Chapter 4. Results Bioactivity guided fractionation of E. crassipes root extract Column chromatography

Transcription

1 CHAPTER 4 Results

2 54 Chapter 4 Results 4.1. Bioactivity guided fractionation of E. crassipes root extract Column chromatography The pulverised root material of E. crassipes was extracted with acetone in a soxhlet apparatus for 72 h. Methanol was evaporated from the extract by rotary evaporator up to dryness under reduced pressure, which yielded a thick residue. This crude extract was fractionated through Column chromatography using different solvents in increased polarity order. The fractionation of crude extract with the step wise gradient of hexane: ethyl acetate and methanol yielded 102 fractions of 50 ml. These fractions were collected and the similar ones were pooled to thirteen groups F1-F13 according to their Rf values determined by thin layer chromatography (TLC). Among these fractions F5 showed toxicity, this is further purified by TLC. The fraction showed three visible spots on TLC performed with 10% ethyl acetate in chloroform as a mobile phase. The plate was sprayed with CuSO4 solution in cadmium disulphide developed brown colour and orange red colour with Uranyl nitrate in CS2 indicating alkaloid in the crude active fraction with Rf value = Structure elucidation of principal bioactive compound Bioassay-guided fractionation of the E. crassipes root extract afforded an active constituents identified by spectroscopic analyses, which includes EIMS, IR and NMR. Electron Interference Mass Spectroscopy (EIMS) showed the presence of molecular ion EIMS (70ev) M+ m/e at 127 and other fragment ions at 126, 98, 58, 41 and 39 corresponds to molecular formulae C8 H17 N. FTIR: cm , , 1459 and HNMR spectra showing δ CDCl t. CH3; m. CH2 X 5; m, CH2N; 3.8 m, CHN.

3 55 Based on these results the compound was identified and conformed as Coniine (2-npropyl piperidine) with the molecular formulae of C 8 H 17 N after comparison with published data (Mody and Coworkers, 1976) E. Crassipus Crude extract Hexane Chloroform Ethyl Acetate Methanol Inactive Inactive Inactive Active Fractionation ethyl acetate in hexane 10% EA 30% EA 40% EA 50% EA 60% EA 70% EA 80% EA 100% EA Active All other fractions except 10% EA were Inactive Structural analysis by NMR, EIMS, FTIR, GC and GCMS 2-propyl piperidinre (Coniine) Chemical Charecterization FTIR, TEM, XRD, Particle size, Contolled release, Shelf life and Dispertion Formulated with Silica nanoparticle Bioassays against agricultural pests Antifeedant, Topical toxicity and growth regulatory Fig 8: Schematic representation of the isolation procedure for principal bioactive compounds from E. crassipes plant

4 Characterization of Coniine/SiO 2 nanoformulation FTIR analysis of the Coniine nanoformulation FTIR spectroscopy was used to analyze the structure and interaction between Coniine and silica nanoparticles. Even though infrared spectroscopy, frequencies are ranged from 400 to 4000cm-1, silica based materials showed typical absorption band in certain regions. The assignment of each region is as follows: i cm -1 - Stretching vibrations of hydroxyl groups. This region associated with H-bridging hydroxyl (-Si OH.O Si-) groups, isolated silanol (-Si OH) and adsorbed molecular water. ii cm -1 Si O CH3 symmetric stretching and C-H stretching iii and 800 cm -1 Si O Si and vibration of SiO2 network iv cm -1 H O H for adsorbed molecular water v cm -1 Si O, vibration of silica network vi. 800 cm -1 Si O Si vibrational mode vii. 948, 460 cm -1 Si O Si deformation The FTIR spectra of Coniine and their bonding with silica nanoparticles were shown in Fig. 9. Fig. 9(a) represented the FTIR spectra of pure silica nanoparticles. The corresponding bands appear at 950 and 1090 cm -1, due to the vibrations of Si-OH and Si- O-Si bands, respectively. These bands are very intense and correspond to the formation of the SiO2 network. Fig. 9(f) shows the FTIR spectra of pure Coniine. The peak at cm -1 indicates the presence of C-H stretching in Coniine compound. Fig. 9(b-e) shows the FTIR spectra of different Coniine -silica hybrid compounds. The peak shift to lower wave number of OH group position in silica shows the presence of hydrogen bonding between Coniine and SINPS.

5 57 Fig.9: FTIRspectra of (a) Native silica nanoparticles, (b) 0.025% CONSI (c) 0.05% CONSI (d) % CONSI (e) 0.1% CONSI and (f) pure Coniine TEM analysis of the Coniine nanoformulation The transmission electron microscope (TEM) images of the silica nanoparticles and Coniine formulated silica nanoparticles were shown in the Fig. 10. Natural agglomeration was observed in the native silica nanoparticles whereas the agglomeration is less in the Coniine formulated silica nanoparticles.

6 58 b C Fig.10: TEM micrographs of (a) native silica nanoparticles (b and c) nanoformulated Coniine XRD analysis of the Coniine nanoformulation The X-ray diffraction curve of silica nanoparticles and nanosilica formulated Coniine was presented in Fig. 11. Native silica nanoparticles peaks were taken as a standard and then compared with those where Coniine added subsequently. The peaks observed in the X- ray diffraction patterns of these silica nanoparticles were broad. SINPs spectrum (Fig. 11) showed a broad peak in the range of (2θ). After formulation with Coniine, silica nanoparticles showed the in the narrowing the peak. The sharp peaks of the formulation indicated the crystalline nature of the silica nanoparticles but due to the lack of long range and order formulation they do not exhibit a regular pattern like crystalline silica polymorphs quartz or tridymite.

7 59 Fig.11: XRD spectra of silica nanoparticles and nano-formulated Coniine Particle size and zeta potential analysis of the Coniine nanoformulation. The silica nanoparticles dispersed in the millipore water indicated by zeta potential value ( 28.4 mv) and hydrodynamic particle size (46.3 nm). The zeta potential of formulations depends on the nanomaterial and the active ingredient content of the framework. The zeta potential value of the Coniine nanoformultion ( 44.7 mv) is higher than that of native silica nanoparticles ( 28.4 mv) Shelf-life analysis of the Coniine nanoformulation. The physical stability of the formulations were analysed by studying its dispersion and controlled release of compounds from formulation both before and after storage. The UV spectrum of the Coniine was not altered due to the storage up to 6 months, which confirms the chemical stability of the compound. There is no significant difference in the dispersion stability of the nanoformulation up to 6 months. Whereas the controlled release property of the formulation is affected on storage. Before storage strong control release property was observed in the nanoformulated Coniine (40 % of the compounds dissolved in the solvent in 20 min). Whereas this property was gradually decreased with

8 60 the time of storage and drastic decrease was observed upon 60 days storage (82% the compound dissolved in the solvent in 20 min). Hence the benefit of controlled release was lost during the storage after 60 days. However, after 60 min, the amount of the drug that has dissolved in both cases (for the nanoformulation before and after storage) is almost the same (80% of the compounds dissolved in 60 min for the mixture before storage, versus 75% of the terpene dissolved in 60 min for the mixture after storage). Hence, formulation of compounds with SINPs helps to maintain their physical stability/shelf life. Fig.12. Effect of storage on controlled release profile of the Coniine from its nano formulations Controlled release properties of the Coniine nanoformulation The controlled release profile of Coniine from nanoformulated Coniine was studied by HPLC and the results were presented in Fig. 13. Controlled release of the compounds from nanoformulations were interesting as 40% of compounds were released in the first 20 minutes, while the other 60% was released in a typical sustained release pattern and dissolved out slowly and evenly for a time period of 24 hours.

9 61 Fig.13: Release profiles of Coniine in normal state and from its nano-formulation in methanol Dispersion stability of the nano-formulated Coniine The dispersion stability of Coniine nanoformulation was studied and the results were presented in the Fig. 14. Dispersion stability of nanoformulation and native silica are similar up to one hour while on keeping the solution idle for 3 days, the particles in the native silica was precipitated with in 30 hours, where as the particles in the nanoformulation were well dispersed for 48 hours. Fig.14: Dispersion stability of the Coniine nanoformulation in comparison with native silica nanoparticles

10 Biological activities against agricultural pests Antifeedant activity of the E. crassipes and fractions and isolated pure compound The antifeedant activities of crude, fractions and pure samples were determined by the free no choice bioassay and the results were presented in Table 2. Acetone extract of the E. crassipes have been shown to have potent antifeedant activity against S. litura and A. janata with LC 50 values and µg cm2 respectively. The antifeedant activity increases with the increase in concentration of the crude. The crude extract showed 22% antifeedancy at 20.0 µg cm -2 and reaches to 100% mortality at 50.0 µgcm -2 against A. janata. Among the fraction collected from the extract, fraction F5 showed potent anifeedant activity against S. litura and A. janata in a dose-dependent manner, with EC 50 values of 9.02 and 7.59 µg cm -2 respectively. Table 2: Antifeedant activity of crude, fraction and isolated compound coniine against S. litura, A. janata and H. armigera S. Compounds/ ED 50 (95% FL a ) µg cm -2 No formulations S. litura A. janata H. armigera 1 Crude 33.79( ) 25.83( ) 39.84( ) 2 F 5 fraction 9.02( ) 7.59( ) 11,43( 6, ) 3 Coniine 6.69( ) 6.09( ) 6.98( ) a Fiducial limits The results showed that the Coniine isolated from bioactive fraction exhibited antifeedant effect on larvae of S. litura, H. armigera and A. janata, and this effect was enhanced with increasing concentrations of the Coniine. The best antifeedant effect was observed against the A. janata, in which the Coniine of five concentrations (5.0, 7.5, 10.0, and 15.0 µg cm 2 ) resulted in antifeedant rates of 29%, 72%, 92% and 100%, respectively with an EC 50 of 6.09 µg cm 2. However same compound showed less antifeedant activity against

11 63 S. litura with ED µg cm 2. It is notable that Coniine did not display any toxicity up to 10 µg/ cm2 applied in the experiments contact toxicity of the E. crassipes and its isolates Contact toxicity of the E. crassipes and its principal bioactive compound, Coniine was assessed by topical bioassay. Application of the crude extract at 50µg/larvae, showed 28% toxicity against A. janata, H. armigera and S. litura. A good dose response was obtained. The insecticidal activity was increased with the concentration of the extract and approached 100% activity at 200 µg/insect with IC 50 value 77 µg /insect. Among the 13 fractions F5 fraction exhibited a potent insecticidal activity with a mortality of >75% at a concentration of 100 µg/insect. Other fractions collected failed to show insecticidal activity against both the insects tested even at the dosage of 100 μg/ insect. The principal bioactive compound isolated from Coniine exhibited promising insecticidal activity with LC 50 values 14.10, and 9.30 µg /insect against S. litura, H. armigera and A. janata respectively. The formulations enhanced the toxicity by two folds. Table.3: Contact Toxicity of E. crassipes crude, fraction and isolated compound coniine against S. litura, A. janata and H. armigera S. Compounds/ LD 50 (95% FL a ) µg/insect No formulations S. litura H. armigera A. janata 1 Crude 78.62( ) ( ) ( ) 2 F 5 Fraction 48.92( ) ( ) ( ) 3 Coniine ( ) ( ) 9.30 ( ) 4 Formulation 7.26 ( ) 7.66 ( ) 6.51 ( ) 5 SINPs(µgcm -2 ) >15 >15 > 15 a Fiducial limits

12 Antifeedant activity of Coniine and its nano-formulations From the results it is evident that nanoformulation of isolated compound Coniine with silica nanoparticles has enhanced the antifeedant activity of the Coniine. Pure compound showed antifeedant activity against S. litura, H armigera and A. janata with ED 50 values of 6.25, 6.98 and 5.53 µl cm -2 respectively. The antifeedant activity of the nanoformulations depends on the dose as well as the concentration of the Coniine present in the formulation. As the concentration of Coniine increased in the formulation from to 0.1% (w/w) the antifeedant activity also increased from ED 50 4 to 2 µl cm -2 against S. litura and H. armigera. In other experiment similar enhanced activity was observed against A. janata. Table. 4: Feeding deterrent activity of coniine and its nanoformulations against S. litura, H. armigera and A. janata S. Compounds/ ED 50 (95% FL a ) µg cm -2 No formulations S. litura H. armigera A. janata 1 Coniine 6.28 ( ) 6.98( ) 5.53 ( ) % CONSI 4.09 ( ) 4.25( ) 3.50 ( ) % CONSI 3.56 ( ) 3.62( ) 2.95 ( ) % CONSI 2.96 ( ) 3.10( ) 1.74 ( ) 5 0.1% CONSI 2.27 ( ) 2.10( ) 1.29 ( ) 6 SINPs (µg cm -2 ) >15 >15 > 15 a Fiducial limits 4.2. Bioactivity guided fractionation of the P. capensis Isolation principal bioactive compounds The roots of P. capensis were collected from Tirumala forest, Tirupati, Andhra Pradesh, India. A voucher specimen was deposited at the herbarium of Indian Institute of Chemical Technology, Hyderabad, India. The shade dried roots of P. capensis were

13 65 powdered in a pulvarizer. The powdered root material (10 kg) was extracted with chloroform in a soxhlet apparatus for 72 h. Chloroform extract was evaporated to dryness under reduced pressure, yielded resulted a syrupy residue (12 g). The chloroform extract was subjected to column chromatography on a silica gel column ( mesh, cm) and eluted with a step wise gradient of hexane/etoac (99:1, 98:2, 92:8, 90:10, to 10:90 by volume) to afford a total of 68 fractions of 50 ml each. Column fractions were analyzed by TLC (Silica Gel 60 F254, hexane/ EtOAc, 85:15), and fractions with similar TLC patterns were combined to give five major fractions (F1 to F5). The fraction F1 (8 g), showed a potent antifeedant activity against tested insects, which was purified by silica gel column chromatography ( mesh, hexane/etoac, 99:1) to give orange needles compound (7.5 g) Structural elucidation of isolated bioactive compound Orange needles from hexane fraction. uv maxima at 264, 436 nm. FTIR 1656, 1647, 1614, 1455, 1360, 1320, 1216, 1026, 900, 782 cm 2. NMR 2.13(3H), 6.66 (1H), (1H),7.07(1H), 7.39(1H) and 7.47 (1H). MS (m/e) (M+. Calcd. for C22H1406: ), and other fragment ions at 346 (C 21 H ), 331 (C 20 H ), 317 (C 20 H , 303 (C 19 H ), 287 (C 17 H ), 250 (C 16 H 10 O 3 ).above all this characterisations conforms the pure compound is Plumbagin. P. Capensis Crude extract Hexane Chloroform Ethyl Acetate Methanol Inactive Active Inactive Inactive Fractionation Chloroform in hexane 20% CHCl 3in hexane 30% 40% 50% 60% 70% 80% 100% All other fractions except 40% Chloroform were Inactive

14 66 Active Structural analysis by NMR, EIMS, IR, GC and GCMS Plumbagin Chemical Charecterization FTIR, TEM, XRD, Particle size, Contolled release, Shelf life and Dispertion Formulated with Silica nanoparticle Bioassays against agricultural pests Antifeedant(active only in feeding deterent mode ) Fig 15: Schematic representation of the isolation procedure for principal bioactive compounds from P.capensis plant Characterization of Plumbagin/SiO2 nanoformulation (PBSI) FTIR analysis of the nano-formulated plumbagin The bonding between plumbagin and silica nanoparticles was shown the Fig. 16. Fig. 16(a) represents the FTIR spectra of native silica and Fig. 16(b, c, d) and (e) represents the plumbagin formulated silica nanoparticles. The band at cm -1 indicates the present of O-H stretching. The peak at cm -1 indicates the present of C-H stretching of methyl group present in the plumbagin molecule. The presence of hydrogen bonding was demonstrated by the shifting of wave number of OH group position in silica to lower wave number.

15 67 Fig.16: FTIRspectra of (a) Native silica nanoparticles, nano-formulations of plumbagin (b) % PBSI (c) 0.05% PBSI (d) 0.075% PBSI and (e) 0.1%% PBSI TEM analysis of Plumbagin nanoformulation Fig. 17 represents the TEM photographs of silica nanoparticles and plumbagin formulated silica nanoparticles. The primary silica nanoparticles were heavily agglomerated. The agglomeration of silica nanoparticles decreased on formulation with plumbagin. This disappearance of agglomeration could be due to the electrostatic repulsion between plumbagin and SINPs b C Fig 17: TEM micrographs of (a) native silica nanoparticles (b & c) nano-formulated plumbagin

16 XRD analysis of Plumbagin nanoformulation The X- Ray diffraction spectra of native silica and plumbagin formulated silica nanoparticles presented in the Fig. 18. A broad peak in the range of (2θ) of native silica represents the amorphous nature of particles. The broadness of this peak was increased in the plumbagin formulated silica nanoparticles demonstrate the formation of additive. Fig.18: XRD spectra of silica nanoparticles and nano-formulated plumbagin Particle size and zeta potential analysis of Plumbagin nanoformulation Particle size and zeta potential of the native silica nanoparticles and Plumbagin nanoformulation measured by dispersing them in Millipore water. Native silica nanoparticles showed average particle size of 46.3nm with the mv zeta potential. The Plumbagin interaction with silica nanoparticles in the formulation caused the reduction in the zeta potential from to mv, which indicates the high stability of nanoformulations. In addition to this the size of the silica nanoparticles were decreased from 46.3 to 36.7nm indicates the dispersion efficiency of the nanoparticles. The methyl group of the Plumbagin might react and de-protonated the hydroxide groups to form Si O which enhances the surface more negatively charged.

17 Dispersion stability of Plumbagin nanoformulation Dispersion studies were carried out for the nanoformulated Plumbagin in order to check the dispersion stability. The dispersion stability of nanoformulated Plumbagin was compared with that of native silica nanoparticles (Fig. 19). Native silica nanoparticles were precipitated within 24 hours, whereas the nanoformulated Plumbagin particles were highly dispersed in the methanol even after 5days. This stable colloidal dispersion is due to the interaction of Plumbagin with silica nanoparticles which hinders the silica particles in agglomeration and the particles were highly dispersed. Fig.19: Dispersion stability of the Plumbagin nanoformulation in comparison with native silica nanoparticles Shelf-life analysis of Plumbagin nanoformulation The physical stability (shelf-life) of the Plumbagin formulated silica nanoparticles was tested both before and after storage. The storage up to 6 months did not affect the UV absorption spectra of Plumbagin nanoformulation. There is no change in the dispersion stability of the Plumbagin formulated silica nanoparticles up to 3 months. However, the control release property of the formulation was lost after 3 months of storage. Newly

18 70 prepared Plumbagin nanoformulation showed good control release property by releasing only 28% of the Plumbagin in to solvent for 20 min. whereas the same formulation after 90 days the control release property of the formulation was lost and release more than 75 % of the Plumbagin in to the solvent with in 20 min. Hence, formulation of compounds with SINPs helps to maintain their physical stability/shelf life. Fig.20: Effect of storage on controlled release profile of the Plumbagin from freshly prepared nano formulations and the nanoformulation stored for 6 months Controlled release properties of Plumbagin nanoformulation HPLC analysis revealed the Plumbagin release profiles from its nanoformulation as shown in the Fig. 21. Non formulated Plumbagin directly dissolved in the solvent and the release rate is 100% within 15 min. After formulating with silica nanoparticles the release rate of the Plumbagin in the solvent is drastically decreased and only 28% of the Plumbagin was released from the formulation in first 20 min and it takes 15 hours to release the 100% of the Plumbagin in solvent. Fig.21: Release profiles of plumbagin in normal state and Coniine from its nanoformulation

19 Antifeedant activity of plumbagin and its nano-formulations Plumbagin showed moderate antifeedant activity towards both the insects tested. After formulation the antifeedant activity of the Plumbagin appreciably increased. The antifeedant activity of the formulations increased with the increase of Plumbagin in the formulation from to 0.1% (w/w) as shown in the Table 5. Silica nanoparticles formulations exhibited advantage of enhancing antifeedant activity of Plumbagin by more than ten folds. Table.5: Effect of P. capensis crude, fraction, isolated pure compound, plimbagin and its nanoformulations against feeding behavior of S. litura, H. armigera and A. janata S.No Compound ED 50 (95% FL a ) µl cm -2 S. litura H. armigera A. janata 1 Crude ( ) ( ) ( ) 2 Fraction ( ) ( ) ( ) 3 Plumbagin ( ) ( ) ( ) % PBSI ( ) ( ) ( ) % PBSI ( ) ( ) ( ) % PBSI ( ) ( ) 64.74( ) % PBSI 24.31( ) 25.28( ) 19.20( ) 8 SINPs > 10 >10 >10 a Fiducial limits

20 Bioactivity guided fractionation of Piper betle L. leaf extract Isolation of pure compounds Piper betle L. was evaluated for bioactivity against S. litura, H. armigera and A. janata. The results of the present study demonstrated that, P. betle leaf extract has high antifeedant activity against the insects tested. The antfeedant assay of the crude extracts showed 20% activity 50µg cm -2 against A. janata, H. armigera and S. litura. Antifeedant activity was increased with the increased the dose employed. The crude extract on subjected to liquid-liquid chromatography yielded hexane, ethyl acetate and methanol fractions. Among the fractions ethyl acetetae fractions showed promising antifeedant activity against insects tested. Bioactive ethyl acetate fractions were further fractionated with ethyl acetate gradient in hexane, which yielded 74 fractions. Similar profile having fractions were pooled on the basis of their TLC profile which resulted seventeen fractions. It is interesting to observe that 3 fractions showed the antifeedant activity against S. litura, H. armigera and A. janata. Hence three fractions were further fractionated, yielded five bioactive compounds. Subsequent chemical characterisation and comparison with previous literature and standards these compounds were identified as Eugenol, α-pinene, Limonene, Ocimene, Trans-caryophyllene Nerolidol and Carene. The major component was Eugenol and remaining compounds were present in lower concentrations. Therefore for further analysis and nanoformulations the above compounds were purchased from sigma-aldrich.

21 73 Piper betle Crude extract Hexane Chloroform Ethyl Acetate Methanol Inactive Inactive Active Inactive Fractionation ethyl acetate in hexane 20% EA 30% EA 40% EA 50% EA 60% EA 70% EA 80% EA 100% EA Active Eugenol α-pinene, linalool Structural analysis Active Limonine Ocimene, Trans-caryophyllene Nerolidol Carene Formulated with Silica nanoparticle Chemical Charecterization FTIR, TEM, XRD, Particle size, Contolled release, Shelf life and Dispertion Bioassays against agricultural pests Antifeedant, Topical toxicity and growth regulatory Fig 22: Schematic representation of the isolation procedure for principal bioactive compounds from P.betle plant

22 74 Linalool Limonene Trans-caryophylline α-pinene Eugenol Ocimene Nerolidol Carene Structure of the isolated compounds Characterization of Eugenol /SiO2 nanoformulation (EUSI) FTIR analysis of the nano-formulated Eugenol Fig. illustrates the FTIRspectra of native nano-sio2 particles and their formulation with Eugenol 23. FIG a represents the FTIRspectra of unmodified nano-sio2 and Fig 23 b-f represent the silica formulation in 0.025, 0.05, and 0.1 % and pure Eugenol respectively. The peak at 3422 cm 1 is attributed to O H stretching mode, shear vibration near 1630cm 1, bending mode near 1390cm 1. The peak at 1103cm 1 corresponds to the Si O Si absorption bands, the asymmetric stretching vibration near810 cm 1 and bending vibration near 487 cm 1. The peak at 2926 is assigned to the methane symmetrical stretch vibration in Eugenol, which confirms the adsorption of the Eugenol on silica surface. Peak at 1635 indicates the presence of moisture in the sample. The decrease in OH bending band (800 cm 1 ) and Si-O bond rocking band (467cm 1 ) was

23 75 observed. A peak of hydroxyl group (between 3200 tom3600 cm 1 ), The Si-OH band stretch (958cm 1 ) and asymmetric Si-O-Si stretching (1090 cm 1 ) does not alter after adsorption. Thus it can be concluded that the Eugenol adsorbed on the silica through strong hydrogen bonding. Fig.23: FTIRspectra of (a) Silica nanoparticles, nano-formulations of Eugenol (b) 0.025% EUSI (c) 0.05% EUSI (d) 0.075% EUSI (e) 0.1% EUSI and (f) pure Eugenol

24 TEM analysis of the nano-formulated Eugenol The morphology of the native and Eugenol formulated silica nanoparticles were resented in the form of TEM images (Fig. 24). The distribution of Eugenol formulated silica nanoparticles was higher compared to the native silica nanoparticles, indicating the surface attached Eugenol cased an advantageous effect and the reduced the aggregation of the silica nanoparticles. Fig.24: TEM micrographs of (a) native silica nanoparticles (SINPs) (b and c) nanoformulated Eugenol (EUSI) Particle size and zeta potential analysis of the nano-formulated Eugenol Particle size and zeta potential of the silica nanoparticles and Eugenol formulated silica nanoformultion were measured by dispersing them in to millipore water indicated by zeta potential value ( 28.4 mv) and hydrodynamic particle size (46.3nm). The Eugenol formulated silica nanoparticles showed well dispersion and the average particle size is 24.7 nm. The native silica nanoparticles showed 46.3 nm, hence the reduction in size demonstrated that the Eugenol has interacted with silica nanoparticles to form an additive which has less agglomeration efficiency. The zeta potential of formulations depends on the nanomaterial and the active ingredient content of the framework. The zeta potential of

25 77 the formulation was higher in negative value ( 39.0 mv) than that of SINPs ( 28.4 mv) demonstrates the stability of the nanoformulation Dispersion stability of the nano-formulated Eugenol The dispersion stability of Eugenol nanoformulation was studied and the results were presented in the Fig. 25. Initial period of 1 hour both the nanoformulation and native silica showed similar dispersion profile while on keeping the solution idle for 3 days, total precipitation of the particles were absorbed in the native silica nanoparticles, where as Eugenol formulated nanoparticles were well distributed and showed stable colloidal dispersion even after 48 hours. Fig. 25: Dispersion stability of the Eugenol nanoformulation in comparison with native silica nanoparticles Shelf-life analysis of the nano-formulated Eugenol Dispersion and controlled release of compounds from formulation are the indication of physical stability. There is no significant difference in the dispersion stability of the nanoformulation up to 6 months. Controlled release study reveals the impact of storage on physical stability of the Eugenol formulation. Freshly prepared Eugenol nanoformulation releases the Eugenol in to the solvent in a time dependent manner. It

26 78 releases 34% of Eugenol in the first 1 hour and it needs 16 hours to release total compound in to the solvent. 3 months storage did not affect the controlled released nature of the Eugenol formulation, while further storage causes drastic damage in the controlled release profile as 79 % of the compound released within one hour. Fig. 26: Effect of storage on controlled release profile of the Eugenol from nano formulations Controlled release properties Eugenol nano-formulations The release profile of the Eugenol from its nano-formulation was studied by HPLC and the results were presented in Fig..27. Unformulated Eugenol dissolves in the methanol very easily with a release rate of 100% within 20 min. After formulation with silica nanoparticles Eugenol release profile in the solvent was controlled and only 34% of Eugenol was released in the first 1 hour, and rest has released slowly and 100% of Eugenol was released after 16 hours. Fig. 27: Release profiles of Eugenol in normal state and Eugenol from its nanoformulation.

27 Antifeedant activity of Eugenol and its nano-formulations The feeding deterrent activity of Eugenol and its nanoformulations was studied against S. litura, H. armigera and A. janata using leaf disc no-choice method and results are presented in the Table 6. The antifeedant activity of the Eugenol and its formulations were presented in the form of antifeedant index. Eugenol showed higher antifeedant activity against with ED 50 < 1 µl cm -2. On formulation and 0.1% showed enhanced bioactivity against S. litura and H. armigera, whereas nanoformulation has reduced the activity of Eugenol against A. janata. Therefore, it may be stated that silica formulation has blocked the active site of the Eugenol against A. janata. Table.6: Feeding behavior of S. litura, H. armigera and A. janata against P. betle crude, isolated pure compound, Eugenol and its nanoformulations against S. ED 50 (95% FL a ) µg cm -2 Compounds No S. litura H. armigera A. janata 1 Crude 51.74( ) 57.34( ) 32.93( ) 2 Eugenol (EU) 0.72( ) 0.71( ) 0.34( ) % EUSI 1.03( ) 2.22( ) 1.49( ) % EUSI 0.87( ) 1.33( ) 0.76( ) % EUSI 0.43( ) 0.62( ) 0.62( ) % EUSI 0.34( ) 0.59( ) 0.38( ) 7 SiINPs > 10 >10 > 10 a Fiducial limits

28 Characterization of α-pinene /SiO2 nanoformulation (APSI) FTIR analysis of the nano-formulated α-pinene FTIR spectroscopy was used to analyze the structure and interaction between α-pinene and silica nanoparticles. The FTIR spectra of α-pinene and their interaction with SINPs were shown in Fig.28. The wave numbers of the α-pinene and their hybrid functional groups were described here. Fig. 28(a) represented the FTIR spectra of pure silica nanoparticles. The corresponding bands appear at 950 and 1090 cm 1, due to the vibrations of Si-OH and Si-O-Si bands, respectively. These bands are very intense and correspond to the formation of the SiO2 network. Fig. 28 (f) shows the FTIR spectra of pure α-pinene. The peak at cm 1 indicates the presence of C-H stretching in α- pinene compound. Fig. 28(b-e) shows the FTIR spectra of different α-pinene-silica hybrid compounds. The peak shift to lower wave number of OH group position in silica shows the presence of hydrogen bonding between α-pinene and SINPS. Fig. 28: FTIRspectra of (a) Native silica nanoparticles, nano-formulations of α- Pinene (b) (c) 0.05 (d) (e) 0.1% APSI and (f) pure α-pinene

29 TEM analysis nano-formulated α-pinene The morphological information of silica nanoparticles and nano-formulations were obtained by TEM analysis in the form of images as shown in following Fig. 29. The agglomeration was observed in both native and α-pinene formulated silica nanoparticles. But the intensity of the agglutination is less in the formulation. This suggests a bonding either physical or chemical occurs between the α-pinene and silica nanoparticles, which reduces the surface free energy and controls the agglomeration. b C Fig. 29: TEM micrographs of (a) native silica nanoparticles (SINPs) (b and c) nanoformulated α-pinene Particle size and zeta potential analysis of nano-formulated α-pinene. Silica nanoparticles and α-pinene formulated silica nanoparticles were dispersed in the millipore water and their particle size and zeta potentials were measured. Native silica nanoparticles showed an average particle size of 43.6 nm with a zeta potential value of 28.4 mv. After formulation α-pinene interacted with the hydroxide group of the silica nanoparticles enhanced the dispersion of silica nanoparticles. Due to their interaction the size of the silica nanoparticles comes to 32.6nm and showed the zeta potential value in higher negative values with 39.7 mv than that of Silica NPs ( 28.4 mv).

30 Dispersion stability of nano-formulated α-pinene The dispersion stability of α-pinene nanoformulation was compared with silica nanoparticles and the results were presented in Fig. 30. Native silica nanoparticles precipitated quickly and total precipitation was observed within 24 days, whereas the α- pinene stops the precipitation of silica nanoparticles up to 72 hours. After formulation α- pinene has changed the surface charge of silica which resulted in the stable colloidal particles even after 3 days. Fig. 30: Dispersion stability of the α-pinene nanoformulation in comparison with native silica nanoparticles Shelf-life analysis of nano-formulated α-pinene. The physical stability (shelf-life) of the nanobioformulations was tested both before and after storage. The UV spectrum of the compounds was not altered due to the storage up to 6 months, which confirms the chemical stability of the compound. The physical stability of the formulations were analysed by studying its dispersion and controlled release of compounds from formulation both before and after storage. There is no significant difference in the dispersion stability of the nanoformulation up to 6 months. Whereas the controlled release property of the formulation is affected on storage.

31 83 Before storage strong control release property was observed in the nanoformulations (40 % of the compounds dissolved in the solvent in 20 min). Whereas this property was gradually decreased with the time of storage and drastic decrease was observed upon 60 days storage (82% the compound dissolved in the solvent in 20 min). Hence the benefit of controlled release was lost during the storage after 60 days. However, after 60 min, the amount of the drug that has dissolved in both cases (for the nanoformulation before and after storage) is almost the same (80% of the compounds dissolved in 60 min for the mixture before storage, versus 75% of the α-pinene dissolved in 60 min for the mixture after storage). Hence, formulation of compounds with silica nanoparticles helps to maintain their physical stability/shelf life. Fig. 31: Effect of storage on controlled release profile of α-pinene from formulation Controlled release properties of nano-formulated α-pinene α-pinene rapidly dissolves in the solution as 100% of the compound was dissolved in the solution within 15 minutes. This on formulation with silica nanoparticles reduced the dissolving capacity of the α-pinene to keep the compound available for longer duration. HPLC studies revealed that the release rate of α-pinene from formulation was controlled and the release with time. 24% α-pinene was released within 15 minutes and it reaches gradually to 100% after 19 hours as shown in the Fig. 32.

32 84 Fig. 32: Release profiles of α-pinene in normal state and from formulation in methanol Antifeedant activity of α-pinene and its nano-formulations The isolated compound α-pinene exhibited antifeedant activity against S. litura with ED 50 >1 µl cm -2. After silica nanoformulation α-pinene exhibited 25 folds enhanced antifeedant activity with ED 50 value µl cm -2. The nanoformulated α-pinene showed 100 percentage feeding deterrence at 0.1 µl cm -2, while nonformulated α-pinene failed to show absolute deterrence even at 2 µl cm -2. However the same formulations exhibited distinct results against A. janata. The antifeedant activity of α-pinene against A. janata was found less in comparison to that of the S. litura on formulating with silica nanoparticles. Overall, these results imply that the mode of perception as well as the structure-activity relationship of the nanoformulations, like free methyl groups in the isopreme units differs considerably between the insect species examined in this study. α- pinene may have more than one active site and the active site responsible for the antifiidant activity against the A. janata may be masked with the silica nanoparticles while the actives sites against S. litura will be free. Native SINPs alone did not produce any toxicity or antifeedant activity to A. janata or S. litura even at higher concentration tested (5 µg cm -2 ). The results on the antifeedant activity of the formulations having different doses of α-pinene (0.025, 0.05, and 0.1 % α-pinene in silica nanoparticles) were shown in Table 7. The activity of nanoformulations had increased with the increase of α-pinene concentration in the formulations against S. litura and H. armigera. Similar results were obtained against A.

33 85 janata up to (0.075 % terpene concentration) and then remained more or less constant even after increasing the α-pinene concentration in the formulations. Table.7: Feeding inhibitory effects of P. betle, isolated pure compound, α-pinene and its nanoformulations against S. litura, H. armigera and A. janata S.No Compounds ED 50 (95% FL a ) µl cm -2 S. litura H. armigera A. janata 1 Crude 51.74( ) 57.34( ) 32.93( ) 2 α-pinene (AP) ( ) 0.980( ) ( ) % APSI ( ) 0.080( ) ( ) % APSI ( ) 0.085( ) 0.283( ) % APSI ( ) 0.045( ) 0.129( ) 6 0.1% APSI ( ) ( ) ( ) 7 SINPs >10 >10 >10 a Fiducial limits 4.5. Characterization of Linalool /SiO 2 nanoformulation (LLSI) FTIR analysis of the nano-formulated Linalool The FTIR spectra of Linalool and its interaction with SINPs were shown in Fig. 33. The wave numbers of the Linalool and their hybrid functional groups were described here. Fig. 33 (f) shows the FTIR spectra of pure Linalool. The band at cm -1 indicates the present of O-H stretching. The peak at cm -1 indicates the present of C-H stretching in Linalool compound. Fig. 33(b-e) shows the FTIR spectra of different Linalool-silica hybrid compounds. The chemical interactions between Linalool molecules and silica surface sites are clearly demonstrated by hydrogen bonding. The peak shift to lower wave number of OH group position in silica shows the presence of hydrogen bonding. A strong absorption band in the range cm- 1 shows the asymmetric stretching vibrations of the Si-O-Si bonds of the silica component in all the Linalool-silica hybrid formulations. The band at

34 cm -1 show the Si-OH vibration, the intensity is decreased with increasing the Linalool concentration. Fig. 33: FTIRspectra of (a) SINPs, nano-formulations of Linalool (b) 0.025%LLSI (c) 0.05%LLSI (d) 0.075%LLSI (e) 0.1%LLSI and (f) pure Linalool TEM analysis nano-formulated Linalool TEM photographs illustrate the influence of Linalool on the morphology of the silica nanoparticles. TEM image of the silica nanoparticles showed agglutination. Linalool formulated silica nanoparticles showed less agglutination than that of the native silica nanoparticles. b C Fig. 34: TEM micrographs of (a) native silica nanoparticles (b and c) nanoformulated Linalool

35 Particle size and zeta potential analysis. The zeta potential of formulations depends on the nanomaterial and the Linalool in the framework. The formulation exhibited high negative zeta potential (-39.7 mv) than that of the native silica ( 28.4 mv). Similar to that pinene formulation Linalool nanoformulation also showed higher dispersion stability due to change in zeta potential. It is interesting to note that particular size of the formulation has slightly higher (48.6nm) than that of native silica nanoparticles. The interaction of Linalool might not change the agglomeration character of the silica particles Dispersion stability of nano-formulations Similar to pinene formulation Linalool formulation also showed higher dispertion stability. Native silica nanoparticles were used for comparing dispersion stability. Native silica nanoparticles precipitated within 24 hours whereas nanoformulation showed colloidal suspension up to 48 hours. Fig. 35: Dispersion stability of the Linalool nanoformulation in comparison with native silica nanoparticles

36 Shelf-life analysis of nano-formulations. Shelf-life of the Linalool formulated silica nanoparticles were assessed by its dispersion stability and controlled release of linolool from formulation studies both before and after storage. The UV spectral analysis results conforms chemical stability as absorption profile of the Linalool was similar in both before and after storage up to 6 months. The dispersion stability was similar and there is no significant difference up to 3 months. However the duration of dispersion stability was reduced to 30 hours. The release profile of Linalool from formulation was altered on storage. Newly prepared Linalool nanoformulation showed well controlled released behaviour by releasing 40 % of the Linalool in to the solvent in 20 minutes and attains 100% only after15 minutes. The same formulation after storing for 3 months the controlled release property was lost as 79% of Linalool released in to solvent within one hour. Fig. 36: Effect of storage on controlled release profile of Linalool from its nano formulations Controlled release properties nano-formulations The release profile of the Linalool from nanoformulation was measured and presented in Fig. 37. HPLC Results suggest that Linalool showed 100 % release rate within 15 minutes whereas the silica formulated Linalool showed controlled release profile of Linalool. 39% Linalool was released within 20 minutes and the release rate reaches to 100% after a period of 14 hours.

37 89 Fig. 37: Release profiles of Linalool in normal state and from its nano-formulation in methanol Antifeedant activity of Linalool and its nano-formulations Linalool showed higher antifeedant activity against A. janata with ED 50 value of 0.01 µl cm -2 while more than 1 µl cm -2 of the same compound was required to get 50 % antifeedant activity against S. litura and H. armigera. nanoformulation improved the antifeedant activity Linalool against S. litura and H. armigera. However, same formulation failed to exhibit enhanced antifeedant activity against A. janata. In spite of that the antifeedant activity of Linalool against A. janata was found decrease upon formulating with silica nanoparticles. SINPs did not produce any toxicity or antifeedant activity to insects tested even at higher concentration tested (10 µg cm -2 ). The antifeedant activity of the formulations comprising different concentrations of Linalool (0.025, 0.05, and 0.1 % LLSI) were shown in Table 8. The antifeedant activity of Linalool formulations had increased with the increase of Linalool concentrations in the formulations against insects tested. None of the compounds and formulations showed mortality in treated insects.

38 90 Table.8: Efficiency of linalool and its nanosilica hybrids in inhibiting the feeding activity of S. litura, H. armigera and A. janata S. Compounds ED 50 (95% FL a ) µg cm -2 No S. litura H. armigera A. janata 1 Crude ( ) 57.34( ) 32.93( ) 2 Linalool (LL) ( ) 1.26( ) 0.010( ) % LLSI ( ) 0.22( ) 0.655( ) % LLSI ( ) 0.14( ) 0.614( ) % LLSI ( ) 0.09( ) 0.323( ) % LLSI ( ) 0.05( ) 0.249( ) 7 SINPs >10 >10 >10 a Fiducial limits 4.6. Characterization of Limonene /SiO2 nanoformulation(lisi) FTIR analysis of the nano-formulated Limonene The FTIR spectra of Limonene and its nano formulation with silica nanoparticles were explained through FTIR spectra in this Fig. 38. Fig. 38(d) shows the FTIR spectra of pure Limonene. The band at cm -1 indicates the present of O-H stretching. The peak at 2919 cm -1 indicates the presence of C-H stretching of Limonene in the formulation. Fig. 38 (b and c) shows the FTIR spectra of different formulations of Limonene-silica composites. The peak shift to lower wave number of OH group position in silica shows the presence of hydrogen bonding.

39 91 Fig. 38: FTIRspectra of (a) Native silica nanoparticles, nano-formulations of Limonene (b) 0.05 % LISI (c) 0.1%LISI and (d) pure Limonene TEM analysis nano-formulated Limonene TEM images of Limonene/Silica nanoparticles were shown in Fig. 39. The images evidently showed the agglutination in silica nanoparticles and the agglutination is less in the case the case of Limonene formulated silica nanoparticles. b C Fig. 39: TEM micrographs of (a) native silica nanoparticles (b and c) nanoformulated Limonene

40 Particle size and zeta potential analysis of nano-formulated Limonene Millipore water dispersed silica nanoparticles showed zeta potential value ( 28.4 mv) and hydrodynamic particle size (46.3nm). After formulation, Limonene silica nanoparticles did not show change the size of the silica particles (40.4nm), but exhibited higher negative value of zeta potential (-57.7 mv) than that of the native silica nanoparticles ( 28.4 mv). The change in the zeta potential resulted in stable colloid suspension of the Limonene formulated silica nanoparticles Dispersion stability of nano-formulated Limonene In compared with α-pinene and Linalool nanoformulations, Limonene formulation showed higher dispersion stability. The dispersion stability of nanoformulations was compared with silica nanoparticles and the results were presented in Fig. 40. Native nanosilica showed complete precipitation within 24 hours, while nanoformulations have a stable colloidal dispersion even after 4 days. This dispersion efficiency is due to the high negative zeta potential value. Fig. 40: Dispersion stability of the Limonene nanoformulation in comparison with native silica nanoparticles

41 Shelf-life analysis of nano-formulated Limonene. Dispersion stability and controlled release of Limonene from Limonene nanoformulation were studied and results were presented in Fig. 41 in of order to assess the shelf life of the Limonene nanoformulation. Store of Limonene formulation upto 2 months did not affected the dispersion stability. However longer period storage reduces the suspension stability and the formulation precipitated after 2 days. Controlled release profile of the Limonene formulation was less in compared with other formulations where newly prepared formulation release 55% Limonene with in 1 hour and rest within 8 hours of duration. This controlled released behaviour was observed only up to six months of storage. After storage of one month Limonene formulation lost benefit of controlled release property and releases 85% of the Limonene within a period of 1 hour. Fig. 41: Effect of storage on controlled release profile of Limonene from its nano formulations Controlled release properties of nano-formulated Limonene Limonene release from nanoformulation was measured by HPLC and results were presented in Fig. 42. In compared with other formulations, Limonene formulation showed less controlled release profile. 35% of Limonene was released in the first 1 hour, while the other 65% was released within 15 hours. Fig. 42: Release profiles of Limonene from its nano-formulation in methanol

42 Antifeedant activity of Limonene and its nano-formulation. The antifeedant activity of the Limonene was presented in the Table 9 in the form of antifeedant index. Limonene did not show any toxic property against tested larvae, S. litura and A. janata even at higher concentrations tested. However Limonene showed promising antifeedant activity against S. litura, H. armigera and A. janata with ED 50 values of 1.37, 1.57 and 0.97 respectively. On nanoformulation Limonene showed enhanced antifeedant activity against both the insects. However % formulation does not alter activity of the Limonene while the antifeedant activity constantly increased from and 0.1 %. The antifeedant activity of the Limonene enhanced along with the increase in concentration of the Limonene in formulation up to 0.1 % and further addition of the Limonene in the formulation does not alter the activity. 0.1 % formulation has exhibited 3 folds enhancement in the Limonene activity against all the tested insects. Table.9: Effect of Limonene and its silica nanoparticle conjugates on feeding behavior of S. litura, H. armigera and A. janata S. No Compounds ED 50 (95% FL a ) µg cm -2 S. litura H. armigera A. janata 1 Crude 51.74( ) 57.34( ) 32.93( ) 2 Limonene 1.37( ) 1.57( ) 0.97( ) % LISI 1.52( ) 1.36( ) 0.94( ) % LISI 0.86( ) 1.46( ) 0.73( ) % LISI 0.87( ) 1.24( ) 0.35( ) % LISI 0.49( ) 0.64( ) 0.36( ) 7 SINPs (µg cm -2 ) > 5 >5 > 5 a Fiducial limits

43 Characterization of Ocimene /SiO2 nanoformulation (OCSI) FTIR analysis of the nano-formulated ocimene Nanoformulations of Ocimene also showed week hydrogen bonding with SINPs. The FTIR spectra of Ocimene, wave number and their interaction with silica nanoparticles were described in this Fig. 43. Native silica nanoparticles showed peak at 950 and 1090 cm -1, due to the vibrations of Si-OH and Si-O-Si bands, respectively. Fig. 43 shows the FTIR spectra of pure Ocimene. The methyl group of Ocimene has involved the formation of the additive as the peak at cm -1 in the nanoformulations. This peak has intensified as the concentration of the Ocimene increased in the formulation as evident in the FTIR spectra of different Ocimene -silica hybrid compounds. The peak broadening and shifting to lower wave number of OH group position in silica shows the presence of hydrogen bonding between Ocimene and SINPS. Fig. 43: FTIRspectra of Native silica nanoparticles, nano-formulations of Ocimene in 0.025%OCSI, 0.05%OCSI, %OCSI, 0.1%OCSI and pure Ocimene

44 TEM analysis of nano-formulated Ocimene TEM images of Ocimene formulated silica nanoparticles and native silica particles were presented in Fig. 44. The result explains the agglomeration of the silica nanoparticles naturally while the formulated silica nanoparticles were less agglutinated. The images also showed that the nanoparticles were well dispersed. b C Fig. 44: TEM micrographs of (a) native silica nanoparticles (SINPs) (b and c) nanoformulated Ocimene Particle size and zeta potential analysis of the nano-formulated ocimene Native silica nanoparticles and Ocimene nanoformulations were studied for their zeta potential and particular size. Native silica nanoparticles in millipore showed a zeta potential value of 28.4 mv with 46.3nm hydrodynamic particle size. Ocimene reduced the size of the silica nanoparticles to 22.4nm on formulation this indicates the Ocimene hinders the agglomeration of silica nanoparticles. In the similar way Ocimene formulation also showed zeta potential with higher negative value (-44.6mV) indicating the higher dispersion stability.

45 Dispersion stability of the nano-formulated Ocimene The dispersion stability of Ocimene nanoformulation was compared with silica nanoparticles and the results were presented in Fig. 45. Ocimene showed higher suspension stability and the dispersion efficiency of the formulation is stands for 4 days. While, native silica nanoparticles were precipitated within 30 hours. Fig. 45: Dispersion stability of the Ocimene nanoformulation in comparison with native silica nanoparticles Shelf-life analysis of the nano-formulated Ocimene The physical stability of the formulations were analysed by studying its dispersion and controlled release of compounds from formulation both before and after storage. Higher dispersion stability of the Ocimene formulation was noted up to 6 months. Improved controlled release was exhibited by Ocimene nanoformulation more over 4 months storage did not affected the controlled release behaviour of the formulation. Freshly prepared Ocimene formulation showed highest controlled release profile, where 18% of the Ocimene was released in to the solvent in 1 hour and total release was shown only after 24 hours. Concurrently, storage of Ocimene formulation for four months does not affect the controlled release profile where 37% of Ocimene was released at fist one hour

46 98 and 100% of compound released only after 18 hours. Hence, formulation of compounds with SINPs helps to maintain their physical stability/shelf life. Fig. 46: Effect of storage on controlled release profile of Ocimene from its nano formulations Controlled release properties of the nano-formulated Ocimene The controlled release profile of Ocimene from its nanoformulation was studied by HPLC and the results were presented in Fig. 47. Native Ocimene showed 100 % release within 15 minutes. When formulated with silica nanoparticles, controlled release profile was observed where 18% of the Ocimene was released in to the solvent in 1 hour followed by sustained release of Ocimene and total release was shown only after 16 hours. Fig. 47: Release profiles of Ocimene in normal state and from its nano-formulation in methanol

47 Antifeedant activity of Ocimene and its nano-formulations The feeding deterrent activity of Ocimene and its nanoformulations was studied compared and presented in the Table 10. The antifeedant index ED 50 values of the OC and OCSI against S. litura, H. armigera and A. janata after 24 hr of feeding are presented in Table 10. Ocimene exhibited 50% feeding deterrent activity against S. litura, H. armigera and A. janata at 3.95, 4.12 and 5.89 µl cm² respectively. It is interesting to note that the activity of the formulations has drastically enhanced and showed highest activity at 0.1% formulation against both the insects tested , 0.05 and 0.075% formulations showed more or less similar antifeedant activity against S. litura and H. armigera. In the similar way all Ocimene formulations showed equal activity against A. janata. Antifeedant activity of Ocimene has increased more than 10 folds on nanoformulation. Table.10: Feeding activity of S. litura, H. armigera and A. janata on treatment with Oimene and its nanoconjugates. S. No Compounds ED 50 (95% FL a ) µg cm -2 S. litura H. armigera A. janata 1 Crude 51.74( ) 57.34( ) 32.93( ) 2 Ocimene 3.95( ) 4.12( ) 5.89( ) % OCSI 0.89( ) 1.02( ) 0.34( ) % OCSI 0.69( ) 0.63( ) 0.40( ) % OCSI 0.79( ) 0.79( ) 0.42( ) % OCSI 0.28( ) 0.30( ) 0.45( ) 7 SINPs > 5 >5 > 5 a Fiducial limits

48 Characterization of Trans-caryophyllene /SiO2 nanoformulation (TCSI) FTIR analysis of the nano-formulated Trans-caryophyllene The surface groups of SiO 2 nanoparticles before and after terpene adsorption was characterized by infrared spectroscopy Fig. 48 a represents the FTIR spectra of nonadsorbed and adsorbed SiO2 nanoparticles Fig. 48 (b e) represent Trans-caryophyllene nanoformulations and fig 48 f represents the pure TC. In Fig. 48 (b e), the peak at 2962 cm-1 corresponds to the long alkyl chain TC in the nano-formulation. The decrease in OH bending band (800 cm -1 ) and Si-O bond rocking band (467cm) was observed. A peak of hydroxyl group (between 3200 to 3600 cm1), The Si-OH band stretch (958) and asymmetric Si-O-Si stretching (1090) does not alter after adsorption. Fig. 48: FTIRspectra of (a) SINPs, nano-formulations of Trans-caryophyllene (b) (c) 0.05 (d) (e) 0.1% TCSI and (f) pure trans caryophyllene

49 TEM analysis of nano-formulated trans- caryophyllene The TEM images of nanoformulations were shown in the Fig.49. Agglomeration was observed in both the Trans-caryophyllene and unformulated silica nanoparticles. b C Fig. 49: TEM micrographs of (a) native silica nanoparticles (SINPs) (b and c) nanoformulated Trans-caryophyllene (TCSI) Particle size and zeta potential analysis of the nano-formulated Transcaryophyllene The particle size and zeta potential of the TCSI were measured and compared with that of native SINPs. Millipore water dispersed native SINPs shows the zeta potential value of 28.4 mv with 43.6nm hydrodynamic particle size. Among the formulations TCSI showed highest zeta potential negative value (-67.5mV) indicating the well dispersion stability. The TCSI also showed the smaller particle size (38.4nm) in comparison with native silica nanoparticles. The TCSI remained highly dispersed, whereas native SINPs were agglomerated.

50 Dispersion stability of the nano-formulated trans-caryophyllene The TCSI showed high dispersion stability in compared with all the other nanoformulations. Both the formulated and native silica nanoparticles initially dispersed equally in the methanol. After a period of 24 hours native silica nanoparticles precipitated whereas the TCSI showed stable suspension even after 7 days. Interaction of hydroxyl groups ( OH) of SI NPs with compounds played a vital role in reducing the agglutination of the inorganic nano-particles for a longer period. Fig. 50: Dispersion stability of the Trans-caryophyllene nanoformulation in comparison with native silica nanoparticles Shelf-life analysis of the nano-formulated trans-caryophyllene The shelf-life (dispersion stability and controlled release property) of the Transcaryophyllene formulated silica nanoparticles (TCSI) was determined both before and after storage. Among the formulations TCSI displayed elevated dispersion stability and freshly prepared TCSI reveals the longer dispersion capability up to 7 days. In a same way 6 months stored TCSI also displays dispersion stability for 5 days. Coming to controlled release property of the TCSI assorted results were identified where the formulation release higher amount (58 %) of TC in first one hour, at the same time it need longer time duration to release the left over compound and 100% of the compound

51 103 released only after 16 hours. Formulation conserve the similar profile until 6 months storage further storage leads to the loss of controlled release behaviour and released 50 % of the compound with in 1 hour Fig. 51: Effect of storage on controlled release profile of the Trans-caryophyllene from its nano formulations Controlled release properties of the nano-formulated trans-caryophyllene The controlled release pattern of the Trans-caryophyllene was studied and the results were presented in Fig.52 HPLC analysis showed interesting results, where 58% of the TC was released within 25 minutes. Remaining amount of TC was released very slowly and total TC released only after 15 hours. Fig. 52: Release profiles of Trans-caryophyllene in normal state and from its nanoformulation in methanol

52 Antifeedant activity of Trans-caryophyllene and its nano-formulations Trans-caryophyllene (TC) exhibited considerable antifeedant activity against S. litura, H. armigera (ED 50 >2µl cm -2 ) and A. janata (ED 50 value of 1.51 µl cm -2 ). TC after formulating with SINPS demonstrated an enhanced activity against S. litura, H. armigera and A. janata with the ED 50 value 0.53, 0.42 and 0.30µl cm -2 respectively, being less than four times that of non formulated trans-caryophyllene. These nanoformulated transcaryophyllene(tcsi) showed 100 percentage feeding deterrence at 1µl cm -2, in contrast to parent trans-caryophyllene, which failed to show absolute deterrence even at 4 µl cm -2. The results on the antifeedant activity of the other formulations having different doses of silica nanoparticles (0.025, 0.05, and 0.1% TCSI) were shown Table 11. The activity had increased linearly with the increase of Trans-caryophyllene concentration in the formulations. Table.11: Feeding responses of S. litura, H. armigera and A. janata against Transcaryophyllene and its nanoformulations S. No Compounds ED 50 (95% FL a ) µg cm -2 S. litura H. armigera A. janata 1 Crude 51.74( ) 57.34( ) 32.93( ) 2 Transcaryophyllene 2.59( ) 2.46( ) 1.51( ) % TCSI 1.01( ) 1.14( ) 0.73( ) % TCSI 0.86( ) 0.80( ) 0.58( ) % TCSI 0.63( ) 0.63( ) 0.40( ) % TCSI 0.53( ) 0.42( ) 0.30( ) 7 SINPs > 5 >5 > 5 a Fiducial limits

53 Characterization of nanosilica conjugated nerolidol FTIR analysis of the nano-formulated Nerolidol Interaction between Nerolidol and SINPs were confirmed by FTIR spectroscopy and the FTIR spectra of formulation and native SINPs were shown in Figs 53. The wave numbers of the Nerolidol and its conjugated SINPs functional groups are described in this section. FTIR spectra of pure SINPs showed the corresponding bands appear at 950 and 1090 cm - 1, due to the vibrations of Si-OH and Si-O-Si bands respectively. These bands are very intense and correspond to the formation of the SiO 2 network. The FTIR spectra of Nerolidol/silica formulation showed the peak at cm -1 indicates the presence of C-H stretching in Nerolidol compound. The peak shift to lower wave number of OH group position in silica shows the presence of hydrogen bonding between Nerolidol and SINPs. Fig. 53: FTIRspectra of (a) Native silica nanoparticles, (b) 25% Nerolidol/nanosilica formulation

54 TEM analysis of the nano-formulated Nerolidol TEM results in the form of images were presented in Fig. 54. The obvious agglomeration was present in the formulations, while the formulations exhibited much less agglutination in comparison with the native silica nanoparticles. This suggests a bonding either physical or chemical occurs between the terpene and SINPs, which reduces the surface free energy and controls the agglomeration. The above results indicate that adsorption of terpene has the advantage that besides being less agglutination it also has got the high dispersion stability. Fig. 54: TEM micrographs of (a) native silica nanoparticles (SINPs) (b) 25% Nerolidol/nanosilica formulation XRD analysis of the nano-formulated Nerolidol X-ray diffraction images showed a broad peak in the range of (2θ) and the broadness of peak was increased on formulating with Nerolidol confirm the formation of additive (Fig. 55). The peaks in spectrum, indicates an amorphous structure and lacks long range order. Primary building units of amorphous silica, SiO 4 tetrahedra, were connected to each other and do not exhibit a regular pattern like crystalline silica polymorphs quartz or tridymite.

55 107 Fig. 55: XRD spectra of silica nanoparticles, hybrids of Nerolidol and silica nanoparticles Particle size and zeta potential analysis of the nano-formulated Nerolidol The native silica nanoparticles dispersed in the millipore water, exhibited a hydrodynamic particle size (32.68 nm) and zeta potential value of mv. A slight change in the particle size was observed NSI with 54.3nm in compared with native silica naoparticles. The formulations exhibited higher zeta potential value (-39.7 mv) than that of SINPs (-28.8 mv) as shown in Fig. 4. Interaction of Nerolidol might cause easy deprotonated of hydroxide groups (Si OH terpene) to form Si O which enhances the surface more negatively charged Dispersion stability analysis of the nano-formulated Nerolidol Formulations displayed high dispersion stability in compared with native SINPs. Complete precipitation was observed in SINPs within 2 days; while NESI have a stable colloidal dispersion even after 7days. Interaction of hydroxyl groups (-OH) of SINPs with terpene played a vital role in reducing the agglutination of the silica nanoparticles for a longer period.

56 Antifeedant bioassay of nano-formulated Nerolidol The antifeedant activity of the formulations is directly proportional to the treated dosage as well as the concentration of terpenes present in the formulations. Antifeedant activity of the nano-formulations was compared with that of parent compounds and presented in Table. 12. I have calculated the exact amount of terpene present in the treated dose of formulations. The parent terpene, Nerolidol showed antifeedant activity against A. janata and S. litura, but the A. jantha was more susceptible to the treatment than S. litura with ED 50 values between 7.21 µl cm -2. After formulating these two compounds the activity has been increased (with ED 50 value between 3.16 µl cm -2 ). The quantity of the terpene in the formulations played a very important role in executing the bioactivity and the bioactivity increased with the increase in the terpene quantity (Table 12). Interestingly, the SINPs alone did not produce any toxicity or antifeedant activity to the test insects even at higher concentration tested (15 µg cm -2 ). Table. 12: Feeding deterrent activity of nerolidol and its nanosilica conjugates against major agricultural pests, S. litura, H. armigera and A. janata. S. No Compounds ED 50 (95% FL a ) µg cm -2 S. litura H. armigera A. janata 1 Crude 51.74( ) 57.34( ) 32.93( ) 2 Nerolidol 7.21 ( ) 7.34( ) 6.13 ( ) 3 10% NSI 4.12 ( ) 4.59( ) 3.68 ( ) 4 25% NSI 3.16 ( ) 3.27 ( ) 2.80 ( ) 5 SINPs > 5 >5 > 5 a Fiducial limits

57 Characterization carene nanohybrids FTIR analysis of the nano-formulated Carene FTIR spectroscopy demonstrated the interactions between silica nanoparticles and Carene as shown in Fig. 56. The wave numbers of the terpenes and their hybrid functional groups are described in this section. Fig. 50 represented the FTIR spectra of pure SINPs. The corresponding bands appear at 950 and 1090 cm -1, due to the vibrations of Si-OH and Si- O-Si bands respectively. These bands are very intense and correspond to the formation of the SiO 2 network. FTIR spectra of Carene formulation showed the band at cm - 1 indicates the present of O-H stretching. The peak at cm -1 indicates the present of C-H stretching in Carene compound. The chemical interactions between Carene molecules and silica surface sites are clearly demonstrated by hydrogen bonding. The peak shift to lower wave number of OH group position in silica shows the presence of hydrogen bonding. A strong absorption band in the range cm - 1 shows the asymmetric stretching vibrations of the Si-O-Si bonds of the silica component in all the Carene-silica hybrid formulations. The band at cm -1 show the Si-OH vibration, the intensity is decreased with increasing the Carene concentration. Fig. 56: FTIRspectra of (a) Native silica nanoparticles, (b) Carene/ nanosilica formulation.

58 TEM analysis of the nano-formulated Carene TEM results in the form of images were presented in Fig. 57. The obvious agglomeration was present in all the formulations, while the formulations exhibited much less agglutination in comparison with the native silica nanoparticles. This suggests a bonding either physical or chemical occurs between the terpene and SINPs, which reduces the surface free energy and controls the agglomeration. The above results indicate that adsorption of terpene has the advantage that besides being less agglutination it also has got the high dispersion stability. Fig. 57: TEM micrographs of (a) native silica nanoparticles (SINPs) (b) Carene/ nanosilica formulation XRD analysis of the nano-formulated Carene X-ray diffraction images showed a broad peak in the range of (2θ) and the broadness of peak was increased on formulating with Carene confirms the formation of additive (Fig.58). The peaks in spectrum, indicates an amorphous structure and lacks long range order. Primary building units of amorphous silica, SiO 4 tetrahedra, were connected to each other and do not exhibit a regular pattern like crystalline silica polymorphs quartz or tridymite.