Formulation of Effervescent Granules from Bangkal Tree (Nauclea orientalis) Leaf Extract: A Potential

Larvicide Against Aedes Aegypti

Marin, Anthony R.*, Baccay, Princess Nicole M., Cruz, Honeylene M., Tibayan, Ghiesel Anne B., Tria, Lorna M.

Department of Pharmacy, St Dominic College of Asia, Bacoor. Cavite, Philippines

*Correspondence: amarin@sdca.edu.ph

DOI: https://doi.org/10.71431/IJRPAS.2026.5618 

INTRODUCTION

Even today, diseases carried by mosquitoes continue to cause serious harm to millions of people worldwide. What should be mentioned first of all is that mosquito-transmitted diseases occur when mosquitoes bite people who are infected with certain viruses or parasites (World Health Organization [WHO], 2022). The diseases usually carried by mosquitoes include malaria, dengue fever, chikungunya, and Zika virus infection. There has been an increased occurrence of dengue fever in Southeast Asia. The Philippines has had the greatest number of cases among all the countries located in the region. In 2023, the number of cases of dengue fever recorded in the country was 195,603 As such, the number of cases of dengue in the Philippines is higher compared to those recorded in other neighboring states including Vietnam, Thailand, Malaysia, Indonesia, Laos PDR, and Cambodia (Olana et al., 2025).

WHO (2020) states that herbal medicines may be considered as an appropriate substitute if proven by scientific studies for their effectiveness and safety. According to WHO, active components such as alkaloids, tannins, flavonoids, and phenolics have medicinal importance due to their therapeutic and biological activity. As a result, there arises an interest in medicinal plants like Nauclea orientalis which may contribute to eco-friendly mosquito repellent management.

The primary problem addressed in this research is the need to rigorously evaluate the potential larvicidal efficacy of Nauclea orientalis (Bangkal) leaf extract against Aedes aegypti in the form of effervescent granules. The study aims to answer the following questions:

·         How can Nauclea orientalis leaf extract be formulated as an effervescent granule?

·         What is the percent mortality rate of Aedes aegypti in crude extract and various effervescent granules formulation of Nauclea orientalis leaf extract?

·         Is there a significant difference in the larvicidal activity using different treatment groups against Aedes aegypti?

The research project has a defined scope aimed at assessing the potential Larvicidal activity of Nauclea orientalis leaf extract and its effervescent granule form in a controlled laboratory setting. However, it also acknowledges limitations inherent to experimental research and the need for further studies to validate and apply the findings in broader contexts.

MATERIALS AND METHODS

Research Design

The research is an analytical experimental study of the larvicidal activity of the leaf extract of Nauclea orientalis (Bangkal) in accordance with the protocol described by the World Health Organization for testing mosquito larvicides in laboratories.

Research Locale

The plant sample Nauclea orientalis (Bangkal) is collected from Malagasang, Imus City of Cavite. The plant sample is identified and authenticated by the Jose Vera Santos Memorial Herbarium (PUH), Institute of Biology, University of the Philippines Diliman. The extraction process are conducted in the College of Pharmacy Laboratory at St. Dominic College of Asia located at Bacoor City of Cavite. 

Extraction of Plant Materials

Fresh leaf of Nauclea orientalis (Bangkal) were washed, sliced and allowed to dried in room temperature for two weeks until crispy. Further, the leaves were separately pulverized in electric grinder. The powdered plant material was then subjected to extraction using the method of maceration. Hence, 100 g each of the leaves powder was subjected to soaking in ethanol (700mL) for 24 hours in room temperature. Subsequently, the extract was filtered and subjected to drying through rotary evaporator to give crude extract. Parajuli-Baral, K. (2023)

Formulation of Effervescent Granules

The formulation of the effervescent granules is carried out according to the method used by Kalpana Parajuli-Baral (2023) with slight modifications. The Nauclea orientalis leaves that were extracted previously will be used as the active ingredient. Other ingredients that will be used are the effervescent agents-sodium bicarbonate, tartaric acid, and citric acid.

Table 1. General Ingredients of Effervescent Granules

Herbal effervescent granules were prepared by wet granulation method. The Nauclea orientalis leaf extract (active ingredient) 2.5 g, citric acid 12 g, tartaric acid 24 g and sodium bicarbonate 41.28 g were triturated in a mortar and pestle to make a fine powder. Then sufficient alcohol was added to make a damp mass. This mass was passed through sieve no. 10 to get granules and these granules were dried in a hot air oven at 40°C and then they were packed in an air-tight container.

Collection and Preparation of Larvae

The test organisms Aedes aegypti will be used in the experimental procedure. The eggs of Aedes aegypti were identified and obtained in the University of the Philippines, Los banos, Laguna. The newly acquired Aedes aegypti eggs will be reared in a plastic microwaveable container – 23cm x 15cm x 6cm filled with pre-boiled water (cooled at room temp) at a depth of at least 1 inch and aerated through lid punctures. The paper strip with eggs is submerged to the container and waited for 24 h or until most of the eggs have hatched into larvae. Once first instar larvae were hatched the next day, they were fed intermittently once a day with yeast tablets (ratio 1:2 w/w). Formation of residual food artifacts detrimental to the larvae development was closely monitored and periodically removed. (Areshi S. et.al 2023). Once the third instar larvae were hatched and obtained for the larvicidal experiments in the present study, they will be transferred into a plastic cup container according to their different groups.

Larvicidal Bioassay Test

The larvicidal activity of the Nauclea orientalis extracts was tested against the Aedes aegypti larvae in accordance with the protocol of the World Health Organization with slight modifications (WHO 2005). The Aedes aegypti larvae were exposed to varying concentrations in each experimental group. In experimental group 1, which utilizes Bangkal leaf extract, trials were conducted with varying concentrations to be exposed to the mosquito larvae. In experimental group 2, which utilized Bangkal effervescent granules, trials were conducted with varying concentrations. In experimental group 3, standard larvicide was used. Thus, in the control group, the mosquito larvae were exposed to tap water.  In each trial, 25 Aedes aegypti larvae were used. The toxicity of the plant extract based on their concentration was determined using the mortality rate with the formula. This was observed after 24 hours time estimation for Aedes aegypti larvae exposure. (Pam Euza A. Bayon et.al., 2023)

Statistical Analysis

One-way Analysis of Variance (ANOVA) was used in order to determine if the means of mortality between the experimental and control groups were significantly different based on the concentrations exposed to the mosquito larvae, it was used to compare the two experimental groups' various concentrations. (Pam Euza A. Bayon et.al., 2023

RESULT AND DISCUSSION

This chapter presents the results obtained from the larvicidal bioassay experiments conducted to evaluate the efficacy of Bangkal (Nauclea orientalis) leaf extract and its formulated effervescent granules against Aedes aegypti larvae. The findings are presented through descriptive statistics and analyzed to determine the larvicidal potential of the plant extract at various concentrations and exposure times.

How can Nauclea orientalis leaf extract be formulated as an effervescent granule?

Figure 1. Formulation of Nauclea orientalis leaf extract as an effervescent granule

The formulation of the effervescent granules is carried out according to the method used by Kalpana Parajuli-Baral (2023) with slight modifications. The Nauclea orientalis leaves that were extracted previously will be used as the active ingredient. Other ingredients that will be used are the effervescent agents-sodium bicarbonate, tartaric acid, and citric acid.

What is the percent mortality rate of Aedes aegypti in crude extract and various effervescent granules formulation of Nauclea orientalis leaf extract?

Table 2. Larvicidal Activity of Bangkal (Nauclea orientalis) Leaf Extract Against Aedes aegypti

Table 2 presents the mortality rates of Aedes aegypti larvae exposed to crude Bangkal leaf extract at three different concentrations (0.25g, 0.50g, and 1.0g) over three observation periods (1 hour, 12 hours, and 24 hours).

The findings prove that the extract of Bangkal leaves can be used as a larvicide against Aedes aegypti since its effectiveness depends on both concentration and contact time. The one-hour exposure did not yield any mortality regardless of the concentrations used, thus, indicating that the compounds need to have more contact time with the larvae in order to cause toxicity.

During the 12-hour exposure, there was noticeable larvicidal action for higher concentrations. There was 12% mortality for the 0.50g concentration (3 larvae) and 20% mortality for the 1.0g concentration (5 larvae). It was apparent that lower concentrations did not show effectiveness during this contact time, thus, indicating that a certain concentration level was needed for effective action.

Mortality was evident during the 24-hour exposure regardless of the concentrations used, which further confirms the dependence of the larvicidal action of the extract from the contact time. While the lowest concentration caused 28% mortality, the medium concentration yielded 40% mortality and the highest concentration produced 56% mortality.

Table 3. Larvicidal Activity of Effervescent Granules F1 (2000 ppm)

Table 3 shows that formulation F1 containing 2000 ppm of Bangkal extract showed no larvicidal activity at 1 hour. However, at 12 hours, mortality ranged from 8% to 40% depending on the concentration used. At 24 hours, the highest concentration (1.0g) achieved 68% mortality, representing a significant improvement over the crude extract.

Table 4. Larvicidal Activity of Effervescent Granules F2 (4000 ppm)

Table 4 indicates that formulation F2 with 4000 ppm demonstrated enhanced larvicidal activity compared to F1. At 12 hours, mortality rates ranged from 20% to 48%, showing earlier onset of action. At 24 hours, the formulation achieved 80% mortality at the highest concentration, indicating superior efficacy compared to both the crude extract and F1 formulation.

Table 5. Larvicidal Activity of Effervescent Granules F3 (6000 ppm)

Table 5 shows that formulation F3 containing the highest concentration (6000 ppm) exhibited the most potent larvicidal activity among all formulations tested. At 12 hours, mortality rates ranged from 32% to 60%, demonstrating rapid action even at lower application rates. At 24 hours, F3 achieved the highest mortality rate of 92% at 1.0g concentration, approaching the efficacy of the positive control while offering a natural alternative to synthetic larvicides.

Table 6. Larvicidal Activity of Temephos Sand (Positive Control)

Table 6 reflects that the temephos sand, a widely used synthetic organophosphate larvicide, served as the positive control. The results demonstrate rapid and complete larvicidal activity, with 32-76% mortality within 1 hour and 100% mortality achieved by 12 hours across all concentrations. This confirms the sensitivity of the test organisms and validates the experimental methodology

Table 7. Mortality of Aedes aegypti Larvae in Tap Water (Negative Control)

Table 7 indicates that the negative control group, in which Aedes aegypti larvae were exposed to tap water only, showed zero mortality across all observation periods (1, 12, and 24 hours). This confirms that tap water alone does not possess any larvicidal properties and that the observed mortality in experimental groups was directly attributable to the Bangkal extract and its formulations.

Is there a significant difference in the larvicidal activity using different treatment groups against Aedes aegypti?

One-Way Analysis of Variance (ANOVA) - Complete Breakdown

One-way ANOVA was conducted to test the null hypothesis that there is no significant difference in mean mortality rates among the six treatment groups (crude extract, F1, F2, F3, temephos, tap water) at the highest concentration (1.0g) after 24 hours.

Table 8. One-Way ANOVA Summary Table (1.0g concentration, 24 hours)

            Interpretation: The F-statistic value of 1,148.00 and p-value < 0.001 indicate very high proof of rejection of the null hypothesis. This is demonstrated by the high sum of squares between groups, which is 45,920.00 compared to the sum of squares within groups, which is 96.00, hence 99.8% of the variations are explained by the difference in groups. As the p-value is much lower than 0.001 (α = 0.05), we reject null hypothesis.

Tukey's Honestly Significant Difference (HSD) Post-Hoc Test

Following the significant ANOVA result, Tukey's HSD post-hoc test was conducted to identify which specific pairs of formulations differ significantly from each other. Tukey's HSD controls for Type I error rate across multiple comparisons

Table 9. Tukey's HSD Post-Hoc Pairwise Comparisons (1.0g, 24 hours)

            Note: *** indicates p < 0.001 (highly significant), ** indicates p < 0.01 (very significant). 95% CI = 95% Confidence Interval for the mean difference. All botanical formulations differ significantly from each other and from both controls (p < 0.001), establishing a clear efficacy hierarchy: Tap Water < Crude Extract < F1 < F2 < F3 < Temephos.

CONCLUSION

From the results of the above-stated study, the following conclusions could be drawn regarding the larvicidal efficacy of Bangkal (Nauclea orientalis) leaf extract.

·         The crude ethanolic extract of Nauclea orientalis leaves showed concentration-dependent and time-dependent larvicidal properties with a positive correlation between extract concentration, exposure duration, and mortality.

·         Formulation of Bangkal leaf extract as effervescent granules increases its larvicidal efficacy in comparison with the crude extract due to better dispersion and absorption of bioactive compounds leading to increased mortality rates.

·         From the three formulated extracts, F3 containing 6000 ppm of plant extract exhibited high larvicidal efficacy (mortality rate 92% after 24 hours). In terms of effectiveness, the extract approached that of temephos while having many environmental and health benefits.

·         There is a significant difference between the larvicidal activity of Nauclea orientalis leaf extract formulated as effervescent granules and Aedes aegypti larvae, which rejects the null hypothesis and confirms the alternative one.

·         Compounds identified in Nauclea orientalis (alkaloids, flavonoids, terpenoids, tannins) were the sources of larvicidal activity due to their physiological and metabolic interference with larval functions.

·         This study confirms the use of Bangkal as an organic larvicide, offering scientific support for its potential use in vector control programs. The preparation of effervescent granules presents a feasible approach to implement in community level dengue disease control campaigns in locations characterized by the presence of Aedes aegypti breeding sites.

·         The plant-based larvicide discovered in this study constitutes an environmentally friendly alternative to chemical compounds and can contribute to sustainable mosquito control methods without causing harm to the environment and avoiding the emergence of pesticide-resistant mosquitoes. Nonetheless, it should be acknowledged that although F3 formulation was highly effective in inducing 92% mortality, it failed to meet the 100% mortality rate exhibited by temephos but still produced a high mortality rate, showing its potential to be an effective alternative.

CONFLICT OF INTEREST

The authors declare that they have no known conflict of interest that could have appeared in the influence of work reported in this manuscript.

ACKNOWLEDGEMENT

We would like to express our sincerest gratitude to the people that offered us support and encouragement throughout the course of our manuscript making.

We would like to give our heartfelt appreciation to our ever-supportive program chair, Prof. Anthony R. Marin, RPh, MSPharm, for his continuous motivation, guidance, patience, insightful critiques, and unwavering encouragement towards the completion of this study. His expertise, imparted knowledge, and deep commitment to academic excellence and meticulous attention to detail have significantly shaped this paper.

We owe our deepest and most sincere gratitude to our family for being our constant source of strength, comfort, and inspiration. Their unconditional love, endless patience, unwavering belief in us, financial support, and even prayers made the most challenging  moments in the making of this manuscript be more bearable. We could never have reached this point without all of you. We dedicate this work to all of you with all our love and gratitude.

REFERENCES       

1.        Ahmed, M., et al. (2023). Synergistic effects of plant secondary metabolites in mosquito control.

2.        Allen, L. V. (Ed.). (2020). Remington: The science and practice of pharmacy (23rd ed.). Pharmaceutical Press.

3.        Allen, L. V., Popovich, N. G., & Ansel, H. C. (2021). Ansel’s pharmaceutical dosage forms and drug delivery systems (12th ed.). Wolters Kluwer.

4.        Becker, N. (2021). Microbial control of mosquitoes: Bacillus thuringiensis israelensis and its applications. Current Opinion in Insect Science, 48, 1–7.

5.        Brunton, L. L., Hilal-Dandan, R., & Knollmann, B. C. (Eds.). (2018). Goodman & Gilman’s the pharmacological basis of therapeutics (13th ed.). McGraw-Hill Education.

6.        Clements, A. N. (2012). The biology of mosquitoes (Vol. 1). CABI Publishing.

7.        Dewick, P. M. (2009). Medicinal natural products: A biosynthetic approach (3rd ed.).

8.        Dixit, U. C. (2020). Plant secondary metabolites. CRC Press.

9.        Evans, W. C. (2009). Trease and Evans pharmacognosy (16th ed.). Saunders Elsevier

10.    Hershan, A. A. (2023). Dengue virus: Molecular biology and recent developments in control strategies, prevention, management, and therapeutics.

11.    Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2021). Brock biology of microorganisms (16th ed.). Pearson.

12.    Merrill, E. D. (1923). A dictionary of Philippine plant names. Bureau of Printing.

13.    Service, M. W. (2012). Medical entomology for students (5th ed.). Cambridge University Press.

14.    Tadeo, J. L., Sánchez-Brunete, C., Albero, B., & García-Valcárcel, A. I. (2019). Analysis of pesticides: Methods and applications. CRC Press.

15.    Wilson, A. L., & Carson, R. T. (2023). Public health implications of pyrethroid resistance in mosquitoes. Vector-Borne and Zoonotic Diseases.

16.    World Health Organization. (2017). Global vector control response 2017–2030. World Health Organization.

17.    Harborne, J. B. (1998). Phytochemical methods: A guide to modern techniques of plant analysis (3rd ed.). Springer. https://doi.org/10.1007/978-94-009-5570-7

18.    Isman, M. B. (2020). Botanical insecticides in the twenty-first century. Annual Review of Entomology, 65, 233–249. https://doi.org/10.1146/annurev-ento-011019-025010

19.    Kurek, J. (2019). Alkaloids: Their importance in nature and human life. IntechOpen. https://doi.org/10.5772/intechopen.85400

20.    Moyes, C. L., et al (2021). Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations. Parasites & Vectors, 14(1), Article 115. https://doi.org/10.1186/s13071-021-04609-5

21.    Pardi, N., & Krammer, F. (2024). mRNA vaccines for infectious diseases—Advances, challenges and opportunities. Nature Reviews Drug Discovery, 23, 838–861. https://doi.org/10.1038/s41573-024-01042-y

22.    Rajput, A., Sharma, R., & Bharti, R. (2021). Pharmacological activities and toxicities of alkaloids on human health. In Materials Today: Proceedings. https://doi.org/10.1016/j.matpr.2021.09.189

23.    Ruxton, G. D., & Colegrave, N. (2011). Experimental design for the life sciences (3rd ed.). Oxford University Press.

24.    Sparks, T. C., & Nauen, R. (2021). Insecticide chemistry and mode of action. Pest Management Science, 77(2), 403–406. https://doi.org/10.1002/ps.6070

25.    World Health Organization. (2024). Global vector control response and integrated vector management guidelines. https://www.who.int

26.    World Health Organization. (2024). Global vector control response and insecticide resistance management guidelines. https://www.who.int

27.    Zulfa, R., Lo, W.-C., Cheng, P.-C., Martini, M., & Chuang, T.-W. (2022). Updating the insecticide resistance status of Aedes aegypti and Aedes albopictus in Asia: A systematic review and meta-analysis. Tropical Medicine and Infectious Disease, 7(10), 306. https://doi.org/10.3390/tropicalmed7100306

28.    Adhikari, K., & Khanikor, B. (2021). Gradual reduction of susceptibility and enhanced   detoxifying enzyme activities of laboratory-reared Aedes aegypti under exposure of temephos for 28 generations. Toxicology Reports, 8, 1883–1891. https://doi.org/10.1016/j.toxrep.2021.11.013

29.    Aleonar, B. C. (2024). Analysis of the commercialized traditional medicinal plants and their therapeutic claims sold on the sidewalks in Iligan City, Philippines. Himalayan Journal of Applied Medical Sciences and Research, 5. https://doi.org/10.47310/hjamsr.2024.v05i01.013

30.    Alkaloids, D., Fowsiya, J., & Madhumitha, G. (2020). A review of bioinsecticidal activity and mode of action of plant extracts. Research Journal of Pharmacy and Technology, 13(2), 963. https://doi.org/10.5958/0974-360X.2020.00181.X

31.    Araniti, F., Landi, M., Laudicina, V. A., & Abenavoli, M. R. (2021). Secondary metabolites and eco-friendly techniques for agricultural weed/pest management. Plants, 10(7), 1418. https://doi.org/10.3390/plants10071418

32.    Aryal, P., et al. (2022). Potential therapeutic applications of plant-derived alkaloids against inflammatory and neurodegenerative diseases. Evidence-Based Complementary and Alternative Medicine, 2022, 1–18. https://doi.org/10.1155/2022/7299778

33.    Aryal, S., Kondhare, K. R., & Giri, A. P. (2021). Diversity in chemical structures and biological properties of plant alkaloids. Molecules, 26(11), 3374. https://doi.org/10.3390/molecules26113374 

34.    Aryaprema, V. S. (2023). A systematic review of published literature on mosquito control action thresholds across the world. PLoS Neglected Tropical Diseases, 17(3), e0011173. https://doi.org/10.1371/journal.pntd.0011173

35.    Balingit, J. C., Carvajal, T. M., Saito-Obata, M., et al. (2020). Surveillance of dengue virus in individual Aedes aegypti mosquitoes collected concurrently with suspected human cases in Tarlac City, Philippines. Parasites & Vectors, 13, 594. https://doi.org/10.1186/s13071-020-04470-y 

36.    Barbehenn, R. V., & Constabel, C. P. (2011). Tannins in plant–herbivore interactions. Phytochemistry, 72(13), 1551–1565. https://doi.org/10.1016/j.phytochem.2011.01.040 

37.    Besharati, M., et al. (2022). Tannin in ruminant nutrition: Review. Molecules, 27(23), 8273. https://doi.org/10.3390/molecules27238273

38.    Cordero, C., Ligsay, A., & Alejandro, G. (2020). Ethnobotanical documentation of medicinal plants used by the Ati tribe in Malay, Aklan, Philippines. Journal of Complementary Medicine Research, 11(1), 170. https://doi.org/10.5455/jcmr.2020.11.01.20

39.    Dias, M. C., Pinto, D. C. G. A., & Silva, A. M. S. (2021). Plant flavonoids: Chemical characteristics and biological activity. Molecules, 26(17), 5377. https://doi.org/10.3390/molecules26175377

40.    Dutta, U., & Dey, S. (2023). Bioassay of larvicidal efficacy of selected plant extracts against mosquito larvae Anopheles culicifacies and Aedes aegypti. Bulletin of Pure & Applied Sciences-Zoology, 42(1), 170–189. https://doi.org/10.48165/bpas.2023.42a.1.16

41.    Gan, S. J., et al. (2021). Dengue fever and insecticide resistance in Aedes mosquitoes in Southeast Asia: A review. Parasites & Vectors, 14, 315. https://doi.org/10.1186/s13071-021-04785-4

42.    Harapan, H., Ryan, M., Yohan, B., et al. (2020). Dengue: A minireview. Viruses, 12(8), 829. https://doi.org/10.3390/v12080829

43.    Ioannou, C. S., et al. (2021). Resistance development in Aedes albopictus under diflubenzuron and Bacillus thuringiensis israelensis selection pressure. Pest Management Science, 77(7), 3130–3140. https://doi.org/10.1002/ps.6350

44.    Iqbal, N., Agrawal, A., & Kumar, J. (2021). Development of effervescent tablet formulation for rapid control of mosquito problem in early stages from different breeding sites. Arabian Journal of Chemistry, 14(4), 103082. https://doi.org/10.1016/j.arabjc.2021.103082

45.    Jiang, L., Liu, Y., Su, W., Cao, Y., Jing, Q., Wu, X., & Yang, Z. (2022). Circulation of genotypes of dengue virus serotype 2 in Guangzhou over a period of 20 years. Virology Journal, 19(1), 47. https://doi.org/10.1186/s12985-022-01773-7 

46.    Kanchanapoom, T., Sahakitpichan, P., Chimnoi, N., Petchthong, C., Thamniyom, W., Nangkoed, P., & Ruchirawat, S. (2021). Monoterpene alkaloid glycosides from the leaves of Nauclea orientalis. Phytochemistry Letters, 41, 83–87. https://doi.org/10.1016/j.phytol.2020.11.016 

47.    Kumar, S., & Pandey, A. K. (2013). Chemistry and biological activities of flavonoids: An overview. The Scientific World Journal, 2013, 162750. https://doi.org/10.1155/2013/162750

48.    Kurek, J. (2019). Introductory chapter: Alkaloids—Their importance in nature and for human life. In Alkaloids - Their Importance in Nature and Human Life. IntechOpen. https://doi.org/10.5772/intechopen.85400

49.    Liu, N. (2019). Insecticide resistance in mosquitoes: Impact, mechanisms, and research directions. Annual Review of Entomology, 60, 537–559. https://doi.org/10.1146/annurev-ento-010814-020828

50.    Martínez-Mercado, J. P., et al. (2022). Temephos, an organophosphate larvicide for residential use: A review of its toxicity. Critical Reviews in Toxicology, 52(2), 113–124. https://doi.org/10.1080/10408444.2022.2065967

51.    Ngau, T. H., Phuong, N. M., & Bien, L. D. (2024). Phytochemicals, antioxidant, α-amylase inhibitory and antihyperglycemic potentials of ethanol extract from Nauclea orientalis bark. Tạp Chí Khoa Học Đại Học Công Thương, 24(5), 19–32. https://doi.org/10.62985/j.huit_ojs.vol24.no5.128

52.    Parajuli-Baral, K., et al. (2023). Formulation and evaluation of quality parameters of effervescent granules from Ocimum tenuiflorum. Evidence-Based Complementary and Alternative Medicine, 2023, 2050846. https://doi.org/10.1155/2023/2050846

53.    Pathak, V. M., et al. (2022). Current status of pesticide effects on environment, human health and its eco-friendly management as bioremediation: A comprehensive review. Frontiers in Microbiology, 13, 962619. https://doi.org/10.3389/fmicb.2022.962619

54.    Pavela, R., Maggi, F., Iannarelli, R., & Benelli, G. (2019). Plant extracts for developing mosquito larvicides: From laboratory to the field, with insights on the modes of action. Acta Tropica, 193, 236–271. https://doi.org/10.1016/j.actatropica.2019.01.019 

55.    Rahaman, M., Gope, A., Khanrah, J., & Rawani, A. (2024). A review on recent advances of natural products as larvicides in vector control management. Jordan Journal of Pharmaceutical Sciences, 17(1), 78–104. https://doi.org/10.35516/jjps.v17i1.1792

56.    Sandamali, J. A. N., Hewawasam, R. P., Jayatilaka, K. A. P. W., & Mudduwa, L. K. B. (2022). Nauclea orientalis bark extract protects rat cardiomyocytes from doxorubicin-induced oxidative stress, inflammation, apoptosis, and DNA fragmentation. Oxidative Medicine and Cellular Longevity, 2022(1). https://doi.org/10.1155/2022/1714841

57.    Silvério, M. R. S., Espindola, L. S., Lopes, N. P., & Vieira, P. C. (2020). Plant natural products for the control of Aedes aegypti: The main vector of important arboviruses. Molecules, 25(15), 3484. https://doi.org/10.3390/molecules25153484

58.    Songoen, W., Brunmair, J., Traxler, F., Wieser, V. C., Phanchai, W., Pluempanupat, W., Brecker, L., & Schinnerl, J. (2022). Yellow Twig (Nauclea orientalis) from Thailand: Strictosamide as the key alkaloid of this plant species. Molecules, 27(16), 5176. https://doi.org/10.3390/molecules27165176 

59.    Songoen, W., Brunmair, J., Traxler, F., Wieser, V. C., Phanchai, W., Pluempanupat, W., Brecker, L., & Schinnerl, J. (2022). Yellow twig (Nauclea orientalis) from Thailand: Strictosamide as the key alkaloid of this plant species. Molecules, 27(16), 5176. https://doi.org/10.3390/molecules27165176 

60.    Sparks, T. C., Crossthwaite, A. J., Nauen, R., et al. (2020). Insecticides, biologics and nematicides: Updates to IRAC’s mode of action classification. Pesticide Biochemistry and Physiology, 167, 104587. https://doi.org/10.1016/j.pestbp.2020.104587

61.    Ukoroije, R. B., & Otayor, R. A. (2020). Review on the bio-insecticidal properties of some plant secondary metabolites: Types, formulations, modes of action, advantages and limitations. Asian Journal of Research in Zoology, 3(4), 27–60. https://doi.org/10.9734/ajriz/2020/v3i430099 

62.    Tudi, M., Daniel Ruan, H., Wang, L., Lyu, J., Sadler, R., Connell, D., Chu, C., & Phung, D. T. (2021). Agriculture development, pesticide application and its impact on the environment. International Journal of Environmental Research and Public Health, 18(3), 1112. https://doi.org/10.3390/ijerph18031112

63.    Wang, Y., Wang, X., Brown, D. J., An, M., Xue, R.-D., & Liu, N. (2023). Insecticide resistance: Status and potential mechanisms in Aedes aegypti. Pesticide Biochemistry and Physiology, 195, 105577. https://doi.org/10.1016/j.pestbp.2023.105577 

64.    Yang, K. J., Lee, J., & Park, H. L. (2020). Organophosphate pesticide exposure and breast cancer risk: A rapid review of human, animal, and cell-based studies. International Journal of Environmental Research and Public Health, 17(14), 5030. https://doi.org/10.3390/ijerph17145030 

65.    Yang, Y., et al. (2020). Advances in pharmacological activities of terpenoids. Natural Product Communications, 15(3). https://doi.org/10.1177/1934578X20903555

66.    Ahsan, Z., Wu, Z., Lin, Z., Ji, T., & Wang, K. (2025). Sublethal effects of neonicotinoids on honeybees. Biology, 14(8), 1076. https://doi.org/10.3390/biology14081076

67.    Alves, G. B., Melo, F. L., Oliveira, E. E., Haddi, K., Costa, L. T. M., Dias, M. L., et al. (2020). Comparative genomic analysis and mosquito larvicidal activity of four Bacillus thuringiensis serovar israelensis strains. Scientific Reports, 10, 666. https://doi.org/10.1038/s41598-020-60670-7

68.    Barbieri, M. V., et al. (2020). Polar pesticide residues in sediments and ecological risk assessment. Science of the Total Environment. 

69.    Centers for Disease Control and Prevention. (2022). Pesticide exposure and long-term health effects. https://www.cdc.gov

70.    Centers for Disease Control and Prevention. (2024). Larvicides and mosquito control safety guidelines. https://www.cdc.gov

71.    Chattopadhyay, A., et al. (2023). Pesticide-mediated collapse in plant–pollinator networks. Environmental Pollution. 

72.    CMMCP. (2024). Microbial larvicides for mosquito control. 

73.    Department of Health (Philippines). (2023). Dengue prevention and mosquito-borne disease information in the Philippines. https://doh.gov.ph

74.    Department of Health (Philippines). (2024). National dengue morbidity and mortality reduction targets. https://doh.gov.ph

75.    Environmental Protection Agency. (2023). Biopesticides and pesticide registration guidelines. https://www.epa.gov

76.    European Environment Agency. (2021). Pesticides and human health risks in Europe. https://www.eea.europa.eu

77.    Food and Agriculture Organization. (2021). International code of conduct on pesticide management. https://www.fao.org

78.    Food and Agriculture Organization. (2023). International regulations on pesticide use. https://www.fao.org

79.    Land, M., Bundschuh, M., Hopkins, R. J., Poulin, B., & McKie, B. G. (2023). Effects of Bacillus thuringiensis israelensis on aquatic ecosystems: A systematic review. Environmental Evidence, 12, 26. https://doi.org/10.1186/s13750-023-00319-w

80.    National Parks Development Committee. (2023). Bangkal tree information. https://www.npdc.gov.ph

81.    PAHO/WHO. (2024). Mosquito-borne disease transmission and vector infection process. https://www.paho.org

82.    Pesticide Action Network. (2023). Effects of insecticides on biodiversity and pollinators. https://www.panap.net

83.    Philippine News Agency. (2025). Dengue cases and deaths in the Philippines. https://www.pna.gov.ph

84.    Stuart Jr., G. (2025). Nauclea orientalis (Bangkal): Medicinal uses and phytochemicals. https://www.stuartxchange.org

85.    Syahrir, M. S., et al. (2025). Toxicological impacts of agricultural pesticides on pollinators. Journal of Chemical Health Risks. 

86.    Valtierra-de-Luis, D., Villanueva, M., Lai, L., Williams, T., & Caballero, P. (2020). Potential of Cry10Aa and Cyt2Ba toxins for control of Aedes aegypti larvae. Toxins, 12(6), 355. https://doi.org/10.3390/toxins12060355

87.    Vox. (2023). Decline of bats and insecticide use. https://www.vox.com

88.    Washington State Department of Health. (2024). Bti larvicide safety information. https://doh.wa.gov 

89.    World Bank. (2020). Pesticides, public health, and socio-economic implications. https://www.worldbank.org