Optimization of N-hexane and Methanol Solvents on Total Phenol Levels of Gnetum gnemon Peel Extract in the Production of Biopesticide

. The excessive use of chemical pesticides has led to pest resistance and the accumulation of residues. Pest resistance occurs when pests become immune to pesticides and require higher doses or stronger pesticides for control. This study aims to identify the optimal solvent for dissolving secondary metabolite compounds that selectively and safely kill or inhibit the growth of plant pests. The biopesticide used is derived from melinjo bark extract through the process of soxhlet extraction using methanol and N-hexane solvents. The study employed a two-factor factorial design with three variables. The findings revealed that methanol solvent was more effective in dissolving phenolic compounds, resulting in a phenol content of 34.23 mg/equivalent Gallic Acid. The optimal solvent ratio was achieved at a ratio of 600 ml. The highest yield, reaching 56.27%, was obtained when using methanol as the solvent. Moreover, the pest mortality rate was found to be 100% at the specified concentration.


. Introduction
One of the factors that cause a type of plant to be used as a biopesticide is the content of secondary metabolites present in plants.Secondary metabolites are chemical substances produced in secondary metabolic processes.Secondary metabolites have more functions than just the self-defense system in plants.Plants produce secondary metabolites when stressed.When they feel threatened, competition between other plant species, depletion of food ingredients, and long dry seasons to UV light exposure can play a role in the formation of secondary metabolites through the long evolution of plant biochemical systems [1].Secondary metabolites produced by plants are classified into three major groups: alkaloids, terpenoids, and phenols.The benefits of phenol include: The flavonoid compounds in phenol can act as stomach poisoning or stomach poison, causing a decrease in nerve function and damage to the respiratory system of insects, as an antioxidant, anti-inflammatory, anticarcinogenic, and other biological properties [2].Antioxidants are substances that can inhibit the chain reaction of atoms or atomic groups of a radical compound [3].Many indigenous fruits in Indonesia can be potential sources of phenol, inclusing Gnetum Gneom [4].Gnetum gnemon L is one of the plants found in Indonesia and can grow well in almost all parts of Indonesia.Gnetum gnemon L bark has secondary metabolites that may be used as vegetable pesticides, namely phenolic compounds, flavonoids, tannins, saponins, and terpenoids.In a study conducted by [5] the phenolic content was 18.64 mg GAE/g extract, flavonoid content 15.83 mg QE/g extract, lycopene 248.9 mg/kg, beta carotene 25.96 mg/100g, and vitamin C 53.35 mg/100g.Based on the calculation of the percentage of free radical scavenging shows that the skin of red Gnetum gnemon L has an antioxidant activity of 61.0875%.Meanwhile, in the research [6] conducted entitled "The Effect of Long melinjo Skin Extraction (Gnetum gnemon L.) Assisted by Ultrasonic Waves on Yield, Phenolics, Flavonoids, Tannins, and Antioxidant Activities".The best time for extracting the skin of red melinjo with ultrasonic waves is 30 minutes (P3) with a yield of 17.86%, phenolic 2.74 mg GAE/g, flavonoid 2.30 mg QE/g, tannin 2.92 mg TAE/g, and antioxidant activity of 74.33%.The processing of melinjo skin as a raw material for biopesticides is a novelty in this research.In the skin of melinjo there are secondary metabolites, namely phenolic compounds, flavonoids, saponins, and terpenoids, these compounds act as bioactive, the bioactive compounds contained in plants can be cytotoxic, the heating treatment using the soxhletation method is expected to increase the solvent ability so that it can extract these compounds.compounds that are insoluble at room temperature (maceration) [7].Biopesticides made from melinjo skin are expected to be an alternative to the use of synthetic pesticides [8].To find out the potential of melinjo skin using the soxhlet method, further research is needed on the use of melinjo peel which has the potential to be developed into biopesticides.In this study using the soxhlet extraction method using two types of solvents, namely n-hexane and methanol which are expected to be able to extract phenolic compounds in melinjo skin.

. Materials and Methods
The methods of experiment in this research can be explain with below points.

Experimental Stage
This research was conducted in several consecutive stages to achieve the objectives of the study.The research was carried out in a laboratory and consisted of 4 stages: Stage I: Preparation of melinjo skin extract through peeling, crushing, and drying processes, aiming to facilitate the extraction of melinjo.Stage II: Extraction process using the soxhlet method with predetermined variables and conditions.Stage III: Separation of the extraction solvent through distillation to obtain a solvent-free solution.

Tools and Materials
The equipment used in this research includes Measuring cylinders, 3 sizes (25ml, 50ml, and 100ml), Extractor tubes, Condenser, Thermometer, Beaker, Hot plate, Test tube, pipets, Oven, Blender, Static clamp, Three-neck flask, Digital balance, Watch glass, Filter paper, Stopwatch, Aluminum foil, Basin, Baking tray, Knife, Spray bottle, 100-mesh sieve.All of these equipment items play important roles in various laboratory experiments and processes to ensure the success of the research or analysis being conducted.The materials used in this research are Melinjo fruit obtained directly from the Batang region, Pekalongan, Central Java, Indonesia.Other materials used include N-hexane solvent, Methanol solvent, Sodium chloride (NaCl), Ethanol solvent, Sodium hydroxide (NaOH), Hydrochloric acid (HCl), Chloroform (CHCl3), Sulfuric acid (H2SO4), Magnesium powder, Acetic acid (CH3COOH), Mayer's reagent.  .2, it is known that the methanol solvent yielded the highest average yield which was significantly different from the treatment with the n-hexane solvent, which was 56.27%, while the lowest yield was obtained from the n hexane solvent treatment of 31.05%.The difference in yield results is based on differences in the type of solvent, solvent ratio, and the variable extraction time used.In Figure 2 The yield of Melinjo Bark Extract can be seen the differences between the discovery variables and the extraction time used respectively.To find out the most influential variables, a Normal Probability Yield table was made which can be seen in Table .4 .The results based on the phytochemical screening contained in Table .3. it is known that melinjo peel contains flavonoids, alkaloids, sterols, triterpenoids, tannins, phenols, and coumarins.The test results on the flavonoid content showed positive results which were indicated by the formation of red foam.It means, melinjo also contain natural antioxidants which are beneficial for health, especially against degenerative disease [9].The addition of concentrated HCL in the test was used to hydrolyse flavonoids into aglycones so that a red color was formed in the sample.The results of phytochemical screening of alkaloid content using Dragendroff's reagent on melinjo peel extract showed positive results.This indicates the formation of alkaloid deposits that occur due to the covalent presence of nitrogen with K+ which is a metal ion.Meanwhile, the Mayer reagent did not detect any alkaloids in the sample.The test results on sterol+ triterpenoids showed positive results which were indicated by the formation of a violet-red ring using CHCl3 and H2SO4 reagents on melinjo peel extract.This event indicates an oxidation reaction in the sterol group of compounds [10].Phytochemical examination of the tannin content using FeCl3 1% added to melinjo peel extract showed a positive test result, which resulted in a blue-black color.The tannins contained in the extract react with Fe 3+ ions from the reagents to form complex compounds [11] .The results of phytochemical screening of phenol content showed positive results which were indicated by the formation of a green color due to the reaction with FeCl3.The test results for the coumarin content in the sample are indicated by positive signs.The results of this study are in line with previous research which states that melinjo peel extract contains phenols, flavonoids, and tannins, triterpenoids, and tannins [7].In another study, it was stated that saliara extract also contains saponins [12].Differences in the results of phytochemical screening, namely the presence of alkaloids, sterols, and coumarins in melinjo peel can be caused by differences in the solutions used in dissolving melinjo peel, differences in testing methods, and differences in the environment where melinjo.

Most Influential Variable
The initial experiment, calculations were made to determine the most influential variable on total phenol content.For the most influential variables, the Experimental Normal Probability Table is made in Table 5. ) the type of solvent-solvent ratio interaction.So it can be said that the most influential variable on the extraction of melinjo peel is the type of solvent-ratio interaction variable.After the calculation, then the optimization calculation obtained equation: Y= 0.1661x-0.7739 From the optimization equation above, it can be seen that the interaction in the types of solvent solutions has a positive (+) effect on the total phenol content produced.This is because the use of polar methanol content can dissolve phenol better so that the total phenol content is higher, while the interaction ratio of solvent to phenol the greater the number of solvent ratios used causes the total phenol produced to be higher and increase.there is a significant effect between parameters, the greater the amount of solvent used, the greater the ability to dissolve the compounds contained in the material so that more compounds can be extracted by the solvent.This is following the results of Amalia's research (2019), phenolic compounds increased with a large ratio, so the more contact between powder particles and phenolic compounds the more extracted and the higher the phenol content.

Optimum Operating Conditions
From the results of the Normal Probability calculation, it can be seen that the most influential variables are the type of solvent and the ratio of the solvent.Thus, the methanol solvent variables were selected with the solvent ratio used as the independent variable in the optimization.While the dependent variable used is the temperature according to the boiling point of methanol 64.5°C and the extraction time variable is 3 hours.The solvent variables used were 400 ml, 500 ml, 600 ml, 700 ml and 800 ml.The value of this positive (+) effect is used as a guide in the selection variable in optimization.The positive effect value (+) means that the solvent variable used is a variable that is in the upper middle range.From Fig. 3.The relationship between Solvent Volume and Total Phenol Concentration shows that for sample 3, the solvent ratio of 600 ml has an inflection point on the graph.The point is the optimum point of the solvent used in the optimization extraction.the solvent ratio will increase the extraction yield of phenol increase.In the extraction process, there is diffusion of solvent molecules (methanol) into the pores of the melinjo skin so that there is contact between the methanol molecules and the melinjo skin molecules.This intermolecular contact causes the phenol to dissolve in the methanol solvent.
If the ratio of the solvent ratio is greater, the amount of phenol dissolved in methanol will be more and more.However, after looking at the 600 ml solvent ratio, it did not increase the phenol yield again (constant yield) because the extraction process had already achieved results.Therefore, it can be said that the optimum solvent ratio was achieved at a solvent ratio of 600 ml with a phenol content of 34.23 Mg/GAE.After the optimum feed solvent ratio is reached, the total phenol content produced is constant.In line with research on the extraction of the skin of Durio zibethinus Murr [5] and leaves of Averrhoa bilimbi L [13].The larger volume of solvent allows more contact of the activating bubble to break up the cells so that the number of phenolic increases [14.However, use that exceeds the optimum limit can cause dissolution by the dispersing phase in the form of an extract that is too dilute and the concentration of phenol is reduced [15].

Biopesticide Test Against Pests In Vitro
The results of the optimization of melinjo peel extraction were then applied to crickets to be tested for mortality or mortality rates by varying the concentration of biopesticides, namely concentrations (0%, 25%, 50%, 75%, and 100%) each of which was dissolved in 25 ml of air.Then 3 crickets were put into a petri dish and sprayed 3 cc, the variables were observed for 1 hour, 2 hours and 3 hours, each done 3 times.After observing the results in Table  x 100 %.The average percentage of crickets mortality at a concentration of 25% was 22.22%; at a concentration of 50% the mortality of crickets was 37.03%; at a concentration of 75%, the mortality of crickets was 48.10%, while at a concentration of 100% or without the addition of a dilution using biopesticide, melinjo skin had a mortality ability of 100%.The results of the observational data obtained in Factors The use of raw materials is very influential on the content of secondary metabolism obtained.One of the factors that causes a type of plant to be used as a biopesticide is the content of secondary metabolites present in plants.Secondary metabolites are chemical substances produced in the process of secondary metabolism.The components or compounds in the red seed coat of melinjo contain flavonoids, alkaloids, sterols, triterpenoids, tannins, and phenols [16].The flavonoid compounds in phenol can act as stomach poisoning and can cause decreased nerve function and damage to the insect's respiratory system.
The use of the type used in the extraction can affect the compound to be dissolved.The use of methanol as a solvent because it has a good ability to attract various polar and nonpolar compounds as well as in the extraction process of phenol compounds.Methanol can well extract phenolic compounds from various as well as methanol has toxic properties, methanol is often used as an additive.The addition of this "poison" will make methanol toxic to pests [17].

Conclusions
As a conclusion soxhletation extraction method has the highest value in the use of methanol solvent of 56.27% and phenol content of 11.02 Mg/GAE.Optimization is achieved at a solvent ratio of 600 ml with a phenol content of 34.23 Mg/GAE.After the optimum feed solvent ratio is reached, the total phenol content produced is constant.Phytochemical screening showed that melinjo peel contains flavonoids, alkaloids, sterols, triterpenoids, tannins, phenols.Biopesticide from melinjo skin with a dose of 100% has the most effective activity in killing crickets in vitro.

7 .
Pest Mortality Test Results.Parameters observed in this experiment is to use the formula M=

Table 1 .
Yield Extract Result

Table 2 .
Yield Extract Result

Table 3 .
Results of Phytochemical Screening

Table 5 .
Normal Probability Graph Initial Trial Probability From fig. 2. it can be seen that the point that has the highest value from the normal line is 0.46 point.It can be read in the Initial Experiment Normal Probability Table, that the point is a variable ( 13 Table. 6.In Table.6.The results of the extraction optimization show the results obtained after the optimization experiment for the largest total phenol content in sample 3 with a solv ent ratio of 600 ml, namely 34.23 Mg/GAE.The lowest error occurred in sample 5 with a solvent ratio of 800 ml, which was 16.91 Mg/GAE.Graph Effect of Solvent Volume with Total Phenol Level

Table 8 .
Table. 8 were then analyzed using single factor ANOVA in Table.8.Mortality test table with ANOVA The test results are based on the calculation results in Table.8.The results of the MortalityTest with ANOVA showed that the biopesticide of melinjo skin had an effect on the death of crickets.This is indicated by the results of the analysis of variance above the 5% level, where the Fcount value > Ftable (40.68 > 2.605) indicates that there is an effect of the use of biopesticide concentrations on the mortality of crickets in vitro.