Marrubium vulgare Extract Improves Spatial Working Memory and Oxidative Stress Damage in Scopolamine-Treated Rats

Abstract

Background:

The cholinergic neuronal loss in the basal forebrain and increasing brain oxidative stress are one of the main features of the brain suffering from Alzheimer’s disease. Marrubium vulgare (M. vulgare), commonly known as ‘white horehound,’ possesses a variety of valuable properties, such as antioxidative, anti-inflammatory, and antidiabetic activities. Moreover, it possesses neuromodulatory properties that could potentially impact short-term memory functions.

Objective:

The present study was undertaken to investigate the preventive effects of water M. vulgare extract on working memory, cholinergic neurotransmission, and oxidative stress in rats with scopolamine (Sco)-induced dementia.

Methods:

Male Wistar rats (200–250 g) were divided into four experimental groups. The plant extract was administered orally for 21 days, and Sco (2 mg/kg) was administered intraperitoneally for 11 consecutive days. The behavioral performance of the animals was evaluated by the T-maze test. The effect of the extract on acetylcholinesterase (AChE) activity and antioxidant status in cortex and hippocampus were also monitored.

Results:

Our experimental data revealed that treatment with M. vulgare significantly increased the percentage of correct choices of rats with Sco-induced dementia in the T maze test (by 38%, p < 0.05). Additionally, it reduced AChE activity in the hippocampus (by 20%, p < 0.05) and alleviated oxidative stress induced by Sco, particularly in the cortex.

Conclusions:

M. vulgare water extract demonstrated working memory preserving effect in rats with Sco-induced dementia, AChE inhibitory activity and in vivo antioxidant potential, and deserve further attention.

Keywords

Acetylcholinesterase Alzheimer’s disease oxidative stress scopolamine-induced dementia T-maze test

Introduction

Alzheimer’s disease (AD) is the most common form of dementia worldwide. Amyloid-β peptide (Aβ) accumulation, oxidative stress, neuroinflammation, affected synaptic function, and neuronal loss are the main neuropathological features of the disease and are closely related with learning and memory impairment [1 –7]. AD patients typically experience loss of working, spatial, and anterograde memory [8].

Increasing oxidative stress in the brain is a key pathological hallmark in the multicomponent etiology of AD. It is widely accepted that inflammation caused by the Aβ accumulation is a major source of free radical formation [9, 10]. The overproduction of reactive oxygen species such as hydrogen peroxide, superoxide anione radicals, and hydroxyl radical, leads to peroxidation of cell membranes, damage to DNA and RNA, and altered proteins, ultimately causing mitochondrial dysfunction. The accumulated damage in turn, disturb the redox balance in cells and provoke neurodegeneration [11, 12]. In this way the use of antioxidants as free radical scavengers in the disease treatment, could potentially prevent or reduce the intensity of inflammatory processes and oxidative stress damage.

Main affected from neurodegenerative process in AD are the cholinergic neurons in the basal forebrain, which provide most of the cholinergic input to the cortex and hippocampus [13, 14]. The cholinergic neurons utilize the neurotransmitter acetylcholine (ACh) and play a crucial role in cognitive functions like memory and learning. The severity of degenerative changes in AD correlates with cognitive deficits in patients [15, 16]. One of the most effective pharmacological strategies for AD treatment involves the use of acetylcholinesterase (AChE) inhibitors such as donepezil, rivastigmine, and galantamine [17]. AChE is a key enzyme responsible for the hydrolysis of ACh to choline and acetate in cholinergic synapses [18, 19]. The reduction of enzyme activity leads to an increase in ACh levels and alleviation of cognitive symptoms in the disease. However, AChE inhibitors hardly exhibit any antioxidant potential and therefore do not interfere with the inflammatory processes. Moreover, the non-selectivity of these drugs, along with their limited efficacy and poor bioavailability, adverse cholinergic side effects in the periphery, narrow therapeutic ranges, and hepatotoxicity, are some of the various limitations to their therapeutic success [20, 21].

Despite the breakthrough in AD treatment with passive immunotherapy antibodies in early 2023, the treatment of the disease remains ineffective.

Numerous studies have been conducted to explore diverse natural sources with the aim of discovering more effective AChE inhibitors, as well as potent antioxidants, anti-inflammatory agents, and enhancers of cognitive function [22 –24].

Marrubium vulgare L. (M. vulgare) (Lamiaceae) known as “white horehound” naturally grows in Northern and Southern USA but is also found in Europe and Asia. It possesses antidiabetic, antioxidant, antibacterial [25 –27], gastroprotective [28], and anti-inflammatory [29] properties. M. vulgare contains a variety of active phytochemicals including polyphenols, tannins, flavonoids, diterpenes (marrubiin, marrubinic acid, and marrubenol) and phenylpropanoid esters (arenarioside, acteoside, forsythoside B, and ballotetroside), which make the species rich source of antioxidants [25 , 30–37]. Based upon a long-standing use in European Union, M. vulgare is approved as traditional herbal medicinal product for the following indications: 1) as an expectorant in cough associated with cold; 2) for symptomatic treatment of mild dyspeptic complaints such as bloating and flatulence; and 3) for temporary loss of appetite [38]. Nidhi et al. (2022) reported that hydroalcoholic extract from M. vulgare could improve the short-term loss in memory of mice in in vivo model of traumatic brain injury [39]. Furthermore, the extract was shown to possess antioxidant properties affecting various endogenous oxidative stress markers such as glutathione, malondialdehyde (MDA), and catalase (CAT). The possibility of the extract to modulate inhibitory (GABA) and excitatory (Glutamate) neurotransmitters was also reported [39]. The imbalance between these neurotransmitters is widely recognized as a contributing factor to neurodegeneration in the brain [40 –42], as elevated levels of glutamate can induce excitotoxicity [43]. The antioxidant and hepatoprotective effects of M. vulgare extract have also been demonstrated in rats with cyclophosphamide-induced liver toxicity [44 –46]. The present study aimed to investigate whether water extract from M. vulgare exerts the preventative effect on scopolamine (Sco)-induced memory impairment in a rat model. We monitored its anti-dementia potential, antioxidant capacity, and AChE inhibitory activity. The memory performance of the animals was evaluated by the T-maze test. The effect of horehound on lipid peroxidation level, activity of antioxidant enzymes (SOD, glutathione peroxidase (GPx), and CAT) and AChE were determined in the cortex and hippocampus of the experimental animals.

Materials and Methods

Plant material

Marrubium vulgare plants were cultivated of the experimental fields near Sofia city (Elin Pelin district, at 558 m above sea level). Aerial parts of the plants were collected in summer (June) of 2022 year, dried under shade and used for extract preparation.

Preparation of freeze-dried extract

Dried aerial parts were grounded to fine powder with laboratory mill. Five g of the powder were added to 200 ml water (90°C) and incubated for 15 min. The slurry was centrifuged (6,000 x g) and supernatant was collected and freeze dried for 96 h in an Alpha 1–4 LD plus laboratory freeze drier (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany).

Determination of total polyphenol and total flavonoid contents

Total polyphenols were determined according to the method of Singleton and Rossi (1965) [47] with gallic acid as a calibration standard. Results were expressed in mg gallic acid equivalents (GAE) per 100 g dry weight (DW)±SD. The total flavonoid content was determined according to Chang et al. (2002) [48] using AlCl₃ reagent and calibration curve built with quercetin dihydrate (10–200 mg/L) as a standard compound. Results are expressed as mg quercetin equivalents (QE) per 100 g DW±SD.

HPLC determination of phenolic compounds

HPLC analyses were performed on an UHPLC system Nexera-i LC2040 C Plus (Shimadzu Corporation, Kyoto, Japan) with a UV-VIS detector at λ=280 nm and a binary pump according to Atanasova et al. [49]. The column was Poroshell 120 EC-C18 (3 mm×100 mm, 2.7μm), thermostated at 26°C. The flow rate was 0.3 mL/min and the injection volume 5μL. The mobile phase consisted of A: 0.5% acetic acid and B: 100% acetonitrile. The gradient condition started with 14% (B), between 6 and 30 min, linearly increased to 25% (B), and then to 50% (B) at 40 min. The identification of compounds was confirmed by a comparison of retention times utilizing standard solutions, and standard calibration curves of different phenolics (gallic acid, neochlorogenic acid, 3,4-di-hydroxy benzoic acid, chlorogenic acid, catechin, vanillic acid, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, rutin, ellagic acid, quercetin-3-galactoside, quercetin-3-glucoside, naringin, rosmarinic acid, myricetin, cinnamic acid, quercetin, luteolin, naringenin, apigenin and kaempferol). The results for individual phenolic compounds were expressed in mg per 100 g DW±SD.

Antioxidant activity assays

Oxygen radical absorbance capacity (ORAC) and hydroxyl radical averting capacity (HORAC) were measured according to the methodology of Ou et al. (2001) [50] and Ou et al. (2002) [51] with details reported by Denev et al. (2010) [52]. Results were expressed as micromole Trolox equivalents (μmol TE) and micromole gallic acid equivalents (μmol GAE) per gram dry plant extract for ORAC and HORAC, respectively. The measurements were carried out on a FLUOstar OPTIMA plate reader (BMG Labtech, Germany).

Animals

Male Wistar rats (200–250 g) purchased from Erboj, Slivniza, Sofia were used. Rats were housed three per cage under constant laboratory conditions (25±3°C, 12/12 h light/dark cycle) with food and water available ad libitum with habituation period of 5 days before start of the experiment. The experimental protocol was in accordance with requirements of European Communities Council Directive (86/609/EEC) and Bulgarian Food Safety Agency (Approval for working with laboratory animals No. 340/13.12.22).

Experimental design

The animals were divided randomly into four experimental groups (n = 12 per group): 1) Control group; 2) Sco group; 3) Sco+M. vulgare group, and 4) M. vulgare group.

The control and Sco groups received distilled water (0.5 ml/100 g body weight) orally for 21 consecutive days, starting 10 days before and continuing 11 days simultaneously with i.p. administration of 0.9% NaCl and Sco respectively.

In the M. vulgare and Sco+M. vulgare groups the plant extract (200 mg/kg, per os) [25] was applied for 21 consecutive days, 10 days before and 11 days simultaneously with i.p. administration of Sco. The M. vulgare extract was added 1 h before Sco.

The control and M. vulgare groups received intraperitoneal (i.p) injections (0.5 ml/ 100 g bw) of 0.9% NaCl for 11 consecutive days.

The Sco and Sco+M. vulgare groups were injected with Sco hydrobromide at a dose of 2 mg/kg i.p for 11 consecutive days.

Sco was dissolved in distilled water ex tempore before each administration. The dose and duration of Sco treatment was selected based on our previous studies [53 –55] as well as on literature data [56 –58].

Animals from all groups were subjected to Hole- board and T-maze behavioral tests 11 and 12 days after the first Sco treatment. Behavioral observations were conducted in a dimly lit room from 9 a.m. to 12 p.m. One hour after the last test, the animals were euthanized. Biochemical analyses were performed on brain homogenates from cortex and hippocampus (Fig. 1).

Fig. 1

Schematic timeline of the experimental paradigm.

T-maze test

The test is commonly used to assess spatial working memory [59, 60]. The T-maze apparatus (Stoelting Co) consists of a start arm and two arms arranged in a T-shape. Rats were tested in the T-maze rewarded alternation paradigm. The objective was to train the animals to alternate between arms, relying on their memory of the previously visited arms [61]. To achieve this, the arms of the maze were sequentially baited with food, and the animals were mildly food-deprived. A modified protocol by Hussein et al. [62] was used. Training Session: Following five days of acclimatization, which included handling and habituation in the T-maze apparatus, the animals underwent task training. During the habituation period, rats were permitted to explore the maze and discover food rewards, initially throughout the entire maze and then only at the ends of the two T-shaped arms. At the start of the training session, each animal was placed in the starting area box and released after 10 s. The first trial was a forced one—only the right arm was open and contained a food reward. In subsequent trials, both arms were open, but only the arm opposite to the one used in the forced trial was baited. Each animal completed a total of 10 trials. A choice was considered correct if the animal visited a baited arm. Training continued until each set achieved 90% positive responses. The training session was conducted prior to the treatments. Throughout the entire training session, the rats were food-deprived to 90–95% of their free-feeding body weight and kept without any food for the last 12 h before the test session. Test Session: The T-maze test was conducted 12 days after the first Sco treatment. The animals were allowed to perform 10 trials with the first attempt forced towards the right arm. The results are presented as a percentage: (number of correct choices / (number of total trials – 1)) X 100.

Hole board test

The hole-board apparatus is a raised platform (50×50 cm) with 16 symmetrically positioned circular holes, each measuring 3 cm in diameter. The floor is divided into nine equal squares. Each rat was placed at the center of the board, and its behavior was assessed over a 3-min period. Parameters such as the count of squares traversed (reflecting locomotion) and the number of head dips (indicative of direct exploration and anxiety) were observed [63]. The test was conducted 11 days after the first Sco treatment.

Brain dissection technique

After behavioral assessments, the rats were decapitated, and their brains were carefully removed from the skull. The skin covering the skull was incised along the midline and removed to expose the dorsal skull plates. A pair of scissors was used to split the plates along the midline, and they were then twisted and turned across the lateral border to facilitate brain exposure. The brain was extracted using a spatula. To isolate the hippocampus, two short microspatulas were employed. The tip of one spatula was secured just above the cerebellum near the junction with the cortex. Simultaneously, the tip of the other spatula was positioned near the same junction, and the cortical hemisphere was gently peeled laterally, revealing the hippocampus. While securing the brain with one spatula, the other spatula was carefully placed just below the caudal tip of the hippocampus, and gently “scooped” it laterally. Subsequently, the frontal cortex was dissected. The tissue was periodically rinsed with fresh ice-cold saline (0.9% NaCl) using a pipette.

Determination of acetylcholinesterase activity

AChE activity in two brain’s structures, related to memory (cortex and hippocampus) was measured by spectrophotometric method of Ellman et al. [64]. According to the method, the enzyme activity is measured by following the increase in the yellow color produced from thiocholine, when it reacts with the dithiobisnitrobenzoate ion. Thiocholine is produced under the catalytic action of AChE, when the substrate acetylthiocholine is added. In our experiments 10% brain homogenates, prepared in 0.1 M phosphate buffer (pH 8; 1000 g), were centrifuged at 4,500 g for 10 min. In a test tube, 2,900μl of 0.1M phosphate buffer, 100μl of 0.1M DTNB, 20μl of 0.075M freshly prepared acetylthiocholine iodide, and 100μl of supernatant were added. 500μl of the reaction mixture was injected into the Semiauto Chemistry Analyzer and the absorbance was measured at 412 nm every 10 s for 3 min.

Determination of oxidative stress parameters

Tissue preparation

After the behavioral tests were conducted, the animals were decapitated, brains were quickly removed on ice and both structures cortex and hippocampus were separated. A post nuclear fraction was obtained—10% -homogenate in 0.15 M KCl-10 mM potassium phosphate buffer (pH 7.4) Potter-Elvehjem glass homogenizer with a Teflon pestle, centrifuged for 10 min at 3000 rpm. Thus, the received preparation was used for quantitative measurement of the levels of lipid peroxidation and the content of total glutathione. Part of this post nuclear homogenate was centrifuged for 20 min at 12,000 rpm. The resulting post-mitochondrial supernatant was used for determination of the antioxidant enzyme activities (temperature control, between 0° and+4°C)

Determination of protein content

The method of Lowry et al. [65] was used for the identification of protein content. The reaction was carried out in an alkaline medium—a complex compound of copper with the aromatic amino acids tyrosine and tryptophan was formed. The resulting complex gave blue coloration with a Folin reagent. The intensity of staining is proportional to the amount of protein in the sample at 700 nm. Protein content was determined using a calibration curve obtained with bovine serum albumin (Pentex USA).

Using commercially available kits oxidative stress biomarkers were measured spectrophotometrically, following strictly the manufacturer’s working instructions: Lipid Peroxidation (MDA) Assay Kit MAK085; Superoxide dismutase Assay Kit-WST 19160; Catalase Assay Kit CAT100; Glutathione Peroxidase Cellular Activity Assay, Cat. No. CGP 1 (Sigma-Aldrich Co. LLC, USA).

Statistics

Descriptive statistics, using the statistical program GraphPad Prism 8.0, Shapiro–Wilks test of normality, and One-Way ANOVA with Tukey’s post hoc test, were applied. The results are expressed as mean±SEM. p < 0.05 was considered to indicate a statistically significant result.

Results

Polyphenol content and antioxidant activity of the M. vulgare extract

Polyphenols and particularly flavonoids are the compounds mostly responsible for the antioxidant properties of plants. Therefore, we determined their content in M. vulgare freeze-dried extract supplemented to dementia mice, as well as its antioxidant activity (Table 1). The total content of polyphenols and flavonoids was high (8,990.3±121.7 mg GAE/100 g DW and 188.6±16.8 mg GAE/100 g DW, respectively), which is an indicator for the high antioxidant activity of the used extract – 1,516.7±49.9μmol TE/g DW and 538.5±6.5μmol GAE/g DW, determined by the ORAC and HORAC assays, respectively. The main phenolic compounds present in the extract were rosmarinic acid (261.4±21.1 mg/100 g DW) and quercetin-3-galactoside (85.4±3.0 mg/100 g DW).

Table 1

Content and composition of main polyphenolic compounds and antioxidant activity of M. vulgare freeze-dried extract

Caffeic acid, mg/100 g DW	25.1±2.3
p-Coumaric acid, mg/100 g DW	12.4±0.9
Ferulic acid, mg/100 g DW	81.2±5.6
Rosmarinic acid, mg/100 g DW	261.4±21.1
Quercetin-3-galactside, mg/100 g DW	85.4±3.0
Quercetin-3-glucoside, mg/100 g DW	6.5±0.5
Quercetin, mg/100 g DW	3.1±0.4
Apigenin, mg/100 g DW	21.4±0.9
Total polyphenols, mg GAE/100 g DW	8,990.3±121.7
Total flavonoids, mg QE/100 g DW	188.6±16.8
ORAC, μmol TE/g DW	1,516.7±49.9
HORAC, μmol GAE/g DW	538.5±6.5

Effects of the M. vulgare extract in the T-maze test

The T-maze test was employed to evaluate changes in spatial working memory of test animals. The effect of water extract of M. vulgare on short-term memory is shown in Fig. 2.

Fig. 2

Effect of M. vulgare extract on the spontaneous alternation in percentage in T-maze test. Each bar represents mean±SEM (n = 12 animals per group). Significance versus saline-treated group: *p < 0.05; significance versus Sco-treated group: ^#p < 0.05.

Eleven consecutive days Sco administration significantly reduced the percentage of correct choices (by 33%, p < 0.05, n = 12) compared to the control, indicating impaired spatial working memory in the experimental animals (Fig. 2). Water extract of M. vulgare significantly increased the percentage of correct choices (by 38%, p < 0.05, n = 12) in rats with cognitive deficit induced by Sco, but did not have significant effect in healthy rats after 21 days treatment.

Effects of the M. vulgare extract in the Hole board test

The effects of M. vulgare extract on locomotion, exploratory activity, and anxiety-like behavior in healthy rats and rats treated with Sco were evaluated by measuring two parameters: the number of line crossings and head dips over a 3-min period (Fig. 3A, B). Our results indicate that 11 days of Sco treatment had no significant impact on the locomotion of rats (Fig. 3A) but did decrease the number of head dips by 36.91% (p < 0.05, n = 6) compared to saline-treated animals (Fig. 3B). M. vulgare treatment in rats with induced dementia did not reverse the reduction in head dips caused by Sco, and it did not significantly alter the line crossing numbers compared to the untreated Sco group. In healthy animals, M. vulgare treatment increased locomotor activity by 83.09% (p < 0.001, n = 6) (Fig. 3A) and exploratory activity by 49.21% (p < 0.001, n = 6) (Fig. 3B) compared to the control group.

Fig. 3

Effect of M. vulgare extract on the number of line crossings (A) and head dips (B) in a hole-board test. Each bar represents mean±SEM (n = 6 animals per group). Significance versus saline-treated group: **p < 0.01; significance versus Sco-treated group: ^# # #p < 0.001.

Effect of M. vulgare extract on brain AChE activity

The effect of the water extract of M. vulgare on brain AChE activity is depicted in Fig. 4. The alterations in enzyme activity were assessed in the cortex and hippocampus, as these structures are associated with cognitive functions.

Fig. 4

Effect of M. vulgare extract on brain AChE activity on Sco-treated and healthy rats after 21 days administration. Each bar represents mean±SEM (n = 6 animals per group). Significance versus saline-treated group: *p < 0.05; **p < 0.01; significance versus Sco-treated group: ^#p < 0.05.

Sco administration led to a significant increase in brain AChE activity compared to the control group. The elevation of enzyme activity in the cortex was by 31% (p < 0.01, n = 6) and in the hippocampus by 35% (p < 0.01, n = 6) compared to the control.

Water extract of M. vulgare significantly reduced AChE activity in hippocampus (by 20%, p < 0.05, n = 6) and did not significantly change it in cortex of dement animals compared to untreated Sco group.

In healthy animals, water extract of M. vulgare did not significantly change the control level of enzyme activity in hippocampus and increased it in cortex (by 30%, p < 0.05, n = 6) after 21 days administration (Fig. 4).

These results show that administration of water extract of M. vulgare suppress the Sco-induced increase in AChE activity in the hippocampus.

Effect of M. vulgare extract on brain lipid peroxidation and antioxidant enzymes activity

Effect on lipid peroxidation

The effect of water extract of M. vulgare on lipid peroxidation levels in cortex and hippocampus is shown in Fig. 5.

Fig. 5

Effect of the M. vulgare extract on lipid peroxidation levels in cortex and hippocampus on Sco-treated and healthy animals after 21 days administration. Each bar represents mean±SEM (n = 6 animals per group). Significance versus saline-treated group: *p < 0.05; significance versus Sco-treated group: ^#p < 0.05.

Eleven days of Sco administration caused oxidative stress in experimental animals, demonstrated by significantly increase of brain lipid peroxidation levels (Fig. 5) and altered activity of main antioxidant enzymes (Figs. 6–8). Our results showed that in cortex and hippocampus of Sco-treated rats the MDA concentration increased by 45% (p < 0.05, n = 6) and 31% (p < 0.05, n = 6), respectively compared to the control.

Fig. 6

Effect of the M. vulgare extract on SOD activity in cortex and hippocampus on Sco-treated and healthy rats after 21 days administration. Each bar represents mean±SEM (n = 6 animals per group). Significance versus saline-treated group: **p < 0.01; ***p < 0.001; significance versus Sco-treated group: ^# # #p < 0.001.

Fig. 7

Effect of the M. vulgare extract after 21 days administration on CAT activity in cortex and hippocampus of Sco-treated and healthy animals. Each bar represents mean±SEM (n = 6 animals per group). Significance versus saline-treated group: *p < 0.05; ***p < 0.001; significance versus Sco-treated group:^#p < 0.05.

Fig. 8

Effect of the M. vulgare extract, after 21 days administration, on GPx activity in cortex and hippocampus of Sco-treated and healthy animals. Each bar represents mean±SEM (n = 6 animals per group). Significance versus saline-treated group: *p < 0.05; **p < 0.05; significance versus Sco-treated group:^#p < 0.05.

Water extract of M. vulgare reduced the content of tiobarbituric-acid substances in the brain of Sco-treated and healthy animals after 21 days administration. The effect was significant in cortex, where the lipid peroxidation levels in rats with cognitive deficit decreased by 29% (p < 0.05, n = 6) as compared to the Sco group and by 25% (p < 0.05, n = 6) in healthy animals compared to the controlgroup.

The results show that administration of water extract of M. vulgare suppressed the increased by the Sco MDA brain levels. The effect was more pronounce in the cortex.

Effect on SOD activity

Our results show that Sco treatment increased significantly SOD activity in the cortex (by 15%, p < 0.01, n = 6) and in the hippocampus (by 12%, ns) compared with the controls (Fig. 6).

Water extract of M. vulgare decreased SOD activity in cortex of Sco-treated and healthy animals after 21 days administration. The decline of enzyme activity of rats with cognitive deficit was by 44% (p < 0.001, n = 6) as compared to the Sco group and by 26% (p < 0.001, n = 6) in healthy animals compared to the control group.

In hippocampus, neither Sco nor M. vulgare treatment significantly altered the control value of SOD activity.

These results demonstrate that the application of the water extract of M. vulgare effectively suppress the Sco-induced increase of SOD activity in the cortex.

Effect on CAT activity

As it is shown in Fig. 7, Sco treatment increased CAT activity in cortex by 25% (p < 0.001, n = 6) and in the hippocampus (by 9%, ns) compared to the control. After 21 days of M. vulgare treatment, CAT activity in the cortex was reduced by 13% (p < 0.05, n = 6) in dementia rats compared to the Sco group, and by 8% (p < 0.05, n = 6) in healthy rodents compared to the control.

In hippocampus, neither Sco, nor M. vulgare treatment significantly altered the control value of CAT activity.

These results demonstrate that the administration of the water extract of M. vulgare effectively suppress the Sco-induced increase of CAT activity in the cortex.

Effect on CPx activity

The effect of the water extract of M. vulgare administration on GPx activity in the cortex and hippocampus of Sco-treated and healthy animals is shown in Fig. 8.

Eleven days of Sco treatment did not significantly change the brain GPx activity. Twenty-one days M. vulgare extract administration suppressed the enzyme activity in cortex of rats with cognitive decline by 19% (p < 0.05, n = 6) and by 27% (p < 0.05, n = 6) in healthy animals compared to the Sco and control groups, respectively.

In hippocampus the GPx activity was elevated by M. vulgare treatment. The enzyme activity increased by 28% in the dementia rats (p < 0.05, n = 6), and by 29% in the healthy rats (p < 0.05, n = 6), compared to the Sco and control groups, respectively.

Manifested results demonstrate that the administration of the water extract of M. vulgare suppress the Sco-induced increase of GPx activity in thecortex.

DISCUSSION

In this study, the preventive effect of water extract of M. vulgare in Sco-model of dementia was investigated. We have elucidated the beneficial effects of the extract on spatial working memory in rats with Sco-induced memory impairment. Moreover, we meticulously evaluated the extract’s capabilities as an AChE inhibitor and antioxidant, while highlighting its distinct effects on both the cortex and hippocampus regions of the tested animals

Sco is commonly used as a pharmacological tool for induction of experimental model of AD. As a nonselective muscarinic antagonist, it hinders cholinergic signaling and leads to significant impairments in learning, acquisition, and short-term retention of spatial memory tasks [55 , 66–68]. Increased AChE activity, decreased ACh levels, and elevated oxidative stress in the cortex and hippocampus of the experimental animals are also part of its neurotoxic effects [55 , 68].

Decline in spatial memory is a primary characteristic of the cognitive impairments observed in AD patients [69].

In the present study the effect of M. vulgare treatment on spatial working memory in Sco-treated and healthy animals was assessed by a T-maze test where the behavior known as “spontaneous alternation” is viewed as an indicator of spatial short-term memory impairment [70]. This behavior reflects the animals’ inherent tendency to switch arm visits during consecutive trials [71]. Switching arms indicates that rats recall the previous arm they visited, and when they do so correctly, it is considered as making the correct choices. Sco treatment impaired spontaneous alternation behavior in experimental animals, which is consistent with the results from our previous studies [55 , 72]. However, the treatment of dementia animals with M. vulgare water extract for 21 consecutive days significantly reversed the Sco-induced cognitive deficit. The number of correct choices in this group was very close to that observed in the control group. In healthy animals, the plant extract did not have any significant impact on memory performance. If we take into consideration the fact that the normal interaction between the medial prefrontal cortex and hippocampus is absolutely essential for facilitating of working memory, as a cognitive process [73], we can assume that Sco disrupts this interaction, while M. vulgare protects the functionality of the underlying neuronal circuits and molecular signaling cascades involved in this process.

To rule out the possibility that differences in the animals’ memory performance in the T-maze test were linked to a failure in their motor skills, we conducted the hole-board test. This test provides an independent measurement of locomotion, exploratory activity, and anxiety-like behavior [74]. Our results showed that Sco treatment, whether in the presence or absence of M. vulgare, did not significantly alter the locomotor activity of rats. However, there was a decrease in the number of head dips, indicating reduced exploratory activity and an anxiety-like effect of the muscarinic antagonist [75]. Notably, M. vulgare treatment exhibited an anxiolytic-like behavioral effect in healthy animals, as evidenced by the increased head dipsnumbers.

It is well known that the neurodegenerative process in AD patients mainly affects the cholinergic neurons in the basal forebrain and their projections to cortex and hippocampus [13, 14]. Furthermore, the learning and memory deficit is closely related with affection of cholinergic transmission by alteration in the levels of ACh or AChE activity in the brain [76]. AChE is an enzyme responsible for the degradation of ACh to acetate and choline at the synaptic cleft level. To date, the inhibition of AChE has been widely employed as a primary strategy for treating AD [23, 77]. By increasing the level of ACh in the brain, it partially reverses the cognitive deficit in patients suffering from the disease [78]. Thus, AChE can be regarded as a protein that functions as a specific marker of physiological activity of cholinergic neurons and plays an important role in the homeostasis of neuronal ACh [79]. Estimating AChE activity can provide valuable information on cholinergic function, which can be correlated with cognitive function.

Neuroprotective properties of phenolic compounds are well known and widely documented. For example, rosmarinic acid, which was the major constituent of the extract used in the current study could reveal its neuroprotective effect by several mechanisms including modulation of the Nrf2 pathway, reduction of gene and protein expression of iNOS and COX-2, reduction in the levels of reactive oxygen and nitrogen species, inhibition of monoamine oxidases and catechol-O-methyltransferase enzymes, etc. [80]. Besides phenolic compounds, horehound contains the diterpenoid lactone marrubiin, which has shown neuroprotective potential after traumatic brain injury in experimental mice [39] and potent AChE inhibitory activity [81].

In order to assess impact of M. vulgare water extract on cholinergic neurotransmission, we evaluated the AChE activity in cortex and hippocampus in Sco treated and healthy animals. Our results show that Sco treatment increased enzyme activity in the cortex and hippocampus of the rats, an indication for a cholinergic deficit, which is in line with our previous studies [55, 67]. The plant extract administration for three weeks affected AChE activity in the rats’ brain. The inhibitory AChE activity of M. vulgare was observed in the hippocampus of the rodents subjected to Sco-induced memory impairment.

Oxidative stress has been implicated in the pathogenesis of AD [82, 83] and accumulated data has shown that the unique biochemical characteristics of the brain render it susceptible to oxidative damage, ultimately leading to dysfunction, neurodegeneration, and neuronal death [84]. Furthermore, several studies have demonstrated that severe oxidative stress occurs in the early stages of AD even before significant Aβ accumulation. The critical role of reactive oxygen species in the pathogenesis of AD is attributed to their deleterious effects on molecules and energy metabolism [85]. Antioxidants play a crucial role in maintaining redox homeostasis and controlling various physiological functions of the body [86]. Essentially, antioxidants have the capability of neutralizing highly destructive free radicals and non-radical molecules. While they may not operate through the same mechanisms, they all contribute to reducing cerebral stress and defending the brain from degeneration. In recent years, researchers have been focused on searching for natural substances as antioxidant therapeutic strategies, modulating free radical production, for the control and prevention of neurodegenerative disorders. Thus, MDA content is indicative for lipid peroxidation level, and the activities of SOD, CAT, and GPx, which are components of the cellular enzyme antioxidant defense system, have been used as biomarkers to assess the level of oxidative stress in the brain.

In our research we found that the brain of Sco-treated rats exhibited alterations in the primary oxidative stress indicators. The notable elevation in MDA levels and increased activities of SOD and CAT strongly suggested the presence of induced oxidative stress. Increased oxidative stress in brains is one more suggested mechanism for memory disruptive effect of Sco [87]. M.vulgare treatment for 21 days reduced lipid peroxidation levels and activity of antioxidant enzymes (SOD, CAT, GPx) both in Sco-treated and healthy animals. The effect was significant and more pronounced in cortex. These findings strongly indicate that the plant extract possesses in vivo antioxidant properties and has the potential to mitigate the effects of reactive oxygen species in the rat brain.

We suggest that the manifested antioxidant potential of the herbal extract could be due to the presence of the phenolics and flavonoids, major phytochemical molecules with antioxidant properties.

Conclusion

The current study demonstrates the favorable impact of M. vulgare water extract on cognitive impairment induced by Sco, particularly in terms of spatial working memory. The observed preventive memory effect, as evidenced by the T-maze behavioral test, aligns with the extract’s demonstrated AChE inhibitory and antioxidant properties. Notably, the AChE inhibitory activity of the horehound extract was more prominently observed in the hippocampus, while its antioxidant capacity was more marked in the cortex.

AUTHOR CONTRIBUTIONS

Maria Lazarova (Conceptualization; Formal analysis; Investigation; Methodology; Visualization; Writing – original draft; Writing – review & editing); Elina R. Tsvetanova (Methodology); Almira P. Georgieva (Methodology); Miroslava O. Stefanova (Methodology); Diamara N. Uzunova (Methodology); Petko N. Denev (Methodology; Writing – review & editing); Krasimira N. Tasheva (Project administration; Resources).

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This research was funded by Bulgarian National Science Fund, Grant number KP-06-N 56/16.

CONFLICT OF INTEREST

All the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be considered as a potential conflict of interest.

Maria I. Lazarova is an Editorial Board Member of this journal but was not involved in the peer-review process of this article nor had access to any information regarding its peer-review.

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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