“LARVICIDAL POTENTIALS OF ESSENTIAL OILS FROM THE FLOWERS OF NELUMBO NUCIFERA AND TAGETES ERECTA AGAINST AEDES AEGYPTI” By K.SREE LEKHA A Dissertation Submitted to RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA in partial fulfillment of the requirements for the degree of MASTER OF PHARMACY IN PHARMACOGNOSY under the guidance of Dr. K. GIRIJA (Asst. Prof.) DEPARTMENT OF PHARMACOGNOSY P.E.S. COLLEGE OF PHARMACY BANGALORE-560050 MAY-2014 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA DECLARATION BY THE CANDIDATE I hereby declare that this dissertation entitled “Larvicidal potentials of essential oils from the flowers of Nelumbo nucifera and Tagetes erecta against Aedes aegypti” is a bonafide and genuine research work carried out by me under the guidance of Dr. K. GIRIJA, Assistant Professor, P.E.S. College of Pharmacy, Bangalore. Date: Place: Bangalore (K Sree lekha) PES COLLEGE OF PHARMACY Hanumanthanagar, Bangalore – 560 050 CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled “Larvicidal potentials of essential oils from the flowers of Nelumbo nucifera and Tagetes erecta against Aedes aegypti” is a bonafide research work done by K Sree lekha in partial fulfillment of the requirement for the degree of Master of Pharmacy in Pharmacognosy. Such materials as obtained from other sources have been duly acknowledged in this thesis. This report is now ready for examination. Date: Place: Bangalore (Dr.K. Girija) PES COLLEGE OF PHARMACY Hanumanthanagar, Bangalore – 560 050 ENDORSEMENT BY THE HOD, PRINCIPAL OF THE INSTITUTION This is to certify that the dissertation entitled “Larvicidal potentials of essential oils from the flowers of Nelumbo nucifera and Tagetes erecta against Aedes aegypti” is a bonafide research work done by K Sree lekha under the guidance of Dr. K. GIRIJA, P.E.S. College of Pharmacy, Bangalore. (Prof. Dr. K. Lakshman) (Prof. Dr. S. Mohan) Date: Date: Place: Bangalore Place: Bangalore COPYRIGHT DECLARATION BY THE CANDIDATE I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka will have the rights to preserve, use and disseminate this dissertation in print or electronic format for academic / research purpose. Date: Place: Bangalore (K Sree lekha) © Rajiv Gandhi University of Health Sciences, Karnataka. ACKNOWLEDGMENT “The will to achieve something is the best thrust of motivation.” I would like to express my sincere gratitude and appreciation at this stage to my guide, Asst.Prof. Dr. K.Girija, Department of Pharmacognosy. I express my deep sense of gratitude towards Dr.S.K Ghosh, Scientist G and Officer-in charge, National Institute Of Malarial Research, ICMR, Bangalore, for extending whole hearted support towards my work. I would also like to thank Mr. Devaiah for helping me actively during the course of research work. My sincere thanks to our beloved principal Prof. Dr. S.Mohan for providing me full facilities and being a constant source of inspiration. I am also grateful to Dr. K. Lakshman, Head of the department and Mr.G. Murugananthan, Asst. Professor and Mrs. Vinutha Bhat, Lecturer, Department of Pharmacognosy, Dr. Nagaraj, Department of Pharmaceutical Analysis, P.E.S college of Pharmacy for lending their kind support ,valuable advice and friendly help. I wish to extend my thanks to Mr. Anand, Mr. Anil and Mrs. Nirmala, nonteaching staff of P.E.S college of Pharmacy, who helped me directly or indirectly in completion of my dissertation. I wish to extend my warmest and sincere thanks to all my batch mates. My special thanks to all my juniors for their cheerful support. My gratitude won’t be justified, without my special thanks to my family members who have been a continuous source of support, encouragement, unconditional love and patience. Date: Place: Bangalore. K. Sree lekha LIST OF ABBREVIATIONS LIST OF ABBREVIATIONS Ae. aegypti Aedes aegypti cm Centimeter conc Concentrated DDT Dichlorodiphenyltrichloroethane DMSO Dimethyl sulphoxide ER Effective Repellency ft Feet g Grams GC-MS Gas chromatography-Mass spectrometry H Hours HCl Hydrochloric acid H2SO4 Sulfuric acid LC50 Lethal Concentration 50 LC90 Lethal Concentration 90 LCL Lower Confidence Limits LD50 Lethal Dose 50 LD90 Lethal Dose 90 M Molarity MIC Minimum Inhibitory Concentration mg/L Milligram per Liter mg/mL Milligram per MilliLiter Larvicidal potentials LIST OF ABBREVIATIONS min Minute mL MilliLiter N Normality NaOH Sodium hydroxide ODI Oviposition Deterrent Index OAI Oviposition Activity Index Ppm Parts per million Rf Retention factor rpm Revolutions per minute TCA Tri chloro Acetic acid TLC Thin Layer Chromatography UCL Upper Confidence Limits UV- Vis Ultraviolet-Visible spectrophotometer Z. rhetsa Zanthoxylum rhetsa μg Microgram μg/mL Microgram per Milliliter μL Microliter o degree Celsius C Larvicidal potentials Chapter-1 Introduction Chapter I Introduction 1.INTRODUCTION Mosquitoes are responsible for transmitting the most important vector-borne diseases, namely Malaria, Lymphatic filariasis, Dengue, Japanese encephalitis as well as yellow fever and other forms of encephalitis1.The mosquito vector Aedes aegypti is known to carry dengue and yellow fever virus. Mosquitoes are responsible for spread of many diseases than any other group of arthropod2. Urbanization and changed lifestyles mainly contributed to the proliferation of larval habitats resulting in the disease epidemics3. WHO has declared the mosquito “Public enemy number one”. Mosquitoes are the vectors for the dreadful diseases of mankind, of all the insects that transmit diseases, mosquitoes represent greatest menace4. Most people consider mosquitoes as an annoyance; these tiny assassins have the potential and lethal capacity to kill more than a million victims a year around the world5. Prevalence of Mosquito borne diseases are one of the world’s most health hazardous problems. Many approaches have been developed to control mosquito menace6. It is estimated that every year at least 500 million people in the world suffer from one or the other tropical diseases that include malaria, lymphatic filariasis, schistosomiasis, dengue, trypanosomiasis and leishmaniasis. Of late chikungunya, a serious mosquito borne epidemic has gained momentum in India. These diseases not only cause high levels of morbidity and mortality, but also inflict great economic loss and social disruption on developing countries7. Dengue fever is endemic over large areas of tropics and subtropics. The etiological agent is an arbovirus and the major vector is the Aedes aegypti mosquitoe. A four fold Larvicidal potentials 1 Chapter I Introduction increase in dengue fever incidence has occurred since 1970 and presently nearly half the world’s population is at risk. In 1990,almost 30% of the world population lived in regions where the estimated risk of dengue transmission was greater than 50%8. Chikungunya virus infection, transmitted by the vector mosquito Aedes aegypti was originally reported in Africa in 1950 and its emergence has led to the ongoing outbreak that has involved >1.5 million patients8. One of the effective methods to control these diseases is to target the vectors for interrupting the disease transmission. The control effort can be targeted at all stages of the mosquito life cycle, but the focus has mainly been on the adult stage and larval stage, especially in Sub-Saharan Africa1. Vector control using synthetic insecticides had been favorable so far, because of their speedy action and easy application9. However, the extensive and unbalanced usage of synthetic insecticides such as organochlorides, organophosphates and carbamates10 has disrupted natural biological control systems, led to the development of resistance as in case of Malathion and Deltamethrin resistance in adult A.aegypti11, adversely affected the nontarget flora and fauna inhabiting the same aquatic habitat12 and fostered environmental and human health concerns13, which paved the way to search for alternative vector control measures. The control of mosquito at the larval stage is necessary in integrated mosquito control strategies. As the mosquitoes are relatively immobile in immature stage1, it is much easier to control the mosquito population in the larval stages compared to adults. Moreover, focusing the larval stage has the advantage of controlling the vector prior to acquisition of the disease and interrupting the life cycle before transmission of the disease14. Therefore, the ideal Larvicidal potentials 2 Chapter I Introduction control method would be the systematic treatment of their breeding places through larvicides15. Disease transmission can be interrupted by controlling the vectors using various methods. One of the methods available for the control of mosquitoes is the use of insecticides. Chemical control using synthetic insecticides had been favorable so far, because of their speed action and easy application16. Synthetic insecticides are toxic and adversely affect the environment by contaminating soil, water and air17. However, the extensive and unbalanced use of chemical insecticides have created problems like enhancing resistance, adverse effects on the non-target flora and fauna inhabiting the same aquatic habitat18. The present proliferation of these diseases is not only due to higher number of breeding places in urban agglomeration, but also due to increasing resistance of mosquitoes to current commercial insecticides such as organochlorides, organophosphates and carbamates10. Oviposition is one of the most important events in the life cycle of mosquitoes. Mosquito population can be reduced by disrupting its oviposition19. As majority of the compounds are chemicals it steadily causes problems, demanding an intensive research for new products that are environmentally safe, target specific and degradable19. Since botanicals are less likely to cause ecological damage, a large number of plants have been screened for their insecticidal activities against mosquitoes20. Botanical pesticides are promising in that they are effective, environment – friendly, easily biodegradable and also inexpensive21. Larvicidal potentials 3 Chapter I Introduction Plants are considered as a rich source of bioactive chemicals and they may be an alternative source of mosquito control agents22. The co-evolution of plants with insects has equipped them with a plethora of chemical defenses which can be used against insects. Since botanicals are less likely to cause ecological damage, a large number of plants have been screened for their insecticidal activities against mosquitoes and some of these have been found to possess promising effects23. The development of resistance by pests and vectors against botanicals has not been reported so far. Active principles from many plants have been recognized, isolated, purified and formulated as insecticides24. Botanical pesticides have been used traditionally by human communities in many parts of the world against pest species of insects25. Natural source has provided many effective larvicides as exemplified by Pyrethrins from Chrysanthemum species, Azadirachtin from Azadirachta species, Rotenone from Derris species, and Ryanodine from Ryania speciosa 26. Also a large number of plants like Azadirachta indica, Cymbopogan species, Mentha species and Eucalyptus maculata have exhibited mosquito repellent activities27. Besides essential oils from plants like Citronella, eucalyptus, neem, geranium and lemon grass have furnished active ingredients of many commercial repellents28. Although many plant products are used for insecticidal, repellent and oviposition deterrent properties against mosquitoes, the search for effective natural products as alternative vector control agents is continuing. Therefore, the present study was aimed to explore larvicidal potentials of essential oils from the flowers of Nelumbo nucifera and Tagetes erecta against mosquito vector Aedes aegypti. Larvicidal potentials 4 Chapter-2 Objectives Chapter II Aims and Objectives 2. AIM AND OBJECTIVE The aim of the present study was to evaluate the Larvicidal potentials of essential oils from the flowers of nelumbo nucifera and tagetes erecta. The work was carried out at P.E.S college of Pharmacy and National Institute of Malarial Research, Indian Council of Medical Research (ICMR), Bangalore The main objectives of present study were:  Procurement of Essential oils of flowers of Nelumbo nucifera and Tagetes erecta from Falcon Essential oils, Bangalore.  Preliminary phytochemical studies of essential oils.  Screening for larvicidal activity on Aedes aegypti vector. Lavicidal potentials 5 Chapter-3 Review of Literature Chapter III Review of literature 3. REVIEWOF LITERATURE 3.1. Plant Profile of Nelumbo nucifera Fig no.1 Nelumbo nucifera flower 3.2 Synonyms : Nelumbium speciosum wild. Nymphaea nelumbo 3.3 Vernacular names : 30 English – Lotus, Sacred lotus Hindi – Kamal, Pundarika, Padma Sanskrit – Sarsija, Pankeruha, Sharada Larvicidal potentials 6 Chapter III Review of literature Telugu – Tamara, Erra-tamara Tamil – Chenthaamarai, Tamarai Kannada – Tavare-gadde. 3.4 Scientific classification: TAXONOMY : 29 Kingdom Plantae Subkingdom Superdivision Tracheobionta Spermatophyta Division Magnoliophyta Class Magnoliopsida Subclass Magnoliidae Order Nymphaeales Family Genus Species Larvicidal potentials Nelumbonaceae Nelumbo Adans. Nelumbo nucifera Gaertn. 7 Chapter III Review of literature 3.5 General Description31: Nelumbo nucifera is an perennial aquatic herb, Root stock stout, cylindrical. Leaves peltate, radiately nerved. Flowers large, solitary, fragnant, ovoid, fleshy, sunk separately in cavities of receptacle, maturing into nut-like achenes and typically grows 3-6’ tall in shallow water and spreads by thickened rhizomes rooted in the mud. This is a marginal aquatic perennial that features rounded, parasol-like, upward-cupped, waxy green leaves (to 2’ across) that appear above the water on long petioles which attach at the middle of the leaf underside. 3.6 Parts used : Roots, leaves, flowers, seeds. 3.7 Chemical constituents32 : Leaves contain alkaloids: nuciferine, roemerine, flavanoid: quercetin. The plumules yield proteins, sugars and vitamins. The receptacles contain quercetin. Several flavonoids have been identified in the stamens of N.nucifera.These include kaempferol and its glycosides: kaempferol 3-O-β-D-galactopyranoside, kaempferol 3-O- β-D- glucopyranoside, kaempferol 7-O-β-D-glucopyranoside, It also contains two isorhamnetin glycosides: isorhamnetin 3-O-D-glucopyranoside and isorhamnetin 3-O-a-L-rhamnopyranosyl(1,6)-D-glucopyranoside. Larvicidal potentials 8 Chapter III Review of literature Properties of Kaempferol 34: Kaempferol (38): R1 = R2 = H Kaempferol 3-O-_-D-galactopyranoside (39): R1 = Gal; R2 = H Kaempferol 3-O-_-D-glucopyranoside (40): R1 = Glc; R2 = H Kaempferol 7-O-_-D-glucopyranoside (41): R1 = H; R2 = Glc Kaempferol 3-O-_-L-rhamnopyranosyl-(1,6)- _-D-glucopyranoside (42): R1 = Rha-(1®6)-Glc; R2 = H Kaempferol 3-O-_-L-rhamnopyranosyl-(1_2)- _-D-glucopyranoside (43): R1 = Rha-(1_2)-Glc; R2 = H Kaempferol 3-O-_-L-rhamnopyranosyl-(1_2)- _-D-glucuronopyranoside (44): R1 = Rha-(1_2)- Gln; R2 = H Kaempferol 3-O-_-D-glucuronopyranoside (45): R1 = Gln; R2 = H Kaempferol 3-O-_-D-glucuronopyranosyl Methylester (46): R1 = Gln-Me; R2 = H Larvicidal potentials 9 Chapter III Review of literature IUPAC Name 33 : 3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one Molecular Formula : C15H10O6 Molecular Weight : 286.24 Physical State : Yellow crystalline powder Solubility : Slightly soluble in water, very soluble in ethanol. Melting Point : 276-278oc Boiling Point : 610oc Stability : Stable under normal conditions Assay : 98.0% min Loss on drying : 2.0% min Kaempferol is a strong antioxidant and helps to prevent oxidative damage of our cells, lipids and DNA. Kaempferol seems to prevent arteriosclerosis by inhibiting the oxidation of low density lipoprotein and the formation of platelets in the blood. Kaempferol acts as a chemopreventive agent, which means that it inhibits the formation of cancer cells. Larvicidal potentials 10 Chapter III Review of literature NUCIFERINE((3) – R1=R2=R3=CH3 ) Quercetin 3-O-β-D-glucopyranoside 3.8 Modern and Traditional uses34: The astringent qualities of Lotus may be efficacious in treating hemorrhoids and dysentery. Lotus is helpful in controlling the burning sensation, due to its cold potency. The leaf paste is applied to the body in case of fever and inflammatory skin conditions. Larvicidal potentials 11 Chapter III Review of literature The flower stalk, mixed with other herbs, is used to treat bleeding from the uterusons. Roots and Rhizomes: To treat smallpox, throat conditions, loss of skin pigmentation, coughs, diarrhea, and dysentery. Rhizomes boiled with sesame oil and rubbed on the head to cool the head, including the eyes. Leaves and Stems: Prepared many ways to treat hemorrhoids (piles), leprosy, parasites and vomiting, The leaves are also used in treating sunstroke, diarrhea, dysentery, dizziness and vomiting of blood. Various parts of the flower, including petals: For diarrhea, cholera, fever, liver conditions, bronchitis, skin eruptions, snakebites, and scorpion stings. Dried flowers are prepared into a syrup to treat coughs. Fruits and seeds: To soothe inflamed mucous membranes, lower fever, and alleviate halitosis. Seeds taken orally with a rice wash for seven days can increase female fertility. 3.9 PHARMACOLOGICAL INVESTIGATION: 1. Hypoglycaemic activity35 : Sun-dried flower powder of N.nucifera, as well as the aqueous and alcoholic extract of the flower, produced significant hypoglycaemia in fasting normal albino rabbits. There was no significant difference in the activities of 1000 mg/kg of the test drug (sun-dried powder of the flower) and equivalent amounts of the extracts. Glucose tolerance studies with normal rabbits showed that oral doses of both extracts, equivalent to 1000 mg/kg of the test drug, produced significant depression of the peak rise in fasting blood sugar after glucose load; the effects of both extracts were 50% to that produced by 250 mg/kg of tolbutamide. A study of glucose tolerance curves shows that the duration of hyperglycaemia was also notably reduced compared with the controls. In normal rabbits, the Larvicidal potentials 12 Chapter III Review of literature extract at a dose of 1000 mg/kg significantly lowered hyperglycaemia induced by subcutaneous injection of 0.5 mg/kg adrenaline hydrochloride. 2. Aldose reductase inhibitory activity36 : Two glycosides, namely kaempferol 3-O-a-L-rhamnopyranosyl- (1,6)-_-D glucopyranoside and isorhamnetin 3-O-a- Lrhamnopyranosyl-( 1!6)-_-D-glucopyranoside, isolated from the methanol extract of stamens of N. nucifera exhibited a high degree of inhibitory activity against rat lens aldose reductase in vitro, with IC50 values of 5.6 and 9.0 mm, respectively. 3. Antioxidant activity37 : The potential of N. nucifera stamens to scavenge 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radicals and peroxynitrites (ONOO–), and the inhibition of total ROS generation by kidney homogenates using 2v,7’-dichlorodihydrofluorescein diacetate (DCHF-DA) was examined[44].The methanol extract showed strong antioxidant activity in the ONOO– system and marginal activity in the DPPH and total ROS systems. Similarly, seven known flavonoids were isolated from lotus stamens, most of which also showed potent antioxidant activity[44]. The glycosides nelumboroside A, nelumboroside B, isorhamnetin glycoside and isorhamnetin rutinoside isolated from N. nucifera stamens showed potent antioxidant activity in DPPH and ONOO– assays. „Yunyupju’, a liquor made from the blossoms and leaves of lotus, has been reported to have antioxidant activity. The maximum scavenging activity on hydroxyl radicals (40%) could be achieved when lotus liquor was more than 500 μg. Lotus liquor also has a potent superoxide radical scavenging activity with value of 0.93 unit mg−1 as superoxide dismutase equivalents with an IC50 value of 1.07 ± 0.04 mg. 4. Antiplatellet activity38 : The antiplatelet activity of hydroethanolic extract of N.Nucifera flowers using platelet-rich plasma in different concentrations (100 -500μg/ ml) was reported. N.nucifera flower extracts showed Larvicidal potentials 13 Chapter III Review of literature dosedependent effective antiplatelet activity with maximum activity at 500μg/ml concentration; prevention of platelet aggregation was 50% of that achieved with standard aspirin. 5. Larvicidal activity39 : i. The larvicidal potential of the hexane, chloroform, ethyl acetate, acetone, methanol, and aqueous leaf extracts of Nelumbo nucifera Gaertn. (Nymphaeaceae) and synthesized silver nanoparticles using aqueous leaf extract against fourth instar larvae of Anopheles subpictus Grassi and Culex quinquefasciatus Say (Diptera: Culicidae). ii. Nanoparticles are being used in many commercial applications. It was found that aqueous silver ions can be reduced by aqueous extract of plant parts to generate extremely stable silver nanoparticles in water. Larvae were exposed to varying concentrations of plant extracts and synthesized silver nanoparticles for 24 h. iii. All extracts showed moderate larvicidal effects; however, the maximum efficacy was observed in crude methanol, aqueous, and synthesized silver nanoparticles against the larvae of A. subpictus (LC(50) = 8.89, 11.82, and 0.69 ppm; LC(90) = 28.65, 36.06, and 2.15 ppm) and against the larvae of C. quinquefasciatus (LC(50) = 9.51, 13.65, and 1.10 ppm; LC(90) = 28.13, 35.83, and 3.59 ppm), respectively. 6. Antibacterial Activity40 : The hydroethanolic extract of flowers of N.nucifera Gaertn invitro was reported to possess antibacterial activity. The antibacterial activity was screened against different bacterial strains like Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Bacillus Subtilis, Staphylococcus aureus by detecting zone of inhibition and minimum inhibitory concentration (MIC). The maximum zone of inhibition was exhibited by N.nucifera flowers against Escherichia coli (14mm), Bacillus Subtilis (13mm) and Staphylococcus aureus (11mm). The moderate zone of Larvicidal potentials 14 Chapter III Review of literature inhibition was found against Klebsiella pneumonia (10mm) and Pseudomonas aeruginosa (8mm). Gram-negative bacteria were more susceptible to the N.nucifera flower extracts than gram-positive bacteria which contradict the previous reports that plant extracts are more active against grampositive bacteria than gram-negative bacteria. These results were compared with the standard antibiotic chloramphenicol (30μg/ml). PHYTOCHEMICAL INVESTIGATION : 1. Phytochemical investigation of the MeOH extract of the leaves of Nelumbo nucifera resulted in the isolation of five norsesquiterpenes, four flavonoids, two triterpenes and one alkaloid. Their chemical structures were characterized by spectroscopic methods to be (E)-3-hydroxymegastigm-7en-9-one (1), (3S,5R,6S,7E)-megastigma-7-ene-3,5,6,9-tetrol (2), dendranthemoside B (3), icariside B2 (4), sedumoside F1 (5), luteolin (6), quercetin 3-O-β-D-glucuronide (7), quercetin 3-O-β-Dglucoside (8), isorhamnetin 3-O-rutinoside (9), alphitolic acid (10), maslinic acid (11), and Nmethylasimilobine (12). Norsesquiterpenoids (1-5) and triterpenes (10-11) were isolated for the first time from this plant. Compounds 6 and 10-12 exhibited considerable cytotoxicity against four human cancer cell lines in vitro using a SRB bioassay41. 2. Phytochemical constituents, free radical scavenging activity and total antioxidant activity of various extracts of Nelumbo nucifera flowers were carried out in the study. Phytochemicals were extracted from Nelumbo nucifera flowers using various solvents such as aqueous, benzene, chloroform, ethanol, ethyl acetate and methanol and petroleum ether. Screening of phytochemicals showed positive results for the presence of flavanoids, alkaloids, phenols, glycosides, carbohydrates and tannins42. Larvicidal potentials 15 Chapter III 3. Review of literature Nelumbo nucifera leaf and flower solvent extracts were evaluated for phytochemical analysis, anticancer activity. Phytochemical analysis revealed major active phytoconstituents such as alkaloids, flavonoids, phenols, tannins, steroids and glycosides. Anticancer activity of methanol and acetone leaf extracts showed less activity against human breast cancer cell (MCF7). The lotus leaf showed outstanding water repellency (super-hydrophobicity) particularly on its upper side, which is more robust and less sensitive to mechanical damage than the underside. Leaf treated with 0.9% NaCl showed complete rupture of the cells and that treated with glucose showed no complete rupture of the cells43. 4. A phytochemical investigation of N. nucifera leaves led to the isolation of 13 megastigmanes (1-13), including a new megastigmane, nelumnucifoside A (1), and a new eudesmane sesquiterpene, nelumnucifoside B (14), eight alkaloids (15-22), and 11 flavonoids (23-33). Therefore, the leaves of N. nucifera have potential as an anti-obesity agent by inhibiting pancreatic lipase and adipocyte differentiation44. 5. Fresh rhizomes of nelumbo nucifera contain 31.2% starch, which shows no characteristic taste or odour.The methanol extract of the rhizome has been found to possess a steroidal triterpenoid – betulinic acid. Fresh rhizome contains 83.80% water, 0.11% fat, 1.56% reducing sugar, 0.41% sucrose, 2.70% crude protein, 9.25% starch, 0.80% fibre, l.10% ash and 0.06% calcium. The vitamins thiamine (0.22 mg/100 g), riboflavin (0.6 mg/100 g), niacin (2.10 mg/100 g) and ascorbic acid (1.5 mg/100 g) and an asparagine-like amino acid (2%) are also present in the rhizomes. The oxalate content of rhizome was found to be 84.3 mg/100g34. Larvicidal potentials 16 Chapter III Review of literature REVIEW OF LITERATURE 3.10. Plant profile of Tagetes erecta : Fig no.2 Tagetes erecta flower 3.11 Synonyms : African marigold Aztec marigold 3.12 Vernacular Names46 : English : African marigold Sanskrit : Jhandu, sthulapushpa, zandu, zanduga Hindi : Genda, hajara, hajari, hajri, jhandu Telugu : Banthi Kannada : Chandu hoo, chandu mallige, seeme shavantige Tamil : Kancappucceti, totika, totikavantippa. Larvicidal potentials 17 Chapter III Review of literature 3.13 Scientific classification TAXONOMY45 : Kingdom Plantae Subkingdom Superdivision Tracheobionta Spermatophyta Division Magnoliophyta Class Magnoliopsida Subclass Asteridae Order Asterales Family Genus Species Larvicidal potentials Asteraceae Tagetes L. Tagetes erecta L. 18 Chapter III Review of literature 3.14 General description47 : Erect annual herb up to 180 cm tall, glabrous; stem and branches angular to rounded. Leaves opposite in lower part of plant, alternate in upper part, pinnately compound with 9–17 leaflets; leaflets linear to lanceolate, 1–3 cm × 0.5–1.5 cm, acute or acuminate at both ends, margins serrate, bearing scattered oil glands. Ray flowers female, ligulate, 5–8 in wild types, in cultivars often very numerous, ligule broadly obovate, 1–2.5 cm × 1–2 cm, bright yellow in wild types, lemon-yellow to deep brownred in cultivated types; disk flowers bisexual, tubular, numerous, 8–10 mm long; stamens 5, anthers united into a tube around the style; ovary inferior, 1-celled, style 2-branched. 3.15 Parts used : Flowers, leaves, stem 3.16 Chemical constituents48 : Tagetes erecta contains thiophenes, flavonoids, carotenoids, triterpenoids. Glucoside of Quercetagenin, phenolics, syringic acid, quercetin, thienyl and ethyl gallat. Benzaldehyde, (s)-(-)-limonene α-pinene, camphene, lutein α-phellandrene, α-terpinolene piperitone, piperitenone sabinene, (E) –tagetone, linalool. Larvicidal potentials 19 Chapter III Review of literature Properties of Linalool 49 : Linalool is naturally occurring terpene alcohol chemical found in many flowers and spice plants with many commercial applications. It has other names such as β-linalool, linalyl alcohol, linaloyl oxide, p-linalool, allo-ocimenol, and 2,6-dimethyl-2,7-octadien-6-ol. Linalool has a stereogenic center at C3 and therefore there are two stereoisomers: (R)-(–)-linalool is also known as licareol and (S)-(+)-linalool is also known as coriandrol. In addition, linalool is used by pest professionals as a flea, fruit fly and cockroach insecticide. Linalool is used in some mosquito-repellent products, however the EPA notes that: "A preliminary screen of labels for products containing Linalool (as the sole active ingredient) indicates that efficacy data on file with the Agency may not support certain claims to repel mosquitoes. IUPAC Name : 3,7-dimethylocta-1,6-dien-3-ol Molecular Formula : C18H10O6 Molecular Weight : 154.24 Molar Mass : 154.25 g/mol Density : 0.858- 0.868 g/cm3 Melting Point : < -20oc Larvicidal potentials 20 Chapter III Review of literature Boling Point : 198-199oc Solubility : soluble in water (1.589g/l) PIPERITONE α – TERPINOLENE 3.17 Modern And Traditional uses47 : The flowers of Tagetes erecta are the source of two food colourant products: Marigold meal and Marigold extract. The plant is most effective against the nematode species Pratylenchus penetrans. T. erecta is grown to extract lutein, a common yellow/orange food colour. The essential oil of the flower contains antioxidants. Larvicidal potentials 21 Chapter III Review of literature Fresh and dry flowers can be used to dye wool, silk and cellulose fibres into shades of goldenyellow to orange and olive-green to bronze. The whole herb is considered medicinal with anthelmintic, aromatic, digestive, diuretic, sedative and stomachic properties. It is used internally to treat indigestion, colic, severe constipation, dysentery, cough and fever, and externally to treat sores, ulcers, eczema, sore eyes and rheumatism. 3.18 PHARMACOLOGICAL INVESTIGATION : 1. Antibacterial activity50 : Rhama and Madhavan reported the antibacterial activity of different solvents of tagetes erecta flowers against Alcaligens faecalis, Bacillus cereus, Campylobacter coli, Eschercia coli, Klebsellia pneumoniae, Pseudomonas aeuroginosa, Proteus vulgaris, Streptococcus mutans. The flavanoids posess anti-bacterial activity against all the tested strains and shows maximum zone of inhibition for Klebsellia pneumonia(29.50 mm). The flavanoid-Patulitrin is one of the potential elements for its anti-bacterialactivity. 2. Antimicrobial Activity51 : Flavonoids and esters of phenolic acids were investigated for their antibacterial, antifungal and antiviral activities. All samples were active against the fungal and gram-positive bacterial test strains and most showed antiviral activity. 3 . Antifungal Activity51 : Number of flavonoids isolated from peel of tangerine orange, when tested for fungistatic activity towards Deuterophoma tracheiphila showed promising activity. Chlorflavonin was the first chlorine-containing flavonoid type antifungal antibiotic produced by strains of Aspergillus candidus. Larvicidal potentials 22 Chapter III Review of literature 4. Anti oxidant activity52 : In Vitro antioxidant studies were performed on the ethanolic extract of Tagetes erecta flowers. During the study preliminary phytochemical analysis were carried out on ethanolic extract of flowers of Tagetes erecta and found the presence of Alkaloids, Flavonoids, Proteins, Steroids and tannins. For the study of in Vitro antioxidant activity three different assays like DPPH, reducing power and super oxide radical scavenging activity at different concentrations were used. In all the three assay, Tagetes erecta showed better reducing power than the standard (i.e. ascorbic acid), and superoxide anion scavenging activity and DPPH antioxidant activity showed less than standard. 4. Larvicidal activity53 : The aim of this study was to evaluate the activity of essential oil from Tagetes erecta against 3rd instars of Aedes aegypti and to determine the amounts of larvicidal thiophenes in all plant tissues.. The essential oil was active against larvae of Ae. aegypti, with LC50 of 79.78 microg/ml and LC90 of 100.84 microg/ml. The larvicidal thiophene contents were higher in the roots and flowers as demonstrated by high-performance liquid chromatography analysis. Thus, T. erecta constitutes a good source of varied compounds showing larvicidal activity against Ae. aegypti. 5. Mosquitocidal activity54 : Nikkon et al. repoted the mosquitocidal activity in ethanolic, chloroform and ether extract of tagetes erecta flowers against different instars of culex quinquefasciatus. Among the tested samples the chloroform soluble fractions showed the highest toxicity and consequently, the lowest LC50 values (14.14 µg/mL, 17.06 µg/mL, 36.88 µg/mL and 75.48 µg/mL) for all the instars larvae of Cx. quinquefasciatus. The larvae showed comparative tolerance in the course of increasing age and time. Larvicidal potentials 23 Chapter III Review of literature It can be concluded that the flowers of T. erecta are very effective natural larvicide and could be useful against Cx. quinquefasciatus. PHYTOCHEMICAL INVESTIGATION : 1. Devika and Justin reported various bioactive compounds from Tagetes erecta. The various parts of the plant such as stem, leaf, flower and root were subjected to analysis the qualitative phytochemical constituents with two different solvents (ethyl alcohol and petroleum ether). About sixteen phytochemicals analysis were carried out, out of which six phytochemicals were identified in both the solvents predominantly. The present phytochemical screening of the Tagetes erecta proved to contain flavonoid, alkaloid, quinones, Phenols, triterpenoid and coumarins bioactive compounds which are of medicinal value and have a definite physiological action on the human body55. 2. Marigold (Tagetes erecta L.) flower petal extracts were prepared in the laboratory by extracting with different solvents. Different chromatographic and spectroscopic techniques were adopted for purification and identification of the compounds. Fractions obtained after column chromatography of solvent extracts showed the presence of six compounds. From the mass fragmentation pattern, the molecules identified were dodecanoic acid (I), myristic acid (II), palmitic acid (III), stearic acid (IV), octaeicosane-8-one (V) and triacontane-1-ol (VI). The crude extract, solvent fractions and the purified compounds were subjected to bioassay against two agriculturally important pest Spodoptera litura and Meloidogyne incognita. The hexane extract and the purified compounds showed highest activity against Spodoptera larvae while the Larvicidal potentials 24 Chapter III Review of literature methanol fractions and the purified compounds again yielded best result against Meloidogyne incognita 56. 3. The flowers of African marigold (Tagetes erecta L), a medicinal plant widely cultivated in Thailand, were subjected to evaluation for total phenolics, DPPH scavenging and thiobarbituric acid-reactive substance (TBARs) assays as well as tyrosinase inhibitory activity. In preliminary studies, the ethyl acetate (EA) extract obtained by continuous extraction showed the highest activities with highest phenolic content among all extracts57. 4. Kadam Prasad vijay et al. reported the pharmacognostic and phytochemical investigation of tagetes erecta of powdered flowers and methanolic extract which shows the presence of tannins, flavanoids, phenolic compounds, steroids, triterpenoids, saponins, alkaloids which indicates therapeutic activity. The physicochemical parameters of tagetes erecta were Loss on drying: 7.46%w/w, total ash : 4.95%w/w, acid insoluble ash : 0.2%w/w, sulphated ash: 1.3%w/w, water soluble extractive value: 72%w/w, alcohol soluble extractive value: 16.8%58. Larvicidal potentials 25 Chapter III Review of literature 3.19DESCRIPTION OF VECTOR: Aedes aegypti: Aedes aegypti has been one of the most important mosquito vectors of human disease59. It is a main vector which transmits the viruses that cause dengue, chikungunya and yellow fever. The viruses are passed on to humans through the bites of an infective female Aedes mosquito, which mainly acquires the virus while feeding on the blood of an infected person64,65,66. . Fig.3 Adult Aedes aegypti Scientific classification Kingdom : Animalia Phylum : Arthropoda Class : Insecta Order : Diptera Family : Culicidae Genus : Aedes Species : aegypti Larvicidal potentials 26 Chapter III Review of literature Biting Behavior Ae. aegypti bites primarily during the day. This species is most active for approximately two hours after sunrise and several hours before sunset, but it can bite at night in well lit areas. Ae. aegypti prefers biting people but it also bites dogs and other domestic animals, mostly mammals. Only females bite to obtain blood in order to lay eggs. Life Cycle of Aedes aegypti Ae. aegypti is a so-called holometabolous insect, meaning that the insects goes through a complete metamorphosis with an egg, larvae, pupae, and adult stage. The adult life span can range from two weeks to a month depending on environmental conditions. The life cycle of Ae. aegypti can be completed within one-and-a-half to three weeks63 shown in fig 4. Fig.4 Life Cycle of Aedes aegypti Larvicidal potentials 27 Chapter III Review of literature Egg Stage: After taking a complete blood meal, females produce on average 100 to 200 eggs per batch and can produce up to five batches of eggs during a lifetime64. Eggs are laid on damp surfaces in areas likely to temporarily flood, such as tree holes and man-made containers, and are laid singly, rather than in a mass65. Most often, eggs will be placed at varying distances above the water line, and a female will not lay the entire clutch at a single site, but rather spread out the eggs over two or more sites. Eggs of Ae. aegypti are long, smooth, ovoid shaped, and approximately one millimeter 66 .Ae. aegypti eggs can survive desiccation for months and hatch once submerged in water.64 Fig. 5: Ae. aegypti Eggs Larval Stage : The mosquito larva has a well-developed head with mouth brushes used for feeding, a large thorax with no legs, and a segmented abdomen as shown in Figure5. Larvae breathe through siphon located on the eighth abdominal segment and therefore must come to the surface frequently. The larvae feeds on algae, bacteria, and other microbes in the surface microlayer. Larvae swim either through propulsion with their mouth brushes, or by jerky movements of their entire bodies, giving them the common name of "wigglers" or "wrigglers". Larvae develop through four stages, or instars, as shown in Figure 1.6 after which they metamorphose into pupae67. Larvicidal potentials 28 Chapter III Review of literature Figure 6: Dorsal Veiw of Ae. aegypti larvae Figure 7: Ae. aegypti larvae stage Pupal Stage: The head and thorax of the mosquito pupa are merged into a cephalothorax, with the abdomen curving around underneath as shown in fig 8. The pupa can swim actively by flipping its abdomen, and it is commonly called a "tumbler" because of its swimming action. As with the larvae, the pupae of most species must come to the surface frequently to breathe, which they do through a pair of respiratory trumpets on the cephalothorax. The pupae do not feed. After few days, the pupa rises to the water surface, the dorsal surface of its cephalothorax splits, and the adult mosquito emerges68. Figure 8: Ae. aegypti pupae stage Larvicidal potentials 29 Chapter III Review of literature Adult Stage: The adult mosquito after emerging out of pupal case, it will float on the surface of the water and rest there until its body and wings harden. Once the body has hardened the mosquito will fly off to begin its new life. One of the first things newly emerged mosquitoes do is seek out nectar for a sugar meal to provide energy for flying and mating63. Figure 9: Dorsal View of Ae. aegypti Adult Ae. aegypti is a smallish, dark mosquito with conspicuous white markings and banded legs; the proboscis is all black although the palps are white tipped; the scutum has a dorsal pattern of white scales in the form of a 'lyre' with curved lateral and 2 central stripes contrasting with the general covering of narrow dark scales; wings are dark scaled; hind legs with femur pale scaled for basal three-quarters with dark as shown in Figure 9 69. Generally male mosquitoes emerge a few days before female mosquitoes. This gives the males a chance to mature before the females emerge. After they mate the female will look for a blood meal. Only the adult female mosquito feeds on blood, as it needs the protein from the blood to develop the eggs63. Larvicidal potentials 30 Chapter III Review of literature Aedes aegypti transmitted diseases : Dengue Dengue is the most rapidly spreading mosquito-borne viral disease in the world. In the last 50 years, incidence has increased 30-fold with increasing geographic expansion to new countries and, in the present decade, from urban to rural settings. An estimated 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries. In 2012, it is estimated that 60 million cases per year, and 30000 deaths per year in the world70. In India, 49,602 cases and 241deaths occurred year 201271. Dengue has a wide spectrum of clinical presentations, often with unpredictable clinical evolution and outcome. Changes in the epidemiology of dengue, lead to problems with the use of the existing WHO classification. Symptomatic dengue virus infections were grouped into three categories: undifferentiated fever, dengue fever (DF) and dengue haemorrhagic fever (DHF). The dengue virus (DEN) comprises four distinct serotypes (DEN-1, DEN-2, DEN-3 and DEN-4) which belong to the genus Flavivirus, family Flaviviridae. Among them, “Asian” genotypes of DEN2 and DEN-3 are frequently associated with severe disease accompanying secondary dengue infections61. Yellow fever Yellow fever is an acute viral hemorrhagic disease transmitted by infected mosquitoes. The "yellow" in the name refers to the jaundice that affects some patients. Up to 50% of severely affected persons without treatment will die from yellow fever. There are an estimated 200 000 cases of yellow fever, causing 30 000 deaths, worldwide each year60. Three phases of yellow fever are described. The first, during which virus is present in blood, is characterized by fever, malaise, generalized myalgia, Larvicidal potentials 31 Chapter III Review of literature nausea, vomiting, irritability, dizziness, and a generally toxic appearance. The second phase is characterized by improvement in symptoms. The third phase occurs in 15% of cases and is characterized by the return of fever, nausea, vomiting, jaundice, and bleeding diathesis72. Chikungunya Chikungunya is a viral disease that is spread by mosquitoes. It causes fever and severe joint pain. Other symptoms include muscle pain, headache, nausea, fatigue and rash. Treatment is focused on relieving the symptoms. The disease occurs in Africa, Asia and the Indian subcontinent. In recent decades mosquito vectors of chikungunya have spread to Europe and the Americas. Chikungunya is a mosquito-borne viral disease first described during an outbreak in southern Tanzania in 1952. It is an alphavirus of the family Togaviridae62. There are no specific treatments or licensed or commercial vaccination for these diseases. Treatment is directed primarily at relieving the symptoms60,61,62. The control of these diseases is largely dependent on controlling the vector, Aedes aegypti. Larvicidal potentials 32 Chapter III Review of literature Table 1: Larvicidal efficacy of botanical extracts in controlling/reducing the population of vector mosquitoes Plant Species Family Plant Target Mosquito Lethal Concentrations/ References Species Biological Activity Parts Used Petroleum ether extract Argemone mexicana Eucalyptu s globules Papaveraceae Myrtaceae Leaf, An. seed stephensi Seed, Cx. pipiens leaf Citrus aurantium Rutaceae Fruit peel Cx. quinquefasciatus LC50 = 30.47 and Saktivadive 24.17 ppm; LC90 = 246.33 and et al 184.99 ppm for 73 leaves and seeds (2008) respectively Both the extracts at a dose of 1000 ppm caused 100 Sheeren et al and 80% (2006)74 mortality LC90 = 53.80 and Kassir et al 32.52 ppm (1989)75 Acetone extract Tridax procumbe ns Compositae Ageratum Asteraceae conyzoides Leaf An. subpictus LC50 value 39.98 mg/l Leaf Cx. Potent larvicidal Saxena et al activity was (1992)80 noticed quinquefasciatus Larvicidal potentials of Kamaraj et al (2011)79 33 Chapter III Review of literature An. Ae. Millington ia hortensis Bignoniaceae Leaf stephensi, LC50 values = Kaushik 104.70, 138 and (2008)81 83.18 ppm aegypti and Cx. Respectively quinquefasciatus et al Chloroform extract Latex and Euphorbia tirucalli Plant Species Euphorbiaceae Family stem bark Cx. pipiens LC50 value was 200.76 and LC90 Yadav et al value was 343.515 mg/l (2002)82 Target Mosquito Lethal Concentrations/ References Species Used Biological Activity LC50 value was Bagavan 58.25 & LC90 value (2009)83 was 298.31 ppm Plant Parts Citrus sinensis Rutaceae Fruit peel An. subpictus Aloe ngongensi s Asphodelaceae Leaf An. gambie etal LC50 value was Matasyoh et al 58.25 mg/mL (2008)84 Methanol extract Euphorbia tirucalli Euphorbiaceae Latex and Cx. stem bark pipiens pallens Annona Annonaceae Larvicidal potentials Leaf Ae. LC50 value was Yadav et al 177.14; LC90 value (2002)85 was 513.387 mg/l LC50 value was Das et al 20.26; LC90 value 34 Chapter III Review of literature squamosa albopictus was 86.59 ppm (2007)86 Ethanol Extract Cx. Citrus reticulate Rutaceae Seed quinquefasciatus and Ae. aegypti Azadiracht a indica Meliaceae Leaf Cx. fatigans Larvicidal potentials LC50 value were Sumroiphon 2,267.71, and et al (2006)87 2,639.27 ppm LC50 value Azmi et al was 390 ppm (1998)88 35 Chapter-4 Methodology Chapter IV Methodology 4. METHODOLOGY 4.1. PROCUREMENT OF ESSENTIAL OILS The essential oils from the flowers of Nelumbo nucifera and Tagetes erecta were procured from Falcon Essential oils, Bangalore. 4.2 CHEMICAL TESTS 1. Test for carbohydrates : To 2ml of plant extract, 1ml of Molisch’s reagent and few drops of concentrated sulphuric acid were added. Presence of purple or reddish color indicated the presence of Carbohydrates. 2. Test for Tannins To 1ml of plant extract, 2ml of 5% ferric chloride was added. Formation of dark blue or greenish black indicated the presence of tannins. 3. Test for Flavonoids To 2ml of plant extract, 1ml of 2N sodium hydroxide was added. Presence of yellow color indicated the presence of flavonoids. 4. Test for Alkaloids To 2ml of plant extract, 2ml of concentrated hydrochloric acid was added. Then few drops of Mayer’s reagent were added. Presence of green color or white precipitate indicated the presenceof alkaloids. 5. Test for Quinones To 1ml of extract, 1ml of concentrated sulphuric acid was added. Formation of red color indicated the presence of quinones. Larvicidal potentials 36 Chapter IV Methodology 6. Test for Terpenoids To 0.5ml of extract, 2ml of chloroform was added and concentrated sulphuric acid was added carefully. Formation of red brown color at the interface indicated the presence of terpenoids. 7. Test for Triterpenoids To 1.5ml of extract, 1ml of Libemann – Buchard Reagent (aectic anhydride+concentrated sulphuric acid) was added. Formation of blue green color indicated the presence of triterpenoids. 8. Test for Phenols To 1ml of the extract, 2ml of distilled water followed by few drops of 10% ferric chloride was added. Formation of blue or green color indicated the presence of phenols. 9. Test for Coumarins To 1 ml of extract, 1ml of 10% NaOH was added. Formation of yellow color indicated the presence of coumarins. 10. Steroids and Phytosteroids To 1ml of plant extract equal volume of chloroform was added and subjected with few drops of concentrated sulphuric acid where the appearance of brown ring indicated the presence of steroids and appearance of bluish brown ring indicated the presence of phytosteroids. 11. Shinoda test Four pieces of magnesium fillings (ribbon) are added to the ethanolic extract followed by few drops of concentrated hydrochloric acid. A pink or red colour indicates the presence of flavonoid. Colours varying from orange to red indicated flavones, red to crimson indicated flavonoids, crimson to magenta indicated flavonones. Larvicidal potentials 37 Chapter IV Methodology 12. Mayer's reagent It is an alkaloidal precipitating reagent used for the detection of alkaloids in natural products. Mayer’s reagent is freshly prepared by dissolving a mixture of mercuric chloride (1.36 g) and of potassium iodide (5.00 g) in water (100.0 ml). Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer’s reagent (potassiomercuric iodide solution) to give a cream coloured precipitate. 7. Test for Glycosides To 2ml of plant extract, 3ml of choloroform and 10% ammonia solution was added. Formation of pink color indicated the presence of glycosides. 4.3 THIN LAYER CHROMATOGRAPHY (TLC): Thin layer chromatography is mainly used for qualitative screening of essential oils which serve as a very important tool in the overall phytochemical research studies. Procedure: Slurry of silica gel G was prepared in distilled water and poured over a glass plate to form a thin film the prepared plates were allowed for setting (air drying). After setting, the plates were kept in oven at 100 to 1200c (30 min) for activation. The samples were applied to the activated plates (1 cm above from the bottom). It was then kept in previously saturated developing chamber containing mobile phase and allowed to run 3/4th of the height of the plate. The developed plate was removed, air dried and observed under ultraviolet light and then compared with standard drug spot and calculates the Rf value using following formula. Larvicidal potentials 38 Chapter IV Rf = Methodology Distance travelled by solute Distance travelled by solvent front Selection of Mobile phase: TLC system for identification of constituent’s in the extract and one of the following which showed better separation was selected as mobile phase for the study. 1) Ethyl acetate: methanol: water (9:3:7) 2) petether: ethyl acetate (8:2) 3) Chloroform: methanol (9:1) 4) Ethyl acetate: chloroform (3:7) 4.4 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC): HPTLC is the most simple separation technique available today to the analyst. Currently HPTLC is becoming routine technique for analysis of not only synthetic drugs but also herbal drugs. Here in this study Quantitative densitometric HPTLC analysis was performed to develop the characteristic fingerprint profile for the formulation with respect to essential oils.  Preparation of sample solution : 10 ml of sample solution was taken in a separating funnel and 20 ml of chloroform was added and then the chloroform layer was separated and evaporated for some time. Then the residue was collected in a test tube and then labeled. Mobile phase: chloroform: methanol (9:1) Larvicidal potentials 39 Chapter IV Methodology Instrumentation and chromatographic conditions: HPTLC was performed on 10×10 cm aluminium baked silica gel plates coated with silica gel 60 F254 (Merck, Germany). Sample solutions were applied to the plates as bands on the same chromatographic plate by use of a camag (Muttenz, Switzerland) Linomat V sample applicator equipped with a 100ml Hamilton(USA) syringe. Ascending development was performed at room temperature (28 ±20c) using mobile phase in a camag glass twin-trough chamber previously saturated with mobile phase vapour for 20 min. after development the plates were dried and then scanned at 254 nm with a camag TLC scanner 3 with winCATS software in the fluorescence mode at 365nm for the estimation of chief constituents. The source of radiation utilized was mercury lamp (Hg lamp). 4.5 Rearing and Maintenance of Aedes aegypti Colony Mosquitoes were reared in the insectary of National Institute of Malaria Research, Bangalore. Adult of Ae. aegypti were cultured in the portable mosquito net cage, dimension of 0.5 m × 0.5 m × 0.5 m. The adults of Ae. aegypti were fed with 10% sucrose syrup solution soaked in cotton balls, the moisture is squeezed out and the balls are then placed inside the cage. The cotton balls are changed daily. The female mosquitoes were periodically fed on blood by the mice as it supports the development of eggs. Larvicidal potentials 40 Chapter IV Methodology LARVAE EXPOSED TO ESSENTIAL OILS Eggs are collected in a bowl half filled with water and lined with a 3 wide strip of filter paper. The filter paper will be served as a substrate for mosquito eggs. The filter paper stripe of eggs was brought to dry at room temperature and then kept at 20oC during study or stored in cool and dry humidity for long term up to one year. A number of eggs were collected and allowed to hatch at the same time in a filled water container in order to obtain larvae of the same age within 2-4 days. Typically, the yield of hatching was approximately 80%. Newly hatched larvae were transferred to white enamel trays with breeding source water (16x9x2”). The water is maintained at temperature 27°C and photoperiod of 12: 12 light: dark schedule. Dead larvae and associated debris were removed timely and the larvae are transferred to clean water daily. Later, minimal larval food comprising powdered yeast was supplemented. Larvicidal potentials 41 Chapter IV Methodology The pupae are routinely collected, so that no adults emerge in the larval rearing containers. The pupae were transferred to the net cage. They took 1-2 days to develop into adults. 4.6 Larvicidal Bioassay Larvicidal Test: rd Larvicidal efficacy of N.nucifera and Tagetes erecta oils were tested against late 3 or th early 4 instar stage larvae of Ae. aegypti employing standard WHO procedure89. Experiments o were conducted at 27±2 C, 12 h light/dark regime. Batches of 25 larvae were exposed to known concentration of test solution (1ml of Dimethyl sulphoxide (DMSO) dissolved test extract in 249ml de-chlorinated tap water) in bowls of 500ml capacity. Three replicate sets were tested with a final tally of 75 larvae for each concentration. Solutions containing 1ml of DMSO in 249ml tap water, without plant extract, in 500mL bowls served as Control. No larval feed was provided to the larvae during the test period. Mortality and survival were recorded after 24 h of treatment. The larvae were considered as dead or moribund, if they were not responsive to a gentle prodding with a fine needle. The moribund and dead larvae in replicates were combined and expressed as percentage mortality using the formula. The observed percent mortality was adjusted with the control mortality, wherever required, using Abbott’s formula and expressed as corrected mortality. Percentage mortality: Percentage mortality = (D1 + M1) + (D2 + M2) + (D3 + M3) x 100 .............. No. of Larvae exposed Larvicidal potentials 42 Chapter IV Methodology Where, D1, D2, D3 – No. of dead larvae in replicate 1, 2, 3 respectively. M1, M2, M3 – No. of moribund larvae in replicate 1, 2, 3 respectively. Abbott's formula: Corrected percentage mortality = 1 - Number of dead larvae in treated _ x 100 .... Number of dead larvae in Control Statistical analysis : The obtained mortality values were subjected to regression analysis of probit mortality on log dosage to estimate the LC50 and LC90 as well as their 95% confidence limit. Data were analyzed using a Statistical Software BioStat® 2009, Version 5.8.3.0 (British Columbia, Canada). Larvicidal potentials 43 Chapter-5 Results Chapter V Results 5. RESULTS 5.1 PROCUREMENT OF ESSENTIAL OILS : The essential oils were procured from Falcon essential oils, Bangalore. 5.2 Preliminary Phytochemical Screening of Nelumbo nucifera oil: Preliminary phytochemical screening of N.nucifera oil revealed the presence of different primary and secondary metabolites as shown in Table no.2. Phytoconstituents Tannins Phenolic compounds Larvicidal potentials Observation + + Flavonoids + Steroids + Triterpenoids ++ Saponins + Alkaloids + Carbohydrates + Anthocyanins + Glycosides + 44 Chapter V Results 5.3 Preliminary Phytochemical Screening of Tagetes erecta flowers : Table no.3: Phytoconstituents Tannins Phenolic compounds Larvicidal potentials Observation + + Flavonoids ++ Steroids + Triterpenoids + Alkaloids + Cardiac glycosides ++ Carbohydrates + Quinones + Coumarins + Carotenoids ++ 45 Chapter V Results Figure 10: Preliminary Phytochemical Screening of Essential oils The flower part of both the oils were found to contain terpenoids, tannins, saponins, flavonoids, steroids, glycosides, and alkaloids where as t.erecta shows carotenoids, coumarins, quinones, phenols and cardiac glycosides. Larvicidal potentials 46 Chapter V Results 5.4 TLC Profile of Nelumbo nucifera and Tagetes erecta oils : Fig 11 : pet ether: ethyl acetate (8:2) Fig 12 : chloroform: ethyl acetate (3:7) Table no.4 TLC profile of essential oils using various solvents S.NO Solvent system Sample No. of spots Rf values 1. Nelumbo nucifera 2 0.9, 0.35 Tagetes erecta 1 0.21 Chloroform: ethyl Nelumbo nucifera 2 0.17, 0.64 acetate (3:7) Tagetes erecta 1 0.39 Petroleum ether : ethyl acetate (8:2) 2. Larvicidal potentials 47 Chapter V Results HPTLC Profile of Nelumbo nucifera and Tagetes erecta oils : Fig 13. Ethyl acetate: methanol: water (9:3:7) . Fig no.14 Chromatographic spectra of N.nucifera and T.erecta essential oils at 365nm Larvicidal potentials 48 Chapter V Results No.5 Table representing track and peak values S.no Track Peak Start Start Max Max position height position height Max % End End position height Area Area % 1. 1 1 0.01Rf 0.0AU 0.03Rf 36.3AU 44.28% 0.06Rf 0.1AU 416.2AU 34.27% 2. 1 2 0.18 Rf 7.3AU 0.21 Rf 30.2AU 36.91% 0.23 Rf 2.2 AU 468.6AU 38.58% 3. 1 3 0.72 Rf 5.8AU 0.73 Rf 15.4AU 18.81% 0.77 Rf 2.4 AU 329.8AU 27.15% 4. 2 1 0.03 Rf 3.2AU 0.00 Rf 85.4AU 78.32% 0.02 Rf 2.9 AU 1180.4AU 86.86% 5. 2 2 0.84 Rf 0.1AU 0.85 Rf 23.6AU 21.68% 0.86 Rf 19.8 AU 178.6AU 13.14% Note: (Track 1 : Nelumbo nucifera. Track 2 : Tagetes erecta) Larvicidal potentials 49 Chapter V Results HPTLC Profile of Nelumbo nucifera oil : Fig no.15 Methanol : chloroform (1:9) Table no.6 HPTLC of Nelumbo nucifera oils S.no Solvent system Sample No. of Rf value spots 1.  Methanol: Nelumbo chloroform(1:9) nucifera HPTLC of nelumbo nucifera 4 has shown four spots 0.21,0.49, 0.73,0.85 with sovent system methanol:chloroform. Larvicidal potentials 50 Chapter V Results Table 7: Larvicidal action of Nelumbo nucifera oils SR DOSE NO. OF NO. (ppm) LARVAE NO. OF LARVAE DIED AFTER 24 hrs EXPOSED (EXPERIMENTAL) NO. OF LARVAE DIED AFTER 24 hrs (CONTROL) % CORRECTED MORTALITY E1 E2 E3 E4 AVG. C1 C2 C3 C4 AVG. 1. 50 100 2 3 2 0 1.75 0 0 0 0 0 7% 2. 200 100 6 5 3 4 4.5 0 0 0 0 0 18% 3. 300 100 8 6 7 7 7.0 0 0 0 0 0 28% 4. 400 100 18 17 15 14 16.0 0 0 0 0 0 64% 5. 500 100 20 19 21 19 19.75 0 0 0 0 0 78% 6. 600 100 25 24 24 24 24.25 0 0 0 0 0 97% Larvicidal potentials 51 Chapter V Results Table 8: Larvicidal action of Tagetes erecta oils SR DOSE NO. OF NO. (ppm) LARVAE NO. OF LARVAE DIED AFTER 24 hrs EXPOSED (EXPERIMENTAL) NO. OF LARVAE DIED AFTER 24 hrs (CONTROL) % CORRECTED MORTALITY E1 E2 E3 E4 AVG. C1 C2 C3 C4 AVG. 1. 50 100 2 5 8 3 4.5 0 0 0 0 0 18% 2. 100 100 9 10 12 7 9.5 0 0 0 0 0 38% 3. 150 100 15 11 17 13 14.0 0 0 0 0 0 56% 4. 200 100 18 21 14 16 17.25 0 0 0 0 0 69% 5. 250 100 21 19 18 19 19.25 0 0 0 0 0 77% 6. 300 100 23 21 20 22 21.5 0 0 0 0 0 86% Larvicidal potentials 52 Chapter V Results 5.5 Larvicidal efficacy of essential oils on Aedes aegypti vector Table no. 9 Sl. Extract type No 1 Tagetes erecta Mosquito species employed Ae. aegypti Oil 2. Nelumbo nucifera oil Larvicidal potentials Ae. aegypti LC50 (ppm) LC90 (ppm) CHI-SQUARE (LCL-UCL) (LCL-UCL) VALUE 124.8 403.1 (111.6-138.1) (338.4-511.5) 338.6 591.3 (253.1-421.3) (463.0-1219.9) 1.3 15.2 53 Chapter V Results Fig no.16 Larvicidal efficacy of Nelumbo nucifera oil against Aedes aegypti 100 86 90 77 80 Corrected mortality 69 70 56 60 50 38 40 30 20 18 10 0 50 200 300 400 500 600 Dose (ppm) Larvicidal potentials 54 Chapter V Results Fig no.17 Larvicidal efficacy of Tagetes erecta oil against Aedes Aegypti 100 86 90 77 80 Corrected mortality 69 70 56 60 50 38 40 30 20 18 10 0 50 100 150 200 250 300 Dose (ppm) Larvicidal potentials 55 Chapter-6 Discussion Chapter VI Discussion 6. DISCUSSION: Nelumbo nucifera and Tagetes erecta essential oils were studied for larvicidal action against Aedes aegypti. The chemical tests were performed for both the oils which showed the presence of glycosides, carbohydrates, alkaloids, triterpenoids, tannins and phenolic compounds. No significant mortality was observed in controls, which ensured that the solvent used to dissolve the extract i.e, DMSO did not contribute to the overall mortality during the bioassay. Essential oils showed lethal effect and mortality in positively dose dependent manner against the larvae and adult mosquitoes. Larvicidal activities of plant extracts vary according to the plant species, plant part, geographical location of the plants and the application method. Several plant extracts have been screened for larvicidal activity against mosquito larvae and have shown promising results. Annona squamosa leaf ethanolic extract showed LC 90 values of 76.73 ppm against Aedes albopictus and 31.80 ppm against Culex quinquefasciatus respectively90.Eucalyptus camaldulensis oil extract has also showed effective larvicidal action against Aedes albopictus and Aedes aegypti with LC 90 values of 192.4 and 71.8 μg/ml91. Also, the essential oil of Ziziphora clinopodioides showed effective larvicidal activity against Culex pipiens and Anopheles stephensi larvae with LC90 value of 28.6 and 22.3 μg/ml respectively. The essential oil of Nelumbo nucifera has shown larvicidal activity with LC50 and LC90 of 338.6 and 591.3. The essential oil of Tagetes erecta has shown activity at LC50 and LC90 of 124.8 and 403.1 respectively92. Larvicidal potentials 56 Chapter-7 Conclusion Chapter VII Conclusion 7. CONCLUSION  The present study was aimed to explore the potential of essential oils from flowers of Nelumbo nucifera and Tagetes erecta for larvicidal against Aedes aegypti.  The % yield of Nelumbo nucifera and Tagetes erecta was found to be 0.18% and 0.20%.  The essential oil Tagetes erecta showed an effective larvicidal activity in comparison to the essential oil of Nelumbo nucifera.  The essential oil of Tagetes erecta shown 86% mortality rate at 300 ppm whereas Nelumbo nucifera has shown 97% at 600 ppm.  From this study, it can be concluded that the flower part of both the plants Nelumbo nucifera and Tagetes erecta has larvicidal activity against Aedes aegypti vector.  Further analytical research is required to isolate and determine the chemical compound responsible for the activity.  Also, further research is required for studying the eco-toxicity of its bioactive compounds and derivatives for active implementation as a larvicide. Larvicidal potentials 57 Chapter-8 Summary Chapter VIII Summary 8. SUMMARY Mosquitoes are responsible for transmitting the most important vector-borne diseases, namely malaria, Lymphatic filariasis, dengue, Japanese encephalitis as well as yellow fever and other forms of encephalitis. Aedes aegypti is a vector responsible for causing diseases like dengue, chikungunya and yellow fever. The causative agent is arbovirus for dengue . The female mosquitoes seek for protein from blood of mammals for egg laying. During this period the arbovirus is infected into humans by saliva of female mosquito. Hence, one of the effective methods to control these diseases is to target the life cycle of vectors for interrupting the transmission. Therefore, the present study was undertaken to explore the Larvicidal potentials of essential oils from the flowers of Nelumbo nucifera and Tagetes erecta against Aedes aegypti for their potential towards vector control strategy. Nelumbo nucifera belongs to the family Nelumbonaceae.It is an perennial aquatic herb, Root stock stout, cylindrical. Leaves peltate, radiately nerved. Flowers are large, solitary, fragnant. Tagetes erecta belongs to the family Asteraceae. Erect annual herb, glabrous; stem and branches angular to rounded. Leaves opposite in lower part of plant, alternate in upper part, pinnately compound with 9–17 leaflets; leaflets linear to lanceolate, acute or acuminate at both ends, margins serrate, bearing scattered oil glands. Ray flowers female, ligulate. Both the essential oils contain tannins, alkaloids, triterpenoids, phenolic compounds as common chemical constituents in the flower part. Hence they were subjected to various chemical tests such as mayers test, shinoda test, test for phenols, tannins, test for glycosides and terpenoids to identify the presence of constituents for which they gave positive results. Larvicidal potentials 58 Chapter VIII Summary TLC and HPTLC studies were carried out on Nelumbo nucifera and Tagetes erecta essential oils. Different mobile phases were used for both the oils but low polar solvents have shown maximum retention factors at 365nm. Ethyl acetate, methanol, chloroform, water has been used as solvents. Rf values of Nelumbo nucifera oils at 365nm was found to be 0.06, 0.23 and 0.77 and Rf values of Tagetes erecta oils was found to be 0.02 and 0.86 at 365nm respectively. Essential oils from the flowers of Nelumbo nucifera and Tagetes erecta were tested for larvicidal potency against batches of Aedes aegypti larvae. LC50 and LC90 values of Nelumbo nucifera oils were 338.6 ppm and 491.3 ppm whereas Tagetes erecta oils were 124.8 ppm and 403.1 ppm respectively. The essential oil from the flowers of Tagetes erecta has shown upto 86% mortality at 300 ppm whereas Nelumbo nucifera oils shown 97% mortality rate at 600 ppm. 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