Tangeretin confers neuroprotection, cognitive and memory enhancement in global cerebral ischemia in rats

Abstract

Global cerebral ischemia is commonly associated with neurological deficits, including cognitive and memory impairments. The present study aims to investigate the neuroprotective, cognitive, and memory enhancement effects of Tangeretin, a flavonoid against global cerebral ischemia in rats. Bilateral common carotid artery occlusion (BCCAO) and reperfusion injury method was used to induce global cerebral ischemia in rats. Motor, cognitive, and memory functions were evaluated using rotarod, grip strength, Y-maze, and Morris water maze. Further, acetylcholine esterase (AchE) enzyme activity, acetylcholine (Ach), oxidative stress markers (ROS, SOD, MDA, and CAT), inflammation (IL-6 and TNF-α), and apoptotic markers (cytochrome C, caspase 9, and caspase 3) in BCCAO rats were measured following Tangeretin (5,10, and 20 mg/kg, oral) treatment. Our findings show that Tangeretin treatment significantly improved cognition and memory by enhancing Ach levels through the amelioration of AchE enzyme activity in BCCAO rats. Moreover, Tangeretin exhibited neuroprotective effects through the mitigation of oxidative stress, inflammation, and apoptosis in the BCCAO rats. In summary, the current findings suggested that Tangeretin exhibited neuroprotection, cognitive and memory enhancement against global cerebral ischemia.

Introduction

Cognitive dysfunction and memory impairment are commonly associated with cerebral ischemia (Ramakrishna et al. 2021). Global cerebral ischemia, resulted due to reduced blood flow promotes learning and memory dysfunction (Khoshnam et al. 2017, 2018). Numerous studies have reported that cerebral ischemia triggers oxidative stress and neuroinflammation which eventually causes neuronal cell death (Kim et al. 2023; Jordán et al. 2008) thereby producing behavioral abnormalities including locomotion, cognition, and memory (Daulatzai 2017; Ramakrishna et al. 2022a). Cerebral ischemia accelerates oxidative stress by reducing endogenous antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) and elevation of malondialdehyde (MDA) (Ramakrishna et al. 2022a). Following, cerebral artery occlusion and reperfusion injury, inflammatory markers such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) (Khoshnam et al. 2018; Yan et al. 2021) are elevated and antinflammatory cytokine such as IL-10 levels are decreased (Crack and Wong 2008). Combinedly, oxidative stress and neuroinflammation trigger apoptosis by elevating cytochrome C, caspase 9, and caspase 3, which further aggravate brain injury after a cerebral ischemic event (Wu et al. 2020). Acetylcholine esterase (AchE) regulates the levels of acetylcholine (Ach), which is mainly responsible for cognition and memory (Tripathi et al. 2019). Global cerebral ischemia followed by chronic hypoperfusion accelerates oxidative stress, inflammation, and apoptosis and also promotes the AchE activities leading to decreased Ach levels in the hippocampus (Gutti et al. 2019; Bhanukiran et al. 2023), thereby causing cognitive and memory impaired diseases (Rahmati et al. 2019; Xiao et al. 2023) such as Alzheimer’s disease (Srivastava et al. 2019) and other dementias (Kim et al. 2012; Torre 2012). Indeed, Ach neurons in the hippocampus die due to elevated oxidative stress and inflammation. Therefore, a promising neuroprotective method for treating global cerebral ischemia with simultaneously decreased oxidative stress, inflammation, and apoptosis is sought (Wu et al. 2020).

To date, numerous phytochemicals have been widely studied against memory impairment diseases (Singh et al. 2021). Tangeretin is a O-polymethoxylated flavone that is commonly present in the peels of citrus fruits (Tang et al. 2018). Tangeretin shows potent antioxidant (Wang et al. 2018), anti-inflammatory effects (Lee et al. 2016), and neuroprotection in multiple neurological diseases, including Alzheimer’s disease (AD), Parkinson’s disease (Braidy et al. 2017a), and focal cerebral ischemia (Yang et al. 2020). Additionally, Tangeretin improved the cognitive and memory functions in experimental AD by decreasing and increasing the AchE activity and Ach levels, respectively (Braidy et al. 2017a; Bao et al. 2022). Global cerebral ischemia is one of the major risk factors for dementia. Currently, there are no medications to treat global cerebral ischemia and its associated cognitive and memory impairments. Therefore, there is a need for new potent molecules to combat global cerebral ischemia. Moreover, Tangeretin neuroprotection and its ability to improve cognition and memory in global cerebral ischemia have not been studied. Therefore, the current study was undertaken to evaluate the neuroprotective, cognitive, and memory enhancement effects of Tangeretin against global cerebral ischemic conditions.

Experimental

Chemicals

Tangeretin (Sigma Aldrich), Thiopentone sodium (Neon Labs, India), 2′,7′-Dichlorofluorescin Diacetate (DCFDA, Sigma Aldrich, USA), Betadine solution (Cipla, India), AchE (Sigma Aldrich, USA) were purchased. Inflammation-related markers (IL-6, IL-10, and TNF-1α,), apoptosis markers (cytochrome C, and caspase 9), and Ach ELISA kits were obtained from Krishgen Biosystems, Mumbai, India. A colorimetric kit to estimate caspase 3 was purchased from Abcam, USA. All other chemicals related to measuring oxidative stress were procured from local suppliers.

Experimental animals

Adult male Wistar rats (200–220 g) were procured and kept in clean polypropylene cages under standard laboratory animal environmental conditions. The Institutional Animal Ethical Committee at Mother Teresa Pharmacy College in Sattupalli, Telangana, India accepted the experimental methodology (protocol No. 2018/IAEC/03). The National Research Council US Committee for the Update of the Guide for the Care and Use of Laboratory Animals (2019) recommendations were applied in strict conformity with CPCSEA criteria while conducting the animal experiments.

Experimental design

The experimental animals were allocated into five groups at random: sham (1), bilateral common carotid artery occlusion and reperfusion (BCCAO) (2), BCCAO + Tangeretin (5 mg/kg) (3), BCCAO + Tangeretin (10 mg/kg) (4), and BCCAO + Tangeretin (20 mg/kg) (5). The dose of Tangeretin (5, 10, and 20 mg) was chosen from earlier reports (Yang et al. 2020). The BCCAO method was used to induce global cerebral ischemia in rats. Tangeretin was orally administered to treatment groups immediately after reperfusion and continued once daily for two weeks. Animals were euthanized after the behavioral tests, and the brains were removed. Furthermore, biochemical and ELISA assays were used to examine the indicators for brain inflammation, oxidative stress, and apoptosis. The detailed study design is depicted in Fig. 1.

Fig. 1.

Study design. BCCAO Bilateral common carotid artery occlusion, D day, AchE acetylcholine esterase, and MWM Morris water maze

BCCAO surgery

BCCAO and reperfusion injury method was used to induce global cerebral ischemia in rats (Zhang et al. 2019). Briefly, following the 12-h fasting period rats were anesthetized with an intraperitoneal injection of thiopentone sodium (40 mg/kg) (Ramakrishna et al. 2022b). Then, using surgical blades, a middle neck incision was made and the left and right common carotid arteries were isolated without causing any harm to the vagus nerve. After that, microvascular clips were used to block both carotid arteriesfor 30 min. Microvascular clips were taken out to allow for reperfusion after a 30-min incision was sutured. In the case of sham animals, common carotid arteries were exposed but did not occlude (Zhang et al. 2019). Following surgical procedures, povidone-iodine was applied on sutures to avoid infection, dehydration was prevented by administering a subcutaneous injection of saline, and animals were observed for food consumption.

Assessment of motor activity

The grip strength and rotarod tests were conducted to assess rats motor activities after BCCAO injury. Grip strength: The BCCAO rats neuromuscular stability is assessed by hanging them on a 90 cm long, 1 mm-diameter metal wire that is held by two 60 cm-tall vertical stands. The grip strength performance of BCCAO rats was assessed on the following outcomes: 0: the animal slips off; 1: it hangs on with two forepaws; 2: it hangs on with two forepaws plus one or both hind paws; 4: it clings on with all four paws and tries to climb the string; 5: it manages to elude the wire and land on a level surface (cut-off time: 60 s) (Mishra et al. 2018; Meyer et al. 1979). Rotarod: BCCAO rats motor coordination was evaluated using the rotarod test. To acquire consistent findings, rats were trained twice daily on the rotarod (8 rpm) (IKON Instrument, New Delhi, India). Each rat’s time spent on the moving rod was timed on the probing testing day. Counting was halted as soon as the rat fell off the rod, and the result was saved as the rotarod retention time (Mishra et al. 2018).

Determination of AChE enzyme activity

After the behavioral tests, animals were euthanized, and the brains were quickly removed. Then, the brains were cleaned with phosphate buffer solution (PBS) (10 mM, pH 7.4), and with a fresh PBS solution brains were homogenized. After that, brain homogenates were centrifuged for 15 min at 4 °C and 4360 g. Following centrifugation, supernatants were collected and AChE activity was assessed (Gutti et al. 2023). Briefly, the supernatant (100 µl) was incubated with acetylthiocholine iodide (ATCI) (100 µl,15 mM) for 5 min. Following the addition of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (100 µL, 1.5 mM) absorbance was measured at 415 nm. The rate of hydrolysis was estimated as µM of substrate hydrolyzed/min/mg of protein (Ellman et al. 1961).

Y-maze test

The Y-maze test is used to assess the short-term spatial working memory as well as the ability to adapt to new circumstances of BCCAO rats. Each Y-maze arm is 60 cm long, 30 cm high, and 10 cm wide, with a constant angle of 120° across the board. Every rat was placed in the center of the maze and allowed 5 min to explore. Spontaneous alternation was noted, when the animal entered an arm that was different from the two it had previously entered (for example, ABC, BCA, and CAB), and if the animal went back to either of the two previously visited arms, it was considered as a mistake (ABA and BCB). The BCCAO rats memory was assessed in terms of the relative alternation in the percentage using the following formula: Spontaneous alternation (%) = Number of alternations/Number of total arm entries− 2 × 100 (Gutti et al. 2019).

Morris water maze test

Spatial memory and learning behavior of BCCAO rats were assessed using the Morris water maze test (MWM). Briefly, MWM consists of a pool that is divided into four equal quadrants, one of which includes a hidden platform and is painted black, measuring 62 cm in height, 32 cm in depth, and 121 cm in diameter. Each rat was allowed 120 s to find the platform and had four trials each day for five days, with a 10-min break between each trial. The escape latency was considered when an animal found the platform. On the sixth day, a probing experiment without a platform was conducted, giving the animals a 120-s trial to locate the platform to test their spatial memory (Bhanukiran et al. 2023; D’Hooge et al. 2001).

Assessment of oxidative stress markers

Reactive oxygen species (ROS) levels were calculated using dichloro-dihydro-fluorescein diacetate (DCFH-DA), a fluorescent dye. Briefly, DCFH-DA (10 µM) was incubated with brain supernatants for 30 min at 37 °C in the dark. Then, the absorbance of fluorescence excitation and emission wavelengths was measured at 485 nm and 520 nm, respectively (Liu et al. 2019). The measurement of MDA and its representation in terms of moles of MDA/mg protein were used to determine the degree of lipid peroxidation (Sunderman et al. 1985). The SOD enzyme activity was assessed in the presence of phenazine methosulphate (PMS) and nicotinamide adenine dinucleotide (NADH) using the nitroblue tetrazolium (NBT) reduction method. A wavelength of 560 nm was used to measure the generated blue color caused by the presence of formazan (Kakkar et al. 1984). The reduction in hydrogen peroxide (6%) absorbance at 240 nm for 3 min at 30 s intervals was used to measure the CAT enzyme activity (Beers and Sizer 1952).

Enzyme-linked immunosorbent assay (ELISA)

Brain tissue homogenates were obtained as per manufacturer’s guidelines (Krishgen Biosystems, India), and inflammatory markers such as IL-6, IL-10, and TNF-α, and apoptosis markers such as cytochrome C, and caspase 9 levels were estimated. Caspase 3 levels were measured as per user guidelines (Abcam, USA).

Statistical analysis

The data obtained from the current investigation was analysed using GraphPad Prism, version 8. The mean standard deviation (SD) of all the data is displayed. A one-way ANOVA and Tukey’s post hoc test and two-way ANOVA followed by Bonferroni’s post hoc test were used to assess the data. p value less than 0.05 was used for statistical significance.

Results

Tangeretin improved the motor function in BCCAO rats

Figure 2 shows modifications in motor abilities after Tangeretin treatment in BCCAO rats. The significant differences between the rotarod and grip strength performances were identified by one-way ANOVA. In comparison to sham-operated animals, BCCAO rats performed poorly on the rotarod (p < 0.0001) and grip strength tests (p < 0.0001). Further, Tangeretin-received BCCAO rats show a substantial increase in grip strength (p < 0.0001) and rotarod performance duration (p < 0.0001). These findings show that Tangeretin improved motor function in BCCAO rats.

Fig. 2.

The effect of Tangeretin administration on rotarod and grip strength performances against global ischemia Grip strength and rotarod performances in BCCAO rats were represented in (A) and (B), respectively. BCCAO rats show a significant decrease in motor performance than sham-operated rats. Tangeretin treatment with 20 mg/kg treated rats significantly improved the motor performances than Tangeretin 5 and 10 mg/kg treated rats and the improvement in motor performances is dose-dependent. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All the results are expressed as Median (n = 15). One-way ANOVA followed by Tukey’s post hoc test

Tangeretin improved the memory in spontaneous alternation test

Figure 3 depicts changes in spontaneous memory in BCCAO rats. The spontaneous alternations percentages were significantly lower in BCCAO rats than in sham animals (p < 0.001). Tangeretin treatment increased the spontaneous alternations (%) in BCCAO rats in a dose-dependent manner. These results imply that Tangeretin therapy improved memory in BCCAO rats.

Fig. 3.

The effect of Tangeretin treatment on the percentage of spontaneous alternation. The spontaneous alternations in BCCAO rats were significantly lower than in sham-operated rats and the treatment with Tangeretin 5, 10, and 20 mg/kg treatment in BCCAO rats increased the percentage of spontaneous alternations in a dose-dependent manner. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All the results are expressed as Mean ± SD (n = 15). One-way ANOVA followed by Tukey's post hoc test

Tangeretin improved the spatial memory in global ischemic rats

Figure 4 shows Tangeretin-induced modifications to spatial memory in BCCAO rats. In comparison to sham-operated animals, BCCAO rats dramatically reduced the escape latency (p < 0.0001), number of entries into the platform zone (p < 0.0001), and time spent in the platform zone (p < 0.0001). Further, the number of entries onto the platform zone (p < 0.0001), time spent in the platform zone (p < 0.0001), and escape latency (p < 0.0001) were all considerably higher in Tangeretin-treated BCCAO rats. The BCCAO rats treated with 20 mg/kg of Tangeretin did not differ from the rats that underwent a sham operation. Tangeretin treatment in BCCAO rats significantly enhanced the escape latency (p < 0.0001), the time spent in the platform zone (p < 0.0001), number of entries into the platform zone (p < 0.0001) than BCCAO rats. These results indicated that in BCCAO rats, the Tangeretin therapy substantially enhanced spatial memory.

Fig. 4.

The effect of Tangeretin treatment on spatial memory in MWM test. A, B, and C, represent the escape latency, time spent in the platform zone, and number of entries into the platform zone, respectively. BCCAO rats significantly increased the escape latency time, and decreased the time spent and number of entries into the platform zone compared to sham-operated rats. Tangeretin (5, 10, and 20 mg/kg) treatment significantly reversed the above effects in BCCAO rats in a dose-dependent manner. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All the results are expressed as mean ± SD (n = 8). One-way and two-ANOVA followed by Tukey’s and Bonferroni’s post hoc test

Tangeretin enhanced Ach levels by inhibiting AchE activity

We found a substantial decrease in Ach levels with elevated AchE enzyme activities in BCCAO rats than sham-operated rats (p < 0.0001) (Fig. 5). Further, treatment with Tangeretin alleviated the AchE enzyme activity (p < 0.0001) and increased Ach levels (p < 0.0001) compared to BCCAO rats. These results imply that Tangeretin treatment reduced AchE enzyme activity thus increasing Ach levels.

Fig. 5.

The changes in AchE and Ach levels (%) following Tangeretin treatment in BCCAO rats. A and B, indicate the changes in AchE and Ach levels (%), respectively. BCCAO rats substantially increased the AchE enzyme activity and reduced the Ach levels than sham-operated rats. Tangeretin (5, 10, and 20 mg/kg) treatment inhibited the AchE enzyme activity and improved the Ach levels in BCCAO rats. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All the results are expressed as mean ± SD (n = 6). One-way ANOVA followed by Tukey’s post hoc test

Tangeretin mitigated the oxidative stress

Changes in oxidative stress indicators are shown in Fig. 6 after Tangeretin administration in BCCAO rats. One-way ANOVA and post hoc analysis revealed that there were significant differences in ROS (p < 0.0001), MDA (p < 0.001), SOD (p < 0.0001), and CAT (p < 0.0001) between the groups BCCAO and sham group. In comparison to BCCAO animals, Tangeretin administration significantly reduced ROS (p < 0.0001) and MDA (p < 0.0001) and increased CAT (p < 0.0001) and SOD (p < 0.0001) enzyme levels. These findings, therefore, confirm that Tangeretin acts as an antioxidant.

Fig. 6.

The effect of Tangeretin treatment on oxidative stress in BCCAO rats. A, B, C, and D indicate the levels of ROS, MDA, SOD, and CAT in BCCAO rats after Tangeretin treatment. BCCAO rats significantly elevated the oxidative markers, such as ROSS and MDA, and decreased the levels of SOD, and CAT compared to sham-operated rats. Following treatment with Tangeretin (5, 10, and 20 mg/kg), BCCAO rats reduced the ROS and MDA, and increased the SOD and CAT enzyme activities. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All values are expressed as Mean ± SD (n = 6). One-way ANOVA followed by Tukey post hoc test

Tangeretin attenuated the neuroinflammation

The effect of Tangeretin on neuroinflammatory markers in the brain of BCCAO rats is shown in Fig. 7. We found that BCCAO rats had significantly higher levels of proinflammatory cytokines (TNF-α and IL-6, p < 0.0001) and decreased anti-inflammatory cytokine (IL-10, p < 0.0001) than sham-operated rats. Tangeretin treatment in BCCAO rats significantly elevated TNF-α and IL-6 (p < 0.0001) and elevated the IL-10 levels (p < 0.0001) in a dose-dependent manner. These findings confirm that Tangeretin acts as an anti-inflammatory agent to mitigate BCCAO injury.

Fig. 7.

The effect of Tangeretin treatment on neuroinflammation. A, B, and C represent the changes in TNF-α, IL-6, and IL-10 levels. BCCAO rats exhibited elevated levels of inflammatory mediators (TNF-α, and IL-6), and decreased the anti-inflammatory cytokine (IL-10) than sham-operated rats. Tangeretin (5, 10, and 20 mg/kg) administration to BCCAO rats reversed the above changes in the inflammatory markers in the brain. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All values are expressed as Mean ± SD (n = 6). One-way ANOVA followed by Tukey post hoc test

Tangeretin alleviated apoptosis

We observed significant inhibition of the levels of apoptosis markers in the brains of BCCAO rats following treatment with Tangeretin (Fig. 8). Apoptosis markers such as cytochrome C (p < 0.0001), caspase 9 (p < 0.0001), and caspase 3 (p < 0.0001) were reduced by Tangeretin in a dose-dependent manner compared to BCCAO animals. However, there were no variations in these apoptotic markers between BCCAO animals treated with Tangeretin 20 mg/kg and sham rats. These results demonstrate that Tangeretin shielded the brain from BCCAO-induced apoptotic damage.

Fig. 8.

The effect of Tangeretin treatment on apoptosis against global cerebral injury. A, B, and C represent the changes in Cyt. C, caspase 9, and caspase 3 levels. BCCAO rats were significantly observed with elevated levels of apoptosis markers such as cyt C, caspase9, and caspase 3 compared to sham-operated rats. Tangeretin dose-dependently (5, 10, and 20 mg/kg) mitigated the elevation of apoptosis markers in the brains of BCCAO rats. ^ap vs. sham, ^bp vs. BCCAO, ^cp vs. BCCAO + Tangeretin 5 mg/kg, and ^dp vs. BCCAO + Tangeretin 10 mg/kg. All values are expressed as Mean ± SD (n = 6). One-way ANOVA followed by Tukey post hoc test

Discussion

The present study uncovered that treatment with Tangeretin substantially improved cognition and memory performance in global cerebral ischemic rats could be attributed to its action of inhibiting the enzyme activity of AchE and simultaneously increasing Ach levels. Moreover, Tangeretin demonstrated additional beneficial properties, such as antioxidant, anti-inflammatory, and anti-apoptotic activities against global cerebral ischemia. These novel findings suggest that Tangeretin could be a promising neuroprotective agent agent for the m of management of memory loss and global cerebral ischemia.

Global cerebral ischemia often leads to impaired motor function (Damodaran et al. 2014). In our study, rats subjected to BCCAO exhibited significant motor impairments, including reduced grip strength and impaired performance on the rota rod test. We found that the treatment with Tangeretin in these BCCAO rats showed notable improvements in grip strength and rota rod performance, suggesting that Tangeretin effectively ameliorated locomotion impairments caused by global cerebral ischemia. Numerous studies reported that cerebral ischemia produces significant memory impairment (Ramakrishna et al. 2021; Jin et al. 2014). Ach serves as the neurotransmitter responsible for memory function. It is widely acknowledged that the levels of Ach are controlled by an enzyme known as AchE (Rai et al. 2020, 2021). Studies have reported a significant increase in AchE activity, leading to the degradation of Ach levels. Consequently, inhibiting the function of this enzyme becomes essential for preserving memory retention (Gutti et al. 2019; Bhanukiran et al. 2023). It has been reported that Tangeretin significantly reduced the AchE enzyme activity and improved the Ach levels with improved cognition and memory in multiple neurological conditions (Wu et al. 2018; Braidy et al. 2017b; Lee et al. 2018; Chen et al. 2022; Bao et al. 2022). In our study, we observed a significant elevation in AchE activity along with decreased Ach levels in the brains of BCCAO rats. Upon administering Tangeretin, there was a substantial reduction in AchE enzyme activity with simultaneous increase in Ach levels. These changes were accompanied by remarkable improvements in memory and cognition as evidenced by increased spontaneous alternations in the Y-maze test, reduced escape latency time, and enhanced performance in the MWM test. These findings collectively suggest that Tangeretin treatment enhances cognition and memory in the global cerebral ischemia through its ability to increase Ach levels and decrease AchE activity. These promising results position Tangeretin as a potential memory-enhancing agent against global cerebral ischemic injury.

Neuronal damage in cerebral ischemia is primarily attributed to oxidative stress, a major contributing factor that leads to impaired neurological outcomes. In animal models, such as BCCAO and reperfusion injury, the brain experiences elevated levels of oxidative stress markers, exacerbating the brain injury (Zhang et al. 2011). Previously, Tangeretin was reported to exhibit antioxidant properties against focal cerebral ischemic rats (Yang et al. 2020). Likewise, treatment with Tangeretin increased the levels of antioxidant enzymes such as SOD and CAT, accompanied by a reduction in ROS and MDA levels. These observations emphasize that Tangeretin’s neuroprotective properties are attributed to its effective antioxidant action.

Following ischemic events, neuroinflammatory markers, such as TNF-α and IL-6 substantially raised in the brain alongside a decrease in IL-10 levels (Khoshnam et al. 2018; Ramakrishna et al. 2022a; Yuan and Zhang 2021). Our study shows that BCCAO rats exhibited a noteworthy increase in proinflammatory cytokines (TNF-α and IL-6) and a decrease in anti-inflammatory cytokines (IL-10), indicating the presence of neuroinflammation following global cerebral ischemia. Earlier reports indicate that Tangeretin exhibited anti-inflammatory activities by reducing inflammatory markers in focal cerebral ischemia (Yang et al. 2020). Similarly, we observed a notable decrease in inflammatory markers, such as TNF-α and IL-6, along with a concurrent enhancement of the anti-inflammatory cytokine, IL-10, implying that Tangeretin effectively mitigates neuroinflammation in global cerebral ischemia.

BCCAO and reperfusion injury induce a transient deprivation of energy and nutrients to the brain, resulting in the death of brain cells (Fang et al. 2020). Indeed, apoptosis markers like cytochrome C, caspase 9, and caspase 3 levels are elevated in the global cerebral ischemic brains (Saad et al. 2015). Previous studies found that Tangeretin mitigated neuronal apoptosis (Braidy et al. 2017b; Guo et al. 2017). Similarly, we observed a substantial increase in these apoptosis markers in the brains of BCCAO rats. However, upon treatment with Tangeretin, these apoptotic markers were alleviated, indicating that Tangeretin demonstrated neuroprotective effects in global ischemic conditions. Certainly, oxidative stress and inflammation mutually upregulate, leading to the promotion of apoptosis (Wu et al. 2020; Ramakrishna et al. 2023). In our investigation, Tangeretin treatment demonstrated the amelioration of oxidative stress and inflammation, concomitantly mitigating apoptosis in the context of global cerebral ischemic injury. These findings indicate that Tangeretin exerts multiple modes of neuroprotection, addressing various aspects of neurological damage caused by ischemic injury. However, the present study has some limitations including that the current study did not investigate the molecular inhibitions of oxidative stress, inflammation, and apoptosis pathways. Moreover, the present did not compare the efficacy of Tangeretin with already existing nootropic agents. Hence, future studies need to investigate Tangeretin molecular mechanisms and compare its efficacy with existing nootropic agents.

Conclusion

Our preclinical investigation underscores the promising role of Tangeretin as a memory and cognitive enhancer in the context of global cerebral ischemia. Tangeretin exhibited significant neuroprotection by ameliorating AchE enzyme activity, oxidative stress, inflammation, and apoptosis, thereby enhancing cognition and memory. The observed neuroprotection and cognitive improvement suggest that Tangeretin could be a valuable therapeutic agent for ameliorating the neurological consequences of global cerebral ischemia.

Abbreviations

BCCAO

Bilateral common carotid artery occlusion

AD

Alzheimer’s disease

AchE

Acetylcholine esterase

Ach

Acetylcholine

DCFH-DA

Dichlorodihydrofluorescein diacetate

ROS

Reactive oxygen species

rtPA

Recombinant tissue plasminogen activator

TNF-α

Tumor necrosis factor-α

IL

Interleukin

MWM

Morris water maze

ROS

Reactive oxygen species

MDA

Malondialdehyde

SOD

Superoxide dismutase

PMS

Phenazine methosulphate

NBT

Nitroblue tetrazolium

CAT

Catalase

Author contributions

ANR and SP performed the experiments, collected the data, analysed the data, and wrote the manuscript, SRC designed the study, analysed data, and revised the manuscript, and RK proofread the manuscript. All authors have approved the final manuscript.

Funding

The authors declare that no funds, grants, or other support were received for the execution of the present study.

Data availability

The authors declared that the data supporting the findings of this study are available within the article.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

There were no experiments involving human subjects in the current study. All the experiments involved in animals were approved by the Institutional Animal Ethics Committee, Mother Teresa Pharmacy College in Sattupalli, Telangana, India (Protocol No. 2018/IAEC/03). All the experimental procedures were conducted as per the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication-2011).