Lipophilicity Determination of Highly Lipophilic Compounds by Liquid Chromatography

Different experimental strategies using short columns in both conventional liquid chromatography (HPLC) and ultra‐high pressure liquid chromatography (UHPLC) were evaluated to allow, for the first time with these techniques, the lipophilicity determination of compounds with log P>5. Various organic modifiers, stationary phases, and elution modes were tested on 14 rigid compounds with a CLogP between 5 and 8, and 38 compounds with log P_oct from 0 to 5. The best results in HPLC were obtained with the 20‐mm Discovery ^® RP Amide C16 stationary phase in isocratic mode using MeOH as organic modifier. To improve analysis time, the UHPLC approach was then evaluated. Consequently, a generic method was developed with a 30‐mm Acquity BEH Shield RP18 column in gradient mode using MeOH as organic modifier, allowing a fourfold gain of time compared to the HPLC method, for the highly lipophilic compounds tested. Finally, the most rapid and accurate results were obtained with a 10‐mm Hypersil^TM GOLD Javelin HTS stationary phase in UHPLC, enabling an eightfold gain of time compared to the HPLC method.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

珊瑚来源真菌Parengyodontium album SCSIO SX7W11中聚酮化合物的发现及其生物合成途径分析

海洋来源真菌的天然产物因其独特的结构与生物学活性而备受关注,而利用基因组信息对其代谢产物进行深入挖掘也成为研究策略之一。[目的] 本文以一株南海珊瑚来源的真菌Parengyodontium album SCSIO SX7W11为目标菌株,挖掘其生产聚酮类化合物的潜能。[方法] 本研究利用Illumina Miseq技术对SX7W11菌株进行全基因组扫描测序,运用生物信息学手段对其基因组的生物合成基因簇进行预测和基因功能注释,挖掘可能产生新颖聚酮化合物的基因簇。对SX7W11进行放大发酵后,利用正相色谱、中压反相色谱、Sephadex LH-20凝胶色谱、HPLC半制备等分离手段分离纯化出单体化合物。再利用高分辨质谱（HR-ESI-MS）、¹H NMR、¹³C NMR、X-ray单晶衍射等波谱手段确定化合物的结构,并根据生物合成基因簇对化合物的生物合成途径进行推导。[结果] 全基因组扫描测序结果显示,P.album SCSIO SX7W11基因组大小为34.0 Mb,含有24个生物合成基因簇,包括6个聚酮合酶基因簇以及3个萜烯合酶基因簇。从发酵产物中分离鉴定到3个聚酮类化合物：emodin（1）、alternaphenol B（2）和sydowinin A（3）,其中化合物3获得了单晶结构数据。通过生物信息学方法从菌株基因组中定位到了sydowinin A的生物合成基因簇。结合文献对emodin（1）、alternaphenol B（2）和sydowinin A（3）的生物合成途径进行了分析。[结论] 本研究通过基因组挖掘及培养基优化,发现1株珊瑚来源的真菌P.album SCSIO SX7W11具有生产sydowinins类聚酮类化合物的能力,为该类化合物生物合成机制深入研究奠定了基础。     

High‐performance liquid chromatography study of gatifloxacin and sparfloxacin using erythrosine as post‐column resonance Rayleigh scattering reagent and mechanism study

Herein, a highly selective high‐performance liquid chromatography (HPLC) coupled with resonance Rayleigh scattering (RRS) method was developed to detect gatifloxacin (GFLX) and sparfloxacin (SPLX). GFLX and SPLX were first separated by HPLC, then, in pH 4.4 Britton–Robinson (BR) buffer medium, protonic quaternary ammonia cation of GFLX and SPLX reacted with erythrosine (ERY) to form 1:1 ion‐association complexes, which resulted in a significant enhancement of RRS signal. The experimental conditions of HPLC and post‐column RRS have been investigated, including detection wavelength, flow rate, pH, reacting tube length and reaction temperature. Reaction mechanism were studied in detail by calculating the distribution fraction. The maximum RRS signals for GFLX and SPLX were recorded at λ_ex = λ_em = 330 nm. The detection limits were 3.8 ng ml^?1 for GFLX and 17.5 ng ml^?1 for SPLX at a signal‐to‐noise ratio of 3. The developed method was successfully applied to the determination of GFLX and SPLX in water samples. Recoveries from spiked water samples were 97.56–98.85%.     

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography

Introduction. Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. Objective. To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). Methodology. The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative‐scale separation of lancemasides by CPC using n‐hexane:n‐butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two‐phase solvent system. The upper phase (organic phase) of the two‐phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. Results. The demonstrated separation sequence, hot water extraction, DIAION HP‐20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. Conclusion. The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.     

Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin‐layer chromatography

Introduction – Asparagus officinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin‐layer chromatography or high‐performance liquid chromatography (HPLC‐UV or HPLC‐ELSD), which is time‐consuming and expensive, so we need to find a rapid solution to this problem. Objective – To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantification of diosgenin and sarsasapogenin. Methodology – Samples were prepared by extraction of A. officinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 m hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were verified by HPLC and HPLC‐MS. Results – The retention factor (R_f) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. officinalis extract were 0.27–0.46 and 0.11–0.32%, respectively. Conclusion – The study showed that thin‐layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. officinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of Secondary Metabolites in Flowers of Sanguisorba officinalis L. by HPLC‐DAD‐MSn and GC/MS

The investigations reported here focus on an in‐depth characterization of the secondary metabolite profile of Sanguisorba officinalis flowers. For this purpose, fresh flowers were extracted with MeOH/H₂O and EtOH/H₂O and the resulting crude extracts fractionated using CH₂Cl₂, AcOEt, and BuOH. Individual compounds were characterized by high performance liquid chromatography and gas chromatography coupled with mass spectrometric detection (HPLC‐DAD‐MSⁿ and GC/MS). MeOH/H₂O extraction and LC/MSⁿ investigations revealed the occurrence of flavonoid glycosides (quercetin, kaempferol), ellagitannin glycosides and four anthocyanins. Among the latter, two components, i. e., cyanidin‐malonyl‐glucose and cyanidin‐galloyl‐hexose, have not been reported for S. officinalis so far. Furthermore, phenylethylamine was characterized for the first time in Sanguisorba by pH value dependent extraction with CH₂Cl₂. In addition, AcOEt and BuOH extracts were analyzed by GC/MS both prior to and after acid hydrolysis of secondary metabolites. For this purpose, the extracts were treated with 1 n HCl solution (105 °C, 1 h) and derivatized with BSTFA. Analyses revealed the occurrence of several classes of phenolic compounds, such as gallic acid, hydroxybenzoic acid, hydroxycinnamic acid and ellagic acid derivatives. Additionally, the most prominent ursane‐type triterpenoid (ziyu‐glycoside I) from Sanguisorba and its corresponding aglycone isomers were detected and assigned based on their characteristic fragmentation patterns.     

Microbial transformation of polydatin and emodin-8-β-d-glucoside of Polygonum cuspidatum Sieb. et Zucc into resveratrol and emodin respectively by Rhizopus microsporus

Rhizopus microsporus isolated by our laboratory was able to transform polydatin into resveratrol and emodin-8-β-d-glucoside into emodin, respectively, through the fermentation of Polygonum cuspidatum Sieb. et Zucc. The fermentation products were separated and purified by H1020 resin and silica gel column chromatography. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were used to identify the products and evaluate the transformation efficiency. A variety of parameters of submerged state fermentation, including the growth characteristics, the change of β-glucosidase activity and the amount of polydatin, resveratrol, emodin-8-β-d-glucoside, emodin, and the dissolved oxygen, were monitored simultaneously. The amount of resveratrol yielded increased dramatically from 0.04 g/l at the beginning to the maximum value of 0.34 g/l at 36 h of fermentation, and emodin was from 0.4 g/l to 0.65 g/l at 80 h. The transformation rate of glycosides reached 98% and the purity of both resveratrol and emodin was 95%.     

Inducing and isolation of antibacterial peptides from oriental fruit fly, Bactrocera dorsalis Hendel

One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion‐exchange chromatography, gel filtration chromatography and reverse‐phase high performance liquid chromatography (HPLC). The final purified fraction was checked on the Smart system HPLC and was judged as a pure fraction. The results of physical and biological analysis revealed that this fraction is heat stable and showed strong activities against Gram‐positive bacterial growth. It possesses antibicrobial peptide properties and is worth further investigation.     

Folium Sennae and emodin reverse airway smooth muscle contraction

The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High‐performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine‐evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L‐type voltage‐dependent Ca²⁺ channels, LVDCCs), YM‐58483 (a selective inhibitor of store‐operated Ca²⁺ entry, SOCE), as well as Y‐27632 (an inhibitor of Rho‐associated protein kinase). In addition, LVDCC‐ and SOCE‐mediated currents and cytosolic Ca²⁺ elevations were inhibited by emodin. Emodin reversed acetylcholine‐caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine‐induced respiratory system resistance in mice. These results indicate that EEFS‐induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca²⁺ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.     

Volatile compounds from the symbiotic system Azolla filiculoides‐Anabaena azollae bacteria

Abstract

Azolla filiculoides is an aquatic pteridophyte that may be used as animal food, biofertilizer and phytoremediation. Its volatile composition was never studied although several phytochemical analyses were performed. The volatile composition of A. filiculoides grown outdoors in a pond at the Botanical Garden of Lisbon University (BGLU) or in culture conditions as well as the effect of different harvesting times and the storage type were evaluated. The volatiles isolated by hydrodistillation and distillation‐extraction were analysed by gas chromatography and gas chromatography‐mass spectrometry. The oil of all the A. filiculoides samples studied affords a yellowish colour and an unpleasant odour in a yield of 0.01% (v/fw). Alcohols, aldehydes, alkanes and ketones dominated the culture samples, while aldehydes, alcohols, terpenoids and alkanes represented the main volatiles of the BGLU samples. Some quantitative differences were detected in seasonal and type of storage (fresh, dry or frozen at ?20°C) studies of A. filiculoides from the BGLU. The BGLU and culture volatiles showed qualitative differences: 2‐ethyl‐1‐hexanol was only identified in the fern culture, whereas acetophenone, pentylfuran, acetylpyridine and 2‐octanone were only detected in BGLU samples. The dendrogram showed two distinct clusters (culture and BGLU samples). The possible biological origin and bioactivity of some of the volatile compounds is discussed.

Abbreviations: FID, flame ionization detector; i.d., internal diameter; v/fw, volume by fresh weight; GC, gas chromatography; GC‐MS, gas chromatography‐mass spectrometry; u, unified atomic mass unit     

Detection of Xeljanz Enantiomers in Diethyl Amine Active Pharmaceutical Ingredients and Tablets

A high‐performance liquid chromatography (HPLC) method was established to detect Xeljanz enantiomers in active pharmaceutical ingredients (APIs) and tablets. The separation was achieved on a Chiralpak IC column using a mobile phase of hexane‐ethanol‐diethylamine (65:35:0.1, v/v). The detection wavelength was 289 nm. The peak areas and the enantiomer concentrations in the range of 0.15–2.25 μg?mL^?1 were in high linearity, with correlation coefficients higher than 0.999. The recoveries were 86.44% at the concentrations of 7.5, 18.75, and 37.5 μg?mL^?1. The limit of detection (LOD) and limit of quantification (LOQ) were 0.042 and 0.14 μg?mL^?1, respectively. This HPLC method is suitable for detecting the enantiomers of Xeljanz in its APIs and tablets. Chirality 27:235–238, 2015. © 2014 Wiley Periodicals, Inc.     

NMR,HS‐SPME‐GC/MS,and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by ¹H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSⁿ). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. ¹H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSⁿ allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.     

Dynamic Behavior of Clobazam on High‐Performance Liquid Chromatography Chiral Stationary Phases

Clobazam, a 1,5‐benzodiazepin‐2,4‐dione, is a chiral molecule because its ground state conformation features a nonplanar seven‐membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring‐flipping process. Variable temperature HPLC on the Pirkle type (R)‐N‐(3,5‐dinitronenzoyl)phenylglycine and (R,R)‐Whelk‐O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence‐decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high‐performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on‐column enantiomer interconversion. (enantiomerization) in the column temperature range between T_col = 10°C and T_col = 30°C, whereas on‐column interconversion was absent at temperature close to or lower than T_col = 5°C. Computer simulation of exchange‐deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on‐column enantiomerization and the corresponding free energy activation barriers. At T_col = 20°C the averaged enantiomerization barriers, ΔG^?, for clobazam were found in the range 21.08–21.53 kcal mol^?1 on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents. Chirality 28:17–21, 2016. © 2015 Wiley Periodicals, Inc.     

Emodin O-methyltransferase from Aspergillus terreus

Emodin O-methyltransferase, an enzyme catalyzing methylation of the 8-hydroxy group of emodin, was identified in the mould Aspergillus terreus IMI 16043, a (+)-geodin producing strain. The enzyme catalyzed the formation of questin from emodin and S-adenosyl-l-methionine. By chromatography on DEAE-cellulose, Phenyl Sepharose, Q-Sepharose, Hydroxyapatite, and CM-cellulose, emodin O-methyltransferase was purified to apparent homogeneity. The purified protein had a molecular weight of 322 kDa as estimated by gel filtration and 53.6 kDa as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme was a homohexamer. The enzyme showed pI 4.4 and optimum pH 7–8. Magnesium ion or manganese ion was not an absolute requirement, nor increased the enzyme activity. The enzyme had strict substrate specificity and very low Km values for both emodin (3.4×10^-7 M) and S-adenosyl-l-methionine (4.1×10^-6 M).Abbreviations EOMT emodin O-methyltransferase from A. terreus - SAM S-adenosyl-l-methionine - PAGE polyacrylamide gel electrophoresis     

一株红树林曲霉菌Aspergillus sp.WHUF0343的次级代谢产物研究

【目的】研究分离自三亚亚龙湾红树林根系土壤的真菌Aspergillus sp. WHUF0343的次级代谢产物及其生物活性。【方法】利用硅胶柱层析、Sephadex LH-20凝胶柱层析和半制备高效液相色谱等技术对该菌的发酵产物进行分离纯化;综合利用核磁共振波谱(NMR)和质谱(MS)等现代波谱技术以及与文献数据对照确定化合物的结构;分别采用肉汤微量稀释检测法和MTS法对化合物进行抗菌和肿瘤细胞毒活性测试。【结果】从真菌Aspergillus sp.WHUF0343的发酵产物共分离并鉴定了10个化合物,分别为isoechinulin A (1)、neoechinulin A (2)、neoechinulin E (3)、preechinulin (4)、neoechinulin D (5)、variecolorin J (6)、dehydroechinulin (7)、questinol (8)、emodin (9)和catenarin (10)。活性评价显示化合物2、9和10对幽门螺杆菌(Helicobacter pylori)和金黄色葡萄球菌(Staphylococcus aure...     

Isolation and biological activity of frankiamide

An antibiotic produced by the symbiotic actinomycete Frankia strain AiPs1 was isolated from culture broth using optimized thin-layer chromatography and high-performance liquid chromatography (HPLC) methods. The novel compound that was isolated, dubbed frankiamide, displayed antimicrobial activity against all 14 Gram-positive bacterial strains and six pathogenic fungal strains tested. The pathogenic actinomycete Clavibacter michiganensis and the ooymycete Phytophthora were especially susceptible. In addition to displaying antimicrobial activity, frankiamide also strongly inhibited ⁴⁵Ca²⁺ fluxes in clonal rat pituitary GH₄C₁ tumor cells and was comparable to a frequently used calcium antagonist, verapamil hydrochloride. The results of HPLC analysis, supported by both nuclear magnetic resonance and mass spectroscopy studies, showed that frankiamide has a high affinity for Na⁺ ions. Journal of Industrial Microbiology & Biotechnology (2001) 27, 62–66. Received 26 February 2001/ Accepted in revised form 01 June 2001     

Preparative Enantioseparation of (RS)‐Baclofen: Determination of Molecular Dissymmetry

The present work reports preparative enantioseparation of (RS)‐baclofen using thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC). Diastereomers were synthesized using a new monochloro‐s‐triazine‐based chiral derivatizing reagent (CDR), namely, N‐(4‐chloro‐6‐piperidinyl‐[1,3,5]‐triazine‐2‐yl)‐L‐phenylalanine, under microwave irradiation. Acetonitrile‐0.1% aq. triflouroacetic acid in gradient elution mode and CH₃OH‐CH₂Cl₂ (4:5; v/v) were successful as mobile phase in HPLC and TLC, respectively. The two diastereomers were isolated by preparative TLC. Molecular dissymmetry was established by developing the lowest energy optimized structures of the diastereomers based on Density Functional Theory and with the help of ¹H NMR showing anisotropic effect associated with aromatic ring of s‐triazine (in the CDR). The configuration of diastereomers was established as [L‐Phe‐(R)‐Bac] and [L‐Phe‐(S)‐Bac], where the first notation refers to the configuration of chiral auxiliary (of the CDR) and the second to that of the analyte Bac. Limits of detection were found to be 0.056 and 0.061 ng mL^?1, respectively, for the two diastereomers. Determination of absolute configuration of the two diastereomers lent support to the elution order and separation mechanism.Chirality 27:299–305, 2015. © 2015 Wiley Periodicals, Inc.     

Stereolability of Chiral Ruthenium Catalysts With Frozen NHC Ligand Conformations Investigated by Dynamic‐HPLC

The stereolability of chiral Hoveyda–Grubbs II type ruthenium complexes bearing N‐heterocyclic carbene (NHC) ligands with Syn‐phenyl groups on the backbone and Syn‐ or Anti‐oriented o‐tolyl N‐substituents was studied by resorting to dynamic high‐performance liquid chromatography (D‐HPLC). A complete chromatographic picture of the involved stereoisomers (four for Anti‐ and two for Syn‐complexes) was achieved at very low temperatures (?53°C and ?40°C respectively), at which the NHC‐Ru bond rotations were frozen out. Inspection of the chromatographic profiles recorded at higher temperatures revealed the presence of plateau zones between the couples of either Syn or Anti stereoisomers, attesting to the active interconversion between the eluted species. Such dynamic chromatograms were successfully simulated through procedures based on both theoretical plate and classical stochastic models. The good superimposition achieved between experimental and simulated chromatographic profiles allowed determination of the related isomerization energy barriers (ΔG_isom^#), all derived by rotation around the NHC‐Ru bond. The obtained diastereomerization barriers between the Anti isomers were found in very good agreement with those previously measured by experimental nuclear magnetic resonance (NMR) and assessed through Density Functional Theory (DFT) calculations. With the same approach, for the first time we also determined the enantiomerization barrier of the Syn isomer. Focused changes to the structure of complex Syn, studied by a molecular modeling approach, were found suitable to strongly reduce the stereolability arising from rotation around the NHC‐Ru bond. Chirality 27:685–692, 2015. © 2015 Wiley Periodicals, Inc.     

Impact on fruit removal and seed predation of a secondary metabolite, emodin, in Rhamnus alaternus fruit pulp

There are two contradictory approaches to explaining the presence of secondary metabolites in ripe fruits. One holds that they evolved toward enhancing dispersal success (adaptive approach); the other claims that they evolved primarily to deter herbivores from eating leaves and seeds and that their presence in ripe fruits is a byproduct of that function (non‐adaptive approach). We tested the validity of three hypotheses of the adaptive approach that explain the presence of secondary metabolites in ripe fruits. We explored the current function of a secondary metabolite, emodin, in Mediterranean buckthorn (Rhamnus alaternus, Rhamnaceae) fruits by relating intraspecific variation and seasonal patterns of concentration to fruit removal and seed damage and by conducting feeding trials with captive birds presented artificial fruits that varied in emodin concentration. The concentration of emodin in the pulp of 10–13 Rhamnus plants from the same population was determined by HPLC every month during two fruiting seasons. Fruit removal by birds and seed predation by invertebrates and microbes were determined for the same plants. Emodin concentration rose during the first stages of ripening, reaching a peak before the fruits were ripe, and then decreased to a minimum when the fruits were ripe. No significant correlation between emodin concentration and ripe fruit removal rate among trees was observed in the first year, whereas in the second year the correlation was positive and significant. Thus, the impact of emodin on fruit selection varied between years, suggesting that emodin concentration does not solely govern fruit selection. A significant negative correlation was found in the first year between emodin concentration and seed predation during the first fruiting month. The yellow‐vented bulbul (Pycnonotus xanthopygos), a seed dispersing bird, distinguished between artificial foods that differed in emodin concentration (control, 0.001% and 0.002%), always preferring the lower concentration. In contrast, house sparrows, (Passer domesticus), a seed predator, did not detect such differences in emodin concentration but did distinguish between control foods and food with 0.005% and 0.001% emodin. We suggest that emodin has an ecological role, preventing seed predation by invertebrates and microbes without decreasing fruit removal by avian dispersers.