TH (AgroMas®) was supplied by the Federal Agricultural Marketing Authority, Ministry of Agriculture and Agro-Based Industry, Kedah, Malaysia.

The THSN was prepared using the green synthesis method17. First, TH was synthesized with silver nitrates to obtain the nanoparticle powder. The TH solution was adjusted to pH 8.0 by adding a few drops of sodium hydroxide (NaOH), before adding 0.1 M of silver nitrate solution. A color change from light brown to dark brown was observed, indicating the successful synthesis of THSN. The solution was dried overnight in the oven at 60 °C, and the nanoparticles were collected in powdered form and stored at room temperature. THSN solution was freshly prepared by dissolving the THSN powder in 0.5 mL of distilled water.

Adult male Sprague Dawley rats (weighing approximately 200 – 250 g; aged 8 – 10 weeks) were purchased from the Animal Research and Service Centre, Universiti Sains Malaysia (USM) Health Campus, Kubang Kerian, Kelantan. The animals were housed individually in polypropylene cages and maintained at a temperature of 25 ± 2 °C under a 12 h light/dark cycle. The animals were acclimatized for at least 7 days and provided rat pellets and water ad libitum. The experimental procedure was reviewed and approved by the Animal Ethic Committee of USM [USM/IACUC/2018/(111)(904)]. All experimental procedures involving animals were performed following the Institutional Guidelines for the Care and Use of Animals for Scientific Purposes.

A total of 72 male rats were randomized into six major groups (n = 12 rats per major group). The major groups were established as follows:

Group 1: Control — Rats were orally administered distilled water. Thirty minutes after the last oral treatment, the rats were injected subcutaneously with 0.5 mL of saline solution.

Group 2: THSN 10 mg — Rats were orally administered THSN (10 mg/kg). Thirty minutes after the last oral treatment, the rats were injected subcutaneously with 0.5 mL of saline solution.

Group 3: THSN 50 mg — Rats were orally administered THSN (50 mg/kg). Thirty minutes after the last oral treatment, the rats were injected subcutaneously with 0.5 mL of saline solution.

Group 4: KA only — Rats were orally administered distilled water. Thirty minutes after the last oral treatment, the animals were injected subcutaneously with KA solution (15 mg/kg).

Group 5: THSN 10 mg + KA — Rats were orally administered THSN (10 mg/kg). Thirty minutes after the last oral treatment, the animals were injected subcutaneously with KA solution (15 mg/kg).

Group 6: THSN 50 mg + KA — Rats were orally administered THSN (50 mg/kg). Thirty minutes after the last oral treatment, the animals were injected subcutaneously with KA solution (15 mg/kg).

Each major group was further divided randomly into two subgroups depending on the time of sacrifice (24 h and 5 days after KA induction; n = 6 rats per subgroup). For the THSN pre-treatment, 10 and 50 mg/kg dosages were chosen for this study because of the antidiabetic effect and increased antioxidant activities reported in rat models22, 23. Each group was pre-treated five times with the respective treatments at 12 h intervals.

The dose of KA (15 mg/kg) was reported to be sufficient in eliciting demonstrable damage in different brain regions such as the cerebellum and brainstem5. Moreover, a subcutaneous injection of saline was given to animals in the control group (no KA induction). Since the KA dosage of 15 mg/kg was associated with a high mortality rate, diazepam (10 mg/kg) was injected approximately 90 min after the onset of the first generalized seizure, as a precaution, to increase the survival rate of KA-induced rats5, 9. Finally, the rat hippocampus was used for biochemical analysis, to assess oxidative stress markers.

At the time of sacrifice (24 h or 5 days after KA induction), rats were euthanized with an overdose of sodium pentobarbital (100 mg/kg, intraperitoneal), followed by decapitation using a guillotine. First, the hippocampus from each animal was quickly extracted from the brain, isolated, weighed, and stored at −80 °C until use. Then, the tissue samples were homogenized (10% w/v) in ice-cold 0.1 M phosphate-buffered saline (pH 7.4). Next, the homogenates were centrifuged (10,000 x g) for 10 min, and the supernatants were aliquoted and stored at −80 °C until analysis.

The oxidative stress parameters were determined based on the levels of MDA, NOx, PCO, GSH, TAS, and CAT activity. These markers were determined using commercially available ELISA kits (Qayee, Wuhan) via the double antibody enzyme-linked immunosorbent one-step process. The assays were performed following the manufacturer’s instructions.

The current study investigated the state of oxidative stress in the hippocampus by evaluating MDA, PCO, and NOx levels. MDA is widely used as a convenient biomarker for lipid peroxidation of polyunsaturated fatty acids because it readily reacts with thiobarbituric acid24. In the present study, MDA levels were significantly elevated at 24 h and 5 days following KA administration, indicating that a significant level of lipid peroxidation occurred in the hippocampus. This finding is consistent with other studies25, 26.

Another important lipid peroxidation biomarker is NOx, a messenger molecule in the central nervous system27. A high level of NOx has been associated with a rise in the highly reactive toxic peroxynitrite radical, which may cause cellular damage28. It has been demonstrated that glutamate-KA receptor stimulation induces neuronal NO release, which in turn modulates glutamate transmission29. The current study demonstrated that NOx levels in the rats’ hippocampus were significantly elevated at 24 h after KA administration, but not 5 days after. This time-dependent outcome was consistent with an earlier report, where KA significantly increased NOx levels in the right temporal lobe as early as 60 to 90 min after KA induction, and the NOx levels decreased gradually after 120 min30.

In the present study, the occurrence of protein oxidation was demonstrated by the raised PCO levels at 24 h after KA administration. Protein oxidation involves the covalent modification of amino acid side chains and protein backbones, which results in protein fragmentation or protein-protein cross-linking, altering their conformation, solubility, and enzyme activities31. The use of PCO as a biomarker of oxidative stress has several advantages because of its stability and relatively early formation. The result of the present study is similar to previous studies, which reported KA-induced protein oxidation in different regions of the brain, including the hippocampus5, 32. The current study also demonstrated that PCO levels were remarkably increased at 5 days after KA administration but not at 24 h. Furthermore, Dutra et al. (2018) demonstrated that rats in the epilepsy animal model exhibited higher levels of protein carbonyl in the hippocampus during the early silent phase (3 – 5 days after status epilepticus)33. An elevated PCO level may be caused by excessive protein oxidation or the decreased capacity of an organism to eliminate oxidatively damaged proteins.

The hippocampus is susceptible to oxidative stress because of the high levels of serotonin that promote free radical formation34, high content of polyunsaturated fatty acids and iron, and low antioxidant defenses such as CAT and GSH35. Consistent with previous reports, systemic KA administration affected CAT activity in the brain36, 37. We observed that the CAT activity in the hippocampal tissue was significantly lowered in the KA-induced rats at 24 h and 5 days, which represents an important index of oxidative stress. The present study agrees with Liu et al. (2015), where KA induction led to lower CAT activity in the hippocampus38.

The current study also demonstrated the presence of low GSH levels in the hippocampus at 24 h and 5 days after KA induction, indicating a low GSH antioxidant defense mechanism. GSH is the most abundant intracellular thiol in the brain39, 40, which maintains the redox balance of cells, reduces oxidized particles, and detoxifies reactive oxygen species41. Therefore, the amount of GSH may be a valuable indicator of oxidative stress levels.

The use of exogenous antioxidants to inhibit the production of free radicals is well-recognized in the literature38. In the present study, pre-treatment with THSN prevented the KA-induced increase in MDA, NOx, and PCO while simultaneously enhancing the production of CAT, GSH, and TAS at different time points, which confirms the protective effects of THSN against oxidative stress. The pre-treatments with THSN significantly reduced lipid peroxidation, NOx, and TAS after 24 h and protein oxidation after 5 days of KA administration, indicating the protective effects of these substances against oxidative stress on membrane lipids and cellular proteins. However, THSN did not significantly increase the TAS level or reduce the MDA or NOx levels after 5 days, possibly because of a lower rate of bioavailability, rapid metabolism, and elimination of active ingredients in THSN42, 43.

The increase in GSH and TAS levels by THSN suggested antioxidant properties of THSN possibly by increasing the brain’s endogenous defense. TAS is used to measure the overall antioxidant status of the body, which helps elucidate the protective effect displayed by antioxidants, reflecting their improvement in endogenous defense44. The findings in this study showed that 10 mg and 50 mg of THSN at 24 h after KA administration significantly improved TAS levels in the hippocampus.

The neuroprotective effects of THSN are not surprising because THSN exhibited remarkable antioxidant activity17. Previous studies have demonstrated that the majority of silver nanoparticles synthesized using green methods, particularly plant extracts, have free radical scavenging activity45. The THSN synthesized in the current study contains alcohols, phenols, amide, carboxylate ions, and protein, and exhibited high antioxidant activity, as described previously17. Moreover, TH, the reducing agent used to synthesize the silver nanoparticles, is known to contain antioxidants, such as phenols and flavonoids, with a high total phenolic content (251.7 ± 7.9 mg gallic acid/kg honey), high total antioxidant activity (322.1 ± 9.7 μM Fe(II)), and good antiradical activity (41.30 ± 0.78% inhibition)46.

The attachment of antioxidant molecules (such as phenols and flavonoids) to the surfaces of nanoparticles may create phytoantioxidant-functionalized nanoparticles that can synergistically protect against oxidative damage45. These phytoantioxidant-functionalized nanoparticles are a safer alternative to nanoparticles produced using physical or synthetic chemical methods. Creating nanoparticles using natural antioxidants, like honey, is advantageous because it can increase stability and biocompatibility while diminishing toxicity, in addition to preserving the desirable properties of natural compounds.

Taken together, the present study supports earlier findings showing that KA administration involves oxidative stress pathways5, 47, 48. KA-induced oxidative stress is associated with enhanced lipid peroxidation and protein oxidation while reducing both the enzymatic and non-enzymatic anti-oxidative defenses of the body. Previously, it has been shown that KA-induced oxidative stress is associated with a rise in extracellular glutamate levels in the hippocampus, which is associated with free radicals generation and a reduction in residual antioxidant activity10. Notably, the present study demonstrated that these KA-induced changes can be improved by introducing silver nanoparticles produced using TH, which are non-toxic, environmentally friendly, and economically more sustainable.

The present study concluded that THSN attenuates oxidative stress in the hippocampus of KA-induced rats. THSN has been shown to prevent the KA-induced increase in oxidative stress markers and simultaneously improve the anti-oxidative defenses. Further investigations at the molecular level of the mechanism may further clarify the protective effects of THSN against KA-induced oxidative stress in rats.

CAT: Catalase, ELISA: enzyme-linked immunosorbent assay, GSH: Reduced glutathione, KA: Kainic acid, MDA: Malondialdehyde, NOx: total nitrate/nitrite, PCO: Protein carbonyl, SEM: Standard error mean, SPSS: Statistical package for social sciences, TAS: total antioxidant status, TH: Tualang honey, THSN: Tualang honey-mediated silver nanoparticles

The authors gratefully acknowledge Universiti Malaysia Kelantan, Animal Research and Service Centre (ARASC), and Central Research Laboratory (CRL), Unversiti Sains Malaysia, Health Campus for providing the lab facilities for this research work.

SKNS developed the original idea and designed the study. PVR gave advices regarding on preparation of THSN. SKNS and SM assist the biochemical study. MAA and SKNS supervised the work. HH prepared the THSN, performed the experiment, analysed the results and drafted the article. All authors have read, revised and approved the article submission.

This research was financially supported by the Universiti Sains Malaysia under Research University (Individual) (RUI Grant No: 1001/PPSP/8012249).

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

The experimental procedure was approved by the Animal Ethic Committee of USM [USM/IACUC/2018/(111)(904)]. All experimental and procedures involving animals were performed following the Institutional Guidelines for the Care and Use of Animals for Scientific Purposes. No human volunteers were used.

Not applicable.

The authors declare that they have no competing interests.