Centella asiatica L. Urban protects against cognitive dysfunction in alluminum chloride-induced neurotoxicity in rats via inhibition of acetylcholinesterase level

ABSTRACT

Aluminum (Al) is a ubiquitous element on planet earth and its exposure to human systems is increasing steadily due to industrialization. Its accumulation in the biological systems have deleterious effects such as neurodegeneration and cognitive impairment. Centella asiatica L. (CA) is a herb used in the Ayurveda system of medicine as a neurotonic and for enhancement of memory. This study aimed at assessing the protective role of CA in aluminum chloride (AlCl₃) induced cognitive dysfunction in rats. Results have shown that AlCl₃ toxicity caused significant (p < 0.05) cognitive dysfunction of both spatial and non-spatial memory in rats, histopathological aberration of cerebral cortex and significant (p < 0.05) increase in the level of acetylcholinesterase in their brain. However, administration of CA irrespective of the dose given prevented all the observed changes which are comparable to a standard drug donepezil. CA prevented cognitive impairment and neurodegeneration in rats by decreasing the level of acetylcholinesterase. Hence, CA could be developed as a memory enhancing drug.

Graphical abstract

KEYWORDS:

• Aluminum chloride
• Centella asiatica
• cognitive impairment
• acetylcholinesterase
• neurotoxicity

Background

Aluminum (Al) is an abundant metal on earth’s crust and its exposure into the biological system is constantly increasing due to growing global industrialization [1,2]. Daily lifestyle of humans makes it easy for Al to gain entrance into their bodies, as they are constantly exposed through diet, medicines (adjuvant, antacids, buffered aspirin and allergen), cosmetics and utensils. Hence, it is of utmost importance to understand the effects of Al exposure on humans [3]. Studies have shown, chronic exposure to Al leads to progressive neurodegeneration of different parts of the brain including hippocampus and cerebral cortex. It also resulted into undesirable biochemical changes [4,5]. Although, the role of Al or aluminum chloride (AlCl₃) in aggravating neurodegenerative diseases such as Parkinsonism and Alzheimer’s disease (AD) is debatable, chronic exposure to Al resulted into learning and memory impairments and cognitive dysfunction as observed in both clinical and animal experiments [6,7]. Further, rodents exposed to Al have shown increased activities of acetylcholinesterase and reduced levels of acetylcholine which invariably resulted to memory dysfunction [8–10].

Large-scale research is ongoing in search of medicinal plants with antioxidant properties as well as memory enhancing capabilities [11]. Centella asiatica (L.) (CA) urban, belonging to Apiaceae family, locally known as pegaga in Malay is a perennial herbaceous creeper [12,13]. CA is considered to be an invigorating herb and believed to enhance memory and intelligence in Ayurvedic system of medicine. CA’s medicinal values are attributed to its numerous active constituents which includes madecassic acid, asiaticoside, asiatic acid, braminoside, bramoside, madecassoside and flavonoids [14]. Asiatic acid exerted neuroprotection against AlCl₃-induced apoptosis, oxidative stress and amyloid pathology in rats [15]. Further, it also exhibited a powerful neuroprotective ability against cerebral ischemia in a mouse model [16]. Existing literature has shown that CA extract increased dendritic arborization in neuronal cells [17], has antioxidant ability [18] and has improved mental state of psychologically impaired children [19]. Against this backdrop, this study evaluated the protective potentials of CA against AlCl₃-induced cognitive dysfunction in rat. Motor activity of the rats was evaluated followed by cognitive abilities, afterward the rats were euthanized and the level of acetylcholenesterase in their brains was evaluated besides the histology of their cerebral cortices.

Methods

Ethics statement

Before the commencement of the study, the protocol was revised and approved by the Animals Ethics Committee, Universiti Putra Malaysia (UPM/IACUC/AUP-R096/2016). Thirty female albino rats weighing between 200–250 g, aged 2 months, purchased from a local vendor (Perniagaan Usaha Cahaya, Batu Caves, Selangor), were used in this study. The rats were kept in a Standard Animal House 2–3 per cage, at temperature (22°C ±3°C) and 12/h light /dark cycle. Additionally, the rats were given free access to food and water. They were kept 7 days for acclimation with the laboratory conditions before the commencement of the experiments proper.

Chemicals and equipment

Sigma Aldrich (St. Louis, MO, USA) supplied AlCl₃ and cresyl violet, while ELISA kits for acetylcholinesterase were bought from Wuhan Fine Biotech Co., Ltd. Open field box and T-maze apparatus were locally fabricated from the home laboratory, all other chemicals used were of analytic grade. A standardized 60% hydro-ethanol extract of Centella asiatica (ref. no. AuRins-MIA-1-0) was used [20,21] for the study. Briefly, 60% aqueous ethanol extraction was carried-out with plant material to solvent ratio of 1:10 (weight to volume). Dried powder of CA was soaked in to the solution for 8 h at temperature of 60°C. Afterward it was filtered, concentrated and freeze dried. The yield of the crude extract in relation to the dried weight of the plant material was 16%. HPLC analysis was conducted using Agilent 1290 Infinity II LC [20].

Study design and treatment protocol

The rats were grouped randomly into six (n = 5). Group 1 (control) received distilled water orally and normal saline intraperitoneally, Group 2 received AlCl₃ (70 mg/kg b.w) intraperitoneally for 42 days. Group 3 rats were co-administered with Donepezil (dissolved in distilled water, 1 mg/kg b. w) and AlCl₃ for 42 days. While groups 4, 5 and 6 rats were co-administered with CA extract 200, 400 and 800 mg/kg b. w respectively and AlCl₃ for 42 days. At completion of the treatment regimen, rats were assessed for motor and cognitive behaviors followed by euthanisia and their brains were excised for histological and biochemical analysis (Figure 1).

Figure 1. Experimental design.

Behavioral assessment

Open field test (OFT)

The OFT is centered on overall activities of rodents when placed in a new environment, the apparatus used [22] and the protocol followed were earlier described [23].

T-maze spontaneous alternation test

The T-maze task was design to test the spatial learning and memory in rodents, the apparatus used and the protocol followed was adopted from Deacon [24].

Novel object recognition (NOR) test

The novel object recognition test was carried out to evaluate the cognitive abilities of rats as earlier documented [25]. Hence, the apparatus used and the protocol followed were adopted from previous studies [21,25].

Histological studies

The integrity of the cells on the cerebral cortices of the rats was checked through cresyl violet stain [26], while the scoring of histopathological changes was carried-out by an independent pathologist blinded to the groupings [27]. The slides were viewed using an Olympus BX51 and DP72 under 40x, 100x and 400x magnification with different spots per specimens. The Olympus Cellˆ F Imaging software used to capture images of the slide.

Biochemical studies

Protein determination

The protein concentration of the brain homogenate was ascertained through bovine serum albumin as a standard and according to Bradford method [28].

Determination of AChE activity

In the brain tissue homogenate, AChE activity was assessed using ELISA Kits (Wuhan Fine Biotech Co., Ltd.) according to their manufacturer’s instructions.

Statistical analysis

For biochemical analysis, OFT, T-MAZE and scoring of histology, statistical analyses were conducted using one way ANOVA followed by Tukey’s post hoc test. While for NOR test, two-way ANOVA followed by Tukey’s post hoc test was employed. All data were expressed as the mean ± standard error of mean (SEM). P value of < 0.05 was considered to be statistically significant.

Results

Effects of CA on the locomotor activities of AlCl₃-induced rats in open field test

One way ANOVA did not show any statistically significant differences [F _{(5, 21)} = 2.034, p = ns] in the distances moved among the groups of rats (Figure 2).

Figure 2. Effect of CA on locomotor activities. Control (distilled water + normal salin), Model (AlCl₃ 70 mg/kg. bwt), Donepezil (Donepezil + AlCl₃ 70 mg/kg.bwt), CA 200 (CA 200 mg/kg.bwt + AlCl₃), CA 400 (CA 400 mg/kg.bwt + AlCl₃) and CA 800 (CA 800 mg/kg.bwt + AlCl₃). Data represent as mean ± SEM (n = 5).

Effects of CA on spontaneous alternation in AlCl₃-induced rats during T-maze test

As shown in Figure 3, rats from control group scored 80%, model 52.5%, Donepezil 78%, CA 200 57.5%, CA 400 62.5% and CA 800 68%. One-way ANOVA showed significant differences in the number of alterations [F _{(5, 24)} = 6.135, p = 0.0009]. Tukey’s test further showed a marked of correct alternations in model group (52.5 ± 6.168, p = 0.0026) and CA 200 group (57.5 ± 5.72, p = 0.0172) in comparison to control group (80 ± 7.07). Additionally, there is statistically significant decrease of correct alternations in model group (78 ± 5.72, p = 0.0056) and CA 200 group (57.5 ± 4.598, p = 0.0353), when compared to Donepezil group (78 ± 12.58) of rats.

Figure 3. Effect of Centella asiatica on spontaneous alternation in rats using T-maze. Control (distilled water + normal saline), Model (AlCl₃ 70 mg/kg. bwt), Donepezil (Donepezil 10 mg/kg + AlCl₃ 70 mg/kg.bwt), CA 200 (200 mg/kg.bwt + AlCl₃), CA 400 (400 mg/kg.bwt + AlCl₃) and CA 800 (800 mg/kg.bwt + AlCl₃). Data represent mean ± SEM (n = 5), one way ANOVA. * = p < 0.05, Model and CA 200 vs. Control group. # = p < 0.05, Model and CA 200 vs. Donepezil group.

Effects of Centella asiatica against non-spatial memory deficit in AlCl₃-induced rats in the NOR test.

In the acquisition trial (Session I), two-way ANOVA did not show any statistical significant differences in the time spent by the rats exploring the two familiar objects A1 and A2 (Figure 4a). In the retention trial (Session ll), two-way NOVA revealed statistical significant interactions between the treatment given and recognition time of the rats toward the novel object [F (5, 40) = 344.7 P < 0.0001] (Figure 4b). Bonferroni’s post hoc test confirmed a statistical significant difference (p < 0.05) in the preference for the novel object by all the groups of rats except for the model group.

Figure 4. A and b: Effect of different doses of Centella asiatica on the preference of both familiar objects (Session I) (a) and preference of familiar and novel object after 30 minutes (session II) (b) of introductory session. Data were analyzed using independent sample t-test. *P < 0.05. Control (distilled water + normal saline), Model (AlCl₃ 70 mg/kg. bwt), Donepezil (Donepezil 10 mg/kg + AlCl₃ 70 mg/kg.bwt), CA 200 (200 mg/kg.bwt + AlCl₃), CA 400 (400 mg/kg.bwt + AlCl₃) and CA 800 (800 mg/kg.bwt + AlCl₃).

Effects of Centella asiatica on brain acetylcholinesterase (AChE) level in AlCl₃ induced rats

Figure 5. shows a statistically significant difference in the Model, CA 200 and CA 400 group as compared to control group. CA 800 group shows an effective treatment of CA on the rat’s brain as it reduces the AChE level until it does not show significant difference as compared to control. This figure also showed a statistically significant decreased of AChE level in all treatment groups when compared with model group.

Figure 5. Effect of Centella asiatica on the rat’s brain acetylcholinesterase (AChE) levels. Control (distilled water + normal saline), Model (AlCl₃ 70 mg/kg. bwt), Donepezil (Donepezil 10 mg/kg + AlCl₃ 70 mg/kg.bwt), CA 200 (200 mg/kg.bwt + AlCl₃), CA 400 (400 mg/kg.bwt + AlCl₃) and CA 800 (800 mg/kg.bwt + AlCl₃). Data represent mean ± SEM (n = 5), one way ANOVA. a = p < 0.05, Model, CA 200 and CA 400 vs Control group. b = p < 0.05, Donepezil, CA 200, CA 400 and CA 800 vs Model group.

Histopathological alterations in the brain

Figure 6a – 6f demonstrates the potentials of CA on the brain of AlCl₃-induced rat models. Normal histological appearance of the brain was noticed in control rats (Figure 6a, 7a, 8a), however, the AlCl₃-induced rats showed severe encephalomalacia, diffuse gliosis (Figure 6b), focal gliosis (Figure 7b) and severe precellular edema (Figure 8b). Notwithstanding, AlCl₃-induced treated with Donepezil presented mild encephalomalacia with diffuse gliosis (Figure 6c), mild focal gliosis (Figure 7c) and also mild precellular edema (Figure 8c). On the other hand, administration of CA to AlCl₃-induced ameliorates most of the histological aberrations (Figure 6 d-f, 7d-f and 8d-f) observed in the AlCl₃-induced alone groups. The severity of pathological changes were scored and presented in Table 1.

Table 1. The severity of pathological changes.

Histopathological alterations	Control	AlCl3 (70 mg/kg)	AlCl3 + Donepezil	AlCl3 + CA 200	AlCl3 + CA 400	AlCl3 + CA 800
Encephalomalacia	-	+++	+	+++	++	-
Focal gliosis	-	+++	+	+++	++	+
Precellular edema	-	+++	+	+++	++	+

+++ Severe, ++ Moderate, + Mild, – Nil

Figure 6. Photomicrographs of cerebrum: a) Control group, b) Model group, c) Donepezil, d) CA 200, e) CA 400 and f) CA 800. H & E stain, magnification 40x, n = 3. Encephalomalacia of matrix brain (red arrow), diffuse gliosis (green arrow).

Figure 7. Photomicrographs of cerebrum: a) Control group, b) Model group, c) Donepezil, d) CA 200, e) CA 400 and f) CA 800. H & E stain, magnification 40x, n = 3. Focal gliosis in the cerebral cortex with different severity (black arrows).

Figure 8. Photomicrographs of cerebrum: a) Control group, b) Model group, c) Donepezil, d) CA 200, e) CA 400 and f) CA 800. H & E stain, magnification 40x, n = 3. Precellular edema in the cerebral cortex (black arrows).

Discussion

The present study explored the protective potentials of CA on AlCl₃-induced neurotoxicity in rat model. The AlCl₃ toxicity resulted into diminishing of both non-spatial and spatial memories, cognitive dysfunction, neurodegeneration and increased level of AChE activities in rat model. However, co-administration of CA with AlCl₃ irrespective of the dose given reversed the memory and cognitive dysfunctions, maintained the architecture of the brain cells besides attenuating the levels of AChE. These attributes of CA are comparable to that of donepezil, a commercially available anticholinesterase drug used for AD patients.

In this study, chronic exposure to AlCl₃ alone or AlCl₃ with CA or AlCl₃ with donepezil does not affect the motor behavior of the rats in terms of locomotor activity. Therefore, impairments of motor activity of the rats might not have interfered with the cognitive tests carried-out in the present study, which is largely dependent on the rat’s movements. Similar results were documented in intracerebroventricular streptozotocin [29] and AlCl₃/d-gal [Chiroma 7] mediated cognitive impairments in rats. Hence, the present study continued with different behavioral paradigms to ascertain the cognition enhancing potentials of CA as claimed by folkloric usages [2,13].

T-maze test is used differently to assess the mental behaviors in rodents [30], therefore alternation either rewarded or spontaneous in T-Maze is a superb method for detection of hippocampal impairment, possibly superior to Morris water maze [24] since both procedures identify hippocampal abrasions. In this study, AlCl₃-induced rats showed complete hippocampal lesion because they achieved less than 60% of correct alternation in T-maze spontaneous tasks. Notwithstanding, control, donepezil and CA exposed rat groups scored above 60% of correct alternations in the T-maze tasks. This study is in agreement with Rao [31] and Chiroma [10] who documented similar result on CA’s cognitive enhancing abilities.

This study further assessed the role of CA in enhancing non-spatial memory and learning in rats using NOR test. NOR test is usually desirable since it requires no external reward, motivation or punishment, but rather just a little habituation and training besides it can be completed in a short period of time [32]. The spatial learning and memory dysfunctions seen in AlCl₃ alone exposed group of rats in this study was overcame by CA and donepezil treated rats. As the rats were able to distinguish between familiar and novel objects, because of the increased time they spent exploring the novel objects which signifies improved non-spatial memory. The current study supported the earlier report by Lin [33] who documented asiaitcosite, an active phytochemical isolated from CA improved cognitive abilities in senescence-accelerated mice (SAMP8).

The present study shows increased level of AChE in the cerebrum of the rats, which decreases after supplementation with CA or donepezil. Previous studies reported increased activity of AChE in brain after Al exposure [34,35] and decreased AChE activity after administration of ethanol extract of CA [5]. Acetylcholine (ACh) is an important neurotransmitter for memory and learning and is involved in several neuropsychiatric functions. According to Raina [36], AChE is accountable for the breakdown of ACh in the synaptic cleft. Therefore, mitigating AChE activity can increase the ACh levels in the synaptic cleft and partly recover the cognitive deficit in patients with AD. The cognitive and memory enhancing abilities of CA as revealed by the behavioral studies could be as a result of its potential to decrease the level of AChE, which could be attributed to its phytochemical asiatic acid [15].

The histology of the rat’s cerebrum showed that AlCl₃ facilitates a steady pathological alterations which includes; focal and diffuse gliosis, precellular edema encephalomalacia with neuronal degeneration. Findings such as nuclear degradation, karyorrhexis, dense cytosolic staining and disorganization of pyramidal cells of the hippocampus were also reported by similar studies [2,37,38]. The histopathological aberrations observed in the present study and the others reported could be as a result of increased oxidative stress due to Al exposure [2]. Al is an abundant heavy metal on earth’s crust which is associated with etiology of AD, where it aggravates brain’s oxidative stress. Al exerts its effects on synaptic junction and axons leading to degeneration of cholinergic neurons which are chiefly associated with memory and learning impairments [6]. Notwithstanding, CA administration prevented the neurodegeneration by preserving the cholinergic neurons of the brain which could further be attributed to its AChE inhibition capacity as seen earlier in this study.

Conclusion

Conclusively, CA protects against AlCl₃-induced cognitive impairment and neurodegeneration. As its administrations improved cognition as well as non-spatial learning and memory, which could be attributed to CA’s ability to decrease brain’s level of AChE. Therefore, CA could be developed as cholinesterase inhibitor to enhance memory and as a neuro-protectant. However, further studies on active phytochemical constituents of CA and the molecular pathways involved in its mechanism of action are recommended.

Ethical issues

The protocol was revised and approved by the Institutional Animal Care and Use Committee, Universiti Putra Malaysia, with a reference number UPM/IACUC/AUP-R096/2016

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The research presented in this paper was financially supported by Department of Anatomy, Universiti Putra Malaysia [Grant No.GP-IPS 9535400]