Phytochemical profiling of Artocarpus lakoocha Roxb. leaf methanol extract and its antioxidant, antimicrobial and antioxidative activities

Objective: To explore the phytochemical profile of Artocarpus lakoocha Roxb. leaves both qualitatively and quantitatively, and validate its role as a potent antioxidant and antimicrobial agent. Methods: Extraction and isolation of different compounds were done from the leaves of Artocarpus lakoocha based on solvent fractionation method. Subsequently, quantitative and qualitative phytochemical profiling along with antioxidant, antimicrobial and antioxidative activities were tested following standard protocols. Results: Among the five fractions, methanol fraction of Artocarpus lakoocha exhibited higher content of phytochemical compounds [phenols = (3175.21依290.43) mg GAE/g dry extract, flavonoids = (1173.15依47.52) mg QE/g dry extract and tannins = (923.53依95.21) mg TAE/g dry extract] as compared to other fractions. The methanol fraction showed the highest antioxidant activity in DPPH and ABTS radical scavenging assays with IC50 of (111.98依34.20)μg/mL and (138.26依0.66) μg/mL, respectively, and the best reduction potential with a value of (316.81依2.96) mg QE/g dry extract in reducing power assay. There was significant correlation between the amount of phytochemicals and antioxidant activities. Moreover, the extract successfully protected Lambda phage DNA from damage at 5 and 6 mg/mL concentration and exhibited substantial bactericidal as well as fungicidal activity. The GC-MS analysis of methanol fraction of Artocarpus lakoocha revealed diethyl phthalate as the main phytochemical compound, along with 3,4-dihydroxymandelic acid, 9-octyl eicosane and 7,8-didehydro-3-methoxy-17-methyl-6-methylene morphinan. Conclusions: The methanol fraction of Artocarpus lakoocha could be used as a potent antioxidant and antimicrobial agent for sustainable agriculture and pharmaceutical purposes.

A conjugated polymer-peptide hybrid system for prostate-specific antigen (PSA) detection.

We developed fast and readily applicable microarray chips to detect PSA by designing a novel conjugated polymer (energy donor) and combining it with on-chip peptide synthesis. The selective cleavage of a probing peptide labelled with a dye or a quencher (energy acceptor) produced a fluorescence sensory signal via fluorescent energy resonance transfer (FRET).

Biocompatible Polymers: Structurally Controlled Bio-hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells (Adv. Mater. 48/2009).

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured biohybrid materials, report Joerg Lahann and co-workers on p. 4920. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.

Structurally Controlled Bio-hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells.

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured bio-hybrid materials. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.

Micropatterned fiber scaffolds for spatially controlled cell adhesion.

Because the local microstructure plays a pivotal role for many biological functions, a wide range of methods have been developed to design precisely engineered substrates for both fundamental biological studies and biotechnological applications. However, these techniques have been by-and-large limited to flat surfaces. Herein, we use electrohydrodynamic co-spinning to prepare biodegradable three-dimensional fiber scaffolds with precisely engineered, micrometre-scale patterns, wherein each fiber is comprised of two distinguishable compartments. When bicompartmental fiber scaffolds are modified via spatially controlled peptide immobilization, highly selective cell guidance at spatial resolutions (<10 µm), so far exclusively reserved for flat substrates, is achieved. Microstructured fiber scaffolds may have utility for a range of biotechnological applications including tissue engineering or cell-based assays.

Cytophobic surface modification of microfluidic arrays for in situ parallel peptide synthesis and cell adhesion assays.

A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid-phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence-specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.

Therapeutic use of quercetin in the control of infection and anemia associated with visceral leishmaniasis.

Flavonoids are a broad class of plant phenolics that are known to possess a well-established protective effect against membrane lipoperoxidative damages. Oxidative damage of erythrocytes has been implicated in the reduced survival of erythrocytes during leishmanial infection. This study reveals the efficacy of five naturally occurring flavonoids in arresting the development of anemia during the postinfection period. Among the compounds studied, quercetin was most successful in inhibiting the oxidation of proteins and lipids on the red cell membranes of infected animals. Apart from its antianemic property, quercetin also seemed to be highly potent in lowering the parasite load in the spleen. Combination therapy of quercetin with the antileishmanial drug stibanate produced a better decay of .OH in the erythrocytes of the infected animals compared to that induced by quercetin or drug treatment alone. Similar results were obtained in successful prevention of proteolytic degradation resulting in an aversion to early lysis of red cells after simultaneous treatment with quercetin and stibanate. Subsequent studies demonstrated the therapeutic efficacy of the combination treatment in the abatement of both anemia and parasitemia under the diseased condition.

Dihydrobetulinic acid induces apoptosis in Leishmania donovani by targeting DNA topoisomerase I and II: implications in antileishmanial therapy.

Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 microM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.

Targeting of liposomal flavonoid to liver in combating hepatocellular oxidative damage.

The efficacy of mannosylated liposome formulations with Quercetin (QC, a flavonoid antioxidant isolated from indigenous origin) has been tested in vivo against carbon tetrachloride(CCl(4))-induced liver oxidative damage in rats. Single subcutaneous injection of CCl(4) (40% v/v in olive oil; 1 ml/kg) induces the generation of toxic oxygen radicals and results in hepatocellular damage. The increased serum enzyme levels (glutamate pyruvate transaminase, alkaline phosphatase) and hepatocellular conjugated diene levels by CCl(4) induction were significantly lowered due to pretreatment with mannosylated liposomal QC (MLQ) (0.5 ml liposomal suspension containing 0.27 mg QC), whereas the same amount of free QC was found to be ineffective. In addition, the effectiveness of MLQ on CCl(4)-induced acute liver damage also was evaluated by tissue histopathological examination. Damage produced by CCl(4) in liver reverted to normal with pretreatment of MLQ.

Betulinic acid, a potent inhibitor of eukaryotic topoisomerase I: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives.

BACKGROUND: Betulinic acid, a naturally abundant, plant derived, pentacyclic triterpenoid possesses anti-HIV, anti-malarial and anti-inflammatory properties and has recently emerged as a potent anti-tumor compound. This study explores the mode of action of betulinic acid on eukaryotic topoisomerase I and identifies the major functional group responsible along with more potent derivatives. MATERIAL/METHODS: Topoisomerase I relaxation activity was electrophoretically measured by the decreased mobility of the relaxed monomers followed by ethidium bromide staining. DNA cleavage was studied by electrophoretic separation of the nicked monomers from the relaxed and supercoiled monomers in presence of ethidium bromide. In-vivo DNA cleavage was studied in blasted mouse splenocytes by the SDS-K+ trapping of 3H-DNA-topoisomerase I-camptothecin ternary complex. RESULTS: Betulinic acid exerts its inhibitory effect by preventing topoisomerase I-DNA interaction as a result of which the 'cleavable complex' is not formed. In consequence, it also acts as an antagonist to camptothecin-mediated cleavage. A series of analogues modified at C-3, C-17 and C-20 positions of betulinic acid were subsequently assayed for inhibition of topoisomerase I catalytic activity. Replacement of the 17-carboxylic group reduces the inhibitory effect and decarboxylation leads to the complete loss of inhibitory effect. CONCLUSIONS: This study is the first detail report of betulinic acid as a very potent inhibitior of eukaryotic topoisomerase I and highlights the necessity of the carboxylic functional group. Dihydro betulinic acid is the most potent (IC50=0.5 mM) pentacyclic triterpenoid to inhibit eukaryotic topoisomerase I till date and can be exploited as a strong candidate for anti-tumor drug designing.

Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I.

Luteolin, a naturally occurring flavonoid, is abundant in our daily dietary intake. It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets other than the fact that it induces topoisomerase II-mediated apoptosis. In the present study, we show that luteolin completely inhibits the catalytic activity of eukaryotic DNA topoisomerase I at a concentration of 40 microM, with an IC50 of 5 microM. Preincubation of enzyme with luteolin before adding a DNA substrate increases the inhibition of the catalytic activity (IC50=0.66 microM). Treatment of DNA with luteolin before addition of topoisomerase I reduces this inhibitory effect. Subsequent fluorescence tests show that luteolin not only interacts directly with the enzyme but also with the substrate DNA, and intercalates at a very high concentration (>250 microM) without binding to the minor groove. Direct interaction between luteolin and DNA does not affect the assembly of the enzyme-DNA complex, as evident from the electrophoretic mobility-shift assays. Here we show that the inhibition of topoisomerase I by luteolin is due to the stabilization of topoisomerase-I DNA-cleavable complexes. Hence, luteolin is similar to camptothecin, a class I inhibitor, with respect to its ability to form the topoisomerase I-mediated 'cleavable complex'. But, unlike camptothecin, luteolin interacts with both free enzyme and substrate DNA. The inhibitory effect of luteolin is translated into concanavalin A-stimulated mouse splenocytes, with the compound inducing SDS-K+-precipitable DNA-topoisomerase complexes. This is the first report on luteolin as an inhibitor of the catalytic activity of topoisomerase I, and our results further support its therapeutic potential as a lead anti-cancer compound that poisons topoisomerases.