Bacopa monnieri reduces Tau aggregation and Tau-mediated toxicity in cells.

Alzheimer's disease is a neurodegenerative disease characterized by progressive memory loss and behavioral impairments. In the present study, the ethanolic extract of Bacopa monnieri was studied for its potency to inhibit Tau aggregation and rescuing of the viability of Tau-stressed cells. Bacopa monnieri was observed to inhibit the Tau aggregation in vitro. The cells exposed to Bacopa monnieri were also observed to have a low level of ROS and caspase-3 activity. The immunoblot and immunofluorescence analysis showed that Bacopa monnieri acts as an antioxidant and restored the Nrf2 levels in Neuro2a cells. Bacopa monnieri treatment to Neuro2a cells was observed to reduce the phospho-Tau load in formaldehyde-stressed cells. Furthermore, the treatment of Bacopa monnieri reduced the phosphorylation of GSK-3ß in formaldehyde-stressed cells. Ran and NUP358 are the key proteins involved in nuclear transport. It was observed that formaldehyde treatment impaired the nuclear transport by missorting the NUP358 arrangement in Neuro2a cells. On the contrary, Bacopa monnieri treatment restored the NUP358 arrangement in cells. The overall results of the present study suggested that Bacopa monnieri could be considered a potent herb against Tau phosphorylation and Tau aggregation, which projects it as a promising formulation for Alzheimer's disease.

Improvement in serum amylase and glucose levels in diabetic rats on oral administration of bisdemethoxycurcumin from Curcuma longa and limonoids from Azadirachta indica.

Curcuma longa and Azadirachta indica are traditionally used in Indian cuisine and Ayurvedic medicine as nutraceuticals against diabetes. The crude C. longa isopropanol extract, bisdemethoxycurcumin (BDMC), the purified bioactive component from C. longa, and limonoids azadiradione, gedunin from A. indica, are able to inhibit in vitro the antidiabetic target human pancreatic α-amylase independently. However, no reports on their in vivo efficacy in animal models exist. Thus, the antidiabetic effect of these orally administered human pancreatic α-amylase inhibitors was performed on streptozotocin-induced Sprague-Dawley rats. Initially, the normal rats were treated with test compounds (10-100 mg/kg of body weight) in corn oil (5 ml/kg), and as no lethality was observed in these doses, further studies were carried out with lowest concentration of 10 mg/kg of body weight. A reduction in area under curve (AUC) suggested glucose-lowering effect of these compounds in starch fed diabetic rats. The efficacy study showed a significant improvement in body weight, blood glucose levels, serum amylase, and fructosamine levels as well in other serum parameters associated with diabetes with respect to liver and renal functions. Hence, under in vivo conditions, inhibition of α-amylase activity by BDMC and limonoids affirms it as one of the mechanisms of action resulting in reduction of blood glucose levels. PRACTICAL APPLICATIONS: Bisdemethoxycurcumin from C. longa and limonoids, namely, azadiradione and gedunin, from A. indica are potent inhibitors of the antidiabetic target human pancreatic α-amylase. Oral Starch Tolerance Test (OSTT) and 28-day efficacy study to check the effect of these orally administered inhibitors in diabetic rat models showed significant improvements in serum blood glucose and amylase levels as well as in other diabetes related serum parameters, namely, bilirubin, lipids, lactate dehydrogenase, alkaline phosphatase, and urea. The study contributes to understanding the action and efficacy of these pancreatic α-amylase inhibitors and suggests a potential role for them as nutraceuticals/therapeutics in management of post-prandial hyperglycemia.

Role of Jasmonate in Modulation of Mycorrhizae-Induced Resistance Against Fungal Pathogens.

Symbiotic association of plants with arbuscular mycorrhizal (AM) fungi brings about changes in levels of the phytohormone jasmonate (JA) in root and shoot tissues of a plant. The enhanced JA levels not only play a role in controlling the extent of AM colonization but are also involved in the expression of mycorrhizal-induced resistance (MIR) against pathogens. We describe a method used to study the levels of a volatile jasmonate derivative, methyl jasmonate (MeJA), in tomato plants colonized by AM fungi and in response to subsequent attack by the foliar pathogen Alternaria alternata.

Enhancing epi-cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi-cedrol synthase.

Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi-cedrol synthase (AaECS). The stop codon at the N-terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS-ECS produced sesquiterpene 8-epi-cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K m values for FPPS-ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi-cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi-cedrol in fermentation medium. The strain having fusion plasmid (pET32a-FPPS-ECS) produced 1.084 ± 0.09 mg/L epi-cedrol, while the strain harbouring mixed plasmid (pRSETB-FPPS and pET28a-ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I-TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N- C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.

Camptothecin-producing endophytic bacteria from Pyrenacantha volubilis Hook. (Icacinaceae): A possible role of a plasmid in the production of camptothecin.

BACKGROUND: Camptothecin (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this property, several derivatives of CPT are used as chemotherapeutic agents. CPT is produced by several plant species belonging to the Asterid clade as well as by a number of endophytic fungal associates of these plants. In this study, we report the production of CPT by four bacterial endophytes and show the possible role of a plasmid in the biosynthesis of CPT. METHODS: Endophytic bacteria were isolated from leaves, stems and fruits of Pyrenacantha volubilis Hook. (Icacinanceae). The bacterial isolates were purified and analyzed for production of CPT by ESI-MS/MS and NMR analysis. Bacterial identity was established based on the morphology and 16s rRNA sequence analysis. Crude extracts of the bacterial endophytes were evaluated for their cytotoxicity using colon cancer cell lines. The role of plasmid in the production of CPT was studied by purging the plasmid, using acriflavine, as well as reconstituting the bacteria with the plasmid. RESULTS: Four bacterial isolates, Bacillus sp. (KP125955 and KP125956), Bacillus subtilis (KY741853) and Bacillus amyloliquefaciens (KY741854) were found to produce CPT in culture. Both based on ESI-MS/MS and NMR analysis, the identity of CPT was found to be similar to that produced by the host plant. The CPT was biologically active as evident by its cytotoxicity against colon cancer cell line. The production of CPT by the endophyte (Bacillus subtilis, KY741853) attenuated with sub-culture. A likely role of a plasmid in the production of CPT was established. A 5 kbp plasmid was recovered from the bacteria. Bacterial isolate cured of plasmid failed to produce CPT. CONCLUSION: Our study implies a possible role of a plasmid in the production of CPT by the endophytic bacteria and opens up further work to unravel the exact mechanisms that might be involved.

Acetone and methanol fruit extracts of Terminalia paniculata inhibit HIV-1 infection in vitro.

In this study, we report the in vitro anti-HIV1 activity of acetone and methanol extracts of fruit of Terminalia paniculata. Cytotoxicity tests were conducted on TZM-bl cells and peripheral blood mononuclear cells (PBMC), the CC50 values of both the extracts were ≥260 µg/mL. Using TZM-bl cells, the extracts were tested for their ability to inhibit replication of two primary isolates HIV-1 (X4, Subtype D) and HIV-1 (R5, Subtype C). The activity against HIV-1 primary isolate (R5, Subtype C) was confirmed using activated PBMC and by quantification of HIV-1 p24 antigen. Both the extracts showed anti-HIV1 activity in a dose-dependent manner. The EC50 values of the acetone and methanol extracts of T. paniculata were ≤10.3 µg/mL. The enzymatic assays were performed to determine the mechanism of action which indicated that the anti-HIV1 activity might be due to inhibition of reverse transcriptase (≥77.7% inhibition) and protease (≥69.9% inhibition) enzymes.

Systemic jasmonic acid modulation in mycorrhizal tomato plants and its role in induced resistance against Alternaria alternata.

Tomato plants colonised with the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum show systemic induced resistance to the foliar pathogen Alternaria alternata, as observed in interactions of other AM-colonised plants with a range of pathogens. The role of jasmonic (JA) and salicylic (SA) acid in expression of this mycorrhiza-induced resistance (MIR) against A. alternata was studied by measuring: (i) activity of enzymes reported to be involved in their biosynthesis, namely lipoxygenase (LOX) and phenylammonia lyase (PAL); and (ii) levels of methyl jasmonate (MeJA) and SA. Transcript abundance of some defence genes associated with JA and SA response pathways were also studied. Both LOX and PAL activity increased twofold in response to pathogen application to control plants. AM-colonised plants had three-fold higher LOX activity compared to control plants, but unlike controls, this did not increase further in response to pathogen application. Higher LOX activity in AM-colonised plants correlated with four-fold higher MeJA in leaves of AM-colonised plants compared to controls. Treatment of plants with the JA biosynthesis inhibitor salicylhydroxamic acid (SHAM) led to 50% lower MeJA in both control and AM-colonised plants and correlated with increased susceptibility to A. alternata, suggesting a causal role for JA in expression of MIR against the pathogen. Genes involved in JA biosynthesis (OPR3) and response (COI1) showed six- and 42-fold higher expression, respectively, in leaves of AM-colonised plants compared to controls. AM-colonised plants also showed increased expression of the SA response gene PR1 and that of the wound-inducible polypeptide prosystemin. Our results suggest that the systemic increase in JA in response to AM colonisation plays a key role in expression of MIR against A. alternata.

Way toward "dietary pesticides": molecular investigation of insecticidal action of caffeic acid against Helicoverpa armigera.

Bioprospecting of natural molecules is essential to overcome serious environmental issues and pesticide resistance in insects. Here we are reporting insights into insecticidal activity of a plant natural phenol. In silico and in vitro screening of multiple molecules supported by in vivo validations suggested that caffeic acid (CA) is a potent inhibitor of Helicoverpa armigera gut proteases. Protease activity and gene expression were altered in CA-fed larvae. The structure-activity relationship of CA highlighted that all the functional groups are crucial for inhibition of protease activity. Biophysical studies and molecular dynamic simulations revealed that sequential binding of multiple CA molecules induces conformational changes in the protease(s) and thus lead to a significant decline in their activity. CA treatment significantly inhibits the insect's detoxification enzymes, thus intensifying the insecticidal effect. Our findings suggest that CA can be implicated as a potent insecticidal molecule and explored for the development of effective dietary pesticides.

Andrographolide inhibits osteopontin expression and breast tumor growth through down regulation of PI3 kinase/Akt signaling pathway.

Breast cancer is one of the most common cancers among women in India and around the world. Despite recent advancement in the treatment of breast cancer, the results of chemotherapy to date remain unsatisfactory, prompting a need to identify natural agents that could target cancer efficiently with least side effects. Andrographolide (Andro) is one such molecule which has been shown to possess inhibitory effect on cancer cell growth. In this study, Andro, a natural diterpenoid lactone isolated from Andrographis paniculata has been shown to inhibit breast cancer cell proliferation, migration and arrest cell cycle at G2/M phase and induces apoptosis through caspase independent pathway. Our experimental evidences suggest that Andro attenuates endothelial cell motility and tumor-endothelial cell interaction. Moreover, Andro suppresses breast tumor growth in orthotopic NOD/SCID mice model. The anti-tumor activity of Andro in both in vitro and in vivo model was correlated with down regulation of PI3 kinase/Akt activation and inhibition of pro-angiogenic molecules such as OPN and VEGF expressions. Collectively, these results demonstrate that Andro may act as an effective anti-tumor and anti-angiogenic agent for the treatment of breast cancer.

Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639.

The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20 °C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400 kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25 °C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate.

Identification of two flavin monooxygenases from an effluent treatment plant sludge metagenomic library.

Oxygenases are useful for the production of many industrially important molecules. Screening of an effluent treatment plant (ETP) sludge metagenomic library identified two clones encoding proteins, B1 and B2, with similarity to putative flavin monooxygenases from Mesorhizobium loti and Sphingomonas wittichi, respectively. The deduced amino acid sequences show only 20% identity, but both have a paired Rossman fold and a flavin monooxygenase (FMO) motif. B1 and B2 appear to be members of the flavin-containing monooxygenase and the Baeyer-Villiger monooxygenases subfamilies, respectively. When expressed in Escherichia coli, the two clones produced activities that oxidized indole to a mixture of indigo and indirubin pigments. These results suggest that B1 and B2 have potential as a biocatalyst in indigo/indirubin production.

Effect of ring size in R-(+)-pulegone-mediated hepatotoxicity: studies on the metabolism of R-(+)-4-methyl-2-(1-methylethylidene)-cyclopentanone and DL-camphorone in rats.

R-(+)-Pulegone, a monoterpene ketone, is a potent hepatotoxin. The present study was designed to evaluate whether the reduction of the ring size in R-(+)-pulegone would affect its mode of metabolism and its hepatotoxic potential. Metabolic fate of R-(+)-4-methyl-2-(1-methylethylidene)-cyclopentanone (I) and 5-methyl-2-(1-methylethylidene)-cyclopentanone (DL-camphorone; II) were examined in rats. Compounds I and II were administered orally (250 mg/kg of b.wt./day) to rats for 5 to 7 days. The following metabolites were isolated and identified from the urine of rats dosed with I: 3-methyl-5-(1-methylethylidene)-cyclopent-2-enone (Ie), Z-4-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (Ib), E-4-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (Ia), 3-hydroxy-4-methyl-2-(1-methylethylidene)-cyclopentanone (If), 4-hydroxy-4-methyl-2-(1-methylethylidene)-cyclopentanone (Ic), and E-4-methyl-2-(1-carboxyethylidene)-cyclopentanone (Id). Phenobarbital (PB)-induced rat liver microsomes in the presence of NADPH transformed compound I into metabolites, which were identified as Ia, Ib, Ic, Ie, and If. The following urinary metabolites were isolated and identified from compound II: 5-hydroxy-5-methyl-2-(1-methylethylidene)-cyclopentanone (IIc), 5-hydroxy-5-methyl-2-(1-methylethyl)-cyclopentanone (IIg), Z-5-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (IIb), 5-methyl-2-(1-hydroxymethylethyl)-cyclopentanone (IIf), E-5-methyl-2-(1-hydroxymethylethylidene)-cyclopentanone (IIa), E-5-methyl-2-(1-carboxyethylidene)-cyclopentanone (IId), and 5-methyl-2-(1-carboxyethyl)-cyclopentanone (IIe). PB-induced rat liver microsomes in the presence of NADPH were shown to transform compound II to IIa, IIb, and IIc. Studies carried out in vitro demonstrated that hydroxylation at the tertiary carbon atom or oxidation of the isopropylidene methyl groups in II can be specifically blocked through structural modifications as seen in compounds 2,2-dimethyl-5-(1-methylethylidene)-cyclopentanone (III) and 5-methyl-2-(1-ethyl-1-propylidene)-cyclopentanone (IV). Similar observation was also made when isopropylidene methyl groups in R-(+)-pulegone were replaced by ethyl groups. Intraperitoneal administration of a single dose (250 mg/kg) of I and II to rats did not elicit hepatotoxicity as judged by serum alanine aminotransaminase levels and liver microsomal drug metabolizing enzyme activities.

Role of C-5 chiral center in R-(+)-pulegone-mediated hepatotoxicity: metabolic disposition and toxicity of 5, 5-dimethyl-2-(1-Methylethylidene)-cyclohexanone in rats.

Metabolic disposition of 5, 5-dimethyl-2-(1-methylethylidene)-cyclohexanone (I) was examined in rats. Compound (I) was administered orally (250 mg/kg of body weight/day) to rats for 5 days. The following urinary metabolites were isolated and identified: 4,5,6,7-tetrahydro-3,6, 6-trimethylbenzofuran (III), 3,3-dimethylcyclohexanone (VI), 5, 5-dimethyl-3-hydroxy-2-(1-methylethylidene)-cyclohexanone (X), 5, 5-dimethyl-2-(1-hydroxymethylethyl)-cyclohexanone (IX), 3-hydroxy-5-hydroxymethyl-5-methyl-2-(1-methylethylidene)-cyclo hexano ne (XI), 5,6-dihydro-3,6,6-trimethyl-2(4H)-benzofuranone (VIII), and 5,5-dimethyl-3-hydroxy-2-(1-carboxy ethylidene)-cyclohexanone (XIII). Incubation of compound (I) with phenobarbital (PB)-induced rat liver microsomes in the presence of NADPH resulted in the formation of a metabolite, tentatively identified as a furanoterpene (III) based on proton magnetic resonance, gas chromatography, and gas chromatography-mass spectroscopy analyses. The formation of III was inhibited to a significant extent by carbon monoxide, metyrapone, SKF 525-A, and cytochrome c, suggesting the participation of PB-induced microsomal cytochrome P-450 system in the conversion of I to III. Compound I gave type I spectral change in the PB-induced liver microsomes and the dissociation constant (Ks) for I was 38.5 microM. Intraperitoneal administration of a single dose (250 mg/kg) of I to rats resulted in 26, 23, and 41% decreases in the levels of cytochrome P-450, glucose-6-phosphatase, and aminopyrine N-demethylase, respectively, at the end of 24 h. During this period, a 11-fold increase in serum glutamate pyruvate transaminase level was also observed. However, a decrease in the level of cytochrome P-450 and glucose-6-phosphatase, and an increase in serum glutamate pyruvate transaminase values were comparatively more pronounced when R-(+)-pulegone (250 mg/kg) or CCl(4) (0.6 ml/kg) was administered to rats. Pretreatment of rats with PB potentiated the hepatotoxicity caused by I, whereas pretreatment with 3-methylcholanthrene protected from it. This suggests that PB-induced cytochrome P-450-catalyzed reactive metabolites may be responsible for the toxic effects caused by I.

Transformation of a monoterpene ketone, (R)-(+)-pulegone, a potent hepatotoxin, in Mucor piriformis.

Biotransformation of a monoterpene ketone, (R)-(+)-pulegone (I), a potent hepatotoxin, was studied using a fungal strain, Mucor piriformis. Eight metabolites, namely, 5-hydroxypulegone (II), piperitenone (III), 6-hydroxypulegone (IV), 3-hydroxypulegone (V), 5-methyl-2-(1-hydroxy-1-methylethyl)-2-cyclohexene-1-one (VI), 3-hydroxyisopulegone (VII), 7-hydroxypiperitenone (VIII), and 7-hydroxypulegone (IX), have been isolated from the fermentation medium and identified. GC analysis of the metabolites indicated that II was the major metabolite formed. The organism initiates transformation either by hydroxylation at the C-5 position or by hydroxylation of the ring methylenes, the former being the major activity. On the basis of the identification of the metabolites, pathways for the biotransformation of (R)-(+)-pulegone have been proposed. The mode of transformation of (S)-(-)-pulegone by this organism was shown to be similar to that of its (R)-(+)-enantiomer. When isopulegone (X) was used as the substrate, the organism isomerized it to pulegone (I), which was then transformed to metabolites II-IX.