Marcello Nicoletti

Cymbopogon citratus-synthesized gold nanoparticles boost the predation efficiency of copepod Mesocyclops aspericornis against malaria and dengue mosquitoes

Plant-borne compounds can be employed to synthesize mosquitocidal nanoparticles that are effective at low doses. However, how they affect the activity of mosquito predators in the aquatic environment is unknown. In this study, we synthesized gold nanoparticles (AuN) using the leaf extract of Cymbopogon citratus, which acted as a reducing and capping agent. AuN were characterized by a variety of biophysical methods and sorted for size in order to confirm structural integrity. C. citratus extract and biosynthesized AuN were tested against larvae and pupae of the malaria vector Anopheles stephensi and the dengue vector Aedes aegypti. LC₅₀ of C. citratus extract ranged from 219.32 ppm to 471.36 ppm. LC₅₀ of AuN ranged from 18.80 ppm to 41.52 ppm. In laboratory, the predatory efficiency of the cyclopoid crustacean Mesocyclops aspericornis against A. stephensi larvae was 26.8% (larva I) and 17% (larva II), while against A. aegypti was 56% (I) and 35.1% (II). Predation against late-instar larvae was minimal. In AuN-contaminated environment,predation efficiency against A. stephensi was 45.6% (I) and 26.7% (II), while against A. aegypti was 77.3% (I) and 51.6% (II). Overall, low doses of AuN may help to boost the control of Anopheles and Aedes larval populations in copepod-based control programs.

Old ingredients for a new recipe? Neem cake, a low-cost botanical by-product in the fight against mosquito-borne diseases

Mosquitoes (Diptera: Culicidae) represent an important threat to millions of people worldwide, since they act as vectors for important pathogens, such as malaria, yellow fever, dengue and West Nile. Control programmes mainly rely on chemical treatments against larvae, indoor residual spraying and insecticide-treated bed nets. In recent years, huge efforts have been carried out to propose new eco-friendly alternatives, with a special focus on the evaluation of plant-borne mosquitocidal compounds. Major examples are neem-based products (Azadirachta indica A. Juss, Meliaceae) that have been proven as really effective against a huge range of pests of medical and veterinary importance, including mosquitoes. Recent research highlighted that neem cake, a cheap by-product from neem oil extraction, is an important source of mosquitocidal metabolites. In this review, we examined (i) the latest achievements about neem cake metabolomics with special reference to nor-terpenoid and related content; (ii) the neem cake ovicidal, larvicidal and pupicidal toxicity against Aedes, Anopheles and Culex mosquito vectors; (iii) its non-target effects against vertebrates; and (iv) its oviposition deterrence effects on mosquito females. Overall, neem cake can be proposed as an eco-friendly and low-cost source of chemicals to build newer and safer control tools against mosquito vectors.

Commentary: Making Green Pesticides Greener? The Potential of Plant Products for Nanosynthesis and Pest Control

The resurgence of interest on natural substances and their progressive affirmation in the market open doors for novel marketed products, with intrinsic original approaches. Evident examples are in the food supplements sector as well as in mix of synthetic drugs and natural substances, as novel drugs. A decisive key role will be played by technology and the capacity of throw novel opportunities out of the normal landscape. As in other fields, most of the future technology developments rely on nanotechnology. In this Commentary, after the discussion focused on the novel roles of natural products, we report the recent results in the application of nanotechnology in one of the most relevant challenge for mankind, consisting in the resurgence of important vector-borne diseases and emergence of new epidemic alerts. In particular, the green synthesis of nanopesticides is cheap and environmental friendly, since it does not require the employment of highly toxic chemicals or elevate energy inputs. In the conclusions, a brief agenda summarizing the challenges for nano-based pest control is outlined.

Characterization and biotoxicity of Hypnea musciformis-synthesized silver nanoparticles as potential eco-friendly control tool against Aedes aegypti and Plutella xylostella

Two of the most important challenges facing humanity in the 21st century comprise food production and disease control. Eco-friendly control tools against mosquito vectors and agricultural pests are urgently needed. Insecticidal products of marine origin have a huge potential to control these pests. In this research, we reported a single-step method to synthesize silver nanoparticles (AgNP) using the aqueous leaf extract of the seaweed Hypnea musciformis, a cheap, nontoxic and eco-friendly material, that worked as reducing and stabilizing agent during the biosynthesis. The formation of AgNP was confirmed by surface plasmon resonance band illustrated in UV-vis spectrophotometer. AgNP were characterized by FTIR, SEM, EDX and XRD analyses. AgNP were mostly spherical in shape, crystalline in nature, with face-centered cubic geometry, and their mean size was 40-65nm. Low doses of H. musciformis aqueous extract and seaweed-synthesized AgNP showed larvicidal and pupicidal toxicity against the dengue vector Aedes aegypti and the cabbage pest Plutella xylostella. The LC50 value of AgNP ranged from 18.14 to 38.23ppm for 1st instar larvae (L1) and pupae of A. aegypti, and from 24.5 to 38.23ppm for L1 and pupae of P. xylostella. Both H. musciformis extract and AgNP strongly reduced longevity and fecundity of A. aegypti and P. xylostella adults. This study adds knowledge on the toxicity of seaweed borne insecticides and green-synthesized AgNP against arthropods of medical and agricultural importance, allowing us to propose the tested products as effective candidates to develop newer and cheap pest control tools.

Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study.

The purpose of this study was to investigate the effects of six lichen metabolites (diffractaic acid, lobaric acid, usnic acid, vicanicin, variolaric acid, protolichesterinic acid) on proliferation, viability and reactive oxygen species (ROS) level towards three human cancer cell lines, MCF‐7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and HCT‐116 (colon carcinoma). Cells were treated with different concentrations (2.5–100 μM) of these compounds for 48 h. In this comparative study, our lichen metabolites showed various cytotoxic effects in a concentration‐dependent manner, and usnic acid was the most potent cytotoxic agent, while variolaric acid did not inhibit the proliferation of any of the three cell lines used. All tested lichen compounds did not exhibit free radical scavenging activity using the 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay. The lichen metabolites did not significantly increase the intracellular ROS level and did not prevent oxidative injury induced by t‐butylhydroperoxide in HeLa cells. To better clarify the mechanism(s) of cytotoxic effect induced by protolichesterinic acid in HeLa cells, we investigated apoptotic markers such as condensation and fragmentation of nuclear chromatin and activation of caspase‐3, 8 and 9. Our results revealed that the antiproliferative activity of 40 μM protolichesterinic acid in HeLa cells is related to its ability to induce programmed cell death involving caspase‐3, 8 and 9 activation. Copyright

Nutraceuticals and botanicals: overview and perspectives

The discovery, development and marketing of food supplements, nutraceuticals and related products are currently the fastest growing segments of the food industry. Functional foods can be considered part or borderline to these products and may be defined as foods or food ingredients that have additional health or physiological benefits over and above the normal nutritional value they provide. This trend is driven by several factors, mainly due to the current consumer perceptions: the first and dominant being ‘Natural is good’, and other secondary, such as the increasing cost of many pharmaceuticals and their negative secondary effects, the insistent marketing campaign, the increasing perception of the need of a healthy diet and its importance in the health and homeostasis organism conditions. However, the central point is that nutraceuticals, botanicals and other herbal remedies, including the entry of new functional foods, are important because of their acceptance as the novel and modern forms to benefit of natural substances. Due to the rapid expansion in this area, the development of several aspects is considered as it could influence the future of the market of these products negatively: an imbalance existing between the increasing number of claims and products on the one hand, the development of policies to regulate their application and safety on the other, rapid and valuable controls to check the composition, including the plant extracts or adulteration to improve efficacy, like the presence of synthetic drugs. It is interesting to see that, from the negative factors reported by the market analysts, a change in consumers preferences is absent. The functional properties of many plant extracts, in particular, are being investigated for potential use as novel nutraceuticals and functional foods. Although the availability of scientific data is rapidly improving, the central aspect concerns the validation of these products. The first step of this crucial aspect is the security of the composition, obtained by the useful and adapted analytical approach. On the other hand, in the first instance, security is assured by the millenary use as food of the great majority of these plants. The importance and the novelty of functional food are inherent in the possibility to renew the secure use of plants to maintain healthiness of man in novel forms of use adapted to modern times. The market of ‘other substances’, after the emergence of the first period of enthusiastic explosion, is entering into the maturation period, with three important arguments to face: (a) security in composition, production and sale, avoiding easy conversions or convenient approaches and favouring competence and professionalism, (b) definition of influence of metabolic aspects, including scientific validation and (c) regulatory aspects, e.g. the claims definition and relative influences. The last aspect seams to be in primis the most crucial and fundamental to the future of all the sectors. The role of European Food and Safety Authority (EFSA) in the EU market must be considered and consequences if negative decisions on ‘other substances’ claim will be adopted in each countrys legislation.

In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and ΔE model methods.

The in vitro antimicrobial activities of usnic acid were evaluated in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC₉₀, MIC₅₀, as well as MBC₉₀ and MBC₅₀, were evaluated. A synergistic action was observed in combination with gentamicin, while antagonism was observed with levofloxacin. The combination with erythromycin showed indifference, while variability was observed for clindamycin and oxacillin. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index (FICI) and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings, beside confirm the well known antimicrobial activity of usnic acid, suggest, however, that this substance might be a good candidate for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.

Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma, Plasmodium falciparum parasites and malaria mosquitoes.

The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW-AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW-AgNP showed plasmon resonance reduction in UV-vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW-AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW-AgNP were toxic to Anopheles stephensi larvae and pupae, LC(50) were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW-AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW-AgNP IC(50) were 49.3 μg/ml (CQ-s) and 55.5 μg/ml (CQ-r), while chloroquine IC(50) were 81.5 μg/ml (CQ-s) and 86.5 μg/ml (CQ-r). EW-AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW-AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW-AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW-AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies.

Neem cake: chemical composition and larvicidal activity on Asian tiger mosquito

New pesticides based on natural products are urgently needed, in consideration of their environmental care and lower collateral effects. Neem oil, the main product obtained from Azadiractha indica A. Juss, commonly known as neem tree, is mainly used in medical devices, cosmetics and soaps, as well as important insecticide. Manufacturing of neem oil first includes the collection of the neem seeds as raw material used for the extraction. Neem cake is the waste by-product remaining after extraction processes. The quality of the oil, as that of the cake, strictly depends from the quality of seeds as well as from the type of extraction processes used, which strongly influences the chemical composition of the product. Currently, the different types of commercial neem cake on the market are roughly identified as oiled and deoiled cake, but several other differences can be detected. The differences are relevant and must be determined, to obtain the necessary correlation between chemical constitution and larvicidal activities. Six different batches of neem cake, marketed by several Indian and European companies, were analyzed by HPLC and HPTLC, and their fingerprints compared, obtaining information about the different compositions, focusing in particular on nortriterpenes, considered as the main active components of neem oil. Therefore, the chemical composition of each cake was connected with the biological activitiy, i.e., the effects of the extracts of the six neem cakes were tested on eggs and larvae of Aedes albopictus (Stegomyia albopicta) (Diptera: Culicidae), commonly known as Asian tiger mosquito. The results confirmed the previously reported larvicide effects of neem cake that, however, can now be related to the chemical composition, in particular with nortriterpenes, allowing in that way to discriminate between the quality of the various marketed products, as potential domestic insecticides.

In vivo and in vitro effectiveness of Azadirachta indica-synthesized silver nanocrystals against Plasmodium berghei and Plasmodium falciparum, and their potential against malaria mosquitoes

Malaria transmission is a serious emergence in urban and semiurban areas worldwide, becoming a major international public health concern. Malaria is transmitted through the bites of Anopheles mosquitoes. The extensive employ of synthetic pesticides leads to negative effects on human health and the environment. Recently, plant-synthesized nanoparticles have been proposed as highly effective mosquitocides. In this research, we synthesized silver nanoparticles (AgNP) using the Azadirachta indica seed kernel extract as reducing and stabilizing agent. AgNP were characterized by UV-vis spectrophotometry, SEM, EDX, XRD and FTIR spectroscopy. The A. indica seed kernel extract was toxic against Anopheles stephensi larvae and pupae, LC50 were 232.8ppm (larva I), 260.6ppm (II), 290.3ppm (III), 323.4ppm (IV), and 348.4ppm (pupa). AgNP LC50 were 3.9ppm (I), 4.9ppm (II), 5.6ppm (III), 6.5ppm (IV), and 8.2ppm (pupa). The antiplasmodial activity of A. indica seed kernel extract and AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of A. indica seed kernel extract were 63.18μg/ml (CQ-s) and 69.24μg/ml (CQ-r). A. indica seed kernel-synthesized AgNP achieved IC50, of 82.41μg/ml (CQ-s) and 86.12μg/ml (CQ-r). However, in vivo anti-plasmodial experiments conducted on Plasmodium berghei infecting albino mice showed moderate activity of the A. indica extract and AgNP. Overall, this study showed that the A. indica-mediated fabrication of AgNP is of interest for a wide array of purposes, ranging from IPM of mosquito vectors to the development of novel and cheap antimalarial drugs.