---

Yashwant Rana , Sushma Sharma

Department of Biosciences,Himachal Pradesh University, Shimla-05, India

Corresponding Author Email: sushmabiosci@rediffmail.com

DOI : https://doi.org/10.51470/AMSR.2024.04.01.13

Abstract

Keywords

Download this article as:

---

With the global population rising rapidly, our food systems are facing mounting pressure. By 2050, agricultural production must increase substantially to meet the growing demand for food [1]. Agriculture, which remains the backbone of food security, now struggles under a host of challenges: shrinking arable land, degrading soil quality, rising pest resistance, labour shortages, and unpredictable climate patterns [2].  In response, modern farming practices have leaned heavily on synthetic agrochemicals, especially pesticides, to sustain crop yields [3]. While these chemicals have proven effective in boosting productivity, their extensive use over the past two decades has triggered serious concerns regarding environmental safety and human health. Alarmingly, nearly 64% of global agricultural land, about 24.5 million km²is now contaminated with pesticide residues [1].  At the same time, aquatic ecosystems in regions like South Africa, China, India, Australia, and Argentina are reporting elevated levels of pesticide contamination [4].

Although pesticides play a key role in safeguarding crops [5], they are also increasingly implicated in widespread environmental degradation. Their chemical resilience allows them to persist in both land and water environments, where they may disrupt non-target species [6].  Surface runoff is one of the main ways these substances enter freshwater systems, disturbing aquatic life and threatening biodiversity [7].

One such pesticide is tebuconazole (TBZ), a widely used triazole fungicide known for its effectiveness against fungal infections in crops such as cereals, vegetables, and fruits [8,9]. TBZ is particularly concerning due to its long environmental half-life, ranging from 300 to 600 days in soil, and its high solubility in water, which facilitates its movement into nearby aquatic systems via agricultural runoff. Consequently, TBZ has been detected in surface waters at concentrations between 0.6 and 200 µg/L [10]. Growing evidence indicates that TBZ is toxic to non-target aquatic organisms. Studies have shown that it can disrupt thyroid function [11], delay development [12], cause genetic damage [13], impair neurological health [14], and negatively affect reproduction [8].  However, despite this body of research, there remains limited understanding of how TBZ impacts fish muscle tissue, a vital system essential for locomotion, respiration, and osmoregulation [15].

Muscle tissue is highly sensitive to environmental pollutants, particularly through oxidative stress, a key mechanism in TBZ-induced toxicity. This occurs when the overproduction of reactive oxygen species (ROS) overwhelms the body’s natural antioxidant defences. Normally, enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) work together to neutralize ROS and protect cells from damage [8]. When these defences falter under chemical stress, it can lead to lipid peroxidation, protein carbonylation, and deterioration of muscle integrity [16].

Because of their ecological significance and sensitivity to waterborne toxins, fish are commonly used as model organisms in environmental toxicology [17]. In the present study, Cyprinus carpio (common carp) was selected due to its ecological and economic value, widespread distribution, and vulnerability to sediment- and water-associated contaminants. As a bottom-dwelling species, it is particularly prone to absorbing pollutants such as TBZ.In recent years, there has been growing interest in the use of natural substances to mitigate the toxic effects of synthetic chemicals. Among these, propolis—a resinous substance collected by honeybees from plant exudates—has gained attention for its strong antioxidant, anti-inflammatory, and tissue-healing properties [18]. Rich in flavonoids and phenolic acids, propolis can help neutralise free radicals, enhance the activity of antioxidant enzymes, and protect tissues from oxidative injury [19].  Previous studies have shown that propolis can reduce pesticide-induced organ damage in fish, although its specific protective effects against TBZ-induced muscle toxicity have not yet been fully elucidated [20,21,22]. This study, therefore, aims to evaluate the protective effects of propolis against tebuconazole-induced muscle damage in Cyprinus carpio through detailed biochemical and histological assessments. By exploring both the extent of TBZ toxicity and the modulatory role of propolis, this research seeks to contribute to the development of eco-friendly strategies for mitigating pesticide impacts in aquatic systems.

Furthermore, the broader ecological implications of pesticide contamination cannot be overlooked. Recent findings have shown that fungicides like TBZ can cause irreversible harm to marine organisms such as Heterostegina depressa and their algal symbionts, reflecting the far-reaching consequences of agrochemical runoff [23]. Such evidence reinforces the urgent need for sustainable pest management and the exploration of natural alternatives like propolis to safeguard environmental and aquatic health.

2. Materials and Methods

2.1 Chemicals and Materials

A commercial-grade tebuconazole (25.9% EC) formulation was used for toxicity testing. Working concentrations were prepared using distilled water. All other chemicals were of analytical grade and sourced from Merck (India).

2.2 Propolis Extraction

Propolis extraction was conducted following the procedure described by Mani et al. (2006) [24]. Briefly, 30 g of propolis was finely ground and mixed with 70% ethanol to a final volume of 100 ml to prepare a 30% ethanolic extract of propolis (EEP). The extraction was carried out at room temperature, shielded from direct light, with continuous moderate shaking. Subsequently, the solvents were evaporated, and the dry weights of the extracts were determined, yielding concentrations of approximately 126 mg/ml for the EEP.

2.3 Fish collection and maintenance

Healthy Cyprinus carpio (20 ± 2 cm; 180–250 g) were obtained from a hatchery in Bilaspur, Himachal Pradesh. Fish were transported in aerated bags and disinfected with 0.2% potassium permanganate for 2–4 minutes. They were acclimatised for 12-15 days in 80 L glass aquaria with filtered and aerated water, partially changed (40%) daily. Fish were fed twice daily, and water quality was maintained at: temperature 22 ± 1.4°C, pH 7.5 ± 0.2, DO 8 ± 1 mg/L, TDS 155 ± 5 ppm, alkalinity 165 ± 8 mg/L, and hardness 120 ± 4 mg/L.

2.4 Sublethal exposure study

Fish were divided into five groups: control (Group 1), 6.47 µl/l TBZ (Group 2), 8.09 µl/l TBZ (Group 3), Propolis only (Group 4), and 8.09 µl/l TBZ propolis (Group 5). Exposure lasted 60 days, with daily renewal of toxicant solutions. Samples from groups 1,2, and 3 were collected at 10, 20, and 30-day intervals, while samples from groups 4 and 5 were collected at intervals of 20-40 and 60 days. No mortality occurred during the experiment.

2.5 Biochemical determinations

Biochemical parameters like total protein were measured by the method of Bradford (1976) [25], total amino acids by Moore and Stein (1954) [26], and total lipid content by the method of Folch et al. (1957) [27].

2.6 Bioenzyme assay

All bioenzyme assessments were carried out using the subcellular fraction extracted from the muscle tissue of C. carpio. The activities of key antioxidant enzymes—catalase (CAT) and superoxide dismutase (SOD)—along with lipid peroxidation (LPO) levels and lactate dehydrogenase (LDH) activity were evaluated based on the protocols outlined by Aebi, (1984) [28], Misra and Fridovich, (1972) [29], Dhindsa et al. (1981) [30] and Borgmannet al., (1974) [31]. Measurements of glutathione (GSH) content, glutathione reductase (GR), and glutathione peroxidase (Gpx) activity followed the methodology described by Moronet al. (1979) [32], Rotruck et al. (1973)[33], and Racker (1955) [34]. Acetylcholinesterase enzyme assay in muscle tissue was specifically measured with the method of Ellman et al. (1961) [35].

2.7 Histopathology

A standardised protocol for tissue handling, processing, and histological examination was carried out for tissue sampling of fish muscle. A mid-portion of the muscle was carefully excised and fixed in 10% neutral buffered formalin for 48 hours. Following fixation, the tissues were washed thoroughly with tap water, then gradually dehydrated through a graded ethanol series. The samples were subsequently processed using routine histological procedures and stained with hematoxylin and eosin for examination under a light microscope.

2.8 Statistical analysis

The results were expressed as mean ± SE. Statistical analysis was conducted using SPSS 20.0 software. Two-way ANOVA was used to assess the effects of treatments and exposure periods, with statistical significance set at p < 0.05. Tukey’s post hoc test was applied to identify differences among group means. Capital letters indicated significant differences within groups across durations, while lowercase letters represented differences between treatments at the same time point; ‘a denoted the lowest difference, with ascending letters indicating progressively higher deviation from the normal value.

Results and discussion

Tebuconazole induced notable biochemical alterations in the muscle tissue of the treated fish, which are summarised in Fig. A (1-3). A significant reduction in protein and total lipid contents was observed, along with a pronounced elevation in total free amino acids, indicating disrupted macromolecular metabolism and stress response. This reduction in protein and total lipid content was significantly lower (P<0.05)in experimental groups compared to the control fish. However, supplementation with propolis markedly reversed these changes, with all parameters showing a trend toward normalisation, closely resembling control values. This highlights the restorative effect of propolis in maintaining biochemical homeostasis under fungicide-induced stress.

Tebuconazole exposure also significantly impaired antioxidant and enzymatic activities, are presented in Fig. B (1-8). There was a decline in SOD, GSH, GPx, and LDH levels, suggesting oxidative stress and compromised cellular integrity. This decline in all the antioxidant enzymes’ activity was significantly lower (P<0.05)in TBZ-treated fish in comparison to the control fish. These findings align with previous studies reporting that TBZ induces oxidative imbalance, DNA damage, and cellular apoptosis through ROS generation and disruption of redox mechanisms in mammalian and aquatic models [36,37]. Such enzymatic suppression reflects the oxidative burden imposed by fungicides on vital organs, as similarly evidenced in pesticide-induced hepatotoxicity and metabolic dysfunction [38]. Collectively, these alterations highlight the systemic oxidative threat posed by TBZ exposure.

Interestingly, CAT activity exhibited a biphasic response—initial elevation followed by a sharp decline—indicating an early compensatory mechanism overwhelmed by prolonged exposure. This pattern aligns with the notion that catalase, while crucial in decomposing hydrogen peroxide during early oxidative stress, can become functionally impaired or downregulated upon prolonged toxicant exposure, leading to cellular vulnerability [39,40]. In contrast, GR and AChE activities were significantly upregulated(P<0.05), reflecting disrupted redox balance and neuromuscular function. Such elevations may reflect compensatory responses to oxidative overload and disturbed mitochondrial homeostasis, as seen in pesticide-exposed aquatic models and oxidative injury-induced neuromuscular dysfunction [41,42]. Notably, co-administration of propolis effectively mitigated these disruptions, restoring all altered enzymatic markers toward normal physiological levels, likely due to its rich antioxidant profile and modulatory effects on cellular redox signalling and metabolic pathways, Fig. B (1-8). [43,44,45]. Collectively, these findings provide strong evidence for the protective role of propolis in counteracting tebuconazole-induced oxidative and metabolic toxicity.

Muscle histopathology revealed significant pathological alterations upon TBZ exposure, including fibre fragmentation, vacuolisation, nuclear malplacement, haemorrhage, fibrosis, and myotomal disintegration, with severity increasing dose- and time-dependently, demonstrated in Fig. C (1-11). Our findings are consistent with established drug-induced muscle toxicity patterns reported in both aquatic and mammalian models [46,47,48]. These changes indicated profound cytoskeletal damage and oxidative stress, aligning with prior studies that demonstrate how oxidative insults disrupt actin filament organisation and compromise muscle cell integrity under toxic or mechanical stress conditions [49,50]. However, co-treatment with propolis markedly ameliorated these effects. Fig. C (8-11), showing progressive structural recovery, reduced vacuolisation, minimised haemorrhage, and restored myofiber organization, consistent with emerging evidence supporting the role of natural bee-derived antioxidants like propolis in preserving muscle architecture and counteracting toxin-induced myopathy [51,52,53].

Conclusion

The present findings underscore the profound biochemical and structural toxicity of tebuconazole on fish muscle, evidenced by oxidative stress, disrupted macromolecular metabolism, and severe histopathological damage. Alterations in enzymatic activity further highlight the systemic oxidative burden imposed by prolonged fungicide exposure. Notably, propolis supplementation demonstrated significant restorative effects, normalising biochemical markers and preserving muscle integrity. This suggests its therapeutic potential as a natural antioxidant intervention. Overall, propolis offers a promising strategy to mitigate pesticide-induced toxicity in aquatic organisms.

Acknowledgment

The authors are grateful to the Chairperson, Department of Biosciences, Himachal Pradesh University, Shimla, for encouragement and for providing necessary facilities. The authors also truly appreciate the faculty and staff for their guidance.

References

1.         Elumalai, P., Gao, X., Parthipan, P., Luo, J., and Cui, J. (2025). Agrochemical pollution: a serious threat to environmental health. Current Opinion in Environmental Science and Health, 100597.

2.         Godfray, H. C. J., Beddington, J. R., Crute, I. R., Haddad, L., Lawrence, D., Muir, J. F., … and Toulmin, C. (2010). Food security: the challenge of feeding 9 billion people. Science, 327(5967), 812-818.

3.         Khan, B. A., Nadeem, M. A., Nawaz, H., Amin, M. M., Abbasi, G. H., Nadeem, M., … and Ayub, M. A. (2023). Pesticides: impacts on agriculture productivity, environment, and management strategies. In Emerging contaminants and plants: Interactions, adaptations and remediation technologies (pp. 109-134). Cham: Springer International Publishing.

4.         Sharma, A., Kumar, V., Shahzad, B., Tanveer, M., Sidhu, G. P. S., Handa, N., … and Thukral, A. K. (2019). Worldwide pesticide usage and its impacts on the ecosystem. SN Applied Sciences, 1, 1-16.

5.         Khoshnood, Z. (2024). A review on the toxic effects of pesticides in Zebrafish, Danio rerio, and common carp, Cyprinus carpio, emphasising Atrazine herbicide. Toxicology Reports, 101694.

6.         Tang, F. H., Lenzen, M., McBratney, A., and Maggi, F. (2021). Risk of pesticide pollution at the global scale. Nature Geoscience, 14(4), 206-210.

7.         Zhou, Q., Wang, S., Liu, J., Hu, X., Liu, Y., He, Y., … & Wu, X. (2022). Geological evolution of offshore pollution and its long-term potential impacts on marine ecosystems. Geoscience Frontiers, 13(5), 101427.

8.         Li, S., Sun, Q., Wu, Q., Gui, W., Zhu, G., and Schlenk, D. (2019). Endocrine-disrupting effects of tebuconazole on different life stages of zebrafish (Danio rerio). Environmental Pollution, 249, 1049-1059.

9.         Li, S., Jiang, Y., Sun, Q., Coffin, S., Chen, L., Qiao, K., … and Zhu, G. (2020). Tebuconazole induced oxidative stress-related hepatotoxicity in adult and larval zebrafish (Danio rerio). Chemosphere, 241, 125129.

10.       Škulcová, L., Chandran, N. N., and Bielská, L. (2020). Chiral conazole fungicides–(Enantioselective) terrestrial bioaccumulation and aquatic toxicity. Science of The Total Environment, 743, 140821.

11.       Yu, L., Chen, M., Liu, Y., Gui, W., and Zhu, G. (2013). Thyroid endocrine disruption in zebrafish larvae following exposure to hexaconazole and tebuconazole. Aquatic toxicology, 138, 35-42.

12.       . Bernabò, I., Guardia, A., Macirella, R., Sesti, S., Crescente, A., and Brunelli, E. (2016). Effects of long-term exposure to two fungicides, pyrimethanil and tebuconazole, on survival and life history traits of the Italian tree frog (Hyla intermedia). Aquatic Toxicology, 172, 56-66.

13.       Castro, T. F. D., da Silva Souza, J. G., de Carvalho, A. F. S., de Lima Assis, I., Palmieri, M. J., Vieira, L. F. A., … and Murgas, L. D. S. (2018). Anxiety-associated behavior and genotoxicity were found in adult Danio rerio exposed to a tebuconazole-based commercial product. Environmental toxicology and pharmacology, 62, 140-146.

14.       Altenhofen, S., Nabinger, D. D., Wiprich, M. T., Pereira, T. C. B., Bogo, M. R., and Bonan, C. D. (2017). Tebuconazole alters morphological, behavioral, and neurochemical parameters in larvae and adult zebrafish (Danio rerio). Chemosphere, 180, 483-490.

15.       Gupta, R. C., Doss, R. B., Dettbarn, W. D., and Milatovic, D. (2019). Skeletal muscle toxicity biomarkers. In Biomarkers in Toxicology (pp. 355-373). Academic Press.

16.       Toni, C., Ferreira, D., Kreutz, L. C., Loro, V. L. and Barcellos, L. J. G. (2011). Assessment of oxidative stress and metabolic changes in common carp (Cyprinus carpio) acutely exposed to different concentrations of the fungicide tebuconazole. Chemosphere, 83(4), 579-584.

17.       Van der Oost, R., Beyer, J., & Vermeulen, N. P. (2003). Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environmental toxicology and pharmacology, 13(2), 57-149.

18.       Siheri, W., Alenezi, S., Tusiimire, J., and Watson, D. G. (2017). The chemical and biological properties of propolis. Bee products-chemical and biological properties, 137-178.

19.       Attia, A. A., ElMazoudy, R. H., and El-Shenawy, N. S. (2012). Antioxidant role of propolis extract against oxidative damage of testicular tissue induced by insecticide chlorpyrifos in rats. Pesticide Biochemistry and Physiology, 103(2), 87-93.

20.       Yonar, M. E., Yonar, S. M., Ural, M. Ş., Silici, S., and Düşükcan, M. (2012). Protective role of propolis in chlorpyrifos-induced changes in the haematological parameters and the oxidative/antioxidative status of Cyprinus carpio carpio. Food and Chemical Toxicology, 50(8), 2703-2708.

21.       Kakoolaki, S., Talas, Z. S., Cakir, O., Ciftci, O., and Ozdemir, I. (2013). Role of propolis on oxidative stress in fish brain. Basic and Clinical Neuroscience, 4(2), 153.

22.       Hamed, H. S., and Abdel-Tawwab, M. (2017). Ameliorative effect of propolis supplementation on alleviating bisphenol-A toxicity: Growth performance, biochemical variables, and oxidative stress biomarkers of Nile tilapia, Oreochromis niloticus (L.) fingerlings. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology, 202, 63-69.

23.       Niță, V., Nenciu, M., and Galațchi, M. (2022). Speciile de Pești de la Litoralul Românesc. Atlas Actualizat/Fish Species of the Romanian Coast. Updated Atlas.

24.       Mani, F., Damasceno, H. C. R., Novelli, E. L. B., Martins, E. A. M., & Sforcin, J. M. (2006). Propolis: Effect of different concentrations, extracts, and intake period on seric biochemical variables. Journal of ethnopharmacology, 105(1-2), 95-98.

25.       Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem., 72(1-2), 248-254.

26.       Moore S, Stein WH (1954). A modified ninhydrin reagent for photometric determination of amino acids and related compounds. J Biol Chem., 211, 907–913.

27.       Folch, J., Lees, M. and Stanley, G. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Bio. chem., 226(1), 497-509.

28.       Aebi, H. (1984). Catalase in vitro. In Meth.  Enzymol.,105, 121-126.

29.       . Misra, H. P. and Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem., 247(10), 3170-3175.

30.       Dhindsa, R. S., Plumb-Dhindsa, P. A. M. E. L. A. and Thorpe, T. A. (1981). Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot., 32(1), 93-101.

31.       Borgmann, U., Moon, T. W. and Laidler, K. J. (1974). Molecular kinetics of beef heart lactate dehydrogenase. Biochem., 13(25), 5152-5158.

32.       Moron, M. S., Depierre, J. W. and Mannervik, B. (1979). Levels of glutathione, glutathione reductase, and glutathione S-transferase activities in rat lung and liver. Biochim. Biophys. Acta Gen. Subj., 582(1), 67-78.

33.       Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G. and Hoekstra, W. (1973). Selenium: biochemical role as a component of glutathione peroxidase. Sci., 179(4073), 588-590.

34.       Racker, E. (1955). Glutathione reductase from yeast and beef liver. J. of Bio. Chem., 217(2), 855-865.

35.       Ellman, G. L., Courtney, K. D., Andres Jr, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7(2), 88-95.

36.       Othmène, Y. B., Hamdi, H., Salem, I. B., Annabi, E., Amara, I., Neffati, F., … and Abid-Essefi, S. (2020). Oxidative stress, DNA damage, and apoptosis induced by tebuconazole in the kidney of male Wistar rats. Chemico-Biological Interactions, 330, 109114.

37.       Ku, T., Zhou, M., Hou, Y., Xie, Y., Li, G., and Sang, N. (2021). Tebuconazole induces liver injury coupled with ROS-mediated hepatic metabolism disorder. Ecotoxicology and Environmental Safety, 220, 112309.

38.       Khan, B. A., Nadeem, M. A., Nawaz, H., Amin, M. M., Abbasi, G. H., Nadeem, M., … and Ayub, M. A. (2023). Pesticides: impacts on agriculture productivity, environment, and management strategies. In Emerging contaminants and plants: Interactions, adaptations and remediation technologies (pp. 109-134). Cham: Springer International Publishing.

39.       Limón-Pacheco, J., and Gonsebatt, M. E. (2009). The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 674(1-2), 137-147.

40.       Jomova, K., Alomar, S. Y., Alwasel, S. H., Nepovimova, E., Kuca, K., and Valko, M. (2024). Several lines of antioxidant defence against oxidative stress: Antioxidant enzymes, nanomaterials with multiple enzyme-mimicking activities, and low-molecular-weight antioxidants. Archives of Toxicology, 98(5), 1323-1367.

41. Das, A., Bank, S., Chatterjee, S., Paul, N., Sarkar, K., Chatterjee, A., … and Ghosh, S. (2023). Bifenthrin disrupts cytochrome c oxidase activity and reduces mitochondrial DNA copy number through oxidative damage in the pool barb (Puntius sophore). Chemosphere, 332, 138848.

42.       . Liu, H., Wu, J., Yao, J. Y., Wang, H., and Li, S. T. (2017). The role of oxidative stress in decreased acetylcholinesterase activity at the neuromuscular junction of the diaphragm during sepsis. Oxidative medicine and cellular longevity, 2017(1), 9718615

43.       . Khan, M. R., and Siddique, F. (2012). Antioxidant effects of Citharexylum spinosum in CCl4-induced nephrotoxicity in rats. Experimental and toxicologic pathology, 64(4), 349-355.

44.       Kitamura, H. (2019). Effects of propolis extract and propolis-derived compounds on obesity and diabetes: knowledge from cellular and animal models. Molecules, 24(23), 4394.

45.       Hossain, S., Yousaf, M., Liu, Y., Chang, D., and Zhou, X. (2022). An overview of the evidence and mechanism of drug–herb interactions between propolis and pharmaceutical drugs. Frontiers in Pharmacology, 13, 876183.

46.       Janssen, L., Allard, N. A., Saris, C. G., Keijer, J., Hopman, M. T., and Timmers, S. (2020). Muscle toxicity of drugs: When drugs turn physiology into pathophysiology. Physiological reviews, 100(2), 633-672.

47.       Loganathan, K., Tennyson, S., and Arivoli, S. (2024). Triazophos toxicity induced histological abnormalities in Heteropneustes fossilis Bloch 1794 (Siluriformes: Heteropneustidae) organs and assessment of recovery response. The Journal of Basic and Applied Zoology, 85(1), 1-25.

48.       Valiyil, R., and Christopher-Stine, L. (2010). Drug-related myopathies of which the clinician should be aware. Current rheumatology reports, 12, 213-220.

49.       Yao, Y., Lacroix, D., and Mak, A. F. (2016). Effects of oxidative stress-induced changes in the actin cytoskeletal structure on myoblast damage under compressive stress: confocal-based cell-specific finite element analysis. Biomechanics and modeling in mechanobiology, 15, 1495-1508.

50.       Chou, H. C., Chen, Y. W., Lee, T. R., Wu, F. S., Chan, H. T., Lyu, P. C., … and Chan, H. L. (2010). Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia–reperfusion injury and treatment. Free Radical Biology and Medicine, 49(1), 96-108.

51.       Ali, A. M., and Kunugi, H. (2020). Apitherapy for age-related skeletal muscle dysfunction (sarcopenia): A review on the effects of royal jelly, propolis, and bee pollen. Foods, 9(10), 1362.

52.       Rolland, Y., Dray, C., Vellas, B., and Barreto, P. D. S. (2023). Current and investigational medications for the treatment of sarcopenia. Metabolism, 149, 155597.

53.       De Spiegeleer, A., Beckwée, D., Bautmans, I., and Petrovic, M. (2018). Pharmacological interventions to improve muscle mass, muscle strength, and physical performance in older people: an umbrella review of systematic reviews and meta-analyses. Drugs and ageing, 35, 719-734.