Heat Shock Factor 1 Inducers from the Bark of Eucommia ulmoides as Cytoprotective Agents

The barks of Eucommia ulmoides (Eucommiae Cortex, Eucommiaceae) have been used as a traditional medicine in Korea, Japan, and China to treat hypertension, reinforce the muscles and bones, and recover the damaged liver and kidney functions. Among these traditional uses, to establish the recovery effects on the damaged organs on the basis of phytochemistry, the barks of E. ulmoides have been investigated to afford three known phenolic compounds, coniferaldehyde glucoside ( 1 ), bartsioside ( 2 ), and feretoside ( 3 ), which were found in the family Eucommiaceae for the first time. The compounds 1 – 3 were evaluated for their inducible activities on the heat shock factor 1 (HSF1), and heat shock proteins (HSPs) 27 and 70, along with four compounds, geniposide ( 4 ), geniposidic acid ( 5 ), pinoresinol diglucoside ( 6 ), and liriodendrin ( 7 ), which were previously reported from E. ulmoides. Compounds 1 – 7 increased expression of HSF1 by a factor of 1.214, 1.144, 1.153, 1.114, 1.159, 1.041, and 1.167 at 3 μM , respectively. Coniferaldehyde glucoside ( 1 ) showed the most effective increase of HSF1 and induced successive expressions of HSP27 and HSP70 in a dose‐dependent manner without cellular cytotoxicity, suggesting a possible application as a HSP inducer to act as cytoprotective agent.     

Development of chromatographic and free radical scavenging activity fingerprints by thin‐layer chromatography for selected Salvia species

Introduction – Plant‐derived free radical scavengers have become the subject of intensive scientific interest. Recently, the concept of coupling chromatographic fingerprints with biological fingerprinting analysis has gained much attention for the quality control of plant extracts. However, identification of free radical scavenging activity of each single compound in a complex mixture is a difficult task. Thin‐layer chromatography with post‐chromatographic derivatisation with the methanol solution of DPPH can be a valuable tool in such analyses. Objective – Development of chromatographic and free radical scavenging fingerprints of nineteen Salvia species grown and cultivated in Poland. Methodology – Chromatography was performed on the silica gel layers with use of two eluents, one for the resolution of the less polar compounds, and the other one for the resolution of the medium and highly polar ones. The plates were sprayed with the vanillin–sulfuric acid reagent to produce chemical fingerprints, and with DPPH solution to generate free radical scavenging fingerprints. Results – With four Salvia species, it was revealed that their strong free radical scavenging properties are not only due to the presence of polar flavonoids and phenolic acids, but also due to the presence of several free radical scavengers in the less polar fraction. Because of the similarities in both the chromatographic and the free radical scavenging fingerprints, S. triloba can be introduced as a possible equivalent of the pharmacopoeial species, S. officinalis. Conclusion – Fingerprints developed in the experiments proved useful for the analysis of complex extracts of the different Salvia species. Copyright © 2010 John Wiley & Sons, Ltd.     

HPLC fingerprints combined with principal component analysis,hierarchical cluster analysis and linear discriminant analysis for the classification and differentiation of Peganum sp. indigenous to China

Introduction – Seeds of wild Peganum harmala Linn., P. multisectum (Maxim) Bobr., P. nigellastrum Bunge and a probable indeterminate species, herein referred to as P. variety, are commonly used in Chinese medicine. These seeds cannot be differentiated based on morphology. Objective – Seeds of P. harmala Linn., P. multisectum (Maxim) Bobr., P. nigellastrum Bunge and P. variety were collected in different provinces in China and their HPLC profiles were recorded for statistical analysis and pattern recognition. Methodology – HPLC chromatograms of seed extracts were recorded under the same conditions. Individual HPLC chromatograms for each species were evaluated against the mean chromatogram for the same species generated using a similarity evaluation computer program. Data from chromatographic fingerprints were also processed using principal component analysis (PCA), hierarchical cluster analysis (HCA) and linear discriminant analysis (LDA). Results – The Peganum sp. seed extracts had similar HPLC fingerprints but with some inter‐specific differences. The chromatographic fingerprints combined with PCA, HCA and LDA could distinguish the seeds of the different species of Peganum investigated. Conclusion – HPLC fingerprints can be used to authenticate and differentiate the seeds of three different species of genus Peganum indigenous to China. The results indicated that the unidentified P. variety might indeed be a new species or variety. Copyright © 2009 John Wiley & Sons, Ltd.     

Microsatellite genetic variation in the Chinese endemic Eucommia ulmoides (Eucommiaceae): implications for conservation

Eucommia ulmoides, a traditional Chinese medicinal plant, is endangered as a consequence of long‐term and widespread harvest in the late 20th century. It has been widely cultivated as a source of herbal medicine and for use in the organic chemical industry in China. In this study, eight microsatellite markers were applied to investigate genetic diversity in E. ulmoides. Three hundred individuals from one semi‐wild population and nine cultivated populations across its main production area were collected. A high level of genetic diversity at population levels (H_E = 0.716) was observed. The highly outcrossed mating system, high longevity of E. ulmoides and seed admixture may be responsible for high genetic variation within populations. A genetic bottleneck was observed in one population. Populations were only slightly differentiated from one another (F_ST = 0.063); this was also supported by AMOVA, which revealed that 94.05% of the total variation resided within populations. This is probably attributable to long‐distance gene flow mediated by the exchange of seeds by local farmers. Implications of these results for the conservation of genetic resources of E. ulmoides are discussed. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 775–785.     

Coniferous litter extracts inhibit the litter decomposition of Catalpa fargesii Bur. and Eucommia ulmoides Oliver

In this study, the litter of Catalpa fargesii and Eucommia ulmoides was treated with the water extracts of 5 types of coniferous litter for a 0.5-year indoor, simulated decomposition experiment, and the effects of plant secondary metabolites (PSMs) from coniferous litter on the mass loss and C, N and P release of the 2 types of litter were detected. The results indicated that the litter extracts of Platycladus orientalis, Pinus tabuliformis, Pinus armandii and Larix principis-rupprechtii significantly reduced the decomposition and overall final nutrient release rates of the litter of C. fargesii and E. ulmoides (in which only the extracts of P. orientalis litter did not affect the mass loss of E. ulmoides litter). Correspondingly, significant decreases in the activity of soil cellulase and polyphenol oxidase were observed during the entire decomposition period, especially during the middle and later decomposition stages, indicating that the PSM release from coniferous litter might inhibit the decomposition and nutrient cycling of C. fargesii and E. ulmoides litter by depressing the activity of lignocellulolytic enzymes during mixed decomposition. In conclusion, more attention should be given to the effects of leached litter PSMs on the decomposition of other types of litter during mixed afforestation or in the mixed transformation of pure forests.     

杜仲基因密码子使用模式分析

为了解杜仲基因密码子使用模式,该文以杜仲基因组密码子为研究对象,运用CodonW软件对杜仲的320个蛋白编码基因进行同义密码子相对使用频率(RSCU)分析、ENC-GC3s关联分析编码基因的密码子ENC值、PR2-plot偏倚分析编码基因的密码子碱基使用频率,并运用CUSP软件与Codon Usage Database软件对杜仲基因密码子的GC含量、使用频率与代表性物种烟草、拟南芥、大肠杆菌和酿酒酵母的密码子GC含量和使用频率进行比较。结果表明:杜仲基因密码子的RSCU>1的密码子有30个,其中18个以G/C结尾、12个以A/U结尾,说明杜仲基因密码子偏好以G/C结尾,且偏好性较强;有效密码子数(ENC)范围为30~60,该范围内的密码子距离标准曲线较远,其ENC值小,偏好性较强;PR2-plot偏倚分析碱基使用频率显示,G>C、U>A;杜仲与代表性物种的GC含量分析显示,杜仲的GC12、GC3以及平均GC含量均高于代表性物种;杜仲与代表性物种的密码子使用频率分析显示,杜仲与烟草、酿酒酵母的密码子偏好较为接近,杜仲与拟南芥、大肠杆菌的密码子偏好差距较大。杜仲是我国特有的珍贵中药材,对其进行密码子使用模式分析,并研究其密码子偏好规律,为杜仲植物基因工程中外源基因的改良及表达提供了理论基础。     

Development and characterization of microsatellite markers in Eucommia ulmoides Oliver (Eucommiaceae)

Twenty‐three microsatellite markers were developed from an AC‐enriched genomic library of Eucommia ulmoides, an economically important tree species for both herbal medicine and organic chemical industry in China. Nineteen microsatellite loci were found polymorphic by testing 36 individuals from 10 populations, with two to 14 alleles per locus. The expected heterozygosity ranged from 0.054 to 0.874. This set of microsatellite markers has provided a useful tool for the ongoing efforts in studying population genetic structure of E. ulmoides.     

Divergence in Female Duetting Signals in the Enchenopa binotata Species Complex of Treehoppers (Hemiptera: Membracidae)

Sexual communication often involves signal exchanges between the sexes, or duetting, in which mate choice is expressed through response signals. With both sexes acting as signalers and receivers, variation in the signals of males and females may be important for mate choice, reproductive isolation, and divergence. In the Enchenopa binotata species complex – a case study of sympatric speciation in which vibrational duetting may have an important role – male signals are species‐specific, females choose among males on the basis of signal traits that reflect species and individual differences, and female preferences have exerted divergent selection on male signals. Here, we describe variation in female signals in the E. binotata species complex. We report substantial species differences in the spectral and temporal features of female signals, and in their timing relative to male signals. These differences were similar in range to differences in male signals in the E. binotata complex. We consider processes that might contribute to divergence in female signals, and suggest that signal evolution in the E. binotata complex may be influenced by mate choice in both sexes.     

Accumulation of chlorogenic acid in cell suspension cultures of Eucommia ulmoides

Suspension cultures of Eucommia ulmoides were developed and shown to accumulate chlorogenic acid. MS medium plus 2.0 mg l^–1 2,4-dichlorophenoxy acetic acid was used for the cell suspension cultures of Eucommia ulmoides. The chlorogenic acid content of suspension cells was analyzed by capillary electrophoresis, and the mean content was 2.15%, approximate to that of Eucommia ulmoides leaves.     

冬虫夏草及杜仲磷脂成分的研究

本文对不同产地的冬虫夏草及杜仲磷脂成分进行了分析研究。以钼蓝比色法测定了它们的总磷脂含量,采用薄层色谱扫描和吸光度比例系数校正法测定了其磷脂组成及相对百分含量。冬虫夏草约含8种磷脂组分,主要成分为磷脂酰胆碱、磷脂酰叽醇、磷脂酰丝氨酸、磷脂酰乙醇胺和磷脂酸。杜仲约含6种磷脂组分,其中以溶血磷脂酰胆碱和磷脂酰胆碱为主。     

Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid,Diaphorina citri

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem‐inhabiting bacterium, Candidatus Liberibacter asiaticus, which is presumed to cause HLB in Florida citrus. Laboratory and field studies were conducted to examine the behavioral responses of male and female D. citri to their cuticular extracts. In olfactometer assays, more male D. citri were attracted to one, five, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted proportionately more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female D. citri revealed differences between the sexes in chemical composition of their cuticular extracts. Laboratory bioassays with synthetic chemicals identified from cuticular extracts indicated that dodecanoic acid attracted more males than clean air. Traps baited with dodecanoic acid did not increase total catch of D. citri as compared with blank traps at the dosages tested; however, the sex ratio of psyllid catch was male biased on traps baited with the highest lure loading dosage tested (10.0 mg).     

Suppression of Splenic Lymphocyte Proliferation by Eucommia ulmoides and Genipin

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.     

Studies on a chlorogenic acid-producing endophytic fungi isolated from <Emphasis Type="Italic">Eucommia ulmoides</Emphasis> Oliver

Eucommia ulmoides Oliver is a traditional medicinal plant of China, and it is one of the main sources of chlorogenic acid. Chlorogenic acid is an ester of caffeic acid, quinic acid, and a phenolic compound that has antibacterial, antifungal, antioxidant, and antitumor activities. The purpose of this study was to determine whether endophytic fungi isolated from Eucommia ulmoides Oliver had the same ability to produce chlorogenic acid. Primary screening was done by antibacterial and antifungal reactions, and the strain reselection was done with high-performance liquid chromatography (HPLC) to identify the fermentation products of the selected strains. Extracts of the leaf and cortex of Eucommia ulmoides Oliver were also deteted by HPLC, then positive results of HPLC were analyzed by GC-MS and LC-MS. In this study, 29 strains were isolated from Eucommia ulmoides Oliver. Most of them had antibacterial activity, and a few of them had antifungal activity. One ingredient of the B5 extract had a retention time identical to that of authentic chlorogenic acid. With GC-MS, other ingredients, isocoumarin and p-chlorocinnamide, were found. With LC-MS, chlorogenic acid and geniposide related to Eucommia ulmoides Oliver were found. The strain B5 was identified as Sordariomycete sp. Thus, endophytic fungi may produce the bioactive compound chlorogenic acid, as their host plant does, and could be used for the production of chlorogenic acid by fermentation in the future.     

Global Profiling of Protein Lysine Malonylation in Eucommia ulmoides Leaves Reveals Its Role in Photosynthesis and Metabolism

The modification of proteins by malonylation plays an important regulatory role in multiple biological processes. Protein malonylation is the reversible addition of malonyl groups to lysine residues to regulate protein localization, enzyme activity, protein stability, and many other biochemical processes. However, little information was available on the malonylation of lysine in Eucommia ulmoides, an important traditional Chinese herb with multiple active substances. In this study, we analyzed the malonylation of lysine to determine the potential regulatory role of lysine malonylation in the growth and development of E. ulmoides using LC–MS/MS combined with immune-based purification. A total of 388 lysine malonylation sites distributed in 256 proteins were identified. A gene ontology (GO) analysis showed that the proteins in which lysine is malonylated are related to a wide range of cellular functions ranging from metabolic processes to responses to stimuli. A bioinformatic analysis showed that malonylated proteins are primarily distributed in the chloroplast (108 proteins, 42.19%), cytoplasm (90 proteins, 35.16%), and nucleus (27 proteins, 10.55%). They are primarily involved in energy metabolism, including photosynthesis, carbon metabolism, glycolysis, and the biosynthesis of secondary metabolites. Our research suggests that the malonylation of lysine is necessary for the life cycle of plants, which are primarily involved in the process of converting solar energy to biomass energy that is stored and converting the energy stored by plants into energy (ATP) available for direct use in E. ulmoides. To our knowledge, this is the first posttranslational malonylation modification of E. ulmoides proteins, which was conducted simultaneously to explore the mechanism of modification groups in protein functions, protein interactions, and biological metabolism, so as to provide a theoretical basis for the production and breeding of E. ulmoides.

     

Analysis of nordihydroguaiaretic acid in Larrea divaricata Cav. extracts by micellar electrokinetic chromatography

Introduction – Larrea divaricata Cav. is a common shrub used in folk medicine to treat a variety of diseases. The main product extracted from this bush is nordihydroguaiaretic acid (NDG), which is a potent antioxidant. Objective – In this paper we propose a novel method for the quantification of NDG in different extracts of Larrea divaricata. The concentration of NDG in two different aqueous extracts (I and D) and an ethanolic extract (Eet) was analysed, in order to evaluate the safe use of the extracts for pharmacological purposes. Methodology – Micellar electrokinetic chromatography (MEKC) was performed under the following conditions: the background electrolyte used consisted of 20 mm phosphate buffer (pH 7.5), 10 mm sodium dodecyl sulphate and 10% acetonitrile. Results – The limits of detection and quantitation of NDG were 4.54 × 10^?4 and 10.6 × 10^?4 mg/mL, respectively. The concentration of this acid in both aqueous extracts was within the safe levels. However, the decoction must be used carefully because the concentration of the acid was almost over the recommended limit. In the case of ethanolic extracts, the amount of NDG was above the safe concentration, which is in agreement with the solubility of the active compound in ethanol. Conclusions – The conclusions of this study demonstrate that most of these plant extracts should be used with care, especially those which are used with medicinal purposes. This is the first research on the quantification of NDG using MEKC in jarilla extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular biology,phytochemistry and bioactivity of three endemic Aloe species from Mauritius and Réunion Islands

Introduction – Aloe tormentorii, A. purpurea and A. macra are used as multipurpose folk medicines in Réunion and Mauritius Islands and are mistaken for the introduced Aloe vera. Objective – To compare the phytochemical, antimicrobial and DNA profiles of Aloe endemic to Mauritius and Réunion with the profiles of A. vera. Methodology – Leaf extracts of these Aloe species were analysed using standard phytochemical screening techniques, TLC and by HPLC. These extracts were also assayed for antimicrobial activity using microdilution techniques. Genetic diversity was studied using RAPD markers. Results – Phytochemical and antimicrobial assays and RAPD analysis showed that Mascarene Aloe species were very different from A. vera. Conclusion – This study is the first report highlighting the differences between Aloe sp.p from Mascarene and Aloe vera at the metabolic and genomic level. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical Characterization of Leaves,Male and Female Flowers from Spontaneous Cannabis (Cannabis sativa L.) Growing in Hungary

Spontaneous forms of hemp (Cannabis sativa L., often reported as Cannabis sativa var. spontanea Vavilov ) with a low content of psychoactive cannabinoids can be considered as a valuable source of other phytoconstituents to be used in nutraceuticals or for their health promoting properties. Chemical data on this hemp variety are rather scarce. In this article, we report a comprehensive phytochemical characterization of leaves, male and female inflorescences of C. sativa growing wild in Hungary. For the purpose, the essential oil along with polar extracts were analyzed using GC/MS, NMR and LC‐DAD‐MS techniques, respectively. The results indicated that female inflorescence essential oil contains high amounts of the CB2 agonists, (E)‐caryophyllene (28.3 %) and cannabidiol (CBD; 24.9 %), whereas leaves and male inflorescence essential oils contained lower amounts of both compounds. HPLC/MS allowed to quantify cannabidiol (CBD) and cannabidiolic acid (CBD?A) in the ethyl acetate extracts from leaves, male and female inflorescences; they were 0.3, 0.8 and 0.9 %, and 0.2, 0.3 and 0.4 %, respectively. Flavonoids of this spontaneous form of hemp were formed by C‐glycosides and glucuronic acids of kaempferol and apigenin with a total content of 3.8, 6.1 and 7.8 mg/g in methanolic extracts from leaves, male and female inflorescences, respectively. Based on these results, spontaneous C. sativa may represent an important source of CB₂ agonists and bioflavonoids to be used in nutraceuticals, cosmetics and pharmaceuticals.     

Ethological Isolation,Sexual Behavior and Pheromones in the Fasciatus Species Group of the Lizard Genus Eumeces

Ethological isolation was found among North American members of the fasciatus species group of Eumeces. Ethological isolation was investigated by staging a series of interspecific and intraspecific heterosexual encounters. No male E. laticeps or E. fasciatus courted heterospecific females. In both species, males courted and copulated with conspecific females in a significantly higher frequency of trials than with heterospecific females. However, male E. inexpectatus courted females of all three species, courting conspecific females and female E. laticeps at similar frequencies, but female E. fasciatus at a significantly lower frequency. No females of any of the three species were sexually receptive to heterospecific males, but forced copulation occurred in two of nine courtships of female E. fasciatus by male E. inexpectatus. Thus, ethological isolation in the fasciatus group appears to be complete with the possible exception of occasional forced copulation between male E. inexpectatus and female E. fasciatus. Preliminary evidence on the role of chemical stimuli in maintaining ethological isolation was obtained from experiments involving interspecific transfers of female odors. Male E. inexpectatus courted female E. fasciatus labelled with the odor of female E. inexpectatus in a significantly greater proportion of trials than they courted such females lacking the conspecific female odor. In a similar experiment, male E. fasciatus did not court female E. inexpectatus even if the females bore odors of female fasciatus.