Existence of ionotropic glutamate receptor subtypes in cultured rat retinal ganglion cells obtained by the magnetic cell sorter method and inhibitory effects of 20-hydroxyecdysone,a neurosteroid,on the glutamate response

Glutamate and neurosteroids are known to exist in retinal ganglion cells (RGC). Therefore, patch clamp studies using the whole-cell recording method were performed to determine whether or not ionotropic glutamate receptor subtypes, i.e., N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors, were present on RGC obtained by the magnetic cell sorter (MACS) method and cultures. In addition, the effects of 20-hydroxyecdysone (20-HE), a neurosteroid, on inward currents induced by NMDA, AMPA and kainate were examined at a holding potential of -60 mV. The current-voltage relationship for NMDA in the presence of glycine and Mg2+-free, as well as those for AMPA and kainate were linear, with a reversal potential of around 0 mV. NMDA-induced currents were blocked by MK-801, while both AMPA- and kainate-induced currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Application of 20-HE in the bath resulted in significant inhibitions on NMDA-, AMPA- and kainate-induced currents. Thus, NMDA, AMPA and kainate receptors were confirmed to exist on MACS-separated cultured RGC. Moreover, 20-HE inhibited NMDA receptor-mediated currents most prominently and AMPA- and kainate-mediated currents moderately, suggesting that neurosteroids may be playing a role in modulating glutamate-mediated transmission in RGC, and 20-HE might be useful for preventing glutamate neurotoxicity.     

β-蜕皮甾酮化学结构修饰研究进展

β-蜕皮甾酮是一种重要的植物甾酮,它参与控制昆虫等无脊椎动物的蜕皮和繁殖过程。药理实验表明β-蜕皮甾酮具有促进蛋白质合成、降低血糖血脂以及改善学习和记忆等多种功效。多年来人们对其化学结构修饰及蜕皮活性的研究取得了一定的进展和成果,该文对历年来β-蜕皮甾酮的化学结构修饰和蜕皮活性研究成果进行概述。     

β-蜕皮甾酮的分离及结构修饰

目的从露水草总甾酮中分离β蜕皮甾酮并对其进行结构修饰。方法采用硅胶柱色谱分离β蜕皮甾酮,并以其为原料通过化学合成的各种手段合成结构类似物,利用光谱分析方法对所得化合物进行结构确证。结果与结论从露水草总甾酮中分离了β蜕皮甾酮(20 hydroxyecdysone)及另一含量较大的筋骨草甾酮C(ajugasterone C)。以分离的β蜕皮甾酮为原料化学合成了5种植物甾酮。分别是2,3,20,22双异丙叉基β蜕皮甾酮(20 hydroxyecdysone 2,3,20,22 diacetonideⅠ)、20,22异丙叉基β蜕皮甾酮(20 hydroxyecdysone 20,22 acetonideⅡ)、2,3,20,22双异丙叉基25乙酰基β蜕皮甾酮(viticosterone E 2,3,20,22 diacetonideⅢ)、2,3异丙叉基β蜕皮甾酮(20 hy-droxyecdysone 2,3 acetonideⅣ)、2,3异丙叉基22羰基β蜕皮甾酮(22 oxo 20 hydroxyecdysone2,3 acetonideⅤ)。     

Ecdysone from roots and seeds of Helleborus species

Ecdysones from Roots and Seeds of Helleborus Species From roots and seeds of Helleborus odorus WALDST. et KIT. four ecdysteroids have been isolated: β-Ecdysone (1) , 5-α-hydroxyecdysone (2) , 5-α-hydroxyecdysone 3-α-D-glucoside (3) , and 5-β-hydroxyecdysone 3-β-D-glucoside (4).     

New Ecdysteroids from Serratula tinctoria

Six new ecdysteroids have been isolated from SERRATULA TINCTORIA; these are: the 2,22- and 3,22-diacetates of 20-hydroxyecdysone, 5beta-hydroxyrubrosterone, 3-epi-poststerone, 3-epi-rubrosterone, and 22-oxo-20-hydroxyecdysone. These minor compounds were found together with the known ecdysteroids, 20-hydroxyecdysone, its 2-, 3-, and 22-monoacetates, rubrosterone, poststerone, polypodine B (5beta,20-dihydroxyecdysone), pterosterone (25-deoxy-20,24-dihydroxyecdysone), and makisterone C (24-ethyl-20-hydroxyecdysone). All these ecdysteroids were isolated by a combination of several chromatographic techniques (liquid chromatography on alumina, DCCC, and HPLC), then identified using standard mass spectrometric and 2D (1)H-NMR techniques.     

Simultaneous determination of eight components in Radix Tinosporae by high-performance liquid chromatography coupled with diode array detector and electrospray tandem mass spectrometry

High-performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ESI-MS-MS) and diode array detection (DAD) was used to identify and simultaneously determine eight major ingredients in Radix Tinosporae. The assay was performed on a Diamonsil C(18) analytical column with a gradient solvent system of A (water containing 0.2% formic acid, 20mM ammonium acetate) and B (methanol/acetonitrile=1/1, v/v). The 217, 248, 270 and 347 nm, respectively, were chosen as the monitoring wavelengths to determine four structural types of components, say columbin, phytoecdysteroids (including 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone 3-O-beta-d-glucopyranoside and 2-deoxy-20-hydroxyecdysone), menisperine and protoberberine alkaloids (including columbamine, jatrorrhizine and palmatine). This method was validated in respect to precision, repeatability and accuracy, and was successfully applied to quantify the eight components in 39 batches of R. Tinosporae for quality control purpose. The results indicated that the proposed method could be readily utilized as a quality control method for traditional Chinese medicine (TCM).     

Structural diversity of ecdysteroids of Lychnis flos-cuculi

Eleven ecdysteroids have been isolated from Lychnis floscuculi; we are the first who report eight ecdysteroids of the eleven compounds in this plant. Two of these ecdysteroids, dihydrorubrosterone and 20-hydroxyecdysone 3-acetate are newly discovered natural products. The success of isolation of these new ecdysteroids has been based on the use of separation methods in a proper order; these separation procedures were completing each others. At the beginning steps of isolation simple separation methods were used, such a solvent-solvent distribution and fractionated precipitation. Two third of the contaminants were removed thereby. High capacity low resolution methods were used then, such as classical adsorption column chromatography and preparative thin-layer chromatography. The major component (20-hydroxyecdyssone) and certain minor ecdysteroids (polypodine B and rubrosterone) were isolated in pure form here. Purification of the further minor components (poststerone, 2-deoxy-20-hydroxyecdysone, vitikosterone E, dihydrorubrosterone, makisterone A, taxisterone, 20-hydroxyecdysone 2-acetate, 20-hydroxyecdysone 3-acetate) required HPLC and other absorption chromatographic methods. Our recent separation scheme means a generally applicable guiding principle for isolation of any plant ecdysteroid, major and minor alike. Structural identification of the known ecdysteroids was based on their spectral data and that of their literature information. Structural elucidation of 20-hydroxyecdysone 3-acetate was done by the help of a standard component prepared by acetylation of 20-hydroxyecdysone. From the mixture of seven acetates the corresponding compound (20-hydroxyecdysone 3-acetate) was isolated, and used for identification. Structural diversity of ecdysteroids of Lychnis flos-cuculi is evaluated, and a tentative explanation is introduced for the formation and biosynthesis of the versatility of phytoecdysteroids.     

20-羟基蜕皮甾酮对H_2O_2诱导SH-SY5Y细胞损伤的保护作用

本文旨在研究一种类固醇激素即20-羟基蜕皮甾酮对H2O2诱导的SH-SY5Y细胞损伤的保护作用及其作用机制。预孵育20-羟基蜕皮甾酮可以明显提高H2O2诱导的SH-SY5Y细胞的生存率,减少乳酸脱氢酶（LDH）漏出率,抑制脂质过氧化水平（MDA）升高,提高超氧化物歧化酶（SOD）活性。此外,20-羟基蜕皮甾酮可以通过降低Bax/Bcl-2的比值,延缓caspase-3的活化来抑制H2O2诱导的细胞凋亡。本研究表明20-羟基蜕皮甾酮对H2O2诱导的SH-SY5Y细胞具有保护作用,有可能用于由氧化应激和凋亡导致的神经退行性疾病的治疗。     

Components of Serratula species; screening for ecdysteroid and inorganic constituents of some Serratula plants]

Ecdysteroid and inorganic components were analyzed from several plant species belonging to Caryophyllaceae family, such as in the cases of Serratula tinctoria, Serratula wolffii, Serratula coronata (20-hydroxyecdysone and inorganic components) and Jurinea mollis, Serratula gmellinii and Leuzea carthamoides (inorganic components, only). The 20-hydroxyecdysone content was determined using thin-layer chromatography after a simple clean up that had been performed by solid-phase extraction. Inorganic constitutents were determined using either ICP or flame photometry. Vegetation dependence of both 20-hydroxyecdysone and inorganic elements was studied. Serratula coronata shows remarkable high 20-hydroxyecdysone (2.3%, in April) and the studied Serratula plants gave a minimum of 20-hydroxyecdysone in June. Favorable period for harvesting is suggested as July and August, through the blossoming of these plants. Potassium and 20-hydroxyecdysone content gave a similar tendency considering their vegetation dependence, while the magnesium content moved toward opposite direction. The calcium contents of Serratula tinctoria, Serratula wolffii and Serratula coronata were found between 2.2% and 3.8%, which values are high relative to the other medicinal plants. At the same time, the Cu content of the ecdysteroid producing (and screened) Caryophyllaceae plants is low. The Fe, Mn and Mg contents of Serratula coronata are high, even higher than that of the Leuzea carthamoides. Our results have suggested the importance of analysis and control of inorganic constituents of crude plant extracts used for medicinal and recreational purposes.     

Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects

In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.     

Release of chitobiase as an indicator of potential molting disruption in juvenile Daphnia magna exposed to the ecdysone receptor agonist 20-hydroxyecdysone

During arthropod molting, the old exoskeleton is degraded and recycled by the molting fluid. Chitobiase, a major chitinolytic enzyme in the molting fluid, has been widely used as a biomarker to indicate endocrine disruption of molting in arthropods under environmental stress. Although release of chitobiase was extensively studied in organisms exposed to molting-inhibiting chemicals, enzymic association with molting and response of the molting hormone receptor, ecdysone receptor (EcR), is not well understood. The present study was therefore conducted to identify potential linkages between release of chitobiase, molting frequency, and EcR activation in a freshwater crustacean Daphnia magna after short-term (96 hr) exposure to endogenous molting hormone 20-hydroxyecdysone (20E). A suite of bioassays was used for this purpose, including the chitobiase activity, molting frequency, viability, and in vitro EcR activation. Effect concentrations were compared between different assays analyzed. Results showed that exposure to 20E reduced chitobiase release and molting frequency in a concentration-dependent manner. Exposure to as low as 250 nM 20E significantly decreased release of chitobiase after 72 hr exposure, whereas adverse effects on molting frequency and incomplete molting-associated mortality required higher 20E exposure concentrations. The EcR reporter assay further demonstrated that as low as 100 nM 20E may activate EcR in vitro. Data suggest that release of chitobiase may be employed as a sensitive indicator of potential molting disruption in crustaceans after exposure to EcR agonists such as 20E.     

Nitric oxide inhibitor activity of 2-amino-2-thiazoline derivatives

A series of 2-amino-2-thiazoline derivatives have been synthesized and characterized with respect to NOS-inhibitor activity in vivo. It was established that the dimensions of the substituents in 2-N-mono-and 2-N,N-disubstituted 2-amino-2-thiazolines are not significant for the biological activity of the products. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 41, No. 12, pp. 18–20, December, 2007.     

Detection of C8-(1-hydroxyethyl)guanine in liver RNA and DNA from control and ethanol-treated rats

Alcohol consumption is associated with an increased risk of cancer by mechanisms that remain unknown but have been suggested to involve radical metabolites. We have previously shown that the 1-hydroxyethyl radical produced from ethanol oxidation in vitro is able to alkylate nucleic acids to produce C8-(1-hydroxyethyl)guanine [C8-(1-HE)gua] among other products. To assess if this adduct is produced in vivo, we developed a sensitive HPLC-MS/MS method for its detection and analyzed hydrolysates of liver RNA and DNA from control and ethanol-treated rats. Unexpectedly, C8-(1-HE)gua was found to be present in both RNA and DNA from the liver of control Sprague-Dawley rats, and its levels increased slightly, but not significantly, after an acute ethanol dose (5 g/kg). In rat liver, C8-(1-HE)gua endogenous levels were about 10 times higher in RNA (35 +/- 5/10(7) guanine) than DNA (3.7 +/- 1.1/10(7) guanine). These levels were also found in commercial RNA (calf liver and yeast) and DNA (calf thymus), further indicating the endogenous source of the adduct. DNA basal levels of C8-(1-HE)gua were similar to those reported for other 2C guanine adducts such as N7-(2-hydroxyethyl)guanine and N2-ethyl-2'-deoxyguanosine. We speculate that all of these adducts may be generated from DNA attack by products of basal lipid peroxidation. The higher RNA levels of C8-(1-HE)gua are in agreement with the higher accessibility of RNA and nucleotides to reactive intermediates because they are not as protected or as localized as DNA. Chemical modification of RNA has been receiving increasingly attention as an important event in genotoxic mechanisms. Comparison of RNA basal levels of C8-(1-HE)gua, N7-(2-hydroxyethyl)guanine, and N2-ethyl-2'-deoxyguanosine may provide clues about their endogenous sources and biological significance. Yet, the marginal increase of DNA C8-(1-HE)gua upon ethanol administration argues against this adduct playing a major role in the carcinogenic effects of ethanol.     

Analytical strategy for the detection of ecdysterone and its metabolites in vivo in uPA(+/+)-SCID mice with humanized liver,human urine samples,and estimation of prevalence of its use in anti-doping samples

Ecdysteroids are of interest as potential sport performance enhancers, due to their anabolic effects. The current study aimed to analyze levels of the most abundant ecdysteroid, ecdysterone (20-hydroxyecdysone, 20-OHE) in easily available dietary supplements, and, outline an analytical strategy for its detection, and that, of its metabolites, (1) following administration of pure 20-OHE to uPA(+/+)-SCID mice with humanized liver, (2) in a human volunteer after ingestion of two supplements, one with a relatively low, and the other a high, concentration of 20-OHE, and, (3) to estimate the prevalence of use of 20-OHE in elite athletes (n = 1000). Of the 16 supplements tested, only five showed detectable levels of 20-OHE, with concentrations ranging from undetectable up to 2.3 mg per capsule. Urine of uPA(+/+)-SCID urine showed the presence of 20-OHE and its metabolite, 14 deoxy ecdysterone, within 24 hours (hr) of ingestion. In humans, both the parent and the metabolite were detectable within 2 to 5 hr of ingestion, with the metabolite being detectable for longer than the parent. After ingestion of a low dose supplement, the parent and metabolite were detectable for 70 and 48 hr, while following the higher dose it was 96 and 48 hr, respectively. Analysis of urines from athletes (n = 1000) confirmed four positives for 20-OHE, suggesting a prevalence of use of 0.4%. Prevalence of its use by elite athletes was relatively low, however, this needs to be confirmed in other populations, and with other related ecdysteroids.     

Measurement of oxidative DNA damage by catechol estrogens and analogues in vitro

The growth-promoting effects of estrogens in hormone-dependent tumor tissues involve receptor-mediated pathways that are well-recognized; however, the role of estrogens in tumor initiation remains controversial. Estrogen metabolites, primarily the catechol estrogens (CE's), have been implicated in tumor initiation via a redox cycling mechanism. We have developed metabolically stable CE analogues for the study of receptor versus redox cycling effects on DNA damage. Comparisons between hydroxy estradiols (HE2's), methoxy estradiols (ME2's), and hydroxymethyl estradiols (HME2) in potentiometric and DNA damaging studies were made. DNA damage was assessed in calf thymus DNA using 8-oxo-2'-deoxyguanosine (8-oxo-dG) as a genotoxic marker for oxidative stress. Increases in the number of 8-oxo-dG/10(5) dG were significant for each 2-HE2 and 4-HE2. Cu(II)SO4, a transition metal known to catalyze the redox cycling of o-quinones, substantially increased the amount of DNA damage caused by both CE's. However, DNA damage was only observed at concentrations of 10 microM or higher, much greater than what is found under physiologic conditions. Furthermore, the presence of endogenous antioxidants such as glutathione, SOD, and catalase drastically reduced the amount of DNA damage induced by high concentrations of 2-HE2. There was no DNA damage observed for the non-redox cycling HME2's, making these compounds useful probes in the study of receptor-mediated carcinogenesis. Thus, both 2-HE2 and 4-HE2 are capable of producing oxidative DNA damage at micromolar concentrations in vitro. However, since the amount of CE's has not been shown to surpass nanomolar levels in vivo, it is unlikely that free radical production via redox cycling of CE's is a causative factor in human tumorigenesis.     

Synthesis and in vitro anti-Helicobacter pylori activity of 2-[(chlorobenzyl)thio]-5-(5-nitro-2-furyl)-1,3,4-thiadiazoles

A new series of 2-[(chlorobenzyl)thio]-5-(5-nitro-2-furyl)-1,3,4-thiadiazoles (6a–h) were synthesized and evaluated by the disc diffusion method against Helicobacter pylori. Four compounds which exhibited strong anti-H. pylori activity at concentration of 8–32 μg/disc (average of inhibition zone >20 mm) were further tested against 20 clinical isolates of H. pylori at lower concentrations. The averages of inhibition zone diameters indicated that all selected compounds exhibit better anti-H. pylori activity profile against clinical isolates of H. pylori with respect to standard drug metronidazole. Compound 6c, containing the 3-chlorobenzylthio moiety, was the most potent compound tested.     

Synthesis and antimicrobial activity of 2-aryloxy-N,N-diethylethanamine derivatives

A series of 2-aryloxy-N,N-diethyletanamine hydrochlorides were synthesized using the Williamson reaction. The antimicrobial activity of the synthesized compounds was evaluated by the double serial dilution technique with respect to S. aureus and E. coli. The tested substances exhibited a weak antimicrobial activity. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 41, No. 4, pp. 33–36, April, 2007.     

2-Methoxyestradiol and 2-ethoxyestradiol retard the progression of renal disease in aged, obese, diabetic ZSF1 rats

The metabolic syndrome is a main cause for cardiovascular disease and for the accelerating epidemic of chronic renal failure. Previous studies show that 2-hydroxyestradiol (2-HE), an estradiol metabolite with little estrogenic activity, decreases obesity and arterial blood pressure and attenuates the development of renal disease in young, obese, diabetic ZSF1 rats. In humans, however, diabetic renal disease is more frequent and severe in older patients. In vivo, 2-HE is readily converted to 2-methoxyestradiol (2-ME), an estradiol metabolite with no estrogenic activity. Accordingly, one purpose of this study was to determine whether 2-ME would provide benefit in aged rats with a very severe form of diabetic renal disease. Another objective was to determine whether synthetic analogs of estradiol metabolites might be beneficial in diabetic renal disease. To achieve these objectives we examined the effects of 2-ME and its analog 2-ethoxyestradiol (2-EE) in aged (35-week-old), obese ZSF1 rats. Animals were treated for 9 weeks with vehicle (PEG-400, 0.5 microL per hour), 2-ME or 2-EE (18 microg/kg per hour). Metabolic and renal function were measured at weeks 0, 3, 6, and 9, and renal hemodynamics and excretory function were assessed at week 9. Aged ZSF1 rats had elevated levels of glycosylated hemoglobin; increased renal cortical expression of proliferating cell nuclear antigen (PCNA), nuclear factor kappa B (NF-kappaB), and vascular endothelial growth factor (VEGF); glycosuria, hypertension; and proteinuria. 2-ME and 2-EE did not affect obesity or hypertension and had variable effects on glucose homeostasis, yet they attenuated proteinuria; increased renal blood flow and glomerular filtration; and reduced renal cortical expression of PCNA, NFkappaB, and VEGF. We conclude that 2ME and 2EE are strikingly renoprotective even in aged animals with severe diabetic renal disease. The present study warrants further investigation of 2-ME and analogs of estradiol metabolites for treatment of kidney disease associated with the metabolic syndrome.