{"id":61323,"date":"2024-09-30T11:28:55","date_gmt":"2024-09-30T11:28:55","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=61323"},"modified":"2024-10-09T18:04:42","modified_gmt":"2024-10-09T18:04:42","slug":"extract-of-angelica-keiskei-leaves-attenuates-spatial-memory-impairment-on-the-d-galactose-model-of-brain-aging-in-mice","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/extract-of-angelica-keiskei-leaves-attenuates-spatial-memory-impairment-on-the-d-galactose-model-of-brain-aging-in-mice\/","title":{"rendered":"Extract of Angelica keiskei Leaves Attenuates Spatial Memory Impairment on the D-galactose Model of Brain Aging in Mice"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Aging is a natural\nprocess that will be experienced by all living things, including humans.\nImpaired memory is one of the most frequent symptoms of aging, which can be\ncaused by oxidative stress.1<\/sup> Many studies have shown that the pathogenesis of degenerative diseases and\naging processes involves the formation of reactive oxygen species (ROS).2<\/sup> Age-related memory\nimpairments stem from the build-up of oxidative damage and the concomitant\ndecline in antioxidant defenses, which lowers acetylcholine levels.3<\/sup> Iron overload in childhood that induces oxidative\nstress may lead to cognitive impairments.4<\/sup> Sleep deprivation, via oxidative stress, also\ncontributes to cognitive impairments.5<\/sup> Excessive amounts of radicals and low concentrations\nof antioxidants in the brain can cause cell damage.6<\/sup> The hippocampus\nand cerebellum are the most vulnerable parts of the brain due to their low\nantioxidant capacity.7<\/sup> Study\nreported that oxidative stress in the brain or hypothalamus results in\ndecreased memory.8<\/sup> Other\nfactors that can affect memory function are aging, impaired brain perfusion,\ninfectious and systemic diseases, chemical intoxication, head injury, and\nmental health conditions (depression and\/or anxiety).9<\/sup><\/p>\n\n\n\n

Antioxidants compounds that is capable of preventing\nthe oxidation of other molecules, is thought to play a role in improving memory.10<\/sup> Lately,\nthere has been growing evidence in the potential of phytochemical compounds to\nimprove memory, learning and cognitive abilities.11<\/sup> A prospective\ncohort study among Healthy Aging in Neighborhoods of Diversity across the Life\nSpan with a total sampel of 1947 participants showed that regular flavonoid\nsupplementation has a positive effect on improving cognitive function.12<\/sup> A recent\nmeta-analysis also highlights a strong indications that consuming flavonoids on\na regular basis has a positive effect on neurocognitive performance.13<\/sup><\/p>\n\n\n\n

One of the natural ingredients with high flavonoid content is Angelica keiskei<\/em>, especially the leaf parts. There have been many studies on the benefits of Angelica keiskei<\/em> as anti-obesity,14<\/sup> anti-diabetes,15<\/sup> anti-hyperpigmentation,16<\/sup> antithrombotic,17<\/sup> anti-inflammation,18<\/sup> and anti-myophaty.19<\/sup> However, its effect on spatial memory in mice with brain aging has yet to be investigated. Herein, we explored the effects of an ethanol extract from the leaves of A. keiskei<\/em> (EELAK) on spatial memory in mice with impaired memory functions due to D-gal. <\/p>\n\n\n\n

Materials And Methods<\/strong><\/p>\n\n\n\n

Animals<\/strong><\/p>\n\n\n\n

Thirty experimentally naive mice, strain BALB\/c, male,\n3-month-old, weighing 20\u201330 g was purchased from Faculty of Medicine, Udayana\nUniversity, Indonesia. Throughout the experiment, the animals were housed in a\nstandard experimental animal housing with a consistent temperature of 22\u201324 \u00b0C,\na 12-hour light\u2013dark cycle, 45\u201365% humidity, and unrestricted access to a standard\nfood and water (ad libitum). Prior to the experiment, the animals were given a\nweek to get used to the circumstances of the animal facility. After that, they\nwere randomly assigned to three groups, with ten animals each group. The\nvehicle was administered by orogastric gavage to the healthy control group (HC\ngroup). The D-galactose-induced brain aging group (BA group) received 300 mg\/kg BW\/day of D-gal (Merck, Darmstadt,\nGermany) orally for a duration of 28 days to mimic aging and were orally treated with the same\nvolume of vehicle.20,21<\/sup> The EELAK-treated\ngroup (EELAK group) received 300 mg\/kg BW D-gal in physiological saline and 20\nmg\/kg BW\/day of EELAK dissolved in distilled water orally for a duration of 28\ndays.22<\/sup> <\/p>\n\n\n\n

Preparation EELAK<\/strong><\/p>\n\n\n\n

The Angelica keiskei<\/em> leaves were prepared using earlier techniques.22<\/sup> In brief, dried A. keiskei <\/em>leaves were ground into a coarse powder and then passed through a 40-mesh filter (425 \u00b5m). Five liters of 70% ethanol were mixed with the powdered A. keiskei<\/em> leaves, and the mixture was kept for 48 hours. The mixture was then filtered through Whatman No. 1 filter paper, and the extracts were collected and the solvent was removed using a rotary evaporator. Then, the ethanol extract from A. keiskei<\/em> leaves (EELAK) was kept at -20 \u00b0C.<\/p>\n\n\n\n

Morris Water Maze <\/strong><\/p>\n\n\n\n

We tested the\nmice’s spatial memory using the established Morris Water Maze.23,24<\/sup> The maze was\nmade up of a recording system and a circular stainless-steel tank that measured\n100 cm in diemeter and 60 cm in height. The tank held water with a temperature\nof 20\u00b11\u00b0C and around 30 cm deep. The tank was split into four quadrants\ngeographically. One hidden circular black escape platform measuring 10 cm in\ndiameter and secured one centimeter below the water’s surface was located in\none of these quadrants. For four days in a row, oriented navigation trials were\nconducted twice a day for sixty seconds each, with a thirty-minute break in\nbetween. On the fifth day, the platform was removed. The ratio of time spent in\nthe target quadrant to escape latency before reaching the platform were\nrecorded as Probe test results, and the number of target crossings over the\nprevious location of the target platform were recorded as the number of\nplatform crossings.<\/p>\n\n\n\n

Analysis of Acetylcholinesterase (AChE) Activity<\/strong><\/p>\n\n\n\n

The activity of acetylcholinesterase (AChE) was assessed from the hippocampus homogenates after the spatial memory assasement. Mice were euthanized by cervical dislocation under anasthesia with 40 mg\/kg sodium pentobarbital (I.P.); the intact brains were harvested and and placed into ice-cold PBS (pH 7.4). The cerebellum was removed using a surgical blade, and then the hippocampus was collected from the brain. The hippocampus was either stored frozen at -80 \u00b0C or used immediately. To examine the AChE activity, the hippocampus was homogenized in Dulbecco’s Modified Eagle Medium (Wako), centrifuged at 10,000 rpm at 4\u00b0C for 20 minutes, and the obtained supernatant was subjected to examination using a colorimetric enzyme-linked immunosorbent (ELISA) test (Cat. No. ab138871, Abcam, Cambridge, UK). <\/p>\n\n\n\n

Examination of Neuroinflammatory Markers<\/strong><\/p>\n\n\n\n

The levels of p65 NF-kB (Cat. No. ab176648, Abcam), NO (ab285318, Abcam), and\nTNF-alpha (ab208348, Abcam) were quantified using commercially available mouse\nELISA kits in strict accordance with the manufacturer\u2019s instruction.<\/p>\n\n\n\n

SOD Activity Assay <\/strong><\/p>\n\n\n\n

Superoxide dismutase (SOD) activity was measured using a\nmodified version of an earlier methodology.25<\/sup> Ice-cold\nethanol (0.15 ml) was added after 0.1 ml of hippocampus tissue supernatant and\n0.25 ml of ice-cold chloroform were added. For ten minutes, the mixture was\ncentrifuged at 4 \u00b0C and 3000 rpm. Then, the supernatant was mixed with\ndistilled water (2 ml), EDTA (1.25 ml), and carbonate buffer (3.75 ml). To\nstart the reaction, 1 mL epinephrine (Wako, Osaka, Japan) was added. The\nabsorbance was observed at 480 nm. The SOD activity was then normalized to the\ntotal protein in the sample quantified by Lowry\u2019s method and shown as U\/mg\nprotein.<\/p>\n\n\n\n

Measurement of Malondialdehyde Level<\/strong><\/p>\n\n\n\n

We used the standard calorimetric methods for thiobarbituric acid reactive substances (TBARS) to quantified the level of hippocampus malondialdehyde (MDA).26<\/sup> In brief, one gram of hippocampal tissue was homogenized in Tris-HCl buffer (pH 7.5) and centrifuged for ten minutes at 1000 g. The supernatant (0.1 mL) was mixed with two milliliters of the MDA working solution (thiobarbituric acid 0.37%, 0.25 N HCl, and 15% TCA).  The mixture was allowed to cool to room temperature and centrifuged for 10 minutes at 4 \u00b0C and 1500 rpm after being incubated for 15 minutes at 100 \u00b0C. After that, 96-well plates were filled with the clear supernatant, and the absorbance at 535 nm was determined. Lowry’s method was used to quantify the protein content, and the hippocampal MDA levels were adjusted.<\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

All data were shown as mean \u00b1 S.D. A one-way analysis of variace (ANOVA)\nwas used to determine statistical significance, and Tukey’s post-hoc test was\nthen performed. The findings were considered significant at p < 0.05.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

EELAK Enhances Spatial Memory and Reduces Hippocampal AChE Activity in D-gal-induced Mice <\/strong><\/p>\n\n\n\n

Reduced learning and memory abilities and cognitive impairment are the two most important clinical indicators of aging.27<\/sup> We used the Morris Water Maze task to assess spatial memory in this study. A significant difference was seen between treatment groups in the ratio of time spent in the target quadrant (F2,29<\/sub> = 76.887, p < 0.001) and the number of target platform crossings (F2,29<\/sub> = 50.011, p < 0.001), according to the one-way ANOVA analysis of the data. Ratios of the time spent in the target quadrant and the number of target platform crossings were lower in mice treated with D-gal alone (BA group) than in the control group (p < 0.001 and p < 0.01, respectively). These findings lend credence to the established fact that D-gal intervention can significantly impair the capacity of spatial memory and is being used as a model for brain aging. In contrast, the ELAAK group saw fewer target platform crossings and a lower ratio of time spent in the target quadrant when compared to the BA group. Notably, the EELAK group showed improvements in both the number of target platform crossings and the ratio of time spent in the target quadrant, by 48% and 62%, respectively (p < 0.001). Next, as AChE is the specific serine hydrolase of Ach, its levels in the hippocampus were measured. Since AChE is essential for memory, a decrease in its activity is anticipated to cause an increase in ACh levels. A one-way ANOVA analysis revealed a significant variation in AChE activity between the groups (F2,29<\/sub> = 62.554, p < 0.001). The group treated with D-gal showed an increase in AChE activity in the hippocampus (p < 0.001), and the increase in AchE activity was reduced (p < 0.01) by 20 mg\/kg BW\/day of EELAK (Figure 1). In conclusion, EELAK can significantly enhance mice’s spatial memory with brain aging, which may be connected to a depletion in AChE activity.<\/p>\n\n\n\n\n\n
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Figure 1: EELAK affects AChE activity and spatial memory of mice treated with D-galactose.<\/strong> <\/strong><\/p>\n

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EELAK Decreases Hippocampal NF-\u03baB and Inflammatory Markers in D-gal-stimulated Mice<\/strong><\/p>\n\n\n\n

Several studies have indicated that D-gal activates NF-kB pathway, which in turn causes hippocampal cytokine storms, such as TNF-\u03b1, and releases other inflammatory factors, including nitric oxide (NO).28<\/sup> In the present study, ELISA was used to measure hippocampal NF-kB (canonical p65), NO, and TNF-\u03b1 levels to establish if EELAK treatment influences inflammation in the hippocampus. ANOVA revealed significant differences between group effects for p65 NF-kB (F2,29 <\/sub>= 76.633, p < 0.001), NO (F2,29 <\/sub>= 72.509, p < 0.001), and TNF-\u03b1 (F2,29 <\/sub>= 39.386, p < 0.001). Subsequent posthoc analyses revealed that D-gal treatment alone to the BA group significantly increased p65 NF-kB (p < 0.001), NO (p < 0.01), and TNF-\u03b1 (p < 0.01) levels by 245%, 130%, and 66%, respectively, compared to healthy control group (HC group), which is consistent with several previous reports.29,30<\/sup> In the EELAK-treated group, the levels of p65 NF-kB (p < 0.001), NO (p < 0.01), and TNF-\u03b1 (p < 0.05), in the hippocampus were notably downregulated compared to D-gal only-treated group (BA group) (Figure 2). Together, these data demonstrated that EELAK mediates the inhibition of hippocampal inflammation in the D-gal-treated mice.<\/p>\n\n\n\n\n\n
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Figure 2: Effects of EELAK on hippocampal levels of inflammatory markers on D-galactose-induced brain aging in mice.<\/strong><\/p>\n

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D-gal Leads to Reduce SOD Activity and Increase MDA Level in Hippocampus, and EELAK Attenuates these Changes <\/strong><\/p>\n\n\n\n

To determine the antioxidant-related protective ability of EELAK on D-gal-induced aging brain, we quantified the activity of antioxidant enzyme in mouse hippocampus samples. <\/strong>ANOVA revealed significant between group effects for SOD activity (F2,29<\/sub>= 18.027, p < 0.001) and MDA levels (F2,29<\/sub>= 169.820, p < 0.001). As indicated in Figure 3A, our result demonstrated that D-gal significantly affected the SOD activity and MDA content in the hippocampus of mice (p < 0.01 and p < 0.001, respectively). However, co-treatment with EELAK significantly reversed the reduced SOD activity (p < 0.05) and elevated MDA levels (p < 0.001) caused by D-gal. These results indicate that EELAK enhances the antioxidant capacity of the hippocampus in D-gal-induced mice, which may be related to its memory-enhancing mechanism.<\/p>\n\n\n\n\n\n
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Figure 3: Effects of EELAK on redox status biomarkers on D-galactose-induced brain aging in mice.<\/strong><\/p>\n

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Discussion<\/strong><\/p>\n\n\n\n

Aging is a progressive and\nirreversible natural process that can be delayed. The primary cause of\nincapacity and dependence among the elderly is dementia and\/or cognitive\nimpairment, which is why dementia is designated as a global public health\npriority.31<\/sup> It has been recognized that humans are susceptible to brain aging\nproblems, and the number of persons with dementia is expected to double every\n20 years.32<\/sup> Synthetic drugs normally have a lot of toxicities and side effects.33<\/sup> Angelica keiskei<\/em>, a member of the Umbelliferae family,\ndemonstrated an extensive range of pharmacological actions and safety, which led\nto its widespread use as a functional food or medicinal herb.34<\/sup> Based on the currently available information, Angelica keiskei<\/em> is\nbelieved to be able to alleviate physiological decline associated with aging.35<\/sup> The most prevalent bioactive ingredients in Angelica keiskei<\/em> are\ncoumarins, chalcones, and flavonoids.36<\/sup> In this article, we found for the first time that ethanol extract from the leaves of A. keiskei<\/em>\n(EELAK) minimized D-gal-induced oxidative stress,\nneuro-inflammation, and memory impairment in a mouse model of aging.<\/p>\n\n\n\n

In the present study, we induced\naging in mice using D-galactose, which has been widely used in various studies\nfor aging modeling in experimental animals and is considered the most effective\nto mimics physiological aging, particularly to artificially create brain\nsenescence.28<\/sup> D-gal causes oxidative stress on various tissues, including brain, by\nincreasing the production of ROS and Advance Glycation Endproduct (AGEs) due to\ngalactose oxidase activityand nonenzymatic glycosylation reaction, respectively,\nwhich also occur in the natural aging process.37<\/sup> In addition, several studies indicated that D-gal results in neuroinflammation\nand significant cognitive deterioration.38,39<\/sup> The effect of D-gal on cognitive function is mediated by decreased\nbrain-derived neurotrophic factor (BDNF) expression, neuronal apoptosis, and\ndysfunction of synaptic proteins in the brain.40<\/sup> The current work\neffectively established and used an aging rat model driven by D-gal to\ninvestigate the memory-enhancing properties and potential mechanism of EELAK. Mice administered with D-gal in the present study for 28\ndays displayed significant deficits in spatial memory, the activated NF-kB\npathway (increased NO and TNF-\u03b1), and reduced antioxidant capacity (impaired\nSOD activity and elevated MDA levels). Additionally, a study showed that D-gal-injected mice exhibit the aging\nphenotype such as a dull appearance, a slight bow, and sluggish behavior.41<\/sup> <\/p>\n\n\n\n

The transcription\nfactor family NF-\u03baB is crucial for controlling the inflammatory response.42<\/sup> Two components of NF-\u03baB called p50 and p65 are\ncytoplasmically sequestered alongside their inhibitor protein, I\u03baB\u03b1, in a physiological\nstate.43<\/sup> Once TLR4 and MyD88 are activated by D-gal, they\ntrigger the phosphorylation, ubiquitination, and destruction of I\u03baB\u03b1.44<\/sup> After translocating to the nucleus, free NF-\u03baB\ndimers increase the expression of genes that code for various proinflammatory\nproteins or enzymes. These include interleukin-6, interleukin-1\u03b2, and TNF-\u03b1, as\nwell as COX2 and NO.45<\/sup> To bolster this, we detected changes\nin NF-\u03baB, NO, and TNF-\u03b1 in this study. Next, the biomarkers of oxidative\nstress, an end product of lipid peroxidation (MDA), and the activity of\nantioxidative defense enzymes (SOD) were altered by D-gal. MDA is a significant\nbiomarker of membrane lipid peroxidation under oxidative stress and is widely\nused as a gauge of the severity of aging. Important natural enzymes in the\nantioxidative system that efficiently lower free radicals, such as SOD, have\nalso been used as markers for oxidative damage in various tissues and to\npredict the severity of aging-related phenotypes.26,46<\/sup><\/p>\n\n\n\n

Since oxidative stress influences the development\nof various neurodegenerative disorders, including Alzheimer\u2019s disease, the use\nof radical scavengers or antioxidant agents is a major strategy for managing\nthe progression of neurodegenerative disorders.47<\/sup> Antioxidant\ncompounds, whether synthetic or natural, have previously been shown experimentally\nas effective treatment choices against aging in experimental animal models,\nincluding oxidative stress-related brain aging induced by D-gal.39,48<\/sup> In the\npresent study, we examined the protective role of EELAK on D-gal-induced brain\naging in mice. Our findings demonstrated the potent anti-inflammatory,\nantioxidant, and memory-enhancing activities of EELAK. EELAK effectively\nreversed the detrimental effects of D-gal on spatial memory and biochemical\nparameters of the hippocampus. This finding aligned with earlier research,\nindicating that administering EELAK to mice could shield them from\nD-gal-induced inflammation and oxidative stress through its anti-inflammatory\nand antioxidant properties. <\/p>\n\n\n\n

The EELAK used in the present study contains a\nhigh amount of flavonoid, reaching a concentration of 11523.66 mg QE-Eq\/g. Its\nantioxidant capacity is 28294 mg\/L GAEAC (data not shown). A study showed that flavonoids can improve acetylcholine (ACh) activity by\ninhibiting acetylcholinesterase (AChE), a specific serine hydrolase for ACh\nresponsible for the termination of neuronal transmission and signaling between\ncholinergic synapses.49<\/sup> The role of ACh on the memory process is related to the\nmuscarinic acetylcholine receptor (mAChR) called M1. mAChR activities\nfacilitate the process of synaptic plasticity in learning and memory.50<\/sup> Flavonoids act\nas potent AChE inhibitors that inhibit the hydrolysis of ACh.49,51<\/sup> At the\nmolecular level, flavonoid interacts with AChE molecules through hydrophobic\ninteraction, halogen bonding, and aromatic stacking interactions.49<\/sup> In addition to their AChE inhibitor activity,\nflavonoids activate signaling pathways required for controlling synaptic\nplasticity, promote vascularization, stimulate the growth of new nerve cells,\nand improve the recovery of damaged neurons in the central nervous system.52,53<\/sup> These biological functions of flavonoids are\nessential for the neuronal healing process following D-gal treatment.54<\/sup> AChE inhibitors\napproved by the FDA for the treatment of AD, including donepezil, rivastigmine,\nand galantamine, are among the frequently prescribed drugs.55<\/sup> Two of\nthese three medications are secondary metabolites found in plants, which is why\nwe examined the EELAK in this study. In this study, EELAK showed a significant\neffect, reducing AChE enzymatic activity by 25%. <\/p>\n\n\n\n

In line with our finding, many studies have reported\nthe anti-inflammatory and antioxidant activities of EELAK. A study reported\nthat A. keiskei<\/em> leaves extract (10 \u03bcg\/mL) ameliorates the elevated synthesis of nitric oxide (NO) and the elevation of\ninflammatory cytokines such as TNF-\u03b1 and IL-1B in lipopolysaccharide-stimulated macrophage cell line.56<\/sup> Another study found that the production of NO\nsynthase (iNOS) in macrophage-induced toll-like receptors (TLR) is inhibited by\nthe isobavachalcone of A. keiskei<\/em>.57<\/sup> Regarding the antioxidant capacity, a study showed\nthat a methanol extract of A. keiskei<\/em> leaves exhibit an IC50<\/sub>\nvalue of 129.40 \u00b1 7.36 ppm, indicating moderate antioxidant activity.58<\/sup> In the present study, we found that the ethanol\nextract had an IC50<\/sub> value of 80.16 ppm. Additionally, previous study\ndemonstrated an IC50<\/sub> value of 7.73 ppm.59<\/sup> Together, the current study and others suggest\nthat ethanol is a better solvent to prepare A. keiskei<\/em> leaves extract. The\nmajor and specific flavonoid in A. keiskei <\/em>leaf is chalcones that have\nbeen documented for their diverse pharmacological actions, such as their\nantioxidative functions.34<\/sup> It is believed that the \u03b1,\u03b2-double bond and free\nhydroxyl groups present in chalcones contribute to their antioxidant activity.60<\/sup> Chalcones display bith direct and indirect\nantioxidant action, reducing the generation of ROS, RNS, and superoxide to\nprevent oxidative stress.61<\/sup><\/p>\n\n\n\n

Several plant-based compounds have\nalso been reported to attenuate spatial memory deficits. Persimmon (Diospyros kaki<\/em>) leaves extract with a flavonoid\ncontent of 957.75 mg\/100 g was proven to inhibit cognitive impairment in\nD-galactose-induced mice.20<\/sup> Rosemary extract containing 176.5 mg\/100 g flavonoid has been shown to\nincrease spatial memory in middle-aged mice.62<\/sup> Furthermore, administration of green tea extract with a flavonoid content\nof 255.96 mg\/100 g increases spatial memory in ischemic mice by modulating\noxidative and inflammatory stress responses.63<\/sup>When compared with other plants, the flavonoid content of\nEELAK is relatively higher. Therefore, it is expected that EELAK would be more\neffective in preventing brain aging.<\/p>\n\n\n\n

Natural\nfoods and nutrients are abundant in the world and have shown consistent\nbeneficial effects for human health.64<\/sup> Because they contain bioactive\ningredients, plant extracts are becoming more and more significant additions to\nmedicine these days, and their interventions are still seen as potential\nsolutions to prevent and\/or treat several diseases. 65<\/sup> In this context, based on the\npresent study, we could suggest that EELAK is a potential candidate to be used\nas a sole or supportive therapy for age-related neurodegenerative diseases in\nclinical settings. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Our study indicates that Angelica keiskei<\/em> leaves extract ameliorated D-gal-induced impaired spatial\nmemory in BALB\/c mice. Considering that A.\nkeiskei<\/em> leaves extract reverse the D-Galactose-induced increases in\nhippocampal AChE activity, inflammation, and oxidative stress, the present\nfinding is the first evidence that A.\nkeiskei <\/em>enhances the cholinergic system through its anti-inflammatory and\nantioxidant activities, thereby improving cognitive function. In conclusion,\nthese results suggest A. keiskei<\/em> as a\npromising natural product for the prevention of memory disorders and age-related neurodegenerative disorders such as\nAlzheimer\u2019s disease. Since we artificially\naccelerated brain aging using oral D-galactose, further\nstudies using naturally old mice are needed to prove the memory-enhancing effects of A. keiskei<\/em> leaves extract.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

We thank the Animal Laboratory Unit, Faculty of Medicine, Udayana University, Indonesia for providing facilities to perform animal care and experimental treatment.<\/p>\n\n\n\n

Conflict of Interest<\/strong> <\/p>\n\n\n\n

The authors declare that there are no competing interests. <\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

This study was supported by Grant-in-Aid from Atma\nJaya Catholic University of Indonesia (Hibah Dosen Pemula tahun 2024 to F.M.S.\nand Hibah Desentralisasi tahun 2024 to M.D.N.H), and a Postdoctoral Grant from\nWCU UNDIP Batch IV-2023 (to F.M.S.).<\/p>\n\n\n\n

Data Availability Statement<\/strong><\/p>\n\n\n\n

This statement does not apply to this article. <\/p>\n\n\n\n

Ethical\nApproval<\/strong><\/p>\n\n\n\n

The\nexperimental protocols used in this study comply with international guidelines\nfor humane animal treatment and were backed by the Ethics Committee on\nthe Use of Animals of Udayana University, Indonesia. <\/p>\n\n\n\n

Informed Consent Statement<\/strong><\/p>\n\n\n\n

This study did not involve human participants, and therefore, informed consent was not required. <\/p>\n\n\n\n

Author Contributions<\/strong><\/p>\n\n\n\n

Conception and design of study: FMS, IMTW; Acquisition of data: FMS,\nIMTW, MDNH, RD; Analysis and\/or interpretation of data: FMS; Drafting the\nmanuscript: FMS, NN; Revising the manuscript critically for important\nintellectual content: MDHN, ALE, JLM. All authors have read and approved the\nversion of the manuscript to be published.<\/p>\n\n\n\n

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