Virendra K. Dua

Larvicidal activity of neem oil ( Azadirachta indica ) formulation against mosquitoes

BackgroundMosquitoes transmit serious human diseases, causing millions of deaths every year. Use of synthetic insecticides to control vector mosquitoes has caused physiological resistance and adverse environmental effects in addition to high operational cost. Insecticides of botanical origin have been reported as useful for control of mosquitoes. Azadirachta indica (Meliaceae) and its derived products have shown a variety of insecticidal properties. The present paper discusses the larvicidal activity of neem-based biopesticide for the control of mosquitoes.MethodsLarvicidal efficacy of an emulsified concentrate of neem oil formulation (neem oil with polyoxyethylene ether, sorbitan dioleate and epichlorohydrin) developed by BMR & Company, Pune, India, was evaluated against late 3rd and early 4th instar larvae of different genera of mosquitoes. The larvae were exposed to different concentrations (0.5–5.0 ppm) of the formulation along with untreated control. Larvicidal activity of the formulation was also evaluated in field against Anopheles, Culex, and Aedes mosquitoes. The formulation was diluted with equal volumes of water and applied @ 140 mg a.i./m2 to different mosquito breeding sites with the help of pre calibrated knapsack sprayer. Larval density was determined at pre and post application of the formulation using a standard dipper.ResultsMedian lethal concentration (LC50) of the formulation against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was found to be 1.6, 1.8 and 1.7 ppm respectively. LC50 values of the formulation stored at 26°C, 40°C and 45°C for 48 hours against Ae. aegypti were 1.7, 1.7, 1.8 ppm while LC90 values were 3.7, 3.7 and 3.8 ppm respectively. Further no significant difference in LC50 and LC90 values of the formulation was observed against Ae. aegypti during 18 months storage period at room temperature. An application of the formulation at the rate of 140 mg a.i./m2 in different breeding sites under natural field conditions provided 98.1% reduction of Anopheles larvae on day 1; thereafter 100% reduction was recorded up to week 1 and more than 80% reduction up to week 3, while percent reduction against Culex larvae was 95.5% on day 1, and thereafter 80% reduction was achieved up to week 3. The formulation also showed 95.1% and, 99.7% reduction of Aedes larvae on day 1 and day 2 respectively; thereafter 100% larval control was observed up to day 7.ConclusionThe neem oil formulation was found effective in controlling mosquito larvae in different breeding sites under natural field conditions. As neem trees are widely distributed in India, their formulations may prove to be an effective and eco-friendly larvicide, which could be used as an alternative for malaria control.

Antiprotozoal activities of traditional medicinal plants from the Garhwal region of North West Himalaya, India.

ETHNOPHARMACOLOGICAL RELEVANCE In a search for new plant-derived biologically active compounds against protozoan parasites, an ethnopharmacological study was carried out to evaluate extracts from selected 17 traditional medicinal plants which were used by healers from the Garhwal region of North West Himalaya for the treatment of protozoal infections and fever including malaria. MATERIALS AND METHODS In vitro activity against erythrocytic stages of Plasmodium falciparum was determined using a modified [3H]-hypoxanthine incorporation assay with the chloroquine- and pyrimethamine-resistant K1 strain. Activity against Trypanosoma brucei rhodesiense was performed on the STIB 900 strain and activity against Trypanosoma cruzi on infected rat skeletal myoblasts (L6 cells) seeded in 96-well microtitre plates while amastigotes of Leishmania donovani strain MHOM/ET/67/L82 were used to assess activity against Leishmania donovani. Cytotoxicity assays were performed against rat skeletal myoblasts (L6-cells). RESULTS AND CONCLUSIONS Extracts of Artemisia roxburghiana, Roylea cinerea, Leucas cephalotes, Nepeta hindostana and Viola canescens showed good antiplasmodial activity (IC50<5 μg/ml). The chloroform extract of Artemisia roxburghiana was the most active (IC50 value of 0.42 μg/ml) and the most selective (SI=78) extract for Plasmodium falciparum among all plants extracts examined. The chloroform extract of Leucas cephalotes and the petroleum ether extract of Viola canescens exhibited substantial activities against Leishmania donovani with IC50 values of 3.61 μg/ml (SI=8) and 0.40 μg/ml (SI=30), respectively. The petroleum ether extract of Viola canescens exhibited activity against Trypanosoma cruzi with an IC50 value of 1.86 μg/ml (SI=7). Methanol and water extracts from all plants under investigation were found inactive against all parasites tested. These results support investigation of components of traditional medicines as potential new antiprotozoal agents. On the other hand since herbalism has become the main stream throughout the world, investigation demonstrates that these non-polar plant extracts of six of the plants examined in this study could play an important role in herbal formulations for the treatment of vector borne protozoal diseases.

Anti-malarial activity of Holarrhena antidysenterica and Viola canescens, plants traditionally used against malaria in the Garhwal region of north-west Himalaya

BackgroundThe increasing number of multidrug-resistant Plasmodium strains warrants exploration of new anti-malarials. Medicinal plant research has become more important, particularly after the development of Chinese anti-malarial drug artemisnin from Artemisia annua. The present study shows evaluation of anti-malarial effects of two plants commonly used against malaria in the Garhwal region of north-west Himalaya, in order to discover the herbal-based medicine.MethodsIn vitro anti-plasmodial sensitivity of plant extracts was assessed using schizont maturation and parasite lactate dehydrogenase (pLDH) assay. Cytotoxic activities of the examined extracts were determined on L-6 cells of rat skeletal muscle myoblast. The 4-day test for anti-malarial activity against a chloroquine sensitive Plasmodium berghei NK65 strain in Swiss albino mice was used for monitoring in vivo activity of plant extracts.ResultsChloroform extract of H. antidysenterica (HA-2) and petroleum ether extract of V. canescens (VC-1) plants significantly reduced parasitaemia in P. berghei infected mice. The extract HA-2 showed in vitro anti-plasmodial activity with its IC50 value 5.5 μg/ml using pLDH assay and ED50 value 18.29 mg/kg in P. berghei infected Swiss albino mice. Similarly petroleum ether extract of V. canescens (VC-1) showed in vitro anti-plasmodial activity with its IC50 value 2.76 μg/ml using pLDH assay and ED50 15.8 mg/kg in P. berghei infected mice. The extracts coded as HA-2 at 30 mg/kg and VC-1 at 20 mg/kg exhibited parasite inhibition in mice: 73.2% and 63.0% respectively. Of these two plant extracts, petroleum ether extract of V. canescens was found slightly cytotoxic.ConclusionThe present investigation reflects the use of these traditional medicinal plants against malaria and these plants may work as potential source in the development of variety of herbal formulations for the treatment of malaria.

In vitro antiprotozoal activity of some xanthones isolated from the roots of Andrographis paniculata.

Four xanthones isolated from the roots of Andrographis paniculata were tested in vitro for antiprotozoal activity against Trypanosoma brucei brucei, Trypanosoma cruzi and Leishmania infantum. Compound TDR13011 (1,2‐dihydroxy‐6,8‐dimethoxy‐xanthone) showed good activity against T. b. brucei and L. infantum with a 50% inhibitory concentration (IC50) of 4.6 µg/ml and 8 µg/ml respectively. Xanthones from the roots of Andrographis paniculata exhibited promising anti‐protozoal activity and these compounds could be chemically modified to obtain a more potent product. Copyright

Insecticidal and genotoxic activity of Psoralea corylifolia Linn. (Fabaceae) against Culex quinquefasciatus Say, 1823

BackgroundIndiscriminate use of synthetic insecticides to eradicate mosquitoes has caused physiological resistance. Plants provide a reservoir of biochemical compounds; among these compounds some have inhibitory effect on mosquitoes. In the present study the larvicidal, adulticidal and genotoxic activity of essential oil of Psoralea corylifolia Linn. against Culex quinquefasciatus Say was explored.MethodsEssential oil was isolated from the seeds of P. corylifolia Linn. Larvicidal and adulticidal bioassay of Cx. quinquefasciatus was carried out by WHO method. Genotoxic activity of samples was determined by comet assay. Identification of different compounds was carried out by gas chromatography- mass spectrometry analysis.ResultsLC50 and LC90 values of essential oil were 63.38±6.30 and 99.02±16.63 ppm, respectively against Cx. quinquefasciatus larvae. The LD50 and LD90 values were 0.057±0.007 and 0.109±0.014 mg/cm2 respectively against adult Cx. quinquefasciatus,. Genotoxicity of adults was determined at 0.034 and 0.069 mg/cm2. The mean comet tail length was 6.2548±0.754 μm and 8.47±0.931 μm and the respective DNA damage was significant i.e. 6.713% and 8.864% in comparison to controls. GCMS analysis of essential oil revealed 20 compounds. The major eight compounds were caryophyllene oxide (40.79%), phenol,4-(3,7-dimethyl-3-ethenylocta-1,6-dienyl) (20.78%), caryophyllene (17.84%), α-humulene (2.15%), (+)- aromadendrene (1.57%), naphthalene, 1,2,3,4-tetra hydro-1,6-dimethyle-4-(1-methyl)-, (1S-cis) (1.53%), trans- caryophyllene (0.75%), and methyl hexadecanoate (0.67%).ConclusionEssential oil obtained from the seeds of P. corylifolia showed potent toxicity against larvae and adult Cx. quinquefasciatus. The present work revealed that the essential oil of P. corylifolia could be used as environmentally sound larvicidal and adulticidal agent for mosquito control.

Molecular epidemiology of Plasmodium vivax anti-folate resistance in India

BackgroundSulphadoxine and pyrimethamine are anti-folate drugs that show synergistic anti-malarial effect. Point mutations in dihydrofolate reductase (dhfr) and dihydropteorate synthatase (dhps) cause anti-folate drug resistance phenotype in human malaria parasites. This study presents pattern of point mutations in dhfr/dhps genes among Indian sub-continent.MethodsMicroscopically diagnosed one hundred Plasmodium vivax field isolates were collected from five widely separated geographical regions of India. Dhfr and dhps genes were PCR amplified and sequenced. Previously published mutations data were collected and analyzed using Chi square test to identify geographical cluster of mutant/wild type genotypes.ResultsSequence analysis revealed single (S58R), double (S58R/S117N) and quadruple (F57L/S58R/T61M/S117T/) point mutations at dhfr and single (A383G) to double (A383G/A553G) mutations at dhps in P. vivax field isolates. In addition, three new mutations were also observed at dhfr. Both, dhfr and dhps genes revealed tandem repeat variations in field isolates. Dhps revealed very low mutation frequency (14.0%) compared to dhfr (50.70%). Comparative analysis revealed a progressive increase in frequency of quadruple mutant dhfr genotype (p < 0.001) within five years in north-eastern state (Kamrup, Assam). Frequency of dhfr genotypes revealed three distinct geographical clusters of wild (northern India), double mutant (southern India), and quadruple mutant (north-eastern and island regions of India) on the Indian sub-continent.ConclusionStudy suggests that SP may be susceptible to P. vivax in India, except Andaman and north-eastern state. The distinction of geographical regions with sensitive and resistant parasite phenotypes would be highly useful for designing and administering national anti-malarial drug policy.

Anti-malarial property of steroidal alkaloid conessine isolated from the bark of Holarrhena antidysenterica

BackgroundIn the face of chronic and emerging resistance of parasites to currently available drugs and constant need for new anti-malarials, natural plant products have been the bastion of anti-malarials for thousands of years. Moreover natural plant products and their derivatives have traditionally been a common source of drugs, and represent more than 30% of the current pharmaceutical market. The present study shows evaluation of anti-malarial effects of compound conessine isolated from plant Holarrhena antidysenterica frequently used against malaria in the Garhwal region of north-west Himalaya.MethodsIn vitro anti-plasmodial activity of compound was assessed using schizont maturation and parasite lactate dehydrogenase (pLDH) assay. Cytotoxic activities of the examined compound were determined on L-6 cells of rat skeletal muscle myoblast. The four-day test for anti-malarial activity against a chloroquine-sensitive Plasmodium berghei NK65 strain in BALB/c mice was used for monitoring in vivo activity of compound. In liver and kidney function test, the activity of alkaline phosphatase (ALP) was examined by p-NPP method, bilirubin by Jendrassik and Grof method. The urea percentage was determined by modified Berthelot method and creatinine by alkaline picrate method in serum of mice using ENZOPAK/CHEMPAK reagent kits.ResultsCompound conessine showed in vitro anti-plasmodial activity with its IC50 value 1.9 μg/ml and 1.3 μg/ml using schizont maturation and pLDH assay respectively. The compound showed cytotoxity IC50= 14 μg/ml against L6 cells of rat skeletal muscle myoblast. The isolated compound from plant H. antidysenterica significantly reduced parasitaemia (at 10 mg/kg exhibited 88.95% parasite inhibition) in P. berghei-infected mice. Due to slightly toxic nature (cytotoxicity = 14), biochemical analysis (liver and kidney function test) of the serum from mice after administration of conessine were also observed.ConclusionThe present investigation demonstrates that the compound conessine exhibited substantial anti-malarial property. The isolated compound could be chemically modified to obtain a more potent chemical entity with improved characteristics against malaria.

Development and validation of a reversed phase HPLC method for simultaneous determination of curcumin and piperine in human plasma for application in clinical pharmacological studies.

Abstract A sensitive and specific HPLC method was developed for the simultaneous determination of curcumin and piperine in human plasma in view of the potential therapeutic application of curcumin and piperine in various diseases. The HPLC method consisted of isocratic elution with acetonitrile-methanol-trifluoroacetic acid-water (17.6:35.3:0.1:47.0, v/v/v/v) with a flow rate of 2.5 mL min−1 on a Chromolith® SpeedROD RP-18 (50 × 4.6 mm) column at an ambient temperature. Ultraviolet detection was performed in programe mode at 415 nm for curcumin, 335 nm for piperine, and 280 nm for β-17-estradiol acetate (internal standard). Curcumin, piperine, and internal standard were extracted from plasma using ethyl acetate-propanol (9:1 v/v). Mean extraction recoveries for curcumin, piperine, and internal standard were 91.3, 91.4, and 92.9%, respectively. The assay was linear over the therapeutic concentration range (10–500 ng mL−1) for both drugs with correlation coefficients of r2 > 0.99. Limit of detection and limit of quantification were 1 ng mL−1 and 10 ng mL−1 for both curcumin and piperine. The method was applied to the determination of the concentrations of curcumin and piperine in healthy volunteers after treatment with 1500 mg curcumin and 500 mg piperine, and should find an applications in pharmacokinetic studies of these compounds.

Antigenic repertoire of Plasmodium vivax transmission-blocking vaccine candidates from the Indian subcontinent

BackgroundGenetic polymorphism is an inevitable component of a multistage infectious organism, such as the malaria parasite. By means of genetic polymorphism, parasite opts particular polymorph and reveals survival advantage. Pvs25 and pvs28 are sexual stage antigen genes, expressed at the ookinete stage inside the mosquito gut, and considered as potential transmission-blocking vaccine candidates. This study presents sequence variations in two important transmission blocking antigen genes pvs25 and pvs28 in the field isolates of P. vivax from the Indian subcontinent.MethodsOne hundred microscopically diagnosed P. vivax isolates were collected from five geographical regions of India. Pvs25 and pvs28 genes were PCR amplified and sequenced to assess sequence variation among field isolates.ResultsA total of 26 amino acid substitutions were observed in Pvs25 (10) and Pvs28 (16) among field isolates of P. vivax. Tandem repeat polymorphism observed in pvs28 shows 3-6 tandem repeats in the field isolates. Seven and eight novel amino acid substitutions were observed in Pvs25 and Pvs28, respectively in Indian isolates. Comparison of amino acid substitutions suggests that majority of substitutions observed in global isolates were also present in Indian subcontinent. A single haplotype was observed to be major haplotype among isolates of Delhi, Nadiad, Chennai and Panna except in isolates of Kamrup. Further, population comparison analyses suggest that P. vivax isolates inhabiting in north-eastern region (Kamrup) were distantly related with the isolates from remaining parts of the country. Majority of the amino acid substitutions observed in Indian isolates were more identical to the substitutions reported from isolates of Thailand and Bangladesh.ConclusionStudy uncovered many new amino acid substitutions as well as a predominance of single haplotype in Indian subcontinent except in north-eastern region of the country. The amino acid substitutions data generated in this study from different geographical regions of the Indian subcontinent could be helpful in designing a more effective anti-malarial transmission-blocking vaccine.

Population cytogenetic and molecular evidence for existence of a new species in Anopheles fluviatilis complex (Diptera: Culicidae)

Anopheles fluviatilis James, an important malaria vector in the Oriental region has been established as a complex of at least three cryptic species which vary in their biological characteristics and malaria transmission potential. The sibling species S, T and U of Fluviatilis Complex can be identified by examination of species-specific fixed inversions in the polytene chromosomes and can also be differentiated by an allele-specific PCR assay based on differences in the D3 region of 28S ribosomal DNA (rDNA) of these species. Here we report a new An. fluviatilis population from villages under Laksar Community Health Centre, District Haridwar (Uttarakhand state), India which differs from the three sibling species of Fluviatilis Complex by two fixed paracentric inversions, s(1) and S in polytene chromosome arms 2 and 3 respectively. Longitudinal study carried out in study villages showed that the new cytotype was sympatric with species T and U in all the collections and no inversion heterozygotes were observed between them. Thus presence of two fixed paracentric inversions in polytene chromosomes with total absence of inversion heterozygotes demonstrates reproductive isolation which unequivocally establishes this cytological variant as a new species, provisionally designated as species V in the Fluviatilis Complex. Analysis of DNA sequences of D3 domain of 28S rDNA and ITS 2 region has also shown that species V is distinctly different from species S, T and U. With the discovery of new species in the Fluviatilis Complex, in-depth studies are required to know its distribution pattern and biological characteristics and to ascertain its role in malaria transmission.