Hrev_master Healthcare in Low-resource Settings 2024; volume 12:11772 Ethanolic extract of red dragon fruit inhibits growth of mosquito larvae Dhina Ayu Susanti, Fitria Meliana Putri Milyunier, Galuh Maulidatin Nufus, Lita Aulia Pramesti, Nia Laela Nur Khamimi, Wima Anggitasari, Lindawati Setyaningrum, Inna Armandari Bachelor Pharmacy Study Program, Faculty of Health Sciences, Universitas Dr. Soebandi, Jember, Indonesia Abstract Controlling the vector by eradicating mosquito larvae is fre- quently done by spreading Abate powder containing 1% Temephos as a larvicide agent. However, the use of Abate powder will possibly increase pesticide residue contamination in the water. This study aimed to explore the potential use of ethanolic extract from red dragon fruit skin to kill mosquito larvae. An experimental laboratory study was performed to examine the lar- vicidal activity of ethanolic extract from red dragon fruit skin. Maceration method was used to extract active compounds from the fruit skin. Subsequently, the viscous extract was formulated using the wet granulation method to obtain powder form. The lar- vicide test was done by comparing the lethal concentration (LC50) of mosquito larvae after treatment using the extract and the refer- ence larvicide agent, 1% Temephos Abate. The treatment groups consisted of four different extract concentrations of 1%, 2%, 3%, and 4%. The LC50 of each treatment groups was then calculated against the negative control group. Phytochemical screening showed that the ethanol extract of red dragon fruit skin contains alkaloids, flavonoids, saponins, triterpenoids, and tannins. The lar- vicide test on Aedes aegypti mosquito larvae showed that the extract treatment hardly killed the Aedes aegypti mosquito larvae. However, on Culex mosquito larvae, the ethanolic extract of red dragon fruit skin was able to kill mosquito larvae, with LC50 value of of 19.06%. The ethanolic extract of red dragon fruit skin was effective on killing the Culex mosquito larvae but not the Aedes aegypti mosquito larvae. Introduction Vector-based infectious diseases account for over 17% of all infectious diseases, resulting in more than 400,000 deaths annual- ly and afflicting 219 million people worldwide. In Indonesia, these diseases are prevalent health issues in various regions and cities, often leading to outbreaks with a high risk of mortality. Being a tropical country, Indonesia experiences relatively high humidity and rainfall, creating favorable conditions for the proliferation of vector populations.1,2 Mosquitoes can transmit several vector- based infectious diseases, with some of the most prevalent ones in Indonesia being Dengue Hemorrhagic Fever (DHF),3 Japanese encephalitis, and filariasis. These diseases require particular atten- tion due to their high incidence in the country. The vector responsible for transmitting Dengue Hemorrhagic Fever (DHF) is the Aedes aegypti mosquito, while Japanese encephalitis (JE) is transmitted by the Culex mosquito. In the case of filariasis, it can be transmitted by various mosquito species, including Mansonia, Anopheles, Culex, Aedes, and Armigeres mosquitoes. Several studies have emphasized that global climate change has had an impact on the risk of disease vector transmis- sion, particularly in the case of mosquitoes.4–7 As of the end of 2022, Indonesia had reported approximately 143,000 cases of Dengue Hemorrhagic Fever (DHF), while there were an average of 35,000 Japanese encephalitis (JE) cases in Asia each year. In 2021, Indonesia reported 9,354 cases of filariasis. These vector- based infectious diseases have the potential to cause severe phys- Correspondence: Dhina Ayu Susanti, Bachelor Pharmacy Study Program, Faculty of Health Sciences, Universitas dr. Soebandi, Jember, Indonesia. E-mail: dhina.apt@gmail.com Key words: Aedes Aegypti, culex, granule, larvicidal activity, LC50, maceration. Contributions: DAS conceptualization, data curation, formal analysis, methodology, validation, visualization, writing – original draft, review and editing; FMPM conceptualization, investigation, methodology, vali- dation, and writing – original draft, review and editing; GMN conceptu- alization, methodology, formal analysis, validation, and writing – origi- nal draft, review and editing; HH methodology, visualization, writing – review and editing; LAP resources, investigation, and writing –review and editing; NLNK formal analysis, validation, writing – review and editing; WA resources, supervision, and writing –review & editing; LS resources, investigation, and writing –review and editing; ia resources, investigation, and writing –review and editing. Conflict of interest: the authors declare no conflict of interest. Ethics approval and consent to participate: this research has received eth- ical approval from the Health Research Ethics Commission Universitas Dr. Soebandi Jember, based on ethical certificate 300/KEPK/UDS/IX/2022 during the research, the researcher pays atten- tion to the ethical principles of information to consent, respect for animal rights, and non-maleficence. Patient consent for publication: written informed consent was obtained for anonymized patient information to be published in this article. Funding: this research was supported by the Direktorat Pemebelajaran dan Kemahasiswaan affairs through the Program Kreativitas Mahasiswa (PKM) 2022. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Acknowledgement: we would like to thank Universitas dr. Soebandi for their valuable insights and contributions to this study. Received: 12 September 2023. Accepted: 17 November 2023. Early access: 22 December 2023. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2024; 12:11772 doi:10.4081/hls.2023.11772 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organi- zations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [page 100] [Healthcare in Low-resource Settings 2024;12:11772] Non -co mmerc ial us e o nly ical and mental disabilities and can increase mortality.8–10 Given these facts, disease prevention measures are crucial to prevent their spread and reduce mortality. One of the most efficient preventive actions is the control of disease vectors by eradicating mosquito larvae of Aedes aegypti and Culex using larvicidal agents, such as 1% Temephos in the form of Abate powder. It has been in use since 1976 and has been proven effective in killing Aedes aegypti and Culex mosquito lar- vae. However, the accumulation of Abate residue from its long- term use may lead to water contamination, especially in drinking water, and may create selection pressure that induces larvae to become resistant to the larvicidal agent.11–13 Therefore, there is an urgent need for an alternative larvicide that is safer, more selective, less toxic for the environment, and yet has a similar effect to Abate. Red dragon fruit skin is a natural ingredient that has the same function as abate. Commonly, red dragon fruit skin is often dis- carded despite containing various active compounds, including flavonoids, tannins, alkaloids, and saponins.14–16 Flavonoids are known to be toxic to insects’ digestive and respiratory tracts.17,18 Tannins can inhibit insects in the process of food metabolism.17 Alkaloids have the ability to degrade insect cells,19 and saponins can reduce metabolic work and protein work in insects.17 There is a lack of research exploring red dragon fruit skin as a mosquito lar- vicide agent. This study aimed to explore the larvicidal activity of red dragon fruit skin (Hylocereus polyrhizus) ethanolic extract against Aedes aegypti and Culex mosquito larvae. Materials and Methods This study was an experimental laboratory research that using Aedes aegypti and Culex mosquito larvae as subjects. The material used in this study was red dragon fruit peel, ethanol 96%, HCl 2N, dragendorf reagen, mayer reagen. Magnesium powder, chloro- form, acetic acid, sulfuric acid, FeCl3, PGA, aquadest, and amy- lum. The independent variables were the compound content in the ethanol extract of red dragon fruit peel and variations in herbal Abate concentrations. The dependent variable was the mortality of Aedes aegypti and Culex mosquito larvae, determined by counting the number of mosquito larvae that survived after treatment. Red dragon fruits (Hylocereus polyrhizus) were obtained from Jember area, East Java. The fruits were then cleaned and separated from their fruit flesh. Subsequently, the fruit skin was dried and grounded to form powder. The maceration process was carried out using 96% ethanol to obtain thick extract followed by wet granule formulation to get powder form. Besides, the viscous extract was subjected for phytochemical screening to asses its active ingredi- ents. The larvicidal activity of red dragon skin ethanolic extract against mosquito larva was determined using lethal concentration (LC50). Briefly, the Aedes aegypti and Culex mosquito larvae were treated using different concentrations of powdered extract at 1%, 2%, 3%, and 4%. The 1% Temephos Abate was used as a positive control. The dead larva populations after incubation were count- ed20–23. Subsequently, the LC50 was determined using the probit lin- ear regression test24. Results Plant determination was carried out to determine the tribe and type of the red dragon fruit (Hylocereus polyrhizus) plant. The determination results showed that the sample used was a red drag- on fruit plant (Hylocereus polyrhizus). Phytochemical screening test of red dragon fruit The results showed that the ethanolic extract of red dragon fruit peel contains alkaloids, flavonoids, saponins, triterpenoids, and tannins (Table 1). Differences in the content of plant secondary metabolites can occur due to differences in light, temperature, pH, altitude, and soil conditions in the sample planting areas. Unfortunately, we hardly detected steroid compounds using non- specific color changes testing as well as the quantitative testing using. This may occur due to low steroid content in the extract. Based on the phytochemical screening, red dragon fruit skin extract contains potential compounds that can be used as a biolar- vicidal agent for Aedes aegypti and Culex mosquitoes. Larvicidal activity test The larvicidal activity test of red dragon fruit skin ethanolic extract of was carried out Aedes aegypti and Culex mosquito lar- Transforming Healthcare in Low-Resource Settings: a Multidisciplinary Approach Towards Sustainable Solutions Table 1. Phytochemical screening results. Secondary metabolites Test result Alkaloid (+) Flavonoid (+) Saponin (+) Triterpenoid and steroid Triterpenoid (+) Steroid (-) Tanin (+) Table 2. Data on observation results of effectiveness tests on Culex mosquito larvae. No. Rep. Number of dead larvae Negative Red dragon fruit skin extract Positive control control concentration (1% Themefos Abate) 1% 2% 3% 4% 1 I 5 7 16 21 24 25 2 II 4 13 15 20 25 22 3 III 6 9 18 15 24 26 Amount 15 29 49 56 73 73 Average 5 9,7 16,3 18,7 24,3 24,3 Percentage (%) 19 36 60 69 90 90 Profit value 4.12 4.64 5.25 5.50 6.28 6.28 [Healthcare in Low-resource Settings 2024;12:11772] [page 101] Non -co mmerc ial us e o nly vae. Selection of third instar larvae because these larvae have a larger size. In addition, third-instar mosquito larvae have relatively good resistance to the external environment and mechanically stronger resistance when transferring the larvae. Each test group, 25 mosquito larvae were used in 100 mL of water. The test was car- ried out for 24 hours with three replications. Our results revealed hat the ethanolic extract of red dragon fruit skin could not kill the Aedes aegypti mosquito larvae. However, the larvicidal activity of the extract on Culex mosquito larvae are shown in the Table 2. Based on Table 2, it can be seen that 1% ethanolic extract of red dragon fruit peel can kill 36 % of the population Culex mosquito larvae. The 2% concentration of ethanolic extract killed about 60% of the Culex mosquito larvae and at 3% extract, about 69% of the population Culex mosquito larvae died. At 4% extract concentration 90% of the population Culex mosquito larvae were died, the same death percentage as seen in the positive control. It can be concluded that the higher concentration of the ethanolic extract of red dragon fruit skin, the higher the death precentage of Culex mosquito larvae. The probit linear regression test showed that the LC50 value of the ethanolic extract of red dragon fruit peel against Culex mosquito larvae was 19.06% and proven to be toxic for Culex mosquito larvae. Discussion This research was conducted to determine the effectiveness of red dragon fruit peel extract (Hylocereus polyrhizus) against Aedes aegypti and Culex mosquito larvae. Previous studies using soursop and Angsana leaf extract had LC50 values of 0.736% and 0.83%, respectively, on Culex mosquito larvae.25,26 From the results of this study, the LC50 value of the ethanol extract of red dragon fruit peel against Culex mosquito larvae was 19.06%. This shows that the herbal abate 96% ethanol extract of red dragon fruit peel is more toxic to Culex mosquito larvae than soursop and angsana leaf extract. Compounds that may be toxic from the ethanol extract of red dragon fruit peel (Hylocereus polyrhizus) are alkaloids, flavonoids, saponins, triterpenoids, and tannins. Alkaloids are salts that can degrade cell membranes so that they damage cells and can also interfere with the larval nervous system by inhibiting the action of the acetylcholinesterase enzyme.24 Flavonoids can interfere with energy metabolism in mitochondria by inhibiting the electron transport system so that ATP production is inhibited and causes a decrease in oxygen usage by mitochondria. This can inhibit the respiratory chain, oxidative phosphorylation, and break the chain between the respiratory chain and oxidative phosphorylation so that flavonoids can work as respiratory inhibitors in mosquitoes.17 Saponins can reduce the surface tension of the mucous membrane of the larvae’s digestive tract so that the larvae’s walls become cor- rosive and eventually damaged.27 Triterpenoids as stomach poi- sons.28 Tannin compounds can bind to protease enzymes by bind- ing to enzymes by tannins, so the work of these enzymes will be hampered so that cell metabolic processes can be disrupted, and the larvae will lack nutrition.27 This finding can be used in developing an abate product from red dragon fruit peel extract, which is environmentally friendly and has almost the same effectiveness as temefos abate. The herbal abate of red dragon fruit peel is expected to replace the role of temefos abate as a larvicide so that it can reduce environmental pollution, especially water pollution. The limitation of this study is that it only examined one herbal abate formula and only at concen- trations of 1%, 2%, 3%, and 4%. Further research can be carried out so that the herbal abate of red dragon fruit peel can be even bet- ter in terms of formulation, concentration, and appearance and can be used as a larvicide on all mosquito larvae that act as disease vec- tors. This study has a limitation namely only examining the chem- ical compound content ethanol extract of red dragon fruit peel and proving the effectiveness ethanol extract of red dragon fruit peel against Aedes aegypti and Culex mosquito larvae by calculating the LC50 value. Conclusions The phytochemical screening revealed the presence of alka- loids, flavonoids, saponins, triterpenoids, and tannins in the red dragon fruit peel. However, the ethanol extract of red dragon fruit peel (Hylocereus polyrhizus) did not demonstrate effectiveness as a larvicide against Aedes aegypti larvae. Conversely, it was effec- tive against Culexmosquito larvae, with an LC50 value of 19.06%. Future research should investigate the larvicidal efficacy of the ethanol extract of red dragon fruit peel on different mosquito species. References 1. Santoso S, Yahya Y, Suryaningtyas NH, et al. Studi Bioekologi Nyamuk Mansonia Spp Vektor Filariasis Di Kabupaten Tanjung Jabung Timur, Provinsi Jambi. Vektora J Vektor dan Reserv Penyakit 2016;8:71–80. 2. Mulyatno KC, Kotaki T, Yotopranoto S, et al. Detection and serotyping of dengue viruses in aedes aegypti and aedes albopictus (Diptera: Culicidae) collected in Surabaya, Indonesia from 2008 to 2015. Jpn J Infect Dis 2018;71:58–61. 3. Ananta LK, Efendi F, Makhfudli, et al. Social Support and its Correlation with “3M Plus” Behavior in the Prevention of Dengue Hemorrhagic Fever. Indian J Public Heal Res Dev 2019;10:2681. 4. Nadifah F, Farida Muhajir N, Arisandi D, D. Owa Lobo M. Identifikasi Larva Nyamuk Pada Tempat Penampungan Air Di Padukuhan Dero Condong Catur Kabupaten Sleman. J Kesehat Masy Andalas 2017;10:172. 5. Adnyana IMDM, Sumarya IM, Sudaryati NLG. Efficacy and Toxicity of Parasayu incense ash as a Larvicide for the Eradication of Aedes aegypti (Diptera: Culicidae) Mosquito Larvae. J Res Pharm 2022;26:1805–13. 6. Wahidah FF, Hamidah, Rosmanida. Essential oil characteriza- tion of plant as breeding site of aedes aegypti and aedes albopictus. Ann Biol 2020;36:245–7. 7. Buchori D, Mawan A, Nurhayati I, et al. Risk Assessment on the Release of Wolbachia-Infected Aedes aegypti in Yogyakarta, Indonesia. Insects 2022;13(10). 8. Indonesia KKR. Membuka Lembaran Baru. Laporan Tahunan 2022 Deman Berdarah Dengue. Germas 2022. 1–36 p. 9. Rampengan NH. Japanese Ensefalitis. J Biomedik 2018;8:10– 22. 10. Kementerian Kesehatan RI. Direktorat Pencegahan dan Pengendalian Penyakit Menular Kementerian Kesehatan. Kementerian Kesehatan Republik Indonesia. 2018;3:1–119. 11. Hanafiah E, Syuhriatin S, Meidatuzzahra D, Swandayani RE. Efektivitas Penggunaan Abate dan Bactivec Terhadap Kematian Larva Nyamuk Aedes sp. di Kabupaten Lombok Transforming Healthcare in Low-Resource Settings: a Multidisciplinary Approach Towards Sustainable Solutions [page 102] [Healthcare in Low-resource Settings 2024;12:11772] Non -co mmerc ial us e o nly Barat. Lomb J Sci 2019;1:38–41. 12. Utami RW, Haqi DN. Hubungan Faktor Predisposisi Dengan Keberadaan Jentik Nyamuk Aedes Aegypti. J Promkes 2018;6:227. 13. Lusno MFD, Yudhastuti R, Haksama S, Dwirahmadi F, Prayoga D, Farid AF, et al. Integration of climate, transmis- sion, and spread of dengue hemorrhagic fever in endemic areas. J Public Health Africa 2023;14(S2). 14. Sari DN, Wahdaningsih S, Kurniawan H. Analisis Gugus Fungsi Ekstrak Kulit Buah Naga Merah (Hylocereus polyrhizus). J Mhs Farm Fak Kedokt UNTAN 2021;5:1–5. 15. Diyatri I, Juliastuti WS, Ridwan RD, Ananda GC, Waskita FA, Juliana N V, et al. Antibacterial effect of a gingival patch con- taining nano-emulsion of red dragon fruit peel extract on Porphyromonas gingivalis, Aggregatibacter actinomycetem- comitans, and Fusobacterium nucleatum assessed in vitro. J Oral Biol Craniofacial Res 2023;13:386–91. 16. Lim TW, Lim CJ, Liow CA, et al. Studies on the storage sta- bility of betacyanins from fermented red dragon fruit (Hylocereus polyrhizus) drink imparted by xanthan gum and carboxymethyl cellulose. Food Chem 2022;393. 17. Sigit M, Rahmawati I, Candram AYR, Prasetyo FB. Pengaruh Pemberian Ekstrak Dayn Lidah Buaya (Aloe vera) Terhadap Mortalitas Larva Nyamuk. J Vitek Bid Kedokt Hewan 2022;12:17–21. 18. Sok Yen F, Shu Qin C, Tan Shi Xuan S, et al. Hypoglycemic Effects of Plant Flavonoids: A Review. Evidence-based Complement Altern Med 2021;2021. 19. Ahmad A, Adriyanto A. Efektivitas Serbuk Biji Pepaya (Carica Papaya L.) Terhadap Kematian Jentik (Larva) Culex sp. J Med (Media Inf Kesehatan) 2019;6:104–12. 20. La EOJ, Sawiji RT, Yuliawati AN. Skrining Fitokimia Dan Analisis Kromatografi Lapis Tipis Ekstrak Etanol Kulit Buah Naga Merah. Indones J Pharmacy Nat Prod 2020;03:45–58. 21. Noor MI, EY dan Z. Identifikasi Kandungan Ekstrak Kulit Buah Naga Merah Menggunakan Fourier Transform Infrared ( FTIR ) dan Fitokimia Identification Content of the Red Dragon Fruit Extract Skin Using Fourier Transform Infrared ( FTIR ) and Phytochemistry. J Aceh Phys Soc 2016;5:14–6. 22. Pujiastuti E, El’Zeba D. Perbandingan Kadar Flavonoid Total Ekstrak Etanol 70 % Dan 96 % Kulit Buah Naga Merah ( Hylocereus). Cendekia J Pharm 2021;5:28–43. 23. Suhaimi, Kartikasari D. Uji aktivitas larvasida granul ekstrak batang seledri (Avium graveolens) pada larva instar 3 Aedes aegypti. J Insa Farm Indones 2018;1:260–7. 24. Utami WW, Ahmad AR, Malik A. Uji Aktivitas Larvasida Ekstrak Daun Jarak Kepyar (Ricinus communis L.) Terhadap Larva Nyamuk Aedes aegypti. J Fitofarmaka Indones 2016;3:141–5. 25. Kewa M, Almet J, Laut MM. Median Lethal Concentration (Lc50) Ekstrak Daun Sirsak (Annona muricata Linn) Terhadap Larva Culex sp di Kota Lampung. J Kaji Vet 2020;4:791–2. 26. Abdurrozak MI, Syafnir L, Sadiyah ER. Uji Efektivitas Ekstrak Etanol Daun Angsana (Pterocarpus Indicus Willd) sebagai Biolarvasida terhadap Larva Nyamuk Culex Sp. J Ris Farm 2021;1:33–7. 27. Ramayanti I, Febriani R. Uji Efektivitas Larvasida Ekstrak Daun Pepaya (Carica papaya Linn) terhadap Larva Aedes aegypti. Syifa’ Med J Kedokt dan Kesehat 2016;6:79. 28. Wulansari. Analisis Senyawa Metabolit Sekunder dan Uji Aktivitas Larvasida Alami Pada Ekstral Etanol Daun Bidara (Ziziphus mauritiana Lamk.) Terhadap Larva Aedes aegypti. Skripsi. Islam Negeri Maulana Malik Ibrahim, Malang; 2022 Vol. 3. Transforming Healthcare in Low-Resource Settings: a Multidisciplinary Approach Towards Sustainable Solutions [Healthcare in Low-resource Settings 2024;12:11772] [page 103] Non -co mmerc ial us e o nly