A Specific Herbal Mixture of Lavandula, Cloves, Rosa, Astragalus, and Cornus Mas (LCRAC) Improves Cognitive Function in a Rat Model of Streptozotocin-Induced Mild Cognitive Impairment

Abstract

Background:

In this study, researchers evaluated the effects of a plant-based mixture called LCRAC (containing Lavandula angustifolia Mill., Syzygium aromaticum (L.) Merr. and L. M. Perry, Rosa damascena Herrm., Astragalus membranaceus (Fisch.) Bunge, and Cornus mas L.) on cognition and depression in a rat model of mild cognitive impairment (MCI).

Materials and Methods:

Sixty male Wistar rats were randomly assigned to six groups and MCI was induced by intracerebroventricular injection of streptozotocin (STZ). LCRAC (20, 40, or 80 mg/kg/day) was administered by gavage for 14 days. Spatial learning and memory, working memory, and avoidance learning were assessed using the Morris water maze (MWM) test, Y-maze continuous alternation task (Y-CAT), and passive avoidance test, respectively. Depressive-like behaviors were examined using the forced swim test (FST). Comprehensive biochemical and histopathological analyses were performed to assess potential toxicity.

Results:

LCRAC treatment significantly improved spatial learning and memory, working memory, and avoidance learning in the STZ-induced MCI rats. LCRAC also exhibited antidepressant-like effects, decreasing immobility time in the FST. Importantly, LCRAC administration did not induce any significant changes in serum biochemical markers or histopathological alterations in the kidney, liver, or hippocampus.

Conclusion:

The study suggests that LCRAC shows promise as a therapeutic agent for enhancing cognitive function and alleviating depressive-like symptoms in MCI without evident toxicity, warranting further clinical investigation.

INTRODUCTION

As the population ages, there is an increasing demand for effective treatment strategies to mitigate age-related cognitive decline. One promising approach involves the use of natural products, including herbal compounds, due to their potential multiple mechanisms of action. In the present study, we evaluated the impact of a plant mixture consisting of Lavandula angustifolia Mill., Syzygium aromaticum (L.) Merr. and L. M. Perry, Rosa damascena Herrm., Astragalus membranaceus (Fisch.) Bunge, and Cornus mas L. (LCRAC) on cognitive functions, memory, and learning abilities in a male rat model of streptozotocin (STZ)-induced MCI. This study investigates whether the combination of these herbs provides a synergistic or additive benefit in mitigating cognitive impairment compared to what is known about the effects of each herb individually.

The selection of LCRAC was based on previous reports demonstrating their positive effects on memory and cognitive abilities in animal and human studies. Lavandula enhances memory and learning in mice via cholinergic-dependent mechanisms,[1] and a clinical trial showed that it alleviates anxiety in postmenopausal women.[2] Cloves improve schizophrenic behaviors in mice through CREB-dependent mechanisms in the hippocampus[3] and modulate the function of the adenosine system.[4] Rosa damascena has been shown to enhance learning and memory in laboratory animals through modulation of the serotonergic system and promotion of neurogenesis and synapses.[5,6] Astragalus exhibits cognitive-enhancing effects in humans[7] and laboratory animals.[8] Cornus mas contains anthocyanins, which protect against protein oxidation in the brain and improve cognitive abilities and memory through regulation of the noradrenergic system.[9,10] Additionally, Cornus mas has been shown to regulate iron metabolism in the brain.[11] Each of these herbs has demonstrated cognitive-enhancing properties through various mechanisms. The combination of these herbs may lead to a synergistic effect on synaptic plasticity by simultaneously targeting multiple neurotransmitter systems. However, it is also possible that some components of LCRAC could have overlapping mechanisms, rendering their combination redundant, or that certain compounds could interfere with each other’s absorption or activity.

We selected this specific combination of herbs based on their complementary mechanisms of action and their potential to address multiple pathological pathways involved in MCI. Lavandula and Rosa may improve neurotransmitter function, while Astragalus and Cornus mas may provide neuroprotection and regulate iron metabolism. By combining these herbs, we aimed to create a multi-targeted therapeutic approach that addresses the complex etiology of MCI more effectively than any single herb alone.

In this study, we not only investigate the cognitive-enhancing potential of the herbal mixture LCRAC but also address a critical question: How do the interactions among these herbs influence their overall efficacy? While each herb has been individually reported to improve memory and cognition via distinct mechanisms (e.g. cholinergic enhancement, serotonergic modulation, neuroprotection), their combination raises important uncertainties. Certain compounds may produce redundant effects if their mechanisms overlap, while others might counteract each other’s benefits. Therefore, our study is designed to determine whether the combined effects of these herbs surpass the effects observed with individual components, thus providing a rationale for a novel, integrative therapeutic approach to MCI.

MATERIALS AND METHODS

Animal subjects

Sixty male Wistar rats weighing 250–300 g were procured from the animal facility of Rafsanjan University of Medical Sciences. The rats were housed in a temperature-controlled room (23 ± 2°C) under a 12-h light/12-h dark cycle, with free access to food and water. Efforts were made to minimize animal suffering throughout the experimental procedures. Body weights of the animals were recorded weekly during the study period.

Experimental design

A total of 60 male Wistar rats were initially randomized (n = 10 per group) into the following six experimental groups

Sham/Control

On Day 1, rats received an intracerebroventricular (i.c.v.) injection of saline (the vehicle for STZ). Beginning 2 weeks later, these rats were administered 3 mL of distilled water (the vehicle for LCRAC) by oral gavage daily for 26 days.

STZ

On Day 1, rats received a single i.c.v. injection of STZ (3 mg/kg). Starting two weeks post-injection, they received 3 mL of distilled water by oral gavage daily for 26 days.

STZ + LCRAC (20 mg/kg)

Rats received a single i.c.v. injection of STZ on Day 1. Two weeks later, they were treated by oral gavage with LCRAC at a dose of 20 mg/kg, administered in a final volume of 3 mL daily for 26 days.

STZ + LCRAC (40 mg/kg)

As above, but LCRAC was given at 40 mg/kg in 3 mL via oral gavage for 26 days starting two weeks after STZ administration.

STZ + LCRAC (80 mg/kg)

As above, with LCRAC administered at 80 mg/kg in 3 mL via oral gavage for 26 days starting two weeks after the STZ injection.

Saline + LCRAC

Rats received an i.c.v. injection of saline on Day 1 and, beginning two weeks later, were administered LCRAC at 80 mg/kg (in 3 mL of vehicle) by oral gavage daily for 26 days.

For groups receiving LCRAC, due to its poor water solubility, the compound was first dissolved in a minimal volume of ethanol and then diluted with distilled water containing 1% Tween 80. This process produced a uniform suspension that was administered in a final volume of 3 mL per rat, with the ethanol content kept below 2% to avoid any vehicle-related effects. To ensure consistency across groups, the Sham/Control and STZ groups received 3 mL of the corresponding vehicle (distilled water, with ethanol/Tween 80).

Behavioral testing began 14 days after treatment initiation (Day 29) and was conducted in the following order: – Morris Water Maze: Days 29–32, Y-Maze: Day 34, Passive Avoidance: Day 36 and forced swim test: Day 38. A 48-h recovery period was provided between tests to allow for normalization of the HPA axis, and daily handling with environmental enrichment was maintained throughout the study. A schematic diagram summarizing the drug treatment schedule and study design is presented in Figure 1.

Figure 1.

A schematic diagram of drug treatment schedule and protocol design. I.C.V. STZ; STZ intracerebroventricular, LCRAC: an herbal compound, FST: force swimming test, MWM: Morris water maze, PAT: passive avoidance test, Y-CAT: Y-maze continuous alternation task

Due to spontaneous recovery and attrition during the study, the final numbers of rats included in the analysis were: 7 in the Sham/Control group, 9 in the STZ group, 8 in each of the STZ + LCRAC groups (20, 40, and 80 mg/kg), and 9 in the Saline + LCRAC group, resulting in a total of 49 rats completing the study.

Preparation of the LCRAC compound

The herbal components of the LCRAC compound were procured from the Isfahan Botany Herbarium (specimen no. 40058) in March 2021 and processed by Barij Essential Pharmaceutical Company, Iran. The detailed extraction methods are as follows:

Lavandula (angustifolia) extraction

One thousand and three hundred grams of powdered aerial parts were percolated with 6 liters of 50% ethanol at room temperature. After 48 h, the resulting 4 liters of hydroalcoholic extract were concentrated using a vacuum rotary evaporator at 50–55°C to recover most of the alcohol. The concentrated extract was then spray-dried to obtain a powdered extract with a final moisture content of 7.6% (w/w on a dry basis).[12]

Clove (Eugenia caryophylata) extraction

Five hundred grams of powdered clove buds were percolated with 2 liters of 50% ethanol at room temperature. The 1000 mL of hydroalcoholic extract collected after 48 h was concentrated using a vacuum rotary evaporator at 50–55°C, and the resulting concentrated extract was spray-dried to obtain a powdered extract with a final moisture content of 11.9% (w/w on a dry basis).[13]

Rose petal (Rosa damascena) extraction

Two hundred grams of powdered rose petals were percolated with 2 liters of 50% ethanol at room temperature. After four days, the 1200 mL of hydroalcoholic extract was concentrated using a vacuum rotary evaporator at 50–55°C, and the concentrated extract was then dried using a spray dryer to obtain a powdered extract with a final moisture content of 6.1% (w/w on a dry basis).[14,15]

Astragalus preparation: Dried Astragalus was powdered and used directly.[7]

Cornus mas extraction

One kilogram of dried Cornus mas fruit was soaked in 2 liters of 30% ethanol for about 3 h. After softening, the fruits were crushed by squeezing to separate the kernels. The mixture was left for 48 h with occasional shaking, and then the solids were allowed to settle, and the supernatant was filtered. The liquid extract was then poured into a tray and the solvent was evaporated in an oven at 45–50°C, resulting in a final moisture content of 5.6% (w/w on a dry basis).[16]

The final LCRAC mixture was prepared by combining the dried extracts and Astragalus powder in equal weight proportions (each component contributing 20% to the final formulation).

LCRAC standardization

To ensure batch-to-batch consistency, the mixture was standardized by HPLC analysis, which revealed that the final preparation contained 0.916% rosmarinic acid (used as a marker for Lavandula) and 0.14% quercetin (used as a marker for Rosa damascena). Although the active compounds from Clove, Astragalus, and Cornus mas were not individually quantified, their equal incorporation in the formulation ensures consistent contribution from each herb [Figure 2].

Figure 2.

HPLC analysis of Rosa damascena (A) and Lavandula (B) extractions in LCRAC

Intracerebroventricular injection of streptozotocin and perioperative care

STZ was administered as a single bilateral i.c.v. injection at a total dose of 3 mg/kg (1.5 mg/kg per ventricle) to induce mild cognitive impairment (MCI). This protocol was chosen based on previous studies, such as Reeta et al. (2017),[17] which demonstrated that a single injection of STZ at this dose reliably produces an insulin-resistant state, oxidative stress, and subtle cognitive deficits that mimic early Alzheimer-type pathology. The use of a single injection minimizes complications that may arise from repeated injections—such as exacerbated inflammatory responses or excessive neurodegeneration—thereby preserving the mild nature of the induced cognitive impairment. STZ was freshly prepared in saline immediately before use. Following anesthetization with a ketamine/xylazine mixture (60/4 mg/kg, i. p.), rats were secured in a stereotactic frame, and bilateral burr holes were drilled using the following coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to the sagittal suture, and 3.6 mm ventral from the brain surface. A volume of 5 μL saline per hemisphere was used to administer the STZ solution. The selected dose is optimized to induce deficits in spatial learning and memory—evidenced by performance in the Morris water maze—without causing extensive systemic toxicity or overt neuronal loss. This approach ensures that any observed cognitive improvements following LCRAC treatment can be attributed to therapeutic effects rather than recovery from severe neurodegeneration. Compared to protocols employing multiple injections, the single i.c.v. injection protocol offers a more controlled and reproducible method for modeling MCI.[18]

Morris water maze

The Morris water maze (MWM) test was employed to evaluate hippocampus-dependent spatial learning and memory. The MWM apparatus consisted of a black circular tank (60 cm height, 140 cm diameter) filled with water (22 ± 1°C) to a depth of 35 cm. The tank was divided into four conceptual quadrants, and a black platform (10 cm diameter) was submerged 2 cm below the water surface in the center of one of the quadrants (the target quadrant).

The MWM was positioned in the center of a room with four distinct visual cues placed on the walls surrounding the tank to provide spatial references for the animals. Over four consecutive days, the rats underwent four trials per day, with each trial starting from a different quadrant. During each 60-second trial, the animals were placed in the water facing the tank wall and allowed to swim freely to locate the hidden platform. The latency to reach the platform (escape latency) and the distance traveled (path length) were recorded using an automated video tracking system (Ethovision software, version 7.1, Noldus Information Technology, the Netherlands) Twenty-four h after the last training session, a spatial probe test was conducted. In this test, the hidden platform was removed, and the rats were allowed to swim freely for 60 seconds. The time spent swimming in the target quadrant (where the platform was previously located) and the swimming speed were measured to assess the animals‘ spatial memory retention.[19]

Swimming speed (path length/escape latency) was used to assess the motoric activity of rats in this task.

Passive avoidance learning

The passive avoidance task was evaluated using a shuttle box apparatus, as described previously.[20] The shuttle box consisted of two equal-sized compartments (25 × 25 × 25 cm) - a light compartment and a dark compartment - separated by a guillotine door. The floor of the apparatus was made of a metal grid, and the walls were constructed of Plexiglas.

Prior to the training session, each rat was individually placed in the apparatus and allowed to freely explore the two compartments for 5 min to become habituated to the environment. During the training session, the animal was placed in the light compartment for 1 min. Upon opening the guillotine door, the rat was allowed to enter the dark compartment. Once the rat had fully entered the dark chamber, the door was closed, and a mild electric foot shock (0.5 mA, 50 Hz) was delivered through the grid floor for 2 seconds. After 20 sec, the rat was returned to its home cage.

Twenty-four h after the training session, the test session was conducted. Each rat was again placed in the light compartment, and the latency to enter the dark compartment (step-through latency) was recorded as a measure of memory performance. A maximum cutoff time of 100 seconds was set for the test session.

The step-through latency in the test session was used as a positive index of the animal’s memory, with longer latencies indicating better memory retention.

Y-Maze Continuous Alternation Task (Y-CAT)

The Y-CAT was employed to evaluate the animals’ working memory performance, as described previously.[21] The Y-maze apparatus consisted of three identical arms (40 × 4.5 × 12 cm) made of opaque polyethylene plastic, arranged at 120-degree angles. At the start of the test, each rat was placed in one of the maze arms and allowed to freely explore the apparatus for 8 min. The number of arm entries was recorded, and a successful alternation was defined as consecutive entries into three different arms without revisiting a previously explored arm. The percentage of correct alternations was calculated using the following formula: Percentage of correct alternations = [(Number of alternations)/(Total number of arm entries - 2)] ×100. This metric served as an index of the animal’s working memory performance, with higher percentages of correct alternations indicating better working memory. After each trial, the Y-maze was thoroughly cleaned with a 50% ethanol solution to remove any residual odor or markings that could influence the next animal’s behavior.

Animals showing >15% weight regain between Days 14 and 21 or baseline cognitive performance exceeding 75% of sham controls during preliminary MWM screening (Day 14) were excluded from final analysis to account for spontaneous recovery.

Forced swim test

The forced swim test (FST) was employed to assess depression-like behaviors and evaluate the effects of LCRAC on stress responses in rats. While the standard rat FST protocol typically includes a pre-test habituation swim 24 h prior to the test session, in this study, we employed a single-session FST without prior habituation.[21] The FST apparatus consisted of a transparent cylindrical tank (50 cm height, 25 cm diameter) filled with tap water to a depth of 35 cm, maintained at a temperature of 23–25°C. Each rat was individually placed in the tank and allowed to swim freely for a 6-min session. Immobility was defined as the absence of active swimming movements, with the animal remaining motionless and floating in the water, save for the small movements necessary to keep its head above the water surface. The duration of immobility during the 6-min test session was recorded as the primary outcome measure. Due to the absence of pre-test habituation, our FST results primarily reflect the acute responses to LCRAC rather than chronic antidepressant effects. The immobility time was interpreted as an indicator of acute behavioral despair and stress response, with shorter immobility durations suggesting a modulation of these acute responses.

Terminal procedures protocol

Following completion of all behavioral testing (Day 38 post-STZ), animals were prepared for terminal sample collection:

Fasting period

Animals were food-deprived for 12 h prior to sacrifice to standardize metabolic parameters, with water available ad libitum.

Anesthesia induction

Rats were deeply anesthetized using ketamine/xylazine (100/10 mg/kg, i. p.) with depth of anesthesia confirmed by the absence of pedal withdrawal reflex.

Blood collection

While under complete anesthesia, cardiac puncture was performed using a 22-G needle inserted into the left ventricle, collecting 5–7 mL of blood into serum separator tubes. Serum was separated by centrifugation (3000 × g, 15 min, 4°C) and stored at -80°C until analysis.

Comprehensive biochemical and histopathological assessments

To thoroughly investigate the potential toxicity of LCRAC, a comprehensive evaluation of hepatorenal function, biochemical parameters, and histopathological indices was conducted. In the biochemical analysis, serum levels of key liver enzymes, such as alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST), were measured. Additionally, renal function markers, including creatinine (Cr) and blood urea nitrogen (BUN), as well as blood glucose (BS), cholesterol, triglycerides, high-density lipoproteins (HDL), and low-density lipoproteins (LDL), were assessed using a biochemical auto-analyzer (MINDRAY, Guangzhou, China) and commercially available kits (Pars Azmoon Co., Tehran, Iran).

Histopathological examination

Histopathological examinations of the kidney, liver, and brain tissues were performed. The fixed rat organs were immersed in 10% formaldehyde for a period of 24 h, following standard tissue processing procedures. The processed tissues were then carefully embedded in paraffin, and 4-μm-thick sections were created using a microtome. These sections were stained using the hematoxylin and eosin (HandE) method. Kidney sections were meticulously examined for any signs of glomerular atrophy, inflammatory cell infiltration, or tubular necrosis, which are indicative of kidney damage. Liver sections were assessed for congestion and pyknosis, whereas hippocampal sections were analyzed for neuronal nucleus shape and density, providing insights into neuronal health. Two experienced pathologists, blinded to the experimental conditions, conducted these assessments to ensure unbiased analysis.

Statistical analysis

The data are presented as mean ± standard error of the mean (SEM). The differences among the experimental groups were evaluated using one-way analysis of variance (ANOVA), followed by the Tukey post hoc test. The differences in escape latency in the Morris water maze (MWM) test were analyzed using two-way repeated measures ANOVA (RMA), followed by the Tukey test. A P value less than 0.05 was considered statistically significant.

RESULTS

LCRAC enhances spatial learning and memory

The repeated measures ANOVA (RMA) analysis on the latency time to find the hidden platform (escape latency) in the Morris water maze (MWM) test revealed a significant effect for groups [F (5, 168) = 7.054, P < 0.0001], time [F (3, 168) = 52.28, P < 0.001], and the interaction between groups and time [F (15,168) = 1.184, P = 0.2883]. Specifically, the STZ-treated animals exhibited a significantly longer latency time to find the hidden platform compared to the control rats (P = 0.0016), indicating impaired spatial learning performance due to the STZ treatment. Interestingly, treatment with LCRAC at doses of 20 (P = 0.0042), 40 (P = 0.0006), and 80 (P = 0.0029) mg/kg effectively decreased the latency time in the STZ-induced MCI rats [Figure 3].

Figure 3.

The effect of LCRAC administration on spatial learning. Each line represents the average of escape latency to find the hidden platform in the MWM test for four consecutive trial days. Each value is the mean ± SEM. n = 7–9 per group. ***P = 0.0016 versus control (non-STZ treated) group. ^##P = 0.0042 and ^###P < 0.001 versus STZ group

The probe trial results further corroborated the beneficial effects of LCRAC on spatial memory. As shown in Figure 4a, the STZ-treated animals spent significantly less time in the target quadrant compared to the saline-treated control group [ANOVA, F (5, 32) = 5.302, P = 0.0012; Tukey’s test, P = 0.0197), indicating impaired spatial memory. However, the STZ-induced MCI rats treated with 20 (P = 0.0016), 40 (P = 0.0038), and 80 (P = 0.0061) mg/kg of LCRAC spent more time in the target quadrant compared to the STZ group. Importantly, the swimming speed did not differ significantly among the experimental groups [ANOVA, F (5, 47) = 1.227, P = 0.3113), suggesting that the observed improvements in spatial learning and memory were not confounded by potential locomotor impairments [Figure 4b].

Figure 4.

The effect of LCRAC treatment on spatial memory. The percentage of time spent in the target quadrant (a) and the swimming speed (b) in the probe task in the MWM test. Each value is the mean ± SEM. n = 7-9 per group. *P = 0.0197 versus control/sham group; # all P < 0.01 compared to STZ group

LCRAC enhances working memory

The analysis of the Y-maze continuous alternation task (Y-CAT) data revealed that STZ administration significantly decreased the percentage of correct alternations compared to the control group [ANOVA, F (5, 41) = 4.55, P = 0.0022; Tukey’s test, P = 0.0329]. Notably, the administration of LCRAC to the STZ-induced MCI animals at doses of 20 (P = 0.0356), 40 (P = 0.0225), and 80 (P = 0.0022) mg/kg effectively increased the percentage of correct alternations compared to the STZ group [Figure 5].

Figure 5.

The effect of LCRAC on continuous alternation task. Each value is the mean + SEM. The result from the immobility time was analyzed by one-way ANOVA followed by Tukey’s post hoc test. n = 7–9 per group. *P < 0.05 versus control (non-STZ treated) group; ^#P < 0.05, ^##P < 0.01 compared to STZ group

LCRAC enhances avoidance learning

The results from the passive avoidance test showed that the retention latency in the STZ-treated rats was significantly shorter than that of the control animals [ANOVA, F (5, 34) = 7.52, P < 0.0001; Tukey’s test, P = 0.0013), indicating impaired avoidance learning. Importantly, the administration of LCRAC to the STZ-induced MCI animals at doses of 20 (P = 0.0009), 40 (P = 0.0032), and 80 (P < 0.001) mg/kg significantly increased the retention latency compared to the STZ group [Figure 6].

Figure 6.

The effect of LCRAC treatment on avoidance learning. Each value is the mean ± SEM. n = 7–9 per group. **P = 0.0013 versus control (non-STZ treated) group; ## all P < 0.001 compared to STZ group

Forced Swim Test (FST) results

The impact of LCRAC on acute stress responses was evaluated using the FST. As our protocol lacked a pre-test habituation swim, which is typically recommended for rat FST, our interpretation focuses on the acute behavioral responses to the test.

The STZ-treated group exhibited a significantly higher immobility time compared to the Sham/Control group (145 ± 12 s vs. 98 ± 10 s, P < 0.01), indicating increased acute behavioral despair in response to the stressful situation. Treatment with LCRAC at all three doses (20, 40, and 80 mg/kg) resulted in a significant reduction in immobility time in the STZ-treated rats [Figure 7]. Specifically: STZ + LCRAC 20 mg/kg: 115 ± 9 s (P < 0.05 vs. STZ), STZ + LCRAC 40 mg/kg: 102 ± 8 s (P < 0.01 vs. STZ), STZ + LCRAC 80 mg/kg: 95 ± 7 s (P < 0.001 vs. STZ). The Saline + LCRAC group (80 mg/kg) did not show a significant difference in immobility time compared to the Sham/Control group (92 ± 8 s vs 98 ± 10 s, P > 0.05). These findings suggest that LCRAC treatment modulated the acute behavioral response to the FST in STZ-induced MCI rats, leading to reduced immobility. While we cannot definitively conclude an antidepressant effect due to the lack of habituation, the results indicate that LCRAC may possess stress-buffering or anxiolytic properties that warrant further investigation.

Figure 7.

The effect of LCRAC on depressive-like behavior which was evaluated by the forced swimming test. Each value is the mean ± SEM. n = 7–9 per group. *P = 0.0041 versus control (non-STZ treated) group; # all P < 0.01 compared to STZ group

Comprehensive evaluation of serum biochemical and histopathological parameters

To thoroughly investigate the potential hepatorenal toxicity of LCRAC, a comprehensive analysis of relevant serum biochemical parameters was conducted. The data revealed that LCRAC administration did not significantly affect the serum levels of key liver enzymes, such as ALT, ALP, and AST, or renal function markers, including BUN and creatinine (Cr) [Figure 8, P > 0.05]. Furthermore, no significant changes were observed in the BS levels and lipid profile, including cholesterol, triglycerides, HDL, and LDL, across the experimental groups [Figure 8, P > 0.05].

Figure 8.

The effects of LCRAC on serum biochemical parameters. Serum glucose (a), Blood urea (b), Serum creatinine (c), Cholesterol (d), Triglycerides (e), HDL cholesterol (f), LDL cholesterol (g), SGOT (h), SGPT (i), ALP (j). Each value is the mean + SEM. n = 7–9 per group

The histopathological examination of the kidney [Figure 9 and Table 1] and liver [Figure 10 and Table 1] tissues also did not reveal any significant changes in the experimental groups. In the hippocampus, a moderate density in the neuronal nuclei was observed in the STZ-treated group, while the hippocampal neurons appeared normal in the other groups [Figure 11 and Table 1].

Figure 9.

Histopathological examination showed a normal kidney in experimental groups. Arrowhead shows glomerulus. Scale bar 200 µm

Table 1.

The histopathological changes of rat liver, kidney, and hippocampus sections

Groups	Histological criteria
	Liver		Kidney			Hippocampus
	Congestion	Pyknosis	Leukocyte infiltration	Glomerular atrophy	Tubular necrosis	Congestion	Necrosis
Sham/Control	0.3±0.08	0	0.3±0.01	0.2±0.04	0.3±0.01	0.1±0.02	0.3±0.05
STZ	0.1±0.02	0	0.1±0.001	0.1±0.06	0	0.9±0.12	0.4±0.09
STZ + LCRAC (20 mg/kg)	0.1±0.01	0.3±0.01	0.1±0.006	0.2±0.01	0.1±0.006	0.1±0.009	0.1±0.06
STZ + LCRAC (40 mg/kg)	0	0.1±0.01	0	0	0.1±0.009	0.2±0.01	0
STZ + LCRAC (80 mg/kg)	0.1±0.04	0	0	0.3±0.02	0	0.3±0.007	0.1±0.02
Saline + LCRAC	0.1±0.009	0.1±0.05	0.2±0.02	0	0.3±0.05	0.2±0.01	0

Values are expressed as mean±SEM. In each group, the differences were not statistically significant when compared to the Sham/Control group. STZ: Streptozotocin, LCRAC: Lavandula, cloves, rosa, astragalus, and cornus Mas

Figure 10.

Histopathological examination showed a normal liver in experimental groups. White arrowhead shows central vein of hepatic lobule. Black arrowhead shows hepatocyte nucleus. Scale bar 100 µm

Figure 11.

Histopathological examination showed a moderate density in neuronal nucleus hippocampus of STZ treating group. In other groups, hippocampus was normal. Scale bar 200 µm

Collectively, these findings demonstrate that LCRAC does not exert any serious adverse effects on the histopathological and functional parameters of the kidney and liver in the rat model. The lack of significant changes in the serum biochemical markers (P > 0.05) and the preservation of normal tissue architecture in the vital organs suggest that LCRAC is well tolerated and does not pose a substantial toxicological risk under the experimental conditions.

Effects of LCRAC on body weight

A two-way repeated measures ANOVA revealed a significant main effect of Group [F (5,48) = 5.32, P = 0.001], indicating differences in overall weight across groups. The STZ group exhibited significantly lower average weights compared to the Sham/Control group, underscoring the detrimental effect of STZ on weight. Additionally, a significant Group × Time interaction [F (20,192) = 3.67, P = 0.003] showed that weight change patterns over time varied between groups. The Sham/Control group displayed steady weight gain, while the STZ group showed weight loss or reduced weight gain. In contrast, LCRAC-treated groups, particularly at higher doses, demonstrated mitigated weight loss or weight gain, suggesting a dose-dependent protective effect of LCRAC on weight in STZ-treated rats [Table 2].

Table 2.

Means and standard errors of the mean (SEM) for weight (grams) by group and time point

Group	Baseline	Week 1	Week 2	Week 3	Week 4
Sham/Control	259.4±6.08	262.6±6.80	269.1±6.02	275.2±6.24	284.8±7.31
STZ	249.3±11.83	255.1±11.84	262.6±11.44	268.8±11.67	274.0±12.01
STZ + LCRAC20	244.4±6.46	249.9±6.00	250.3±6.09	255.9±6.19	262.0±6.91
STZ + LCRAC40	244.5±9.46	245.9±9.76	252.9±10.53	258.4±11.16	265.8±11.63
STZ + LCRAC80	255.0±8.17	258.1±7.84	265.0±8.50	270.7±9.15	279.9±9.95
Saline + LCRAC	257.6±8.23	261.4±8.03	268.3±8.71	272.1±9.21	281.6±9.84

STZ: Streptozotocin, LCRAC: Lavandula, cloves, rosa, astragalus, and cornus Mas

Mortality analysis

Our study experienced 18.3% total attrition (11/60 rats), with the following group-specific mortality [Table 3].

Table 3.

Mortality analysis

Group	Initial number	Final number	Mortality rate	Primary mortality window
Sham/Control	10	7	30%	Days 1–3 post-surgery
STZ	10	9	10%	Days 2–5 post-injection
STZ + LCRAC (all doses)	30	24	20%	Days 1–7 post-treatment

STZ: Streptozotocin, LCRAC: Lavandula, cloves, rosa, astragalus, and cornus Mas

Spontaneous recovery monitoring

Three STZ-treated animals that complete MWM performance recovery by Day 14 were excluded from analysis. The remaining STZ group showed stable cognitive deficits throughout the testing period (Days 15–36), with no late spontaneous recovery observed.

DISCUSSION

The present findings demonstrate that LCRAC, a polyherbal formulation comprising lavender, clove, rose, Astragalus, and Cornus mas, exerts dose-dependent improvements in spatial learning, working memory, and avoidance learning in STZ-induced MCI rats.

The observed cognitive benefits are likely related to the ability of LCRAC to counteract the pathological effects of STZ on the brain. STZ induces cognitive impairment through several mechanisms, including: disruption of insulin signaling, leading to glucose hypometabolism and energy deficits; increased production of reactive oxygen species (ROS), causing oxidative damage to neurons; impairment of cholinergic neurotransmission, which is critical for learning and memory; and triggering of inflammatory responses in the brain, contributing to neuronal damage.[22,23,24,25]

LCRAC may counteract these STZ-induced changes through several mechanisms. The antioxidant compounds in LCRAC (e.g. rosmarinic acid from Lavandula, anthocyanins from Cornus mas) may reduce oxidative stress, protecting neurons from damage.[26,27] Lavandula and other herbs may enhance cholinergic neurotransmission, compensating for STZ-induced cholinergic deficits.[28,29] Furthermore, some of the herbs in LCRAC may possess anti-inflammatory properties, reducing neuroinflammation.[8] Finally, Cornus mas may regulate iron metabolism, preventing iron-induced oxidative damage.[30]

The observed cognitive and antidepressant-like effects of LCRAC are likely mediated by a complex interplay of mechanisms. We hypothesize that the mixture exerts its beneficial effects through a multi-faceted approach. First, the combination of antioxidant compounds from multiple herbs (e.g. rosmarinic acid from Lavandula, anthocyanins from Cornus mas) may result in synergistic antioxidant activity, reducing oxidative stress and neuronal damage more effectively than individual components alone. For instance, the observed improvement in spatial learning in the MWM [Figures 3 and 4] appeared as a more pronounced and faster trend compared to the effects of individual antioxidant herbs tested in separate studies,[26,27] hinting at a potential synergistic effect where the blend amplifies neuroprotection beyond individual capacities. Specifically, the LCRAC treated animal reached control animal level by Day 3 trial in MWM test. This suggests that the combination of herbs in LCRAC may be producing a synergistic effect on antioxidant activity, as well as neuronal function resulting in faster learning activity.

Second, LCRAC may modulate multiple neurotransmitter systems simultaneously, with Lavandula and Rosa potentially influencing cholinergic and serotonergic pathways, respectively, and Cornus mas affecting the noradrenergic system. The improvements in working memory in the Y-maze may reflect an additive effect of multiple herbs targeting similar pathways. For instance, both Lavandula[29] and Rosa[28] have been shown to enhance cholinergic function, and their combined presence in LCRAC may lead to a greater overall enhancement of cholinergic neurotransmission in this model of STZ induced cognitive decline. However, the improvements in Y-maze test were only partial, comparing to control animals, and were not as significant as MWM and passive avoidance tests. This suggests that the synergistic effects of the herb components were lower in working memory. Third, the combined neuroprotective effects of Astragalus and Cornus mas, potentially through regulation of iron metabolism and promotion of neurogenesis, may contribute to neuronal survival and repair. While the histopathological examination [Figures 9–11 and Table 1] did not reveal significant differences in neuronal density or morphology (likely due to the relatively mild nature of the MCI model and the limited duration of treatment), the LCRAC-treated groups showed a trend toward reduced neuronal damage in the hippocampus compared to the STZ group, supporting the potential for neuroprotective effects. However, it is important to consider that Astragalus enhances iron absorption,[11] while Cornus mas may regulate iron metabolism.[30] The interaction between these two herbs could potentially lead to a complex effect on iron homeostasis in the brain, which might explain the lack of a clear dose-dependent effect in our study, and mild changes in histopathological examination. Further studies are needed to investigate these potential interactions and to determine the optimal combination of herbs for maximizing therapeutic efficacy.

While several studies have examined the individual herbs, limited research exists on the combined effects of these natural products. A recent study by He et al.,[18] evaluated a mixture of Carthamus tinctorius L. seed and Taraxacum coreanum in an Alzheimer’s disease model, reporting improvements in spatial learning and memory, corroborating our findings with LCRAC. However, their study did not assess working memory or depressive-like behaviors.

One of the notable findings of our study was that LCRAC treatment reduced immobility time in the FST in STZ-induced MCI rats. However, the interpretation of this result requires careful consideration due to our use of a single-session FST without prior habituation. Because our FST protocol lacked the pre-swim habituation, the results primarily reflect the acute effect of LCRAC on the behavioral response to an inescapable stressor rather than a definitive indication of antidepressant-like activity. The observed reduction in immobility time may indicate that LCRAC modulates the acute stress response or alters coping strategies in the face of an unavoidable stressor. It is possible that LCRAC possesses stress-buffering or anxiolytic properties that contribute to this acute behavioral change. The potential stress-buffering effects further support the idea of anxiolytic properties that should be addressed through other test for validation. However, the FST results align with previous reports showing anxiolytic and antidepressant effects from Lavandula,[12] and Rosa damascena.[5]

The comprehensive biochemical and histopathological assessments performed in this study provide insights into the safety and toxicity profile of LCRAC, which is crucial for potential clinical applications. Our findings align with previous reports on the individual constituent herbs, which have generally been considered safe and well tolerated.[2,31]

This study has some limitations. First, we only used male Wistar rats in this study. Future studies are needed to address any differences in the effects of LCRAC between male and female subjects. Second, future studies should evaluate different interactions (synergistic, additive, and indifference) between LCRAC ingredients in MCI. Third, in some animals that received LCRAC, mild diarrhea was observed. However, it generally took two to three days for the diarrhea to resolve, and no decrease in weight was observed during the study period.

In conclusion, this investigation contributes novel insights to the field of MCI management by introducing LCRAC as a potential therapeutic agent. Its efficacy in enhancing cognitive performance, its dual action on cognition and stress, and its encouraging safety profile make it a compelling candidate for further study. Future research should continue to explore the mechanisms behind LCRAC’s action and evaluate its long-term efficacy and safety, ultimately informing its potential translation into clinical practice.

Ethics approval and consent to participate

All experimental procedures in this study were approved by the Ethics Committee of Rafsanjan University of Medical Sciences (Ethical Code: IR.RUMS.REC.1400.021) in accordance with the United States NIH Guide for the Care and Use of Laboratory Animals (Publication No. 85–23).

Author contribution

NJ and TT performed the experiments. JH, MA, MH, and MM contributed reagents/materials/analysis tools. AA and AS conceived the idea, designed the experiments and wrote the article. All authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript.

Data availability

The data supporting the findings of this study are available on request from the corresponding author.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was supported financially and technically by the Physiology and Pharmacology Research Center of Rafsanjan University of Medical Sciences (Grant No. 99285).