Abstract
This present study quantified 5-hydroxymethylfurfural (HMF) in ATH samples of different storage durations using UV-visible spectroscopy and evaluated the effects of these samples on the reproductive health of female Sprague Dawley (SD) rats. Rats with regular oestrous cycles were divided into 11 treatment groups (n=10), receiving either distilled water (control), fresh TH (FTH), ATH (24-, 36-, and 48-months old), purified HMF (low, medium, and high doses), or combinations of FTH and HMF. The rats were subsequently administered with test substances via oral gavage on the first day of dioestrus for four consecutive weeks. On the first dioestrus day upon completion of the four-week testing period, the rats (fasted overnight) were anaesthetised followed by blood collection and laparotomy. Blood samples were subjected to biochemical and hormonal analyses, and visceral reproductive organs were collected. The length of the oestrous cycles in control and treatment groups, in both pre (H-statistic=6.50, p=0.77) and during (H-statistic=11.48, p=0.32) treatment period showed no significant differences when evaluated using the Kruskal Wallis test. In addition, the one-way ANOVA test showed no significant differences in the body weights throughout the experimental period (F-statistic=0.195–0.978, p=0.468–0.996). As for the absolute and relative organ weights which were analysed using one-way ANOVA or Kruskal-Wallis analysis, only the absolute (H-statistic=18.87, p=0.042) and relative (H-statistic=20.62, p=0.024) weights of the right fallopian tube showed significant differences. Through the Kruskal-Wallis analysis, significant differences were observed in albumin (H-statistic=22.66, p=0.012), albumin/globulin A/G ratio (H-statistic=20.02, p=0.029), gamma-glutamyl transferase (GGT) (H-statistic=42.41, p<0.0001), and oestrogen (H-statistic=20.49, p=0.025) levels. These findings indicate that elevated HMF exposure may influence targeted biochemical parameters, regulation of oestrogen, and reproductive organ weight, even in the absence of marked changes in oestrous cyclicity and body weight. The inclusion of FTH may not attenuate high HMF effects, suggesting potential reproductive risks from prolonged honey storage.
References
Abd Wahab, M.S., Othman, N., Othman, N.H.I., Jamari, A.A., & Ali, A.A. (2017). Exploring the use of and perceptions about honey as complementary and alternative medicine among the general public in the state of Selangor, Malaysia. Journal of Applied Pharmaceutical Science, 7(12): 144-150.
https://dx.doi.org/10.7324/JAPS.2017.71220
Ahmed, S., & Othman, N.H. (2013). Review of the medicinal effects of tualang honey and a comparison with manuka honey. The Malaysian Journal of Medical Sciences, 20(3): 6-13.
https://pmc.ncbi.nlm.nih.gov/articles/PMC3743976/
Ahmed, S., Sulaiman, S.A., Ibrahim, M., Rasul, A., Yasir, M., Waqas, A.A., Afzaal, M., Ahmed, M.Z., & Hayati, N. (2018). Effect of daily supplementation of Malaysian Jungle Tualang Honey and Australian/New Zealand Manuka honey on hematological and some biochemical variables in female rats. Annals of Life Sciences, 2(5): 10-22.
Andritoiu, C.V., Ochiuz, L., Andritoiu, V., & Popa, M. (2014). Effect of apitherapy formulations against carbon tetrachloride-induced toxicity in Wistar rats after three weeks of treatment. Molecules, 19(9): 13374-13391.
https://doi.org/10.3390/molecules190913374
Azman, K.F., Aziz, C.B.A., Zakaria, R., Ahmad, A.H., Shafin, N., & Ismail, C.A.N. (2021). Tualang honey: a decade of neurological research. Molecules, 26(17): 5424.
https://doi.org/10.3390/molecules26175424
Bauer-Marinovic, M., Taugner, F., Florian, S., & Glatt, H. (2012). Toxicity studies with 5-hydroxymethylfurfural and its metabolite 5-sulphooxymethylfurfural in wild-type mice and transgenic mice expressing human sulphotransferases 1A1 and 1A2. Archives of Toxicology, 86(5): 701-711.
https://doi.org/10.1007/s00204-012-0807-5
Bogdanov, S., Martin, P., & Lullmann C. (2002). Harmonised methods of the international honey commission. Swiss Bee Research Centre, FAM, Liebefeld, 5.
https://ihc-platform.net/ihcmethods2009.pdf
Cavia, M.M., Fernández-Muiño, M.A., Gömez-Alonso, E., Montes-Pérez, M.J., Huidobro, J.F., & Sancho, M.T. (2002). Evolution of fructose and glucose in honey over one year: influence of induced granulation. Food Chemistry, 78(2): 157-161.
https://doi.org/10.1016/S0308-8146(01)00393-4
Chen, Y., Chen, Y., Zhao, L., He, H., Wei, L., Lai, W., Yuan, J., Hong, X., Liu, L., Wang, B., & Nandakumar, K.S. (2021). Albumin/Globulin Ratio as Yin–Yang in rheumatoid arthritis and its correlation to inflamm-aging cytokines. Journal of Inflammation Research: 5501-5511.
https://doi.org/10.2147/JIR.S335671
Cheraghi, M., Ahmadvand, H., Maleki, A., Babaeenezhad, E., Shakiba, S., & Hassanzadeh, F. (2019). Oxidative stress status and liver markers in coronary heart disease. Reports of Biochemistry & Molecular Biology, 8(1): 49-55.
https://pmc.ncbi.nlm.nih.gov/articles/PMC6590939/
Chew, C.Y., Chua, L.S., Soontorngun, N., & Lee, C.T. (2018). Discovering potential bioactive compounds from Tualang honey. Agriculture and Natural Resources, 52(4): 361-365.
https://doi.org/10.1016/j.anres.2018.10.011
Cianciosi, D., Forbes-Hernández, T.Y., Afrin, S., Gasparrini, M., Reboredo-Rodriguez, P., Manna, P.P., Zhang, J., Bravo Lamas, L., Martínez Flórez, S., Agudo Toyos, P., Quiles, J.L., Giampieri, F., & Battino, M. (2018). Phenolic compounds in honey and their associated health benefits: a review. Molecules, 23(9): 2322.
https://doi.org/10.3390/molecules23092322
Craig, Z.R., Wang, W., & Flaws, J.A. (2011). Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling. Reproduction, 142(5): 633.
http://dx.doi.org/10.1530/REP-11-0136
Ding, T., Yan, W., Zhou, T., Shen, W., Wang, T., Li, M., Zhou, S., Wu, M., Dai, J., Huang, K., Zhang, J., Chang, J., & Wang, S. (2022). Endocrine disrupting chemicals impact on ovarian aging: Evidence from epidemiological and experimental evidence. Environmental Pollution, 305: 119269.
https://doi.org/10.1016/j.envpol.2022.119269
Dozortsev, D., Pellicer, A., & Diamond, M.P. (2020). Progesterone is a physiological trigger of ovulatory gonadotropins. Fertility and Sterility, 113(5): 923-924.
https://doi.org/10.1016/j.fertnstert.2019.12.024
El Bohi, K.M., Ghoniem, M.H., Azab, H.H., Ali, H., & Farag, M.R. (2020). Extra virgin olive oil enhances the hepatic antioxidant defense and inhibits cytogenotoxic effects evoked by 5-hydroxymethylfurfural in mice. Environmental Science and Pollution Research, 27(11): 11882-11891.
https://doi.org/10.1007/s11356-020-07659-x
Elmaoğulları, S., Kadan, E., Anadol, E., Gökçeoğlu, A., Çetinkaya, S., Yarım, G.F., Uçaktürk, S.A., & Aycan, Z. (2020). Effects of 5-hydroxymethylfurfural on pubertal development of female wistar rats. Journal of Clinical Research in Pediatric Endocrinology, 12(1): 79-85.
https://dx.doi.org/10.4274/jcrpe.galenos.2019.2019.0080
Fakhlaei, R., Selamat, J., Khatib, A., Razis, A.F.A., Sukor, R., Ahmad, S., & Babadi, A.A. (2020). The toxic impact of honey adulteration: a review. Foods, 9(11): 1538.
https://doi.org/10.3390/foods9111538
Fan, L., Wang, F., Yao, Q., Wu, H., Wen, F., Wang, J., Li, H., & Zheng, N. (2021). Lactoferrin could alleviate liver injury caused by Maillard reaction products with furan ring through regulating necroptosis pathway. Food Science & Nutrition, 9(7): 3449-3459.
https://doi.org/10.1002/fsn3.2254
Fuad, W.E.M., Jaafar, H., & Sulaiman, S.A. (2017). The effects of Malaysian herb, Labisia pumila var. alata on oestrous cyclicity and reproductive parameters of nulliparous rats. Sains Malaysiana, 46(10): 1721-1726.
https://doi.org/10.17576/jsm-2017-4610-07
Haron, M.N., Rahman, W.F.W.A., Sulaiman, S.A., & Mohamed, M. (2014). Tualang honey ameliorates restraint stress-induced impaired pregnancy outcomes in rats. European Journal of Integrative Medicine, 6(6): 657-663.
https://doi.org/10.1016/j.eujim.2014.07.001
Haron, H., Talib, R.A., Subramaniam, P., Arifen, Z.N.Z., & Ibrahim, M. (2022). A comparison of chemical compositions in Kelulut honey from different regions. Malaysian Journal of Analytical Sciences, 26(3): 447-456.
http://irep.iium.edu.my/id/eprint/107590
Hashem, N.M., Hassanein, E.M., & Simal-Gandara, J. (2021). Improving reproductive performance and health of mammals using honeybee products. Antioxidants, 10(3): 336.
https://doi.org/10.3390/antiox10030336
Hasim, H., Abd Aziz, C.B., Suhaimi, S.Q.A., Mohamed, M., Long, I., & Zakaria, R. (2020). Effects of Tualang honey on pain behaviour and oxidative stress in the thalamus of prenatally stressed rat offspring. Malaysian Journal of Medicine and Health Sciences, 16(3): 85–92.
https://medic.upm.edu.my/upload/dokumen/2020090810182814_MJMHS_0002.pdf
Hasan, S., Rahim, R.A., Alias, M.A., Muhammad, N., Abdullah, N., & Razak, R.N.H.A. (2022). Tualang honey’s spermatoprotective effects in a model of high cholesterol diet-induced animals. Pharmacophore, 13(1-2022): 27-35.
https://doi.org/10.51847/BBTwqvL1og
Hou, Y., Zhang, X., Liu, X., Wu, Q., Hou, J., Su, P., & Guo, Q. (2022). Comparison of the effects of 5-Hydroxymethylfurfural in milk powder matrix and standard water on oxidative stress system of Zebrafish. Foods, 11(12): 1814.
https://doi.org/10.3390/foods11121814
Hussein, S.Z., Yusoff, K.M., Makpol, S. & Yusof, Y.M. (2014). Does gamma irradiation affect physicochemical properties of honey. La Clinica Terapeutica, 165(2):e125-133.
https://doi.org/10.7471/ct.2014.1695
Ismail, N.I., Kadir, M.R.A., Zulkifli, R.M. & Mohamed, M. (2021). Comparison of physicochemical, total protein and antioxidant profiles between Malaysian Apis and Trigona honeys. Malaysian Journal of Analytical Sciences, 25(2):243-256.
https://mjas.analis.com.my/mjas/v25_n2/pdf/Norjihada_25_2_7.pdf
Ismail, N.H., Osman, K., Zulkefli, A.F., Mokhtar, M.H. & Ibrahim, S.F. (2021). The physicochemical characteristics of gelam honey and its outcome on the female reproductive tissue of sprague–dawley rats: a preliminary study. Molecules, 26(11):3346.
https://doi.org/10.3390/molecules26113346
Jiang, Y., Zhong, Z., Wang, M., & Zhang, X. (2022). 5-Hydroxymethyl-2-furaldehyde induces developmental toxicology and decreases bone mineralization in zebrafish larvae. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 254(2022): 109254.
https://doi.org/10.1016/j.cbpc.2021.109254
Khalil, M.I., Sulaiman, S.A., & Gan, S.H. (2010). High 5-hydroxymethylfurfural concentrations are found in Malaysian honey samples stored for more than one year. Food and Chemical Toxicology, 48(8-9): 2388-2392.
https://doi.org/10.1016/j.fct.2010.05.076
Kishore, R.K., Halim, A.S., Syazana, M.N., & Sirajudeen, K.N.S. (2011). Tualang honey has higher phenolic content and greater radical scavenging activity compared with other honey sources. Nutrition Research, 31(4): 322-325.
https://doi.org/10.1016/j.nutres.2011.03.001
Kishore, R.K., Nasar, S.A., Viji, P.T., Lim, C.K., & Halim, A.S. (2014). Physicochemical and antioxidant properties of Malaysian Tualang honey on long term storage. Compendium of Oral Science, 1: 13-24.
https://doi.org/10.24191/cos.v1i0.17530
Koenig, G., & Seneff, S. (2015). Gamma‐glutamyltransferase: a predictive biomarker of cellular antioxidant inadequacy and disease risk. Disease Markers, 2015(1): 818570.
https://doi.org/10.1155/2015/818570
Marcondes, F.K., Bianchi, F.J., & Tanno, A.P. (2002). Determination of the estrous cycle phases of rats: some helpful considerations. Brazilian Journal of Biology, 62: 609-614.
https://doi.org/10.1590/S1519-69842002000400008
Melin, V.E., Melin, T.E., Dessify, B.J., Nguyen, C.T., Shea, C.S., & Hrubec, T.C. (2016). Quaternary ammonium disinfectants cause subfertility in mice by targeting both male and female reproductive processes. Reproductive Toxicology, 59: 159-166.
https://doi.org/10.1016/j.reprotox.2015.10.006
Mikhael, S., Punjala-Patel, A., & Gavrilova-Jordan, L. (2019). Hypothalamic-pituitary-ovarian axis disorders impacting female fertility. Biomedicines, 7(1): 5.
https://doi.org/10.3390/biomedicines7010005
Ministry of Health, Malaysia. (2014). Food Regulations 1985 (Amendment 2014), under the Food Act 1983, Malaysia.
Mizushina, Y., Yagita, E., Kuramochi, K., Kuriyama, I., Shimazaki, N., Koiwai, O., Uchiyama, Y., Yomezawa, Y., Sugawara, F., Kobayashi, S., & Sakaguchi, K. (2006). 5-(Hydroxymethyl)-2-furfural: a selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase. Archives of Biochemistry and Biophysics, 446(1): 69-76.
https://doi.org/10.1016/j.abb.2005.11.015
Mohd Kamal, D.A., Ibrahim, S.F., Kamal, H., Kashim, M.I.A.M., & Mokhtar, M.H. (2021). Physicochemical and medicinal properties of tualang, gelam and kelulut honeys: A comprehensive review. Nutrients, 13(1): 197.
https://doi.org/10.3390/nu13010197
Moniruzzaman, M., Khalil, M.I., Sulaiman, S.A., & Gan, S.H. (2013). Physicochemical and antioxidant properties of Malaysian honeys produced by Apis cerana, Apis dorsata and Apis mellifera. BMC Complementary and Alternative Medicine, 13(1): 43.
https://doi.org/10.1186/1472-6882-13-43
Mosavat, M., Ooi, F.K., & Mohamed, M. (2014). Stress hormone and reproductive system in response to honey supplementation combined with different jumping exercise intensities in female rats. BioMed Research International, 2014(1): 123640.
https://doi.org/10.1155/2014/123640
National Toxicology Program. (2010). NTP toxicology and carcinogenesis studies of 5-(Hydroxymethyl)-2-furfural (CAS No. 67-47-0) in F344/N rats and B6C3F1 mice (gavage studies). National Toxicology Program Technical Report Series, 554: 7.
https://ntp.niehs.nih.gov/publications/reports/tr/tr554
Özkök, D., & Silici, S. (2016). Effects of honey HMF on enzyme activities and serum biochemical parameters of Wistar rats. Environmental Science and Pollution Research, 23(20): 20186-20193.
https://doi.org/10.1007/s11356-016-7218-8
Papadimitriou, K., Mousiolis, A.C., Mintziori, G., Tarenidou, C., Polyzos, S.A., & Goulis, D.G. (2024). Hypogonadism and nonalcoholic fatty liver disease. Endocrine, 86(1): 28-47.
https://doi.org/10.1007/s12020-024-03878-1
Qiu, Y., Lin, X., Chen, Z., Li, B., & Zhang, Y. (2022). 5-Hydroxymethylfurfural exerts negative effects on gastric mucosal epithelial cells by inducing oxidative stress, apoptosis, and tight junction disruption. Journal of Agricultural and Food Chemistry, 70(12): 3852-3861.
https://doi.org/10.1021/acs.jafc.2c00269
Rajindran, N., Wahab, R.A., Huda, N., Julmohammad, N., Shariff, A.H.M., Ismail, N.I., & Huyop, F. (2022). Physicochemical properties of a new green honey from Banggi Island, Sabah. Molecules, 27(13): 4164.
https://doi.org/10.3390/molecules27134164
Ranneh, Y., Akim, A.M., Hamid, H.A., Khazaai, H., Fadel, A., Zakaria, Z.A., Albujja, M., & Bakar, M.F.A. (2021). Honey and its nutritional and anti-inflammatory value. BMC Complementary Medicine and Therapies, 21(1): 30.
https://doi.org/10.1186/s12906-020-03170-5
Ruslee, S.S., Zaid, S.S.M., Bakrin, I.H., Goh, Y.M., & Mustapha, N.M. (2020). Protective effect of Tualang honey against cadmium-induced morphological abnormalities and oxidative stress in the ovary of rats. BMC Complementary Medicine and Therapies, 20(1): 160.
https://doi.org/10.1186/s12906-020-02960-1
Sabireen, S.N., Sulaiman, S.A., & Fuad, W.E.M. (2022). The subacute toxicity effects of aged Tualang (Koompassia excelsa) honey with high 5-hydroxymethylfurfural content in rats. International Food Research Journal, 29(1): 10–22.
https://doi.org/10.47836/ifrj.29.1.02
Sabireen, S.N., Sulaiman, S.A., Salleh, R.M., & Fuad, W.E.M. (2020). Evaluation of 5-hydroxymethylfurfural (HMF) concentration and physicochemical properties in fresh and aged Tualang honey. Asian Journal of Medicine and Biomedicine, 4(2): 10-16.
https://doi.org/10.37231/ajmb.2020.4.2.356
Severin, I., Dumont, C., Jondeau-Cabaton, A., Graillot, V., & Chagnon, M.C. (2010). Genotoxic activities of the food contaminant 5-hydroxymethylfurfural using different in vitro bioassays. Toxicology letters, 192(2): 189-194.
https://doi.org/10.1016/j.toxlet.2009.10.022
Shapla, U.M., Solayman, M., Alam, N., Khalil, M.I., & Gan, S.H. (2018). 5-Hydroxymethylfurfural (HMF) levels in honey and other food products: effects on bees and human health. Chemistry Central Journal, 12(1): 35.
https://doi.org/10.1186/s13065-018-0408-3
Sommer, Y., Hollnagel, H., Schneider, H., & Glatt, H.R. (2003). Metabolism of 5-hydroxymethyl-2-furfural (HMF) to the mutagen 5-sulfoxymethyl-2-furfural (SMF) by individual sulfotransferases. Naunyn-Schmiedebergs Archives of Pharmacology, 367: R166-R166.
https://doi.org/10.1007/s00210-003-0739-y
Sujanto, I.S.R., Ramly, N.S., Tang, J.Y.H., Abd Ghani, A., Alias, N., Bakar, M.F.A., & Ngah, N. (2021). The physicochemical properties and nutritional value of Stingless bee honey at Gelam Forest in Telaga Papan, Terengganu, Malaysia. Journal of Agrobiotechnology, 12(2): 108-120.
https://doi.org/10.37231/jab.2021.12.2.251
Tsiapara, A.V., Jaakkola, M., Chinou, I., Graikou, K., Tolonen, T., Virtanen, V., & Moutsatsou, P. (2009). Bioactivity of Greek honey extracts on breast cancer (MCF-7), prostate cancer (PC-3) and endometrial cancer (Ishikawa) cells: Profile analysis of extracts. Food Chemistry, 116(3): 702-708.
https://doi.org/10.1016/j.foodchem.2009.03.024
Ullah, S., Ahmad, S., Guo, X., Ullah, S., Ullah, S., Nabi, G., & Wanghe, K. (2023). A review of the endocrine disrupting effects of micro and nano plastic and their associated chemicals in mammals. Frontiers in Endocrinology, 13: 1084236.
https://doi.org/10.3389/fendo.2022.1084236
Voipio, H.M., & Nevalainen, T. (1998). Improved method for vaginal plug detection in rats. Scandinavian Journal of Laboratory Animal Science, 25(1).
https://doi.org/10.23675/sjlas.v25i1.818
Xu, T., Xia, R., He, F., Dong, E.H., Shen, J.M., Xu, C.C., Ji, M.H., & Xu, Q. (2023). 5-Hydroxymethylfurfural induces mice frailty through cell senescence-associated sarcopenia caused by chronic inflammation. Heliyon, 9(2): e13217. https://doi.org/10.1016/j.heliyon.2023.e13217
Ya’akob, H., Norhisham, N.F., Mohamed, M., Sadek, N., & Endrini, S. (2019). Evaluation of physicochemical properties of trigona sp. stingless bee honey from various districts of Johor. Jurnal Kejuruteraan, 2(1): 59-67.
https://doi.org/10.17576/jkukm-2019-si2(1)-08
Yamada, J., Tomiyama, H., Yambe, M., Koji, Y., Motobe, K., Shiina, K., Yamamoto, Y., & Yamashina, A. (2006). Elevated serum levels of alanine aminotransferase and gamma glutamyltransferase are markers of inflammation and oxidative stress independent of the metabolic syndrome. Atherosclerosis, 189(1): 198-205.
https://doi.org/10.1016/j.atherosclerosis.2005.11.036
Zaid, S.S.M., Kassim, N.M., & Othman, S. (2015). Tualang honey protects against BPA-induced morphological abnormalities and disruption of ERα, ERβ, and C3 mRNA and protein expressions in the uterus of rats. Evidence-Based Complementary and Alternative Medicine, 2015: 202874.
https://doi.org/10.1155/2015/202874
Zaid, S.S.M., Othman, S., & Kassim, N.M. (2014). Potential protective effect of Tualang honey on BPA-induced ovarian toxicity in prepubertal rat. Complementary and Alternative Medicine, 14: 509.
http://www.biomedcentral.com/1472-6882/14/509
Zaid, S.S.M., Sulaiman, S.A., Othman, N.H., Soelaiman, I.N., Shuid, A.N., Mohamad, N., & Muhamad, N. (2012). Protective effects of Tualang honey on bone structure in experimental postmenopausal rats. Clinics, 67: 779-784.
https://doi.org/10.6061/clinics/2012(07)13
Zaid, S.S.M., Sulaiman, S.A., Sirajudeen, K.N., & Othman, N.H. (2010). The effects of Tualang honey on female reproductive organs, tibia bone and hormonal profile in ovariectomised rats-animal model for menopause. BMC Complementary and Alternative Medicine, 10(1): 82.
https://doi.org/10.1186/1472-6882-10-82
Zaitsev, A.N., Simonian, T.A., & Pozdniakov, A.L. (1975). Hygienic standards for hydroxymethylfurfural in food products. Voprosy Pitaniia, 1: 52-55.
https://pubmed.ncbi.nlm.nih.gov/54991/
Zarei, M., Fazlara, A., & Alijani, N. (2019). Evaluation of the changes in physicochemical and antioxidant properties of honey during storage. Functional Foods in Health and Disease, 9(9): 593-605.
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