Immunomodulation of streptozotocin induced Type 1 diabetes mellitus in mouse model by Macrophage migration inhibitory factor-2 (MIF-2) homologue of human lymphatic filarial parasite, Wuchereria bancrofti.

Helminth parasites modulate the host immune system to ensure a long-lasting asymptomatic form of infection generally, mediated by the secretion of immunomodulatory molecules and one such molecule is a homologue of human host cytokine, Macrophage migratory Inhibitory Factor (hMIF). In this study, we sought to understand the role of homologue of hMIF from the lymphatic filarial parasite, Wuchereria bancrofti (Wba-MIF2), in the immunomodulation of the Streptozotocin (STZ)-induced Type1 Diabetes Mellitus (T1DM) animal model. Full-length recombinant Wba-MIF2 was expressed and found to have both oxidoreductase and tautomerase activities. Wba-MIF2 recombinant protein was treated to STZ induced T1DM animals, and after 5 weeks pro-inflammatory (IL-1, IL-2, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and gene expressions were determined in sera samples and spleen respectively. Pro-inflammatory and anti-inflammatory cytokine levels were significantly (p<0.05) up-regulated and down-regulated respectively, in the STZ-T1DM animals, as compared to treated groups. Histopathology showed macrophage infiltration and greater damage of islets of beta cells in the pancreatic tissue of STZ-T1DM animals, than Wba-MIF2 treated STZ-T1DM animals. The present study clearly showed the potential of Wba-MIF2 as an immunomodulatory molecule, which could modulate the host immune system in the STZ-T1DM mice model from a pro-inflammatory to anti-inflammatory milieu.

Diethylcarbamazine citrate-fortified salt for lymphatic filariasis elimination in India.

Lymphatic filariasis (LF) is a vector-borne neglected tropical disease, causing permanent disability. The disease is debilitating and widespread, leading to tremendous productivity and economic loss. The Government of India (GOI) prioritized the elimination of LF through the annual mass drug administration (MDA) programme in 2004 and continued with a single dose of diethylcarbamazine citrate (DEC), 6 mg/kg of body weight, plus albendazole annually over a period of 5-6 years. The GOI had set the target to achieve LF elimination by 2015 and now by 2030. The progress so far has been suboptimal. Much remains to be done as about 84 per cent of the total 328 endemic districts are still under MDA. The major challenge in implementing MDA is poor compliance. It is necessary to have a feasible alternative strategy addressing the above challenge to achieve the desired goal of LF elimination. At this juncture, a well-researched approach, i.e. the use of DEC-fortified salt, also advocated by the World Health Organization, as a unique form of MDA, is proposed. As per this strategy, a low dose of DEC (0.2% w/w) is added to the cooking salt at the manufacturing facility of iodized salt and consumed by the LF-endemic communities for about two years. Many examples of successful use of this strategy for LF elimination in small- and large-scale trials have been documented in India and several other endemic countries in the world. Implementing DEC-iodine-fortified salt is a safe, less expensive, more efficient and prompt approach for achieving the elimination of LF in India. Adverse effects are none or minor and self-limiting. The DEC-fortified salt strategy can easily piggyback on the existing countrywide deployment of iodized salt under the National Iodine Deficiency Disorders Control Programme (NIDDCP), which has achieved a great success in reducing iodine-deficiency disorders such as hypothyroidism. This existing robust programme can be leveraged to launch DEC-fortified salt for the community. If implemented appropriately, this strategy will ensure the complete cessation of LF transmission within two years from its introduction. If the said strategy is implemented in 2022, it is expected that India will be able to achieve the LF elimination by 2024, much before the global target of 2030.

Nation-wide vector surveillance on Zika and Dengue did not indicate transmission of the American lineage-pandemic Zika virus in India.

OBJECTIVES: Following the Public Health Emergency of International Concern declared on Zika by the World Health Organization during 2016, the Indian Council of Medical Research carried out nationwide vector surveillance for Zika and Dengue viruses (ZIKV and DENV) in India as a preparedness measure in 2016-19. METHODS: High-risk zones distributed to 49 Districts in 14 states/union territories were included in the study. Seven ICMR institutions participated, following a standard operating protocol. Aedes specimens sampled weekly were processed by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) for ZIKV/DENV and random samples crosschecked with real-time RT-PCR for ZIKV. RESULTS: Altogether, 79 492 Aedes specimens in 6492 pools were processed; 3 (0.05%) and 63 (0.97%) pools, respectively, were found positive for ZIKV and DENV. ZIKV infections were recorded in Aedes aegypti sampled during the 2018 sporadic Zika outbreak in Jaipur, Rajasthan. However, these belonged to the Asian lineage of the virus, already circulating in the country. Both Ae. aegypti and Aedes albopictus distributed to 8 states/union territories were found to be infected with DENV. Both sexes of Ae. albopictus were infected, indicating transovarial transmission. CONCLUSION: This investigation evinced no active transmission of the American lineage-pandemic Zika virus in India during the pandemic period.

Operationalising the "One Health" approach in India: facilitators of and barriers to effective cross-sector convergence for zoonoses prevention and control.

BACKGROUND: There is a strong policy impetus for the One Health cross-sectoral approach to address the complex challenge of zoonotic diseases, particularly in low/lower middle income countries (LMICs). Yet the implementation of this approach in LMIC contexts such as India has proven challenging, due partly to the relatively limited practical guidance and understanding on how to foster and sustain cross-sector collaborations. This study addresses this gap by exploring the facilitators of and barriers to successful convergence between the human, animal and environmental health sectors in India. METHODS: A mixed methods study was conducted using a detailed content review of national policy documents and in-depth semi-structured interview data on zoonotic disease management in India. In total, 29 policy documents were reviewed and 15 key informant interviews were undertaken with national and state level policymakers, disease managers and experts operating within the human-animal-environment interface of zoonotic disease control. RESULTS: Our findings suggest that there is limited policy visibility of zoonotic diseases, although global zoonoses, especially those identified to be of pandemic potential by international organisations (e.g. CDC, WHO and OIE) rather than local, high burden endemic diseases, have high recognition in the existing policy agenda setting. Despite the widespread acknowledgement of the importance of cross-sectoral collaboration, a myriad of factors operated to either constrain or facilitate the success of cross-sectoral convergence at different stages (i.e. information-sharing, undertaking common activities and merging resources and infrastructure) of cross-sectoral action. Importantly, participants identified the lack of supportive policies, conflicting departmental priorities and limited institutional capacities as major barriers that hamper effective cross-sectoral collaboration on zoonotic disease control. Building on existing informal inter-personal relationships and collaboration platforms were suggested by participants as the way forward. CONCLUSION: Our findings point to the importance of strengthening existing national policy frameworks as a first step for leveraging cross-sectoral capacity for improved disease surveillance and interventions. This requires the contextual adaptation of the One Health approach in a manner that is sensitive to the underlying socio-political, institutional and cultural context that determines and shapes outcomes of cross-sector collaborative arrangements.

Prevalence and spectrum of mutations causing G6PD deficiency in Indian populations.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human erythroenzymopathy affecting around 10% of the world population. India is endemic for malaria and antimalarial drugs are known to induce haemolysis in G6PD deficient individuals. Here we report the prevalence as well as the molecular diversity of G6PD deficiency in geographical regions of India. METHODS AND RESULTS: A total of 20,896 individuals (11,838 males and 9058 females) were screened by DPIP dye decolorisation method followed by quantitation of G6PD enzyme activity on the suspected samples. Molecular analysis was undertaken in a total of 350 G6PD deficient individuals by PCR-RFLP and DNA sequencing. A structural characteristic of the novel variant was deduced by using DynaMut web-server. The prevalence rate of G6PD deficiency varied between 0.8 and 6.3% with an overall prevalence of 1.9%. A total of twelve mutations were identified. Of the total deleterious alleles detected G6PD Orissa (56.5%) was found to be the most predominant variant followed by G6PD Mediterranean (23.6%). G6PD Mediterranean, G6PD Kaiping and G6PD Mahidol were found to be severely deficient variant and 14.1% of them showed undetectable activity. A novel mutation c.544C➔G (R182G) in exon 6 was identified in one tribal male where substitution of arginine by glycine, likely causes the alteration in the alpha helix leading to disruption of secondary structure of the protein. CONCLUSION: There are large differences in the distribution of G6PD causal variants between Indian states, and this may have implications for the treatment in the malaria endemic areas.

Functional studies of Plasmodium falciparum's prohibitin1 and prohibitin 2 in yeast.

Prohibitins (PHBs) are evolutionarily conserved mitochondrial integral membrane proteins, shown to regulate mitochondrial structure and function, and can be classified into PHB1 and PHB2. PHB1 and PHB2 have been shown to interact with each other, and form heterodimers in mitochondrial inner membrane. Plasmodium falciparum has orthologues of PHB1 and PHB2 in its genome, and their role is unclear. Here, by homology modelling and yeast two-hybrid analysis, we show that putative Plasmodium PHBs (Pf PHB1 and Pf PHB2) interact with each other, which suggests that they could form supercomplexes of heterodimers in Plasmodium, the functional form required for optimum mitochondrial function.

Functional studies of Plasmodium falciparum putative SURF1 in Saccharomyces cerevisiae.

BACKGROUND AND OBJECTIVES: The mitochondrial electron transport chain (mtETC) of Plasmodium falciparum is an important drug target. Identification and functional validation of putative mitochondrial proteins of the mtETC is critical for drug development. Many of the regulatory subunits and assembly factors of cytochrome c oxidase readily identifiable in humans and yeast are missing in P. falciparum. Here, we describe our efforts to identify and validate the function of putative Pfsurf1, a key assembly factor of complex IV of the mtETC. METHODS: Multiple sequence alignment of SURF 1/Shy 1 was carried out in Clustal X 2.1. Phylogenetic tree was constructed using "Draw tree" option in Clustal X, and was analyzed using interactive Tree of Life software. To identify the conserved sequences, domain search was done using Jalview version 2.8.2 (BLOSUM 62 scoring). The haploid Saccharomyces cerevisiae strain (BY4741) containing the null allele shy1 (Orf: YGR112w) (shy1::Kan) was complemented with putative Pfsurf1 to study its ability to rescue the growth defect. RESULTS: Similarity searches of PfSURF1-like protein in the Pfam shows statistically significant E = 4.7e-10 match to SURF1 family. Sequence alignment of PfSURF1 with other SURF1-like proteins reveals the conservation of transmembrane domains, α-helices and ß-pleated sheets. Phylogenetic analysis clusters putative PfSURF1 with apicomplexan SURF1-like proteins. Yeast complementation studies show that Pfsurf1 can partially rescue the yeast shy1 mutant, YGR112w. INTERPRETATION & CONCLUSION: Bioinformatics and complementation studies in yeast show that P. falciparum's SURF1 is the functional ortholog of human SURF1 and yeast Shy1.

G6PD A- is the major cause of G6PD deficiency among the Siddis of Karnataka, India.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human erythroenzymopathy affecting more than 400 million people worldwide. G6PD deficiency was reported in India more than 50 years ago and the prevalence rate varies from 5.7% to 27.9% in different caste and tribal groups.Aim: To study the prevalence of, and the mutations causing, G6PD deficiency among the Siddis of Karnataka.Subjects and methods: A total of 755 individuals were screened using the DPIP dye decolorisation method and the deficiency was further confirmed by quantitative assay. Molecular characterisation was performed by PCR-RFLP method and DNA sequencing. Biochemical characterisation was performed as per WHO criteria.Results: Of the 755 individuals, 71 individuals (9.4%) were found to be G6PD deficient with an enzyme activity ranging from 0.02 to 3.83 IU/gm Hb. Mutational analysis could be performed on 49 G6PD deficient individuals and 45 (91.8%) of them showed the presence of the G6PD A- variant while the remaining 4 (8.2%) had the G6PD Kerala-Kalyan mutation. Microsatellite analysis in G6PD A- individuals showed the presence of 166/195 bp, AC/CTT alleles.Conclusions: G6PD deficiencies among the Siddis are predominantly due to G6PD A- mutation. Furthermore, biochemical parameters and the microsatellite repeat markers in the Siddi A- chromosome confirmed they are African descendants with Indian admixture.

Thiosemicarbazone derivatives: Design, synthesis and in vitro antimalarial activity studies.

Different types of thiosemicarbazone derivatives were designed and tested for their Drug-Like Molecular (DLM) nature by using Lipinski and Veber rules. Subsequently, compounds with DLM properties were synthesized and characterized by spectral methods. In vitro antimalarial activity studies of the synthesized thiosemicarbazone derivatives have been carried out against Plasmodium falciparum, 3D7 strain using fluorescence assay method and found that the compounds, (E)-2-(1-(4-fluorophenyl)ethylidene)hydrazine-1-carbothioamide (6), (E)-2-(1-(3-bromophenyl) ethylidene) hydrazine-1-carbothioamide (15) and (E)-2-(3,4,5-trimethoxybenzylidene) hydrazine-1-carbothioamide (29) showed notable antimalarial activity with EC50 values of 13.54 µM, 15.83 µM and 14.52 µM respectively.

RNA interference in mosquito: understanding immune responses, double-stranded RNA delivery systems and potential applications in vector control.

RNA interference (RNAi) refers to the process of post-transcriptional silencing of cellular mRNA by the application of double-stranded RNA (dsRNA). RNAi strategies have been widely employed to regulate gene expression in plants and animals including insects. With the availability of the full genome sequences of major vector mosquitoes, RNAi has been increasingly used to conduct genetic studies of human pathogens in mosquito vectors and to study the evolution of insecticide resistance in mosquitoes. This review summarizes the recent progress in our understanding of mosquito-pathogen interactions using RNAi and various methods of dsRNA delivery in mosquitoes at different stages. We also discuss potential applications of this technology to develop novel tools for vector control.

Facile fabrication of eco-friendly nano-mosquitocides: Biophysical characterization and effectiveness on neglected tropical mosquito vectors.

Mosquito (Diptera: Culicidae) vectors are solely responsible for transmitting important diseases such as malaria, dengue, chikungunya, Japanese encephalitis, lymphatic filariasis and Zika virus. Eco-friendly control tools of Culicidae vectors are a priority. In this study, we proposed a facile fabrication process of poly-disperse and stable silver nanoparticles (Ag NPs) using a cheap leaf extract of Ichnocarpus frutescens (Apocyanaceae). Bio-reduced Ag NPs were characterized by UV-vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The acute toxicity of I. frutescens leaf extract and green-synthesized Ag NPs was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Compared to the leaf aqueous extract, Ag NPs showed higher toxicity against A. subpictus, A. albopictus, and C. tritaeniorhynchus with LC50 values of 14.22, 15.84 and 17.26µg/mL, respectively. Ag NPs were found safer to non-target mosquito predators Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 values ranging from 636.61 to 2098.61µg/mL. Overall, this research firstly shed light on the mosquitocidal potential of I. frutescens, a potential bio-resource for rapid, cheap and effective synthesis of poly-disperse and highly stable silver nanocrystals.

Scope of detectability of circulating antigens of human lymphatic filarial parasite Wuchereria bancrofti with smaller amount of serum by Og4C3 assay: its application in lymphatic filariasis elimination programme.

Filarial antigen detection is an appropriate epidemiological indicator for mapping lymphatic filariasis and impact evaluation of filariasis elimination programme in view of low sensitivity of parasite detection. Monoclonal antibody-based Og4C3 immunological test requires 100 µl serum, which is difficult to collect by finger prick method during community based surveys. Hence, we tested lesser volume of serum compared to standard volume of 100 µl to compare its sensitivity and specificity in detecting the circulating filarial antigens. Blood samples were collected from individuals who tested positive [with titer groups 4 (border line positives), 6 (medium positives), and 8 (high positives)] and negative (titre group 3) for Og4C3 assay. Different volumes of serum samples were used to make-up required volume (100 µl) with appropriate dilutions and subjected to Og4C3 assay. The results showed that known negative samples tested negative at all the serum volumes tested. All positives (titer groups 6 and 8) showed positivity at all reduced volumes of serum sample. However one of the medium positive sample showed negative reaction in 5 µl volume of serum and two of the border line positives showed negative at all the serum volume tested. The results thus showed as less as 15 µl serum is adequate for use in Og4C3 assay. So the test can be performed without losing its sensitivity even with 5 µl serum samples at high titre of antigen (titre group 8) and 15 µl for other groups and this method has scope in programme evaluation.

Single-step biosynthesis and characterization of silver nanoparticles using Zornia diphylla leaves: A potent eco-friendly tool against malaria and arbovirus vectors.

Mosquitoes (Diptera: Culicidae) are vectors of important pathogens and parasites, including malaria, dengue, chikungunya, Japanese encephalitis, lymphatic filariasis and Zika virus. The application of synthetic insecticides causes development of resistance, biological magnification of toxic substances through the food chain, and adverse effects on the environment and human health. In this scenario, eco-friendly control tools of mosquito vectors are a priority. Here single-step fabrication of silver nanoparticles (AgNP) using a cheap aqueous leaf extract of Zornia diphylla as reducing and capping agent pf Ag(+) ions has been carried out. Biosynthesized AgNP were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction analysis (XRD). The acute toxicity of Z. diphylla leaf extract and biosynthesized AgNP was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Both the Z. diphylla leaf extract and Ag NP showed dose dependent larvicidal effect against all tested mosquito species. Compared to the leaf aqueous extract, biosynthesized Ag NP showed higher toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with LC50 values of 12.53, 13.42 and 14.61µg/ml, respectively. Biosynthesized Ag NP were found safer to non-target organisms Chironomus circumdatus, Anisops bouvieri and Gambusia affinis, with the respective LC50 values ranging from 613.11 to 6903.93µg/ml, if compared to target mosquitoes. Overall, our results highlight that Z. diphylla-fabricated Ag NP are a promising and eco-friendly tool against larval populations of mosquito vectors of medical and veterinary importance, with negligible toxicity against other non-target organisms.

Facile synthesis of mosquitocidal silver nanoparticles using Mussaenda glabra leaf extract: characterisation and impact on non-target aquatic organisms.

Plant-borne compounds have been proposed for extracellular synthesis of mosquitocidal nanoparticles. However, their impact against mosquito natural enemies has been scarcely studied. Here, we synthesised silver nanoparticles (Ag NPs) using Mussaenda glabra leaf extract as reducing and stabilising agent. Biofabricated Ag NPs were characterised by UV-vis spectrophotometry, X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Compared to the leaf aqueous extract, biosynthesised Ag NPs showed higher toxicity against mosquito vectors Anopheles subpictus, Aedes albopictus and Culex tritaeniorhynchus with LC50 of 17-19 µg/mL, respectively. Ag NPs were found safer to non-target organisms Diplonychus indicus and Gambusia affinis, with respective LC50 values ranging from 1446 to 8628 µg/mL. Overall, M. glabra-fabricated Ag NPs are a promising and eco-friendly tool against larval populations of mosquito vectors of medical and veterinary importance, with negligible toxicity against other non-target aquatic organisms.

An alternative strategy to generate coding sequence of macrophage migration inhibitory factor-2 of Wuchereria bancrofti.

BACKGROUND & OBJECTIVES: Different developmental stages of Wuchereria bancrofti, the major causal organism of lymphatic filariasis (LF), are difficult to obtain. Beside this limitation, to obtain complete coding sequence (CDS) of a gene one has to isolate mRNA and perform subsequent cDNA synthesis which is laborious and not successful at times. In this study, an alternative strategy employing polymerase chain reaction (PCR) was optimized and validated, to generate CDS of Macrophage migration Inhibitory Factor-2 (wbMIF-2), a gene expressed in the transition stage between L3 to L4. METHODS: The genomic DNA of W. bancrofti microfilariae was extracted and used to amplify the full length wbMIF-2 gene (4.275 kb). This amplified product was used as a template for amplifying the exons separately, using the overlapping primers, which were then assembled through another round of PCR. RESULTS: A simple strategy was developed based on PCR, which is used routinely in molecular biology laboratories. The amplified CDS of 363 bp of wbMIF-2 generated using genomic DNA splicing technique was devoid of any intronic sequence. INTERPRETATION & CONCLUSIONS: The cDNA of wbMIF-2 gene was successfully amplified from genomic DNA of microfilarial stage of W. bancrofti thus circumventing the use of inaccessible L3-L4 transitional stage of this parasite. This strategy is useful for generating CDS of genes from parasites that have restricted availability.

One-step synthesis of polydispersed silver nanocrystals using Malva sylvestris: an eco-friendly mosquito larvicide with negligible impact on non-target aquatic organisms.

The synthesis of eco-friendly nanoparticles is evergreen branch of nanoscience with a growing number of biomedical implications. In this study, we investigated the synthesis of polydisperse and stable silver nanoparticles (AgNP) using a cheap leaf extract of Malva sylvestris (Malvaceae). Bio-reduced AgNP were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The acute toxicity of M. sylvestris leaf extract and green-synthesized AgNP was evaluated against larvae of the malaria vector Anopheles stephensi, the dengue vector Aedes aegypti and the filariasis vector Culex quinquefasciatus. Compared to the leaf aqueous extract, AgNP showed higher toxicity against A. stephensi, A. aegypti, and C. quinquefasciatus with LC50 values of 10.33, 11.23, and 12.19 µg/mL, respectively. Green-synthesized AgNP were found safer to non-target organisms Diplonychus indicus and Gambusia affinis, with respective LC50 values ranging from 813.16 to 1044.52 µg/mL. Overall, this research firstly shed light on the mosquitocidal potential of M. sylvestris, a potential bio-resource for rapid, cheap and effective synthesis of polydisperse and highly stable silver nanocrystals.

Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus (Diptera: Culicidae).

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. However, the use of synthetic insecticides to control Culicidae may lead to resistance, high operational costs and adverse non-target effects. Nowadays, plant-borne mosquitocides may serve as suitable alternative in the fight against mosquito vectors. In this study, the mosquito larvicidal activity of Origanum vulgare (Lamiaceae) leaf essential oil (EO) and its major chemical constituents was evaluated against the malaria vectors Anopheles stephensi and An. subpictus, the filariasis vector Culex quinquefasciatus and the Japanese encephalitis vector Cx. tritaeniorhynchus. The chemical composition of the EO was analyzed by gas chromatography-mass spectroscopy. GC-MS revealed that the essential oil of O. vulgare contained 17 compounds. The major chemical components were carvacrol (38.30%) and terpinen-4-ol (28.70%). EO had a significant toxic effect against early third-stage larvae of An. stephensi, An. subpictus, Cx. quinquefasciatus and Cx. tritaeniorhynchus, with LC50 values of 67.00, 74.14, 80.35 and 84.93 µg/ml. The two major constituents extracted from the O. vulgare EO were tested individually for acute toxicity against larvae of the four mosquito vectors. Carvacrol and terpinen-4-ol appeared to be most effective against An. stephensi (LC50=21.15 and 43.27 µg/ml, respectively) followed by An. subpictus (LC50=24.06 and 47.73 µg/ml), Cx. quinquefasciatus (LC50=26.08 and 52.19 µg/ml) and Cx. tritaeniorhynchus (LC50=27.95 and 54.87 µg/ml). Overall, this research adds knowledge to develop newer and safer natural larvicides against malaria, filariasis and Japanese encephalitis mosquito vectors.

Simultaneous determination of vasicine and vasicinone by High-performance liquid chromatography in roots of eight Sida species.

INTRODUCTION: Sida L. is a medicinally important genus widely used in conventional systems of medicine in India. AIM: The present study aims toward simultaneous determination of two bioactive compounds vasicine and vasicinone in root extracts of eight Sida spp. from Western Ghats, India. MATERIALS AND METHODS: Determination of vasicine and vasicinone was undertaken in methanolic root extracts (10% w/v) of Sida acuta, Sida cordata, Sida cordifolia, Sida rhombifolia, Sida spinosa, Sida indica, Sida retusa and Sida mysorensis by high performance liquid chromatography (HPLC) method. The standards were prepared with the concentration of mg/mL. Data were expressed as mean values of three reading and relative standard deviations. The separation was achieved on a Waters, Nova-Pack, C18 (250 mm × 4.6 mm, 5 µ) column, with acetonitrile - 0.1 M phosphate buffer-glacial acetic acid (15: 85: 1, v/v/v) as solvent system at a flow-rate of 1.0 mL/min. The effluent was monitored using ultraviolet detection at a wavelength of 300 nm. RESULTS: Both calibration curves of standard showed good linear regression (R2 > 0.994). The limit of detection and the limit of quantification for vasicine was 0.110 and 0.333 µg/mL and for vasicinone was 0.059 and 0.179 µg/mL respectively. The vasicine content was highest in S. cordifolia (9.891 ± 0.495 µg/100 mg) and vasicinone content was rich in S. cordata (33.013 ± 1.651 µg/100 mg.) The content of vasicinone was higher than vasicine. CONCLUSION: HPLC method provides simple, accurate, and reproducible quantitative analysis for simultaneous determination of vasicine and vasicinone. Among the selected Sida species, S. cordifolia and S. cordata were found to be rich in the vasicine and vasicinone contents, respectively.

Eugenol, α-pinene and ß-caryophyllene from Plectranthus barbatus essential oil as eco-friendly larvicides against malaria, dengue and Japanese encephalitis mosquito vectors.

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. Eco-friendly mosquitocides are a priority. In Ayurvedic medicine, Plectranthus species have been used to treat heart disease, convulsions, spasmodic pain and painful urination. In this research, we evaluated the acute toxicity of essential oil from Plectranthus barbatus and its major constituents, against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. The chemical composition of P. barbatus essential oil was analyzed by gas chromatography-mass spectroscopy. Nineteen components were identified. Major constituents were eugenol (31.12%), α-pinene (19.38%) and ß-caryophyllene (18.42%). Acute toxicity against early third-instar larvae of An. subpictus, Ae. albopictus and Cx. tritaeniorhynchus was investigated. The essential oil had a significant toxic effect against larvae of An. subpictus, Ae. albopictus and Cx. tritaeniorhynchus, with 50% lethal concentration (LC50) values of 84.20, 87.25 and 94.34 µg/ml and 90% lethal concentration (LC90) values of 165.25, 170.56 and 179.58 µg/ml, respectively. Concerning major constituents, eugenol, α-pinene and ß-caryophyllene appeared to be most effective against An. subpictus (LC50 = 25.45, 32.09 and 41.66 µg/ml, respectively), followed by Ae. albopictus (LC50 = 28.14, 34.09 and 44.77 µg/ml, respectively) and Cx. tritaeniorhynchus (LC50 = 30.80, 36.75 and 48.17 µg/ml, respectively). Overall, the chance to use metabolites from P. barbatus essential oil against mosquito vectors seems promising, since they are effective at low doses and could be an advantageous alternative to build newer and safer mosquito control tools.