BOTANICAL INSECTICIDE NANOEMULSION MADE BY Tephrosia vogelii J. D. HOOKER (LEGUMINOSAE) AND TRIAL TO CABAGE Crocidolomia pavonana F. (LEPIDOPTERA : CRAMBIDAE)

1. Students of Plant Protection, Post Graduated Program, Andalas University, Pascasarjana Building, Limau Manis Padang. 25163. Indonesia. 2. Agroecotechnology Department, Faculty of Agriculture, Universitas Andalas, Limau Manis Padang. 25163.Indonesia. 3. Pharmacy Department, Faculty of Pharmacy, Universitas Andalas, Limau Manis Padang. 25163.Indonesia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 30 November 2019 Final Accepted: 31 December 2019 Published: January 2020

Nanoemulsion is a material which consists of oil and water phases with particle size in the range of 20-200 nm. The purpose of this research is to obtain a botanical insecticide nanoemulsion made from T. vogelii which is active against C. pavonana. Nanoemulsion made using low energy method whch is spontaneous emulsification using a magnetic stirrer. This research produced 12 formulas. Four best formulas chosen for PSA (Particle Size Analizer) analysis. Four formulas was selected based on the toxicity of each formula. Nanoemulsion was analyzed for PSA using Malvern's Zetasizer Nano (ZN). The analysis results showed nanoemulsion A.1, A.2, B.1.a and B.1.b had particle sizes in a row of 134nm, 156nm, 1292nm, and 1286nm. Formulas A.1 and A.2 have fulfilled the nanoemulsion requirements and criteria, otherwise formulas B.1.a and B.1.b have not classified as nano-sized material, so they cannot be categorized as nanoemulsions. Formulas A.1, A.2, B.1.a and B.1.b have values is 1.09%, 3.15%, 0.45% and 2.26%.

Tephrosia vogelii extraction:
The powdered T. vogelii leaves were taken as much as 100 grams then put into an erlenmeyer flask and 1000 ml of ethyl acetate solvent were added. Comparative composition of ethyl acetate with T. vogelii powder refers to previous studies by Lina et al. (2014). Soaking process is left for 2 × 24 hours. Then the extracted liquid was filtered twice, first using a glass funnel (9 cm in diameter) on a regular filter paper on the second using a glass funnel (4 cm in diameter) based on whatman filter paper number 41 on the second screening. The filter is collected in a vaporizer flask, then evaporated with a rotary evaporator at a temperature of 50ºC and a pressure of 240 mbar. The solution obtained from evaporation is used to re-soak the extracts of the plant up to three times soaking. The extract was then stored in a refrigerator at 4ºC until used for testing.

Making nanoemulsions:
The process of making nanoemulsion involves the aqueous phase and the organic phase which is homogenized using a magnetic stirer. The organic phase consists of T. vogelii extract and solvent while the water phase consists of sterile aquades and tween 80. The first thing to do is the water phase is homogenized using a hot plate magnetic strirer at a speed of 2,500 rpm while stirring constantly using strirer seeds for 35 minutes. Then made an organic phase in erlemeyer, in the form of extract of T. vogeli dissolved with 96% ethanol.
After the water phase is finished homogenized using a magnetic stirer then an organic phase /oil phase drop is carried out into the water phase using a dropper pipette, the dropping process is carried out slowly (drop by drop). When dripping the organic phase/oil phase into the water phase, the water phase must still be stirred using a magnetic stirrer. After the organic phase has been dropped, the homogenisation process using magnetic strirer is continued for another 45 minutes.
To get the best nanoemulsion composition, nanoemulsion was made using 4 different steps/methods, namely: the first step, making nanoemulsion of Formula A and B. The composition of Formula A and B are shown in Tables 1  and 2. Second step, making nanoemulsion by manipulating composition refers to the formula A, the third step, making nanoemulsion by composition manipulation refers to formula B, and the fourth step making nanoemulsion refers to Putu's method, 2018. The first step nanoemulsion composition, namely making nanoemulsion Formula A and B are: Table 1:-Composition of Nanoemulsoin of Formula A and B.
To get better nanoemulsion, then optimization of formulas A and B. Optimization is done by making a new nanoemulsion but the composition of extracts, solvents, and emulsifiers is arranged as in (Table. 2), in order to produce more optimal nanoemulsions. Optimization is made in 8 of them, 4 formulas refer to formula A consisting of 10% oil phase (second step) and 4 formulas refer to formula B consisting of 20% oil phase (third step). The nanoemulsion optimization composition of the second step, which refers to formula A, is:  To get a more optimal nanoemulsion formula, Formula B.1 nanoemulsion was re-optimized using the Putu method, 2018 by means of active ingredients, emulsifiers and solvents homogenized using magnetic strirers simultaneously for 60 minutes. Furthermore, it is dropped with distilled water while homogenizing again for 35 minutes. Nanoemulsion, the fourth step, is made in 2 formulas including: The process of making nanoemulsion Formula B.1.a and B.1.b is expected to be able to produce nanoemulsion that is more optimal than the previous formula. Overall the composition of the oil phase used is 20%. The ratio of extract versus solvent in the oil phase is 1: 3 in each formula. Agristic emulsifiers were used 3% in Formula B.1.a and Tween 80 emulsifiers were used 3% in Formula B.1.b.

Nanoemulsion toxicity test:
Broccoli (4x4) leaf pieces are dipped one by one into nanoemulsion until they are evenly wet and then air dried, the leaves will be used as treatment feed, Control leaves are dipped in the appropriate control solution. The cuttings of the treatment feed and control leaves were placed separately in a petri dish (9 cm diameter) with a tissue. Petri dishes are placed upside down. A tissue mat is placed on the lid of the cup and the bottom of the cup is closed on the tissue. Thus, the lid and base of the dish are insulated with tissue so that the test larvae cannot escape from the cup.
The 15 instar larvae of C. pavonana newly replaced skin were put into each petri dish which contained treatment feed or control leaves, then the larvae were allowed to eat for 24 hours. Each treatment and control was repeated 5 times. After 24 hours the new treatment feed and control leaves were added again. Twenty-four hours later, the treated leaves were replaced with untreated leaves, dead larvae were counted and removed from the cup while the living ones were kept until the fourth instar larvae. Data from observations of toxicity test were analyzed by probit using POLO PC software then imago mortality data were analyzed by ANOVA using statistical software 8 and followed by Least Significant Different (LSD) test at 5% level.

Nanoemulsion evaluation:
Nanoemulsion evaluation is carried out to determine the nature of nanoemulsion that has been produced and obtain nanoemulsion that is stable, safe to use and efficient in application.
Particle Size Analizer Analysis: nanoemulsion samples were analyzed by PSA using the Zetasizer Nano ZS Malvern located in the Bogor Post Harvest Center. This tool has a sensitivity value of 3-10,000 nm and is able to measure particles or molecules which have a range of 0.15-10µ. Nanoemulsion samples were put into vials with screw caps then labeled and then samples were packed and sent to the Bogor Post Harvest Center. Jalan Tentara Pelajar Student No.12, Cibogor, Bogor Tengah District, Bogor City, West Java 16122.

Zeta Potential Nanoemulsion:
The zeta value of nanoemulsion potential was obtained together with the results of the PSA (Particle Size Analizer) analysis sent from the Insect Bioecology Laboratory, Faculty of Agriculture, Andalas University to the Bogor Post Harvest Center.

PH Test:
PH measurements are carried out using a PH meter that has been calibrated according to the calibration instructions. Measurements were made aimed at determining the PH value and acidity level of nanoemulsion so that the safety of 1048 the nanoemulsion is known to human skin and has no side effects or irritation. PH measurements are carried out at room temperature.

Manoemulsion toxicity test:
Twelve nanoemulsion formulas were tested preliminary, then four formulas with the highest mortality values were selected for further testing. Preliminary tests produce larval mortality as follows:   Table (Table 6). Further tests consisted of five levels of concentration based on preliminary test observations. The results of further tests of the four formulas are as follows:   conveyed other advantages of nanoparticle-sized insecticides, namely: accelerating the solubility of active substances, kinetic stable so as to prevent creaming, and aggregation during storage. With a very small size nanoemulsion is easier to penetrate plant tissue, so as to minimize the insecticide wasted at the time of application and allows quickly kill the target pest. [7] also believes that the small grain size makes it easy to spread. It is therefore clear that nanoemulsion is more effective and efficient compared to non-sized nanoparticle formulas.
The difference in the duration of larval development that survived due to nanoemulsion treatment compared to control was that nanoemulsion treatment delayed the development of larvae to the next instar for about 1 day compared to control. The occurrence of developmental disorders of larvae treated by nanoemulsion may be caused by active compounds contained in T. vogelii. The active compound contained in T. vogelii is rotenone. Rotenon is a toxic cellular respiration that is likely to influence the survival time of larvae that survive.
Probit regression analysis was used to determine the relationship of T. vogelii nanoemulsion concentration of Formula A. 68,00 ± 0,46 C 3,00 ± 0,79 5,88 ± 1,18 0,00 (Kontrol) 00,00 ± 0,00 D 2,96 ± 0,06 5,48 ± 0,10 1,29 ± 0,87 0,12 2,26 b = slope of the regression; SE = standard error Larvae mortality due to nanoemulsion A.1 concentration of 0.1 was 56.67%, whereas the results of larvae mortality due to the treatment of a single extract of T. vogelii instead of nanoemulsion were tested by [5]was 88.89%. If you only see the mortality value, nanoemulsion has a lower value than single extracts rather than nanoemulsion. It is not comparable, if the comparison is made according to LC 95 of each preparation, there is a saving of extract raw material in nanoemulsion compared to single extract rather than nanoemulsion. The LC 95 nanoemulsion A.1 value was 1.09 (Table. 11) while the LC 95 single extract of T. vogelii was not nanoemulsion by [5] is 0.16. Nanoemulsion A.1 has a LC 95 value of 1.09 which means that to kill larvae at 95% it takes nanoemulsion at 0.272ml, in 0.275ml (v⁄v) nanoemulsion there is a T. vogelii extract of 0.013 gram, while a single non-nanoemulsion extract to kill larvae is equal to 95% require an extract of 0.040 grams. There are three times the saving of extract raw material in nanoemulsion compared to non-nanoemulsion extract. This proves the theory of one of the properties of nanoemulsion which has a good penetration ability, thus saving the use of active substances.

Nanoemulsion evaluation:
This research resulted in 12 nanoemulsion formulas, 4 of the best formulas were chosen to be tested for PSA analysis, zeta potential, and pH test. The criteria for selecting the four best formulas were selected according to the toxicity of each formula in the preliminary test (Table. 6). Four formulas are:   (Table. 2). The oil phase is a homegene solution consisting of a combination of extract and 96% ethanol solvent in different ratios in each formula. In accordance with the writings of [9] in his scientific journal that is to produce nano emulsions, it is sufficient to use 3-10% oil phase, in contrast to macroemulsions which require high concentrations in the oil phase of around 20%. Furthermore [10] concluded that the concentration of the oil phase can affect the size distribution of nano emulsion particles. In addition to the oil phase, the composition and type of emulsifier also affect nanoemulsion particle size. [11] reports that in the process of nanoemulsion formation, emulsifiers play an important role in the process of solving droplets / particle size. Monodispersion system shows the distribution of particle size which tends to be narrow which indicates that the monodispersion system has a good uniformity level (homogeneous). A Polydispersity index (PDI) of greater than 0.7 indicates a very broad particle size distribution indicating that it does not have uniformity in other words, that is, heterogeneous.
Based on the overall potential zeta value of the resulting nanoemulsion charge the negative value (-) shows that the formula has a globul surface which is negatively charged (-) as well and is relatively stable because it is less than (-) 30mV. [13] confirms the negative potential zeta value (-) shows the surface charge of globally negatively charged (-) as well, and vice versa the positive zeta value (+) indicates the globul surface charge which is positively charged (+) as well. Added by [14] zeta potential values above (+) 30 mV or below (-) 30 mV indicate a stable colloidal system.
PH test results showed overall nanoemulsion was acidic. This means that the four selected formulas are relatively unsafe to use by farmers, because PH that is too acidic can irritate the skin while PH that is too alkaline can make skin flaky. [15] argue that the PH that is suitable for human skin is 4.6 -6.5. Besides that [16] believes that good pH tolerance used for skin is 5.5 -6.5. Pesticides are generally acidic. Nanoemulsion produced in this research was acidic, presumably due to autocidation that occurred in tween 80 and agristic which caused a decrease in PH value, according to the control solution which also had a low pH and was acidic. [17] expressed a decrease in PH value of nanoemulsion preparations usually caused by oxidation in the presence of oxygen from the atmosphere and light, and the presence of microorganisms. The PH value can increase due to the release of hydroxyl ions slowly. Temperature is one of the factors that can affect the PH value of nanoemulsion. [18] explained the water PH used at the time of the application affected the effectiveness of pesticide spraying. Water PH that has acidic properties is good for pesticide applications because it will extend the duration of effective use of pesticides. Pesticides will be more effectively applied if they are acidic.