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Prof. Vladimir I Kalinin Vladivostok, Russia

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Prof. Sylvia Urban Melbourne, Australia

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HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

axuf64@dsl.pipex.com

Chemoinformatics Approach to Antibacterial Studies of Essential Oils Dragoljub L. Miladinovića,*, Budimir S. Ilića and Branislava D. Kocićb aDepartment of Pharmacy, Faculty of Medicine, University of Niš, 18000 Niš, Serbia

bCenter for Microbiology, Institute for Public Health, 18000 Niš, Serbia

dragoljubm@gmail.com

Received: December 28th, 2014; Accepted: January 22nd, 2015

The chemical composition and antibacterial activity of Nepeta nuda (Lamiaceae) essential oil were examined, as well as the association between it and standard antibiotics: tetracycline and streptomycin. The antibacterial activities of 1,8-cineole, the main constituent of N. nuda oil, individually and in combination with standard antibiotics were also determined. The interactions of the essential oil and 1,8-cineole with antibiotics toward five selected strains were evaluated using the microdilution checkerboard assay in combination with chemoinformatics methods. Oxygenated monoterpenes were the most abundant compound class in the oil (57.8%), with 1,8-cineole (46.0%) as the major compound. The essential oil exhibited in vitro antibacterial activity against all tested bacterial strains, but the activities were lower than those of the standard antibiotics. The combinations N. nuda oil-antibiotic and 1,8-cineole-antibiotic produced a predominantly antagonistic interactions. Chemoinformatics survey confirms the antagonistic interactions as a consequence of membrane potential/proton motive force dissipation. These data indicate cytochrome c oxidase as a target for 1.8-cineole toxicity action mechanisms. Keywords: Nepeta nuda, 1,8-Cineole, Checkerboard assay, Essential oil, Molecular docking. The association of essential oils with antibiotics is one of the novel way to combat bacterial resistance. Essential oils are combined with antibiotics in order to improve the antimicrobial effect and to reduce the required antibiotic concentration [1]. Elucidation of the mechanism of antibacterial action based on the synergism of essential oil/antibiotic combination is not simple. Especially it is not easy to explain the mechanism of the antagonistic interaction of the essential oil/antibiotic combination [2]. To help explain this question, a cheminformatics approach was chosen. Molecular docking is a cheminformatics method which predicts the preferred orientation of one ligand when bound in an active site to form a stable complex. Docking can be used to propose binding affinity between two molecules, which has the objective of providing an explanation of how the ligands inhibit the target [3]. The genus Nepeta includes 280 species, which have been used in folk medicine because of their antiseptic, antispasmodic, expectorant, diuretic, antitussive, antiasthmatic and febrifuge activities [4]. The Serbian flora recognizes three species of Nepeta [5]. Given the importance of N. nuda as a useful antibacterial remedy, the aim of the present study was to examine the chemical composition and antibacterial effect of the essential oil of N. nuda, as well as the association between it and the conventional antibiotics tetracycline and streptomycin. The antibacterial activity of 1,8-cineole, the main constituent of N. nuda oil, individually and in combination with the same antibiotics was also determined. In order to explain the mechanism of action of the combination of investigated substances and selected antibiotics, molecular docking was employed. The yield of N. nuda essential oil was 0.6%, w/w. Based on GC and GC-MS analysis of the essential oil, 46 components were identified that represented 92.5% of the total detected constituents (Table 1). Oxygenated monoterpenes were the most abundant compound class in the oil (57.8%), and they were dominated by 1,8-cineole (46.0%). The sesquiterpene hydrocarbons (20.4%) were dominated by germacene D (6.8%).

The examined essential oil possessed antimicrobial activities against all the tested micro-organisms with MIC values ranging from 1332.8 to 21324.8 g/mL and MBC values from 5331.2 to 42649.6 g/mL (Table 2). 1,8-Cineole was active with MIC and MBC values ranging from 5900.8 to 94412.8 g/mL. The reference antibiotics were active in the following ranges of concentration: tetracycline, 16.0 to 256.0 g/mL, and streptomycin, 4.0 to 64.0 g/mL. The possible interactions between the essential oil and 1,8-cineole with reference antibiotics are given in Table 3. From all the tested combinations of N. nuda essential oil-tetracycline 7 (15.6%) showed synergism, 10 (22.2%) had an additive and 28 (62.2%) an antagonistic effect. From 45 combinations of N. nuda essential oil-streptomycin, 14 (31.1%) showed synergism, while 5 (11.1%) had an additive, and 26 (57.8%) an antagonistic effect. The combination profiles of 1,8-cineole with selected antibiotics are presented in Table 3. From 45 tested combinations of 1,8-cineole-tetracycline, 13 (28.9%) showed synergism, 12 (26.7%) had an additive and 20 (44.4%) an antagonistic effect. Out of the 45 combinations of 1,8-cineole-streptomycin, 7 (15.6%) showed synergism, while 6 (13.3%) had an additive and 32 (71.1%) an antagonistic effect. In order to predict binding affinity between the examined antibacterials and the bacterial target molecules, molecular docking was used. The results of substance partition, penetration and accumulation through the cell membrane are given in Table 4. Molecular docking data regarding interactions between the researched antibacterials and appropriate receptors are presented in Table 5. The dominant component of N. nuda essential oil from southeast Serbia is 1,8-cineole, which is in agreement with literature data [6]. The antibacterial activity of N. nuda essential oil and 1,8-cineole remained lower than those of the standard antibiotics. Alim et al. [7] demonstrated that the essential oil of N. nuda exhibited low activity against E. coli and S. aureus, while the oil has no activity against P. aeruginosa. In a study of the antibacterial activity of the

NPC Natural Product Communications 2015 Vol. 10 No. 6

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1064 Natural Product Communications Vol. 10 (6) 2015 Miladinović et al.

Table 1: Composition of the essential oil of N. nuda.

Component AILa AIEb (%) Monoterpene hydrocarbons 8.5 α-Pinene 932 932 1.4 Sabinene 969 972 1.7 β-Pinene 974 978 4.0 Myrcene 988 989 0.8 α-Terpinene 1014 1017 0.1 trans-β-Ocimene 1044 1046 0.2 γ-Terpinene 1054 1058 0.2 Terpinolene 1086 1085 0.1 Oxygenated monoterpenes 57.8 1,8-Cineole 1026 1026 46.0 cis-Sabinene hydrate 1065 1071 0.6 α-Campholenal 1122 1127 0.1 Nopinone 1135 1139 0.1 trans-Pinocarveol 1135 1141 0.2 Citronellal 1148 1152 0.1 α-Phellandren-8-ol 1166 1172 1.4 4-Terpineol 1174 1181 0.5 α-Terpineol 1186 1195 3.4 trans-Dihydro carvone 1200 1201 0.2 trans-Carveol 1215 1219 0.1 Nerol 1227 1226 0.1 Neral 1235 1238 0.1 Geranial 1264 1268 0.1 4aα,7α,7aα-Nepetalactone 1357 1364 4.3 4aα,7β,7aα-Nepetalactone 1391 1397 0.5

Sesquiterpene hydrocarbons 20.4 α-Copaene 1374 1376 0.2 β-Bourbonene 1387 1385 2.4 β-Elemene 1389 1389 0.6 α-Gurjunene 1409 1407 0.1 β-Caryophyllene 1417 1421 4.8 γ-Elemene 1434 1430 0.3 trans-β-Farnesene 1454 1454 2.2 α-Humulene 1452 1456 1.0 Germacrene D 1484 1483 6.8 Bicyclogermacrene 1500 1495 0.2 α-Selinene 1498 1498 0.1 trans- α-Farnesene 1505 1504 0.1 β-Bisabolene 1505 1508 0.7 δ-Cadinene 1522 1518 0.3 β-Sesquiphellandrene 1521 1524 0.6 Oxygenated sesquiterpenes 5.2 Hedycaryol 1546 1552 0.3 Spathulenol 1577 1578 0.4 Caryophyllene oxide 1582 1584 4.0 Ledol 1602 1604 0.2 α-Cadinol 1652 1655 0.3 Phenolic compounds / Others 0.6 trans-2-Hexenal 846 852 0.1 1-Octen-3-ol 974 984 0.5 Total 92.5 a) AIL=Arithmetic index-literature and library data; b) AIE=Arithmetic index experimentally determined on HP-5MS column.

Table 2: Antibacterial activity of N. nuda essential oil, 1,8-cineole and reference antibiotics (μg/mL).

Bacterial species N. nuda 1,8-Cineole Tetracycline Streptomycin

MIC MBC MIC MBC MIC MBC MIC MBC Escherichia coli ATCC 25922 5331.2 21324.8 5900.8 5900.8 128.0 256.0 8.0 8.0 Klebsiella pneumoniae ATCC 700603 5331.2 21324.8 5900.8 5900.8 128.0 256.0 64.0 64.0 Proteus mirabilis ATCC 12453 21324.8 42649.6 5900.8 11801.6 16.0 32.0 16.0 16.0 Pseudomonas aeruginosa ATCC 27853 10662.4 10662.4 94412.8 94412.8 128.0 256.0 16.0 16.0 Staphylococcus aureus ATCC 29213 1332.8 5331.2 94412.8 94412.8 128.0 256.0 4.0 32.0

essential oil of Cinnamomum longepaniculatum and its constituents, the authors found MIC and MBC values for 1,8-cineole of 3.12 L/mL against E. coli and 6.25 L/mL against S. aureus [8]. Comparing the antibacterial properties of the essential oil with those of 1,8-cineole alone confirmed the importance of other constituents contained within the oil, in relation to the MIC/MBC values obtained. It is obvious that the MIC/MBC concentrations of 1,8-cineole were generally greater than those of the oil. Therefore it appears that while 1,8-cineole may be largely responsible for the antimicrobial activity associated with the oil, other minor constituents contribute [9]. Hendry et al. [10] reported similar results for the antimicrobial properties of crude eucalyptus oil and its main component 1,8-cineole. In the present study, the combinations of chosen oil with selected antibiotics exhibited a predominantly antagonistic effect. The eucalyptus essential oil and its main component 1,8-cineole exhibited synergistic activity with chlorhexidine digluconate against E. coli and S. aureus [10]. As already stated, a correlation of the antibacterial activity of the oil and its chemical composition suggests that the activity of the oil could be attributed to the presence of significant concentrations of 1,8-cineole. Therefore, the antibacterial activity of 1,8-cineole individually and in combination with selected antibiotics has been studied also. The antibacterial activity of combinations of N. nuda essential oil-antibiotic are very similar to those for combinations of 1,8-cineole-antibiotic. A similarity was found in the total number of antagonistic interactions. That number is 54 for the combination of N. nuda essential oil-antibiotic, and 52 for 1,8-cineole-antibiotic combinations. In order to explain this situation, molecular docking was used. The pharmacokinetics and pharmacodynamics of tetracycline and streptomycin have been reviewed recently [11]. Bacterial sensitivity to tetracycline and streptomycin involves multi-step active transport of antibiotics across the cell membrane and their cytoplasm

accumulation. These antibiotics are known to bind to a high-affinity binding site in the 30S ribosomal subunit, causing the inhibition of bacterial protein synthesis. Several binding sites have been observed, including the primary sites at 16S rRNA, protein S7 and protein S12 (Table 5). The first concentration-dependent step in the active transport of tetracycline and streptomycin requires binding of their cationic structures to anionic components in the cell membrane. The subsequent steps are energy-dependent and involve the transport of the polar, highly positive charged structures across the cytoplasmic membrane, followed by interaction with the ribosomes [11]. The driving force for this energy-dependent transfer of antibiotics is membrane potential and proton gradient regulated by the cytochrome c oxidase and ATP synthase (Table 5). A large amount of data is available on the mode of antibacterial action of essential oils and their components. The most frequently reported mechanisms are disruption of bacterial membranes, damage of membrane proteins (e.g. enzymes), cell content leakage, depletion of the proton motive force and coagulation of the cytoplasm [12]. One of the most important mechanisms of cell membrane toxicity by organic molecules is to diminish the energy status of the cell. The effects of molecules on the energy transduction of cell membranes were tested with cytochrome c oxidase as a proton motive force generating mechanism. The presence of the solvent tetraline caused a decrease in both the proton gradient and the membrane potential by 80% and 50%, respectively [13]. The decrease in the proton motive force is not the only reason for lower energy levels of cells in the presence of organic compounds. Additionally, the presence of molecules leads to an impaired ATP synthesis, due to a partial inhibition of the ATP synthase activity and other proteins engaged in the energy transducing process [15]. Also, the high concentration of organic molecules in the cell membrane could produce a passive flux of ions, leading to dissipation of the proton gradient, membrane potential and proton motive force [13].

Nepeta nuda essential oil Natural Product Communications Vol. 10 (6) 2015 1065

Table 3: Antibacterial activity of N. nuda oil-antibiotic and 1,8-cineole-antibiotic combinations, represented by FIC and FICA indices of examined substances.

Bacterial species FICA indices of N. nuda oil and 1,8-cineole

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90

E. coli ATCC 25922

1* 0.21 0.32 0.43 0.54 0.65 0.76 0.87 0.98 1.09 2* 1.20 1.40 1.60 1.80 2.00 2.20 2.06 2.06 2.42 3* 0.21 0.32 0.43 0.54 0.65 0.76 0.87 0.98 1.09 4* 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

K. pneumoniae ATCC 700603

1* 1.09 1.40 1.47 1.80 2.00 1.56 1.55 1.16 1.47 2* 0.32 0.44 0.43 0.68 0.65 0.76 0.87 0.98 1.09 3* 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 4* 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

P. mirabilis ATCC 12453

1* 1.09 1.04 0.95 1.24 0.95 1.08 1.04 1.16 1.47 2* 0.43 0.44 0.69 0.68 0.65 0.76 0.87 0.98 1.09 3* 0.87 0.92 1.08 1.24 1.85 1.40 1.55 1.70 2.04 4* 1.20 1.40 1.47 1.52 1.55 1.72 1.55 1.70 2.04

P. aeruginosa ATCC 27853

1* 1.20 1.40 1.60 1.80 1.70 1.56 1.04 1.16 2.23 2* 1.20 1.40 1.60 1.80 1.70 1.40 1.04 1.16 1.85 3* 0.65 0.68 0.82 0.82 0.80 0.92 1.04 1.16 1.47 4* 1.20 1.04 0.82 0.82 0.80 0.92 1.04 1.16 1.47

S. aureus ATCC 29213

1* 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 2* 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3* 1.20 1.40 0.95 0.96 0.95 0.92 1.04 1.16 1.09 4* 0.87 1.04 0.69 0.82 0.80 0.92 1.04 1.16 1.47

* Examined combinations: oil-tetracycline (1*); oil-streptomycin (2*); 1,8-cineole-tetracycline (3*); 1,8-cineole-streptomycin (4*).

Table 4: Parameters of antibacterial partition, penetration and accumulation throughout cell membrane.

Antibacterials log Po/w

a

(mM) log Pm/b

b

(mM) Sc

(mg/L) MMCd (mM)

Tetracycline -2.0 -2.6 2.3×102 607.5×10-3 Streptomycin -8.0 -8.4 1×106 4.0×10-3 1,8-Cineole 2.5 1.8 3.3×102 20.5×103

a) log Po/w=Partition coefficient octanol/water provided by (http://pubchem.ncbi.nlm.nih.gov/); b) log Po/w=Partition coefficient membrane/buffer calculated according to Sikkema et al. [13]; c) S=Solubility in water at 25°C provided by (http://pubchem.ncbi.nlm.nih.gov/); d) MMC=Maximum membrane concentration calculated according to Neumann et al. [14].

Table 5: Calculated ligand-receptor interaction energies (kcal/mol) of the first ranked Autodock Vina pose.

Receptor PDB entry Tetracycline Streptomycin 1,8-Cineole

Ribosome 30S

16S rRNAa 1HNW -6.8 -6.1 -3.5 Protein S7 1HUS -6.0 -5.9 -4.2 16S rRNAb 1FJG -7.1 -7.0 -4.1 Protein S12 2OM7 -6.6 -6.1 -4.4

Cell membrane Cytochrome c oxidase 1QLE -5.0 -4.1 -6.1 ATP synthase 1C17 -5.2 -4.2 -6.1

a) 16S rRNA moiety coordinated with tetracycline; b) 16S rRNA moiety coordinated with streptomycin.

To improve the efficacy of antibiotics, it is necessary to find methods of improving their diffusion across bacterial membranes and/or to hinder the efflux pumps that are a general resistance mechanism in Gram-negative bacteria [16]. Although the site of action of individual essential oil components has been established in many cases, the mechanism is still not completely understood; filling these gaps in scientific knowledge will assist in finding synergistic combinations and avoiding antagonistic ones [12]. In the present study, we investigated the combining effects of N. nuda oil and 1,8-cineole with ribosome 30S inhibitors, in order to clarify antagonistic behavior in some antibiotic-essential oil or antibiotic-substance combinations [12]. It was determined that the cell membrane concentration of 1,8-cineole was a minimum of 3.4×104 times greater than the concentration of used antibiotics (Table 4). Also the results showed that the binding energy of 1,8-cineole to ribosome 30S receptors was much lower compared with antibiotics (Table 5). These computational results, combined with experimental data (Table 3), suggest that antagonistic interactions in examining systems were not a consequence of the antimicrobial action at the same target [12], but an outcome of membrane potential/proton motive force dissipation [13]. This hypothesis was confirmed by the high binding energies of 1,8-cineole to cytochrome c oxidase and ATP synthase (Table 5). The binding affinity of 1,8-cineole to cytochrome c oxidase (Table 5) was comparable with the calculated affinity (-7.2 kcal/mol) of the specialized cytochrome c oxidase inhibitor-antimycin [17]. Having

in mind that ATP-energy is not quite enough for streptomycin transport to ribosomes [11], and that ATP-synthesis is directly driven by the respiratory chain, we assume that cytochrome c oxidase would be the preferably target of 1,8-cineole. This hypothesis was also confirmed by Vina score results of 1,8-cineole to ATP synthase (Table 5), which was much lower compared with a calculated score of ATP synthase inhibitor-oligomycin (-14.8 kcal/mol). Finally, in the present study, the chemical composition and antibacterial activity of N. nuda essential oil were examined, as well as the association between it and standard antibiotics: tetracycline and streptomycin. The antibacterial activities of 1,8-cineole, the main constituent of N. nuda oil, individually and in combination with standard antibiotics were also determined. It was shown that oxygenated monoterpenes, with 1,8-cineole as the dominant constituent, were the most abundant compound class of the essential oil of N. nuda from southeast Serbia. The researched essential oil exhibited in vitro antibacterial activity against all the tested bacterial strains, but the activities were lower than those of the standard antibiotics. The combinations N. nuda oil-antibiotic and 1,8-cineole-antibiotic produced predominantly antagonistic interactions. The chemoinformatics survey confirms the antagonistic interactions as a consequence of membrane potential/proton motive force dissipation. These data indicate cytochrome c oxidase as a target for 1,8-cineole toxicity action mechanisms.

1066 Natural Product Communications Vol. 10 (6) 2015 Miladinović et al.

Experimental

Plant material and chemicals: The aerial parts of Nepeta nuda were collected in August 2011 from natural populations at the Suva planina mountain, Southeast Serbia. A voucher specimen, with the accession number 16524, is deposited at the Herbarium of the Department of Botany, Faculty of Biology, University of Belgrade-Herbarium Code BEOU. All chemicals, reagents and standards were of analytical reagent grade and were purchased from the Sigma-Aldrich Chemical Company. Extraction and isolation of oil: The aerial parts of the plant (dried and ground) were subjected to hydro-distillation for 4 h, using a Clevenger-type apparatus to obtain the oil. The resulting essential oil was dried over anhydrous sodium sulfate and stored at 4°C. Gas chromatography, gas chromatography/mass spectrometry and identification of compounds: The GC, GC-MS analyses and identification of oil compounds were performed as previously described [1]. Antibacterial testing: The activity of the essential oil samples was tested towards 5 different bacteria: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus

aureus ATCC 29213 using the micro-well dilution assay and microdilution checkerboard assay as previously described [1]. Chemoinformatics methods: Docking calculations were carried out using AutoDock Vina [18]. Vina was used to dock tetracycline, streptomycin and 1,8-cineole against appropriate PDB receptor files (Table 5). The structures of the studied antibacterials were taken from their respective SDF files (http://pubchem.ncbi.nlm.nih.gov/) and the initial PDB structures were generated using Open Babel [19]. AutoDockTools (ADT) was used to prepare the formatted ligands/receptors PDBQT-structures and to determine the "search space". Docking was carried out by Command Prompt commands, giving a Vina score, i.e., calculated ligand-receptor interaction energies (kcal/mol) of the first ranked Autodock Vina pose. A more negative score indicates that antibiotic or 1,8-cineole (ligand) is more likely to dock with the structure (receptor) and achieve more favorable interactions. Maximum membrane concentration of the studied antibacterials was calculated according to Neumann et al. [14]. Acknowledgements - This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. OI 171025 and TR 31079).

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Natural Product Communications Vol. 10 (6) 2015 Published online (www.naturalproduct.us) A New Aromatic Compound from the Stem Bark of Terminalia catappa David Pertuit, Anne-Claire Mitaine-Offer, Tomofumi Miyamoto, Chiaki Tanaka, Stéphanie Delemasure, Patrick Dutartre and Marie-Aleth Lacaille-Dubois 1005

Effect of Non-psychotropic Plant-derived Cannabinoids on Bladder Contractility: Focus on Cannabigerol Ester Pagano, Vittorino Montanaro, Antonio di Girolamo, Antonio Pistone, Vincenzo Altieri, Jordan K. Zjawiony, Angelo A. Izzo and Raffaele Capasso 1009

In Cell Interactome of Oleocanthal, an Extra Virgin Olive Oil Bioactive Component Chiara Cassiano, Agostino Casapullo, Alessandra Tosco, Maria Chiara Monti and Raffaele Riccio 1013

Synthesis of -Viniferin Glycosides by Glucosyltransferase from Phytolacca americana and their Inhibitory Activity on Histamine Release from Rat Peritoneal Mast Cells

Hiroki Hamada, Hatsuyuki Hamada and Kei Shimoda 1017

Stability of the Ellagitannin Fraction and Antioxidant Capacity of Varietal Pomegranate Juices Pedro Mena and Cristina García-Viguera 1019

Phthalide Anions in Organic Synthesis. A Direct Total Synthesis of Furomollugin George A. Kraus and Pengfei Dong 1025

Absolute Configuration Assignment of 3',4'-di-O-acylkhellactones Using Vibrational Circular Dichroism Exciton Chirality Abigail I. Buendía-Trujillo, J. Martín Torrres-Valencia, Pedro Joseph-Nathan and Eleuterio Burgueño-Tapia 1027

Antifouling Compounds from the Marine-Derived Fungus Aspergillus terreus SCSGAF0162 Xu-Hua Nong, Xiao-Yong Zhang, Xin-Ya Xu and Shu-Hua Qi 1033

Goji Berry: Quality Assessment and Crop Adaptation of Plants Cultivated in Tuscany (Italy) by Combination of Carotenoid and DNA Analyses Giada Capecchi, Emanuele Goti, Elena Nicolai,Maria Camilla Bergonzi, Roberto Monnanni and Anna Rita Bilia 1035

Activity of Vitis vinifera Tendrils Extract Against Phytopathogenic Fungi Daniele Fraternale, Donata Ricci, Giancarlo Verardo, Andrea Gorassini, Vilberto Stocchi and Piero Sestili 1037

Long-chain Glucosinolates from Arabis turrita: Enzymatic and Non-enzymatic Degradations Ivica Blažević, Sabine Montaut, Gina Rosalinda De Nicola and Patrick Rollin 1043

Aroma of Turmeric: Dependence on the Combination of Groups of Several Odor Constituents Toshio Hasegawa, Kenta Nakatani, Takashi Fujihara and Hideo Yamada 1047

Terpenoids Preserved in Fossils from Miocene-aged Japanese Conifer Wood Agnieszka Ludwiczuk and Yoshinori Asakawa 1051

Can Ozone Alter the Terpenoid Composition and Membrane Integrity of in vitro Melissa officinalis Shoots? Francesca D’Angiolillo, Mariagrazia Tonelli, Elisa Pellegrini, Cristina Nali, Giacomo Lorenzini, Luisa Pistelli and Laura Pistelli 1055

Composition and Chemical Variability of Ivoirian Xylopia staudtii Leaf Oil Thierry Acafou Yapi, Jean Brice Boti, Antoine Coffy Ahibo, Sylvain Sutour, Ange Bighelli, Joseph Casanova and Félix Tomi 1059

Chemoinformatics Approach to Antibacterial Studies of Essential Oils Dragoljub L. Miladinović, Budimir S. Ilić and Branislava D. Kocić 1063

Chemical Composition of Nardostachys grandiflora Rhizome Oil from Nepal – A Contribution to the Chemotaxonomy and Bioactivity of Nardostachys Prabodh Satyal, Bhuwan K. Chhetri, Noura S. Dosoky, Ambika Poudel and William N. Setzer 1067

Chemical Composition and Biological Activity of Essential Oils from Wild Growing Aromatic Plant Species of Skimmia laureola and Juniperus macropoda from Western Himalaya Iris Stappen, Nurhayat Tabanca, Abbas Ali, David E. Wedge, Jürgen Wanner, Vijay K. Kaul, Brij Lal, Vikas Jaitak, Velizar K. Gochev, Erich Schmidt and Leopold Jirovetz 1071

Comparative Chemical Composition and Antioxidant Properties of the Essential Oils of three Sideritis libanotica Subspecies Carmen Formisano, Filomena Oliviero, Daniela Rigano, Nelly Apostolides Arnold and Felice Senatore 1075

Asplenioideae Species as a Reservoir of Volatile Organic Compounds with Potential Therapeutic Properties Didier Froissard, Sylvie Rapior, Jean-Marie Bessière, Bruno Buatois, Alain Fruchier, Vincent Sol and Françoise Fons 1079

Composition and Comprehensive Antioxidant Activity of Ginger (Zingiber officinale) Essential Oil from Ecuador Martina Höferl, Ivanka Stoilova, Juergen Wanner, Erich Schmidt, Leopold Jirovetz, Dora Trifonova, Veselin Stanchev and Albert Krastanov 1085

Chemical Components of Four Essential Oils in Aromatherapy Recipe Sarin Tadtong, Narisa Kamkaen, Rith Watthanachaiyingcharoen and Nijsiri Ruangrungsi 1091

Accounts/Reivews Recent Advances in the Synthesis of Stemona Alkaloids Xiao-Yu Liu and Feng-Peng Wang 1093

Flavonoid Properties in Plant Families Synthesizing Betalain Pigments (Review) Tsukasa Iwashina 1103

Phytochemistry and Pharmacology of the Genus Tovomita Francesco Epifano, Maria Carmela Specchiulli, Vito Alessandro Taddeo, Serena Fiorito and Salvatore Genovese 1115

Fungal Phytotoxins with Potential Herbicidal Activity to Control Chenopodium album Alessio Cimmino, Marco Masi, Marco Evidente and Antonio Evidente 1119

Essential Oils as “A Cry for Help”. A Review Christine Zitzelsberger and Gerhard Buchbauer 1127

Natural Product Communications Vol. 10 (6) 2015 Published online (www.naturalproduct.us)

Anti-Acetylcholinesterase Alkaloids from Annona glabra Leaf Shoei-Sheng Lee, Dong-Yi Wu, Sheng-Fa Tsai, and Chien-Kuang Chen 891

Increased Oxidative Stress in Cultured 3T3-L1 Cells was Attenuated by Berberine Treatment Shi-fen Dong, Naomi Yasui, Hiroko Negishi, Aya Kishimoto, Jian-ning Sun and Katsumi Ikeda 895

Synthesis and Antimicrobial Activities of 3-Methyl-β-Carboline Derivatives Jiwen Zhang, Longbo Li, Wenjia Dan, Jian Li, Qianliang Zhang, Hongjin Bai and Junru Wang 899

A Novel One-step Synthesis of Quinoline-2(1H)-thiones and Selones by Treating 3-Aryl-3-(2-aminophenyl)-1-propyn-3-ols with a Base and Elemental Sulfur or Selenium Kazuaki Shimada, Hironori Izumi, Koki Otashiro, Kensuke Noro, Shigenobu Aoyagi, Yuji Takikawa and Toshinobu Korenaga 903

Normonanchocidins A, B and D, New Pentacyclic Guanidine Alkaloids from the Far-Eastern Marine Sponge Monanchora pulchra Ksenya M. Tabakmakher, Tatyana N. Makarieva, Vladimir A. Denisenko, Alla G. Guzii, Pavel S. Dmitrenok, Aleksandra S. Kuzmich and Valentin A. Stonik 913

Computational and Investigative Study of Flavonoids Active Against Trypanosoma cruzi and Leishmania spp Frederico F. Ribeiro, Francisco J.B.M. Junior, Marcelo S. da Silva, Marcus Tullius Scotti and Luciana Scotti 917

Two New Secondary Metabolites from Tephrosia purpurea Yin-Ning Chen, Yan Peng, Cheng-Hai Gao, Tao Yan, Zhi-Fang Xu, Samuel X. Qiu, Wen-Hao Cao, Ligao Deng and Ri-Ming Huang 921

Regioselective Glycosylation of 3-, 5-, 6-, and 7-Hydroxyflavones by Cultured Plant Cells Kei Shimoda, Naoji Kubota, Daisuke Uesugi, Yuuya Fujitaka, Shouta Okada, Masato Tanigawa and Hiroki Hamada 923

Unusual Flavonoid Glycosides from the Hawaiian Tree Metrosideros polymorpha Benjamin R. Clark, Swapan Pramanick, Norman Arancon and Robert P. Borris 925

Anti-inflammatory Flavonoids Isolated from Passiflora foetida Thi Yen Nguyen, Dao Cuong To, Manh Hung Tran, Joo Sang Lee, Jeong Hyung Lee, Jeong Ah Kim, Mi Hee Woo and Byung Sun Min 929

Clovamide and Flavonoids from Leaves of Trifolium pratense and T. pratense subsp. nivale Grown in Italy Aldo Tava, Łukasz Pecio, Anna Stochmal and Luciano Pecetti 933

Water Extract of Mentha × villosa: Phenolic Fingerprint and Effect on Ischemia-Reperfusion Injury Silvia Fialova, Lucia Veizerova, Viera Nosalova, Katarina Drabikova, Daniela Tekelova, Daniel Grancai and Ruzena Sotnikova 937

Distribution and Taxonomic Significance of Secondary Metabolites Occurring in the Methanol Extracts of the Stonecrops (Sedum L., Crassulaceae) from the Central Balkan Peninsula Gordana S. Stojanović, Snežana Č. Jovanović and Bojan K. Zlatković 941

In vitro Xanthine Oxidase Inhibitory Studies of Lippia nodiflora and Isolated Flavonoids and Phenylethanoid Glycosides as Potential Uric Acid-lowering Agents Lee-Chuen Cheng, Vikneswaran Murugaiyah and Kit-Lam Chan 945

Enzymatic Synthesis of Quercetin Monoglucopyranoside and Maltooligosaccharides Ryo Yasukawa, Natsumi Moriwaki, Daisuke Uesugi, Fuya Kaneko, Hiroki Hamada and Shin-ichi Ozaki 949

Polyurethane Microstructures-a Good or Bad in vitro Partner for the Isoflavone Genistein? Corina Danciu, Florin Borcan, Codruta Soica, Istvan Zupko, Erzsébet Csányi, Rita Ambrus, Delia Muntean, Camelia Sass, Diana Antal, Claudia Toma and Cristina Dehelean 951

Chemical Constituents of the Underground Parts of Iris florentina and their Cytotoxic Activity Akihito Yokosuka, Yoshikazu Koyama and Yoshihiro Mimaki 955

Synthesis of Arecatannin A1 from Dimeric Epicatechin Electrophile Manato Suda, Kohki Takanashi, Miyuki Katoh, Kiriko Matsumoto, Koichiro Kawaguchi, Sei-ichi Kawahara, Hiroshi Fujii and Hidefumi Makabe 959

Anthocyanin Profile and Antioxidant Activity of Various Berries Cultivated in Korea Hong-Sook Bae, Hyun Ju Kim, Jin Hee Kang, Rika Kudo, Takahiro Hosoya, Shigenori Kumazawa, Mira Jun, Oh-Yoen Kim and Mok-Ryeon Ahn 963

Metabolite Fingerprinting of Eugenia jambolana Fruit Pulp Extracts using NMR, HPLC-PDA-MS, GC-MS, MALDI-TOF-MS and ESI-MS/MS Spectrometry Ram Jee Sharma, Ramesh C. Gupta, Arvind Kumar Bansal and Inder Pal Singh 969

Flavonoids and Phenolic Acids in Methanolic Extracts, Infusions and Tinctures from Commercial Samples of Lemon Balm Agnieszka Arceusz, Marek Wesolowski and Beata Ulewicz-Magulska 977

RP-HPLC-DAD-MSn Analysis and Butyrylcholinesterase Inhibitory Activity of Barbacenia blanchetii Extracts Jósquia S Barbosa, Verônica M Almeida, Rosilene M Marçal and Alexsandro Branco 983

Flavonoids and Other Phenolic Compounds in Needles of Pinus peuce and Other Pine Species from the Macedonian Flora Marija Karapandzova, Gjose Stefkov, Ivana Cvetkovikj, Jasmina Petreska Stanoeva, Marina Stefova and Svetlana Kulevanova 987

Anti-inflammatory, Antioxidant and Antimicrobial Activity Characterization and Toxicity Studies of Flowers of “Jarilla”, a Medicinal Shrub from Argentina Alejandra Moreno, Gabriela Nuño, Soledad Cuello, Jorge E. Sayago, María Rosa Alberto, Catiana Zampini and María Inés Isla 991

Synthesis of Resveratrol Glycosides by Plant Glucosyltransferase and Cyclodextrin Glucanotransferase and Their Neuroprotective Activity Kei Shimoda, Naoji Kubota, Hatsuyuki Hamada and Hiroki Hamada 995

Anti-austeritic Constituents of the Congolese Medicinal Plant Aframomum melegueta Dya Fita Dibwe, Suresh Awale, Hiroyuki Morita and Yasuhiro Tezuka 997

The Lignan-containing Extract of Schisandra chinensis Berries Inhibits the Growth of Chlamydia pneumoniae Elina Hakala, Leena L. Hanski, Teijo Yrjönen, Heikki J. Vuorela and Pia M. Vuorela 1001

Natural Product Communications 2015

Volume 10, Number 6

Contents

Editorial i

Preface iii

Original Paper

Chemical and Genetic Diversity of Ligularia hodgsonii in China Chiaki Kuroda, Kou Inagaki, Xun Chao, Kyosuke Inoue, Yasuko Okamoto, Motoo Tori, Xun Gong, and Ryo Hanai 823

Constituents of Ligularia brassicoides Collected in China: A New Diels-Alder Adduct of Eremophilan-10-ol and Methacrylic Acid Mizuho Taniguchi, Katsuyuki Nakashima, Yasuko Okamoto, Xun Gong, Chiaki Kuroda and Motoo Tori 827

Four New Sesquiterpenoids from Ligularia subspicata Collected in China; Isolation of a Bakkane-type Lactone, an Eremophilane-type Lactone, and Two Ortho Esters Yoshinori Saito, Takanori Otsubo, Yuko Iwamoto, Katsuyuki Nakashima, Yasuko Okamoto, Xun Gong, Chiaki Kuroda and Motoo Tori 831

Natural Caryophyllane Sesquiterpenoids from Rumphella antipathies Hsu-Ming Chung, Wei-Hsien Wang, Tsong-Long Hwang, Yang-Chang Wu and Ping-Jyun Sung 835

Bioactive Compounds in Wild, In vitro Obtained, Ex vitro Adapted, and Acclimated Plants of Centaurea davidovii (Asteraceae) Antoaneta Trendafilova, Milka Jadranin, Rossen Gorgorov and Marina Stanilova 839

New Laurene-type Sesquiterpene from Bornean Laurencia nangii Takashi Kamada and Charles Santhanaraju Vairappan 843

New Furanone and Sesquiterpene from the Pericarp of Calocedrus formosana Tzong-Huei Lee, Ming-Shian Lee, Horng-Huey Ko, Jih-Jung Chen, Hsun-Shuo Chang, Mei-Hwei Tseng, Sheng-Yang Wang, Chien-Chih Chen and Yueh-Hsiung Kuo 845

The Importance of the 5-Alkyl Substituent for the Violet Smell of Ionones: Synthesis of Racemic 5-Demethyl--ionone Serena Chierici, Serena Bugoni, Alessio Porta, Giuseppe Zanoni and Giovanni Vidari 847

Antiproliferative Activity of seco-Oxacassanes from Acacia schaffneri J. Martín Torres-Valencia, Virginia Motilva, J. Jesús Manríquez-Torres, Sofía García-Mauriño, Miguel López-Lázaro, Hanaa Zbakh, José M. Calderón-Montaño, Mario A. Gómez-Hurtado, Juan A. Gayosso-De-Lucio, Carlos M. Cerda-García-Rojas and Pedro Joseph-Nathan 853

neo-Clerodane Diterpenoids from Ajuga macrosperma var. breviflora Amaya Castro, Josep Coll, Anil K. Pant and Om Pakrash 857

Three New C20-Diterpenoid Alkaloids from Aconitum tanguticum var. trichocarpum Zhong-Tang Zhang, Xiao-Yu Liu, Dong-Lin Chen, and Feng-Peng Wang 861

Manoalide-related Sesterterpene from the Marine Sponge Luffariella variabilis Toshiyuki Hamada, Daisuke Harada, Mitsunobu Hirata, Keisuke Yamashita, Kishneth Palaniveloo, Hiroaki Okamura, Tetsuo Iwagawa, Naomichi Arima, Toshiyuki Iriguchi, Nicole J. de Voogd and Charles S. Vairappan 863

Oxygenated Terpenes from Indo-Pacific Nudibranchs: Scalarane Sesterterpenes from Glossodoris hikuerensis and 12-Acetoxy Dendrillolide A from Goniobranchus albonares I. Wayan Mudianta, Andrew M. White and Mary J. Garson 865

Germinating Seeds of Citrus aurantium a Good Source of Bioactive Limonoids Marta R. Ariza, M. Mar Herrador del Pino and Alejandro F. Barrero 869

Chemical Constituents of Lecythis pisonis (Lecythidaceae) – A New Saponin and Complete 1H and 13C Chemical Shift Assignments Rennê C. Duarte, Carlos R. R. Matos, Raimundo Braz-Filho and Leda Mathias 871

Oleanane-type Triterpene Saponins from Glochidion glomerulatum Vu Kim Thu, Nguyen Van Thang, Nguyen Xuan Nhiem, Hoang Le Tuan Anh, Pham Hai Yen, Chau Van Minh, Phan Van Kiem, NanYoung Kim, Seon Ju Park and Seung Hyun Kim 875

Cucumarioside E from the Far Eastern Sea Cucumber Cucumaria japonica (Cucumariidae, Dendrochirotida), New Minor Monosulfated Holostane Triterpene Pentaoside with Glucose as the Second Monosaccharide Residue Alexandra S. Silchenko, Anatoly I.Kalinovsky, Pavel S. Dmitrenok, Vladimir I. Kalinin, Andrey N. Mazeika, Natalia S. Vorobieva, Nina M. Sanina and Edward Y. Kostetsky 877

Structure Revision of Two Polyoxygenated Sterols from the Marine Sponge Neofibularia nolitangere Yasunori Yaoita, Masao Kikuchi and Koichi Machida 881

Ergosterol of Cordyceps militaris Attenuates LPS Induced Inflammation in BV2 Microglia Cells Neeranjini Nallathamby, Lee Guan-Serm, Sharmili Vidyadaran, Sri Nurestri Abd Malek, Jegadeesh Raman and Vikineswary Sabaratnam 885

Two Novel Spirostene Glycosides from Selaginella chrysocaulos and their Chemotaxonomic Significance Olaf Kunert, Rumalla Chidananda Swamy, Bobbala Ravi Kumar, Achanta Venkata Narasimha Appa Rao, Owi Ivar Nandi and Wolfgang Schuehly 887

Continued inside backcover