Abstract: Background and Objective: Diseases caused by microbes vectored by mosquitoes are still a health problem in tropical countries today. DHF and Malaria are the two primary diseases vectored by mosquitoes, the morbidity and mortality rates have increased in low countries until now. However, the best way to control these two diseases is to control vectors, namely mosquitoes. Research has been conducted to determine the bioactive content and larvicidal activity of local plant extracts of North Sulawesi. Materials and Methods: The clove and trumpet flower samples were obtained from Minahasa, while the nutmeg samples were obtained from Sitaro Regency. Empirically, people use plant parts to repel mosquitoes. Extraction of plant simplicia was carried out by the maceration method. Qualitative and quantitative methods carried out the phytochemical content analysis. Qualitative analysis uses Harborne’s (1996) method while qualitative analysis uses the UV Vis Spectrophotometer method. Toxicity tests were carried out on mosquito larvae developed in the laboratory. Results: The results showed that combining clove leaf extract, nutmeg flesh extract and trumpet flower synergistically increased the bioactive content. Flavonoids increased in the combination of extracts compared to partial extracts. The combination of extracts showed the highest toxicity to mosquito larvae (LC50: 22.541 mg L–1), while the lowest was the partial extract of clove leaves with LC50 (54.965 mg L–1). Conclusion: The combination of extracts showed the best toxicity activity on mosquito larvae. Research on bioactive characteristics and toxicity in adult mosquitoes needs to be carried out in the future.
INTRODUCTION
Mosquito population control is still a global public health priority. Mosquitoes are vectors of various infective diseases with high fatality rates such as Malaria, febrile fever, chikungunya, yellow fever, Zika and West Nile, affecting a significant proportion of the world population1-3. These diseases contribute significantly to an estimated 17% of the global vector-borne disease burden of all infectious diseases, accounting for >1 billion new cases and >1 million deaths annually4. Mosquito-borne diseases contribute significantly to the worldwide burden of disease, mortality, poverty and social disadvantage5,6.
Malaria and DHF are endemic diseases in Indonesia, which until now have increased morbidity and mortality7. In 2020, in the COVID-19 pandemic situation, there was an increase in dengue cases in North Sulawesi, Indonesia, compared to the previous year. Climate change has affected the adaptation of mosquitoes to the environment8-11. Besides, the use of synthetic insecticides against mosquitoes also raises resistance and resurgence12,13. Based on research reports and empirically, several liquid synthetic insecticides have decreased toxicity to adult mosquitoes. Besides, residues of synthetic insecticides can be toxic and carcinogenic to humans14.
The use of plants as bioactive insecticides in the tropics is based on biological interactions between plants and insects15-17. Plants produce certain compounds as a form of self-defence from microbial attack or multicellular organisms, especially insects18. Some plants reported containing bioactive insecticide compounds include Acer rubrum L. (Aceraceae), Betula alleghaniensis Britton (Betulaceae), Betula papyrifera Britton, Carya cordiformis K. Prunus serotina Ehrh (Rosaceae), etc.19, langsat seeds20. Furthermore, Nicotiana tabacum, Ocimum basilicum, Datura stramonium, Chenopodium album and Cassia fistula have strong larvicidal activity against Culex quinquefasciatus. Acetate extract of Acacia auriculiformis fruit showed high mortality against mosquito larvae at a concentration of 300 ppm21. Ricinus communis seed extract has better larvicidal activity than a leaf. Both extracts can be used as an effective larvicide against mosquitoes22.
As a malaria-endemic area, the Minahasa people of North Sulawesi, Indonesia, have long recognized and used certain plant species as botanical insecticides. Clove oil is used as a mosquito repellent by the Minahasa tribal community. Nutmeg leaves are also used as a mosquito repellent by the Sanger tribe. In contrast, the trumpet flower has a distinctive aroma that is not liked by mosquitoes. However, research on the toxicity of partial extracts and combinations of these three plant species, specifically from the Wallacea Zone, is still very little reported.
Controlling mosquito populations at the larval stage is more likely than adult mosquitoes23. The use of partial plant extracts has been widely carried out. However, the activity of the combination of plant extracts as larvicides is still reported. The combination of plant extracts in this study was carried out on several ethnomedical plants used by the local community of North Sulawesi as a source of bioactive mosquito repellent for generations. These plants are clove leaves, nutmeg flesh and trumpet flowers.
Research has been carried out to obtain effective concentrations of partial plant extracts and combinations of plant extracts against mosquito larvae.
MATERIALS AND METHODS
Study area: The research was conducted at the Bioactivity and Biopharmaceutical Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Manado State University, Indonesia. The research was conducted from March to September, 2021.
Plant samples: Nutmeg (Myristica fragrans L.) was obtained from Siau, Sitaro District, North Sulawesi. Clove leaves (Syzygium aromaticum L.) were obtained from Kombi District, Minahasa Regency, North Sulawesi. White trumpet flower (Brugmansia suaveolens L.) was obtained from West Tondano District, Minahasa Regency, North Sulawesi. The samples obtained were labelled and preserved in the sample box.
Tools and materials: The tools used in this research include the Blender Philips (Belanda), Kern analytical balance, Rotary Evaporator Buchi (Switzerland) to evaporate the solvent and get the crude extract, Eppendorf centrifuge (Germany), Eppendorf micropipette (Germany), PerkinElmer UV Vis spectrophotometer (USA), Hirox KH8700 microscope (Japan), Mummert hot plate (Germany), glassware Pyrex and others. While the ingredients used are clove leave extract, nutmeg flesh extract, trumpet flower extract, ethanol pa Merck, chloroform pa Merck, ethyl acetate pa Merck, Butanol pa Merck, ion-free water, standard quercetin Brand, Dragendorff reagent, Mayer’s reagent, Wagner’s reagent, HCl, Mg metal, Na2CO3, FeCl3, H2SO4 and acetic anhydride, etc.
Research procedure: The research was carried out in four stages: The preparation of mosquito test objects, plant extraction, analysis of bioactive content, formulation of test extracts and larvicide testing.
Preparation of mosquitoes as larvicide test objects
Catching mosquitoes in the field (Sayono et al.23): Mosquito catching is carried out in their natural habitat (resting place), namely irrigation canals and swamp areas protected from sunlight, in the morning from 05.00-08.00 and around cattle pens from 22.00-24.00. Identification of mosquitoes was carried out by piercing the Book Reid (1968) and then reared individually into the first generation (F1)/iso female line. The first generation (F1) of early IV instar larvae was used for treatment and biochemical tests.
Mosquito maintenance in the laboratory: The captured mosquitoes are then spawned in the laboratory individually/iso female line. This individual maintenance is that each mosquito is placed separately from one another to lay eggs. After the eggs hatched into larvae, each was transferred to a rearing area, namely, a tray measuring 26 and 15 cm long. Every day the larvae are given food in the form of fish pellets. The subjects that will be used are mosquito larvae of Anopheles sp stage 3 and 4. After becoming mosquitoes, they are fed with a mixture of bran powder and meat in a ratio of 10:4 as much as 75-200 mg.
Extraction and analysis of bioactive content
Plant extraction: Extraction has been done by the maceration method by Kaunang and Semuel24 and Semuel et al.25. The ratio of solvent and solute is 1:4 (250 g of plant extract is macerated in 1000 mL of solvent). Extraction was carried out at room temperature for 3×24 hrs. The solvent used is 70% ethanol.
Bioactive content analysis
Phytochemical analysis (Kaunang and Semuel)24
Alkaloid test: A total of 0.1 g of the extract was added to 3 mL of chloroform and three drops of ammonia. The chloroform fraction was separated and acidified with ten drops of 2 M H2SO4. The acid fraction was taken and then Meyer and Wagner’s reagent was added. The presence of alkaloids was indicated by the formation of a white precipitate by the Meyer reaction and a brown precipitate by the Wagner reaction. As a comparison, use the blood footprint.
Saponin and flavonoid test: A total of 1 g of extract was put in a beaker, then added to 100 mL of hot water and boiled for 5 min, after that, it was filtered and the filtrate was used for testing. The saponin test was carried out by shaking 10 mL of the filtrate in a closed test tube for 10 sec and then leaving for 10 min. The formation of stable foam indicates the presence of saponins. Another 10 mL of the filtrate was added with 0.5 g of magnesium powder, 2 mL of carbohydrate alcohol (a mixture of 37% HCl and 95% ethanol in a ratio of 1:1) and 20 mL of amyl alcohol then shaken vigorously. The formation of red, yellow and orange colours on the amyl alcohol layer indicates the presence of flavonoids.
Tannin test: A total of 0.1 g of the extract was added to 2 mL of water and then boiled for several minutes. Then filtered and the filtrate was added with one drop of 1% FeCl3 (w/v). Dark blue or greenish-black colour indicates the presence of tannins.
Triterpenoid and steroid test: A total of 0.1 g of the extract was added to 2 mL of 30% ethanol, then heated and filtered. The filtrate was evaporated and then 1:1 ether was added. The ether layer was added with Lieberman Burchard’s reaction (3 drops of acetic anhydride and one drop of concentrated H2SO4). Red and green colours indicate the presence of triterpenoids and green colours indicate the presence of steroids.
Quantitative analysis of flavonoid content
Determination of the maximum wavelength (max) of Quercetin: The maximum wavelength of Quercetin by running the quercetin solution at a wavelength of 400-450 nm. The result of running is the maximum wavelength of the quercetin standard, which is 435 nm. The wavelength was used to measure the absorbance of the combination of clove leaf extract:nutmeg pulp:trumpet flower (1:1:1).
Quercetin standard curve: A total of 25 mg of standard Quercetin (Merck) was dissolved using 25 mL of absolute ethanol. The stock solution was pipetted as much as 1 mL and the volume was made up to 10 mL with ethanol to obtain a concentration of 100 ppm. From a standard solution of 100 ppm quercetin, then several concentrations were made, namely 6, 8, 10, 12 and 14 ppm. From each concentration of the standard solution of Quercetin, 1 mL was pipetted. Then 1 mL of 2% AlCl3 and 1 mL of 120 mM potassium acetate were added. Samples were incubated for one hour at room temperature. The absorbance was determined using the UV-Vis spectrophotometric method at a maximum wavelength of 435 nm26.
Determination of the total flavonoid content of the extract: Weighed 15 mg of extract, dissolved in 10 mL of ethanol, to obtain a concentration of 1500 ppm. About 1 mL of this solution was pipetted and then 1 mL of 2% AlCl3 solution and 1 mL of 120 mM potassium acetate were added. Samples were incubated for 1 hr at room temperature. The absorbance was determined using the UV-Vis spectrophotometric method at a maximum wavelength of 435 nm. Samples were made in three replications for each analysis and the average value of absorbance was obtained26.
Larvicide test
Exploration test: An exploratory or preliminary test was conducted to determine the treatment concentration that caused the death of 50% of the total population of larvae tested. The criteria for death are test animals or larvae that do not move and, if touched, do not react.
Larvicide activity analysis: Anopheles sp., larvae, as the target test animals, have integuments that are easily damaged24. Therefore, the method used is the dipping method. The test animals are immersed or put into the test solution. The test is carried out at room temperature.
Data analysis: The data from the extraction and analysis of phytochemical groups were analyzed descriptively. The flavonoid content was determined by regression analysis. Lethal Concentration 50 (LC50) was determined based on probit analysis of data from the larvicide test results.
RESULTS
Plant extraction: Fresh samples of clove leaves were obtained from the Minahasa Clove Kombi plantation, Minahasa Regency, North Sulawesi, Indonesia. Nutmeg samples were obtained from Siau Sitaro Regency, North Sulawesi, Indonesia. While the trumpet flower samples were obtained from Kasuang, Minahasa Regency, North Sulawesi, Indonesia. The weight of fresh samples that have been mashed is 250 for each type of plant Simplicia is macerated with 70% ethanol for 2×24 hrs. Evaporation of the solvent with a rotary evaporator produces a blackish-brown extract. Each type of extract has a distinctive aroma, such as cloves, nutmeg and flowers (Fig. 1a-c). The highest average weight of the extract was shown by the nutmeg flesh extract, which was 11.57 with a yield percentage of 4.63. While the lowest average weight of the extract was indicated by the extract of the trumpet flower, which was 4.85 g with a percent yield of 1.94 (Table 1).
Phytochemical content analysis: Based on the intensity of the colour and precipitate obtained from the analysis of the content of the phytochemical groups, it was obtained that the combination of extracts showed the content of the groups with better intensity than the partial extracts. However, the three types of extracts showed flavonoid content with the same intensity as the combination of extracts. Trumpet flower and nutmeg flesh showed relatively the same intensity of alkaloid and triterpenoid content (Table 2).
| Table 1: | Extract weight and percent yield of different extracts | ||||||
| Extract |
Replication |
Samples weight (g) |
Solvent volume (mL) |
Extract weight (g) |
Yield (%) |
Average extract weight (g) |
Average yield (%) |
| Clove leaves |
1 |
250 |
1000 |
10.09 |
4.04 |
9.87 |
3.95 |
|
2 |
250 |
1000 |
10.21 |
4.08 |
|||
|
3 |
250 |
1000 |
9.32 |
3.73 |
|||
| Nutmeg flesh |
1 |
250 |
1000 |
11.16 |
4.46 |
11.57 |
4.63 |
|
2 |
250 |
1000 |
12.32 |
4.93 |
|||
|
3 |
250 |
1000 |
11.24 |
4.50 |
|||
| Trumpet flower |
1 |
250 |
1000 |
4.56 |
1.82 |
4.85 |
1.94 |
|
2 |
250 |
1000 |
4.45 |
1.78 |
|||
|
3 |
250 |
1000 |
5.53 |
2.21 |
| Table 2: | Qualitative test results (colour and precipitate) extract | ||||
| Types of phytochemical test |
Clove leaves |
Trumpet flower |
Nutmeg flesh |
Extract combination (1:1) |
Test indicators |
| Alkaloid |
+ |
++ |
+ |
++ |
Meyer’s reaction: White precipitate |
| Dragendorff’s reaction: Orange precipitate | |||||
| Wagner’s reaction: Brown precipitate | |||||
| Flavonoid |
++ |
++ |
++ |
++ |
Reddish colour after the addition of magnesium powder and followed by the addition of 10 drops of 5 M. HCl |
| Tanin |
+ |
+ |
+ |
++ |
Addition of gelatin and FeCl3, brownish green |
| Saponin |
+ |
+ |
+ |
++ |
Stable foam for 5-10 min |
| Triterpenoid |
+ |
++ |
++ |
++ |
Red or purple colour indicates the presence of triterpenoids) |
| Steroid |
+ |
+ |
++ |
++ |
Green or blue colour indicates the presence of steroids) |
| +: Contains a group of phytochemical compounds indicated by colour indicators and/or precipitates formed and ++: Contains a group of phytochemical compounds in high intensity indicated by colour indicators and/or precipitates formed | |||||
Total flavonoid content: In this study, the standard flavonoid Quercetin (Merck) was used. Quercetin belongs to the flavonoid group of flavonols with a keto group at atomic C number 4 and an OH group at atomic C number 3 and atomic C number 5. Maximum absorption is determined at a wavelength distribution of 400-455 nm. The results of running the optimum absorption of quercetin standard at a wavelength of 435 nm. This wavelength was used to measure the uptake of the extract samples (Table 3).
The standard yield of Quercetin obtained was plotted between the concentration and absorbance. A linear regression equation was obtained, namely, y = 0.049x-0.006 with an R2 value of 0.9976 and an R-value of 0.997. The quercetin calibration curve equation can be used as a comparison to determine the concentration of total flavonoid compounds in the sample extract (Fig. 2). As a control, a blank aqua dest solution was used. Based on the determination of the total flavonoid content of each extract, the highest flavonoid content was obtained in the combination of extracts with an average of 10.835 mg L–1. The partial extract that showed the highest average flavonoid content was cleft leaf extract, 6.158 mg L–1. Meanwhile, the extract with the lowest moderate flavonoid content was trumpet flower extract, 4.268 mg L–1 (Table 4).
Larvicide test: After 24 hrs of giving the extract to the test larvae, it showed the highest mortality rate of the three extracts at the test concentration of 1000 ppm. The trumpet flower extract indicated the lowest mean mortality at a concentration of 10 ppm (Table 5).
The combined extract mortality test was performed three times. Each concentration was repeated three times. The highest mortality rate was at a concentration of 1000 ppm, while the lowest was ten ppm (Table 6).
The determination of the lethal concentration 50, i.e., the total population of 50% dead larvae, was carried out by probit analysis. The highest LC50 was shown in the extract combination (1:1:1), 22.541 mg L–1, while the lowest was in the clove extract, 54.965 mg L–1 (Fig. 3).
| Table 3: | Results of the absorbance measurement of the standard solution of Quercetin at a maximum wavelength of 435 nm |
| Concentration | Absorbance (nm) |
| 6 | 0.294 |
| 8 | 0.386 |
| 10 | 0.475 |
| 12 | 0.576 |
| 14 | 0.689 |
| Table 4: | Results of the determination of the total flavonoid content % (w/w) in the extract using the standard Quercetin | |||
| No | Extract |
Replication |
Total flavonoid level (mg L–1) |
Average |
| 1 | Trumpet flower |
1 |
3.657 |
4.268 |
|
2 |
3.453 |
|||
|
3 |
5.695 |
|||
| 2 | Nutmeg flesh |
1 |
6.967 |
5.508 |
|
2 |
5.706 |
|||
|
3 |
3.851 |
|||
| 3 | Clove leaf |
1 |
4.842 |
6.158 |
|
2 |
7.720 |
|||
|
3 |
5.912 |
|||
| 4 | Combination of extracts (1:1:1) |
1 |
11.069 |
10.835 |
|
2 |
10.616 |
|||
|
3 |
10.822 |
| Table 5: | Mosquito larvae mortality ethanol extract | ||||||
|
Number of dead mosquito larvae |
|||||||
| Extract |
Concentration (ppm) |
Number of larva |
1 |
2 |
3 |
Total dead |
STDEV |
| Clove leaf |
1000 |
10 |
10 |
9 |
9 |
28 |
0.577 |
|
500 |
10 |
7 |
9 |
8 |
24 |
1.000 |
|
|
100 |
10 |
7 |
7 |
8 |
22 |
0.577 |
|
|
10 |
10 |
3 |
2 |
3 |
8 |
0.577 |
|
| Nutmeg flesh |
1000 |
10 |
10 |
10 |
9 |
29 |
0.577 |
|
500 |
10 |
8 |
8 |
9 |
25 |
0.577 |
|
|
100 |
10 |
8 |
7 |
6 |
21 |
1.000 |
|
|
10 |
10 |
2 |
2 |
3 |
7 |
0.577 |
|
| Trumpet flower |
1000 |
10 |
10 |
10 |
10 |
30 |
0.000 |
|
500 |
10 |
9 |
9 |
9 |
27 |
0.000 |
|
|
100 |
10 |
8 |
8 |
8 |
24 |
0.000 |
|
|
10 |
10 |
3 |
3 |
3 |
9 |
0.000 |
|
| Fig. 1(a-c): | Extraction of (a) Nutmeg flesh, (b) Clove leaves and (c) Trumpet flower ethanol extract |
| Fig. 2: | Quercetin standard curve for total flavonoid compounds in the sample extract |
| Table 6: | Mortality of mosquito larvae in extract combinations | ||||||
|
Number of dead mosquito larvae |
|||||||
| Extract replication |
Concentration (ppm) |
Number of Larva |
1 |
2 |
3 |
Number of death |
STDEV |
| U1 |
1000 |
10 |
10 |
10 |
10 |
30 |
0.000 |
|
500 |
10 |
9 |
10 |
9 |
28 |
0.577 |
|
|
100 |
10 |
9 |
9 |
9 |
27 |
0.000 |
|
|
10 |
10 |
5 |
5 |
3 |
13 |
1.155 |
|
| U2 |
1000 |
10 |
10 |
10 |
10 |
30 |
0.000 |
|
500 |
10 |
8 |
9 |
9 |
26 |
0.577 |
|
|
100 |
10 |
8 |
8 |
8 |
24 |
0.000 |
|
|
10 |
10 |
1 |
0 |
1 |
2 |
0.577 |
|
| U3 |
1000 |
10 |
10 |
10 |
9 |
29 |
0.577 |
|
500 |
10 |
9 |
8 |
9 |
26 |
0.577 |
|
|
100 |
10 |
9 |
9 |
8 |
26 |
0.577 |
|
|
10 |
10 |
5 |
5 |
1 |
11 |
2.309 |
|
| Fig. 3: | Combination of extracts with a ratio of 1:1:1 showed the best larvicidal activity y-axis: Variety of extract |
DISCUSSION
Based on the percent yield, nutmeg pulp extract showed the highest yield percentage compared to clove leaf extract and trumpet flower extract. Ethanol solvent was effective in attracting secondary metabolites contained in nutmeg flesh. The results of the analysis of phytochemical groups found that the content of phenolic compounds was evenly distributed in the three types of extracts. However, there are variations in the content of steroids and triterpenoids in the three types of extracts.
Extraction of plant material is an essential step in obtaining secondary plant metabolites used as raw materials for drugs, cosmetics and insecticides. There are many methods for extracting plant material, including percolation, soxhletation and steam distillation methods27,28. The percolation method is only suitable for soluble organic compounds, while soxhletation is only good for heat-resistant compounds. Therefore, the maceration method was chosen to maximize the isolation of secondary metabolites from plant extracts.
Of the various plant extraction methods, maceration is the most widely used in extraction to develop drugs and insecticides29,30. Maceration was chosen because this Extraction provides time for the solvent to attract secondary metabolites in the Simplicia and is carried out at room temperature so that the compound is not damaged or its activity decreases due to exposure to high temperatures. The most important factors affecting the extraction results are solvent, plant samples, time and temperature25,29.
Based on the content analysis of phytochemical groups, it was found that all phytochemical test groups were found in all types of extracts. Because Simplicia was extracted with a polar solvent, namely ethanol, the polar compounds were attracted quite well, indicated by the colour intensity of the high phytochemical content test. All extracts showed high levels of flavonoid content. However, after the extracts of nutmeg pulp, clove leaves and trumpet flowers were combined in a 1:1:1 (w/v), it resulted in high-intensity content of all phytochemical groups. This shows that the mixing of the three types of extracts increases the content of the phytochemical group.
Based on the results of quantitative analysis using the standard flavonoid quercetin, it also strengthens the results of the study of the content of qualitative phytochemical groups in the combination of extracts. The highest quercetin content was found in the combination of extracts compared to other partial extracts. Flavonoids and other phenolic compounds are widely reported to have pharmacologic and insecticidal activity.
The test concentration of 1000 ppm showed the highest larval mortality in all types of extracts and combinations of extracts. The highest LC50 was shown by trumpet flower extract, this indicates the highest toxicity level of trumpet flower extract to mosquito larvae. Furthermore, the lowest toxicity was based on the LC50 value of the nutmeg pulp extract. However, overall the best LC50 was shown in the combination of extracts. Thus, the combination of extracts increased the toxicity of mosquito larvae.
Observations on the toxicity test included larvae often appearing on the surface. The frequency was very long, which indicated that the need for oxygen dissolved in the water was reduced. The larvae often came to the surface to meet their oxygen needs, another symptom was a reduced response to stimuli marked by reduced aggressiveness when the test bottle was touched. This situation is an active symptom caused by the bioactive compounds in the test extract. The content of phytochemical groups was found evenly in all types of extracts. However, the phenolic compound group showed the highest intensity of content both in the extract of nutmeg flesh, trumpet flower, clove leaf and combination of extracts.
Several phytochemicals such as alkaloids, flavonoids, tannins, terpenoids, saponins and steroids have been reported cytotoxic effects in vitro31,32. These compounds can enter the larval body, which has a thin cell membrane. Toxic compounds enter through several parts of the body, including the body’s surface, the respiratory tract and the digestive tract. The body surface wall is the outermost part of the larva’s body that can absorb large amounts of insecticide because this part is directly related to insecticides33,34. In larval respiration, air and oxygen enter the trachea by diffusion with the help of abdominal movements and toxic substances in the extract can also enter the respiratory system in the form of gas or fine granules carried to living tissue. In this study, toxic substances enter the mouth of the larvae through the respiratory system in the form of spiracles on the body’s surface and cause wilting of the nerves and damage to the spiracles larvae cannot breathe and eventually die. One group of plant phytochemicals reported causing nerve wilting is saponins. Saponins can inhibit the action of the enzyme acetylcholinesterase. Acetylcholine formed by the central nervous system serves to deliver impulses from nerve cells to muscle cells. After the impulse is transmitted the enzyme acetylcholinesterase stops the process, which breaks down acetylcholine into acetyl Co-A and choline. The presence of insecticidal compounds (alkaloids, flavonoids and saponins) will inhibit the work of this enzyme resulting in a buildup of acetylcholine which will cause chaos in the impulse delivery system to muscles and can result in muscle spasms, paralysis and eventually death35-37.
One of the mechanisms of flavonoid toxicity is also a strong respiratory inhibitor. Components that interfere with energy metabolism have been identified from either natural or synthetic sources. Energy metabolism disorders occur in the mitochondria by inhibiting the electron transport system or by blocking the coupling between the transport system and ATP production. Inhibition of the electron transport system blocks ATP production and causes a decrease in mitochondrial oxygen consumption. One of them is also a strong respiratory inhibitor. Respiratory inhibitors work by inhibiting the respiratory chain, inhibiting oxidative phosphorylation, or uncoupling the respiratory chain with oxidative phosphorylation. Electron transport inhibitors at the site I work by inhibiting Coenzyme Q reductase (NADH oxidase inhibitor). Inhibitor at site II by inhibiting Cytochrome b-c complex38-41.
Impaired energy metabolism and loss of ATP cause slow-acting toxicity and affect all components, including paralysis and larval death. Kaempferol, Myricetin and Quercetin are included in one of the flavonoid groups, namely flavonols. Flavonoids are good reducing agents so that they can inhibit oxidation reactions, both enzymatically and non-enzymatically42. Flavonoids enzymatically inhibit the oxidation process by acting as ATPase inhibitors, NADH-oxidase-inhibitors (Coenzyme Q reductase inhibitors) and cytochrome inhibitors43,44. The presence of NADH-oxidase-inhibitor (Coenzyme Q reductase inhibitor) causes blockage of electron flow from NADH to CoQ (Coenzyme Q). Cytochrome inhibitors block the flow of electrons from cytochrome b to c1. By blocking the flow of electrons from cytochrome b to c1, all electron acceptors before cytochrome b are reduced. The ATPase inhibitors act at site V by inhibiting the ATP synthesis catalyst from ADP45.
The larvicidal mechanism of tannins is related to their ability to inactivate adenosine, enzymes and cell proteins46,47. Hydrolyzed tannins are usually amorphous compounds, hygroscopic, yellow-brown in colour and soluble in water (boiling water) to form colloidal solutions instead of natural solutions. The purer the tannin, the less soluble it is in water and the easier it is to obtain it in crystalline form. The interaction of tannins with proteins is characteristic and depends on the structure of the tannins48,25. Some tannins have been shown to have an activity to inhibit reverse transcriptase and DNA topoisomerase enzymes46. Tannins bind to polysaccharides and are soluble in water. Catechin tannins play an essential role as larvicides because they cause damage to cell membranes so that mosquito larvae die. The part of flavonoid larvicides occurs through the mechanism of inhibition of nucleic acid (DNA) synthesis of larvae, which causes the death of the larvae49.
The results of this study provide a new alternative to the use of ethnobotanical plants as a source of bioactive mosquito larvicides. However, vector control is more effective than disease control. Furthermore, larvae are a more sensitive stage for controlling mosquito populations. However, further research on larvicidal formulations and large-scale testing are needed to obtain the best formulation of ethnomedical plant larvicides.
CONCLUSION
The combination of clove leaf extract, nutmeg flesh extract and trumpet flower synergistically increases the bioactive content. Flavonoids increased in the combination of extracts compared to partial extracts. High phenolic compounds in all types of extracts were cytotoxic to mosquito larvae. The combination of extracts showed the highest toxicity to mosquito larvae (LC50: 22.541 mg L–1), while the lowest was a partial extract of clove leaves with LC50 (54.965 mg L–1).
SIGNIFICANCE STATEMENT
This study strengthens the empirical and ethnomedical use of medicinal plants for mosquito control. North Sulawesi as a malaria-endemic area has ethnomedical medicinal plants as mosquito bioinsecticides which are passed down from generation to generation. However, this study scientifically proves the bioactive potential of the combination of ethnobotanical plant extracts as larvicides. This research opens the field of research on the use of bioactive medicinal plants for mosquito repellent materials, liquid mosquito repellents and other forms of mosquito control in the future.
ACKNOWLEDGMENTS
We want to thank the Directorate of Research and Community Service, Ministry of Education, Culture, Research and Higher Education, the Republic of Indonesia for funding this research through the Basic Research Scheme for Higher Education Excellence in 2021. This research was funded through a superior basic research scheme of higher education with decree number 129/E4.1/AK.04.PT/2021 and Contract Agreement Number 441/UN41.9/TU/2021. We thank the leadership and staff of the Laboratory of Bioactivity and Molecular Biology, State University of Manado and the Laboratory of PSB Biopharmaca IPB Bogor for assisting the analysis.