Title of Invention

A PROCESS OF FORMULATION OF NEEM (AZADIRACHTA INDICA) ROOT BASED NATURAL FUNGICIDAL PRODUCT

Abstract

Collecting neem root from Banaras Hindu University Campus and drying it at room temperature(25±2°C). Crushing 4 Kg air dried roots in a pastel and mortar to make powder. Extracting twice with 8 litres of methanol in a soxhlet at (50 ± 5°C). Evaporating the solvent by steam distillation to get dry extract. Dissolving the crude extract in methanol in the proportion of 1:5(W/V). Treating the solvent with charcoal and filtering the supernatant through whatman nol. filter paper. Evaporating the excess solvent and finally drying it under vaccum. Passing the so obtained extract of neem root through si-gel column for clean up after washing the column through methanol to get refined neem root extract. Evaporating the solvent under vacuum to get semi solid yellowish brown neem root extract. Formulating this extract in water containing 0.1% tween 20 in the concentration of 250,500,1000,2000,2500 and 3000 ppm(Parts per million) for the purpose of testing its fungicidal effect against phytopathogenic and saprophytic fungi and also powdery mildew disease of pea( Pisum Sativum ).

Full Text

3. Field of Invention A process of formulation of Neem (Azadlrachta Indica) root based natural fungicidal product. This invention relates to the development of fungicide natural product formulation from neem(Azadirachta indica) prepared from the constituents of its roots having potential fungicidal activity against various phytopathogenic fungi. Natural products from higher plants showing fungicidal activity are known in the arl since long. It is generally regarded that plants are the biggest laboratory of natural chemicals, continuously synthesizing diversified chemicals. Many of these biochemicals are considered to provide defence to the plants producing them against pests and diseases. Since plants are regularly exposed to varied environmental conditions, they are likely to produce numerous biochemicals to circumvent the stresses caused due to the biotic and abiotic factors. Neem (Azadirachta indica) belonging to the family Meliaceac, is highly esteemed societal tree of Indian origin having a vast potential for the development of pharmacological and agrochemical agents. Although almost all the aerial parts of this tree have been investigated for their agrochemical potential in the laboratory and field conditions, the underground parts of the tree i.e., its vast roots have never been investigated so far. The underground root continuously remains in the dark and therefore, is supposed to possess unique biochemicals evolved due to certain metabolic activities different from the aerial parts of the tree. Present invention deals with the unmasked fungicidal properties of the extract of Neem root, which further provided the basis for the development of a fungicidal formulation. Parts of Neem tree have been a constant source of insecticidal and antifeedent natural products since long. Diverse class of compounds including the most potent tetranortripenoids, have been isolated from various parts of the tree, especially from the oil. Almost all parts of the tree including leaves, bark, seeds, fruits and inflorescence are known to process unique chemicals, but in the past, basically Neem oil and seed has been the central core of the developments related to agrochemical formulations. Azadirachtin(s), the most potent and novel antifeedant and growth regulatory compound isolated from the seed kernel has constituted the basis for developing many Neem-based insecticidal formulations in the past. Furthermore, neem oil owes very significant fungicidal property against numerous phytopathogenic fungi causing diseases to variety of crops. Formulations of neem oil have been developed and are now being used against the diseases of house-hold and garden plants as well as field crops. Inhibitory effect of aqueous extracts of several parts of neem tree has also been reported against fungal spore germination. Neem leaves in the form of green manure also help in controlling soil-borne fungal and bacterial diseases. Despite several investigations on the biological properties associated with the aerial parts of neem, the underground part, i.e., root has never been examined so far for activities against pests and plant disease causing organisms. The present invention describes the formulation of methanolic extract of neem root as a potential fungicide. Description A process of formulation of Neem (Azadirachta indica) root based natural fungicidal product The objective of this invention is to provide experimental proof regarding the presence of fungicidal activity in the underground part of Neem, i.e., root. Alternatively, this invention is aimed at proposing the development of a "Neem root-based fungicidal formulation" for the purpose of plant disease control. Accordingly the present invention provides a process of formulation of natural fungicidal product from neem root comprising the steps of:- 1. Collecting Neem root from Banaras Hindu University Campus and drying it at room temperature (25±2°C) 2. Crushing 4 Kg air dried roots in a pastel and mortar to make powder. 3. Extracting twice with 8 liters of methanol in a soxhlet at (50±5°C). 4. Evaporating the solvent by steam distillation to get dry extract. 5. Dissolving the crude extract in methanol in the proportion of 1:5(W/V) 6. Treating the solvent with charcoal and filtering the supernatant through whatman nol. filter paper. 7. Evaporating the excess solvent and finally drying it under vacuum. 8. Passing the so obtained extract of Neem root through si-gel column for clean up after washing the column through methanol to get refined Neem root extract. 9. Evaporating the solvent under vacuum to get semi solid yellowish brown neem root extract. 10. Formulating this extract in water containing 0.1% tween 20 in the concentration of 250,500,1000,2000,2500 and 3000 ppm(Parts per million). The steps of obtaining neem root extract are summarised in Scheme -1. Thus obtained different concentrations of the extract were tested against spore germination of different phytopathogenic and saprophytic fungi (Table -1). They were also tested against the disease intensity of powdery mildew causing fungus ( Erysiphe pisi) on pea ( Pi.sum Sativum). Water containing 0.1 % tween 20 was used in comparable volumes as control for bioassay against spore germination of the fungi used (Table 1). Sulphur fungicide (the most common type of synthetic fungicide used to control powdery mildew disease in field condition) was used as control in testing against powdery mildew of pea. The results showed comparable effects of natural product neem root formulation (Table 2) Referring in particular to the fig.l, it is evident that the extract isolated from neem root shows strong fungicidal activity as observed by spore germination test against several devastating phytopathogenic fungi namely Alternaria brassicae, Curvularia lunaia, Hcterosporium sp., Helminthosporium pennisetti, Erysiphe polygoni, Erysiphe cichor acearum, Erysiphe sp., Alternaria solani and Fusarium udum. Conida and spores of the fungi when observed under microscope after treatment with different concentrations of neem root extract failed to germinate, while in control (untreated) sets, significant spore germination has been observed. At lower concentrations, where the germination was observed in treated fungi, the mycelial development was fairly low as compared to the untreated fungi (Fig.l). Scheme 1. Preparation of neent root extract based formulation (Flow Chart Removed) Tab le 1. Effect of methanolic extract of neem (Azadirachta indica) roots on spore germination of some fungi (Table Removed)* Solution prepared in 0.1% Tween-20 Data with ** vary significantly at p Table 2. Effect of fungicidal neem root formulation on powdery mildew (Erysiphe pisi) of pea (Pisum sativum)(Table Removed) Sulfur was taken as reference fungicide for comparison with neem root formulation TW-20 = Tween-20 We claim 1. A process of formulation of neem ( Azadirachta indica ) root based natural fungicidal product comprising the steps of :- 1. Collecting neem root from Banaras Hindu University Campus and drying it at room temperature(25±2°C) 2. Crushing 4 Kg air dried roots in a pastel and mortar to make powder. 3. Extracting twice with 8 liters of methanol in a soxhlet at (50± 5°C). 4. Eivaporating the solvent by steam distillation to get dry extract. 5. Dissolving the crude extract in methanol in the proportion of 1:5(W/V) 6. Treating the solvent with charcoal and filtering the supernatant through whatman nol. filter paper. 7. Evaporating the excess solvent and finally drying it under vacuum. 8. Passing the so obtained extract of Neem root through si-gel column for clean up after washing the column through methanol to get refined Neem root extract. 9. Eivaporating the solvent under vacuum to get semi solid yellowish brown Neem root extract. 10. Formulating this extract in water containing 0.1% tween 20 in the concentration of 250,500,1000,2000,2500 and 3000 ppm(Parts per million). 2. A process of formulation of neem root based natural fungicidal product as claimed in claim 1 which possesses potent antifungal property against a wide range of fungal phytopathogens and prominent disease inhibitory potential against powdery mildew (E. pi si) of pea, from methanol extract of Neem (Azadirachta indica) root.

Documents:

1116-del-2004-abstract.pdf

1116-del-2004-claims.pdf

1116-del-2004-correspondence-others.pdf

1116-del-2004-correspondence-po.pdf

1116-del-2004-description (complete).pdf

1116-del-2004-drawings.pdf

1116-del-2004-form-1.pdf

1116-del-2004-form-13.pdf

1116-del-2004-form-2.pdf

1116-del-2004-form-3.pdf

1116-del-2004-form-5.pdf