Nanoparticles as elicitors and harvesters of economically important secondary metabolites in higher plants: A review.

Nanoparticles possess some unique properties which improve their biochemical reactivity. Plants, due to their stationary nature, are constantly exposed to nanoparticles present in the environment, which act as abiotic stress agents at sub-toxic concentrations and phytotoxic agents at higher concentrations. In general, nanoparticles exert their toxicological effect by the generation of reactive oxygen species to which plants respond by activating both enzymatic and non-enzymatic anti-oxidant defence mechanisms. One important manifestation of the defence response is the increased or de novo biosynthesis of secondary metabolites, many of which have commercial application. The present review extensively summarizes current knowledge about the application of different metallic, non-metallic and carbon-based nanoparticles as elicitors of economically important secondary metabolites in different plants, both in vivo and in vitro. Elicitation of secondary metabolites with nanoparticles in plant cultures, including hairy root cultures, is discussed. Another emergent technology is the ligand-harvesting of secondary metabolites using surface-functionalized nanoparticles, which is also mentioned. A brief explanation of the mechanism of action of nanoparticles on plant secondary metabolism is included. Optimum conditions and parameters to be evaluated and standardized for the successful commercial exploitation of this technology are also mentioned.

Enhancement of secondary metabolites in Bacopa monnieri (L.) Pennell plants treated with copper-based nanoparticles in vivo.

The study aims to document the effect of starch-stabilised copper-based nanoparticles (CuNPs) on the biosynthesis of pharmaceutically valuable secondary metabolites, especially saponins, of the reputed nootropic herb Bacopa monnieri (L.) Pennell. CuNPs were synthesised chemically by the reduction of cupric sulphate pentahydrate with ascorbic acid using starch as the capping agent. They were characterised by UV-visible spectrophotometry, Fourier-transform infra-red spectroscopy, X-ray diffraction, high-resolution transmission electron microscopy and zeta potential. The nanoparticles consisted of cuprous oxide and metallic copper, were approximately spherical, polydispersed with diameter <20 nm. Hydroponically grown B. monnieri plants were treated in vivo with the CuNPs between the concentrations of 0-100 mg l-1. Spectrophotometric estimation of the total contents of saponins, alkaloids, phenolics, flavonoids and DPPH radical scavenging capacity from the methanolic extracts of the whole plants showed a hormetic increase in the content of secondary metabolites in a concentration-dependent manner from 5 mg l-1 until it declined at toxic metabolic concentration. This was accompanied by an increase in ROS markers hydrogen peroxide and malondialdehyde as well as a hormetic effect on activities of phenylalanine ammonia lyase and antioxidant enzymes catalase, ascorbate peroxidase and superoxide dismutase. CuNPs at sub-toxic concentrations were found to enhance secondary metabolism and antioxidant capacity in Bacopa monnieri through ROS-mediated defence response.

Critical evaluation of the therapeutic potential of bassic acid incorporated in oil-in-water microemulsions and poly-D,L-lactide nanoparticles against experimental leishmaniasis.

Bassic acid, an unsaturated triterpene acid isolated from Mimusops elangii, was tested for its antileishmanial properties both in vitro and in vivo. The in vitro antileishmanial activity of bassic acid being encouraging, its activity in vivo was evaluated in hamster models of visceral leishmaniasis, both in free form, as well as incorporated in two different delivery systems, viz microemulsions and polylactide nanoparticles. The delivery systems were prepared by published protocols. The percentage intercalation of bassic acid in nanoparticles and microemulsion was found to be about 50 and 100, respectively, when determined at its absorption maxima (lambda(max)) 285 nm (epsilon(m) = 2.3 x 10(2) M(-1) cm(-1)). At an equivalent dose of 2 mg kg(-1) body weight, when injected subcutaneously for a total of six doses in 15 days, bassic acid was found to reduce spleen parasite loads by 45, 62 and 78% in free, microemulsion-incorporated and nanoparticle-incorporated forms, respectively. A comparison of specific biochemical tests related to normal liver and kidney functions revealed that the nanoparticulate form was successful in significantly reducing the hepatotoxicity and nephrotoxicity of the free drug, but the microemulsion delivery system was less effective and toxic to liver and kidney to some extent. Confocal microscopic images of Leishmania donovani promastigotes treated with bassic acid revealed that the drug induced necrotic cell death due to non-specific membrane damage. Because of its high efficacy as well as non-hepatotoxicity and non-nephrotoxicity, the nanoparticulate form of bassic acid may be considered for clinical application in humans rather than the microemulsion incorporated form.

Targeted delivery of arjunglucoside I using surface hydrophilic and hydrophobic nanocarriers to combat experimental leishmaniasis.

The purpose of the present study was to investigate the therapeutic efficacy of the indigenous drug arjunglucoside I (AG) against in vivo models of experimental leishmaniasis by incorporating it in surface hydrophilic co-polymeric nanogel particles of size less than 100 nm diameter and to compare its efficacy with that of the free drug as well as the drug encapsulated in hydrophobic poly-dl-lactide (PLA) nanoparticles. The drug AG, having glucose at the terminal end of the glycosidic chain, was isolated from an indigenous source. Drug-incorporated ultra-low-sized nanogels (approximately 90 nm in diameter) composed of cross-linked random co-polymer of N-isopropylacrylamide (NIPAAM) and N-vinyl pyrrolidone(VP) were prepared, characterized and used as delivery vehicles to combat experimental leishmaniasis in hamster models. For comparison, drug-encapsulated hydrophobic nanoparticles (approximately 250 nm in diameter) made from PLA were used as a control. The drug AG was incorporated in these nanocarriers and these drug-nanocarrier complexes were physically characterized. The efficacy of lowering spleen parasite load by the free drug, as well as that incorporated in nanogels and PLA nanoparticles were examined in vivo in equimolar concentration against hamsters undergoing experimental leishmaniasis. The reduction of drug toxicity by the nanogels and PLA nanoparticles was also assessed. The efficacy in the lowering of spleen parasite load with the free drug was found to be only 38% but was much higher when the drug was incorporated in co-polymeric nanogels (79%) or in polymeric nanoparticles (75%). Both the nanocarriers were found to be effective in reducing hepatotoxicity and nephrotoxicity nearly to the same extent. It was apparent that in addition to a smaller size and better drug release profile, the contribution of other parameters, e.g. overall surface hydrophilicity or hydrophobicity of the vehicles, also play an important role in the macrophage uptake of the drug. However, whatever be the exact mechanism, being highly efficient, non-hepatotoxic and non-nephrotoxic, AG in either of the two nanoparticulate forms may have useful application in humans

Macrophage specific drug delivery in experimental leishmaniasis.

Macrophage-specific delivery systems are the subject of much interest nowadays, because of the fact that macrophages act as host cells for many parasites and bacteria, which give rise to outbreak of so many deadly diseases(eg. leishmaniasis, tuberculosis etc.) in humans. To combat these deadly diseases initially macrophage specific liposomal delivery system were thought of and tested in vivo against experimental leishmaniasis in hamsters using a series of indigenous or synthetic antileishmanial compounds and the results were critically discussed. In vitro testing was also done against macrophages infected with Leishmania donovani, the causative agent for visceral leishmaniasis. The common problem of liposome therapy being their larger size, stability and storage, non-ionic surfactant vesicles, niosomes were prepared, for their different drug distribution and release characteristics compared to liposomes. When tested in vivo, the retention capacity of niosomes was found to be higher than that of liposomes due to the absence of lipid molecules and their smaller size. Thus the therapeutic efficacy of certain antileishmanial compounds was found to be better than that in the liposomal form. The niosomes, being cheaper, less toxic, biodegradable and non-immunogenic, were considered for sometime as suitable alternatives to liposomes as drug carriers. Besides the advent of other classical drugs carriers(e.g. neoglycoproteins), the biggest challenge came from polymeric delivery vehicles, specially the polymeric nanoparticles which were made of cost effective biodegradable polymers and different natural polymers. Because of very small size and highly stable nature, use of nanoparticles as effective drug carriers has been explored in experimental leishmaniasis using a series of antileishmanial compounds, both of indigenous and synthetic origin. The feasibility of application in vivo, when tested for biological as well as for other physicochemical parameters, the polymeric nanoparticles have turned out to be the best and thus may be projected for effective use in the clinics.

Harmine: evaluation of its antileishmanial properties in various vesicular delivery systems.

Harmine, a beta-carboline amine alkaloid isolated from Peganum harmala, was tested for its antileishmanial properties both in vitro and in vivo. In vitro antileishmanial activity of harmine was encouraging and prompted us to confirm the activity in vivo in hamster models. Harmine was tested both in free form and in different vesicular forms viz. liposomes, niosomes and nanoparticles. The different vesicles were prepared by the published protocols. The percent intercalation of harmine in liposomes, niosomes and nanoparticles was found to be 65, 60 and 20, respectively, when determined at 325 nm (epsilon(M) =2.33 x 10 M(-1) cm(-1)). At an equivalent dose of 1.5 mg/kg body weight, injected subcutaneously (SC) for a total of six doses in 15 days, harmine was found to reduce spleen parasite load by approximately 40, 60, 70 and 80%, respectively in free, liposomal, niosomal and nanoparticular forms. An inverse relationship could be established between the efficacy in the lowering of spleen parasite load and the size of the vesicles. Specific biochemical tests related to normal liver and kidney functions revealed that the toxicity of the drug was reduced in the vesicular forms in the same order as their efficacy and the same was confirmed by the histopathological studies of splenic sections. Cell cycle analysis studies using flow cytometry suggested that although harmine interferes in the cell division stage, it does not induce apoptosis in Leishmania donovani promastigotes. The results using Confocal Microscopy supported that the cell death could be attributed to necrosis due to non-specific membrane damage. Even then, because of its appreciable efficacy in destroying intracellular parasites as well as non-hepatotoxic and non-nephrotoxic nature, harmine, in the vesicular forms, may be considered for clinical application in humans.