Structure and biological activity of a new rotenoid from Pongamia pinnata

Pongarotene (1), a new rotenoid and karanjin (2), a known flavonol, were isolated from the seeds of Pongamia pinnata. The structure determination of these compounds were based on spectral analyses including 2D-NMR. The antifungal, antibacterial and phytotoxicity results of pure compounds 1 and 2 as well as of the methanol (M) and ethyl acetate (E) crude extracts are also being reported.     

The coumarin psoralidin enhances anticancer effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Coumarins are a very common type of secondary plant metabolites with a broad spectrum of biological activities. Psoralidin is a naturally occurring furanocoumarin isolated from Psoralea corylifolia possessing anticancer and chemopreventive properties. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in cancer cells with no toxicity toward normal tissues. Endogenous TRAIL plays an important role in immune surveillance and defence against cancer cells. Coumarins can modulate TRAIL-mediated apoptosis in cancer cells. We examined the cytotoxic and apoptotic activities of psoralidin in combination with TRAIL on HeLa cancer cells. The cytotoxicity was measured by MTT and LDH assays. The apoptosis was detected using annexin V-FITC staining and mitochondrial membrane potential was evaluated using DePsipher staining by fluorescence microscopy. Death receptor (TRAIL-R1/DR4 and TRAIL-R2/DR5) expression was analyzed using flow cytometry. Psoralidin enhanced TRAIL-induced apoptosis in HeLa cells through increased expression of TRAIL-R2 death receptor and depolarization of mitochondrial membrane potential. Our study indicated that psoralidin augmented the anticancer effects of TRAIL and confirmed a potential use of coumarins in cancer chemoprevention.     

红树植物水黄皮pongamia pinnata中一个倍半萜类化合物的结构解析

本研究应用葡聚糖凝胶LH-20,硅胶柱层析等手段,从红树植物水黄皮中分离纯化了1个倍半萜类化合物,结合多种波谱方法(ESI MS, 1D NMR, 2D NMR, UV),确定它的结构为:6,7-Epoxy-3(15)-caryophyllen-12-ol(化合物1).该化合物为首次从红树植物水黄皮中分离得到.     

TLC Based Method for Standardization of Pongamia pinnata (Karanj) Using Karanjin as Marker

Pongamia pinnata Linn. (Papilionaceae) seeds have gained great commercial and industrial importance owing to their high oil content. Presently, there is no appropriate TLC based method available for standardization of P. pinnata. A simple, robust and reproducible TLC method for the determination of Karanjin is reported in the seeds of P. pinnata. The method involves separation of components by TLC on pre-coated silica gel G 60 F₂₅₄ plates developed on toluene: ethyl acetate (7:3 v/v) and detection at 260 nm in absorbance mode. The sensitivity of the method was found to be 100 ng. The linearity range was 50–300 ng. Four samples of P. pinnata from different geographical locations were tested for their karanjin content using the developed method. The proposed method was found to be robust, precise, and accurate, it therefore holds potential for detection, monitoring and quantification of karanjin in Pongamia pinnata.

     

Development of Flow Cytometric Protocol for Nuclear DNA Content Estimation and Determination of Chromosome Number in Pongamia pinnata L., a Valuable Biodiesel Plant

The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays ‘CE-777’ as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and β-mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for P. pinnata leaves from different altitudes (37–117 m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root–tip cells and was found to be 2n?=?22 corresponding to the diploid level (x?=?11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.     

Development of a polymer system for the delivery of daunorubicin to tumor cells to overcome drug resistance

Method for the synthesis of polymeric nanoparticles (NP) with encapsulated daunorubicin (DNR) was developed on the basis of double emulsion solvent evaporation technique using biodegradable poly(lactide-co-glycolide) (PLGA), which is aimed at customization of pharmacokinetic properties of the preparation, enhanced accumulation of DNR in tumor cells and prolongation of its action. The obtained polymer nanoparticles (DNR-PLGA) had average size ranging around 138±36 nm, with zeta-potential of –25.3 mV and the polydispersity index (PDI) of 0.072. The release kinetics of DNR from polymer nanoparticles at pH 7.4 and 5.0 has been studied. In vitro studies showed similar specific activity of DNR- PLGA in K562 and MCF-7 cancer cell lines together with an increase in activity in K562 Adr and MCF-7 Adr cell lines, which are anthracycline resistant, by 1.6 and 3.4 times. The study demonstrated the efficacy of the developed PLGA-based DNR delivery system in the improvement of antitumor effect of DNR, overcoming multidrug resistance in cancer cells, and also in the decrease in nonspecific toxicity of the preparation.     

Inhibition effect of environmentally benign Karanj (Pongamia pinnata) seed extract on corrosion of mild steel in hydrochloric acid solution

The inhibition of the corrosion of mild steel in hydrochloric acid solution by the seed extract of Karanj (Pongamia pinnata) has been studied using weight loss, electrochemical impedance spectroscopy, potentiodynamic polarization, and linear polarization techniques. Inhibition was found to increase with increasing concentration of the extract. The effect of temperature, immersion time, and acid concentration on the corrosion behavior of mild steel in 1 M HCl with addition of extract was also studied. The adsorption of the extract on the mild steel surface obeyed the Langmuir adsorption isotherm. Values of inhibition efficiency calculated from weight loss, potentiodynamic polarization, and electrochemical impedance spectroscopy are in good agreement. Polarization curves showed that Karanj (P. pinnata) seed extract behaves as a mixed-type inhibitor in hydrochloric acid. The activation energy as well as other thermodynamic parameters for the inhibition process was calculated. The adsorbed film on mild steel surface containing Karanj (P. pinnata) seed extract inhibitor was also measured by Fourier transform infrared spectroscopy. The results obtained showed that the seed extract of Karanj (P. pinnata) could serve as an effective inhibitor of the corrosion of mild steel in hydrochloric acid media.     

Utilisation of Non-Edible Source (Pongamia pinnata Seeds Shells) for Producing Methyl Esters as Cleaner Fuel in the Presence of a Novel Heterogeneous Catalyst Synthesized from Waste Eggshells

Waste eggshells were considered for synthesising a precursor (CaO) for a heterogeneous catalyst, further impregnated by alkali caesium oxide (Cs₂O). The following techniques were used to characterise the synthesised catalysts: X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and Temperature Programmed Desorption (CO₂-TPD). The synthesised catalyst revealed its suitability for transesterification to produce biodiesel. The biodiesel production process was optimised, and it showed that the optimal biodiesel yield is 93.59%. The optimal set of process parameters is process temperature 80 °C, process time 90 min, methanol-to-oil molar ratio 8 and catalyst loading 3 wt.%. It has been found that the high basicity of the catalyst tends to give a high biodiesel yield at low methanol-to-oil ratio 8 when the reaction time is also less (90 min). The fuel properties of biodiesel also satisfied the standard limits defined by ASTM and the EN standards. Thus, the synthesised catalyst from waste eggshells is highly active, improved the biodiesel production conditions and PPSS oil is a potential nonedible source.     

Synthesis of oxovanadium complexes and their apoptosis-inducing activity in leukemia cells

Vanadium complexes with different ligands were synthesized and evaluated for antiproliferative activity on U937 cells. The alkyl chain length of the ligands affected the antiproliferative activity, and two complexes-3b and 4-exhibited strong activities with IC(50) values of 6.02 and 3.90 μM respectively. Annexin V staining and DNA ladder formation indicated that these complexes induced apoptosis in U937 cells.     

Development of a liquid chromatography-based screening methodology for proteolytic enzyme activity

A new methodology for the detection and isolation of serine proteases in complex mixtures has been developed. It combines the characterization of crude samples by electrospray tandem mass spectrometry (ESI-MS/MS) in a multi-substrate assay and the differentiated sensitive detection of the responsible enzymes by means of liquid chromatography hyphenated online to biochemical detection (BCD). First, active samples are identified in the multi-substrate assay monitoring the conversion of eight substrates in multiple reaction monitoring in parallel within 60 s. Hereby, the product patterns are investigated and the suitable peptide as substrate for BCD analysis is selected. Subsequently, the active proteases are identified online in the continuous-flow reactor serving as BCD after non-denaturing separation by size-exclusion chromatography and ion-exchange chromatography. For BCD, the selected para-nitroaniline (pNA) labeled peptide is added post-column and is cleaved by eluting proteases under release of the coloured pNA in a reaction coil (reaction time 5 min). The method was optimized and the figures of merit were characterized with trypsin and chymotrypsin serving as the model proteases. For trypsin, a limit of detection in LC–BCD of 0.1 U/mL corresponding to an injected amount of 0.4 ng protein (∼18 fmol) was observed. The BCD signal remained linear for an injected enzyme concentration of 0.3–10 U/mL (1.3–42 ng enzyme). The method was applied to the characterization of the crude venom of the pit viper Bothrops moojeni and the extracellular protease of the pathogenic amoeba Acanthamoeba castellanii. In the two samples, fractions with proteolytic activity potentially interfering with the blood coagulation cascade were identified. The described methodology represents a tool for serine protease screening in complex mixtures by a fast ESI-MS/MS identification of active samples followed by the separation and isolation of active sample constituents in LC–BCD.     

支持向量机法用于4-芳基-4氢-苯并比喃化合物的凋亡诱导活性研究

通过计算获得了39个4-芳基-4氢.苯并吡喃化合物的11个结构特征参数,应用逐步线性回归方法对参数进行筛选.并用支持向量回归(SVR)算法,对4-芳基-4氢-苯并吡喃化合物与其凋亡诱导活性进行定量构效关系(QSAR)研究.通过核函数参数的优化,建立了预测模型,训练集和预测集的实验值与计算值的平方相关系数R2分别为0.997和0.893.研究结果表明,支持向量回归算法可用于小样本数的药物分子设计研究,以合成具有更高生物活性的新药物.     

Evaluation of a two-stage screening procedure in the combinatorial search for serine protease-like activity

A series of peptidosteroid derivatives containing two independent peptide chains in which Ser and His are incorporated were synthesized by solid-phase peptide synthesis. The activity of the different compounds in the hydrolysis of the activated substrate NF31 was assessed in a stepwise fashion. First, the different resin-bound derivatives 6a-l and 6x-z were individually assayed for serine esterification in the absence of water. The use of a colored substrate allowed for a visual identification of the most active compounds. Through the inclusion of control substances, the involvement of histidine in the mechanism for serine acylation was shown. Second, the hydrolysis and methanolysis of the different acylated derivatives 8a-l and 8x were evaluated using UV spectroscopy, again indicating the involvement of histidine. The feasibility of applying the above procedures in a combinatorial context was proven via the screening of artificial libraries, created by mixing the different resin-bound peptidosteroid compounds. In this respect, the use of a photocleavable linker allowed for the unambiguous structural characterization of the selected members via application of single-bead electrospray tandem mass spectrometry.     

Identification of a selectivity determinant for inhibition of tumor necrosis factor-alpha converting enzyme by comparative modeling

Inhibition of tumor necrosis factor-alpha converting enzyme (TACE) is a widespread objective in the search for disease modifying agents to combat rheumatoid arthritis and other autoimmune diseases. Until recently, most of the inhibitors in the literature have shown concomitant activity against the related matrix metalloproteinases (MMPs), producing undesired side effects. Here we describe the successful search for a TACE selectivity mechanism. We built a homology model based on the crystal structure of the related snake venom protein atrolysin. Comparison of the model with crystal structures of MMPs suggested a uniquely shaped S1' pocket that might be exploited for selectivity. A novel gamma-lactam scaffold was used to explore the activity profile of P1' sidechains, resulting in highly selective compounds consistent with this hypothesis. Transferability of the hypothesis was then demonstrated with five other distinct scaffolds.     

Induction of tumor necrosis factor-alpha by UVB: a role for reactive oxygen intermediates and eicosanoids

UVB irradiation induces nuclear factor-kappaB (NF-kappaB) activation, tumor necrosis factor-alpha (TNF-alpha) expression and reactive oxygen intermediates (ROI) in keratinocytes. We investigated whether ROI play a role in UVB-induced TNF-alpha mRNA expression. The antioxidants N-acetyl cysteine, NAC, epigallocathin gallate, EGCG, butylated hydroxyanisole (BHA) and vitamin C could reduce UVB-induced TNF-alpha mRNA levels to various degrees; vitamin E (alpha-tocopherol) had no effect. BHA was the most potent inhibitor. The oxidant tertiary butylated hydroperoxide could effectively induce TNF-alpha mRNA expression. Nordihydroguaiaretic acid (NDGA) and MK-886, inhibitors of lipoxygenase (LOX), and indometacin and quinacrine, inhibitors of cyclooxygenase (COX) and phospholipase A2, respectively, could also reduce UVB-induced TNF-alpha mRNA expression. Inhibition by NDGA was in concordance with the results for BHA. NDGA, indometacin, quinacrine and BHA could also effectively inhibit the inhibitor of NF-kappaB degradation, thereby maintaining NF-kappaB inactivity. In conclusion, we show that ROI are implicated in the induction of TNF-alpha mRNA by UVB and that not all antioxidants are equally effective inhibitors. COX products and more importantly LOX products, which themselves are products of an oxidative metabolism, are the main ROI implicated in this induction of TNF-alpha expression by UVB probably via activation of NF-kappaB.     

Critical evaluation of a screening test for detection of herbicides by inhibiting photosynthesis of isolated chloroplasts

The possibility to determine herbicides in the aqueous environment by observation of inhibiting effects on the photosynthesis of isolated chloroplasts have been critically evaluated. The photosynthetical activities of freshly isolated chloroplasts from spinach (Spinacia oleracea L.) and peas (Pisum sativum L.) have been compared under well-defined conditions. The standard for the rate of photosynthesis was the oxygen evolution, which has been detected by a Clark-type electrode. The ratio between the oxygen production rate before and after a sample addition describes the chloroplast activity. In the presence of herbicides this ratio decreases. Increasing herbicide concentrations have been determined by a normalization procedure in semilogarithmic scales using a sigmoidal calibration plot.Photosynthesis inhibiting substances, both natural and anthropogenic, can be detected collectively. That is an advantage over enzyme immunoassays which can only detect single herbicides. Clearly disadvantageous are the insufficient detection limits (e.g. 8.9 g/l for atrazine, respectively 2.5 g/l for terbuthylazine) and therefore the necessary preconcentration.     

Kinase-catalyzed modification of gold nanoparticles: a new approach to colorimetric kinase activity screening

Peptide-stabilized gold nanoparticles have been enzymatically biotinylated by a kinase-catalyzed reaction using biotin-ATP as a cosubstrate. Upon mixing with avidin-modified particles, solutions of biotinylated particles change color from red to blue, indicating aggregation of particles. On the basis of this reaction, we have developed a simple colorimetric test to monitor kinase inhibitor activity.     

Development of a high-throughput screening method for racemase activity and its application to the identification of alanine racemase variants with activity towards l-arginine

A high-throughput solid phase screening method has been developed for alanine racemase. Conversion of an l-amino acid to the d-amino acid is detected by a d-amino acid oxidase, which is utilised as a reporter enzyme along with horseradish peroxidase and a colourimetric dye. This system has successfully been used to identify a mutant with increased activity towards arginine. Kinetic analysis and modelling studies have been performed to understand the effect of the mutations found.     

Novel synthetic luteolin analogue-caused sensitization of tumor necrosis factor-alpha-induced apoptosis in human tumor cells

Studies on the sensitization, by novel alkynyl luteolin analogues, of TNF-alpha-induced apoptosis in HeLa and HepG2 cells revealed that LA-12 showed better sensitizing effects on TNF-alpha-induced cell death than luteolin, suggesting great potential for alkynyl luteolin analogues in cancer therapy.     

2-D solid-state assay platform: a tool for screening aldehyde-releasing enzyme activity in colonies

A micro colony chip-based platform for high-throughput screening of proton releasing or oxygen consuming enzyme reactions was introduced recently. Here we present a new assay platform which allows screening for the release of aromatic and aliphatic aldehydes by enzymatic activity. The assay platform consists of 4-hydrazino-7-nitrobenzofurazane (NBD-H) as an indicator embedded in a soft, hydrophobic polymer matrix. NBD-H forms highly fluorescent products with aldehydes via hydrazone formation. The assay was characterized for various aliphatic and aromatic aldehydes and tested with two enzymatic reactions to show its potential. A transesterification reaction under water-free conditions with two esterase mutants and vinyl acetate as acyl donor leads to acetaldehyde as detectable product and a reaction under aqueous conditions with threonine aldolases. The substrates used are phenylserine, which forms benzaldehyde, and threonine, which forms acetaldehyde.