Plant material and obtaining the ethanolic extract

The extract was obtained according to procedures previously described [16]. The aerial parts were dried in a circulating air oven for 7 days. Leaves and stems were ground by hand, obtaining 80.715 g of dry weight. After this, the extract was submitted to two extraction steps with 95% ethanol. The first was a 6-h dynamic maceration at 700 rpm, yielding 3.38 g of extract. The second, using the plant residue, was a 6-day static maceration, yielding 2.49 g. Both extracts were filtered, evaporated at 60°C, dried, and then combined to ensure exhaustive extraction of plant compounds.

Ethical considerations and treatment groups

Male Swiss mice (25–30 g) obtained from the Central Animal Facility of the Universidade do Vale do Itajaí (UNIVALI) were kept in groups with access to food and water ad libitum and under a 12-h light-dark cycle (lights on at 7:00 AM) and controlled temperature (23 ± 1°C). These procedures followed the standards of the UNIVALI Animal Use Ethics Committee (CEUA/UNIVALI), protocol number 022/17, obtained in December 2017 with an addendum in 2023, and are in accordance with the Guide for the Care and Use of Laboratory Animals. Behavioral experiments were conducted from 1:00 PM to 6:00 PM. The animals were divided into six experimental groups: (G1) sham (surgery and vehicle infusion only); (G2) vehicle [streptozotocin (STZ)-induced and treated with phosphate buffered saline (PBS)]; (G3–G5) induced by STZ and treated orally (PO) with EETD at 0.1, 1.0 and 3.0 mg/kg; (G6) positive control [induced by STZ and treated with rivastigmine 0.6 mg/kg, treated intraperitoneally (IP)]. After the behavioral procedures, the animals were euthanized with a deep anesthetic overdose through the IP application of a solution of Xylazine 2% (Xilazin^®) 30 mg/kg and Ketamine (Cetamin^®) 300 mg/kg. Their brains were carefully removed and prepared for biochemical assays. The euthanasia method adopted is based on the Euthanasia Practice guideline document of the National Council for the Control of Animal Experimentation (CONCEA), a federal agency to which all CEUAs are linked in Brazil. This procedure was performed in accordance with the ethics committee for animal use at Univali (CEUA/UNIVALI, which is linked to CONCEA).

Drugs and reagents

The following drugs and reagents were used in this study. STZ and rivastigmine were purchased from Sigma-Aldrich (St. Louis, MO, USA), Ketamine was obtained (Vetbrands, FL, USA), xylazine (Agener União, SP, BR), and lidocaine with epinephrine 2% (Cristália, SP, BR). The reagents used to obtain EETD were obtained from VETEC (RJ, BR) and LABSYNTH (SP, BR). All drugs were dissolved in saline (except STZ, which was dissolved in artificial cerebrospinal fluid prepared as described [18] and infused at room temperature).

Alzheimer’s animal model: intracerebral administration of streptozotocin

This procedure was performed as described by Pinton et al. (2010) [19] with adaptations [18, 20–22]. Mice were anesthetized with a combination of xylazine and ketamine (10 and 100 mg/kg, respectively, via IP route) and received two intracerebroventricular STZ (I.C.V. STZ) injections of (2.5 μL, 2.5 mg/mL), 48 h apart, targeting the cerebral ventricle. Injections were made using a Hamilton syringe and were guided by the bregma. Treatments began on the third day after the first injection and continued until day 24. Behavioral assessments (ambulation, anxiety, and memory) were conducted between days 17 and 24. From the 17th to 24th, mice had the assessment of ambulation, anxiety, and memory according to the experimental design presented in Figure 1.

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Figure 1. Timeline of experimental protocol. Male Swiss mice were administered by intracerebroventricular (I.C.V.) injection with 2.5 mg/mL of STZ. Experimental treatments started 48 h after the last infusion (day 1). A total of 60 animals were used in the study. The animals were divided into groups (n = 10) according to treatment with vehicle, EETD [0.1, 1.0, or 3.0 mg/kg, administered orally (PO)] or rivastigmine [0.6 mg/kg, intraperitoneally (IP)], starting from day 1 to day 17 without interruption and continuing from day 17 until day 24 (period in which the behavioral tests took place). A group of animals (sham operated/n = 10) underwent the same I.C.V. procedure with artificial liquor, instead of STZ, used to control the procedure. Behavioral parameters were assessed with the same animals starting on day 1 until the 24th. Finally, the animals were euthanized 24 h after the last behavior test and had their hippocampus dissected for subsequent biochemical analysis. EETD: Tithonia diversifolia ethanol extract; OFT: open field test; EPM: elevated plus maze; FST: forced swimming test; IAT: inhibitory avoidance test; NOR: novel object recognition; STZ: streptozotocin.

Open field test

To assess possible treatment effects on locomotion, mice were placed in a 50 cm × 50 cm × 39 cm open field divided into nine squares. The test was conducted 60 min after EETD (0.1, 1.0, or 3.0 mg/kg, PO) or vehicle administration and 30 min after rivastigmine (0.6 mg/kg, IP). The number of line crossings was recorded during 6 min [20–22].

Elevated plus maze test

To assess anxiety-related behavior, mice were subjected to the elevated plus maze (EPM) test 60 min after administration of EETD (0.1, 1.0, or 3.0 mg/kg) or vehicle administered PO, and 30 min after rivastigmine (0.6 mg/kg, IP). The apparatus had two open and two closed arms, elevated 70 cm from the floor. Each animal was placed in the center, and total arm entries and time spent in each arm were recorded over 5 min [18].

Forced swimming test

The forced swimming test (FST) was chosen to evaluate behavioral parameters of depression. It is a model developed by Porsolt and collaborators first in rats and later adapted for mice and adapted to our laboratory by Tolardo et al. (2010) [23]. In the test, the animals received the treatments and were subsequently transferred individually to a glass tank (46 cm high × 20 cm diameter) containing 20 cm of water at 24 ± 1°C at a controlled temperature (24 ± 1°C) and forced to swim for a period of 6 min. The behavioral parameter observed was the immobility time. Each animal was considered to be in a state of either floating or immobility when it produced minimal movements necessary to keep its head above water, preventing it from sinking. The test was conducted 60 min after EETD (0.1, 1.0, or 3.0 mg/kg, PO) or vehicle administration, and 30 min after rivastigmine (0.6 mg/kg, IP).

Novel object recognition test

The novel object recognition (NOR) test has been widely used to evaluate the effect of substances on the special memory of animals, especially mice [24, 25]. The experiment was conducted in 3 stages, each lasting 10 min. In the first stage (day 1), the animals were placed in an open field where they remained freely exploring the apparatus. In the second stage (24 h after the first), two objects (A and B) that were completely identical (color and size) were introduced into the apparatus, positioned equilaterally to each other, and the interaction time with each object was timed. In the third stage (24 h after the second), object B was removed and replaced by a new object (different in color and shape to object B, but of the same size), and the interaction time between the previously known object and the new object was timed [20, 24]. RI (%) = [(time spent on the new object/time spent on the already known object + time spent on the new object) × 100%]. It was considered a positive effect on memory results above 50%.

Inhibitory avoidance test

This test evaluates the aversive memory. The apparatus used was an automated box (Insight^®) measuring 50 cm × 25 cm × 25 cm. The base included copper grid bars (1 mm in diameter, spaced 1 cm apart) and a wooden platform. The procedure consisted of two phases: training and testing. During training, each mouse was placed on the platform, and the time taken to step down was recorded. Upon stepping down, the animal received a 2-second foot shock (0.4 mA). After 24 h, the test session was conducted using the same setup, but without shocks. Treatments were administered immediately after the training session, and memory performance was evaluated based on the difference in step-down latency between the two sessions [20, 22, 24].

Tissue preparation, biochemical analysis, and nitrite production

Once the behavioral assays were completed, the animals were euthanized with an overdose of deep anesthetic (as previously described), and the brains were collected and dissected to obtain the hippocampus. As is routinely done in our laboratories, the samples were homogenized in phosphate buffer (pH 7.4) in a dilution ratio of 1:3 (w/v) for immediate quantification of reduced glutathione (GSH) and malondialdehyde (MDA) levels.

For GSH evaluation [26], aliquots of tissue homogenate were deproteinized with 12.5% trichloroacetic acid and centrifuged at 900 × g for 15 min at 4°C. A 10 μL portion of the resulting supernatant was mixed with 280 μL of 0.4 M Tris-HCl buffer (pH 8.9) and 10 μL of 10 mM 5,5ʼ-dithiobis(2-nitrobenzoic acid) (DTNB). After 20 min, absorbance was measured at 405 nm, and GSH concentrations were determined by interpolation against a standard curve (1–10 μg/mL), with results expressed as μg/g [24]. Lipid peroxidation was measured by the MDA quantification. A 100 µL aliquot of the homogenate was mixed with 200 µL of 0.5% thiobarbituric acid in 20% trichloroacetic acid and incubated at 95°C for 60 min. After cooling to room temperature, samples were centrifuged at 3,000 × g for 10 min, and the absorbance of the supernatant was measured at 532 nm. MDA concentrations were calculated using a standard curve generated with known MDA concentrations and expressed as nmol/mg [27].

Moreover, the homogenates were centrifuged at 9,000 × g for 15 min, and the supernatants were used for biochemical analyses of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). For SOD activity measurement, analysis was conducted as described previously [28] with some modifications [24] where the supernatant obtained from homogenized samples was incubated with pyrogallol (1 mM) in a buffer system containing Tris-HCl (1 mM) and EDTA (5 mM), adjusted to pH 8.5. After 20 min of incubation, the reaction was stopped by the addition of hydrochloric acid (1 M), and absorbance was recorded at 405 nm. Enzymatic activity was defined as the amount of SOD required to suppress 50% of pyrogallol auto-oxidation compared to control samples, and results were expressed as mmol·min^–1·mg^–1.

For the CAT assay, performance was conducted in accordance with Aebi [29] with adaptations [30]. For the assay, aliquots of the supernatant were added to a reaction medium consisting of Tris-HCl–EDTA buffer (200 mM, pH 8.5) supplemented with 20 mM hydrogen peroxide, once this activity is determined as the enzymatic capacity to degrade 1 μmol of hydrogen peroxide per minute under standard conditions (25°C, pH 7.0) [29]. Enzymatic activity was quantified by spectrophotometric analysis at 240 nm, and values were expressed as μmol·min^–1·mg^–1.

The GST assay was performed according to Habig et al. (1974) [31] with adaptations [30] using supernatant aliquots in a reaction mixture containing 1 mM 1-chloro-2,4-dinitrobenzene (CDNB), 1 mM GSH, and 100 mM potassium phosphate buffer (pH 6.5). The conjugation of CDNB with GSH was monitored spectrophotometrically at 340 nm over a 90-second interval. Enzymatic activity was calculated based on the molar extinction coefficient of GSH (9.6 mM^–1·cm^–1) and expressed as mmol·min^–1·mg^–1.

GR activity was measured by monitoring the NADPH-dependent reduction of oxidized glutathione (GSSG), as described by Carlberg and Mannervik [32]. Briefly, assays were performed in a phosphate buffer (0.1–0.2 M, pH 7.0) containing 2 mM EDTA, with final reaction concentrations of 0.1 mM NADPH and 1.0 mM GSSG at 25°C. Sample aliquots were added to pre-equilibrated reaction buffer, and the enzymatic reaction was initiated by GSSG addition. The decrease in absorbance at 340 nm, corresponding to NADPH oxidation, was recorded continuously for 1–3 min. Enzyme activity was calculated using the molar extinction coefficient of NADPH (6.22 mM^–1·cm^–1) and normalized to protein content. GR activity was determined by measuring the rate of NADPH oxidation and expressed as μmol·min^–1·mg^–1.

GPx activity was assessed in a 96-well plate by monitoring NADPH oxidation at 340 nm for 1 min (10-second intervals) in a reaction mixture containing sodium azide, β-NADPH, yeast GR, and GSH in sodium phosphate buffer (pH 7.0). The reaction was initiated by the addition of hydrogen peroxide, and results were expressed as nmol·min^–1·mg^–1. The analysis was performed as described previously [33] with adaptations [34]. For all the measurements, protein concentrations were determined using the Bradford method [35].

Finally, nitrite production was determined by the Griess reaction [36]. After centrifugation of hippocampal homogenates, 50 µL of the supernatant was incubated with 100 µL of Griess reagent at room temperature for 10 min. Absorbance was measured at 525 nm using a microplate reader, and nitrite concentrations were determined from a standard curve (0–100 µM) and were expressed as µmol/g.

Determination of inflammatory biomarkers

Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels were measured from hippocampal tissue homogenates previously prepared as previously described. For this purpose, mouse ELISA kits purchased from RayBiotech Inc. (Norcross, GA, USA) and R&D Systems Inc. (Minneapolis, USA), respectively, were used. The procedures were performed according to the manufacturer’s instructions and the results were presented as pg/mg protein for TNF-α and IL-6.

Acetylcholinesterase activity

AChE activity was measured according to the method of Ellman [37] and standardized in our laboratories by Cazarin et al. (2021) [24]. Approximately 900 µL of 24 mM K-phosphate buffer (pH 7.2), 50 µL of 1 mM 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB), 50 µL of supernatant solution S1 (0.2–0.4 mg protein) and different concentrations of TXR (0.45, 0.89, and 1.77 µM) were contained in the assay medium. To start the reaction, 25 µL of 0.8 mM ACh was added and monitored at 412 nm for 4 min at 25°C in a spectrophotometer. AChE activity was expressed in µmol of ACh hydrolyzed/h/mg protein [24].

Cell culture

The procedures for cell culture were adapted from the previously reported protocols by Lee et al. (2023) [38] and Kim et al. (2022) [39]. The BV2 (murine microglial), HT22 (murine hippocampal neuronal), and [Chinese hamster ovary (CHO) cells stably expressing amyloid precursor protein (APP)] APP-CHO cell lines were utilized as described in the original studies [38, 39]. BV2 and HT22 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. In contrast, APP-CHO cells were cultured in RPMI-1640 (cell culture medium commonly used to grow mammalian cells) containing 10% heat-inactivated FBS and 1 μg/mL geneticin. All cell lines were incubated at 37°C in a humidified incubator with 5% CO₂.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

To evaluate cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted. BV2 cells were plated at a density of 2.0 × 10⁴ cells per well in 96-well plates and allowed to adhere for 3 h. HT22 cells were seeded at 1.0 × 10⁴ cells/well and incubated for 24 h, while APP-CHO cells were plated at 2.0 × 10⁴ cells/well and cultured overnight. After the initial incubation, the cells were serum-deprived for 1 h and subsequently treated with test compounds dissolved in dimethyl sulfoxide (DMSO). Following 24 h of treatment, 10 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated at 37°C for an additional 3 h. The supernatant was then discarded, and the resulting formazan crystals were dissolved in 100 μL of DMSO. Absorbance was measured at 540 nm using a BioTek Epoch microplate reader (BioTek Instruments). Each experiment was carried out in triplicate as per the reference protocol [39].

Measurement of nitric oxide production

For the quantification of NO production, BV2 cells were seeded into 24-well plates at a concentration of 2.0 × 10⁵ cells/well and cultured for 24 h. Following incubation, the culture medium was replaced with serum-free DMEM containing test samples (prepared in DMSO). After 1 h of pre-treatment, lipopolysaccharide (LPS) was added at a final concentration of 1 μg/mL, and the cells were incubated for an additional 24 h at 37°C. Subsequently, 100 μL of the culture supernatant from each well was transferred to a 96-well plate and mixed with an equal volume of Griess reagent. The mixture was incubated in the dark at room temperature for 30 min, and the absorbance was measured. All assays were performed in triplicate, following the previously established method [39].

Effect of the extract on Aβ production

APP-CHO cells were seeded in 6-well plates at a density of 6.0 × 10⁵ cells/well and incubated overnight. After serum starvation for 1 h, cells were treated with DMSO-diluted test samples and incubated for 24 h at 37°C. Cells were then rinsed with PBS, lysed in Laemmli buffer, and boiled at 100°C for 10 min. Proteins were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with 5% skim milk in PBS for 1 h. Membranes were incubated overnight at 4°C with primary antibodies: APP/β-amyloid (1:1,000, Cell Signaling Technology), BACE1 (1:1,000, EMD Millipore), and α-tubulin (1:25,000, Sigma-Aldrich). After PBS-T washes (3 × 15 min), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,500, Bio-Rad). Protein bands were visualized using ECL (Western Blotting Substrate) reagent (Menlo Park, CA, USA) and detected using the ChemiDoc™ XRS+ (Bio-Rad) [38].

Determination of tau hyperphosphorylation

HT22 cells were seeded in 6-well plates at a density of 6.0 × 10⁵ cells/well in 1,000 μL of medium and incubated for 24 h. The medium was then replaced with serum-free DMEM, and DMSO-diluted test samples were added. After 1 h, okadaic acid (80 nM) was introduced, followed by 16 h incubation at 37°C. Cells were then rinsed with PBS and lysed in Laemmli sample buffer. Cell lysates were boiled for 15 min at 100°C before analysis by western blotting. For the western blot analysis, proteins were separated via 7.5% SDS-PAGE, transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA), and blocked with 5% bovine serum albumin in TBS for 2 h. Membranes were incubated overnight at 4°C with primary antibodies including AT8 (1:1,000, Thermo Fisher Scientific, Middlesex, MA, USA) and α-tubulin (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA). After TBST washes (3× for 15 min each), membranes were incubated with HRP-conjugated secondary antibodies (1:1,000, Thermo Fisher Scientific) for 1 h at room temperature. ECL detection reagent (ATTA corporation, Tokyo, Japan) was used for visualization, and protein bands were detected using a ChemiDoc™ XRS+ (Bio-Rad). The procedure was followed as previously described [40].

Statistical analysis

The parametric results were expressed as mean ± standard deviation (SD) and statistical significance was obtained by one or two-way analysis of variance (ANOVA), followed by Tukey’s test, when applicable. Kruskal-Wallis test followed by Dunn’s test was used to evaluate non-parametric results, which were expressed as median ± interquartile ranges. The Kolmogorov-Smirnov normality test was applied to verify the data’s normality. Moreover, power analysis was performed to determine all sample sizes. Differences were significant when P < 0.05, by using the GraphPad Prism version 5.00 for Windows (GraphPad Software, California, USA) program. To complement the inferential statistical tests, analyses of effect sizes and statistical power were conducted using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA, USA). Specifically, Cohen’s d was calculated to quantify the magnitude of differences between two means in pairwise comparisons, while Cohen’s f was employed to assess the effect size for analyses involving more than two groups, such as in ANOVA models.

Effects of treatments on parameters of locomotion, depression-like, and anxiety-like behavior in animals with STZ-induced AD

The effects of treatments on locomotor performance, behavioral parameters of anxiety and depression in animals with STZ-induced AD are shown in Figure 2. I.C.V. STZ promoted a significant decrease at open arms entries (P < 0.001, Cohen’s d = –2.59) (Figure 2A) and exploration time (P < 0.01, Cohen’s d = –2.82) (Figure 2B) compared to sham group and mice treated with EETD at all doses used, had a significant increase in the frequency (P < 0.001, Cohen’s d = 2.48; P < 0.001, Cohen’s d = 5.99 and P < 0.001, Cohen’s d = 6.71 respectively for 0.1, 1.0 and 3.0 mg/kg of EETD) and time of permanence (P < 0.001, Cohen’s d = 9.83; P < 0.001, Cohen’s d = 5.19 and P < 0.001, Cohen’s d = 6.89 respectively for 0.1, 1.0 and 3.0 mg/kg) in the open arms when subjected to EPM suggesting anxiolytic-like effect (Figure 2A and B) when compared to the vehicle group. Furthermore, the treatments did not change the locomotor performance (Figure 2C) of the mice, according to the results obtained in the open field test experiment. Besides, the results showed that the EETD has an antidepressant-like effect, which was evidenced by the decrease in the immobility time of the animals when submitted to the FST (Figure 2D) compared to the vehicle group (P < 0.001, Cohen’s d = –1.94; P < 0.001, Cohen’s d = –2.54 and P < 0.0001, Cohen’s d = –4.57 respectively for 0.1, 1.0 and 3.0 mg/kg). Rivastigmine at the dose used also significantly promoted anxiolytic and antidepressant-like effects when compared to the vehicle group (P < 0.001 and P < 0.0001, Cohen’s d = 13.81 and Cohen’s d = –3.90, respectively).

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Figure 2. Behavioral effects of EETD 0.1, 1.0, or 3.0 mg/kg treatments on anxiety- and depression-like parameters and locomotory activity of I.C.V. STZ-induced AD in mice. (A) Frequency of entries into open arms; (B) time to explore in open arms; (C) number of crossings; (D) immobility time. Effect of EETD [0.1, 1.0, or 3.0 mg/kg, administered orally (PO)] or rivastigmine [0.6 mg/kg, intraperitoneally (IP)] in mice submitted to STZ 2.5 mg/mL intracerebroventricular (I.C.V.) injection. Behavior parameters assessed on the Elevated Plus Maze (EPM) are shown in Figure 2A and B (open arms frequency entries and open arms time exploration, respectively); open field test (OFT) in Figure 2C (number of crossings) and forced swimming test (FST) in Figure 2D (immobility time). Results are presented by mean ± SD, n = 10 per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared to the vehicle group. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the sham group (animals that received no I.C.V. STZ administration). EETD: Tithonia diversifolia ethanol extracts; STZ: streptozotocin.

Effect of treatment on aversive memory

The findings indicate that administering STZ (2.5 mg/mL, I.C.V.) caused a decline in cognitive function in the animals, evidenced by the vehicle group’s consistent descent latency during both training and testing sessions (Figure 3A). However, groups with STZ-induced AD that received EETD treatment at doses of 1.0 and 3.0 mg/kg (the highest doses used) exhibited a statistically significant improvement in cognitive deficits (P < 0.001 for both doses, Cohen’s d = 7.24 and 6.13, respectively) (Figure 3A and B). This indicates that EETD increased the time it took for animals to descend from the platform in the inhibitory avoidance test (IAT), suggesting a beneficial effect on memory compared to the vehicle group. Additionally, rivastigmine (0.6 mg/kg, IP) significantly enhanced memory in animals with AD (P < 0.001, Cohen’s d = 13.03) when compared to the vehicle group, as expected.

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Figure 3. Behavioral effects of EETD 0.1, 1.0, or 3.0 mg/kg treatments on aversive memory of I.C.V. STZ-induced AD mice submitted to inhibitory avoidance test (IAT). (A) Results from training and testing sessions, respectively; (B) results refer to the test session only. Behavior evaluation on IAT. In panel A, the first column of each treatment represents the training session and the second column represents the testing session. Panel B shows differences among groups for step-down latency in the test session. In each column, data are expressed as medians followed by interquartile ranges (25–75). Data were submitted to analysis of variance (ANOVA) with Dunn’s test and showed ***P < 0.001 compared to the vehicle group. ###P < 0.001 compared to sham group (animals that received no I.C.V. STZ administration) for the average descent index (Mann-Whitney test). n = 10 per group. Cohen’s f = 3.09. EETD: Tithonia diversifolia ethanol extract; RIVAST: rivastigmine; STZ: streptozotocin.

Effect of treatment on spatial memory

Figure 4 shows the results related to NOR. In panel A, it can be observed that the group that received STZ and was treated with vehicle had a significant reduction (P < 0.05) in the time spent exploring the new object (B) when compared to the sham group. However, animals treated with EETD (1.0 and 3.0 mg/kg) as well as with rivastigmine had a longer exploration time on the new object (P < 0.05 and P < 0.001, P < 0.01, respectively, compared to the vehicle (Figure 4A). The beneficial effects of treatments with EETD and rivastigmine are best observed in panel B, which represents the recognition index of the new object. The results demonstrate that the administration of STZ resulted in a decline in the cognitive function of the animals, as demonstrated in the vehicle group (P < 0.001, Cohen’s d = –4.85 compared to the sham group), where the old object that was already familiar to them was not recognized. However, in the groups with AD induced by STZ and treated with EETD at doses of 1.0 and 3.0 mg/kg, it was possible to observe a reduction in the cognitive deficit, through an increase in the recognition index of the new object in a statistically significant way when compared to the vehicle group (P < 0.001, Cohen’s d = 5.55 and 5.77, respectively).

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Figure 4. Behavior evaluation of EETD 0.1, 1.0, or 3.0 mg/kg treatments on recognition index of I.C.V. STZ-induced AD in mice submitted to novel object recognition (NOR). (A) Exploration time of objects A and B; (B) recognition index. Effect of EETD [0.1, 1.0, or 3.0 mg/kg, administered orally (PO)] or rivastigmine [0.6 mg/kg, intraperitoneally (IP)] in mice submitted to STZ 2.5 mg/mL intracerebroventricular (I.C.V.) injection. Behavior evaluation on NOR demonstrates the differences in exploration time among familiar and new objects in Figure 4A. Figure 4B shows the recognition index for groups. Results are represented by mean ± SD, n = 10 per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to vehicle group. ###P < 0.001 compared to sham group (animals that received no I.C.V. STZ administration). Cohen’s f = 2.17, indicating a substantial effect size. EETD: Tithonia diversifolia ethanol extract; STZ: streptozotocin.

Biochemical markers of oxidative and nitrosative stress

The results are shown in Figure 5, where it is demonstrated that I.C.V. STZ injection indicates a decrease in CAT (Figure 5A), SOD (Figure 5B), GST (Figure 5C), GR (Figure 5D), GPx (Figure 5E) and GSH (Figure 5F) compared to sham group (P < 0.0001). The 3.0 mg/kg EETD treatment highly significantly elevated levels of GSH (P < 0.0001, Cohen’s d = 2.87), increased CAT (P < 0.05, Cohen’s d = 2.50), GPx (P < 0.01, Cohen’s d = 1.91) and SOD activity (P < 0.001, Cohen’s d = 4.54) compared to the vehicle-treated group. It was also observed in the experiments that the treatment of animals with rivastigmine promoted a significant increase (P < 0.01 or P < 0.0001) in these markers (except for GSH) compared to the vehicle group. Additionally, the results also showed that STZ was able to significantly increase the levels of MDA (P < 0.0001, Cohen’s d = 40.45) (Figure 6A) and nitrite (P < 0.01, Cohen’s d = 2.02) (Figure 6B) in the brain of the animals. Interestingly, the treatment with EETD and rivastigmine were able to decrease MDA and nitrite levels induced by STZ (P < 0.0001, Cohen’s d = –11.30 and Cohen’s d = –19.78 for MDA; P < 0.05, Cohen’s d = –1.94 and P < 0.01, Cohen’s d = –2.90 for nitrite; EETD and rivastigmine, respectively).

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Figure 5. Antioxidant effects of EETD 3.0 mg/kg treatment in the hippocampus of mice submitted to I.C.V. STZ-induced AD. (A) CAT activity evaluation; (B) SOD activity; (C) GST evaluation; (D) GR levels; (E) GPx evaluation; (F) GSH levels. Effect of 3.0 mg/kg EETD treatment administered orally (PO) or 0.6 mg/kg rivastigmine intraperitoneally (IP) in the hippocampus of mice submitted to intracerebroventricular (I.C.V.) STZ 2.5 mg/mL injection. Results are represented by mean ± SD, n = 6 per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 compared to vehicle group. ####P < 0.0001 compared to sham group (animals that received no I.C.V. STZ administration). CAT: catalase; SOD: superoxide dismutase; GST: glutathione S-transferase; GR: glutathione reductase; GPx: glutathione peroxidase; GSH: reduced glutathione; EETD: Tithonia diversifolia ethanol extract; STZ: streptozotocin.

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Figure 6. Lipidic peroxidation and nitrite levels in the hippocampus of mice submitted to I.C.V. STZ-induced AD and administered with 3.0 mg/kg EETD. (A) Lipid peroxidation; (B) nitrite levels. Effect of 3.0 mg/kg EETD treatment administered orally (PO) or 0.6 mg/kg rivastigmine intraperitoneally (IP) in the hippocampus of mice submitted to intracerebroventricular (I.C.V.) STZ 2.5 mg/mL injection. Panels A and B show, respectively, the modulation of lipid peroxidation and nitrite levels. Results are represented by mean ± SD, n = 6 per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ****P < 0.0001 compared to vehicle group. ##P < 0.01, ###P < 0.001, and ####P < 0.0001 compared to sham group (animals that received no I.C.V. STZ administration). MDA: malondialdehyde; EETD: Tithonia diversifolia ethanol extract; STZ: streptozotocin; AD: Alzheimer’s disease.

Effect of treatment on acetylcholinesterase activity

The results presented in Table 1 show that AChE activity was significantly reduced in animals treated with EETD at doses of 1.0 mg/kg (P < 0.05) and 3.0 mg/kg (P < 0.001), as well as in those treated with rivastigmine 0.6 mg/kg (P < 0.0001), compared to the group that received STZ and vehicle control.

Determination of inflammatory biomarkers

The results presented in Table 2 demonstrate that the increased hippocampal levels of cytokines such as TNF-α and IL-6 by STZ administration were significantly reduced by treatments with EETD and rivastigmine (P < 0.001, P < 0.0001).

Effect of EETD on neuroinflammation

The effect of EETD on neuroinflammation was assessed by measuring LPS-induced NO production in BV2 cells. Treatment with the extract at concentrations exceeding 12.5 μg/mL reduced BV2 cell viability to below 80% compared to the DMSO-treated control group, indicating cytotoxicity, as shown in Figure 7A. Therefore, non-cytotoxic concentrations below 6.25 μg/mL were used for further analysis. The effect of the extract on NO production was evaluated using the Griess reagent. As shown in Figure 7B, EETD significantly reduced LPS-induced NO production in a dose-dependent manner compared to the LPS-treated group.

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Figure 7. Effect of EETD on neuroinflammation in BV2 cells. (A) Cell viability; (B) NO production. In panel A, the previous results for cell viability and choice of treatment doses with the extract are shown. In panel B, the results related to NO production are shown. Results are expressed as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 compared to the LPS group. CTR: control; LPS: lipopolysaccharide; NO: nitric oxide; EETD: Tithonia diversifolia ethanol extract.

Effect of EETD on tau hyperphosphorylation

The impact of EETD on tau hyperphosphorylation induced by okadaic acid was assessed via western blot analysis using the AT8 antibody, which detects phosphorylated Tau at Ser202 and Thr205. The viability of HT22 cells remained unaffected by extract treatment at concentrations up to 12.5 μg/mL.

Treatment with okadaic acid significantly increased tau hyperphosphorylation, detected by the AT8 antibody. However, treatment with EETD did not alter tau hyperphosphorylation levels (Figure 8).

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Figure 8. Effect of EETD on tau hyperphosphorylation in HT22 cells. Results are expressed as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 compared to the okadaic acid group. CTR: control; OA: okadaic acid; EETD: Tithonia diversifolia ethanol extract.

Effect of EETD on Aβ production

The effect of EETD on Aβ production was evaluated by measuring soluble amyloid precursor protein beta (sAPPβ), a fragment generated by β-secretase cleavage of APP in APP-CHO cells. Extract concentrations up to 10 μg/mL did not affect cell viability, indicating an absence of cytotoxicity.

To assess Aβ production, western blot analysis was performed. As shown in Figure 9, EETD had minor effects on Aβ production, though the changes were not statistically significant.

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Figure 9. Effect of EETD on Aβ production. Effect of EETD on Aβ production in APP-CHO cells. Results are expressed as the mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 compared to the control group. CTR: control; p.c.: positive control; EETD: Tithonia diversifolia ethanol extract; sAPPβ: soluble amyloid precursor protein beta.