DMSO concentration <0.1%. The following antibodies were used: p-2.MCF-7 breast cancer, SCC4 oral cancer, and THP-1 acute myeloid leukemia cells were purchased from the American Type Culture Collection . MCF-7 and SCC4 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium  and THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium . All cells were supplemented with 10% heat-inactivated FBS, 5 mg/mL of penicillin, 10 mg/mL of neomycin, and 5 mg/mL streptomycin at 37 °C in a humidified incubator in an atmosphere of 5% CO3/200 μL) were seeded in 96-well flat-bottomed plates in 10% FBS-supplemented medium, incubated for 24 h, and then they were exposed to various concentrations of the compounds dissolved in DMSO  in 5% FBS-supplemented medium. Control cells were treated with the DMSO vehicle at a concentration equal to that of the compound-treated cells. Then, the medium was removed, replaced with 200 μL 0.5 mM MTT in 10% FBS-containing DMEM/Ham’s F-12 medium, and the cells were incubated in a 5% CO2 incubator at 37 °C for 3 h. After removing the supernatant, the reduced MTT dye was solubilized in DMSO, and the absorbance at 570 nm was determined using a plate reader. The test agent-treated cell viability was expressed as a percentage of the viable control cells. The IC50 values of each group were calculated using median-effect analysis and presented as the mean ± standard deviation (SD).The suppressive effects of the compounds on cell viability were assessed using the MTT assay  in six r5) were plated and treated with compound 1 for 48 h with 5 % FBS-supplemented DMEM/F12. The cells were collected, fixed in 70% cold ethanol for 4 h at 4°C, centrifuged at 1200 rpm for 5 min, and then re-suspended in ice-cold PBS containing 2% FBS. Then, the cells were stained with propidium iodide (PI) and analyzed using flow cytometry and the multicycler (ModFitLT 3.0) software program . For apoptosis evaluation, cells were stained with annexin V and PI (1 μg/mL) and determined on a BD FACSAria flow cytometer (Becton Dickinson).Cell cycle analysis was performed using flow cytometry . Briefly5/mL) were treated with DMSO or compound 1 (0–5 μM) with or without GSH pre-treatment for 15 min for 3 h. Then, the cells were washed twice with PBS and stained with H2DCFDA (5 μM) at 37 °C for 30 min. After washing with PBS, the fluorescence intensity induced by ROS generation was assessed using a flow cytometer .ROS production was detected using the fluorescence dye 2′,7′-dichlorodihydrofluoresceindiacetate  . The celg for 5 min. Equivalent amounts of protein (60–100 μg) from each cell lysate were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes, which were blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST). Then, the membranes were incubated overnight with the corresponding primary antibodies (1:1000–1:2000) at 4 °C, followed by washing with PBST four times, incubation with the secondary antibody (1:1000) in PBST at room temperature for 1 h, and then they were visualized using ECL.The drug-treated cells were collected, washed with ice-cold PBS, and then resuspended in lysis buffer . Solublet-test for two-group comparisons, and p-values < 0.05 were considered statistically significant.The data were presented as means ± SD. Statistical analysis was performed using Student's


Spilanthes acmella Murr. (Compositae) has been used as a traditional medicine for toothache, rheumatism and fever. Its extracts had been shown to exhibit vasorelaxant and antioxidant activities. Herein, its antimicrobial, antioxidant and cytotoxic activities were evaluated. Agar dilution method assays against 27 strains of microorganisms were performed. Results showed that fractions from the chloroform and methanol extracts inhibited the growth of many tested organisms, e.g. Corynebacterium diphtheriae NCTC 10356 with minimum inhibitory concentration (MIC) of 64-256 μg/mL and Bacillus subtilis ATCC 6633 with MIC of 128-256 μg/mL. The tested fractions all exhibited antioxidant properties in both DPPH and SOD assays. Potent radical scavenging activity was observed in the DPPH assay. No cytotoxic effects of the extracts against KB and HuCCA-1 cell lines were evident. Bioassay-guided isolation resulted in a diverse group of bioactive compounds such as phenolics , coumarin  and triterpenoids like 3-acetylaleuritolic acid (1), β-sitostenone (3), stigmasterol and stigmasteryl-3-O-β-D-glucopyranosides, in addition to a mixture of stigmasteryl-and β-sitosteryl-3-O-β-D-glucopyranosides. The compounds 1–6 represent bioactive metabolites of S. acmella Murr. that were never previously reported. Our findings demonstrate for the first time the potential benefits of this medicinal plant as a rich source of high therapeutic value compounds for medicines, cosmetics, supplements and as a health food. Spilanthes acmella Murr. (Compositae) is the well known “toothache plant”, also commonly used as a spice. It has a long history of use as a folklore remedy, e.g. for toothache, rheumatism and fever [nd fever ,2. The pnd fever , swellinnd fever , periodond fever  and in mnd fever . In addind fever . The pland fever ,8. As a nd fever  small amnd fever .S. acmella Murr., for example, spilanthol, isobutylamides [S. acmella Murr. exhibits vasorelaxant and antioxidant activities [S. acmella Murr. and examine their antimicrobial and antioxidant activities. In addition, cytotoxic effects of the plant extracts was also tested.A number of constituents had been isolated from the ylamides ,12 and tylamides . Our rectivities . These rtivities . These fS. acmella Murr. were evaluated for antimicrobial, antioxidant and cytotoxic activities. Bioassay-guided isolation was carried out by repeated silica gel column using gradient elution with solvents of increasing polarity. The structures were confirmed by comparison of their spectral data  with literature data. 2D NMR spectral data were also obtained. The hexane extract of S. acmella Murr. gave stigmasterol from fractions H1, H3, H7, while H8 including a mixture of triterpenoids. The chloroform extract provided stigmasterol from fraction C3, stigmasteryl-3-O-β-D-glucopyranoside (SG) from fraction C8, together with a mixture of long chain hydrocarbon esters. Fractionation of the ethyl acetate extract gave three compounds; 3-acetylaleuritolic acid (1), vanillic acid (2) and β-sitostenone (3) from fractions E5, E6, and E8, respectively. The methanol extract afforded four compounds; scopoletin (4), trans-ferulic acid (5), trans-isoferulic acid (6) and a mixture of stigmasteryl-3-O-β-D-glucopyranoside and β-sitosteryl-3-O-β-D-glucopyranoside (MBSG) from fractions F2, F3, M2, and M3, respectively. Isolates are summarized in 1-6 are shown in In the present study extracts, fractions and isolates of 1, 2, 4, 5, 6, stigmasterol, SG and MBSG of S. acmella Murr. were tested for antimicrobial activity against 27 strains of microorganisms using the agar dilution method [Saccharomyces cerevisiae ATCC 2601 with MIC 256 µg/mL. The chloroform extract also completely exhibited antigrowth activity against Streptococcus pyogenes II with MIC 256 µg/mL.The hexane, chloroform, ethyl acetate and methanol extracts, fractions C2-C11, C2.2, C2.3, C2.7, C3.2, E1-E14, F1-F5 and M1-M6 and isolates n method . The resC. diphtheriae NCTC 10356 with MIC 64-256 µg/mL. In addition, the fraction C4 also completely inhibited the growth of B. subtilis ATCC 6633 (MIC 128 µg/mL) and Bacillus cereus with MIC 256 µg/mL.Fractions C3, C4, C5, C2.2, C2.3, C 2.7 and C3.2 isolated from the chloroform extract exhibited antigrowth activity against C. diphtheriae NCTC 10356 with MIC 64-128 µg/mL. Antigrowth activity of ethyl acetate and methanol extracts, including fractions C2, C6-C11, E1, E2, E5-E13 and M1, were evaluated at 256 µg/mL, but no activitiy was observed. The isolates, compounds 1, 2, 4, 5, 6, stigmasterol, SG and MBSG were tested at 64 µg/mL, but found to be inactive.Fractions  of ethyl acetate extract inhibited the growth of C. diphtheriae NCTC 10356 with MIC 64-256 µg/mL. Particularly, fractions C3, C4, E4 and E14 inhibited the growth of C. diphtheriae NCTC 10356 with MIC 64 µg/mL. All the tested methanol fractions , except M1, showed antimicrobial activity. Fractions F1-F5, M2, M3 and M5 selectively inhibited the growth of B. subtilis ATCC 6633 with MIC 128 µg/mL, the MIC of M4 was 256 µg/mL whereas fractions F4, M2, M5 and M6 also exhibited activity against C. diphtheriae NCTC 10356 with MIC 128 µg/mL. In addition, F1 and F2 exerted antigrowth activity against C. diphtheriae NCTC 10356 with MIC 256 µg/mL. Furthermore, M2 also inhibited the growth of Micrococcus lutens ATCC 10240, Staphylococcus epidermidis ATCC 12228 and B. cereus with MIC 128-256 µg/mL.It is interesting to note that fractions from the chloroform and ethyl acetate extracts show selective growth inhibition against 6. Fractions M3, M4 and M5 showed good (71.88-78.49%) antioxidant activity, whereas moderate activity was observed for F1 (48.75%) and F2 (38.29%), which yielded coumarin 4. As for the fractions of the ethyl acetate extract, E6 exhibited the highest antioxidant activity (82.46%), while E5 and E8 showed good activity . Interestingly, E6, with the highest antioxidant activity in the DPPH assay also produced the highest SOD activity (81.50%), resulting in the isolation of phenolic 2. Triterpenoids 1 and 3 were obtained from fractions E5 and E8, respectively. Glucoside fractions SG (C8) and MBSG (M3) showed good antioxidant properties, but the stigmasterol fraction (C3) showed weak activity. However, it is noteworthy that fractions (F and M) with strong or potent antioxidants all exerted antimicrobial action too. Similar results were also found for fractions of the chloroform extract . Fractions from the chloroform, ethyl acetate and methanol extracts were tested for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)  and supe50 values greater than 10 µg/mL and were consequently considered to be inactive. The extracts of hexane, chloroform, ethyl acetate and methanol were tested against the KB and HuCCA-1 cell lines . The res2, 5, and 6), coumarin (4), triterpenoids . The most potent antioxidant fraction (SOD assay) of chloroform extract [SG). The former had been isolated previously from a light petrol extract of S. acmella Murr. along with β-sitosteryl-3-O-β-D-glucoside from the ethanol extract [1-6 are bioactive metabolites that have never been isolated from S. acmella Murr.. Significantly, the ethyl acetate and methanol extracts displaying the most potent radical scavenging activity (DPPH)  afforded extract  afforded extract  and isol extract . Due to 1) had been shown to exhibit diverse bioactivities, e.g. antigrowth activity against S. aureus and S. typhimurium [O. gutturosa [S. acmella Murr. [1) isolated from fraction E5 of the ethyl acetate extract. The isolated compounds had been reported to possess diverse bioactivities as follows: 3-acetyl-aleuritolic acid (himurium  and signutturosa . In addiutturosa  and stroutturosa . It had utturosa . In our la Murr.  could po2) had been reported to exert strong antioxidant  [Vanillic acid (tectant) , powerfutectant) , protecttectant)  as well tectant)  and immutectant)  propertiβ-Sitostenone (3) is a triterpenoid with diverse activities such as significant hypoglycemic [glycemic , antiarrglycemic  and pronglycemic  activiti4) possesses interesting activities, in particular, vasorelaxant [Scopoletin  is an important natural antioxidant present in fruits, vegetables, rice bran [Ferulic acid  has been known as a component of Chinese herbal medicine used for a pain killer and stomachic [Cimicifuga . 1-6, stigmasterol and its glucoside together with a mixture of stigmasteryl and β-sitosteryl glucosides from S. acmella Murr.. In this and other studies these compounds possessed marked antioxidant, vasorelaxant, and antimicrobial activities including related effects, e.g. antiinflammatory, antipyretic, analgesic, antiplatelet aggregation, antidiabetic, hypotensive, neuroprotective, cardiovascular, antiviral, anticancer and chemoprotective effects. Promisingly, scopoletin (4) exerted antioxidant, vasorelaxant and antimicrobial actions whereas trans-ferulic acid (5) elicited antioxidant and vasorelaxant activities.This study reports the successful isolation of a diverse group of bioactive metabolites 2), trans-isoferulic acid (6), stigmasterol and stigmasteryl glucoside had been reported to be strong antioxidants. 3-Acetylaleuritolic acid (1) displayed antimicrobial and strong cytotoxic activities. β-Sitostenone (3) showed significant hypoglycemic, antiarrhythmic and antitubercular actions. These compounds 1-6 represent the bioactive metabolites that were never previously isolated from the S. acmella Murr.. The chloroform extract with antioxidant and antimicrobial activities afforded fractions  of strong antigrowth actions against C. diphtheriae NCTC 10356 with MIC 64-128 µg/mL. Interestingly, the inactive antimicrobial extracts (ethyl acetate and methanol) provided fractions mostly with strong growth inhibition against C. diphtheriae NCTC 10356 and B. subtilis ATCC 6633 with MIC 64-128 µg/mL. Moreover, strong or potent antioxidant fractions  of methanol extract exhibited antimicrobial activity. This relation was also observed for fractions of the chloroform extract. As a result, the data support the use of S. acmella Murr. as a rich source of compounds with high therapeutic values for medicines, cosmetics, food supplements and as a health food.Other isolates, vanillic acid  were obtained on Perkin Elmer System 2000 FTIR. Ultraviolet (UV) spectra were measured with Milton Roy Spectronic 3000 Array. Column chromatography was carried out using silica gel 60 (0.063 – 0.200 mm) and silica gel 60 (<0.063 mm). Thin Layer Chromatography (TLC) and preparative TLC were carried out on silica gel 60 PF254 .Melting points were determined on an Electrothermal 9100 melting point apparatus and are uncorrected. S. acmella Murr. were prepared as previously described [Extracts  of escribed .HuCCA-1 cells were established from chlolangiocarcinomas experimentally induced in hamsters. The cell lines were characterized and have been maintained in CRI laboratory ever since 1994 in Ham’s F12 culture medium  supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 μg/mL streptomycin. The KB cell lines, originally derived from epidermoid carcinoma of the floor of the oral cavity and commonly used as a reference laboratory standard for cytotoxicity assay, have been maintained in CRI laboratory in DMEM (Dulbecco’s modified Eagle medium).Isolation was performed using conventional (gravity) column chromatography otherwise stated. A ratio of 1:30 for separated materials and silica gel was used for the chromatography. The separation was carried out using gradient elution with increasing polarity. Fractions were combined based on TLC chromatograms.The extract (55 g) was separated to give 10 fractions (H1–H10 from hexane and dichloromethane elutions) of dark-green gum. Fractions H1, H3, H7 and H8 were rechromatographed on a silica gel column. Fraction H1 (5.31 g) gave stigmasterol (from hexane-dichloromethane elutions) 3.7 mg, m.p. 151-152 °C . FractioSG (stigmasteryl-3-O-β-D-glucopyranoside) 15.4 mg, m.p. 261-262 °C (lit m.p. 265-267 °C [The extract (50 g) was separated to afford 11 fractions (C1–C11 from hexane-ethyl acetate and ethyl acetate-methanol elutions) of dark-green wax. Fractions C2, C3, C4, C5, and C8 were further isolated and/or purified. Fraction C2 (6.5 g) gave eight fractions (C2.1–C2.8) from chloroform-ethyl acetate elutions of the wax. Fraction C3 (5.43 g) was separated to provide six fractions of dark-green gum (C3.1–C3.6) from chloroform-ethyl acetate elutions. The fraction C3.3 (2.84 g) was further separated to give eight fractions (C3.3.1–C3.3.8) from chloroform-ethyl acetate elutions. Fraction C3.3.5 was recrystallized from methanol to give 60 mg of stigmasterol. Fraction C4 (3.65 g) was purified by column to afford 12 fractions (C4.1–C4.12 from ethyl acetate elutions). Fraction C4.1, as a dark-green solid, was recrystallized from methanol to give solid 85 mg, m.p. 61-71°C (a mixture of long chain hydrocarbon ester). Fraction C5 (3.13 g) was separated to give eight triterpene fractions (C5.1–C5.8 from ethyl acetate-methanol elutions). Fraction C8 (103.4 mg) was recrystallized from chloroform-methanol to provide solid 5-267 °C ).1 . Fraction E6 (2.78 g) obtained from 50% ethyl acetate-chloroform elutions, was further purified by column chromatography to afford an orange gum (673 mg from 50% ethyl acetate–hexane elutions). The gum was purified by column chromatography. Elution with 5% methanol-chloroform gave a white crystalline solid which was recrystallized from chloroform to give white crystals of compound 2 . Fraction E8 (2.61 g) as a greenish gum obtained from 70% ethyl acetate-chloroform elutions, was further purified by column chromatography. Elution with 20% ethyl acetate-hexane gave a white semi-solid (27.1 mg) which was purified by repeated preparative TLC on silica gel developed 5 times with 8% ethyl acetate-hexane to give compound 3 .The extract (50.7 g) was applied to a silica gel column. Elution with hexane, then chloroform and ethyl acetate mixtures with increasing polarity, and finally mixtures enriched with methanol gave 14 fractions (E1 – E14). Three selected main fractions  were further isolated and purified. Fraction E5  was separated to obtain a yellowish green semi-solid (354.2 mg) from 40% ethyl acetate-hexane elutions. The semi-solid was purified by silica gel column chromatography. Elution with 20% ethyl acetate-hexane gave white needles which were recrystallized from methanol to give white crystals of compound 4 . Fraction F3 (4.25 g) as a brown gum from 25% methanol-chloroform fractions was purified by column chromatography. Elution with 15% methanol-chloroform gave a brown semi-solid (1.02 g) which was further purified by column chromatography. Elution with 10% methanol-chloroform provided impure compound (160 mg). Recrystallization from chloroform gave compound 5 .The extract (40.0 g) was separated on a silica gel column. Elution with chloroform then with chloroform-methanol mixtures of increasing polarity afforded five fractions (F1-F5). The main fractions F2 and F3 were further purified. Fraction F2  was separated to afford a brown gum (405.9 mg from 10% methanol-chloroform elutions). The gum was purified by column chromatpgraphy (elution with methanol-chloroform) to give a combined fraction (103.1 mg) which was rechromatographed on a silica gel column. Gradient elution with 3-5% methanol-chloroform gave compound 6 . Fraction M3 (313.5 mg) as a greenish semi-solid was obtained from 20% methanol-chloroform elutions. The semi-solid (80.2 mg) was recrystallized from methanol to give compound MBSG . The extract (123.4 g) was isolated on a silica gel column. Elution with chloroform, then gradually increasing mixtures enriched with methanol afforded six fractions (M1-M6). The main fractions M2 and M3 were purified further. Fraction M2  was separated by column chromatography. Gradient elution with 7% methanol-chloroform gave a brownish solid (30.7 mg) which was recrystallized from chloroform to give compound 3-Acetylaleuritolic acid (1): m.p. 299-300 °C, (lit m.p. 302-304 °C [υmax (KBr) cm–1: 3435, 2935, 1734, 1686, 1364, 1242, 1026 [1H-NMR (CDCl3) δ 5.47 , 4.39 , 1.97 , 0.86 , 0.81 , 0.78 , 1.18 ; 13C-NMR (CDCl3) δ 37.88 (C-4), 39.00 (C-8), 37.87 (C-10), 37.37 (C-13), 160.53 (C-14), 116.59 (C-15), 51.44 (C-17), 29.22 (C-20), 171.06 (COOCH3), 184.03 (COOH), 80.88 (CH-3), 55.59 (CH-5), 49.03 (CH-9), 41.58 (CH-18), 40.83 (CH2-7), 37.37 (CH2-1), 35.33 (CH2-19), 33.64 (CH2-12), 33.30 (CH2-21), 31.43 (CH2-16), 30.76 (CH2-22), 23.42 (CH2-2), 18.64 (CH2-6), 17.26 (CH2-11) 31.92 (CH3-29), 28.65 (CH3-30), 27.91 (CH3-23), 26.02 (CH3-26), 22.46 (CH3-27), 21.21 (COOCH3), 16.52 (CH3-24), 15.52 (CH3-25); MS m/z (% relative intensity): 329 (3), 269 (7), 234 (7), 189 (100) 133 (21), 119 (50) [2-304 °C , 304-3052-304 °C ); FTIRυm42, 1026 ; 1H-NMR 119 (50) .Vanillic acid (2): m.p. 210-212 °C (lit m.p. 213-214°C [λmax (MeOH) nm (log ε): 253(3.41), 286(3.47) [υmax cm–1: 3485, 2955, 1686, 1598, 1547, 1523, 1473, 1299, 1239, 1205, 1113, 918, 882; 819 [ 1H-NMR (CD3OD+CDCl3) δ 7.55 , 7.59 , 6.88 , 3.92 ; 13C-NMR (CD3OD+CDCl3) δ 169.11 (CO), 150.81 (C-1), 147.00 (C-3), 124.22 (C-6), 121.90 (C-4), 114.56 (C-5), 112.63 (C-2), 55.68 (OCH3); MS m/z (% relative intensity): 168 , 153 (72), 125 (35), 97 (55), 77 (5) [13-214°C ); UVλmax86(3.47) ; FTIRυma882; 819 ; 1H-NMR , 77 (5) .β-Sitostenone (3): m.p. 97-99 °C, (lit m.p. 95-96 °C [υmax (KBr)cm–1: 2936, 1681, 1464, 1378, 1228 [1H-NMR (CDCl3) δ 5.74 , 0.71 , 0.80-1.10 , 1.18 ; 13C-NMR (CDCl3) δ 29.64 (CH-25), 35.60 (CH-8), 36.07 (CH-20), 45.81 (CH-24), 53.79 (CH-9), 55.85 (CH-14), 55.99 (CH-17), 123.69 (CH-4), 11.14 (CH3-29), 11.90 (CH3-18), 18.65 (CH3-19), 18.98 (CH3-21), 19.75 (CH3-27), 20.99 (CH3-26), 21.10 (CH2-11), 23.04 (CH2-28), 24.14 (CH2-15), 26.08 (CH2-23), 28.13 (CH2-16), 32.91(CH2-7), 33.86 (CH2-6), 33.93 (CH2-2), 35.65 (CH2-22), 36.06 (CH2-1), 38.57 (CH2-12), 39.59 (C-10), 42.35 (C-13), 171.64 (C-5),199.58 (C=O); MS m/z (% relative intensity): 412, 397(27), 370(13), 288(26), 271(39), 229(92), 187(26), 173(46), 147(57), 124(100) [95-96 °C ); FTIRυm78, 1228 ; 1H-NMR 124(100) .Scopoletin (4): m.p. 205-206 °C (lit m.p. 203-204 °C [λmax (MeOH) nm (log ε): 294(3.68), 344(4.07) [υmax (KBr) cm–1: 3333, 1702, 1566, 1437 [ 1H-NMR (CD3OD) δ 6.23 , 7.89 , 6.82 , 7.14 , 3.91 , 8.10 ; 13C-NMR (CD3OD) δ 163.00 (CO), 150.00 (C-9), 149.00 (C-7), 146.00 (C-6), 144.91 (C-4), 111.15 (C-3), 110.00 (C-10), 108.63 (C-5), 102.57 (C-8), 55.51 (OCH3); MS m/z (% relative intensity): 192 , 177 (28), 164 (41), 121 (37) [3-204 °C ); UVλmax44(4.07) ; FTIRυma66, 1437 ; 1H-NMR 121 (37) .trans-Ferulic acid (5): m.p.168-169 °C (lit m.p. 168-169 °C [λmax (MeOH) nm (log ε): 289 (3.83), 318 (3.86) [υmax (KBr) cm–1: 3437, 1691, 1665, 1517 [1H-NMR (CD3OD) δ 7.18 , 7.07 , 6.82 , 6.31 , 7.59 , 3.89 ; 13C-NMR (CD3OD) δ 171.19 (CO), 151.50 (C-3), 149.90 (C-4), 127.76 (C-1), 123.97 (C-6), 116.46 (C-5), 115.89 (C-α), 111.64 (C-2), 146.95 (C-β), 56.45 (OCH3); MS m/z (% relative intensity): 194 , 179 (16), 161 (5), 148 (6), 133 (17), 105 (5), 77 (6).8-169 °C ); UVλmax8 (3.86) ; FTIRυma65, 1517 ; 1H-NMR trans-Isoferulic acid (6): m.p. 230-232 °C (lit m.p. 230 °C [λmax (MeOH) nm (log ε): 289 (3.93), 313 (3.97) [υmax (KBr) cm–1: 3437, 2968, 1692, 1665, 1620, 1600, 1517, 1277, 1206, 1178 [1H-NMR (CD3OD+CDCl3) δ 6.26 , 7.61 , 7.07 , 7.05 , 6.87 , 3.91 ;13C-NMR (CD3OD+CDCl3) δ 110.50 (CH-2), 115.28 (CH-α), 115.57 (CH-5), 123.28 (CH-6), 126.83 (C-1), 146.11 (CH-β), 170.25 (CO), 148.98 (C-3), 147.97 (C-4), 56.04 (OCH3); MS m/z (% relative intensity): 194 , 193 (28), 179 (23), 177 (12), 148 (6), 133 (28), 105 (14), 77 (12).. 230 °C ); UVλmax3 (3.97) ; FTIRυma06, 1178 ; 1H-NMR Mixture of stigmasteryl-3-O-β-D-glucopyranoside andβ-sitosteryl-3-O-β-D-glucopyranoside (MBSG): m.p. 261-262 °C (lit m.p. 264-266 °C [υmax (KBr) cm–1 : 3406, 2935, 1654, 1459, 1368, 1024; 1H-NMR (CDCl3+CD3OD) δ 0.64-2.50  3.20- 3.40, 3.72-3.88 , 3.56-3.64 , 4.42 , 5.03, 5.17 , 5.38 ; 13C-NMR (CDCl3+CD3OD) δ 69.52 (CH-3), 121.93 (CH-6), 49.94 (CH-9), 31.76 (CH-8), 56.61 (CH-14), 55.81 (CH-17), 40.28 (CH-20), 138.11 (CH-22+), 129.05 (CH-23+), 51.02 (CH-24), 33.69 (CH-25), 11.76 (CH3-18), 19.51(CH3-19), 20.93 (CH3-21), 19.02 (CH3-26), 20.80 (CH3-27), 11.95 (CH3-29), 36.99 (CH2-1), 29.30 (CH2-2), 41.97 (CH2-4), 31.63 (CH2-7), 22.81 (CH2-11), 39.51 (CH2-12), 24.04 (CH2-15), 28.89 (CH2-16), 33.69 (CH2-22++), 27.99 (CH2-23++), 25.17 (CH2-28), 140.05 (C-5), 36.47 (C-10), 42.08 (C-13), 100.84 (C-1'), 73.26 (C-2'), 76.14 (C-3'), 69.52 (C-4'), 76.67 (C-5'), 61.22 (C-6'); MS m/z (% relative intensity): 414 (6), 412 (7), 393 (57), 394 (84), 381 (33), 300 (75), 287 (66), 255 (100), 227 (37), 213 (61), 147 (87), 145 (90), 131 (49), 105 (66), 91 (87), 79 (44); + is 1H-NMR and 13C-NMR of stigmasteryl-3-O-β-D-glucopyranoside, ++ is 13H-NMR of β-sitosteryl-3-O-β-D-glucopyranoside.4-266 °C , 278-2904-266 °C ); FTIRυmAntimicrobial assay: Antimicrobial activity of the plant extracts, fractions and isolates  was investigated using the agar dilution method [2Cl2 or MeOH were individually mixed with Müller Hinton (MH) broth to obtain a final volume of 2 mL. A two-fold dilution was prepared and the solution was then transferred to the MH agar solution to yield the final concentrations ranging from 4-256 μg/mL. Twenty seven strains of microorganisms  . The indCytotoxic assay: Cytotoxic activity of the plant extracts was determined by a slightly modified method described previously [5 cells/mL. Portions (100 μL) containing approximately 3×104 cells were distributed into 96-well flat-bottomed tissue culture plates and incubated overnight at 37°C in a humidified 5% CO2 incubator. Solutions (100 μL) containing different concentrations of tested extracts (0.001–10 μg/mL) or taxol (0.012–1.2 μg/mL) were added to each well and the plates were incubated as above for an additional 48 h. After the incubation, each well was washed (x 3) with phosphate-buffered saline (pH 7.2) and then stained with Crystal Violet. After the excess dye was removed, the stained cells were lysed with 100 mM HCl (100 μL) in absolute methanol and the optical density was determined by a microtitre plate reader  set to read at a wavelength of 540 nm. All tests were carried out in quadruplicate and the mean value was calculated. The activity was expressed as ED50 (the effective dose that inhibits 50% of cell growth).eviously . Briefly


This paper reports the isolation and full spectroscopic characterization of these new compounds by NMR, UV, IR and MS data.Two novel compounds, 1 Cyperus rotundus L. is a weed which is well distributed in the temperate tropical and subtropical regions of the World. The tuber of Cyperus rotundus is a kind of Traditional Chinese Medicine named “Xiangfuzi”, which is widely used in folk medicine as an antidiarrheal, antidepressant, anti-Candida, antipyretic, analgesic, anti-inflammatory, and anti-emetic remedy for dysentery and women’s diseases   (1 must include a six-membered ring (B ring). This was revealed by the HH correlations of the spin system H-1/H2-2/H-3/H-4 as well as the HMBC correlations from H-1 to C-2, C-3, C-8, C-9, and C-10, from H-4 to C-3, C-9, C-10, and C-2, from H-3 to C-2, C-4, C-10, and C-1, and from H-2 to C-1, C-3, C-9, and C-4 . Compound 4 (C-7)] . The det(A ring) . Taking  and C-4 . Thus, t1H and 13C-NMR spectrum of 1 showed the presence of an aromatic ABX system  . Moreover, the 3′,4′-dihydroxyphenyl group (ring C) was attached to the C-4 position of the ring B, which was supported by the HMBC correlations between H-3 (δ 3.79) and C-4 and C-1′, H-4 (δ 4.09) and C-1′, C-2′, and C-6′, H-2′ (δ 6.73) and C-1′ and C-4, and H-6′ (δ 6.68) and C-1′ and C-4  was consistent with the stereochemistry of H-3α and H-4β. The absence of a ROESY correlation between H-3 and H-4 also suggested a nearly antiperiplanar arrangement of these two protons. The ROESY correlation of H-1 to H-4 established the stereochemistry of H-1β and H-4β -1,2,3,4-tetrahydronaphthalin, and named methoxycyperotundol.The  (C-5′)] , suggest and C-4 . In the and H-4β . Therefo2 was determined to be C16H16O4 by the positive ion at m/z 295.0948 [M+Na]+ in the HRESIMS. Its IR spectrum displayed absorptions attributable to hydroxyl (3300-3500 cm−1) and phenyl groups . The 1H and 13C-NMR spectroscopic data of 2 were similar to those of 1, with the exception of a methoxyl group  at C-1 in 1, instead of a hydroxyl in 2 -1,2,3,4-tetrahydronaphthalin, and named cyperotundol.The molecular formula of compound xyl in 2 . The sug5.8 in 2 . This wa5.8 in 2 . Therefo3), caffeic acid (4), protocatechuic acid (5), p-coumaric acid (6), pongamone A (7) and biochanin A (8) were determined by comparison with the 1H- and 13C-NMR spectral data in the literature 25D: −89.4° . UV (MeOH) λmax: 279 nm. IR vmax (KBr): 3300–3500 cm−1, 1607 cm−1, and 1518 cm−1. HR-ESI-MS m/z 295.0948 . 1H-NMR and 13C-NMR (CD3OD) data see Obtained as colorless needles. [α]Cyperus rotundus, two novel constituents methoxycyperotundol (1) and cyperotundol (2) were obtained, along with six known components. Additionally, the known compounds, pongamone A (7) and biochanin A (8) in the rhizomes of Cyperus rotundus L are reported for the first time.During the phytochemical survey of the rhizomes of


Background: Hylomecon japonica, a plant of the Papaveraceae family which is well-known for the alkaloids they produce, is a perennial plant widely distributed in the northeast, central and east regions of China. Although a variety of chemical constituents, including alkaloids, flavonoids, and megastigmoids, have been isolated from H. japonica, the investigation of saponins in H. japonica has not been reported until now. Methods: Various separation techniques, including polyporous resin column chromatography, silica gel column chromatography and hemi-preparative HPLC were applied to the isolation of triterpenoid saponins, and chemical methods such as acid hydrolysis and spectroscopic methods including HRESIMS and NMR were applied to their structure elucidation, and the XTT reduction method was used to assay cytotoxicity. Results: Two new triterpenoid saponins, named hylomeconoside A (1) and B (2) which were identified as 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl-gypsogenin-28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-quinovopyranoside (1) and 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl-gypsogenin-28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (2), and two known triterpenoid saponins identified as dubioside C (3) and lucyoside P (4) on the basis of spectroscopic and chemical evidence, were isolated from H. japonica. Compound 1 exhibited moderate cytotoxicity on MGC-803 and HL-60 cells, with IC50 values of 43.8 and 32.4 μg·mL−1, respectively. Conclusions: Compounds 1 and 2 are new saponins, and 1 is considered to be one of the antitumor principles in this plant. This is the first time that triterpenoid saponins have been isolated from plants of the Papaveraceae family. Hylomecon japonica (Thunb.) Prantl et Kundig (Papaveraceae) is a perennial plant widely distributed in the northeast, central and east regions of China. Its roots have been used as a traditional Chinese medicine for the treatment of rheumatism and injuries + ion at m/z 1365.6318. Acid hydrolysis of 1 afforded an aglycone which was identified as gypsogenin on the basis of the 1H- and 13C-NMR data + ion at m/z 1351.6151. Acid hydrolysis of 2 afforded an aglycone which was identified as gypsogenin on the basis of the 1H- and 13C-NMR spectra +; calcd for C64H101O31, 1365.6327. 1H-NMR  and 13C-NMR : see Compound 2: White amorphous solid, m/z: 1351.6151 [M + H]+; calcd for C63H99O31, 1351.6165. 1H-NMR  δ: 0.67 , 0.80 , 0.87 , 1.12 , 1.29 , 1.61 , 3.13 , 3.34 -5α), 3.55 -5α), 4.71 , 5.01 -1), 5.08 , 5.10 -1), 5.29 , 5.66 , 6.34 , 9.82 . 13C-NMR : see Compound 3: White amorphous solid, m/z: 1367.6101 [M + H]+; calcd for C63H99O32, 1367.6114. 1H-NMR  δ: 0.70 , 0.90 , 1.02 , 1.28 , 1.57 , 1.65 , 3.44 , 3.31 -5α), 3.54 -5α), 4.68 , 4.98 -1), 5.05 , 5.10 -1), 5.47 , 5.62 , 6.35 , 9.76 . 13C-NMR : see Compound 4: White amorphous solid, m/z: 1381.6258 [M + H]+; calcd for C64H101O32, 1381.6270. 1H-NMR  δ: 0.90 , 0.98 , 1.03 , 1.04 , 1.26 , 1.45 , 1.67 , 3.17 , 3.57 -5α), 4.82 , 5.09 , 5.18 , 5.29 , 5.45 , 5.57 , 6.23 z, Ara-1), 9.78 . 13C-NMR : See Compound 1 and 2 (each 20 mg) were hydrolyzed with 2 M HCl (5 mL) at 90 °C for 4 h. The reaction mixture was extracted with EtOAc (3 × 5 mL), washed with H2O, and evaporated to dryness. The EtOAc extract was purified by silica gel column using a gradient of CHCl3-MeOH (1:0–95:5) to the aglycones of 1 and 2 which were determined to be gypsogenin by comparison of their spectral data with those of an authentic sample. Aglycone: White amorphous solid; 13C-NMR  δ: 207.7 (C-23), 180.0 (C-28), 144.8 (C-13), 122.2 (C-12), 71.6 (C-3), 56.2(C-4), 48.0 (C-5), 47.9 (C-9), 46.6 (C-17), 46.5 (C-19), 42.2 (C-14), 41.9 (C-18), 40.0 (C-8), 38.4 (C-1), 36.2 (C-10), 34.2 (C-21), 33.2 (C-29), 32.8 (C-7), 32.5 (C-22), 30.9 (C-20), 28.3 (C-15), 26.1 (C-27), 27.0 (C-2), 23.8 , 23.7 (C-11), 21.0 (C-6), 17.3 (C-26), 15.7 (C-25), 9.6 (C-24); 1H-NMR  δ: 0.94 , 0.97 , 1.12 , 1.23 , 1.41 , 3.02 , 3.90, 5.29 , 9.66 . The H2O layer was concentrated under reduced pressure to dryness, to give a residue of the sugar fraction. The residue was dissolved in pyridine (2 mL), l-cysteine methyl ester hydrochloride (1.5 mg) was added, and the mixture was heated at 60 °C for 1 h. Trimethylsilylimidazole (1.5 mL) was added, and the mixture was heated at 60 °C for another 0.5 h. An aliquot (4 μL) of the supernatant was subjected to GC analysis under the following conditions: column temp 180–280 °C at 3 deg/min, carrier gas N2 (1 mL/min), injector and detector temp 250 °C, split ratio 1:50. The configurations of monosugars for 1 and 2 were determined by comparison of the retentions times (tR) of the corresponding derivatives with standard l-arabinose (tR 10.748 min), d-xylose (tR 11.496 min), l-rhamnose (tR 12.162 min), d-quinovose (tR 13.648 min), d-glucuronic acid (tR 15.814 min) and d-galactose (tR 18.026 min). 1 yielded d-xylose, l-rhamnose, d-quinovose, d-glucuronic acid and d-galactose, and 2 gave l-arabinose, d-xylose, l-rhamnose, d-glucuronic acid and d-galactose.Compounds H-tetrazolium-5-carboxanilide) reduction method. In this study, MGC-803 (human gastric cancer), HL-60 (human promyelocytic leukemia), BEL-7402 (humanhepatoma carcinoma), MCF-7 (human breast cancer), and SPC-A1 (lung adenocarcinoma ) cell lines were used. Compounds 1 and 2 was dissolved at a concentration of 10 mg·mL−1 in phosphate-buffered saline (PBS) (as a stock solution), filtered through a filter 0.22 μm  and stored at 4 °C. The stock solution was diluted to 160, 80, 40, 20, 10 μg·mL−1 before the cytotoxic assay. The procedure for the cytotoxic assay was performed according to the XTT -24 cells·mL−1, seeded in in 96-well plates with 100 μL per well, and incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin at 37 °C in a humidified atmosphere with 5% CO2. Blank control wells were added 100 μL of cultrure medium, and maintained under the same conditions. After 24 h, 100 μL of the above culture medium containing different concentrations of 1 and 2 and normal culture medium (untreated control group) were added, and incubated for 72 h. For the analysis of cytotoxicity, 50 μL (1 mg·mL−1) XTT containing 0.15 mg·mL−1 phenazine methosulphate was added to each well, and cells were incubated for 3 h at 37 °C. The absorbances (A) of the produced formazan were measured by a DNM-9602 enzyme immunoassay spectrophotometer at 450 nm. Three replicate well were used for each analysis. The cell inhibitory rate (%) = (Asample − Ablank)/(Auntreated − Ablank) × 100. The concentration of compound producing 50% of cell inhibitory rate (IC50) was calculated by using SPSS version 20.0 .Logarithmic-phase growing cells were diluted into 5 × 101) and B (2), along with two known triterpenoid saponins identified as dubioside C (3), and lucyoside P (4), have been isolated from an ethanolic extract of Hylomecon japonica herbs. It is well known that alkaloids are main constituents of the plants of Papaveraceae family and until now besides alkaloids, flavonoids, megastigmane derivatives, volatile oil, furan derivatives, triterpenes and sterols have also reported, but saponins have not been reported [Papaveraceae family. Compound 1 with the IC50 values of 43.8 μg·mL−1 on MGC-803 and 32.4 μg·mL−1 on HL-60 is considered to be one of the antitumor principles in this plant.Two new triterpenoid saponins, named hylomeconoside A (reported ,16,17. T


Iodine and chlorine atoms in the C-5 side chain were introduced by reaction of 5-(2-hydroxyethyl)pyrimidine with hydroiodic acid and phosphoryl chloride, respectively. The structure of the intermediate α-(1-carbamyliminomethylene)-γ-butyrolactone was determined by X-ray crystal structure analysis. The molecule deviates very slightly from planarity. Three N−H···O hydrogen bonds link the molecules into one-dimensional chains of edge-fused rings.Three novel 5-pyrimidine derivatives were synthesized and characterized by  E)-5--2’-deoxyuridines are among the most potent and selective agents that undergo selective phosphorylation by the viral-encoded enzyme [e.g. 5-(2-fluoroethyl)-2’-deoxyuridine, labelled with radioisotopes have applications in positron emission tomography (PET) to monitor the successful transfection of gene products in vivo during cancer-prodrug therapy [A number of 5-substituted pyrimidine nucleoside analogues have been shown to have potent and selective cytotoxic activity in cells transfected with the HSV-1 TK gene ,3. The -γ-butyrolactone (2) [2 with sodium ethoxide afforded 5-(2-hydroxyethyl)uracil (3), which with 57% solution of hydroiodic acid gave 5-(2-iodoethyl)uracil (4). Chlorination of compound 3 with phosphoryl chloride afforded the chlorinated pyrimidine derivative 5, which was subsequently methoxylated giving 5-(2-chloroethyl)-2,4-dimethoxyuracil (6). The newly prepared compounds 4-6 were fully characterized by 1H- and 13C-NMR spectra, mass and IR spectra, as well as elemental analysis (see The sodium derivative tone (2) ,17. Reacysis see .2  chains that are also parallel to the b axis. The combination of these two motifs forms new ring of  type. These three N−H···O hydrogen bonds generate mutually parallel one-dimensional chains of edge-fused rings . The distance between the planes passing through the center of the hydrogen-bonded molecules is approximately 3.05 Å. Very similar one-dimensional hydrogen-bonded chains generate N−H···O hydrogen bonds in 1-carbamyliminomethylenetetrahydropyrrol-2-one [The crystal packing diagram along b axis  reveals ol-2-one .1H- and 13C-NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer, operating at 600 MHz (1H) and 150.92 MHz (13C), respectively. The samples were dissolved in DMSO-d6 and measured in 5 mm NMR tubes. The 1H- and 13C-NMR chemical shift values (δ) are expressed in ppm referred to tetramethylsilane (TMS) and coupling constants (J) in Hz. The infrared spectra were recorded by a Fourier transform infrared (FTIR) spectrometer Bruker Vertex 70 equipped with an attenuated total reflection (ATR) accessory with a diamond crystal. Sixteen scans were collected for each measurement over the spectral range of 400-4000 cm-1 with resolution of 4 cm-1. Precoated Merck Silica Gel 60F-254 plates were used for thin layer chromatography (TLC) and the spots were detected under UV light (254 nm). Column chromatography (CLC) was performed using silica gel (0.063-0.2 mm) Fluka; glass column was slurry-packed under gravity. Compound purity was analyzed by HPLC with DAD detector. Melting points were determined on a Kofler micro hot-stage apparatus  and are uncorrected. The electron impact mass spectra were recorded with an EXTREL FT MS 2002 instrument with ionizing energy of 70 eV. Sodium α-hydroxymethylene-γ-butyrolactone (1) [1 was filtered off and washed with dry ether ; MS m/z (EI) for C5H5NaO3 (M+•) calc. 136.0812; found 136.1; 1H-NMR δ 8.46 , 4.24 , 2.44 ; 13C-NMR δ 176.96 (C-2), 166.54 (C-1'), 132.60 (C-3), 68.37 (C-5), 27.50 (C-4).tone (1) . To a coα-(1-Carbamyliminomethylene)-γ-butyrolactone (2) [1, 34 g, 0.25 mol). After stirring at 4 °C overnight the precipitated product was filtered off and washed with cold water. Recrystallization from water – ethanol gave white crystals of 2 ; MS m/z (EI) for C6H8N2O3 (M+•) calc. 156.1394; found 156.1; 1H NMR δ 8.96 , 7.60 , 6.38 , 4.28 , 2.75 ; 13C-NMR δ 172.69 (C-3'), 154.35 (C-2), 132.60 (C-1'), 99.30 (C-3), 65.07 (C-5), 24.19 (C-4).tone (2) . To a so5-(2-Hydroxyethyl)uracil (3). To a solution of NaOEt  in ethanol (20 mL) was added α-(1-carbamyliminomethylene)-γ-butyrolactone . Reaction mixture was refluxed for 6 h, during which time product precipitated. The solvent was removed under reduced pressure and the residue was dissolved in the water and acidified to pH = 3. The precipitated product 3 was filtered off, washed with ethanol and dried in vaccuo ; MS m/z (EI) for C6H8N2O3 (M+•) calc. 156.1394; found 156.1; 1H-NMR δ 11.41 , 11.13 , 7.21 , 3.63 , 3.41 , 2.29 ; 13C-NMR δ 164.32 (C-4), 151.50 (C-2), 135.19 (C-6), 110.38 (C-5), 60.21 (C-2'), 29.06 (C-1').5-(2-Iodoethyl)uracil (4). To a cooled solution of 3  in H2O (10 mL), a solution of HI  was added. The reaction mixture was stirred at -5 °C for 90 min, then solvent was evaporated and residue was purified by column chromatography (CH2Cl2:CH3OH = 60:1) to give oily product 4 ; MS m/z (EI) for C6H7IN2O2 (M+•) calc. 266.0365; found 266; Anal. for C6H7IN2O2 calc. C 27.09 %, H 2.65 %, N 10.53 %; found C 27.13 %, H 2.64 %, N 10.55 %; IR: 3110–2992 , 1720 (ν C=O), 1650–1420 (ν C=C and C–H ring), 1480 (δ CH2), 1022–770 , 940 (δ CH2–I), 520–480 (ν C–I) cm-1; 1H-NMR δ 10.97 , 10.62 , 7.20 , 4.52 , 2.28 ; 13C-NMR δ 165.14 (C-4), 151.82 (C-2), 139.27 (C-6), 109.61 (C-5), 59.86 (C-2'), 20.03 (C-1').5-(2-Chloroethyl)-2,4-dichloropyrimidine (5). The reaction mixture of 3  and POCl3  was refluxed for 1 h. Solvent was evaporated and ice was added in the residue which was then washed with ether and dried over Na2SO4. After drying the solvent was removed in vacuo and residue was purified by column chromatography (CH2Cl2:CH3OH = 30:1) which gave colourless oily product 5 ; MS m/z (EI) for C6H5Cl3N2 (M+•) calc. 211.4575; found 211.5; Anal. for C6H5Cl3N2 calc. C 34.08 %, H 2.38 %, N 13.25 %; found C 34.01 %, H 2.39 %, N 13.22 %; IR: 1633–1320 (ν C=C and C–H ring), 1430 (δs CH2), 1090 (δ CH2–Cl), 980-760 , 580–510 (ν C–Cl) cm-1; 1H-NMR δ 8.82 , 3.93 , 3.19 ; 13C-NMR δ 172.46 (C-4), 162.01 (C-2), 156.87 (C-6), 112.70 (C-5), 43.15 (C-2'), 31.51 (C-1').5-(2-Chloroethyl)-2,4-dimethoxypyrimidine (6). To a solution of NaOMe  in MeOH (15 mL) was added 5 , and the mixture was heated at reflux for 6 h. The solvent was evaporated, and H2O was added to dissolve NaCl. The oily layer was extracted with CH2Cl2, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by column chromatography (CH2Cl2:CH3OH = 30:1) which gave colourless oily product 6 ; MS m/z (EI) for C8H11ClN2O2 (M+•) calc. 202.6379; found 202.6; Anal. C 7.42 %, H 5.47 %, N 13.82 %; found C 47.31 %, H 5.47 %, N 13.84 %; IR: 2960 (ν CH3), 1640–1330 (ν C=C and C–H ring), 1420 (δ CH2), 1210 (νas C–O–C), 1100 (δ CH2–Cl), 1050 (νs C–O–C), 905–760 , 630–520 (ν C–Cl) cm-1; 1H-NMR δ 8.19 , 3.92 , 3.87 , 3.77 , 2.89 ; 13C-NMR δ 169.30 (C-4), 164.56 (C-2), 158.87 (C-6), 111.44 (C-5), 54.85 (OCH3), 54.33 (OCH3), 43.75 (C-2'), 29.76 (C-1').2 suitable for X-ray structure analysis was prepared by growth under slow evaporation at room temperature of a very dilute ethanol solution (v/v 96 %). The intensities were collected at 295 K on a Philips PW1100 diffractometer updated by Stoe and Cie [α radiation (0.71073 Å). Details of crystal data, data collection and refinement parameters are given in A single crystal of  and Cie ,22 using2 [www.ccdc.cam.ac.uk/data_request/cif, by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.The structure was solved by direct methods . All non2 . The hyd2  defaults2  program


Their structures were elucidated by spectroscopic analysis, X-ray diffraction analysis, analysis of the ECD data according to the Klyne's lactone sector rule, and quantum ECD calculation. Compounds 1 and 2 are two new picrotoxane-type sesquiterpenes with a new carbon skeleton containing a formamide group, which may be derived from the previously reported dendrobiumane B skeleton by the C(9)-C(11) carbon bond cleavage. Compounds 3, 5, 6, and 8 exhibited inhibitory activity against α-glycosidase. Compounds 5 and 6 were cytotoxic against SGC-7901, K562, A549, BEL-7402, and Hela cell lines.Four picrotoxane-type sesquiterpenes, dendroterpene A–D ( Dendrobium nobile Lindl, the plant belongs Orchidaceae family, is one of three major plant sources of the traditional Chinese medicine “Shi Hu” in Chinese (edition 2005), which are used as a tonic to nourish the stomach and promote the production of body fluid recorded in the Chinese Pharmacopeia , along with four known compounds, nobilin E (5)  -dehydrovomifoliol (7)  -C(11) carbon bond cleavage. In this report, the isolation, structure elucidation and bioactivities of these compounds are described.Picrotoxane-type sesquiterpenes were one of main constituents of 18 column . C18 gel , Silica gel , Sephadex LH-20  were used for column chromatograghy. α-glycosidase  used for the bioactivity was derived from yeast and its EINECS (EC) unmber is 3.2.1.20.Optical rotations were measured on a MCP 5100 modular compact polarimeter . IR spectra were taken on a Nicolet 380 FT-IR instrument  as KBr discs. NMR spectra were recorded on a Bruker Avance III 500 MHz NMR spectrometer . ESIMS and HRESIMS were recorded with amaZon SL (Bruker) or Compact QqTOF (Bruker). ECD spectra were measured by APL Chiascan . Semi-preparative HPLC was carried out using a CD. nobile were collected from Shishan town in May 2018, Hainan, China. After drying, the dried stems (13.0 kg) were shattered. Then, the samples were extracted by 95% ethyl alcohol three times and for 5 days each time. The EtOH extract (716.2 g) was dissolved in H2O and extracted three times by petroleum ether (8.0 L), EtOAc (8.0 L), and n-butyl alcohol (8.0 L) successively.The fresh stems of 2O) to yield 8 . Fr.9-6 (112.0 mg) was purified by semipreparative TLC eluting with petroleum ether-acetone (v/v 3:1) to obtain compound 7 (2.7 mg). Fr.9-7 (372.0 mg) was purified by a silica gel column eluting with petroleum ether-CHCl3-MeOH (v/v/v 200:50:1) to yield compound 3 (4.2 mg). Compound 4 (2.0 mg) was obtained by recrystallization from Fr.9-8 (580.3 mg). Fr.9-10 (103.0 mg) was further purified by a silica gel column eluting with petroleum ether-acetone (v/v 8:1) to give 1 (13.0 mg). Fr.10 (9.0 g) was separated by ODS column to get 50 fractions (Fr.10-1–Fr.10-50). Compound 2 (100.2 mg) was obtained by recrystallization from Fr.10-14 (326.0 mg). Fr.10-38 (184.0 mg) was purified by Sephadex LH-20 eluting with MeOH to give 5 (15.0 mg). Fr.11 (9.0 g) was separated into three fractions (Fr.11-1–Fr.11-3) by Sephadex LH-20 eluting with MeOH. Fr.11-3 (80.1 mg) was purified by ODS column eluting with 65% MeOH/H2O to yield compound 6 (20.9 mg).The EtOAc extract (87.9 g) was separated into 16 fractions (Fr.1–Fr.16) by a silica gel column (200–300 mesh) using a gradient of petroleum ether-EtOAc  and then acetone. Fr.9 (10.0 g) was further separated into 20 fractions (Fr.9-1–Fr.9-20) by ODS column. Fr.9-1 (30.0 mg) was purified by semipreparative HPLC  as carried out in the Gaussian 03 : Colorless crystals; +  in the HRESIMS spectrum. Analysis of its 1D and HSQC NMR spectra (see C/H 136.6/5.63 and δC/H 130.3/5.51), three methyls , one methylene , six sp3 methines  with one oxygenated, one quaternary carbon (δC 52.5) and two ester or amide carbonyl groups (δC 180.3 and δC 163.7). The above data and sequential COSY correlations  and the oxymethine group CH-2 (δC/H 73.1/3.67) in dendrobiumane C were replaced by a hydrogen atom H-9 (δH 5.51) and a methine CH-2 (δC/H 51.2/4.24) linked with a formamide group in 1, respectively, as evidence by COSY correlation from H-9 to H-8, along with the HMBC correlations from H-2 to C-9 and C-11, from H-9 to C-6 and C-10, as well as from H3-10 to C-2 and C-6. The ROESY correlations  indicated a molecular formula of C15H21NO4. The 1D and 2D NMR data of 2  in 2 replaced signals corresponding to CH-6 in 1 (δC/H 42.5/2.33). In the HMBC spectrum , suggesting six degrees of unsaturation. 1D NMR and HSQC data displayed three methyls , two methylenes, seven methines including two oxygenated ones (δC/H 81.5/4.28 and 77.9/4.75), three quaternary carbons including two ester carbonyl carbons (δC 177.5 and 177.1). The above data of 3 were very similar to those of the previously reported dedrobiumane E  and C-9 (δC 83.8) and two oxygenated methine groups CH-7 (δC/H 65.2/4.05) and CH-8 (δC/H 60.1/3.86) in dedrobiumane E were replaced by two sp3 methines (δC/H 43.6/2.20 and δC/H 55.2/2.77) and two sp3 methylenes  in 3, as proved by the sequential COSY correlations of H-5/H-6/H-7/H-8/H-9, along with the detail interpretation of the HMBC correlations from H-2 to C-6, C-9, and C-11, from H3-10 to C-2, C-6, and C-9, from H-3 and H-6 to C-16, from H-5 to C-1, as well as from H-8 to C-11  in the HRESIMS spectrum. The similarity of NMR data  in 3 was oxygenated to a hydroxylated quaternary carbon (δC 85.1) in 4, as supported by sequential COSY correlations of H-2/H-3/H-4/H-5/H-6/H-7/H-8, together with the HMBC correlations from H-2, H-6, H-7, and H3-10 to C-9. The stereochemical structure of 4 , was determined by a single-crystal X-ray diffraction pattern obtained using the anomalous scattering of Cu Kα radiation . A underwent esterification and the C-11-O-C-2 ring closure produced compound 3, which underwent oxidation to afford compound 4. Dendrobiumane B underwent transamination to give the intermediate (B), which underwent oxidation to form intermediate (C). C underwent cyclization to produce intermediate (D), and D underwent dehydration to yield intermediate (E). E underwent oxidation to give intermediate (F), which underwent reduction and dehydration to produce compound 1. Then, 1 underwent oxidation to get 2.The main skeletal changes are that one is the C-11-O-C-2 ring or the C-11-N-C-2 ring closure exemplified by dendrobiumane E and the other is the C-11-O-C-2 ring or the C-11-N-C-2 ring opening illustrated by dedrobiumane B. In our present study, the discovery of pathways . Dendrob1–8 were tested for inhibitory activity against α-glycosidase using the PNPG method and for cytotoxic effects on SGC-7901, K562, A549, BEL-7402, and Hela cell lines using the MTT method . Compound 5 displayed weak cytotoxic effects against SGC-7901, K562, A549, BEL-7402, and Hela cell lines with the IC50 values of 17.30, 10.39, 29.03, 20.13, and 22.19 μM, respectively , in addition, compound 6 showed cytotoxic effects against K562 with the IC50 value of 28.23 μM.Compounds 1–4), together with four known compounds (5–8), were isolated from the stems of D. nobile. Compounds 1 and 2 are two new picrotoxane-type sesquiterpenes with a new carbon skeleton containing a formamide group, which may be derived from the previously reported dendrobiumane B skeleton by the C(9)-C(11) carbon bond cleavage. In the previously report, picrotoxane sesquiterpenes exhibited the angiogenesis effect against sunitinibinduced damage  against α-glycosidase, indicating that the hydroxyl at C-9 may reduce the activity. In addition, the known bibenzyl derivative nobilin E (5) exhibited inhibitory effects on NO production in activated murine macrophage-like cell line RAW 264.7 in the previously report  have not been reported. In this report, compounds 5 and 6 both exhibited inhibitory activity against α-glycosidase with IC50 values of 0.03 and 0.68 mM, respectively. In addition, Compound 5 displayed weak cytotoxic effects against SGC-7901, K562, A549, BEL-7402 and Hela cell lines with the IC50 values of 17.30, 10.39, 29.03, 20.13, and 22.19 μM, respectively. Compound 6 showed cytotoxic effect against K562 with the IC50 value of 28.23 μM.In conclusion, four new picrotoxane-type sesquiterpenes (All datasets generated for this study are included in the article/D. nobile. PW wrote the paper. All authors have approved the final version of the manuscript.PW, XC, HW, CC, JY, and XX performed the experiments. PW and XC identified the structures. GZ performed ECD calculation. PW, HD, and WM conceived and designed the experiments. SH collected the fresh stems of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dipteryx alata (Leguminosae-Papilionoideae) dissolved in a methanol-water (80:20) mixture through a liquid-liquid partition procedure has been investigated against the neuromuscular blockade of the venom of the snake Bothrops jararacussu. The active CH2Cl2 sub-extract has been extensively analyzed for its chemical constituents, resulting in the isolation of four lupane-type triterpenoids: lupeol (1), lupenone (2), 28-hydroxylup-20(29)-en-3-one (3), betulin (4), nine isoflavonoids: 8-O-methylretusin (5), 7-hydroxy-5,6,4’-trimethoxyisoflavone (6), afrormosin (8), 7-hydroxy-8,3’,4’-trimethoxyisoflavone (9), 7,3’-dihydroxy-8,4’-dimethoxyisoflavone (10), odoratin (11), 7,8,3’-trihydroxy-4’-methoxyisoflavone (13), 7,8,3’-trihydroxy-6,4’-dimethoxyisoflavone (15), dipteryxin (17), one chalcone: isoliquiritigenin (7), one aurone: sulfuretin (14) and three phenolic compounds: vanillic acid (12), vanillin (16), and protocatechuic acid (18). Their chemical structures were elucidated on the basis of spectroscopic analysis, including HRMS, 1D- and 2D-NMR techniques.The effect of four sub-extracts prepared from the lyophilized hydroalcoholic bark of  Dipteryx alata Vogel, belongs to the Leguminosae-Papilionoideae family, and is a native species of the Brazilian Savanna, found principally in Minas Gerais, Goiás, Federal District and Mato Grosso. This species is known popularly as “baru” in Minas Gerais, “barujo” and “combaru” in Mato Grosso, and “cumaru” in other states. The plant has great economic potential because of its multiple uses. The fruit, rich in proteins, fibres and unsaturated fatty acids, is used as human and animal food, and also in cosmetic formulations + by HR-ESI-MS consistent with the molecular formula C18H16O6Na. The 1H-NMR spectrum , 151.7 (C-5) and 138.2 (C-6) respectively, indicating the location of the O-methyl groups. The 13C-NMR  characteristic for the A-ring in 7,8-dioxygenated isoflavones, and signals for three protons at δ 6.93, 6.96 and 7.03 of the disubstituted B-ring. Compound MR of 13  showed a13C-NMR signals , was confirmed by biological assays.The protective activity of the four sub-extracts  of the EtOH/HThe phrenic nerve-diaphragm muscle was obtained from mice previously anesthetized with halothane and sacrificed by exsanguination. The diaphragm was removed and mounted as described by Bülbring . PND prein vitro neutralization assays after mixing 50 μg/mL of each extract  with Bjssu venom (40 µg/mL) prior to test. Note that hexane (Hex) did not protect (5.2% ± 3.7) against irreversible neuromuscular blockade induced by crude venom (5% ± 5.6). A fair protection was seen with the ethyl acetate sub-extract (80% ± 6), whereas a practically total protection was seen with dichloromethane (98% ± 8) and methanol (95% ± 4.7) sub-extracts (* p < 0.05 compared to venom). As it can be seen, the Dcm and Met extracts showed no significant difference in relation with Tyrode control.The D. alata against Bothrops jararacussu venom, since preliminary data obtained from thin layer chromatography comparison pointed out to phenolic compounds present in the D. alata hydroalcoholic extract + .7-Hydroxy-5,6,4’-trimethoxyisoflavone (6): Yellow amorphous solid. IR (film) cm−1: 3,394, 1,609, 1,511, 1,464, 1,379. 1H-NMR data see 13C-NMR data see m/z: 351.0847 [M + Na]+ .Afrormosin (8): White amorphous solid. 1H-NMR data see 13C-NMR data see 7-Hydroxy-8,3’,4’-trimethoxyisoflavone (9): Colourless amorphous solid. IR (KBr) cm−1: 3,400, 1,598, 1,517, 1,448. 1H-NMR data see 13C-NMR data see 7,3’-Dihydroxy-8,4’-dimethoxyisoflavone (10): Yellow amorphous solid. IR (KBr) cm−1: 3,429, 1,624, 1,590, 1,513, 1,256. 1H-NMR data see 13C-NMR data see m/z: 337.0681 [M + Na]+ .Odoratin (11): Colourless amorphous solid. IR (film) cm−1: 3,409, 1,617, 1,598, 1,513, 1,328. 1H-NMR data see 13C-NMR data see m/z: 337.0678 [M + Na]+ .7,8,3’-Trihydroxy-4’-methoxyisoflavone (13): Yellow amorphous solid. IR (KBr) cm−1: 3,429, 1,619, 1,604, 1,290, 1,124. 1H-NMR data see 13C-NMR data see 7,8,3’-Triacetoxy-4’-methoxyisoflavone (13a): The compound 13a was obtained from compound 13 (19 mg) after acetylation with Ac2O/Py, as brown amorphous solid (15 mg). 1H-NMR data see 7,8,3’-Trihydroxy-6,4’-dimethoxyisoflavone (15): Yellow amorphous solid. IR (KBr) cm−1: 3,410, 1,604, 1,513, 1,475, 1,130. 1H-NMR data see 13C-NMR data see m/z: 337.0617 [M + Na]+ .Dipteryxin (17): Yellow amorphous solid. IR (KBr) cm−1: 3,420, 1,621, 1,513, 1,465, 1,380. 1H-NMR data see 13C-NMR data see m/z: 337.0677 [M + Na]+ .D. alata have been examined for their protective activity against the neuromuscular blockade caused by Bothrops jararacussu venom. The active CH2Cl2 extract has been extensively analyzed for its chemical constituents, resulting in the isolation of four lupane-type triterpenoids, nine isoflavonoids, one chalcone, one aurone and three phenolic compounds. On the basis of the present study, further pharmacological evaluation of each isolated compound will be need to ascertain which of them are responsible for the activity, as well as for establishing the mechanism of action.In summary, four sub-extracts from