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International Journal of Medical Science and Current Research (IJMSCR) Available online at: www.ijmscr.com Volume2, Issue 3, Page No: 294-301 May-June 2019

To evaluate the effect of Pesticide activity of of nanoformulations of Alstonia scholaris , against pest- Rhyzopertha dominica using HPLC and Fourier transform infrared (FT- IR) spectra analysis

Rohini Kallur1*, Savitha Patil2 Lecturer1*, Lecturer2, Department of Industrial Biotechnology, Nagarjuna college of Engineering and technology, Karnataka

*Corresponding Author:

Rohini Kallur Lecturer1*Department of Industrial Biotechnology, Nagarjuna college of Engineering and technology, Karnataka, India

Type of Publication: Original Research Paper Conflicts of Interest: Nil

ABSTRACT Background: Now many agricultural engineers are seeking knowledge of nano-materials for engineering bio-pesticides, fertilizer coatings and other purposes. These Nanoparticles can be exploited as they offer a greater surface area and circulate more easily. Materials & Methods: Insecticidal activity of Alstonia scholaris Column fractions against stored product pests. Antifeedant Bioassay was done by fumigation method. Separation and fractionation was done by Column chromatography. TEM morphology of nano-sio2 particle was performed on transmission electron micrograph. Statistical analysis: The average larval mortality data were subjected to probit analysis for calculating lethal concentrations LC50 and LC95 and other statistics at 95% fiducial limits of upper confidence limit and lower confidence limit were calculated. Results: The extracts of A. scholaris were moderately toxic and needed at least 2 mg/ ml to obtain 50% mortality. Alstonia scholaris showed high toxicity with a LC50 of 0.8 mg/ml and LC95 of 1.95 mg/ml. Thus it can be concluded that the terpenes adsorbed on the silica through strong hydrogen bonding. All the results above further illustrate that adsorption of terpene has an added advantage that in spite of less agglutination it also exhibit the high dispersion stability. Conclusion: Among the tested plant materials, Alstonia scholaris proved as highly toxic against stored product pests as well as mosquito larvae. This active fraction will be further processed for the pure compound isolation, identification for the principle bioactive compound. The two principle bioactive compounds showed enhanced antifeedant activity against agricultural pests on Nanoformulations..

Keywords: Alstonia scholaris, Rhyzopertha dominica, FT-IR analysis, HLPC, nanopesticide.

INTRODUCTION The Castor Semi-Looper, Achaea janata. Linnaeus, is will attack all the aerial parts of the crop as well as another important pest which feeds on castor bean roots in the soil and the grain, which is stored. and croton plants. Occasional hosts include banana, The yellow fever mosquito, Aedes aegypti, is a cabbage, Chinese cabbage, crown of thorns, Ficus, mosquito that can spread the dengue fever, macadamia, mustard, poinsettia, rose, sugarcane and Chikungunya and yellow fever viruses, and other tomato as well as some legumes, teas, and other diseases. The mosquito can be recognized by white Brassica species.Stored grains are highly damaged by markings on legs and a marking of the form of a lyre insect pests like Rhyzopertha dominica L.,. Almost on the thorax. The mosquito originated from Africa1

all the stages of the insect (adult, larvae, except pupa) but is now found in the tropics worldwide. Direct 294

International Journal of Medical Science and Current Research | May-June 2019 | Vol 2 | Issue 3 Rohini Kallur et al International Journal of Medical Science and Current Research (IJMSCR)

exposure to pesticides has caused mass mortality of reduce the amount of active agent required for many species, but the most hazardous are those, treatment by maintaining an effective concentration which either does not degrade or which degrade very in the target for longer periods of time. They do not slowly in environment. These non-biodegradable cause alteration of gene expression in insect trachea pesticides have been found concentrate in successive and are, thus, qualified for approval as the nano tropic levels in food chain. Basic concepts of food biopesticide in this particular category. Use of security and food self sufficiency logically demand amorphous silica is considered to be safe for human that at least over 70% of the countries food by World Health Organization and US Department of requirement is satisfied domestically. Agriculture. The Indian farmers are in need of effective tools to Materials & Methods: fight against pests. After severe setback arising from Our present study of the objectives are Screening the the use of chemical pesticides on living systems and of plant material for insecticidal property. the environment, the use of eco-friendly biopesticides Formulation of the botanicals with Nanoparticles at is gaining momentum. However, the small farmers in different concentrations to assess the adsorption India are not yet fully aware of the concept, use or compatibility. Evaluating the toxicity of advantages of ecofriendly pest management. Though nanoformulation against agricultural pests. Studies on India has a rich source of plants that could be drug loading/control release of bioactive compound harnessed as botanical pesticides, accentuated from Nanoformulations by using HPLC. Alstonia research on the preparation of biopesticides has not scholaris, were taken for the present work, plant gained ground. material used was leaves. The plant materials were Botanical pesticides are eco-friendly, economic, collected from the premises of Indian Institute of target-specific and biodegradable. For example, Chemical Technology (IICT), Hyderabad.Achaea neem-based botanical pesticides have been used janata L. and Spodopthera lituraF. larvae were traditionally for many years. There are many other collected from infested castor Ricinus communis trees (besides herbs and shrubs) which are also useful plants growing in the laboratory of Indian Institute of as sources of botanical pesticides. However Chemical Technology, Hyderabad, India. The larvae nanosilica have shown excellent efficiency against were collected from the fields and were maintained in this mite. In studies of plants, these nanosilica did not the laboratory at 28 ± 2º C, 70 – 75 % relative show any adverse effect on the plant physiology, fish humidity (RH) and a 16:8 hours light/ dark photo and silkworm physiology. Control of granary weevil period. The larvae were reared on castor leaf.2 The Sitophilus granaries and mustard beetle Phaedon Castor Semi-Looper, Achaea janata is a Noctuid cochleariae can be achieved by the application of moth, the caterpillars of which are termed 'semi- silica based materials Fossil Shield. Immunity in loopers' due to their mode of locomotion and feed on beneficial insects can be developed using nanosilica. Castor Ricinus communis. As they feed off the Castor This was observed in case of Bombyx mori where Oil plant exclusively they are extremely poisonous grasserie disease caused by nuclear plyhedrosis virus and should be avoided at all costs. This is leaf eating (NPV) could be controlled with the help of lipophilic and considered to be one of the important pests of the amorphous silica nanoparticles (LASN)-live BmNPV crop as the larvae defoliate the plants within a short conjugate as drug. The international Agency for period when there is an outbreak. Lesser grain borer, Research on Cancer (IARC) has rated amorphous Rhyzopertha dominica; were maintained at 28 ± 2°C silica dust as non-carcinogenic. and 65-75% r. h. in Indian Institute of Chemical Nanoscale carriers can enhance the drug Technology (IICT)Indian Institute of Chemical bioavailability, reduce drug toxicity, improve the Technology (IICT) The female bores a hole in the timed release of drug molecules, Create high local kernel and deposits an egg, and she can lay up to 400

concentration of the bioactive chemicals in target eggs in her life time. The larva hatches within 72 hrs,

cells and cellular components in order to gain toxic and then starts feeding, within the kernel, and efficiency and enable precisive drug targeting. molting four times before pupating. The white,

legless larva, with a dark head, is only found within

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295 295 295 Controlled release of pesticides can remarkably 295

the seeds they are infesting so you usually don’t see

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them. Silicon dioxide Nanoparticles of average SiO2 particles about 100mg were dispersed in 6ml particle diameter 10 nm, specific surface area 115m2 chloroform, and amount of terpene (25μl, 50μl, 75μl g−1 were procured from sigma-Aldrich. and 100μl) was added into the flask, and subjected to Other reagents were of HPLC grade and used as sonication for 10 minutes and then vacuum drying for received. Eugenol and 1,8-Cineole are the terpenes 24 h. isolated from two plants were provided by Indian Fourier transform infrared (FT-IR) spectra of terpene Institute of Chemical Technology (IICT). Collection adsorbed nanosilica were carried out on spectrometer and preparation of plant material for extraction was (Nicolet Co., Nexus-870, USA) with a resolution of 4 dony by Extraction apparatus (Soxhlet). A rotary cm−1 for which the samples were palletized with evaporator is a device used in chemical and KBr powder and compared with the native nano- biochemical laboratories for the efficient and gentle SiO2. Control release of the bioactive compounds evaporation of solvents. Insecticidal property of the adsorped silica nanoparticles were studied by using botanical extracts against stored product pests, HPLC. 100 mg of terpene adsorbed silica was agricultural pests and yellow fever mosquito was dispersed in 500 ml of dissolution medium (deionized carried out by the following methods. Antifeedant water) in the flask. 2 ml of the suspension was activity against Agricultural pests, Antifeedant sampled, centrifuged and analyzed by a high- Bioassay was done. performance liquid chromatography with a C18 Extracts from Alstonia scholaris was separated by column at the maximum absorbance of 225 nm. column chromatography on silica gel (mesh size The separation of the free terpenes from the terpene 200). Length of the column was one meter, ¾ th of anchored to nano-SiO2 particles was achieved by the column was filled with silica gel. A small plug of dialysis. Typically, 100ml methanol dispersion of glass wool was placed at the bottom to support silica 100mg formulated nano-SiO2 particles was allowed gel. The 20 gm of material was loaded on to the silica to stand at room temperature. After a definite time, gel column and the column was eluted. The mobile 5.0 ml of dispersion liquid was taken out with a phase is allowed to flow through the solid adsorbent. pipette, and the content of nano-SiO2particles A small amount of the sample to be analyzed is dispersed was determined. The stability of dispersion layered the top of the column. The sample mixture was estimated from percentage of dispersed nano- enters the Column of adsorbent material and is SiO2 particles after standing by the following distributed between the mobile phase and the equation. Nano-SiO2 particles dispersed (%) =G/G0 stationary phase. × 100. Where G (gm) and G0 (gm) are the weight of The various components in the sample will have nano-SiO2 dispersed after and before standing, different affinities for the two phases and will move respectively. The morphology analysis was through the column at different rates. Collection of performed on transmission electron micrograph (TEM) analyzer (Jeol 100CX–II JAPAN). Statistical the liquid phase emerging from the column is having separate functions containing the individual analysis: The average larval mortality data were components in the sample. The eluted is collected subjected to probit analysis for calculating lethal and the compound is separated from it by evaporating concentrations LC50 and LC95 and other statistics at the solvent. The compounds dissolved in suitable 95% fiducial limits of upper confidence limit and lower confidence limit were calculated. solvent. Thin layer chromatography was used to identify fractions containing similar compounds. RESULTS: TLC was carried out for each solvent fraction. The Insecticidal activity of crude leaf extract from five bands were visualized by the fumes of iodine. plants was tested on Rhyzopertha dominica. Among Fractions containing the similar compounds were five plants only one plant Alstonia scholaris had mixed. TLC is a simple, quick, and inexpensive insecticidal activity.

procedure that gives the chemist a quick answer as to

how many components are in a mixture. The result on toxicity test of the leaf extract by contact method showed that the compound is

Pure botanical compounds Eugenol and 1,8-Cineole

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296 296 296 moderately toxic to S. oryzae adults. Only the higher 296

were adsorbed on to the nano silica. The native nano-

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Rohini Kallur et al International Journal of Medical Science and Current Research (IJMSCR)

dose produced about 60% mortality after of 96 hours. Table1. Adult mortality was significantly increased The toxicity bioassay of the leaf extracts on adult with increase in concentration and days of exposure. against S. oryzae by contact method is presented in

Table 1: Insecticidal activity of crude leaf extract against Rhyzopertha dominica by Contact method Mean % mortality SINO. (mg/100 gm 24 hrs 48 hrs 72 hrs 96 hrs grains) 1 50 10 30 40 60 2 100 20 40 50 60 3 150 20 40 50 60 4 Control 0 0 0 0

The result on toxicity test of the leaf extract by fumigation method showed that the compound is not very affective in producing mortality against S.oryzae. Adults only the higher dose produced about 80% mortality after of 96 hours. The result on toxicity test of the leaf extract by fumigation method showed that the compound is very affective in producing mortality against Rhyzopertha dominica. At higher dose produced about 100% mortality after 96 hours. Table 2: Insecticidal activity of various crude extracts (mg/ml) to the third instars larvae of A. aegypti Mortality (in %) Sl.No Plants 0.5 1.0 2.0 2 Alstonia scholaris 00 30 70

The effect of various botanical extracts on the mortality of A. aegypti larvae are presented in Table 2. The crude extract of all five plants had insecticidal activity on A. aegypti larvae.From the results it appears that four of the five leaf extracts tested possessed high larvicidal activities in various degrees. The floral extract of Alstonia scholaris at dosage of 2 mg /ml two plant extracts caused 70% mortality in the treated larvae and almost all the extracts were ineffective in producing mortality at lower doses such as 0.5 mg/ ml against A. aegypti larvae. No significant difference in larval mortality was found in less than 0.5mg/ml concentrations compared to the control treatment.

Table 3: LC50 and LC95 with fiducial limits (95%) of tested botanical extracts against larvae of A. aegypti Activity (mg/ml) (95% FL) Sl.No Plants LC50(LCL-UCL) LC95(LCL-UCL) 2 Alstonia scholaris 0.85(0.68-0.99) 2.03(1.67-2.87) FL: Fiducial limit, UCL: upper confidence limit, LCL: lower confidence limit

More accurate data on the toxicity of the plant extracts were obtained by calculation of their LC50 and LC95. In

contrast to the results obtained from screening procedure it was observed that Alstonia scholaris showed

increased mortality rate in the accurate test (Table 3). In the similar way A. scholaris showed the second highest

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mortality with a LC50 at 0.85 mg/ ml and LC95 at 2.03 mg/ml. 297

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Table 4: Toxicity of Nanoformulated botanical compounds (mg/12cm2) against A. janata 3rd instar larva by antifeedancy method Antifeedant index Compounds 0.25mg 0.50 mg 0.75 mg 1.00 mg 2.00 mg 3.00 mg NF Eugenol 30.2 98.1 100 100 100 100 NF 1,8-Cineole 15.3 29.3 49.0 68.0 100 100 *NF: Nanoformulated The effect of Nanoformulated pure compounds on A. janata are presented in Table 4. A gradient increase in Antifeedancy with increase in concentration was observed in both the treatments. At dosage of 0.75mg/12cm2 caused 100% mortality with Eugenol. were as with 1,8-Cineole dosage of 2mg/12cm2 caused 100% mortality. Antifeedancy is doubled in NF Eugenol compared to pure eugenol. In the case of NF 1,8-Cineole there was no increase in antifedancy compared to pure 1,8-Cineole. Table 5: Release profile for 1,8-Cineole S.No. Sample code mVo % Release 1. 1,8-Cineole Standard 1792.03 100 2. NF 1,8-Cineole 25 μl 278.45 15.53 3. NF 1,8-Cineole 50 μl 579.23 32.32 4. NF 1,8-Cineole 75 μl 682.40 38.07 5. NF 1,8-Cineole 100 μl 515.28 28.75

Nanoparticles of various formulations were formulation constant with the increase in compound determined for their cumulative release of botanical in formulation up to at 75 μl /100mg and finally reach pesticide.3,4 Clearly, both of the surface area and the 25.04%, at 100 μl /100mg which has high stability as drug loading ratio could influence the release well as high bioactivity. behavior significantly. A general trend was that the The dispersion stability of terpene adsorbed nano- release rate increased with the increased drug-loading SiO particles in methanol is compared with native ratio for all formulations. Control release studies in 2 nano-SiO2 particles as shown in fig.1. Native nano- case of Eugenol as shown in fig 4.10. and table 4.16. SiO particles have been completely precipitated for Here the formulation at 25 μl /100mg nanosilica lead 2 about 3 days, while the terpene adsorbed SiO2 nano- to 20.22% pesticide release when compared to particle have a stable colloidal dispersion even after 3 standard, this indicated that rest 79.78% of pesticide days. The hydroxyl groups (–OH) from nano-SiO2 is loaded on to the nanosilica. The rest loded particles can interacted with methyl group of terpene pesticide will release subsequently after two to five which play a vital role in reducing the agglutination days, which prevent the subsequent infestation from of the inorganic nano-particles for a long time. pests. The control release efficiency of the

Figure 1: The dispersion stability of nano-SiO2 particles in methanol

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120

100

80 a 60 b c

40 Dispersability (%) Dispersability

0 0 24 48 72 hrs

a - NFPC1 b – NFPC2 c – nativeNP

The morphological information was of native nano-SiO2 and terpene anchored nano-SiO2 suspensions in methanol were obtained by TEM images as shown in fig.2.

Figure 2: TEM morphology of nano-SiO2 particles

(a) Native nano-SiO2 (c) NF 1,8-Cineole 100μl /100mg The agglomeration was observed in both the samples, organisms and the environment, act in many ways on however less agglutination was observed in terpene various types of pest complex as for example the adsorbed nanosilica suggests that physical bonding or neem extracts, and may be applied to the plant in the chemical bonding occurs between the terpene and same way as other agricultural chemicals. Many plant nanosilica which bring mutual exclusion and steric extracts and essential oils are known to possess hindrance effect, thus the surface free energy has ovicidal, repellent and insecticidal activities against been reduced correspondingly and the agglomeration various stored-product insects. In that study, the controlled. All the results above further illustrate that exposure period appeared to be the most important adsorption of terpene has an added advantage that in factor affecting the toxic effects of the vapours of the spite of less agglutination it also exhibit the high C. Alstonia scholaris rather than the dosage. dispersion stability. In our study, strong insecticidal activity against DISCUSSION: adults of R. domanica was obtained with 10% ethyl In the present work, Alstonia scholaris plant extracts acetate fraction from Alstonia scholaris having were tested by fumigation methods against adults of potent insecticidal activity within 1 day after R. domanica. The insecticidal activity varied with treatment against R. domanica adults, This plant

plant-derived material, insect species, and exposure material confirm its usefulness as potent insect-

time. It has been well recognized that some plant- control agents. Many plant-derived materials with derived insecticides affect a limited range of pest high volatility act as fumigants. It has been reported

that many phytochemicals have broad insecticidal

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299 299 299 insects, have little or no harmful effects on non-target 299

activity against agricultural, stored-product and

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medical insect pests, and acts as a fumigant. In our chemical bonding occurs between the terpene and study, Alstonia scholaris was much more effective nanosilica which bring mutual exclusion and steric against adults of R. domanica and in fumigation than hindrance effect, thus the surface free energy has in contact method. These results indicate that the been reduced correspondingly and the agglomeration insecticidal mode of action of the compounds may be controlled. All the results above further illustrate that largely attributable to fumigant action: they may be adsorption of terpene has an added advantage that in toxic by penetrating the insect body via the spite of less agglutination it also exhibit the high respiratory system. dispersion stability. This work demonstrates the potency of Alstonia CONCLUSION: scholaris extract in the control of A. aegypti Among the tested plant materials, Alstonia scholaris mosquito larvae with LC50 value 0.86mg/ml. A. proved as highly toxic against stored product pests as scholaris extract extended the duration of the various well as mosquito larvae. This active fraction will be larval instars and of pupation at very low further processed for the pure compound isolation, concentration tested. Our results corroborates with 5,6 identification for the principle bioactive compound. the results obtained by who stated that the fractions The two principle bioactive compounds showed of Vitex negundo and Syzygium jambolanum enhanced antifeedant activity against agricultural prolonged the larval and pupal duration of Culex pests on Nanoformulations. These Nanoformulations quinquefasciatus and Anopheles stephensi. Till now, are stable and exhibited high compatibility by there has been no promising solution for sustainable releasing the compounds slowly from the control of dengue vectors. The trend for dengue formulation. These formulations will be further vector A. aegypti control in this region has shifted processed for their mode of action against from relying solely on insecticides to biological agricultural pests as insect control agents. control, source reduction and environmental management through community participation. These REFERENCES findings have re-emphasised the need to explore the 1. Mousson L, Dauga C, Garrigues T, Schaffner possibility of using herbal-based larvicides as F, Vazeille M, Failloux AB. "Phylogeography supplementary and complimentary measures for of Aedes (Stegomyia) aegypti (L.) and Aedes malaria control. This will reduce the chemical burden (Stegomyia) albopictus (Skuse) (Diptera: on the environment. Culicidae) based on mitochondrial DNA At dosage of 2μl/12cm2 caused 100% mortality with variations". Genet. Res. 86 (1): 1–11.2005 Eugenol. In case of 1,8-Cineole dosage of 3μl/12cm2 2. Santharam, G. Studies on the nuclear caused about 60% mortality. 1,8-Cineole showed less polyhedrosis virus of the tobacco cutworm, antifeedancy compared to Eugenol. No significant Spodoptera litura (Fabricius) (Noctuidae: difference in antifeedancy was found in less than 0.75 Lepidoptera), PhD Thesis, Tamil Nadu μl/12cm2 concentrations with 1,8-Cineole when Agricultural University, Coimbatore.1985 compared to the control treatment. At dosage of 3. Pushpalatha E, Muthukrishnan J. Larvicidal 1mg/12cm2 caused 100% mortality with Eugenol. activity of a few plant extracts against Culex where as in 1,8-Cineole dosage of 2mg/12cm2 caused quinquefasciatus and Anopheles stephensi. 100% mortality. Antifeedancy is doubled in NF Indian J Malariol 32(1):14–23.1995 Eugenol compared to pure Eugenol. In the case of NF 4. Ulrichs C, Goswami A, Mewis I. Nano- 1,8-Cineole antifedancy is increased by three times of structured silica— physical active pesticides pure 1,8-Cineole. for urban settings. In: Proceedings of the second international symposium on plant The morphological information was of native nano- protection and plant health in Europe, DPG- SiO2 and terpene anchored nano-SiO2 suspensions in BCPC, Berlin, 2007

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