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Toxicity of Miana Leaf ( Coleus blumei ) Extract Against Houseflies ( Musca domestica )

Houseflies (Musca domestica) is an disease-carrying insect that plays an important role in the spread of disease in humans and animals. One way to control houseflies is using Miana leaves (Coleus blumei) as botanical pesticide. The purpose of this study was to determine the chemical compounds contained in Miana leaf as well as determine the optimal concentration of Miana leaf extract in causing knockdown effect in houseflies. Miana leaf fine powder was soaked with methanol (maceration) for 3 days followed by phytochemical screening (chemical reagents and Gas Chromatography Mass Spectrometry) and anti-houseflies toxicity tests. Testing of methanolic extract of Miana leaves was made in the form of spray using the knockdown method at concentrations of 5%, 10%, 20%, 30% and 40% and observed in 5, 10 and 15 minutes. The results of phytochemical screening with chemical reagents showed that the extract contained alkaloids, tannins, flavonoids, saponins and terpenoids; while the results of GCMS analysis showed 8 bioactive compounds. The results of the study revealed that at a concentration of 30% and 40%, methanolic extract of Miana leaves could result in a knockdown effect that was accompanied by death with a percentage value of 100% in the first 5 minutes of observation. This research shows that methanolic extract of Miana leaves can be used as an alternative to botanical pesticides in controlling houseflies (Musca domestica).

the flavonoid content of Ricinus communis leaves is able to kill the houseflies. Whereas Fitriana research (2014) demonstrates that the essential oil content in purple basil leaves (Ocimum sanctum) has the potential as larvacide against M. domestica larvae with a mortality of 52% at a concentration of 20%.
However, the study of Miana (Coleus blumei) leaf as a botanical pesticide against houseflies (Musca domestica) has not been done before. Therefore, this study was conducted to determine the chemical compounds of methanolic extract of Miana leaves and its toxicity against houseflies (Musca domestica) using the knockdown method.
This study aims to explore the benefits of Miana leaves as a potential insect pest control. It demonstrates that the Miana leaves can be used as an alternative botanical pesticides as antihouseflies. It is the kind of repellent that is cheap and environmental friendly. It helps to increase the utilization of the Miana plants, which is commonly regarded as the ornamental plants

METHODS
The materials used in this study included Miana leaves, methanol solvents, houseflies (Musca domestica), filter paper and chemical reagents. While the tools used were glass jars (for maceration), funnels, analytical scales, stirring rods, flies test jars, blenders, glassware, dropping pipettes, measuring pipettes, stoves and Gas Chromatography Mass Spectrometry.
Miana plant samples ( Figure 1) were obtained from the Juanda area of Sidoarjo, East Java. Then the plant was identified at the Biology Service Unit of Airlangga University in Surabaya to ensure that the plant was a Miana plant (Coleus blumei).

Extraction process of Miana leaves
Extraction was done by maceration method. Miana leaves were washed, then dried, cut INTRODUCTION Houseflies (Musca domestica) is one of the insect members of the Diptera order (family Muscidae). They are ubiquitous and able to live in all environment; this makes houseflies cosmopolitan insects (Hastutiek & Fitri, 2007). These insects generally live and often move to a dirty environment, thus carrying various kinds of pathogenic disease-carrying microorganisms. Diseases caused by houseflies include typhus, cholera, diarrhea, parasitic worms, dysentery, trachoma viruses and other digestive infections (Ibrahim et al, 2018).
The most common way to control insect pests (flies) is through the use of synthetic pesticides. However, it has a negative impact on the environment (Gaag, 2000), such as accumulation of chemical residues in the environment and food chain that will disrupt the ecosystems (Damalas & Eleftherohorinos, 2011), water and food contamination (Soares & Porto, 2009) as well as pests resistance (Cole et al, 2000).
To overcome this problem, it is necessary to be able to control flies using natural materials, such as by utilizing the plants (biopesticides). According to Elbermawy et al (2011), plants are used as botanical pesticides because they contain various kinds of chemical compounds that have toxic effects on insect pests, such as repellent (insecticidal), antifeedant (disturbing diet) and the one that disturbing insect growth. Using plants as botanical pesticides have several advantages because raw materials (plants) are easy to get, nontoxic, and their compounds are biodegradable and safer for humans, food and the environment (Moreira et al, 2007).
Miana (Coleus blumei) is a member of the Lamiaceae family. This plant is better known as Mayana and is commonly used as an ornamental plant because it has attractive and striking leaf colors. Miana leaves contain alkaloids, tannins, flavonoids, saponins and terpenoids so that the leaves of Miana are widely used as a remedy for asthma, coughing, bronchitis, neutralizing toxins, diarrhea and worm medicine (Wijayakusuma, 2005). Because Miana leaves contain many of these chemical compounds, therefore this research uses Miana leaves as a houseflies repellent. Yuliana et al's research (2016) shows that the content of alkaloids, flavonoids and saponins in the frangipani leaves (Plumeria acuminate) is able to kill the house flies (Musca domestica) by 77% at a concentration of 60%. Along the similar line, Singh & Kaur's research (2016) shows that into small pieces and blended until became a fine powder. As many as 20 grams of fine powder of Miana leaves were put in a glass jar, then added with 600 ml of methanol solvent. Soaking (maceration) was carried out for 3 days, then the solution was filtered using filter paper and funnel. During maceration, stirring was carried out every day to avoid the compacting of the fine powder. Maceration process was carried out 2 times.

Chemical Compounds Screening of Miana Leaf Methanolic Extract
One hundred ml of extract was taken for phytochemical screening process with reagent and GCMS to determine what chemical compounds contained in the extract. Phytochemical screening with chemical reagent to determine the presence of alkaloids, tannins, saponins, flavonoids, and terpenoids was carried out at the Pharmacognosy Laboratory of the Surabaya Pharmacy Academy. While the bioactive compound test with GCMS was conducted at the Pharmacy Unit Service of Airlangga University.

Making the Various Concentration of Miana Leaf Extract
The concentrations used in the toxicity test were 5%, 10%, 20%, 30% and 40%. In order to get a concentration of 5%, 5 ml of extract was added with 95 ml distilled water and put in a 100 ml sized spray bottle.

Toxicity Test of Miana Leaf Extract Against Houseflies
The toxicity test of houseflies using the knockdown method was carried out in accordance with the research conducted by Kardinan (2007) with several modifications (Figure 2). Holes were made in the lid of the test jar for air circulation so that the houseflies did not die, and the other holes were made to spray the Miana leaf extract.
Each test jar which had been labeled according to the predetermined concentration contained 25 houseflies. Then, the extract was sprayed 2 times (0.5 ml for each spray). The ob-servation was then conducted to count the knocked down houseflies every 5, 10 and 15 minutes. The treatment was carried out with 3 replications.

Calculation of Knocked Down Houseflies
The percentage of houseflies that experienced knockdown was calculated using the equation from Kardinan (2007)

Toxicity Test
Concentrations of extract used in this study were 5%, 10%, 20%, 30% and 40%. The method used was knockdown method, the samples were observed for 5, 10 and 15 minutes. Each test  (E,E)-3,7,11-trimethyl-2,6,10-Dodecatrien-1-ol acetate antibacterial, antifungal (Chandrasekaran et al, 2005), thickener, moisturizer and softener (Lide, 2005), as a nutritious substance and metabolite in living organisms (Cakir, 2004) Table 3 as follows: The effectiveness of Miana leaf extract as a botanical pesticide is indicated by the percentage of knockdown houseflies based on the specified time interval. The higher the percentage, the higher potential of extract as a botanical pesticide.
Based on the method used, the vapors that contain insecticidal chemical compounds entered the body of the houseflies through the respiratory system. That method results in poisoning that occurs in the respiratory system and affects the nervous system in houseflies (Wirawan, 2006). This is in accordance with the statement of Sumita et al (2016), where spiracles located on the body surface of insects play an important role as a cause of knockdown in housefly. Vapor extracts enter the spiracles and quickly enter the insect nervous system, then inhibit the action of the acetylcholinesterase enzyme and finally a knockdown occurs.
In Table 3, it can be seen that the increasing concentration of Miana leaf extract was followed by the increase in the percentage of knockdown in Musca domestica. The higher concentration of extract, then the more chemical compounds contained in it, thus it will shorten the life span of test insects.
The results of this study were supported by Darmadi & Anita (2018), which showed that 15% concentration of Duku (Lansium domesticum) peel extract spray was able to kill Musca domestica within 12 minutes. The results of the study by Yuliana et al (2016), stated that Plumeria acuminate leaves extract spray can killed house flies by 76.7% at a concentration of 60%. Whereas Kumalasari et al (2015) stated that electric mat vaporizer containing Kesum (P. minus) leaf methanol extract at a concentration of 25% resulted in 75% of knockdown of the housefly (Musca domestica).
Result of toxicity test showed that all concentrations of Miana leaf extract in the first 5 minutes gave a real knockdown effect, especially at concentrations of 30% and 40%, where the number of houseflies fell (knockdown) accompanied by death was 100%. At the concentration of the Miana leaf extract of 5% and 10%, there is an increase in the number of houseflies that fall. However, when the concentration level is 5%, this experiment shows a number of flies which still can be recover by 8%. These knockdown flies are still alive; they would respond to any moving and touching triggers. For example, they would make a response to a brush stroke. At the concentration of the Miana leaf of 20%, the effectiveness is almost equal with the one found at the concentration of the Miana leaf of 30% and 40%.
The ability of methanolic extract of Miana leaves as an insect repellent (ie anti-houseflies) is thought to be due to the presence of secondary metabolites which are toxic to insects. The results showed that the methanolic extract of Miana leaves contained alkaloids, tannins, saponins, flavonoids and terpenoids. This is supported by research conducted by Yuliani et al (2005), where alkaloid and terpenoid compounds in Pogostemon cablin's methanol extract have repellent effect against Musca domestica.
Alkaloid compounds are included in toxic compounds. It works through attacking the works of the insect's nervous system. Such attack will slow down the nervous system, create seizure and knockdown and leads to death. Flavonoids are also toxic to insects by weakening the nervous system in spiracles (breathing apparatus in insects) found on the surface of the body, so that the spiracles become damaged. As a result, insects cannot breathe and eventually die (Yuliana et al, 2016). Saponins and terpenoids damage the cell membranes and disrupt insect metabolic processes resulted in the death of insects (Heras & Hortelano, 2009). Tannin damages the permeability of insect cell membranes, so that it disrupts the metabolic process and the insect will eventually experience death (Purwita et al., 2013).
The chemical compound stigma-8(14)en.3.beta.-ol (28.71%) which is the largest component in Miana leaf extract may also have insecticidal properties. This compound belongs to the stigmasterol derivative. Many researches have been done in examining the capacity of stigmasterol as insecticide. Stigmasterol has an insecticidal activities against rabbit ear mites Psoroptes cuniculi (Nong et al., 2017). Stigmast-7-en-3-ol is capable for killing lice Myzus persicae (Diaz et al., 2015).
The content of the active compound in Miana leaf extract propels the fast reaction of the extract in terminating houseflies. There is also a possibility that such fast reaction might be caused by the diffusion of the Miana leaf extract sprayed directly into the test jar, where the flies are placed. The vapor of the Miana leaf extract is immediately absorbed by the insect skin, thus gives effects into the insects nervous system (Sulaiman & Kuswahyuningsih, 2008).
The knockdown process occurs when Miana leaf extract works by inhibiting a very important enzyme in the nervous system called cholinesterase (ChE). This enzyme becomes phosphorylated when bound to organophosphate, and this bond is permanent (irreversible). The effect of the inhibition of the ChE enzyme causes acetylcholine to accumulate at synapses, so that the houseflies experience muscle spasms and eventually become paralyzed or even die (Lee et al, 2017).
The results of this study indicates that the presence of chemical compounds from the methanolic extract of Miana leaf (Coleus blumei) plays an important role in the knockdown effect in houseflies (Musca domestica). This study provides information that Miana leaves can be used as an alternative in minimizing the use of botanical pesticides to control houseflies.
Suggestion furthermore, further research is needed to test the ability of miana leaves to other insect species so that they can become promising alternative plants in the field of integrated pest management in Indonesia.

AKNOWLEDGEMENTS
This research was funded by the Ministry of Research and Technology through the Research Grants of Beginner Lecturers in 2019.