Structural variation underlies functional diversity at methyl salicylate loci in tomato.

Methyl salicylate is an important inter- and intra-plant signaling molecule, but is deemed undesirable by humans when it accumulates to high levels in ripe fruits. Balancing the tradeoff between consumer satisfaction and overall plant health is challenging as the mechanisms regulating volatile levels have not yet been fully elucidated. In this study, we investigated the accumulation of methyl salicylate in ripe fruits of tomatoes that belong to the red-fruited clade. We determine the genetic diversity and the interaction of four known loci controlling methyl salicylate levels in ripe fruits. In addition to Non-Smoky Glucosyl Transferase 1 (NSGT1), we uncovered extensive genome structural variation (SV) at the Methylesterase (MES) locus. This locus contains four tandemly duplicated Methylesterase genes and genome sequence investigations at the locus identified nine distinct haplotypes. Based on gene expression and results from biparental crosses, functional and non-functional haplotypes for MES were identified. The combination of the non-functional MES haplotype 2 and the non-functional NSGT1 haplotype IV or V in a GWAS panel showed high methyl salicylate levels in ripe fruits, particularly in accessions from Ecuador, demonstrating a strong interaction between these two loci and suggesting an ecological advantage. The genetic variation at the other two known loci, Salicylic Acid Methyl Transferase 1 (SAMT1) and tomato UDP Glycosyl Transferase 5 (SlUGT5), did not explain volatile variation in the red-fruited tomato germplasm, suggesting a minor role in methyl salicylate production in red-fruited tomato. Lastly, we found that most heirloom and modern tomato accessions carried a functional MES and a non-functional NSGT1 haplotype, ensuring acceptable levels of methyl salicylate in fruits. Yet, future selection of the functional NSGT1 allele could potentially improve flavor in the modern germplasm.

Hormesis management of Moringa oleifera with exogenous application of plant growth regulators under saline conditions.

The study investigated the adaptability of Moringa oleifera to saline conditions, focusing on its hormesis behavior. It also examined how various plant growth regulators affected growth, physiological parameters, and bioactive compounds of moringa. In the first phase, different NaCl stress levels (0, 50, 100, 150, 200, and 250 mM) were applied. Notably, significant stimulation was observed at 100 mM stress for growth, total phenolics, total flavonoids and total chlorophyll content while 150 mM stress had a marked inhibitory effect, with survival decreasing at 200 and 250 mM NaCl levels. A 38% reduction in root attributes and shoot length, along with a 55% decrease in leaf score, was observed at 150 mM stress. Total phenolics showed a positive correlation with growth attributes. In the second phase, moringa plants grown under 50, 100, and 150 mM NaCl stress were treated with various plant growth regulators, including cytokinin (50 mg L-1), thiourea (5 mM), bezyl amino purine (BAP @50 mg L-1), salicylic acid (50 mg L-1), hydrogen peroxide (H2O2@120 µM), or ascorbic acid (50 mg L-1) to mitigate adverse effects of salinity. Cytokinin, BAP, and salicylic acid applications improved salinity tolerance, enhancing enzymatic, and non-enzymatic antioxidants, and the abundance of kaempferol, quercetin, hydroxybenzoic, and hydroxycinnamic acids. Pearson correlation and principal component analysis manifested relationships among growth parameters, antioxidant activities, flavonoids, and phenolic acids. This study provides new insights into hormesis management for moringa plants and the influence of plant growth regulators on flavonoids and phenolic acid levels in moringa leaves under saline conditions.

This study represents the first exploration of hormesis management in Moringa oleifera dual influence of changing soil conditions and foliar application of plant growth regulators. Additionally, this research fills a gap examining the variations in flavonoids (kaempferol and quercetin), hydroxycinnamic acids and hydroxybenzoic acids in moringa leaves concerning varying salinity levels and the exogenous application of plant growth regulators. Further, the study underscores the correlation among secondary metabolites, antioxidant activities and plant growth behavior.

Development of an RP-HPLC Method for Quantifying Diclofenac Diethylamine, Methyl Salicylate, and Capsaicin in Pharmaceutical Formulation and Skin Samples.

An RP-HPLC method with a UV detector was developed for the simultaneous quantification of diclofenac diethylamine, methyl salicylate, and capsaicin in a pharmaceutical formulation and rabbit skin samples. The separation was achieved using a Thermo Scientific ACCLAIMTM 120 C18 column (Waltham, MA, USA, 4.6 mm × 150 mm, 5 µm). The optimized elution phase consisted of deionized water adjusted to pH = 3 using phosphoric acid mixed with acetonitrile in a 35:65% (v/v) ratio with isocratic elution. The flow rate was set at 0.7 mL/min, and the detection was performed at 205 nm and 25 °C. The method exhibits good linearity for capsaicin (0.05-70.0 µg/mL), methyl salicylate (0.05-100.0 µg/mL), and diclofenac diethylamine (0.05-100.0 µg/mL), with low LOD values (0.0249, 0.0271, and 0.0038 for capsaicin, methyl salicylate, and diclofenac diethylamine, respectively). The RSD% values were below 3.0%, indicating good precision. The overall greenness score of the method was 0.61, reflecting its environmentally friendly nature. The developed RP-HPLC method was successfully applied to analyze Omni Hot Gel® pharmaceutical formulation and rabbit skin permeation samples.

Effect of Leaf Maturity on Host Habitat Location by the Egg-Larval Parasitoid Ascogaster reticulata.

Adoxophyes honmai, a serious pest of tea plants, prefers to lay eggs on mature tea leaves rather than young leaves. Here, we examined a hypothesis that Ascogaster reticulata, an egg-larval parasitoid of A. honmai, increases the likelihood of encountering host egg masses by searching mature tea leaves when host-derived cues are not available. In a dual-choice bioassay using a four-arm olfactometer, A. reticulata preferred odor from intact, mature leaves versus young leaves. Based on volatile analysis with gas chromatography-mass spectrometry (GC-MS), we identified 5 and 10 compounds from mature and young leaf volatiles, respectively. The 5 components in the extract from intact mature leaves included (Z)-3-hexenyl acetate, (E)-ß-ocimene, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and methyl salicylate. When each individual compound, or quaternary and quintenary blends of them, ratios of which were adjusted to match those of mature leaf volatiles, were provided, parasitoids preferred the full mixture and the quaternary blend devoid of DMNT to the solvent control. Methyl salicylate, one of the components of preferred blends, was not detected among young leaf volatiles. We concluded that the volatile composition of tea leaves changes, depending on their maturity, and that this composition affects foraging behavior of the parasitoid, which is closely related to the host herbivore's oviposition preference.

Identification of Salicylates in Willow Bark (Salix Cortex) for Targeting Peripheral Inflammation.

Salix cortex-containing medicine is used against pain conditions, fever, headaches, and inflammation, which are partly mediated via arachidonic acid-derived prostaglandins (PGs). We used an activity-guided fractionation strategy, followed by structure elucidation experiments using LC-MS/MS, CD-spectroscopy, and 1D/2D NMR techniques, to identify the compounds relevant for the inhibition of PGE2 release from activated human peripheral blood mononuclear cells. Subsequent compound purification by means of preparative and semipreparative HPLC revealed 2'-O-acetylsalicortin (1), 3'-O-acetylsalicortin (2), 2'-O-acetylsalicin (3), 2',6'-O-diacetylsalicortin (4), lasiandrin (5), tremulacin (6), and cinnamrutinose A (7). In contrast to 3 and 7, compounds 1, 2, 4, 5, and 6 showed inhibitory activity against PGE2 release with different potencies. Polyphenols were not relevant for the bioactivity of the Salix extract but salicylates, which degrade to, e.g., catechol, salicylic acid, salicin, and/or 1-hydroxy-6-oxo-2-cycohexenecarboxylate. Inflammation presents an important therapeutic target for pharmacological interventions; thus, the identification of relevant key drugs in Salix could provide new prospects for the improvement and standardization of existing clinical medicine.

Effect of different drying methods on product quality, bioactive and toxic components of Ginkgo biloba L. seed.

BACKGROUND: Ginkgo biloba seeds are used as a functional food across Asia. However, the presence of toxic compounds has limited their application. In this study, freeze drying, infrared drying, hot-air drying and pulsed-vacuum drying were used to dry G. biloba seeds. A comprehensive analysis was performed on their product quality, antioxidant activities, bioactive and toxic components. RESULTS: Results showed that the drying methods had a significant influence on product quality with freeze drying being superior due to the minimal microstructural damage, followed by infrared drying and pulsed-vacuum drying. Infrared-dried product possessed the strongest antioxidant activities and higher bioactive compound content than hot-air-dried and pulsed-vacuum-dried product. Toxic compounds in fresh G. biloba seeds (ginkgotoxin, ginkgolic acid and cyanide) were reduced markedly by drying. Ginkgotoxin was reduced fourfold, and the contents of acrylamide, ginkgolic acid and cyanide in dried G. biloba seeds were reduced to the scope of safety. Amongst the four drying methods, infrared drying had the shortest drying time, and its product showed higher quality and bioactive compound content, and stronger antioxidant activities. CONCLUSIONS: These findings will offer salient information for selecting a drying method during the processing of ginkgo seeds. Infrared drying could be considered as a multiple-effect drying method in the processing of ginkgo seeds. © 2020 Society of Chemical Industry.

Submerged fermentation of Ginkgo biloba seed powder using Eurotium cristatum for the development of ginkgo seeds fermented products.

BACKGROUND: Ginkgo biloba seeds are well known for the significant curative effects on relieving cough and asthma. However, the development of products from ginkgo seeds still falls behind at present, resulting in a great waste of ginkgo seeds' resource. In this work, submerged fermentation of ginkgo seed powder using Eurotium cristatum was studied to investigate its feasibility as a new processing method. RESULTS: To promote the growth of E. cristatum, the optimum fermentation medium was 80.0 g L-1 of ginkgo seed powder with addition of 5.0 g L-1 calcium chloride (CaCl2 ), 4.0 g L-1 magnesium sulfate (MgSO4 ), 1.25 g L-1 zinc sulfate (ZnSO4 ) and 0.65 g L-1 iron(II) sulfate (FeSO4 ). The optimum fermentation conditions were pH 5.8 ± 0.1, inoculum size 5.1 × 106 CFU mL-1 , liquid medium volume 100 mL in 250-mL Erlenmeyer flask and fermentation 4 days. Through fermentation, the production of lovastatin in fermentation broth could reach up to 32.97 ± 0.17 µg mL-1 and the total antioxidant capacity was improved by more than two-fold. In addition, 40.15% of the ginkgotoxin in ginkgo seed powder was degraded while the entire degradation of ginkgolic acids was obtained. Moreover, fermented ginkgo seed powder suspension presented pleasant fragrances, and the activities of amylase and protease were enhanced to 11.30 ± 0.10 U mL-1 and 23.01 ± 0.20 U mL-1 , respectively. CONCLUSIONS: Submerged fermentation using E. cristatum could significantly enhance the functional value and safety of ginkgo seed powder, and had great potential to become a novel processing method for the development of ginkgo seeds fermented products. © 2020 Society of Chemical Industry.

Sex Attractant Pheromone of the Japanese Slave-Making Ant, Polyergus samurai.

The objective of our project was to identify the sex attractant pheromone of queens of the slave-making ant Polyergus samurai, which was characterized as a blend of methyl 6-methylsalicylate and methyl 3-ethyl-4-methylpentanoate. Both compounds were identified in volatiles collected from squashed heads of queens, and in field trials, a blend of the two compounds was highly attractive to males. In contrast, males were not attracted to a blend of methyl 6-methylsalicylate with 3-ethyl-4-methylpentanol. Thus, the pheromone blend of this species is analogous to that of species in the Polyergus lucidus complex from eastern North America, rather than that of the Polyergus breviceps complex from western North America and the European species P. rufescens. These results are discussed in the context of speciation within the genus.

Structure and Chemical Analysis of Major Specialized Metabolites Produced by the Lichen Evernia prunastri.

We performed comparative profiling of four specialized metabolites in the lichen Evernia prunastri, collected at three different geographic locations, California and Maine, USA, and Yoshkar Ola, Mari El, Russia. Among the compounds produced at high concentrations that were identified in all three specimens, evernic acid, usnic acid, lecanoric acid and chloroatranorin, evernic acid was the most abundant. Two depsidones, salazinic acid and physodic acid, were detected in the Yoshkar-Ola collection only. The crystalline structure of evernic acid (2-hydroxy-4-[(2-hydroxy-4-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoate) (hmb) revealed two crystallographically and conformationally distinct hmb anions, along with two monovalent sodium atoms. One hmb moiety contained an exotetradentate binding mode to sodium, whereas the other exhibited an exohexadentate binding mode to sodium. Embedded edge-sharing {Na2 O8 }n sodium-oxygen chains connected the hmb anions into the full three-dimensional crystal structure of the title compound. The crystal used for single-crystal X-ray diffraction exhibited non-merohedral twinning. The data suggest the importance of the acetyl-polymalonyl pathway products to processes of maintaining integrity of the lichen holobiont community.

Cyanostar: C-H Hydrogen Bonding Neutral Carrier Scaffold for Anion-Selective Sensors.

Cyanostar, a pentagonal macrocyclic compound with an electropositive cavity, binds anions with CH-based hydrogen bonding. The large size of the cyanostar's cavity along with its planarity favor formation of 2:1 sandwich complexes with larger anions, like perchlorate, ClO4-, relative to the smaller chloride. We also show that cyanostar is selective for ClO4- over the bulky salicylate anions by using NMR titration studies to measure affinity. The performance of this novel macrocycle as an anion ionophore in membrane ion sensors was evaluated. The cyanostar-based electrodes demonstrated a Nernstian response toward perchlorate with selectivity patterns distinctly different from the normal Hofmeister series. Different membrane compositions were explored to identify the optimum concentrations of the ionophore, plasticizer, and lipophilic additive that give rise to the best perchlorate selectivity. Changing the concentration of the lipophilic additive tridodecylmethylammonium chloride was found to impact the selectivity pattern and the analytical dynamic range of the electrodes. The high selectivity of the cyanostar sensors and their detection limit could enable the determination of ClO4- in contaminated environmental samples. This novel class of macrocycle provides a suitable scaffold for designing various anion-selective ionophores by altering the size of the central cavity and its functionalization.

Sex Attractant Pheromones of Virgin Queens of Sympatric Slave-Making Ant Species in the Genus Polyergus, and their Possible Roles in Reproductive Isolation.

Species of the ant genus Polyergus are social parasites that steal brood from colonies of their hosts in the closely related genus Formica. Upon emergence as adults in a mixed population, host Formica workers carry out all the normal worker functions within the Polyergus colony, including foraging, feeding, grooming, and rearing brood of the parasitic Polyergus ants. Some unmated Polyergus gynes (queens) run in the raiding columns of their colonies and attract males by releasing a pheromone from their mandibular glands. There are two Polyergus species groups in North America: an eastern P. lucidus group and a western P. breviceps group. One species of each of these groups, P. lucidus Mayr and P. mexicanus Emery, are sympatric in Missouri. In this study, we characterized the sex pheromones of virgin queens of two species of the P. lucidus group (P. lucidus sensu stricto and P. sanwaldi) and one species of the P. breviceps group (P. mexicanus), and compared these with the previously identified sex pheromone of P. topoffi of the P. breviceps group. We then used sex pheromone blends reconstructed from synthesized components of the two groups to test their efficacy at reproductively isolating these species. We found that methyl 6-methylsalicylate is conserved as the major component of the pheromone blends for both Polyergus species groups; however, methyl (R)-3-ethyl-4-methylpentanoate is the species-specific minor component produced by P. lucidus group queens, and (R)-3-ethyl-4-methylpentan-1-ol is the crucial minor component for P. breviceps group queens. The optimal ratio of the major and minor components for P. lucidus group queens was about 100:1 salicylate to ester. In concurrent field trials in Missouri, males of P. lucidus sensu stricto and P. mexicanus (a member of the P. breviceps group) were attracted almost exclusively to their particular blends of sex pheromone components. To our knowledge, this is the first example of a possible sex-pheromone-based reproductive isolating mechanism in ants.

Measurement of concentrations of four chemical ultraviolet filters in human breast tissue at serial locations across the breast.

The human population is widely exposed to benzophenone-3 (BP-3), octylmethoxycinnamate (OMC), 4-methylbenzilidenecamphor (4-MBC) and homosalate from their use in consumer goods to absorb ultraviolet (UV) light. Their oestrogenic activity and presence in human milk suggest a potential to influence breast cancer development. In this study, high-performance liquid chromatography-tandem mass spectrometry was used to measure concentrations of these UV filters in human breast tissue from three serial locations across the breast from 40 women undergoing mastectomy for primary breast cancer. One or more of these UV filters were quantifiable in 101 of 120 (84%) of the tissue samples and at least one breast region for 38 of 40 women. BP-3 was measured in 83 of 120 (69%) tissue samples and at least one breast region for 33 of 40 women (range 0-26.0 ng g-1 tissue). OMC was measured in 89 of 120 (74%) tissue samples and at least one breast region for 33 of 40 women (range 0-58.7 ng g-1 tissue). 4-MBC was measured in 15 of 120 (13%) tissue samples and at least one breast region for seven of 40 women (range 0-25.6 ng g-1 tissue). Homosalate was not detected in any sample. Spearman's analyses showed significant positive correlations between concentrations of BP-3 and OMC in each of the three breast regions. For ethical reasons cancerous tissue was not available, but as the location of the cancer was known, Mann-Whitney U-tests investigated any link between chemical concentration and whether a tumour was present in that region or not. For the lateral region, more BP-3 was measured when a tumour was present (P = .007) and for OMC the P value was .061. For seven (of 40) women with measurable 4-MBC, six of seven had measurable 4-MBC at the site of the tumour.

Adsorption of selected emerging contaminants onto PAC and GAC: Equilibrium isotherms, kinetics, and effect of the water matrix.

The removal of three emerging contaminants (ECs) (amitriptyline hydrochloride (AH), methyl salicylate (MS) and 2-phenoxyethanol (PE)) dissolved in several water matrices by means of their adsorption onto powdered activated carbon (PAC) and granular activated carbon (GAC) has been investigated. When dissolved in ultrapure water, adsorption of the ECs followed the trend of AH > MS > PE, with a positive effect of the adsorbent dose. According to the analysis of the adsorption isotherms and adsorption kinetics, PAC showed strongly higher adsorption efficiency in both capacity and velocity of the adsorption, in agreement with its higher mesoporosity. Equilibrium isotherm data were fitted by Langmuir and Freundlich models. Pseudo-second order kinetics modeled very successfully the adsorption process. Finally, the effect of the presence of dissolved organic matter (DOM) in the water matrices (ultrapure water, surface water and two effluents from wastewater treatment plants) on the adsorption of the selected ECs onto PAC was established, as well as its performance on the removal of water quality parameters. Results show a negative effect of the DOM content on the adsorption efficiency. Over 50% of organic matter was removed with high PAC doses, revealing that adsorption onto PAC is an effective technology to remove both micro-pollutants and DOM from water matrices.

Concurrent supercritical fluid chromatographic analysis of terpene lactones and ginkgolic acids in Ginkgo biloba extracts and dietary supplements.

Supercritical fluid chromatography was used to resolve and determine ginkgolic acids (GAs) and terpene lactones concurrently in ginkgo plant materials and commercial dietary supplements. Analysis of GAs (C13:0, C15:0, C15:1, and C17:1) was carried out by ESI (-) mass detection. The ESI (-) spectra of GAs simply displayed only the [M-H](-) pseudo-molecular ions, and selected ion monitoring (SIM) for those ions was used for the quantification. Analysis of terpene lactones (ginkgolides A, B, C, J and bilobalide) was complicated by in-source collision-induced dissociation (IS-CID) in the ESI source. Thus, MS analysis could be influenced by the fragmentation pattern produced by the IS-CID. However, it was established that the fragmentation pattern, measured by ion survival yield (ISY), was independent of analyte concentration or matrix at a fixed cone voltage in the ESI source. Therefore, MS with SIM mode was applicable for the analysis of these analytes. The reported method provided consistent and sensitive analysis for the analytes of interest. The LOQs and LODs were determined to be below 100 and 40 ng/mL for GAs and 1 µg/mL and 400 ng/mL for terpene lactones, respectively. Intra- and inter-day precisions were found to be satisfactory with RSDs being below 5.2 %. Analyte recoveries ranged from 87 to 109 %. The developed method was successfully applied to the analysis of 11 ginkgo plant samples and 8 dietary supplements with an analysis time of less than 12 min.

Qualitative and quantitative analysis of amorfrutins, novel antidiabetic dietary natural products, by HPLC.

CONTEXT: Initially isolated from fruits of Amorpha fruticosa L. (Fabaceae), amorfrutins are promising antidiabetic natural products as selective peroxisome proliferator-activated receptor γ-agonists. OBJECTIVE: The objective of this study is to develop a sensitive and convenient HPLC method to analysis amorfrutins in plant materials derived from genera Amorpha and Glycyrrhiza. MATERIALS AND METHODS: The reference compounds were isolated from fruits of A. fruticosa and characterized by UV, HR-ESI-MS, and NMR spectrometric techniques. Three amorfrutins were analyzed by HPLC on a Hypersil BDS C18 column (Waters Corporation, Milford, MA) within a gradient elution of acetonitrile and 0.2% glacial acetic acid. HR-ESI-MS spectra were acquired on a quadrupole-time-of-flight (Q-TOF) mass spectrometer. RESULTS: Three amorfrutins showed good linearity (r(2 )> 0.999) in the ranges of 4.2-84.5, 4.5-90.2, and 4.6-92.7 µg/mL. The amorfrutins were only detected in fruits of A. fruticosa with the contents of 2-carboxy-3,5-dihydroxy-4-geranylbibenzyl, amorfrutin A, and amorfrutin B in the ranges of 1.31-7.43, 0.54-3.52, and 0.64-4.63 mg/g, respectively. No amorfrutin was detected in fruits of Amorpha canescens Pursh and roots of Glycyrrhiza uralensis Fisch. (Fabaceae), Glycyrrhiza inflate Bat., or Glycyrrhiza glabra L. DISCUSSION AND CONCLUSION: A novel HPLC method was developed, validated, and applied to identify and quantify amorfrutins in complex botanic matrixes and the characteristic MS fragmentation behaviors of amorfrutins were revealed for the first time. The analytical method presented in this study could be used for the quality control of related plant materials and amorfrutin-based nutraceuticals.

Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis.

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet-B (UV-B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV-B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV-B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV-B radiation exposure activated antioxidants and stress-responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV-B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV-B irradiation.

To each its own: differential response of specialist and generalist herbivores to plant defence in willows.

Plant-insect food webs tend to be dominated by interactions resulting from diffuse co-evolution between plants and multiple lineages of herbivores rather than by reciprocal co-evolution and co-cladogenesis. Plants therefore require defence strategies effective against a broad range of herbivore species. In one extreme, plants could develop a single universal defence effective against all herbivorous insects, or tailor-made strategies for each herbivore species. The evolution and ecology of plant defence has to be studied with entire insect assemblages, rather than small subsets of pairwise interactions. The present study examines whether specialists and generalists in three coexisting insect lineages, forming the leaf-chewing guild, respond uniformly to plant phylogeny, secondary metabolites, nutrient content and mechanical antiherbivore defences of their hosts, thus permitting universal plant defence strategies against specialized and generalist folivorous insects from various taxa. The extensive data on folivorous assemblages comprising three insect orders and 193 species are linked with plant phylogeny, secondary chemistry (salicylates, flavonoids and tannins), leaf morphological traits [specific leaf area (SLA) and trichome coverage], nutrient (C : N) content and growth form of eight willow (Salix) and one aspen (Populus) species growing in sympatry. Generalists responded to overall host plant chemistry and trichomes, whilst specialists responded to host plant phylogeny and secondary metabolites that are unique to willows and that are capable of being utilized as an antipredator protection. We did not find any significant impact of other plant traits, that is SLA, C : N ratio, flavonoids, tannins and growth form, on the composition of leaf-chewing communities. Our results show that the response to plant traits is differential among specialists and generalists. This finding constrains the ability of plants to develop defensive traits universally effective against herbivores and may lead to diversification of plant defensive mechanisms into several complementary syndromes, required for effective protection against generalists and specialists from multiple insect taxa comprising most leaf-chewing assemblages. These results point to the necessity of broad studies of plant-herbivore interactions, across multiple insect taxa and guilds.

HPLC quantification of all five ginkgolic acid derivatives in Ginkgo biloba extracts using 13 : 0 ginkgolic acid as a single marker compound.

An HPLC quantification method for ginkgolic acid derivatives in Ginkgo biloba leaf extracts was developed. Using 13 : 0 ginkgolic acid as a marker compound, the relative correlation factors of the four other ginkgolic acid derivatives - namely, 15 : 0 ginkgolic acid, 15 : 1 ginkgolic acid, 17 : 1 ginkgolic acid, and 17 : 2 ginkgolic acid - to 13 : 0 ginkgolic acid were determined by HPLC and subsequently used for calculating their contents in ten hydro-ethanolic refined extract samples. In other words, the content of 13 : 0 ginkgolic acid in the extracts was determined using the isolated compound as an external standard. Subsequently the now known concentration of this compound functioned as an internal standard for the quantification of the other four ginkgolic acid derivatives via the described correlation factors. This HPLC method was validated by two independent control measurements, one with an external standard for every individual compound and one based on the present method with the single marker compound alone. The results did not differ significantly in any of the 10 tested extract samples. The protocol presented here thus not only uses the same reference substance for G. biloba extracts as the current Chinese Pharmacopoeia method but also incorporates the advantages of the current European Pharmacopoeia approach. It is simple, reproducible, and can be used to determine the total contents of ginkgolic acid derivatives in G. biloba leaf extracts.

[Determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials by LC-MS/MS].

To develop a LC-MS/MS method for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials, the column was Agilent ZORBAX Eclipse plus C18 (3.0 mm x 50 mm, 1.8 µm), and the mobile phase consisted of methanol-water (containing 0.2% formic acid) (95:5) at a flow rate of 0.5 mL · min(-1). The multiple reaction ion monitoring (MRM) with an ESI interface in the negative ion mode was selected. The results showed that the linear ranges of five kinds of ginkgolic acids were in the range of 0.2-36.0 µg · L(-1) (r ≥ 0.999 5). The lowest limit of quantification (LOQ) of ginkgo acid C13: 0, C15:1, C17:2, C15:0 and C17:1 were 0.18, 0.18, 0.21, 0.10 and 0.20 µg · L(-1), respectively. The average recovery was between 73.28% and 87.56%, and the average content of total ginkgolic acids in three batches of samples was in the range of 0.023-0.028 µg · g(-1), which was much lower than 2 µg · g(-1) prescribed in drug registration standards. This method is simple and rapid with high sensitivity, which can be used for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials.

Salicylic acid and jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana.

Systemic resistance is induced by pathogens and confers protection against a broad range of pathogens. Recent studies have indicated that salicylic acid (SA) derivative methyl salicylate (MeSA) serves as a long-distance phloem-mobile systemic resistance signal in tobacco, Arabidopsis, and potato. However, other experiments indicate that jasmonic acid (JA) is a critical mobile signal. Here, we present evidence suggesting both MeSA and methyl jasmonate (MeJA) are essential for systemic resistance against Tobacco mosaic virus (TMV), possibly acting as the initiating signals for systemic resistance. Foliar application of JA followed by SA triggered the strongest systemic resistance against TMV. Furthermore, we use a virus-induced gene-silencing-based genetics approach to investigate the function of JA and SA biosynthesis or signaling genes in systemic response against TMV infection. Silencing of SA or JA biosynthetic and signaling genes in Nicotiana benthamiana plants increased susceptibility to TMV. Genetic experiments also proved the irreplaceable roles of MeSA and MeJA in systemic resistance response. Systemic resistance was compromised when SA methyl transferase or JA carboxyl methyltransferase, which are required for MeSA and MeJA formation, respectively, were silenced. Moreover, high-performance liquid chromatography-mass spectrometry analysis indicated that JA and MeJA accumulated in phloem exudates of leaves at early stages and SA and MeSA accumulated at later stages, after TMV infection. Our data also indicated that JA and MeJA could regulate MeSA and SA production. Taken together, our results demonstrate that (Me)JA and (Me)SA are required for systemic resistance response against TMV.