Aliivibrio fischeri, Raphidocelis subcapitata and Daphnia magna), to test their acute and chronic toxicity. The results reported that TRA may be classified as a harmful compound to some aquatic organisms whereas its chlorinated product mixture showed no effects on any of the organisms tested. All data suggest however that TRA chlorination treatment produces a variety of DBPs which can be more harmful than TRA and a risk for the aquatic environment and human health.In recent years, many studies have highlighted the consistent finding of tramadol (TRA) in the effluents from wastewater treatment plants (WTPs) and also in some rivers and lakes in both Europe and North America, suggesting that TRA is removed by no more than 36% by specific disinfection treatments. The extensive use of this drug has led to environmental pollution of both water and soil, up to its detection in growing plants. In order to expand the knowledge about TRA toxicity as well as the nature of its disinfection by-products (DBPs), a simulation of the waste treatment chlorination step has been reported herein. In particular, we found seven new by-products, that together with TRA, have been assayed on different living organisms ( According to information available to the International Narcotics Control Board (INCB), the loose legislation in China, India and Sri Lanka allows mass production, not always legal, of tramadol (TRA). Generally, the drug arrives by ship to the Mediterranean where Italy and Greece are important points of disembarkation and distribution, but above all the main flow is towards Libya. The traffic then continues to Egypt and ends up in the main Middle Eastern countries, where in many countries it is not considered illegal. The powerful opioid, which in Europe can only be sold with a non-repeatable medical prescription, is called the “drug of the fighter”, costs a few euros per tablet and militias take it above all to endure the pain of wounds. It is estimated that world traffic drives a turnover of 1.39 billion dollars a year. The total amount of TRA used worldwide in the period from 1990 to 2009 was calculated to be 11,758 million of defined daily doses  ,5.Sarcocephalus latifolia + in the EI mass spectrum and fourteen carbon signals in the 13C-NMR spectrum. In the 1H NMR the four aromatic ring protons seen at δ 6.81, 7.00, 7.05 and 7.29 correlated in the HSQC spectrum to the carbons at δ 111.97, 116.68, 110.88 and 129.26, respectively. The 13C-NMR spectrum, besides the signals of the protonated carbons, showed two quaternary carbons signals at δ 159.52 and 148.48 that HMBC experiments allowed to assign the first one to C-3′ and the latter to C-1′. The DEPT spectrum showed the presence of two methines at δ 2.15 and 4.45, identified as H-2 and H-7, respectively, whose protons were correlated with each other in the COSY spectrum and both correlated to C-1 carbon in the HMBC spectrum, at δ 77.07. In the last spectrum the methine H-2 was correlated to the aromatic carbon C-1′, as well as to the C-3, C-6 and C-7 carbons, at δ 26.23, 40.38 and 67.85 respectively, while the methine H-7 was correlated to C-2 and C-3, at δ 32.70 and 26.23, respectively.BP3 had a pseudo-molecular ion at m/z 234.35 [M]+ in the EI mass spectrum and fourteen carbon signals in the 13C-NMR spectrum, which correspond to the molecular formula C14H18O3. The 1H- NMR indicated the absence of the three carbons bounded to the nitrogen and the presence of two singlet signals at δ 9.61 e 6.65. In the HSQC spectrum these last two signals were correlated to the carbons at δ 192.90 e 150.02, respectively. In the HMBC spectrum the proton a δ 9.61 was correlated to the signals at δ 150.02, 143.50 e 21.44, identified as C-2, C-3 and C-4 carbons, respectively, while the proton a δ 6.65 was correlated to the carbons C-3 and C-4 and to the signals at δ 192.90 and 77.10, identified as carbons C-7 and C-1, respectively.BP4 had a molecular ion at m/z 261.34 [M]+ in the EI mass spectrum and sixteen carbon signals in the 13C-NMR spectrum, which correspond to the molecular formula C16H23NO2. The 1H-NMR indicated the absence of one of the two methyls bound to the nitrogen and the presence of a signal at δ 3.62 and 4.20, correlated in a HSQC spectrum at the carbon δ 82.40 (C-10). The DEPT spectrum confirmed the methylene nature of this signal and the HMBC spectrum indicated the correlation of the protons with the signals at δ 77.10, 56.12 and 40.36, identified as the C-1, C-7 and C-9 carbons, respectively, and the correlation of the corresponding carbon with the H-9 methyl protons at δ 2.01 and with the H-7 protons at δ 2.42 and 2.54.BP5 had a molecular ion at m/z 342.79 [M]+ and 344.80 [M + 2]+ in the EI mass spectrum and seventeen carbon signals in the 13C-NMR spectrum, which correspond to the molecular formula C17H23ClO5. The 1H-NMR showed the presence of four protons of aromatic ring and a methoxyl groups. The 13C-NMR spectrum showed the presence of the signals of the aromatic ring and of its methoxyl group as well as other ten signals, of which two carbonyls at δ 199.69 and 208.13. The remaining height signals in a DEPT experiment were identified as one methyl, five methylenes, one methine and one quaternary carbon, whose corresponding protons were identified by an HSQC spectrum. The carbon at δ 199.69, identified as C-1, was correlated in an HMBC spectrum to the two aromatic protons H-2′ and H-6′, at δ 7.51 and 7.56, respectively, and to the signals at δ 3.04 and 1.83, identified as protons H-2 and H-3, respectively, that were coupled together in a COSY spectrum. The COSY spectrum also showed the correlation between the signals at δ 1.80 and 2.35, of which the first one was identified as relative to the protons H-4, correlated in the same COSY spectrum to the protons H-3, and the second one, identified as relative to protons H-5. The protons H-5 were correlated in a HMBC experiment to signals at δ 96.27 and 74.07, identified as carbons C-6 and C-7, respectively. In the same HMBC spectrum the quaternary carbon at δ 208.13 was correlated with the signal of the methyl protons at δ 2.28, with those of the methylene at δ 2.95 and 3.18 and with the methine at δ 4.39, identified as protons H-10, H-8 and H-7, respectively.BP6 had a molecular ion at m/z 277.38 [M]+ in the EI mass spectrum and sixteen carbon signals in the 13C-NMR spectrum, which could correspond to the molecular formula C16H23NO3. The 1H- NMR indicated the presence of a signal of an aldehyde function at δ 7.79 and the absence of one of the two methyls bound to the nitrogen. The HSQC spectrum showed the correlation of the aforementioned proton at low fields with carbon at δ 163.13 and the HMBC spectra showed its correlations with the signals at δ 50.68 and 29.76, identified as the carbons C-7 and C-9, respectively, confirming that the isolated product was the oxidized TRA to one of the two methyls.BP7 had a molecular ion at m/z 276.31 [M]+ in the EI mass spectrum, beyond that 246.30 and 219.29 due to the loss of NO and NO and HCN; moreover the 13C-NMR spectrum showed fifteen carbon signals, which could correspond to the molecular formula C15H20N2O3. The 1H-NMR indicated the absence of the two methyls bound to the nitrogen, confirmed the presence of the signals of aromatic ring and of methoxyl group and the presence of two pairs of signals at δ 2.66 and 2.70 and at δ 2.73 and 2.75, correlated in an HSQC spectrum to the signals at 67.40 and 54.00, respectively. In correspondence of these last two signals the DEPT spectrum confirmed the presence of two methylenes, as well as two methines at δ 44.53 and 25.81, bound in the HSQC spectra to the protons at δ 2.15 and 1.95, respectively. The methylene protons at δ 2.66/2.70 were correlated in an HMBC spectrum with the signals a δ 25.81, 44.53 and 26.11, identified as the carbons C-3, C-2 and C-4, while the methylene protons at δ 2.73/2.75 were correlated with the carbons C-2 and C-3 and with the signal a δ 77.50, identified as the carbon C-1. A possible mechanism for obtaining the products BP1–BP7 is shown in 50) values up to 100 mg/L. Bergheim et al. -1-(3-methoxyphenyl)cyclohexyl hypochlorite (BP1). White powder. 1H-NMR : δ 1.35 , 1.55 , 1.65 , 1.75 , 1.80 , 1.84 , 2.06 , 2.08 , 2.13 , 2.15 , 2.40 , 3.85 , 6.77 , 7.05 , 7.16 , 7.24 . 13C-NMR : δ 22.24 (C-5), 26.82 (C-3), 27.92 (C-4), 44.73 (C-2), 41.31 (C-6), 55.00 (OCH3), 61.55 (C-7), 77.00 (C-1), 47.72 , 111.25 (C-4′), 110.98 (C-2′), 117.36 (C-6′), 128.94 (C-5′), 152.00 (C-1′), 159.98 (C-3′). ESI-MS (positive ions): m/z calculated for C16H24ClNO2m/z 297.82 [M]+ and 299.82 [M + 2]+; found 297.81 [M]+ and 299.80 [M + 2]+, 262.37 [M − Cl]+, 246.37 [M − OCl]+.2-[Amino(hydroxyl)methyl]-1-(3-methoxyphenyl)cyclohexanol (BP2). White powder. 1H-NMR : δ 1.18 , 1.26 , 1.43 , 1.54 , 1.65 , 1.75 , 1.85 , 2.01 , 2.15 , 3.83 , 4.45 , 6.81 , 7.00 , 7.05 , 7.29 . 13C-NMR : δ 20.91 (C-4), 26.23 (C-3), 26.95 (C-5), 32.70 (C-2), 40.38 (C-6), 55.21 (OCH3), 67.85 (C-7), 77.07 (C-1), 110.88 (C-2′), 111.97 (C-4′), 116.68 (C-6′), 129.26 (C-5′), 148.48 (C-1′), 159.52 (C-3′). ESI-MS (positive ions): m/z calculated for C14H21NO3 251.32 [M]+; found 251.30 [M]+.2-Hydroxy-2-(3-methoxyphenyl)cyclohexanecarbaldehyde (BP3). White powder. 1H-NMR : δ 1.63 , 1.72 , 2.03 , 2.31 , 3.85 , 6.65 , 6.86 , 6.98 , 7.02 , 7.31 , 9.61 . 13C-NMR : δ 18.40 (C-5), 21.44 (C-4), 39.28 (C-6), 55.32 (OCH3), 77.10 (C-1), 111.34 (C-2′), 112.74 (C-4′), 117.61 (C-6′), 129.58 (C-5′), 143.50 (C-3), 150.71 (C-1′), 150.02 (C-2), 159.95 (C-3′), 192.90 (C-7). ESI-MS (positive ions): m/z calculated for C14H18O3 234.29 [M]+; found 234.35 [M]+, 205.37 [M − CHO]+.-8a-(3-Methoxyphenyl)-3-methyloctahydro-2H-benzo[e]oxazine (BP4). White powder. 1H-NMR : δ 1.30 , 1.50 , 1.65 , 1.70 , 1.80 , 1.90 , 1.94 , 2.01 , 2.31 , 2.38 , 2.42 , 2.54 , 3.62 , 3.81 , 4.20 , 6.78 , 6.94 , 6.96 , 7.27 . 13C-NMR : δ 22.07 (C-5), 25.54 (C-3), 27.59 (C-4), 35.13 (C-2), 40.36 (C-9), 42.70 (C-6), 55.20 (OCH3), 56.12 (C-7), 77.10 (C-1), 82.40 (C-10), 111.66 (C-4′), 112.18 (C-2′), 118.25 (C-6′), 129.54 (C-5′), 147.30 (C-1′), 160.00 (C-3′). ESI-MS (positive ions): m/z calculated for C16H23NO2 261.36 [M]+; found 261.34 [M]+.6-Chloro-6,7-dihydroxy-1-(3-methoxyphenyl)decane-1,9-dione (BP5). White powder. 1H-NMR : δ 1.80 , 1.83 , 2.28 , 2.35 , 2.95 , 3.04 , 3.18 , 4.39 , 3.88 , 7.13 , 7.40 , 7.51 , 7.56 . 13C-NMR : δ 23.62 (C-4), 24.47 (C-3), 30.87 (C-10), 38.31 (C-2), 43.60 (C-5), 45.30 (C-8), 55.50 (OCH3), 74.07 (C-7), 96.27 (C-6), 112.40 (C-2′), 119.50 (C-4′), 120.71 (C-6′), 129.62 (C-5′), 138.28 (C-1′), 159.87 (C-3′), 199.69 (C-1), 208.13 (C-10). ESI-MS (positive ions): m/z calculated for C17H23ClO5 342.81 [M]+ and 344.81 [M + 2]+; found 342.79 [M]+ and 344.80 [M + 2]+.N-[(2-Hydroxy-2-(3-methoxyphenyl)cyclohexyl)methyl]-N-methylformamide (BP6). White powder. 1H-NMR : δ 1.31 , 1.41 , 1.58 , 1.60 , 1.70 , 1.75 , 1.90 , 2.26 , 2.59 , 2.71 , 3.08 , 3.79 , 6.73 , 7.01 , 7.18 , 7.26 , 7.79 . 13C-NMR : δ 21.80 (C-5), 25.41 (C-3), 25.63 (C-4), 29.76 (C-9), 41.54 (C-2), 43.30 (C-6), 50.68 (C-7), 55.23 (OCH3), 74.18 (C-1), 111.47 (C-2′), 111.72 (C-4′), 116.85 (C-6′), 129.45 (C-5′), 150.08 (C-1′), 159.74 (C-3′), 163.13 (C-10). ESI-MS (positive ions): m/z calculated for C16H23NO3 277.36 [M]+; found 277.38 [M]+.4-(3-Methoxyphenyl)-2-nitrosooctahydro-1H-isoindol-4-ol (BP7). White powder. 1H-NMR : δ 1.62 , 1.65 , 1.70 , 1.80 , 1.90 , 1.95 , 2.15 , 2.66 , 2.70 , 2.73 , 2.75 , 3.82 , 6.77 , 7.03 , 7.06 , 7.28 . 13C-NMR : δ 21.96 (C-5), 25.81 (C-3), 26.11 (C-4), 44.53 (C-2), 41.62 (C-6), 55.16 (OCH3), 54.00 (C-7), 77.50 (C-1), 67.40 (C-9), 111.59 (C-4′), 110.71 (C-2′), 117.05 (C-6′), 129.13 (C-5′), 149.66 (C-1′), 160.22 (C-3′). ESI-MS (positive ions): m/z calculated for C15H20N2O3 276.33 [M]+; found 276.31 [M]+, 246.30 [M − NO]+, 219.29 [M – NO − HCN]+.Aliivibrio fischeri (NRRL-B-11177), according to ISO 2007 [® analyzer  after 30 min at 15 °C. Tests were carried out in triplicate. Data were analyzed with Microtox Omni software  and the result expressed as percentage of bioluminescence inhibition (%). The chronic growth inhibition test with Raphidocelis subcapitata was carried out according to ISO 2012 [2s). Effect data were expressed as percentage of growth inhibition [TRA and its products were dissolved in 0.01% dimethyl sulfoxide (DMSO) and further diluted in synthetic freshwater to make stock solutions . The acuISO 2007 . The lumISO 2012 . The culhibition .Daphnia magna were carried out according to ISO 2007 [50) along with 95% confidence limit values considering parametric or non-parametric methods. Two-way analysis of variance was carried out followed by Tukey’s post hoc analysis after testing normality and homoscedasticity of data distributions. Statistical analysis and graphs were done using Microsoft® Excel 2013/XLSTAT©-Pro .Acute toxicity tests with ISO 2007 . NewbornISO 2007 . ToxicitThis paper investigated the fate of TRA following disinfection treatment by chlorination and the ecotoxicity of this drug and its disinfection by-products. The reaction was carried out simulating the conditions of a typical wastewater plant, using an excess of sodium hypochlorite. TRA undergoes more than 70% of degradation but the treatment did not give rise the mineralization but rather induces the transformation of TRA itself into at least seven by-products, two of them chlorinated. Consistent with observations of previous studies based on toxic risk associated with hypochlorite treatment ,23,24,40


N-phenylnaphthylamine, N-phenyl-o-phenylenediamine and 1-naphthylamine . These conditions were not successful in the deuteration of triphenylamine or N,N-dimethylaniline, suggesting that these mild conditions are not suitable for the deuteration of tertiary arylamines, but are likely to be applicable for the deuteration of other primary and secondary arylamines. The deuterated arylamines can then be used for synthesis of larger organic molecules or polymers with optoelectronic applications.Deuterated arylamines demonstrate great potential for use in optoelectronic devices, but their widespread utility requires a method for large-scale synthesis. The incorporation of these deuterated materials into optoelectronic devices also provides the opportunity for studies of the functioning device using neutron reflectometry based on the difference in the scattering length density between protonated and deuterated compounds. Here we report mild deuteration conditions utilising standard laboratory glassware for the deuteration of: diphenylamine,  N,N'-di(1-naphthyl)-N,N'-diphenyl--4,4'-diamine (NPD), N,N'-bis(3-methylphenyl)-N,N'-diphenyl-benzidine (TPD) and N,N'-di(1-naphthyl)-N,N'-diphenyl-4,4'-biphenyldiamine (NAD), N-phenylbenzimidizol-2-yl)benzene  isotopologue to allow for accurate estimation of the percentage deuteration. The mean molecular weight was calculated based on the overall percentage of deuteration, and this was used for the calculation of mass yields. Automated medium pressure chromatography was conducted using a Reveleris® flash chromatography system , with specific chromatographic conditions given as details for the relevant compound.2O (~25 mL per gram of arylamine) in a two-neck round bottom flask, to which Pt/C  and Pd/C  were added. The flask was sealed, and the stirring reaction mixture was purged with one balloon of N2(g), followed by one balloon of H2(g), ensuring that the flask was not pressurised. The reaction mixture was heated at 80 °C for 4 h in the case of 1, 2 and 4, and 24 h in the case of 3. The reaction mixture was allowed to cool, CH2Cl2 (~25 mL) was added to the reaction flask, and the mixture filtered over Celite to remove the catalyst, which was then washed with CH2Cl2 (50–100 mL). The filtrate was extracted with 3 × 50 mL CH2Cl2, the combined organics dried over Na2SO4, filtered and evaporated to dryness to yield the deuterated product, characterized as follows:The relevant protonated arylamine (1–12 g) was suspended in D10-Diphenylamined (1). Compound 1 was synthesized according to the general procedure, utilizing 12 g of diphenylamine, 2 g of Pt/C and 2 g of Pd/C. The product was recrystallised from petroleum ether to give cream coloured crystals . 1H-NMR : δ residual protons 7.36 , 7.24 , 7.12 , 6.85 . 2H-NMR : δ 7.28 (br), 7.17 (br), 6.89 (br). 13C-NMR : δ 144.5 (m), 129.5 (m), 120.4 (m), 117.6 (m). ESI-MS m/z: 180.1 [M+H]+. Overall 95.8% D levels with isotopic distribution d8 6.3%, d9 29.9%, d10 63.9%.12-N-Phenylnaphthylamined (2). Compound 2 was synthesized according to the general procedure, utilising 6 g of N-phenylnaphthylamine, 1 g of Pt/C and 1 g of Pd/C. No additional purification was necessary, giving the product as a beige coloured solid . 1H-NMR : δ residual protons 8.18 , 7.89 , 7.56 , 7.48 , 7.41 , 7.39 , 7.23 , 7.08 , 6.85 . 2H-NMR : δ 7.94 (br), 7.67–7.36 (m), 7.28 (br), 7.13 (br), 6.90 (br). 13C NMR : δ 146.1 (s), 140.4 (s), 135.7 (s), 129.4 (m), 128.6 (m), 128.5 (s), 126.4 (m), 126.3 (m), 125.6 (m), 123.2 , 122.4 (m), 120.1 (m), 117.7 (m), 115.3 (m). ESI-MS m/z: 231.1 [M+H]+ overall 87.8% D levels with isotopic distribution d7 1.3%, d8 2.6%, d9 7.8%, d10 27.2%, d11 51.5%, d12 9.4%.9-N-Phenyl-o-phenylenediamined (3). Compound 3 was synthesized according to the general procedure, utilising 1 g of N-phenyl-o-phenylenediamine, 0.2 g of Pt/C and 0.2 g of Pd/C. The reaction was heated for 24 h, with the standard work up employed. The product was purified using automated medium pressure liquid chromatography (40 g silica column) with petroleum ether/ethyl acetate (80:20) as mobile phase to yield a red/brown solid . 1H-NMR : δ residual protons 7.13 , 7.06 , 6.91 , 6.82 , 6.75 , 6.70 , 6.61 , 6.43 , 4.42 . 2H-NMR : δ 7.17 (br), 7.10 (br), 6.95 (br), 6.86 (br), 6.79 (br), 6.66 (br). 13C-NMR : δ 146.5 (s), 143.2 (s), 128.5 (m), 128.2 (s), 124.7 (m), 124.3 (m), 117.7 (m), 117.0 (m), 115.2 (m), 114.5 (m). ESI-MS m/z: 194.1 [M+H]+. Overall 90.1% D levels with isotopic distribution d0 2.0%, d5 1.8%, d6 2.5%, d7 7.9%, d8 35.9%, d9 50.1%.7-1-Naphthylamined (4). Compound 4 was synthesised according to the general procedure, utilising 4 g of 1-naphthylamine, 0.5 g of Pt/C and 0.5g of Pd/C. After heating for 4 h and cooling to room temperature, the reaction mixture was basified by addition of 28% NH3(aq) (20 mL) before the standard work up. The product was purified by recrystallization from petroleum ether to give needle type brown coloured crystals . 1H-NMR : δ residual protons 7.86 , 7.50 , 7.34 , 6.81 , 4.21 . 2H-NMR : δ 7.89 (br), 7.54 (br), 7.39 (br), 6.85 (br). 13C-NMR : δ 142.0 (s), 134.3 (s), 128.1 (m), 125.6 (m), 125.4 (m), 124.4 (m), 123.6 (s), 120.8 , 120.4 (m), 118.5 (m), 109.3 (m). ESI-MS m/z: 151.2 [M+H]+ overall 88.0% D levels with isotopic distribution d3 0.7%, d4 2.4%, d5 10.7%, d6 29.9%, d7 44.2%. 2O at 80 °C utilising Pt/C and Pd/C catalysts. These mild reaction conditions do not require any specialised laboratory equipment and can produce deuterated arylamines in multi-gram quantities, with high (>90%) levels of overall deuteration. The deuteration of organic molecules within optoelectronic devices provides opportunities to study the functioning devices using neutron reflectometry, which is able to exploit the scattering length differences between hydrogen and deuterium. These deuterated arylamines can also be incorporated into optoelectronic devices, which may have better lifetime and efficiency compared to the protonated version. This work describes the H/D exchange of primary and secondary arylamines, performed in D


Viguiera arenaria Baker and two semi-synthetic pimarane derivatives were evaluated in vitro against the following main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); ent-8(14),15-pimaradien-3b-ol; ent-15-pimarene-8b,19-diol; ent-8(14),15-pimaradien-3b-acetoxy and the sodium salt derivative of PA were the most active compounds, displaying MIC values ranging from 2 to 8 μg∙mL-1. Thus, this class of compounds seems promising as a class of new effective anticariogenic agents. Furthermore, our results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new natural compounds that could be employed in the development of oral care products.Seven pimarane type-diterpenes re-isolated from  Streptococcus spp., mainly Streptococcus mutans and S. sobrinus, and Lactobacillus spp. [Dental caries is a common oral bacterial pathology caused by a biofilm consisting of microorganisms present on the tooth surface ,2. It islus spp. ,4.Mechanical removal of the dental plaque is the most efficient procedure in caries prevention; however, the majority of the population may not perform this removal efficiently . MoreoveSeveral antibiotics, such as ampicillin, chlorhexidine, sanguinarine, metronidazole, phenolic-antiseptics, and quaternary ammonium-antiseptics, among others, have been very effective in preventing dental caries , 8. Howeet al. [Myristica fragrans against oral bacteria. Katsura et al. [et al. [The great diversity of the chemical structures of secondary plant metabolites continues to provide new and important leads against several pharmacological targets ,10. Receet al.  have desa et al.  have poi [et al.  have proAspilia foliacea, could be used as a prototype for the discovery of new effective anti-infection agents against pathogens responsible for caries and periodontal diseases [Viguiera arenaria (VaDRE) (Asteraceae), as well as the activity of the mainly isolated and two semi-synthetic pimarane-type diterpenes against some oral microorganisms, including Streptococcus mutans, which is considered one of the primary causative agents of dental caries [Recently, our research group has demonstrated that kaurenoic acid, a diterpene isolated from diseases . The studiseases . In viewl caries . 1,2, 3, 6 and 7 [4 [5 [1H- and 13C-NMR spectral data analysis of the semi-synthetic derivatives, as well as, comparison with literature [The chemical structures of the diterpenes studied in this work are presented in  6 and 7 ,13; 4 [1and 7 [4 ; and 5 [ 7 [4 [5 . The 1H-terature ,13,14 al2, 4, 6, 8 and 9 displayed the highest antibacterial activity , S. sobrinus (13.2 μM), S. mutans (14.9 μM), S. mitis (13.2 μM), S. sanguinis (8.3 μM) and Lactobacillus casei (9.9 μM) with the values previously described for ent-kaur-16(17)-en-19-oic-acid . Samples were dissolved in CDCl3 or CD3OD, and the spectra were calibrated with the solvent signals at 7.26 and 3.30/4.84 (1H) or 77.0 and 49.0 (13C), respectively; the chemical shifts are given in ppm.NMR spectra were run on a Bruker DPX 400 spectrometer (400 MHz for Viguiera arenaria Baker (Asteraceae) was collected by Fernando Batista da Costa from the vicinity of the Washington Luís highway . The plant material was identified by J. N. Nakajima  and E. E. Schilling . A voucher specimen (FBC 60) was deposited in the herbarium of the Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, SP, Brazil, with the code SPFR 4006.V. arenaria (VaDRE), which was prepared in our laboratory in 1999, was suspended in a MeOH/H2O solution (9:1 v/v) and partitioned with n-hexane and dichloromethane (DCM). The n-hexane (2.0 g) and DCM extracts (1.0 g) were re-fractioned using several chromatographic techniques, such as vacuum liquid chromatography, flash chromatography, and preparative thin layer chromatography, as well as recrystallization with methanol, as previously described [ent-8(14),15-pimaradiene (1); 200 mg of ent-pimara-8(14),15-dien-19-oic acid ; 7 mg of 7-keto-ent-pimara-8(14),15-dien-19-oic acid (3); 100 mg of ent-8(14),15-pimaradien-3b-ol (4); 20 mg of ent-8(14),15-pimaradiene-3b,19-diol (5); 5 mg of ent-15-pimarene-8b,19-diol (6); and 20 mg 7b-hydroxy-ent-pimara-8(14),15-dien-19-oic acid (7).An aliquot (8.0 g) of the dichloromethane root crude extract from escribed ,23. Thes4 were treated with excess acetic anhydride in pyridine according to Da Costa et al. [ent-8(14),15-pimaradien-3b-acetoxy (8). This compound was purified through flash chromatography with n-hexane/ethyl acetate (9:1 v/v) as the mobile phase and identified by means of spectrometric analysis and comparison with literature data [1H-NMR: δ 4.52 ; 5.14 , H-14); 5.71 ; 4.90 ; 4.95 ; 0.99 ; 0.88 ; 0.88 ; 0.75 ; 2.05 ); 13C-NMR: δ 38.3 (C-1); 28.7 (C-2); 81.4 (C-3); 37.2 (C-4); 54.6 (C-5); 21.7 (C-6); 35.9 (C-7); 138.3 (C-8); 51.5 (C-9); 38.4 (C-10); 19.5 (C-11); 36.0 (C-12); 39.0 (C-13); 128.7 (C-14); 147.7 (C-15); 113.3 (C-16); 29.8 (C-17); 22.4 (C-18); 17.3 (C-19); 15.2 (C-20); 171.5 (C-21 (COCH3)); 24.5 (C-22 (COCH3)).About 50 mg of a et al. , to givel. [ent-8,15-pimar2, 50 mg) was dissolved in n-hexane (5 mL) and shaken with a NaOH solution  according to Daló et al. [9) as an emulsion, was filtered in a Büchner funnel, yielding 40 mg of the salt derivative after being washed with cold water. The chemical structure of this compound was established using 1H- and 13C-NMR spectral data analysis and comparison with literature data [1H-NMR: δ 5.11 , H-14); 5.70 ; 4.91 ; 4.95 ; 0.96 ; 1.15 ; 0.71 ; 13C-NMR: δ 42.1 (C-1); 21.3 (C-2); 40.6 (C-3); 46.9 (C-4); 58.7 (C-5); 26.9 (C-6); 38.0 (C-7); 141.2 (C-8); 53.2 (C-9); 42.1 (C-10); 22.3 (C-11); 38.9 (C-12); 41.4 (C-13); 129.4 (C-14); 149.5 (C-15); 114.5 (C-16); 31.4 (C-17); 31.0 (C-18); 185.2 (C-19); 15.7 (C-20).PA , Streptococcus sobrinus (ATCC 33478), Streptococcus mutans (ATCC 25275), Streptococcus mitis (ATCC 49456), Streptococcus sanguinis (ATCC 10556), and Lactobacillus casei (ATCC 11578). The samples were dissolved in DMSO (dimethyl sulfoxide) at 1 mg∙mL-1, followed by dilution in tryptic soy broth; concentrations ranging from 80 to 1 mg∙mL-1 were achieved. The final DMSO content was 4% (v/v), and this solution was used as negative control. The inoculum was adjusted for each organism, to yield a cell concentration of 5 x 105 colony forming units (CFU)∙mL-1. One inoculated well was included, to allow control of the adequacy of the broth for organism growth. One non-inoculated well, free of antimicrobial agent, was also included, to ensure medium sterility. Chlorhexidine dihydrochloride was used as positive control. The microplates (96 - wells) were sealed with plastic film and incubated at 37 °C for 24 h. After that, resazurin (30 mL) in aqueous solution (0.02%) was added to the microplates, to indicate microorganism viability. This procedure was based on the methodology described by Palomino et al. [The MIC values (the lowest concentration of the compound capable of inhibiting microorganism growth) of the DCM root extract from h method  in 96-wes ATCC 255, Strept


Ginkgo biloba L. leaves (GBL). In this paper, ginkgo lipids were isolated by extraction with petroleum ether, saponification, and molecular distillation. Eight known compounds: isophytol (1), nerolidol (2), linalool (3), β-sitosterol acetate (4), β-sitosterol (5), stigmasterol (6), ergosterol (7), β-sitosterol-3-O-β-D-glucopyranoside (8) and Ginkgo biloba polyprenols (GBP) were separated from GBL by chromatography and identified mainly by NMR. The separated and identified compounds 1, 2 and 3 are reported here for the first time in GBL. The 3D-DAD-HPLC-chromatogram (190–232 nm) of GBP was recorded. This study provides new evidence as there are no previous reports on antibacterial/antifungal activities and synergistic interactions between GBP and the compounds separated from GBL lipids against Salmonella enterica, Staphylocococus aureus and Aspergillus niger. Nerolidol (2) showed the highest activity among all the tested samples and of all mixture groups tested the GBP with isophytol (1) mixture had the strongest synergistic effect against Salmonella enterica among the three tested strains. A proportion of isophytol and GBP of 38.19%:61.81% (wt/wt) was determined by mixture design as the optimal proportion for the synergistic effect of GBP with isophytol against Salmonella enterica.Polyprenols separated from lipids are promising new components from  Ginkgo biloba L., the sole surviving species of the division Ginkgophyta, is considered as a living fossil due to its survival for over 180 million years. Ginkgo biloba L. leaves (GBL), as a Traditional Chinese Medicine, are commonly used in the clinic in China. This has also been reported in many famous Chinese herbal treatises, such as Shen Nong Ben Cao Jing  and Pen Ts’ao Kang Mu (1596) [β-sitosterol, [et al. used GC/MS to analyze the chemical composition of Ginkgo biloba L. lipids from external and internal leaves and identified some compounds, including several series of phenolic constituents and chainlike alcohols (ketones) [Ginkgo biloba polyprenols (GBP), new natural active components discovered after ginkgo flavonoids and terpene lactones, mainly exist in the form of acetates and are difficult to separate from other lipids. GBP is generally composed of 15 to 21 unsaturated isoprene units and is one type of betulaprenol with an E,E-farnesyl residue at the ω-end of the prenyl chain and terminated by an isoprene unit bearing a primary hydroxyl group [et al. researched the separation of GBP enriched in heavy GBL distillates by molecular short-path distillation [Cao Jing ,800 BC au 1596) . It was osterol, . Nguyen 96 . It wyl group . Reviewsillation . HoweverGinkgo extracts and constituents against bacteria, insects, and fungi were reported [et al. reported that hexane extracts of GBL showed actovoty against five pathogenic strains [Enterococcus (VRE) [in vitro. In this paper, we report that eight known compounds and GBP can be separated from non-saponifiable lipids of GBL by saponification, refrigeration, chromatography and identified by NMR analysis. The antibacterial/ antifungal activities and synergistic interactions between GBP and compounds separated from GBL lipids are examined according to the study route followed  . Therefofollowed .S2) and the light distillates (S3) that were collected from the non-saponifiable lipids of GBL by chromatography and eight compounds were identified using spectroscopic techniques, mainly 1H- and 13C-NMR, as isophytol (1) [2) [3) [β-sitosterol acetate (4) [β-sitosterol (5) [6) [7) [β-sitosterol-3-O-β-D-glucopyranoside (8) [In total eight known compounds were separated from the different polar portion of the frozen sediment (ytol (1) , nerolid (1) [2) , linaloo [2) [3) , β-sitostate (4) , β-sitoserol (5) , stigmas (5) [6) , ergoste [6) [7)  and β-siside (8)  by compa70, C75–C105, C110, C115, C120, GBP (contents over 98%) separated from GBL lipids were determined by HPLC using an external standard method  and all 1), nerolidol (2) and linalool (3) were reported for the first time among the components separated from GBL. Meanwhile, the 3D-DAD-HPLC-chromatogram (190–232 nm) of GBP was established for the first time and the maximum absorption wavelengths corresponding to each polyprenol homolog were recorded from the chromatogram for the first tome too.The present investigation supports that ethnobotanical uses of GBL relying on terpene trilactones and flavonoid glycosides, the active components of GBL might be responsible for their antibacterial activity . CompariS1), the frozen sediment (S2), the light distillates (S3), the heavy distillates (S4) isolated by refrigeration and molecular distillation, the eight individual compounds and GBP was assessed in the present study at 500 μg/mL against three animal and plant pathogenic strains  and their potencies were quantitatively assessed by their inhibition halos (p < 0.05) among all the samples. The results from the diameters of inhibition halos indicated that nerolidol (2) showed the highest activity among all the tested samples and inhibited the growth of all the strains. MIC, MBC and MFC values for the examined strains sensitive  to nerolidol (2) were in the range of 3.9–15.6 μg/mL, 31.3–62.5 μg/mL and 62.5 μg/mL, respectively. GBP  showed the MIC values were 31.3 μg/mL, MBC and MFC were 125 μg/mL. The results from the inhibition halo diameters showed the activity could be ranked from high to low (the same below) in the following order: nerolidol (2), linalool (3), the heavy distillates (S4), the total non-saponifiable lipids (S1), the light distillates (S3), GBP and isophytol (1). These seven samples were effective in inhibiting the growth of all three examined strains. The five samples of β-sitosterol (5), the frozen sediment (S2), β-sitosterol acetate (4), ergosterol (7) and stigmasterol (6) were effective at inhibiting two of the types of bacteria examined in this study. β-Sitosterol-3-O-β-D-glucopyranoside (8) was only effective at inhibiting Staphylocococus aureus.Antibacterial and antifungal activity of the total non-saponifiable lipids  and the heavy distillates (S4) were effective at inhibiting the growth of the three examined strains. None of the samples separated from the frozen sediment (S2) were active against Aspergillus niger.All the samples separated from the light distillates  and in the heavy distillates  of GBL [S4 having higher antibacterial and antifungal activities than the polyprenols from the above results. Therefore, it can be inferred GBP have synergistic inhibitory effects on microorganisms with other lipids, so it was indispensable to research the synergistic inhibitory effect on GBP with other lipids compounds in the next step.Nerolidol showed the highest activity among all the tested samples and inhibited the growth of all the strains used in this study. It was suggested that nerolidol possesses antifungal activity against sruption . Nerolidymyxin B . Consequymyxin B ,26,27. T) of GBL ,28. Besi1–8:GBP, 1:1, wt/wt) by analysis of variance (Tukey’s test at 5% probability) from p > 0.05) compared with the corresponding theoretical values of diameters of inhibition halos among all the mixture groups against all the tested strains. The mixture group C1:GBP had the most significant statistical difference  against Salmonella enterica compared with other mixture groups. The FIC index of C1:GBP against Salmonella enterica was 0.25, that was lowest in all mixture groups against all the tested strains. This result suggested that the GBP with isophytol (1) mixture group had the strongest synergistic effect against Salmonella enterica of all mixture groups against the three tested strains. Isophytol is an important component of many essential oils with reported antimicrobial activity [Salmonella enterica in the next study.The method for assessing synergistic antibacterial/antifungal effects of GBP with separated compounds was by comparing the diameters of inhibition halos between theoretical values and actual values of mixture groups  method  . The result showed the lowest FIC index response value was 0.2452  proportion of isophytol:GBP against Salmonella enterica were 0.25 and 16.5 mm, respectively. The relative error between the actual experimental values and the theoretical values using this design and analysis method was 1.96% (FIC index) and 1.22% , respectively. It can thus be inferred that this design and analysis method was both reasonable and reliable.The optimal proportion determination of synergistic effect on GBP with isophytol against ) method  by ANOVAs 0.2452  and the s 0.2452 . Under tSalmonella enterica ATCC 14028; Staphylocococus aureus ATCC 25923; Aspergillus niger ATCC 16404) bought from the China Center for Type Culture Collection  were used. A Bruker AV-300 NMR instrument was used for compound identification, using CDCl3 or DMSO-d6 as solvents and TMS as internal standard. HPTLC plates (silica gel 60) were obtained from Merck. The standard polyprenols  were purchased from Larodan Fine Chemical Co., Ltd, . HPLC measurements was performed at room temperature with a Shimadzu SPD-20A instrument equipped with DAD detector (210 nm) and a 2.5 μm Thermo BDS HYPERSIL C18 (150 × 4.6 mm) column, using 64/36 isopropanol/methanol solvent mixture as the eluent at 0.5 mL/min for 60 min.The dried GBL were collected in October 2011 from China’s Jiangsu Province. This plant was identified and authenticated by Prof. Cheng-Zhang Wang at the Institute of Chemical Industry of Forestry Products, CAF in China. Three types of strains  which was dissolved in a solvent mixture  for a solid-liquid ratio of 1:6–1:8 (g/mL), then refrigerated for 2 h at −15 °C [S2, 35 g) was obtained by quickly filtering at low temperature the solids from the refrigerated solution and the dissolved matter was concentrated to yield a product as a brown oil (215 g). The brown oil was fractionated by molecular distillation at a feed temperature of 60 °C, distillation temperature of 280 °C, feed flow rate of 180 mL/h, scraper rate of 300 rpm, and operating pressure of 0.1-0.5 Pa to give the light distillates (S3) as a yellow oil (44 g) and the heavy distillates (S4) as a dark brown oil (168 g).Shade air-dried (at room temperature) and pulverized (over 80 mesh sieve) GBL (10 kg) were extracted three times with 30 L  petroleum ether b.p. 60–90 °C) for 24 h at 65–70 °C and concentrated to give an extract (500 g) which was mixed with 5% NaOH-EtOH solution (6 L) for 3 h at room temperature. The hydrolysate was extracted with petroleum ether (6 L) three times. The collected organic phases were washed with water to neutrality and dried with anhydrous Na0 °C for t −15 °C . As a re1 (178 mg) was obtained from the petroleum/ethyl ether  portion that was separated on a HPTLC plate developed with petroleum/ethyl ether . Iodine was chosen for color development. The petroleum/ethyl ether  portion was subjected to gel permeation column chromatography  and eluted with CHCl3/MeOH  in order to purify compound 2 (51 mg). Compound 3 (20 mg) was obtained from the petroleum/ethyl ether  portion that was separated by HPTLC developed by petroleum/ethyl ether . 5% Anisaldehyde sulphuric acid–EtOH solution was chosen for color development.The light distillates were subjected to silica gel column chromatography . The fractions were eluted step by step with petroleum/ethyl ether . Fractions of similar composition as determined by TLC analysis were pooled. Compound S4 by flash column chromatography , using petroleum ether (5 × 250 mL) and 1%, 2%, 3% ethyl ether/petroleum ether (5 × 250 mL) as eluents [GBP were further purified from a portion (10 g) of the heavy distillates  eluents ,9,28. ThS2, 32 g) was dissolved in CHCl3 (30 mL) and the CHCl3solution was subjected to silica gel column chromatography . The fractions were eluted step by step with CHCl3/MeOH . Fractions of similar composition as determined by TLC analysis were pooled. The 100% CHCl3 portion was recrystallized from 2-propanol to yield compound 4 (290 mg). The CHCl3/MeOH  portion was subjected to gel permeation column chromatography  and eluted with CHCl3/MeOH  to isolate in order compounds 5 , 6  and 7 . The CHCl3/MeOH  portion was recrystallized from chloroform at low temperature to yield compound 8 (203 mg). 1H and 13C-NMR data of the structures of the eight compounds were as follows:The frozen sediment (Isophytol (1): 1H-NMR (CDC13) δ (ppm): 5.87–5.96 , 5.17–5.23 , 5.01–5.05 , 1.54–1.56 , 1.45–1.53 , 1.32–1.43 , 1.27 ; The position of this signal varied from 1.03–1.30 ppm for the other saturated methylene protons ; 0.88 , 0.86 , 0.83 . 13C-NMR (CDC13) δ (ppm): 145.3 (C-2), 111.4 (C-1), 73.3 (C-3), 42.7 (C-4), 39.4 (C-14), 37.5 (C-12), 37.4 (C-10), 37.4 (C-8), 37.3 (C-6), 32.8 (C-7), 28.0 (C-15), 27.7 (C-20), 24.8 (C-13), 24.4 (C-9), 22.7 (C-16), 22.6 (C-17), 21.4 (C-5), 19.7 (C-18), 19.6 (C-19).Nerolidol (2): 1H-NMR (CDC13) δ (ppm): 5.91–5.96 , 5.18–5.24 , 5.03–5.07 , 5.10–5.14 , 5.06–5.09 , 1.96–2.10 , 1.68 , 1.60 , 1.51–1.58 , 1.27 . 13C-NMR (CDC13) δ (ppm): 145.0 (C-2), 135.5 (C-7), 131.3 (C-11), 125.0 (C-6), 124.2 (C-10), 111.6 (C-1), 73.4 (C-3), 42.0 (C-4), 39.6 (C-8), 27.8 (C-15), 26.6 (C-9), 25.6 (C-12), 22.6 (C-5), 17.6 (C-13), 15.9 (C-14).Linalool (3): 1H-NMR (CDC13) δ (ppm): 5.86–5.96 , 5.18–5.24 , 5.10–5.14 , 5.04–5.08 , 1.98–2.07 , 1.68 , 1.60 , 1.56–1.59 , 1.53–1.55 , 1.28 . 13C-NMR (CDC13) δ (ppm): 145.0 (C-2), 131.8 (C-7), 124.3 (C-6), 111.6 (C-1), 73.4 (C-3), 42.0 (C-4), 27.8 (C-10), 25.6 (C-8), 22.8 (C-5), 17.6 (C-9).β-Sitosterol acetate (4): 1H-NMR (CDC13) δ (ppm): 5.37, 5.38 , 4.55–4.66 , 2.03 ; The position of other signals overlapped with the corresponding protons of compound 5. The signals of the alkali-hydrolysis product were same as the signals of the corresponding protons of compound 5.β-Sitosterol (5): 1H-NMR (CDC13) δ (ppm): 5.34, 5.36 , 3.48–3.58 ; The position of this signal varied from 1.05–2.33 ppm for the other saturated methylene and methine protons ; 1.01 , 0.91, 0.93 , 0.85, 0.87 , 0.80–0.85 , 0.80, 0.82 , 0.68 . 13C-NMR (DMSO-d6) δ (ppm): 140.7 (C-5), 121.7 (C-6), 71.8 (C-3), 56.2 (C-14), 56.0 (C-17), 50.1 (C-9), 45.8 (C-24), 42.2 (C-4), 39.7 (C-12), 37.2 (C-1), 36.5 (C-10), 36.1 (C-20), 33.9 (C-7), 31.9 , 31.6 (C-2), 29.1 (C-23), 28.2 (C-16), 26.0 (C-28), 24.3 (C-15), 23.0 , 21.0 (C-11), 19.8 (C-19), 19.0 (C-13), 11.8 (C-18), 18.8 , 11.9 (C-29).Stigmasterol (6): 1H-NMR (CDC13) δ (ppm): 5.34, 5.36 , 5.12–5.20 , 4.98–5.06 , 3.47–3.57 . The position of this signal varied from 1.04–2.33 ppm in the other saturated methylene and methine protons ; 1.01, 1.03 , 1.01 , 0.84, 0.86 , 0.81, 0.83 , 0.79–0.84 , 0.70 . 13C-NMR (CDC13) δ (ppm): 37.3 (C-1), 31.7 (C-2), 71.8 (C-3), 42.2 (C-4), 140.8 (C-5), 121.7 (C-6), 31.9 (C-7), 31.9 (C-8), 50.2 (C-9), 36.5 (C-10), 21.1 (C-11), 39.7 (C-12), 42.3 (C-13), 56.9 (C-14), 24.4 (C-15), 28.9 (C-16), 56.0 (C-17), 12.0 (C-18), 19.4 (C-19), 40.5 (C-20), 19.0 (C-21), 138.3 (C-22), 129.3 (C-23), 51.2 (C-24), 31.7 (C-25), 21.1 (C-26), 21.2 (C-27), 25.4 (C-28), 12.2 (C-29).Ergosterol (7): 1H-NMR (CDC13) δ (ppm): 5.56–5.58 , 5.37–5.39 , 5.20–5.27 , 5.13–5.20 , 3.58–3.75 . The position of this signal varied from 1.20–2.50 ppm in the other saturated methylene and methine protons ; 1.03, 1.05 , 0.95 , 0.91, 0.93 , 0.83, 0.85 , 0.81, 0.84 , 0.63 . 13C-NMR (CDC13) δ (ppm): 141.3 (C-8), 139.8 (C-5), 135.6 (C-22), 132.0 (C-23), 119.6 (C-6), 116.3 (C-7), 70.4 (C-3), 55.8 (C-17), 54.6 (C-14), 46.3 (C-9), 42.8 (C-13), 42.8 (C-24), 40.8 (C-4), 40.4 (C-20), 39.1 (C-12), 37.0 (C-10), 38.4 (C-1), 33.1 (C-25), 32.0 (C-2), 28.3 (C-16), 23.0 (C-15), 21.1 (C-21), 21.1 (C-11), 19.9 (C-27), 19.6 (C-26), 17.6 (C-19), 16.3 (C-28), 12.0 (C-18).β-Sitosterol-3-O-β-D-glucopyranoside (8): 1H-NMR (CDC13) δ (ppm): 5.34, 5.36 , 4.40, 4.43 , 3.74–3.87 . The position of this signal varied from 3.30–3.50 ppm in the other protons (4H) attached to carbon connected to oxygen atoms in the glucose ring structure. The position of this signal varied from 1.06–2.44 ppm in the other saturated methylene and methine protons ; 1.00 , 0.91, 0.93 , 0.85, 0.87 , 0.80–0.85 , 0.80, 0.82 , 0.68 . 13C-NMR (DMSO-d6+CDCl3) δ (ppm): 140.4 (C-5), 121.9 (C-6), 101.2 (C-1′), 78.8 (C-3), 77.6 (C-3′), 75.7 (C-5′), 73.8 (C-2′), 71.2 (C-4′), 62.7 (C-6′), 56.7 (C-14), 56.0 (C-17), 50.2 (C-9), 45.8 (C-24), 42.3 (C-13), 39.7 (C-4), 38.9 (C-12), 37.3 (C-1), 36.7 (C-10), 36.1 (C-20), 33.9 (C-22), 31.9 (C-7), 29.7 (C-8), 29.6 (C-2), 29.2 (C-25), 28.1 (C-16), 26.1 (C-23), 24.2 (C-15), 23.0 (C-28), 21.0 (C-11), 19.7 (C-27), 19.3 (C-19), 19.0 (C-26), 18.7 (C-21), 11.9 (C-29), 11.8 (C-18).70), 11.724 min (C75), 13.651 min (C80), 15.966 min (C85), 18.782 min (C90), 22.216 min (C95), 26.393 min (C100), 31.361 min (C105), 37.368 min (C110), 44.663 min (C115) and 53.519 min (C120), respectively. The absorption wavelength range of GBP is 190–232 nm in scanning 190–800 nm with DAD detector and the maximum absorption wavelengths of GBPs are 207 nm (C70), 207 nm (C75), 208 nm (C80), 210 nm (C85), 210 nm (C90), 208 nm (C95), 207 nm (C100), 207 nm (C105), 207 nm (C110), 207 nm (C115) and 206 nm (C120), respectively.The concentration of the GBP sample was 4.38 mg/mL and the injection volume was 5 μL. The retention time of different carbon chain GBPs is 10.127 min  following serial dilution technique. All the wells showing no visible growth of strains were subcultured and incubated at 37 °C  and 28 °C (Aspergillus niger) overnight. The highest dilution showing 100% inhibition was recorded as MBC or MFC [MIC, MBC and MFC were determined using the broth-dilution method. MIC was performed at seven concentrations of samples  option in the Design Expert 7.1.3 Software that generated the experimental scheme (13 standard/run) randomly. All samples were examined at 500 μg/mL . The components A and B were isophytol and GBP, respectively. The responses 1 and 2 were the FIC index and diameters of inhibition halos, respectively.1), nerolidol (2), linalool (3), β-sitosterol acetate (4), β-sitosterol (5), stigmasterol (6), ergosterol (7) and β-sitosterol-3-O-β-D-glucopyranoside (8) were separated from GBL by chromatography and identified by NMR. The separated and identified compounds 1, 2 and 3 were reported for the first time from GBL. The 3D-DAD-HPLC-chromatogram (190–232 nm) of GBP was recorded for the first time. Meanwhile, this study provides the first evidence of the antibacterial/antifungal activities and synergistic effect on GBP with compounds separated from GBL lipids against Salmonella enterica, Staphylocococus aureus and Aspergillus niger. Nerolidol (2) showed the highest activity among all the tested samples, and the GBP with isophytol (1) mixture group had the strongest synergistic effect against Salmonella enterica among all mixture groups against the three tested strains. The proportion of isophytol and GBP of 38.19%:61.81% (wt/wt) was determined as the optimal proportion of synergistic effect on GBP with isophytol against Salmonella enterica. This study provides a new scientific basis for the ethnomedical use of GBL against bacterial and fungal diseases of animals and plants.The eight known compounds isophytol (


R-(+)-5-O-[6'-O-acetyl]-α-d-glucopyranosyl-5-hydroxymellein (1), and a new phenylethanol glycoside, (−)-phenylethyl-8-O-α-l-rhamno-pyranoside (2), were isolated from the ethyl acetate extract of the fungus Xylaria sp. cfcc 87468, together with five known steroids, β-sitosterol (3), stigmast-4-en-3-one (4), ergosterol (5), (22E)-cholesta-4,6,8(14),22-tetraen-3-one (6), and 4α-methyl- ergosta-8(14),24(28)-dien-3β-ol (7). The structures of compounds 1 and 2 were elucidated by MS, extensive 1D and 2D NMR spectroscopy, and the circular dichroism (CD) spectroscopy.A new isocoumarin glycoside, 3 Xylaria produce many types of secondary metabolites -α-d-glucopyranosyl-5-hydroxymellein (1) and (−)-phenylethyl-8-O-α-l-rhamnopyranoside (2), and the five known compounds 3–7 . A molecular formula of C18H22O10 was assigned based on the interpretation of HRESIMS peak at m/z 421.1097 [M+Na]+ . Its IR spectrum showed characteristic hydroxyl group (3397 cm–1), and two ester carbonyl group (1676 and 1737 cm–1) absorptions. The 1H-NMR data of 1  and 7.45 , two methyls at δH 1.98  and 1.52 , and an oxygenated proton at δH 5.35 . The 13C-NMR and DEPT spectra of compound 1 displayed 18 carbon signals, including two methyls, two methylenes (one oxygenated methylene), six methines, six aromatic carbons (four quaternary carbons), and two ester carbonyl groups. One set of proton signals at δH 3.3–4.3, 5.35, and their corresponding carbons resonating at δC 64.9, 71.9, 72.1, 73.2, 74.8, and 100.5, suggested the presence of a hexosyl sugar moiety in the molecule. The 1H-NMR spectrum exhibited protons signals at δH 2.69 , 3.46 , and 4.72 .Compound ata of 1  showed tδH 2.46, 2.69) with C-3 (δC 77.8), C-5 (δC 146.7), C-9 (δC 130.6), and C-10 (δC 109.5); H-6 (δH 6.83) with C-5 (δC 146.7), C-8 (δC 158.5), and C-10 (δC 109.5); H-7 (δH 7.45) with C-5 (δC 146.7), C-8 (δC 158.5), and C-9 (δC 130.6) in the HMBC spectrum as well as the spin systems in the 1H–1H COSY spectrum  indicated the presence of a dihydroisocoumarin skeleton  with C-5 (δC 146.7) implied that the sugar unit was located at C-5 of the dihydroisocoumarin skeleton. In addition, the correlation of H-6'  to C-1' (δC 172.6) indicated that the acetyl group  was located at C-6' of the hexosyl sugar moiety (1 with 4 M aqueous CF3COOH the sugar unit was confirmed to be α-d-glucose -α-d-glucopyranosyl-5-hydroxymellein.Compound d 255 nm . Thus, c2 was obtained as a colorless gum with the molecular formula C14H20O5, as deduced from its HRESIMS peak at m/z 291.1200 [M+Na]+ .  −46 . The IR spectrum of 2 showed absorptions due to hydroxyl (3398 cm−1) and aromatic (1452 and 1497 cm−1) functionalities. The 1H-NMR data of 2 , and a methyl doublet at δH 1.19 , and oxygenated proton at δH 4.65 . The 13C-NMR and DEPT showed six aromatic carbons (one quaternary carbon), two methylenes (one oxygenated), five methine carbons (three oxygenated and one anomeric), and one methyl group. According to the signals of six aromatic carbons and 1H–1H COSY signal at H-8  with H-7 (δH 2.86) indicated compound 2 was a phenylethanol derivative. A series of proton signals in the range of δH 3.3–3.8, 4.65 , and 1.19 , and their corresponding carbons at δC 69.7, 72.2, 72.4, 73.8, 101.5, and 17.9 were observed in the 1H- and 13C-NMR spectra, which indicated the existence of a α-rhamnose moiety. After acid hydrolysis of 2, the sugar moiety was identified to be α-l-rhamnose according to the GC analysis. In the HMBC spectrum, the correlation of H-8  with C-1' (δC 101.5) showed that the α-rhamnose was attached to C-8 (2 was identified as (−)-phenylethyl-8-O-α-l-rhamnopyranoside.Compound ata of 2  showed ad to C-8 , hence c3–7 were identified as β-sitosterol (3) -α-d-glucopyranosyl-5-hydroxymellein (1). A colorless amorphous gum;  +60 ; CD (MeOH): λmax nm (Δε)= 205.0 (+2.33), 209.0 (+2.24), 226.6 (−2.02), 255.0 (−3.75); UV (MeOH) λmax (lg ε) 213 (4.47), 258 (3.70) nm; IR (film) vmax 3397, 2921, 1737, 1676, 1476, 1390, 1248, 1128, 1050, 910, 872 cm−1; 1H and 13C-NMR data, see m/z 421.1097 [M + Na]+ .(−)-Phenylethyl-8-O-α-l-rhamnopyranoside (2). A colorless gum;  −46 ; UV (MeOH) λmax (lg ε) 218 (2.85), 333 (2.13) nm; IR (film) νmax 3398, 2930, 1497, 1542, 1130, 1092, 1053, 981, 806, 749, 699 cm−1; 1H and 13C-NMR data, see m/z 291.1200 [M+Na]+ .Xylaria sp. cfcc 87468. Extensive spectroscopic analysis, chemical methods, and comparison with spectroscopic data in the literature resulted in the isolation of a new isocoumarin glycoside, 3R-(+)-5-O-[6'-O-acetyl]-α-d-glucopyranosyl-5-hydroxymellein (1), and a new phenylethanol glycoside, (−)-phenylethyl-8-O-α-l-rhamnopyranoside (2), along with five known steroids, β-sitosterol (3), stigmast-4-en-3-one (4), ergosterol (5), (22E)-cholesta-4,6,8(14),22-tetraen-3-one (6) and 4α-methylergosta-8(14),24(28)-dien-3β-ol (7). Compounds 3–7 were reported from Xylaria sp. cfcc 87468 for the first time.In this study, we investigated for the first time the chemical constituents of the ethyl acetate extract of


H-benzimidazole (1) was prepared, and hence underwent interesting functionalization reactions to afford several series of benzimidazole-5-(aryldiazenyl)thiazole derivatives: 3a–c, 7a–c, and 8a–c. The antibacterial activities of the synthesized compounds were evaluated by calculation of the inhibition zone diameter (mm) and the determination of minimum inhibitory concentration (µg/mL) against selected pathogenic bacteria Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria).Noticeable efficiency was found based on in vitro screening for their antioxidant activity and cytotoxicity effect against the human liver cancer cell line (HepG2) and human hepatocyte carcinoma cells at relatively high concentrations.The paper describes the synthesis and biological evaluation of some new benzimidazole derivatives as potent clinical drugs that are useful in the treatment of some microbial infections and tumor inhibition. The starting compound 2-(bromomethyl)-1 H-benzimidazol-2-yl)carbamate (mebendazole) as parasitic worm infestations, (RS)-2-pyridin-2-yl]methylsulfinyl)-1H-benzo[d]imidazole (lansoprazole) and (RS)-6-(difluoromethoxy)-2-[methylsulfinyl]-1H-benzo[d]imidazole (pantoprazole) as proton pump inhibitors; 1-[(4-fluorophenyl)methyl]-N-[1-[2-(4-methoxyphenyl)ethyl]-4-piperidyl]benzoimidazol-2-amine (astemizole) as antihistaminic[H-benzoimidazol-2-yl]carbamate  as antimicrobials [H-benzo[d]imidazol-2-amine (enviradine) as antiviral phenyl]methyl)-1H-1,3-benzodiazole-7-carboxylic acid (candesarten) as antihypertensive [N-ribosyl-dimethyl benzimidazole as an axial ligand for cobalt in vitamins [Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) using inhibition zone diameter (mm) and minimum inhibitory concentration methodologies comparing with Kanamycin and Ampicillin, respectively, as standard antibacterial agents. Their antioxidant and antitumor activities against the HepG2 human hepatocyte carcinoma cell line were also evaluated.The promising therapeutic potential of benzimidazole derivatives could be traced back to 1944and Woolley, who speculated that benzimidazole could act similar to purines to elicit some biological responses [istaminic,6; 2-[2-istaminic,6,7; metcrobials ,9,10; 1-ntiviral ,12; antintiviral ,14; 4- proceeded smoothly according to the reported method by the reaction of o-phenylenediamine with bromoacetic acid in the presence of 4 M hydrochloric acid, as shown in Synthesis of the starting 2-(bromomethyl)-1H-benzimidazole (1) revealed intense IR bands at 3372–3025 cm−1 . The 1H-NMR spectrum displayed a singlet signal at δ 4.85 ppm due to the methylene protons , a singlet at δ 10.32 ppm due to the NH–benzimidazole proton, and at δ 7.21–7.60 ppm due to the aromatic protons. The spectral analysis was found to be in good agreement with the literature [The IR spectrum of 2-(bromomethyl)-1terature .H-benzimidazole (1) with 2-aminothiazole was carried out to give the N-((1H-benzimidazol-2-yl)methyl)thiazol-2-amine (2) [N-((1H-benzimidazol-2-yl)methyl)-5-(aryldiazenyl)thiazol-2-amines (3a–c), as shown in The direct condensation of 2-(bromomethyl)-1mine (2) ,which un2 and 3a–c) were secured by their spectral analysis as described in the experimental section, where a characteristic singlet signal at δ 6.90 ppm due to the C4-thiazole proton was displayed.The structures of the isolated compounds (H-benzimidazole (1) with thiourea in acidic medium gave the previously reported derivative 1-((1H-benzimidazol-2-yl)methyl)thiourea (4) [The reaction of thiourea with α-haloalkyl or α-halocarbonyl compounds often proceed through the sulfur atom due to its high nucleophilicity to give thiouronium salts . Whereasurea (4)  through 4 was then incorporated in a set of investigations aiming at exploiting the reactivity of the thioureido moiety to build up the target compounds. Thus, 1-((1H-benzimidazol-2-yl)methyl)thiourea (4) was cyclized into the thiazole derivatives 2-(((1H-benzimidazol-2-yl)methyl)amino)thiazol-4(5H)-one (5) and N-((1H-benzimidazol-2-yl)methyl)-4-phenylthiazol-2-amine (6), by treating with α-halocarbonyl compounds . The aminothiazolyl derivatives (5 and 6) were then allowed to couple with different aromatic diazonium chloride compounds, affording the target arylazo compounds; 2-(((1H-benzimidazol-2-yl)methyl)imino)-5-(4-aryl)diazenyl)thiazolidin-4(5H)-one derivatives (7a–c) and N-((1H-benzimidazol-2-yl)methyl)-5-(4-aryl)diazenyl)-4-phenylthiazol-2-imine derivatives (8a–c) respectively, as shown in The thiourea derivative Staphylococcus aureus (S. aureus) ATCC 25923 as Gram-positive bacteria and Escherichia coli (E. coli) ATCC 25922 as Gram-negative bacteria compared with the standard drugs Kanamycin and Ampicillin, respectively. The results of the assessments are plotted in The antibacterial activities of novel synthesized compounds (100 µg/mL in DMSO), were determined in vitro by using the disc diffusion method  against The Minimum Inhibitory Concentration for the synthesized compounds was determined using a spot-on-lawn assay . The col3c, 8a, and 8c showed activities against E. coli (Gram-negative bacteria), while most of the synthesized compounds exhibited moderate activity against S. aureus (Gram-positive bacteria).The antibacterial study showed that some of the tested compounds exhibited variable antimicrobial activities against both bacteria Gram-negative and Gram-positive bacteria at a concentration of 100 µg/mL. The presence of substituted electron withdrawing groups such as nitro and acetyl groups in compounds 8c showed the highest affect against tested bacterial strains compared with the rest of the synthesized benzimidazoles under consideration due to the presence of an electron-withdrawing group (COR group) on the phenyl ring. It should be noted that the thiazolone moiety resulted in a dramatic reduction of the antibacterial efficiency .Compound 50) values were plotted in 3a and 3c: the highest DPPH value and the lowest EC50 value, respectively). This conclusion is also supported by previously reported results [Antioxidant capacities for the synthesized compounds were screened using a DPPH free radical scavenging assay incorporating a metastable free radical that is capable of accepting hydrogen radicals from antioxidants in solution. The method is based upon different mechanisms to provide complementary insight into the antioxidant activity of the synthesized compounds . The rea results .All of the compounds were screened in vitro for possible potential antitumor molecules against human liver cancer cell line (HepG2) and showed variable anticancer activity. The Neutral Red (NR) technique  was used3) and (8) in common showed higher activity than the other selected compounds that were tested. The presence of the 4-phenyl function with or without attached electron-withdrawing groups increased the antitumor activity due to their high antioxidant and free radical scavenging ability . This also could be explained by the previously reported facts [As shown in ed facts , and is ed facts .All of the chemical reagents that were used were of analytical grade or chemically pure and supplied by Sigma Aldrich Co., Darmstadt, Germany.1H-NMR (300 MHz) and 13C-NMR (75 MHz) spectra were recorded on a Varian Mercury VXR-300 spectrometer  and the chemical shifts were related to that of the solvent DMSO-d6 using tetramethylsilane (TMS) as an internal standard. MS spectra (HRMS) were acquired on TRACE GC Ultra gas chromatograph mass spectrometry, coupled with a THERMO mass spectrometer detector ISQ Single Quadrupole Mass Spectrometer and were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 50–1000.All of the microanalyses and spectral analyses were performed by the Micro Analytical Centers of Taif University-Saudi Arabia , Cairo University , and National Research Center-Egypt (Mass spectra).All of the corrected melting points are in degree centigrade and measured using a Stuart SMP20 melting point apparatus . The infrared spectra were recorded on a PerkinElmer Alpha platinum-ATR spectrometer; the ® Life Technologies, Waltham, MA, USA. The biological tests were performed by the biotechnology unit, faculty of Agriculture-Cairo University, Egypt. Human transformed cell lines from liver (HepG2) were obtained from the Egyptian Company for Vaccine and Serum . The statistical package for social science (SPSS) 1999 program version  was used for all of the analyses [All of the used kits for biochemical parameters were supplied by the Diagnostic Company, Maidenhead, UK. All of the chemicals that were used were supplied by Gibcoanalyses .Kanamycin (30 μg/mL) was aseptically added to the disc and used as a positive standard reference for Gram-positive strains, and Ampicillin (10 μg/mL) was used for Gram-negative strains. The inhibition clear zones (mm) were recorded in all of the samples and equal zero in no-inhibition results.The disc diffusion procedure was used to evaluate the antibacterial activity of the synthesized compounds . Uninocu3 at 30 °C with shaking (120 strokes per min (SPM)) to the early stationary phase, and the initial pH of the medium was adjusted to pH 6.0. Culture fluid was obtained by removing the cells by centrifugation, and was filtered with a cellulose acetate membrane filter . The resulting sample was serially diluted twofold with MRS broth (pH 5.0). Soft nutrient agar (20 mL) was solidified in a sterile dish (140 mm by 100 mm) after the addition of 60 µL of an overnight culture of Listeria monocytogenes SUB635. This strain was used as an indicator in the assay, since a clearer inhibitory zone was obtained with this organism than with B. subtilis. After 30 min of drying, 10 µL of each diluted sample was spotted onto the plate. The resulting plates were incubated at 30 °C overnight, and the titer was defined as the reciprocal of the highest dilution (2n) that resulted in the inhibition of the indicator lawn. Thus, the arbitrary unit (AU) of antibacterial activity per milliliter was defined as 2n × 1000 µL/10 µL.The minimum inhibitory concentration (MIC) of synthesized compounds was assayed by using a spot-on-lawn assay . Cells oTo examine the spectrum of activity of the purified bacteriocin against various bacterial strains, the test described above was performed with soft agar containing cells of test strains grown in a suitable medium to the early stationary phase. The abilities to inhibit a certain bacterial strain were compared by using the highest dilution that resulted in a growth inhibition zone.2, for a further 5 h. After incubation, plates were taken out, gently washed three times with PBS, and 100 μL of NR desorb solution was added per well. Plates were protected from light and shaken on a shaker at 60 rpm for 30 min, followed by keeping the plates still for 5 min. Plates were taken to a 96-well plate reader, and their absorbance was measured in each well at 540 nm. Each value is based on means of the absorbance values acquired from the quadruplet of treatments and normalized to the mean value of untreated control and expressed as % cell death according to the following equation (Equation (1)):Neutral Red (NR) assay  was usedThe data is based on three independent experiments.50 value is defined as the concentration of antioxidant in the reactive system that is necessary to decrease the initial DPPH concentration by 50%, and is calculated from the data obtained.The tested compounds were analyzed for their antioxidant potential according to the reported technique . The sto1) was prepared according to the literature procedure [O-phenylenediamine  was dissolved in 30–40 mL of four M of HCl by heating. The bromoacetic acid  was added thoroughly, and then, the reaction mixture was boiled under reflux for five h. The mixture was neutralized with ammonia, and the solid product was filtered, washed with water, and dried. The obtained product was recrystallized from acetone to give red crystals. M.p.: 60–62 °C , yield:H-benzimidazole 1 , 2-aminothiazole , and potassium iodide KI  in ethanol (50 mL) was heated under reflux for 6 h. Afterwards, an aqueous solution of potassium hydroxide KOH  was added and heating was continued for a further 2 h. The cold reaction mixture was poured onto ice-cold water, and the precipitated solid product was filtered off, dried, and recrystallized from ethanol to give reddish brown crystals of N-((1H-benzimidazol-2-yl)methyl)thiazol-2-amine (2). M.p.: > 250 °C (398 °C [A mixture of 2-(bromomethyl)-1 (398 °C ), yield:2O) to a cold suspension of 10 mmol of different aromatic amines  in concentrated HCl (4–6 mL) with stirring. To a cold solution of N-((1H-benzimidazol-2-yl)methyl)thiazol-2-amine 2  in ethanol (40 mL) and sodium acetate , a cold aqueous solution from the corresponding aromatic diazonium salts was added dropwise with stirring at 0–5 °C for 2 h. The solid products obtained were filtered off, washed with water followed by cold ethanol, dried, and recrystallized from ethanol to give N-((1H-benzimidazol-2-yl)methyl)-5-(aryldiazenyl)thiazol-2-amine (3a–c).The corresponding aryl diazonium chlorides were prepared by adding cold sodium nitrite solution methyl)-5-((4-nitrophenyl)diazenyl)thiazol-2-amine (3a). Brown solid, M.p.: >250 °C, Yield: 2.4 g, 63%. IR: νmax = 3740–2850 , 1620 (C=N), 1451(CH2) and 1335, 1569 (NO2) cm−1. 1H-NMR: δ/ppm = 3.51 , 5.87 , 6.95–7.65 , 10.90 . 13C-NMR: δ/ppm = 43.2, 115.2 (2C), 123.0 (2C), 123.8 (2C), 126.6 (2C), 134.7, 138.8 (2C), 140.7, 141.5, 147.2, 147.8, 163.2. MS : 379 . HRMS calc. for C17H13N7O2S: 379.0851, found: 379.0873.N-((1H-benzimidazol-2-yl)methyl)-5-(p-tolyldiazenyl)thiazol-2-amine (3b). Brown solid, M.p.: >250 °C, Yield: 1.8 g, 54%. IR: νmax = 3740–2850 , 1620 (C=N), 1453 (CH2), and 1389 (CH3) cm−1. 1H-NMR: δ/ppm = 2.49 , 3.40 , 5.87 , 6.92–7.64 , 10.99 . 13C-NMR: δ/ppm = 21.3, 43.2, 115.2 (2C), 123.0 (2C), 125.6, 128.8 (2C), 129.0 (2C), 138.3, 138.8 (2C), 140.7, 141.5, 147.2, 163.2. MS : 348 . HRMS calc. forC18H16N6S: 348.1157, found: 348.1168.1-(4-((2-(((1H-benzimidazol-2-yl)methyl)amino)thiazol-5-yl)diazenyl)phenyl)ethan-1-one (3c).Brown solid, M.p.: >250 °C, Yield: 1.7 g, 47%. IR: νmax = 3740–2850 , 1667 (CO acetyl), 1619 (C=N), 1453 (CH2), and 1388 (CH3) cm−1. 1H-NMR: δ/ppm = 2.44 , 3.43 , 5.87 , 7.16–7.74 , 10.90 . 13C-NMR: δ/ppm = 26.3, 43.3, 115.2 (2C), 123.0 (2C), 128.8 (4C), 133.1, 136.8, 138.8 (2C), 140.7, 141.5, 147.3, 163.2, 197.0. MS : 376 . HRMS calc. for C19H16N6OS: 376.1106, found: 376.1118.H-benzimidazole 1  and thiourea  in ethanol (30 mL) containing catalytic amount of trifluoroacetic acid (0.02 mL), was well stirred under reflux for 2 h. The reaction mixture was allowed to cool, and the solid product that was formed was filtered off, washed with cold ethanol, dried, and recrystallized from ethanol to obtain a brown solid; M.p.: 129–130 °C, Yield: 2 g, 99%. IR: νmax = 3740 (NH2), 3251–3020 , 1601 (C=N) and 1485(CH2) cm−1. 1H-NMR: δ/ppm = 3.39 , 5.87 , 7.18–7.48 , 9.50 , 10.90 , 11.31 . 13C-NMR: δ/ppm = 44.7, 115.2 (2C), 123.0 (2C), 138.8 (2C), 141.5, 182.5. MS : 206 . HRMS calc. for C9H10N4S: 206.0626, found: 206.0649 [A mixture of 2-(bromomethyl)-1206.0649 .H-benzimidazol-2-yl)methyl)thiourea 4  in 20 mL of ethanol containing 0.5 mL of triethylamine (TEA), a chloroacetylchloride  was added over one h at room temperature. The reaction mixture was then heated, and stirring was continued at 60 °C for five h. The mixture was allowed to cool, and the solid product formed was collected, dried, and recrystallized from ethanol to yield orange crystals; M.p.: >250 °C (284 °C [To a well stirred solution of 1-((1 (284 °C ), Yield:H-benzimidazol-2-yl)methyl)thiourea 4  in 20 mL of ethanol containing a few drops of piperidine, and phenacylchloride  was allowed to stir under reflux for six h. The red dish brown solid product formed was collected, washed from methanol, dried, and recrystallized from ethanol; M.p.: 219–220 °C, Yield: 2.7 g, 88%. IR: νmax = 3366–2950 , 1629 (C=N), and 1444(CH2) cm−1. 1H-NMR: δ/ppm = 2.49 , 5.32 , 7.30–7.64 , 7.90 , 10.10 . 13C-NMR: δ/ppm = 43.3, 107.8, 115.2 (2C), 123.0 (2C), 127.5, 128.4 (2C), 134.8 (2C), 136.3, 138.8 (2C), 141.5, 158.2, 161.3. MS : 306 . HRMS calc. for C17H14N4S: 306.0939, found: 306.0960.A mixture of 1-((12O) to a cold suspension of different aromatic amines (10 mmol) in concentrated HCl (4–6 mL) with continuous stirring. To a cold solution of (2-((1H-benzimidazol-2-yl)methyl)amino)thiazo-4(5H)-one 5  and/or N-((1H-benzimidazol-2-yl)methyl)-4-phenylthiazol-2-amine6 in ethanol (20 mL) and sodium acetate , a cold aqueous solution from the corresponding aromatic diazonium salts were added dropwise with stirring at 0–5 °C for two h. The solid products obtained were filtered off, washed with water followed by cold ethanol, dried, and recrystallized from ethanol to give 2-(((1H-benzimidazol-2-yl)methyl)imino)-5-(2-aryldiazenyl)thiazolidin-4-one (7a–c) and N-((1H-benzimidazol-2-yl)methyl)-5-(aryldiazenyl)-4-phenylthiazol-2(3H)-imine (8a–c).The corresponding aryl diazonium chlorideswere prepared by adding cold sodium nitrite solution 0.69 g, 10 mmol in 15 mL of H2-(((1H-benzimidazol-2-yl)methyl)amino)-5-((4-nitrophenyl)diazenyl)thiazol-4(5H)-one (7a). Dark brown solid; M.p.: >250 °C, Yield: 2.9 g, 73%. IR: νmax = 3366–2961 , 1635 (CO), 1585 (C=N), 1438 (CH2) and 1332, 1519 (NO2) cm−1. 1H-NMR: δ/ppm = 2.50 , 4.11 , 7.18–7.89 , 9.70 . 13C-NMR: δ/ppm = 43.5, 74.6, 115.2 (2C), 120.5 (2C), 123.0 (2C), 124.1 (2C), 138.8 (2C), 141.5, 157.4, 157.0, 158.2, 173.3. MS : 395 . HRMS calc. for C17H13N7O3S: 395.0801, found: 395.0822.2-(((1H-benzimidazol-2-yl)methyl)amino)-5-(p-tolyldiazenyl)thiazol-4(5H)-one (7b). Dark brown solid; M.p.: >250 °C, Yield: 1.9 g, 53%. IR: νmax = 3627–2799 , 1654 (CO), 1595 (C=N), 1441 (CH2) and1408 (CH3) cm−1. 1H-NMR: δ/ppm = 2.50 , 3.08, 4.89 , 7.21–8.48  and 8.91 . 13C-NMR: δ/ppm = 21.3, 43.7, 74.6, 115.2 (2C), 122.5 (2C), 123.0 (2C), 129.1 (2C), 138.8 (2C), 141.5, 147.7, 157.7, 158.2, 173.4. MS : 364 . HRMS calc. for C18H168N6OS: 364.1106, found: 364.1131.2-(((1H-benzimidazol-2-yl)methyl)amino)-5-((4-acetylphenyl)diazenyl)thiazol-4(5H)-one (7c). Brown solid; M.p.: >250 °C, Yield: 1.9 g, 49%. IR: νmax = 3627–2990 , 1660 (CO), 1591 (C=N), 1431(CH2) and 1402 (CH3) cm−1. 1H-NMR: δ/ppm = 2.49 , 3.32 , 4.11 , 7.16–7.98  and 9.54 . 13C-NMR: δ/ppm = 26.4, 43.7, 74.6, 115.2 (2C), 122.5 (2C), 123.0 (2C), 129.1 (2C), 133.6, 138.8 (2C), 141.5, 155.1, 158.2, 173.3, 197.0. MS : 393.11 . HRMS calc. for C19H15N6O2S: 392.1055, found: 392.1071.N-((1H-benzimidazol-2-yl)methyl)-5-((4-nitrophenyl)diazenyl)-4-phenylthiazol-2-amine (8a). Brown solid; M.p.: 183–185 °C, Yield: 3.2 g, 72%. IR: νmax = 3366–2950 , 1629 (C=N), 1444 (CH2) and 1322, 1513 (NO2) cm−1. 1H-NMR: δ/ppm = 2.50 , 5.32 , 7.52–8.31, 8.99 . 13C-NMR: δ/ppm = 44.2, 89.6, 115.2 (2C), 123.0 (2C), 123.8 (2C), 126.5 (2C), 127.9, 128.3 (2C), 128.6 (2C), 134.1, 138.8 (2C), 141.5, 147.8, 154.1, 158.2, 161.0. MS : 455 . HRMS calc. for C23H17N7O2S: 455.1164, found: 455.1145.N-((1H-benzimidazol-2-yl)methyl)-4-phenyl-5-(p-tolyldiazenyl)thiazol-2-amine (8b). Brown solid; M.p.: 162–163 °C, Yield 4 g, 95%. IR: νmax = 3385–2964 , 1636 (C=N),1475 (CH2), and 1407 (CH3) cm−1.1H-NMR: δ/ppm = 2.25 , 3.44 , 4.22 , 7.09–7.57 , 10.14 . 13C-NMR: δ/ppm = 21.3, 44.2, 89.6, 115.2 (2C), 123.0 (2C), 127.8, 128.3 (2C), 128.6 (2C), 128.7 (2C), 129.0 (2C), 134.1, 138.4, 138.8 (2C), 141.5, 145.1, 158.2, 161.0. MS : 424 . HRMS calc. for C24H20N6S: 424.1470, found: 424.1452.1-(4-((2-(((1H-benzimidazol-2-yl)methyl)amino)-4-phenylthiazol-5-yl)diazenyl)phenyl)ethan-1-one (8c).Brown solid; M.p.: 130–131 °C, Yield: 4.3 g, 96%. IR: νmax = 3295–2855 , 1656 (CO acetyl), 1588 (C=N), 1474 (CH2), and 1407 (CH3) cm−1. 1H-NMR: δ/ppm = 2.54 , 3.43 , 4.32 , 7.31–8.43  and 10.94 . 13C-NMR: δ/ppm = 26.4, 44.2, 89.6, 115.2 (2C), 123.0 (2C), 128.3 (2C), 128.6 (5C), 128.7 (2C), 134.1, 136.4, 138.8 (2C), 141.5, 152.2, 158.2, 161.0, 197.0. MS : 452 . HRMS calc. for C25H20N6OS: 452.1419, found: 452.1423.3a–c, 7a–c, and 8a–c derived from 2-(bromomethyl)-1H-benzimidazole were prepared in satisfactory yields and fully characterized. They exhibited in vitro variable antimicrobial activities when subjected to the bacterial strains Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) compared with Kanamycin and Ampicillin as standard drugs, respectively. Their inhibition effect against DPPH  compared to Trolox (EC50 = 55.13) was shown as well. They were screened for their antitumor activities against human liver cancer cell line (HepG2) to evaluate the cytotoxic effects of gradient concentrations (100–500 μg/mL) in terms of cell viability percentage and EC50 values. The tested compounds exhibited noticeable efficiency against the tumor cell line under investigation at the higher concentration. The benzimidazole derivatives  and (8b) showed higher activity than the other selected compounds that were tested. Presence of electron withdrawing groups increased the antitumor activities as discussed.In the development of this work, the newly synthesized series of benzimidazole derivatives


We conducted a 1:1 propensity score-matched retrospective cohort study of 87,218 patients with type 2 diabetes  initiating a SGLT2i or a GLP1-RA in Medicare data. We estimated HRs (95% CIs) for a composite cardiovascular endpoint and severe hypoglycemia comparing the two treatments in the entire population and by the CFI-based frailty subgroups. Compared with GLP1-RA, SGLT2i were associated with similar rates of the composite cardiovascular endpoint  and severe hypoglycemia (0.87 [0.71-1.07]) over a mean follow-up of 8.6 months. The rate of composite cardiovascular endpoint was not meaningfully different between SGLT2i and GLP1-RA across non-frail (1.33 [0.80-2.23]), pre-frail (0.96 [0.85-1.08]), and frail patients (0.87 [0.73-1.04]). Similarly, the rate of severe hypoglycemia was not meaningfully different between the two treatments among non-frail (0.97 [0.20-4.80]), pre-frail (0.83 [0.64-1.08]), and frail patients (0.95 [0.67-1.34]).


Kinetic assays and molecular modeling studies confirm that these compounds block both catalytic and peripheral AChE sites. These results are quite interesting since the triazole-quinoline system is a new structural scaffold for AChE inhibitors. Furthermore, the synthetic approach is very efficient for the preparation of target compounds, allowing a further fruitful new chemical library optimization.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide. Currently, the only strategy for palliative treatment of AD is to inhibit acetylcholinesterase (AChE) in order to increase the concentration of acetylcholine in the synaptic cleft. Evidence indicates that AChE also interacts with the β-amyloid (Aβ) protein, acting as a chaperone and increasing the number and neurotoxicity of Aβ fibrils. It is known that AChE has two binding sites: the peripheral site, responsible for the interactions with Aβ, and the catalytic site, related with acetylcholine hydrolysis. In this work, we reported the synthesis and biological evaluation of a library of new tacrine-donepezil hybrids, as a potential dual binding site AChE inhibitor, containing a triazole-quinoline system. The synthesis of hybrids was performed in four steps using the click chemistry strategy. These compounds were evaluated as  N-methyl-d-aspartate (NMDA) receptor antagonist-memantine as palliative treatment for this devastating pathology +; found m/z 303.1235 [M + H]+; 325.1053 [M + Na]+.To a solution of 4-azidoquinoline  in 2 mL of acetone, PCC  was added. The reaction mixture was stirred for 5 min at 60 °C  under microwave irradiation (200 W) and, after completion of the reaction (monitored by TLC), it was quenched with 0.5 mL of methanol before the solvents were removed under reduced pressure. The crude mixture was purified by column chromatography on silica gel in EtOAc as eluent, to support the compound 7 . 1H-NMR : δH ppm 10.06 ; 9.51 ; 9.17 ; 8.25 ; 8.08 ; 8.09–8.04 ; 7.98 ; 7.93  7.78 . 13C-NMR : δC ppm 192.6 (C16); 151.0 (C3); 149.1 (C4); 146.0 (C11); 140.2 (C1); 135.8 (C15); 135.6 (C6); 130.8 (C5); 130.4 (C14); 129.6 (C7); 128.6(C6); 126.0 (C13); 125.4 (C10); 123.1 (C8); 121.6 (C9); 117.0 (C2).To a solution of compound 7  and commercial indanone (8a–h)  in EtOH (1 mL) and DMF (0.5 mL), 150 μL of ethanolic solution 4% KOH was added.Afterwards, the mixture was stirred at room temperature for 3 h. The produced solid was filtered and washed with water and acetone. Compounds 3a–h were obtained as solids that required no further purification.To a solution of compound (E)-5,6-Dimethoxy-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3a). 1H-NMR : δH ppm 9.44 ; 9.18 ; 8.25 ; 8.14 ; 8.06 ; 7.98–7.92 ; 7.79 ; 7.50 ; 7.25 ; 4.09 ; 3.92 ; 3.85 . 13C-NMR : δC ppm 191.8 (C18); 155.4 (C24); 151.0 (C3); 149.4 (C25); 149.1(C4); 146.5 (C12); 145.2 (C19); 140.3 (C20); 136.3 (C15); 135.2 (C1); 131.3 (C13); 130.8 (C6); 130.7 (C17); 130.5 (C16); 130.0 (C9); 129.6 (C5); 128.6 (C7); 125.9 (C14); 125.0 (C10); 124.5 (C3); 123.1 (C8); 121.6 (C11); 116.9 (C2); 108.1 (C23); 104.6 (C22); 56.0 (C27); 55.7 (C26); 31.7 (C21). ESI-MS m/z calcd for C29H22N4O3: 474.1692, found m/z 475.1759 [M + H]+; m/z 497.1579 [M + Na]+.(E)-5,6-Methilenedioxy-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3b). 1H-NMR  δH ppm 9.44 , 9.18 , 8.25 , 8.14 , 8.06 , 7.97–7.90 , 7.79 , 7.49 , 7.21 , 6.21 , 4.09 .(E)-5-Chloro-6-methoxy-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3c). 1H-NMR  δH ppm 9.38 , 9.10 , 8.17 , 8.07 , 7.98 , 7.91–7.84 , 7.74–7.68 , 7.54 , 7.37 , 4.06 , 3.89 . ESI-MS (3c): m/z calcd for C28H19ClN4O2: 478.1197, found m/z 517.0825 [M + K]+.(E)-6-Methyl-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3d). 1H-NMR  δH ppm 9.45 , 9.18 , 8.25 , 8.14 , 8.06 , 7.97–7.92 , 7.78 , 7.62–7.54 , 4.16 , 2.41 . ESI-MS (3d): m/z calcd for C28H20N4O2: 428.1637, found m/z 429.2378 [M + H]+; m/z 467.1268 [M + K]+.(E)-6-Chloro-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3e). 1H-NMR  δH ppm 9.47 , 9.18 , 8.26 , 8.16 , 8.06 , 7.98 , 7.94 , 7.81–7.77 , 7.65 , 4.22 .(E)-4-Methoxy-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3f). 1H-NMR  δH ppm 9.42 , 9.16 , 8.24 , 8.14 , 8.04 , 7.97–7.91 , 7.61 , 7.59 , 7.48 , 7.38 , 7.31 , 4.03 , 3.94 . ESI-MS (3f): m/z calcd for C28H20N4O2: 444.1586, found m/z 483.1152 [M + K]+.(E)-5-Methoxy-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3g). 1H-NMR  δH ppm 9.45 , 9.18 , 8.26 , 8.15 , 8.07 , 7.99–7,93 , 7.80 , 7.76 , 7.53 , 7.24 , 7.07 , 4.17 , 3.92 . ESI-MS (3g): m/z calcd for C28H20N4O2: 444.1586, found m/z 483.1219 [M + K]+.(E)-6-Methoxy-2-(4-(1-(quinolin-4-yl)-1H-1,2,3-triazol-4-yl)benzylidene)-2,3-dihydro-1H-inden-1-one (3h). 1H NMR  δH ppm 9.35 , 9.16 , 8.25 , 8.15 , 8.05 , 7.96–7,93 , 7.89 , 7.78 , 7.62–7.60 , 7.33 , 7.28 , 4.12 , 3.86 . ESI-MS HRMS (ESI) (3h): m/z calcd for C28H20N4O2: 444.1586, found m/z 483.1235 [M + K]+.hAChE EC 3.1.1.7, 1000 units mg−1 from human recombinant, expressed in HEK 293 cells) and butyrylcholinesterase  as lyophilized powder; their substrates acetylthiocholine iodide (ACThI) and butyrylthiocholine iodide (BTChI); 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent or DTNB); donepezil hydrochloride monohydrate; tacrine  were all obtained from Sigma-Aldrich . Buffer components and all of the chemicals used were all analytical grade materials, and were obtained from Sigma, Merck , Synth  or Acros . The water used in all of the preparations was purified with a Millipore Milli-Q® system . Stock solutions (1 mM) of the evaluated inhibitors were prepared and diluted with dimethylsulfoxide (DMSO)/water (50% v/v) to give the desired concentration ranges.The enzymes acetylcholinesterase  and in the absence of inhibitor (A0) according to the expression, % Inhibition = 100 − [(Ai/A0) × 100]. The assays were carried out in duplicate. Tacrine and donepezil were used as standard inhibitors hAChE.The inhibition percentage was obtained by comparing the absorbance in the presence of inhibitor (A50) assay, increasing concentrations of the 25 µL of each inhibitor sample (0.05–50 µM) for standard inhibitors and (25–500 µM) for the ligands were prepared from a 1 mM stock solution. IC50 values were independently determined by performing rate measurements for at least six concentrations of the target inhibitor. The nonlinear regression parameters were calculated, and the IC50 was extrapolated.For the inhibitory potency (ICi) and mechanism of the action assay, ranging concentrations of the 25 µL of ATChI solutions  containing fixed ligands concentrations (from 0.06 to 2.5 µM for standard inhibitors and 55 to 250 µM for the ligands) were carried out. Reciprocal plots of 1/absorbance versus 1/[ACThI] were constructed and the constant Ki can be determined from the replots of primary reciprocal plot data.For the steady-state inhibition constant (K3:MeOH:H2O 65:30:5 (v:v) as the mobile phase. After drying, the plates were sprayed with a solution containing AChE (0.704 mg), BSA (0.025 g), and ACThI (0.00723 g) in Tris , previously incubated at 37 °C for 15 min, followed by addition of Ellman’s reagent prepared in Tris  for the assays with AChE. As for the assays with BChE, BChE itself and its substrate BCThI were used at the same concentrations listed above. This assay was carried out as described in the literature [Each compound sample (2.5 μL) was eluted on a chromatographic silica gel 60 plate using CHClhAChE complexed with donepezil (PDB code 4EY7). Top-ranked orientations were selected by GOLD via the ChemPLP empirical energy function [N-piperidine atom of donepezil.Molecular docking studies were performed using GOLD 5.2 ,35 with function . In eachin vitro studies confirm the therapeutic value of these compounds. Further developments are in progress to optimize these new triazole-quinoline derivatives as potential disease-modifying drug candidates for Alzheimer’s disease treatment in the future.The multi-target treatment for AD is one of the most promising strategies to combat this complex and devastating neurodegenerative disorder urgently in need of an effective treatment. Among them, dual binding site AChE inhibitors showed the advantage of targeting a druggable pharmacological enzyme, modulating both symptomatic and disease-modifying symptoms as potent β-amyloid modulators . Herein,


J. Li et P. K. Hsiao together with four known resveratrol dimer derivatives, including maximol A (3), gnetin C (4), ε-viniferin (5), and pallidol (6). The structures were determined by combined spectroscopic methods and by comparison of their spectral data with those reported in the literature. All the compounds isolated from R. lhasaense were tested for their ability to scavenge1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.Two new resveratrol trimer derivatives, named rheumlhasol A ( Natural resveratrol oligomers, commonly consisting of two to eight resveratrol units, have drawn increasing attention across the world due to their intriguing structures and pharmacological potential ,2,3. ResRheumLinn consists of approximate 60 species and is mainly distributed in sub-alpine and alpine zones of Asia  and two sets of AX2-type signals  that were assigned to two p-substituted phenyl moieties (A1 and C1 rings) and two 1,3,5-trisubstituted aromatic rings (A2 and C2 rings), respectively, characteristic of the two resveratrol structural units. The presence of two sets of mutually coupled methine hydrogen signals  instead of the olefinic proton signals suggesting that the olefinic bond got reduced thereby resulting in trimerisation of these carbons in the two resveratrol structural units. Furthermore, the 1H-NMR spectra of 1 displayed two signals  that were assigned to the cis-coupled olefinic protons in the third resveratrol structural unit. Two aromatic rings  of resveratrol structural units took part in the trimerisation process, as was evident from the 1H-NMR signals as follows: two singlets for m-hydrogens resonating at δH ppm 6.34  and 6.32  were assigned to a 1, 3, 4, 5-tetrasubstituted ring (B1), and two doublets for protons resonating at δH ppm 6.74 , and 7.21  together with a singlet at δH ppm 6.94  were assigned to a 1, 3, 4-trisubstituted ring (B2).Rheumlhasol A  to that of related resveratrol oligomers reported in literature  with relatively low-field chemical shifts and large coupling constants owing to trans-olefinic coupling (instead of cis-olefinic coupling). Thus, rheumlhasol B (2) was characterized as the (E)-geometrical isomer of rheumlhasol A (1) and from these results, the structure of 2 was determined as shown in 3–6 were identified by comparison of their spectroscopic data with literature data.The molecular formula CNMR data  of 2 are1–6 isolated from R. lhasaense were screened for their antioxidant activities by DPPH free radical-scavenging assay that has been widely used for the evaluation of antioxidant activities of natural products. The results obtained in this study are summarized in 2 and 3 exhibited relatively high antioxidant activities with IC50 values of 28.7 and 31.3 µM, respectively, which was comparable to that of α-tocopherol; while 1, 4, and 5 showed moderate activities with IC50 values in the range of 49.7 to 69.8 µM. Compound 6 showed lowest antioxidant activity with IC50 values of 190.2 µM.The resveratrol oligomers 1H-, 13C-, and 2D NMR spectra were recorded on Bruker DRX-500 (500 MHz) spectrometer with TMS as internal standard. The ESI-MS (negative ion mode) and HR-ESI-MS (negative ion mode) spectra were recorded on VG AutoSpe 3000 and API Qstar P ulsar LC/TOF spectrometers, respectively. The UV spectra were measured by using a Shimadzu double-beam 210A spectrophotometer. The IR spectra were recorded on a Bio-Rad FTS-135 spectrometer, in KBr pellets. The optical rotations were measured by using a SEPA-3000 automatic digital polarimeter. The column chromatographic separations were performed on silica gel , or Lichroprep RP-18 gel . The column fractions obtained were monitored by TLC, and spots were visualized by heating the silica gel plates after spraying with 15% H2SO4 in water. The TLC and PTLC separations were performed on silica gel Gf 254 pre-coated plates . The R. lhasaense A. J. Li et P. K. Hsiao plant materials were collected in August 2010 from LhaSa, Tibet Autonomous Region, China, and authenticated by Professor Anjen Li of Institute of Botany, Chinese Academy of Sciences. A voucher of the specimen (No. 2004080203) collected was deposited at School of Chemistry & Biotechnology, Yunnan University of Nationalities.R. lhasaense A. J. Li et P. K. Hsiao were extracted exhaustively with 95% aqueous EtOH (5 × 10 L) at room temperature . The EtOH extract was concentrated in vacuo to yield a brown residue (200 g), which was suspended in water (200 mL), and extracted with EtOAc (3 × 200 mL). The combined organic phase was concentrated to yield a residue (89 g), which was loaded on a silica gel (SiO2) column (2 kg) and eluted with petroleum ether (PE)/acetone gradient to give five fractions (1–5). Fraction 3 eluted with PE/acetone (2:1) was subjected to repeated column chromatography (CC)  to afford 3 (25 mg). Fraction 4 eluted with PE/acetone (1:2) was subjected to repeated CC , followed by PTLC  to afford 4 (27 mg) and 5 (15 mg). Fraction 5 eluted with acetone was subjected to repeated CC  to afford 6 (30 mg) and a sub-fraction containing 1 and 2. This sub-fraction was subjected to repeated CC on RP18 gel eluted by MeOH/water (58:42) to afford 1 (10 mg) and 2 (12 mg). The air-dried powder roots (1 kg) of Rheumlhasol A (1): white powder; [α]D = +10.2262ο ; IR (KBr) νmax 3419, 1603, 1515, 1486, 1449, 1339, 1301, 1233, 1155, 998, and 831 cm−1; UV (MeOH) λmax (log ε) 202 (4.9), 227 (3.3), 285 (2.4), and 300 (2.2) nm; negative ESI-MS [M−H]− at m/z 679; negative HR-ESI-MS [M−H]− at m/z 679.1964 ; 1H and 13C-NMR data (NMR data .RheumlhasolB (2): white powder; [α]D = +5.4321ο ; IR (KBr) νmax 3396, 1600, 1516, 1486, 1450, 1341, 1303, 1235, 1155, 999, 960, and 832; UV (MeOH) λmax (log ε) 202 (4.9), 225 (3.3), 310 (2.3), and 335 (2.4) nm; negative ion ESI-MS [M−H]− at m/z 679; negative ion HR-ESI-MS m/z 679.1943 ; 1H and 13C-NMR spectra ( spectra .Maximol A (3): brown amorphous powder, positive ESI-MS [M]+ at m/z 454; 1H-NMR  δ ppm 7.43 , 7.24 , 7.06 , 6.90 , 6.85 , 6.78 , 6.53 , 6.28 , 6.25 , 6.19 , 5.45 , and 4.46 ; 13C-NMR  δ ppm 160.54 (C-4′), 159.72 (C-11 and C-13), 159.52 (C-11′ and C-13′), 158.40 (C-4), 145.15 (C-9), 140.70 (C-9′), 132.44 (C-1′), 132.14 (C-3′), 131.68 (C-1), 129.04 (C-7′), 128.56 (C-2 and C-6), 128.56 (C-5′), 127.15 (C-8′), 123.86 (C-2′), 116.12 (C-3 and C-5), 110.11 (C-5′), 107.37 (C-10 and C-14), 105.64 (C-10′ and C-14′), 102.68 (C-12′), 102.34 (C-12), 94.00 (C-7), and 57.77 (C-8). These data are consistent with those reported in literature − at m/z 453; 1H-NMR  δ ppm 7.35 , 7.16 , 7.01 , 6.89 , 6.80 , 6.67 , 6.58 , 6.26 , 6.21 , 5.35 , 4.41 ; 13C-NMR  δ ppm 163.36 (C-5′), 159.54 (C-11 and C-13), 158.27 (C-4), 158.09 (C-14), 155.57 (C-3′), 146.71 (C-9), 141.85 (C-1′), 134.31 (C-9′), 130.63 (C-1), 129.56 (C-7′), 129.11 (C-10′ and C-14′), 128.4 (C-2 and C-6), 127.15 (C-8′), 116.6 (C-3 and C-5), 116.51 (C-11′ and C-13′), 115.42 (C-4′), 108.15 (C-2′), 107.40 (C-10 and C-14), 102.26 (C-12), 99.94 (C-6), 94.64 (C-7), and 56.67 (C-8). These data are in accordance with those reported in literature + at m/z 455; 1H-NMR  δ ppm 7.14 , 7.04 , 6.82 , 6.77 , 6.65 , 6.63 , 6.57 , 6.25 , 6.18 , 6.16 , 5.36 , and 4.34 ; 13C-NMR  δ ppm 162.73 (C-3′), 160.05 (C-11 and C-13), 159.75 (C-5′), 158.54 (C-12′), 158.40 (C-4), 147.35 (C-9), 136.90 (C-1′), 133.87 (C-1), 130.36 (C-9′), 130.31 (C-7′), 128.77 (C-2 and C-6), 128.21 (C-10′ and C-13′), 123.66 (C-8′), 120.04 (C-2′), 116.36 (C-3 and C-5), 116.29 (C-11′ and C-13′), 107.44 (C-10 and C-14), 104.30 (C-6′), 102.18 (C-12), 96.83 (C-4′), 94.83 (C-7), and 58.30 (C-8). These data are in accordance with those reported in literature + at m/z 455; 1H-NMR  δ ppm 6.97 , 6.69 , 6.61 , 6.18 , 4.55 , and 3.80 ; 13C-NMR  δ ppm 159.30 (C-11 and C-11′), 156.30 (C-4 and C-4′), 155.28 (C-13 and C-13′), 150.24 (C-9 and C-9′), 137.69 (C-1 and C-1′), 128.98 , 123.18 (C-14 and C-14′), 115.75 , 103.26 (C-10 and C-10′), 102.43 (C-12 and C-12′), 60.45 (C-8 and C-8′), and 53.89 (C-7 and C-7′). These data are in accordance with those reported in literature [terature .Sample stock solutions (1 mM) were diluted to concentrations of 25, 50, 100, 150, 200, and 250 µM in methanol. One milliliter of DPPH methanol solution  was added to 3.0 mL of a MeOH solution of various sample concentrations. The mixtures were shaken vigorously and then kept in the dark at room temperature. After 30 min, the absorbance values were measured at 518 nm and converted into the percentage inhibition of DPPH (Ip) by using the following formula:dl-α-tocopherol solution was used as positive control. The IC50 values obtained represent the concentrations of the tested samples and standards that caused 50% inhibition of DPPH, and were calculated by linear regression of plots where the abscissa represent the concentration of tested compounds and the ordinate represent the average percentage of inhibition from three separate tests. The experiments were done in triplicate, and the results are given as mean ± standard deviation (SD).A mixture of DPPH solution  and methanol (3.0 mL) was used as negative control while 1) and rheumlhasol B (2) were isolated from the roots of Rheum lhasaense A. J. Li et P. K. Hsiao, together with four known dimers 3−6. Apparently, compound 2 is derived from the coupling of gnetin C (4) with another resveratrol unit. The benzofuran ring connects B unit and C unit may formed from condensation of oxygen radical (C12-O·) and carbon C-11 of B2 ring with C-7c and carbon radical (C-8c) of C unit. This is the first time that Rheum plants have been reported to contain resveratrol trimers. In addition, the free radical scavenging activities of all the isolated compounds against DPPH radical have been evaluated in this study. Compounds 1–5 showed moderate antioxidant activities, with IC50 values in the range of 28.7 to 69.8 μM, while compound 6 showed low antioxidant activity with an IC50 value of 190.2 μM.Two new isomeric resveratrol trimers named rheumlhasol A (