Phytochemicals, Anti-Amylase and Anti-Acetylcholinesterase Activities of Honey and Bee Bread of Stingless Bees
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Abstract
Honey and bee bread from stingless bees are widely recognized for their health benefits and are considered promising sources of bioactive compounds for medicinal applications. Inhibition for amylase and acetylcholinesterase activities is an important medicinal property involved in the prevention of type 2 diabetes and Alzheimer’s disease, respectively. In this study, the anti-amylase and anti-acetylcholinesterase activities of stingless bee honey and bee bread, flower extracts of Clitoria ternatea L., and a honey-flower drink were evaluated using colorimetric assays. The phytochemical profiles of these samples were further analyzed by Fourier Transform Infrared (FTIR) spectroscopy and Gas Chromatography-Mass Spectrometry (GC-MS). The findings of this study indicated the presence of anti-amylase and anti-acetylcholinesterase activities in the honey and bee bread, flower extracts of C. ternatea L., and the formulated flower and honey drink. The flower and honey drink exhibited the strongest anti-acetylcholinesterase activity, while ethanol extracts from bee bread demonstrated the highest anti-amylase activity. In addition, these findings indicated that the FTIR showed functional groups of flavonoids, while GC–MS confirmed the presence of several phytochemicals with biological activities. Interestingly, a siginifcant positive correlation was found between anti-acetylcholinesterase activity and FTIR data (β=0.427, p-value <0.05), while a significant negative correlation was found between anti-amylase activity and FTIR data (β=-0.307, p-value<0.05). Honey and bee bread can be developed as healthy drinks.
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References
Almeida-Dias, J. M. V., Morais, M. M., Belinato, J. R., Diogo, Y. R., Miranda-Pinto, P., Batista, N. N. M., Quinete, N., Nascimento, F. S., Francoy, T. M., De Jong, D., & Cappelini, L. T. D. (2025). Botanical origin and volatile organic compounds contents of bee bread from four Brazilian regions. Next Research, 2(2), Article 100341. https://doi.org/10.1016/j.nexres.2025.100341
Alghamdi, B. A., Alshumrani, E. S., Saeed, M. S. B., Rawas, G. M., Alharthi, N. T., Baeshen, M. N., Helmi, N. M., Alam, M. Z., & Suhail, M. (2020). Analysis of sugar composition and pesticides using HPLC and GC-MS techniques in honey samples collected from Saudi Arabian markets. Saudi Journal of Biological Sciences, 27(12), 3720-3726.
Ali, H., Abu Bakar, M. F., Majid, M., Muhammad, N., & Lim, S. Y. (2020). In vitro anti-diabetic activity of stingless bee honey from different botanical origins. Food Research, 4(5), 1421-1426.
Aparna, V., Dileep, K. V., Mandal, P. K., Karthe, P., Sadasivan, C., & Haridas, M. (2012). Anti-inflammatory property of n-hexadecanoic acid: structural evidence and kinetic assessment. Chemical Biology and Drug Design, 80(3), 434-439.
Babacan, S., Pivarnik, L. F., & Rand, A. G. (2002). Honey amylase activity and food starch degradation. Journal of Food Science, 67(5), 1625-1630.
Beizunces, L. P., Tchamitchian, S., & Brunet, J. L. (2012). Neural effects of insecticides in the honey bee. Apidologie, 43, 348-370.
Caunii, A., Pribac, G., Grozea, I., Gaitin, D., & Samfira, I. (2012). Design of optimal solvent for extraction of bioactive ingredients from six varieties of Medicago sativa. Chemistry Central Journal, 6, Article 123. https://doi.org/10.1186/1752-153X-6-123
Colović, M. B., Krstić, D. Z., Lazarević-Pašti, T. D., Bondžić, A. M., & Vasić, V. M. (2013). Acetylcholinesterase inhibitors: pharmacology and toxicology. Current Neuropharmacology, 11(3), 315-335.
Chai, W. M., Liu, X., Hu, Y. H., Feng, H. L., Jia, Y. L., Guo, Y. J., Zhou, H. T., & Chen, Q. X. (2013). Antityrosinase and antimicrobial activities of furfuryl alcohol, furfural and furoic acid. International Journal of Biological Macromolecules, 57, 151-155.
Chen, C. A., Sieburth, S. M., Glekas, A., Hewitt, G. W., Trainor, G. L., Erickson-Viitanen, S., Garber, S. S., Cordova, B., Jeffry, S., & Klabe, R. M. (2001). Drug design with a new transition state analog of the hydrated carbonyl: silicon-based inhibitors of the HIV protease. Chemical Biology, 8(12), 1161-1166.
Chukwu, C. J., Omaka, O. N., & Aja, P. M. (2017). Characterization of 2,5-dimethyl-2,4-dihydroxy-3(2H) furanone, a flavourant principle from Sysepalum dulcificum. Natural Products Chemistry and Research, 5(8), Article 299. https://doi.org/10.4172/2329-6836.1000299
Damodaran, T., Cheah, P. S., Murugaiyah, V. & Hassan, Z. (2020). The nootropic and anticholinesterase activities of Clitoria ternatea Linn. root extract: potential treatment for cognitive decline. Neurochemistry International, 139, Article 104785. https://doi.org/10.1016/j.neuint.2020.104785
Devarajan, S., & Venugopal, S. (2012). Antioxidant and α-amylase inhibition activities of phenolic compounds in the extracts of Indian honey. Chinese Journal of Natural Medicines, 10(4), 255-259.
Divya, A., Anbumalarmathi, J., & Sharmili, S. A. (2018). Phytochemical analysis, antimicrobial and antioxidant activity of Clitoria ternatea blue and white flowered leaves. Advances in Research, 14(5), 1-13.
dos Santos, T. C., Gomes, T. M., Pinto, B. A. S., Camara, A. L., & Paes, A. M. D. A. (2018). Naturally occurring acetylcholinesterase inhibitors and their potential use for Alzheimer’ s disease therapy. Frontiers in Pharmacology, 9, Article 1192. https://doi.org/10.3389/fphar.2018.01192
Geerts, H., Guillaumat, P.-O., Grantham, C., Bode, W., Anciaux, K., & Sachak, S. (2005). Brain levels and acetylcholinesterase inhibition with galantamine and donepezil in rats, mice, and rabbits. Brain Research, 1033(2), 186-193. https://doi.org/10.1016/j.brainres.2004.11.042
Grossberg, G. T. (2003). Cholinesterase inhibitors for the treatment of Alzheimer's disease: getting on and staying on. Current Therapeutic Research, Clinical and Experimental, 64(4), 216-235. https://doi.org/10.1016/S0011-393X(03)00059-6
Gupta, G. K., Chahal, J., & Bhatia, M. (2010). Clitoria ternatea (L.): Old and new aspects. Journal of Pharmacy Research, 3, 2610-2614.
Escher, G. B., Marques, M. B., do Carmo, M. A. V., Azevedo, L., Furtado, M. M., Sant'Ana, A. S., da Silva, M. C., Genovese, M. I., Wen, M., Zhang, L., Oh, W. Y., Shahidi, F., Rosso, N. D., & Granato, D. (2020). Clitoria ternatea L. petal bioactive compounds display antioxidant, antihemolytic and antihypertensive effects, inhibit α-amylase and α-glucosidase activities and reduce human LDL cholesterol and DNA induced oxidation. Food Research International, 128, Article 108763. https://doi.org/10.1016/j.foodres.2019.108763
Fiest, K. M., Roberts, J. I., Maxwell, C. J., Hogan, D. B., Smith, E. E., Frolkis, A., Cohen, A., Kirk, A., Pearson, D., Pringsheim, T., Venegas-Torres, A., & Jetté, N. (2016). The prevalence and incidence of dementia due to Alzheimer's disease: a systematic review and meta-analysis. Canadian Journal of Neurological Sciences, 43, 51-82.
Fratianni, F., Ombra, M. N., Giulio, B. D., d’Acierno, A., & Nazzaro, F. (2025). Lamiaceae honey: polyphenol profile, vitamin C content, antioxidant and in vitro anti-inflammatory, cholinesterase and tyrosinase inhibitory activity. Food Chemistry Advances, 7, Article 100996. https://doi.org/10.1016/j.focha.2025.100996
Hammer, Ø., Harper, D. A. T., & Ryan, P. D. (2001). Past: Paleontological statistics software package for education and data analysis. Paleontologia Electronica, 4(1), 1-9.
Han, W., & Li, C. (2010). Linking type 2 diabetes and Alzheimer's disease. PNAS, 107(15), 6557-6558. https://doi.org/10.1073/pnas.1002555107
Hashemirad, F.-S., Behfar, M., & Kavoosi, G. (2024). Proximate composition, physico-chemical, techno-functional, amino acid profile, fatty acid profile, nutritional quality, antioxidant, anti-amylase and anti-lipase properties of bee bread, royal jelly, and bee propolis. LWT- Food Science and Technology, 200, Article 116190. https://doi.org/10.1016/j.lwt.2024.116190
Hungerford, G., Lemos, M. A., & Chu, B.-S. (2019). Binding of Clitoria ternatea L. flower extract with α-amylase simultaneously monitored at two wavelengths using a photon streaming time-resolved fluorescence approach. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 211, 108-113. https://doi.org/10.1016/j.saa.2018.11.062
Keskin, M., & Özkök, A. (2020). α-Amylase inhibition properties of bee pollen and bee bread (Perga). Hacettepe Journal of Biology and Chemistry, 48, 389-393.
Kieliszek, M., Piwowarek, K., Kot, A. M., Błazejak, S., Chlebowska-Smigiel, A., & Wolska, I. (2018). Pollen and bee bread as new health oriented products: A review. Trends in Food Science & Technology, 71, 170-180.
Kostić, A. Ž., Barać, M. B., Stanojević, S. P., Milojković-Opsenica, D., Tešić, Ž. L., Šikoparija, B., Radišić, P., Prentović, M., & Pešić, M. B. (2015). Physicochemical composition and techno-functional properties of bee pollen collected in Serbia. LWT - Food Science and Technology, 62(1), 301-309. https://doi.org/10.1016/j.lwt.2015.01.031
Kusumah, D., Wakui, M., Murakami, M., Xie, X., Yukihito, K., & Maeda, I. (2020). Linoleic acid, α-linolenic acid, and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus. Bioscience, Biotechnology, and Biochemistry, 84(6), 1285-1290.
Kumar, P. P., Kumaravel, S., & Lalitha, C. (2010). Screening of antioxidant activity, total phenolics and GC-MS study of Vitex nigundo. African Journal of Biochemistry Research, 4, 191-195.
Kumar, A., Singh, V. K., & Kayastha, A. M. (2024). Studies on α-amylase inhibition by acarbose and quercetin using fluorescence, circular dichroism, docking, and dynamics simulations. Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy, 314, Article 124160. https://doi.org/10.1016/j.saa.2024.124160
Mahdavi, A., Torabi, E., Ghasemi, V., Mahdavi, V., & Hasan, F. H. M. (2025). Pesticide residues in nectar, pollen, and beebread matrices from rapeseed (Brassica napus L.): Which matrix is associated with the highest risks to the honey bee (Apis mellifera L.). Environmental Research, 270, Article 121014. https://doi.org/10.1016/j.envres.2025.121014
Mdeni, N. L., Adeniji, A. O., Okoh, A. I., & Okoh, O. O. (2022). Analytical evaluation of carbamate and organophosphate pesticides in human and environmental matrices: A review. Molecules, 27, Article 618. https://doi.org/10.3390/molecules27030618
Montaser, M., Sayed, A. M., Bishr, M. M., Zidan, E. W., Zaki, M. A., Hassan, H. M., Mohammed, R., & Hifnawy, M. S. (2023). GC-MS analysis of honeybee products derived from medicinal plants. Beni-Suef University Journal of Basic and Applied Sciences, 12, Article 63. https://doi.org/10.1186/s43088-023-00396-3
Mutahi, M. W., Nittoli, T., Guo, L., & Sieburth, S. M. (2002). Silicon-based metalloprotease inhibitors: synthesis and evaluation of silanol and silanediol peptide analogues as inhibitors of angiotensin-converting enzyme. Journal of the American Chemical Society, 124(25), 7363-7375. https://doi.org/10.1021/ja026158w
Nagai, T., Inoue, R., Suzuki, N., & Nagashima, T. (2009). Alpha-amylase from persimmon honey: Purification and characterization. International Journal of Food Properties, 12(3), 512-521.
Noh, C. H. C., Azmin, N. F. M., & Amid, A. (2017). Principal component analysis application on flavonoids characterization. Advances in Science, Technology and Engineering Systems Journal, 2(3), 435-440. https://doi.org/10.25046/aj020356
Pasias, I. N., Kiriakou, I. K., & Proestos, C. (2017). HMF and diastase activity in honeys: A fully validated approach and a chemometric analysis for identification of honey freshness and adulteration. Food Chemistry, 229, 425-431.
Peláez-Acero, A., Garrido-Islas, D. B., Campos-Montiel, R. G., González-Montiel, L., Medina-Pérez, G., Luna-Rodríguez, L., González-Lemus, U., & Cenobio-Galindo, A. J. (2022). The application of ultrasound in honey: antioxidant activity, inhibitory effect on α-amylase and α-glucosidase, and in vitro digestibility assessment. Molecules, 27(18), Article 5825. https://doi.org/10.3390/molecules27185825
Pfaff, B., Darrington, J., Stover, J., Satman, M. H., Beckmann, F., Williams, J., Kiefte, M., Kobly, P., & van Son, R. (2013). GNU PSPP. https://www.gnu.org/software/pspp/
Philip, Y., & Mohd Fadzelly, A. B. (2015). Antioxidative and acetylcholinesterase inhibitor potential of selected honey of Sabah, Malaysian Borneo. International Food Research Journal, 22(5), 1953-1960.
Pimentel, T. C., Rosset, M., de Sousa, J. M. B., de Oliveira, L. I. G., Mafaldo, I. M., Pintado, M. M. E., de Souza, E. L., & Magnani, M. (2022). Stingless bee honey: An overview of health benefits and main market challenges. Journal of Food Biochemistry, 46(3), Article e13883. https://doi.org/10.1111/jfbc.13883
Piovesan, B., Padilha, A. C., Morais, M. C., Botton, M., Grützmacher, A. D., & Zotti, M. J. (2020). Effects of insecticides used in strawberries on stingless bees Melipona quadrifasciata and Tetragonisca fiebrigi (Hymenoptera: Apidae). Environmental Science and Pollution Research, 27, 42472-42480.
Pokajewicz, K., Lamaka, D., Hudz, N., Adamchuk, L., & Wieczorek, P. P. (2024). Volatile profile of bee bread. Scientific Reports, 14, Article 6870. https://doi.org/10.1038/s41598-024-57159-y
Ringle, C. M., Wende, S., & Becker, J. M. (2015). SmartPLS 3. Boenningstedt. https://www.smartpls.com
Ranneh, Y., Ali, F., Zarei, M., Akim, A. M., Hamid, H. A., & Khazaai, H. (2018). Malaysian stingless bee and Tualang honeys: A comparative characterization of total antioxidant capacity and phenolic profile using liquid chromatography-mass spectrometry. LWT- Food Science and Technology, 89, 1-9. https://doi.org/10.1016/j.lwt.2017.10.020
Rasmussen, C. (2008). Catalog of the Indo-Malayan/Australasian stingless bees (Hymenoptera: Apidae: Meliponini). Zootaxa, 1935, 1-80.
Schaad, E., Fracheboud, M., Droz, B., & Kast, C. (2023). Quantitation of pesticides in bee bread collected from honey bee colonies in an agricultural environment in Switzerland. Environmental Science and Pollution Research International, 30(19), 56353-56367. https://doi.org/10.1007/s11356-023-26268-y
Sobral, F., Calhelha, R., Barros, L., Dueñas, M., Tomás, A., Santos-Buelga, C., Vilas-Boas, M., & Ferreira, I. C. F. R. (2017). Flavonoid composition and antitumor activity of bee bread collected in Northeast Portugal. Molecules, 22, Article 248. https://doi.org/10.3390/molecules22020248
Szwajgier, D., Baranowska-Wójcik, E., Winiarska-Mieczan, A., & Gajowniczek-Ałasa, D. (2022). Honeys as possible sources of cholinesterase inhibitors. Nutrients, 14(14), Article 2969. https://doi.org/10.3390/nu14142969
Taqui, R., Debnath, M., Ahmed, S., & Ghosh, A. (2022). Advances on plant extracts and phytocompounds with acetylcholinesterase inhibition activity for possible treatment of Alzheimer's disease. Phytomedicine, 2(1), Article 100184. https://doi.org/10.1016/j.phyplu.2021.100184
Topalăa, C. M., Tătarua, L. D., & Ducu, C. (2017). ATR-FTIR spectra fingerprinting of medicinal herbs extracts prepared using microwave extraction. Arabian Journal of Medicinal and Aromatic Plants, 3(1), 1-9.
Thoophom, G. (2025, 29 January). GT test kit protocol. http://www.gttestkit.com/gttestkit_ eng/intro_procedure.htm
Thummajitsakul, S., Thongkerd, N., Pholmeesap, B., Phankham, P., & Silprasit, K. (2020a). Assessment of organophosphate and carbamate insecticides and heavy metal contamination in canal-grown water morning glory (Ipomoea aquatica forssk) in Nakhon Nayok province, Thailand. Applied Environmental Research, 42(1), 26-42.
Thummajitsakul, S., Samaikam, S., Tacha, S., & Silprasit, K. (2020b). Study on FTIR Spectroscopy, total phenolic content, antioxidant activity and anti-amylase activity of extracts and different tea forms of Garcinia schomburgkiana leaves. LWT - Food Science and Technology, 134, Article 110005. https://doi.org/10.1016/j.lwt.2020.110005
Wongsa, P., Phatikulrungsun, P., & Prathumthong, S. (2022). FT-IR characteristics, phenolic profiles and inhibitory potential against digestive enzymes of 25 herbal infusions. Scientific Reports, 12(1), Article 6631. https://doi.org/10.1038/s41598-022-10669-z
Yimdee, J., Uabundit, N., Chaisiwamonkol, K., Iamsaard, S., Hipkaeo, W., & Wattanathorn, J. (2014). Evaluation of cognitive enhancing effects of Clitoria ternatea flowers water extract in normal male mice. KKU Research Journal, 4(3), 59-66.
Yu, X., Zhao, M., Liu, F., Zeng, S., & Hu, J. (2013). Identification of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one as a strong antioxidant in glucose-histidine maillard reaction products. Food Research International, 51, 397-403.
Zafeiraki, E., Kasiotis, K. M., Nisianakis, P., Manea-Karga, E., & Macher, K. (2022). Occurrence and human health risk assessment of mineral elements and pesticides residues in bee pollen. Food and Chemical Toxicology, 161, Article 112826. https://doi.org/10.1016/j.fct.2022.112826
Zakaria, N. N. A, Jaafar, N. N. M., Mad Desa N. N., & Mohamad, A. Z. (2020). Antioxidant, antibacterial and anti-diabetic activities of stingless bee honey from selected areas in Peninsular Malaysia. IOP Conference Series: Earth and Environmental Science, 596, Article 012093. https://doi.org/10.1088/1755-1315/596/1/012093
Zhang, H., Dolan, H. L., Ding, Q., Wang, S., & Tikekar, R. V. (2019). Antimicrobial action of octanoic acid against Escherichia coli O157:H7 during washing of baby spinach and grape tomatoes. Food Research International, 125, Article 108523. https://doi.org/10.1016/j.foodres.2019.108523
Zhao, L., Chen, J., Su, J., Li, L., Hu, S., Li, B., Zhang, X., Xu, Z., & Chen, T. (2013). In vitro antioxidant and antiproliferative activities of 5-hydroxymethylfurfural. Journal of Agricultural and Food Chemistry, 61(44), 10604-10611.
