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Oct 15, 2015 - Abstract: The chemical elucidation of rotenone from Derris elliptica ... Construction of ternary phase diagrams and selection of formulation ..... J. Eccleston, Microemulsions, in: Swarbrick, J., Boylan, J.C., (Eds.), Encyclopedia of.

JCBPS; Section A; August 2015 – October 2015, Vol. 5, No. 4; 3989-3997.

E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online atwww.jcbsc.org

Section A: Chemical Sciences CODEN (USA): JCBPAT

Research Article

Preparation, Characterization and Toxicity of Nano-Emulsion Formulations of Rotenone Extract of Derris Elliptica Norhayu Asib*1, Dzolkhifli Omar1, Rita Muhamad Awang1,Nur Ashikin Psyquay Abdullah2 1

Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia. 2

Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia. Received: 9 June 2015; Revised: 15October 2015; Accepted: 19 October 2015

Abstract: The chemical elucidation of rotenone from Derris elliptica was conducted using Liquid Chromatography Mass Spectrometer. Rotenone and deguelin were found from the elucidation of each accession at retention time of 5.7 and 6.6 minute, respectively. Both have similar mass of 395.1489 with the empirical formula of C23H21O6.The emulsion formulations of rotenone extracts of Derris elliptica KJ579429 (Tuba Merah) were prepared using ternary phase diagrams. The formulations utilised non-ionic surfactant of alkyl polyglucoside and organosillicone, dimethylamide as a carrier and water with rotenone extract as active ingredient. Five formulations were obtained from the isotropic region of the diagrams. All formulations showed a particle size of less than 100nm indicating the prepared formulations were nano-emulsion formulation. The surface tension value of below 30 mN/m. The formulations were also stable at 25°C for two months and the agglomeration was observed for all formulations and stable at temperature 54°C except for formulation F5.The toxicity of rotenone was obtained from Derris elliptica evaluated through dose mortality bioassays against DBM. The leaf-dip technique was used to obtain dose-mortality response of rotenone in the nano-emulsion formulations. All rotenone formulations gave 100% mortality 72 hours after treatment at concentration of 273 µg/mL. Keywords: Rotenone, surfactant, carrier, Derris elliptica.

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INTRODUCTION Commercial pesticides are formulated before use by mixing the active ingredients with inert materials for ease of handling, longer shelf life, safe from undesirable side effects and effectiveness1-4. Formulation of natural products is gaining greater attention due to their lower toxicity to non- target organisms, biodegradability, safe, renewable sources and economical compared to synthetic organic pesticides. The emulsion is the most common commercial formulation and recently nano-emulsion has been introduced. Nano-emulsion comprises a liquid droplet dispersed in another immiscible liquid with droplet size ranging below 400 nm. It composes of an oil phase, surfactant, and an aqueous phase in appropriate ratios5. The nano-emulsion has a very good storage stability over a wide range of temperature (-10°C to 55°C). It has excellent long term dilution properties in water since it is water based and highly capable of solubilizing lipophilic and hydrophilic compounds leading to the use of minimal inert ingredients. The natural product of interest in this study is rotenone. Rotenone is normally extracted from Derris species in South East Asia and in South America. Thus, the study was conducted to prepare and characterize the emulsion formulations and evaluate the biological performance of the formulations against Plutella xylostella, an important pest of cruciferous. MATERIALS AND METHODS Two alkylpolyglycoside surfactants, Agnique® MBL510H and Agnique® MBL530H and palm oil based-carrier, dimethylamide (Agnique® AMD810) were provided by Cognis Oleochemical (M) Sdn. Bhd. The other organosilicone surfactant, Silwet 408, was provided by KC Chemical Sdn. Bhd. Water from the ultra - purification system (ElgaLabwater, 18mΩ) was used for the preparation of nano-emulsion. A rotenone was extracted from Derris elliptica (KJ579429, TubaMerah) obtained from Jerangau,Terengganuis using acetone. Structural elucidation of rotenone from Derris elliptica: The elucidation for the rotenone from the extract was conducted using the Analytical Liquid Mass Spectrometer (BrukermicroTOF-QII) with electrospray ionization (ESI) and positive mode. The HPLC was Model Dionex U3000 and Acclaim® RSLC 120 C18 (2.1mm x 50.0mm, 2.2 mm).The mobile phase was a mixture of methanol: water at ratio of 6:4 (v/v) with a flow rate of 0.3 mL/min at 10 minutes. The solution was degassed in an ultrasonic bath and filtered under vacuum through a membrane (Milipore, PVDF). The isocratic program consisted of 100% mobile phase and a column temperature of 25°C. The signal was acquired and processed using the Microtof Control Software. Construction of ternary phase diagrams and selection of formulation composition: The studies were conducted using three-component phase diagram system6 by aqueous titration method7,8. Mixture of MBL530B and MBL510H was prepared at ratios (w/w) of 50:50 and Silwet 408. The ratio surfactant and oil in the experimental mixture was prepared at ratios (w/w) of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. Appropriate amounts of surfactant and oil according to the ratio were for one minute to attain equilibrium. Water was added by titrating into the oil and surfactant. Mixture at which transition occurred was used to determine the phase domains (isotropic, monophasic, biphasic, or triphasic). Each sample was assessed visually for spontaneous emulsification on the basis of clarity, stability and transparency. The results were used to plot ternary phase diagrams Chemix version 3.5 phase diagram plotter (UK). The percentage ratio of surfactant, oil, and water obtained was marked on the three component phase diagram. Those points which joined together indicated the isotropic area of nano-emulsion in the phase diagram systems. All phase diagram systems were described according their surfactant phase with Agnique AMD 810 as its oil phase and water as its aqueous phase. Samples in the tubes were then centrifuged (3500 rpm, 15 minutes) and observed for their emulsion system stability to retain transparent one-phase appearance at room temperature9. From 3990

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each phase diagram constructed, several points with different oil/surfactant ratios within the isotropic region were selected to be incorporated withthe rotenone extract. Formulations that miscible with rotenone extract and remain one-phase appearance was selected. The experiment on all phase diagram systems were conducted in the laboratory at 26 ± 1°C and 80 ± 15 RH. Characterization of the emulsion formulation Stability test: Selected formulations were centrifuged at 3500 rpm for 30 minutes and kept at room temperature for four weeks8. The formulations were then evaluated for the ability to retain a transparent one-phase appearance after four weeks in order to indicate the presence of a nanoemulsion9. Thermostability test: The selected formulations were stored at room temperature (25°C) for three months and kept at 54°C for 14 days, prescribed by the Food and Agricultural Organization (FAO) as a standard evaluation of agrochemical products to show stability in the tropical climate10. The change in physical appearance of samples was visually observed. Particle size distribution and particle aging: The formulation was dispersed in deionized water in volumetric flask and gently mixed by inverting the flask. One ml of sample was filled into 1 cm² cuvette and put into a thermo stated chamber of Nanophox equipment. The inside dimensions size of cuvette were 10 mm x 10 mm x 44.75 mm with screw caps. Samples were handled carefully to ensure that no bubbles were inside the vial that might interrupt the measurements. The sample cuvette was inserted manually into a basin, keeping the samples at a fixed pre-defined temperature. The position of the sample cuvette was selected by software in order to optimize the count rate. Samples were then observed by PCS (Photon Correlation Spectroscopy) technique. Each formulation sample was investigated directly within 12 - 14 minutes after preparation. The experiments were replicated at least five times for each sample. Particle size growing rates of the formulation were measured on an interval of 15 days for a period of 60 days. Surface tension analysis: Surface tension was measured by using the Du Nuoy ring method through Kruss K6 tensionmeter (Kruss, UK) equipped with a platinum plate. Prior to measurements, calibration was conducted using deionized water with a surface tension of 70.2 mN/m. Sufficient time was allowed to attain equilibrium until no significant changes were observed. The ring was cleaned with methanol, acetone and finally flamed prior to the next measurement. Each run was repeated three times. All measurements were carried out at 25°C Toxicity of the nanoemulsion formulation against Plutella xylostella: Bioassay on the ten larvae DBM was done using the leaf-dip technique method11, 12. Mustard leaf discs (5.0 cm, diameter) were immersed with gentle agitation in the treatment solution for 10 seconds, drained on filter paper and allowed to dry on a corrugated sheet of aluminium foil for approximately 1 hour in the ambient (26 ± 2°C, 80 ± 15% RH) laboratory environment. The treated leaf disc was then transferred to individual glass Petri dishes (5.5 cm, diameter) and sealed with parafilm. The control leaf discs were only immersed in deionized water and ten early third instar larvae (average length, 3.0 mm/3 days after hatching) were placed on each leaf disc. Six concentrations with ten replicates per concentration were tested for each insecticide treatment. The control treatment was deionised water. Larvae were maintained under laboratory conditions at 26 ± 2°C. Assessment of mortality was recorded at 72 hours after treatment. Larvae that failed to respond to gentle prodding with a fine sable brush were considered as dead.

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RESULTS AND DISCUSSION Structural elucidation of rotenone: The original chromatographic system (4.6x 150 mm) wasadapted for a smaller column (2.1x50 mm) by reducing the flow rate from 1 mL/min to 0.3 mL/min. At 60% methanol and 40% deionized water, the larger single peak was separated into two peaks at retention times 5.6 and 6.5 min as shown in Figure 1, and both showed the same m/z of 395.1489. Norman13 reported that there are many compounds in this class and that two compounds in particular share the same formula and mass of 395.1489, namely rotenone and deguelin. Thus, an MS/MS experiment may be useful to differentiate the two. Additional compounds were detected at retention times 4.1 and 4.7 minute. Both peaks gave the identical mass spectra in which the major component was at m/z 433.1212 and the compound at m/z 393.1294 was the minor component. From the SmartFormula, the compound at m/z 393.1294 corresponded to empirical formula C23H21O6 ([M+H] +). Additional compounds were detected at retention times 2.4 and 3.0 min. Both peaks gave different mass spectra. From the SmartFormula, the compounds at m/z 375, 353, 391 and 369 corresponded to empirical formula C23H21O6, C21H21O5, C20H23O8 and C22H25O5 ([M+H] +), respectively.

Fig. 1: MS spectra of compounds Derris elliptica Ternary phase diagrams of nano-emulsion systems: Two ternary phase diagram were constructed (Figures 2 and3) and all phase diagrams showed oneof three phase regions in thenano-emulsion system. Phase diagram of 50% Agnique MBL 510H :50% Agnique MBL 530B /Agnique AMD 810 / water system (39% isotropic region) showed one of three phase regions and phase diagram of Silwet 408 / Agnique AMD 810 / water system showed 33% isotropic region. Five points were selected from their isotropic regions as nano-emulsion formulations and coded as shown in Table 1. All formulations comprise of 10-30% surfactant, 10-30% oil, and 40-70% water after addition to the active ingredient of rotenone extract. Ternary phase diagrams were constructed between the very important components of nano-emulsion systems comprising alkyl polyglucoside (APG) surfactants (Agnique MBL 510H, Agnique MBL 530B and Silwet 408) that greatly affected the solubility and distribution of active ingredient, dimethylamide carrier oil (Agnique AMD 810) which allowed the effective dispersion of the formulations in water. The existence of wider homogenous isotropic nano-emulsion regions in the 3992

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systems prepared by the mixture of two surfactants (Agnique MBL 510H and Agnique MBL 530B) with water suggested a higher efficiency of the surfactant/ oil mixture as compared to one surfactant used alone. Table 1: Composition of rotenone nano-emulsion formulation No.

Compounds

Code name

1

Ag. MBL 530B/Ag. MBL 510H/Ag. AMD 810/H2O (50:50)

F1

2

Ag. MBL 530B/Ag. MBL 510H/Ag. AMD 810/H2O (50:50)

F2

3

Ag. MBL 530B/Ag. MBL 510H/Ag. AMD 810/H2O (50:50)

F3

4

Ag. MBL 530B/Ag. MBL 510H/Ag. AMD 810/H2O (50:50)

F4

5

Silwet 408/Ag. AMD 810/H2O

F5

Fig. 2: Phase diagram of Silwet 408/ Agnique AMD 810/ Water system

Fig. 3: Phase diagram of 50 Agnique MBL 510H: 50 Agnique MBL 530B/ Agnique AMD 810/ Water system 3993

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Silwet 408 showed a poor miscibility when mixed with water where 3-phase regions were observed in the system that caused narrow isotropic regions. These showed that the different inclination of homogeneous regions in phase diagrams was stabilized by the surfactants and was highly influenced by the properties of the surfactants14. APG is a natural, renewal, plant derived surfactant mixture that allowed better solubilization of hydrophobic compounds in nano-emulsions, and this surfactant has shown outstanding biodegradability, excellent dermatological properties, and temperature independence15. In this study, non-ionic surfactant was used due to their compatibility, relatively low toxicity and minimal environment impact. In W/O emulsion, surfactants play a significant role reducing both surface and interfacial tension by accumulating at the interface of the formulations. Characterization of the emulsion formulations: Table 2 shows the characterization of the rotenone formulations. Stability of selected nano-emulsions were observed after centrifugation at ambient temperature without phase separation, creaming and colloid formation at room temperature. The formulations were stable at 25°C for two months. Agglomeration was observed for all formulations. Exposure at 54°C for two weeks, except for the F5, otherformulations were stable. The F5was obtained at the region of Silwet 408. This indicated that the formulations with higher concentration of surfactants relative to oil, water and rotenone extract were more resistant to high temperature. By increasing the temperature, the kinetic motion of molecules was increased to force the surfactants to be loosely adsorbed on the oil-water interface, and thus increasing the probability of collision and coalescence, therefore destabilizing the emulsions system16. The lowest particle size of nano-emulsion was most desirable to be the best formulation. Particle size of selected formulations ranged from 73.40 to 97.49 nm (Table 2). The smallest particle size was detected in F3 (73.40 nm). A good oil in water (o/w) nano-emulsion formulations were considered as being able to hold and load the active ingredient in the nano-emulsion system while having the least effective quantity of inert ingredient with high amount of water. A higher solubility could increase the loading efficiency and could scale up to an economically feasible level. Eccleston, reported, nanoemulsions are the dispersions of oil and water stabilized by an interracial film of surfactant molecules having the droplet size less than 100 nm17. Table 2: Characterization of the emulsion formulations of rotenone Formulation

Particle size (nm)

Surface tension (mN/m)

Stability

Thermostability

F1

81.69

29.00





F2

80.54

29.00





F3

73.40

29.00





F4

95.47

30.27





F5

97.49

27.67



X

At room temperature, the surface tension of all selected formulations was significantly lower as compared to the control (water) with a surface tension of 72 mN/m. F4 was the highest surface tension 30.27 mN/m compared to other formulations. On the other hand, F1 to F3 gave almost same reading at 29 mN/m. The best formulation with the lowest surface tension was observed in F5 (27.67 mN/m). Toxicity against the third instar DBM larvae: The contact and stomach action of the rotenone formulations were evaluated by bioassay in the laboratory using the leaf-dipping technique. Early third instar larvae of the DBM treated with nano-emulsion formulations of rotenone and 3994

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Saphyr(positive control) showed a significant difference in mortality rate at 72 hours after treatment. The LC50 values of the 72 hour mortality after treatment for each formulation are shown in Table 3. Table 3: The LC50 values of the nano-emulsion formulations following leaf-dip bioassay against the third instar DBM larvae No.

Formulation

LC50

95% Fudicial limit Lower

Upper

Slope ± SE

1

Saphyr (+ve control)

82.68

75.25

100.29

2.05 ± 0.13

2

F1

35.54

18.46

44.23

1.13 ± 0.35

3

F2

10.30

5.15

20.32

2.38 ± 0.51

4

F3

0.02

< 0.02

1.25

1.03 ± 0.72

5

F4

61.01

25.52

80.34

0.55 ± 0.25

6

F5

66.39

47.38

80.32

2.47 ± 0.89

These results indicated the potential of plant extracts, formulation, and use of botanical plants in crop protection in line with the sustainable pest management. Maddalena et al. Suggested that the composition of commercial rotenone formulations might vary in rotenoid18 contents depending on the extracts used to organize them. The efficacy of all formulations tested also proved to be different. From the result obtained, composition of surfactant and carrier played an important role in DBM mortality. The highest ratio of mixed surfactant (Agnique MBL 510H: Agnique MBL 530B) and the highest carrier (Agnique AMD 810) gave the highest mortality of DBM larvae and lowest value of F3(0.02 µg/mL). Saphyr was a commercial emulsifiable concentrate (EC) formulation whereas the other formulations tested were nano-emulsion formulations. In this study, we focused on DBM third instar larvae because, due to their high abundance and feeding commitment, the immature stages are responsible for a large part of the direct feeding damage on plants 19. In addition, the feeding damage of the larvae was reduced in a no-choice situation20. Charleston et al.21 provided evidence of a beneficial interaction between botanical extracts and the biological control agents of DBM in the field, which showed that an effectiveness of rotenone formulation was successful in the field trial. CONCLUSION In this study, the formulation nano - emulsion was performed with greener and safer alternative solvents in order to replace harmful solvents used in existing plant protection formulations. Based on determined phase diagrams, thirteen nano-emulsions with selected surfactant/oil weight percent (F1, F2, F3, F4, and F5) were formulated with 29 to 30 mN/m surface tension values. F3 gave smallest particles size measurement at 73.40 nm. All formulations were stable after incubation up to twelve months of storage. These rotenone formulations are affordable to low-income farmers, and, furthermore, they have a non-residue effect in the long term and are considered to be safe. These plant based insecticide formulations has a bright future and potential for use in agriculture, especially with the dramatic need for environmentally friendly pesticides. The advantage of formulating a nanoemulsion is their ability to incorporate hydrophobic drugs into the oil phase, thereby enhancing their solubility, which could help in the enhancement of the oral bioavailability of the poorly soluble drugs22. Furthermore, nano-emulsions also can make the plasma concentration profiles and bioavailability of drugs more reproducible23, 24,25. Based on the present finding, all nano-emulsion formulations with the active ingredient (rotenone) obtained were stable at room temperature for up to two years of storage. 3995

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* Corresponding author: Norhayu Asib, Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia.

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