Acetylcholinesterase inhibitory activity and iron-induced lipid peroxidation reducing potential of Pimpinella stewartii leaves in male wistar rats

2.1 Ethical approval

The study approval was granted by the ethical committee of Centre of Biotechnology and Microbiology, University of Peshawar, Pakistan (No: 119/UOP/Biotech).

2.2 Extraction and fractionation

P. stewartii was collected from District Abbottabad, Pakistan and was identified in Department of Botany, University of Peshawar, Pakistan. Leaves were cleaned, washed, and shade dried at ambient temperature. After drying, leaves were powered using a grinder. Two hundred grams of powder were mixed with 1 L of ethanol (70% v/v) and kept in rotator shaker for a week. After required incubation period, the filtrate was collected and the concentrated product was obtained using a rotary evaporator (Heidolph, HeiVAP Core, Germany). Further, a suspension of ethanolic extract in distilled water was prepared and fractionated with n-butanol, ethyl acetate, and dichloromethane. The aqueous extract was obtained by hot water treatment and was prepared immediately before use. Sterile capped vials were used to store the extracts at 4 °C until further use.

2.3 Animals and experimental design

Male Wistar rats (age – 50 days; weight – 280–300 g) were obtained from National Institute of Health, Islamabad, Pakistan. The animals were housed in steel cages. All the cages were cleaned regularly in order to ensure healthy animals. The animals were allowed free access to the standard pellet diet and also the water was provided ad libitum. The cages were kept in a well aired room having a 12 h light dark cycle. The animals selected for the experiment were divided into 4 different groups that were further divided into various sub-groups (7 rats in each group). Group 1 was assigned as control group, Group 2 was administered with eserine, Group 3 was administered with ethanol only, and Group 4 was administrated with various concentrations of extracts fractions (aqueous fraction with 4 sub-groups: ethyl acetate fraction with 4 sub-groups). There were 7 rats in each group and sub group. Altogether, 17 groups were designed for this study. The treatment was given intra-peritoneal. The mixing of all the compounds was done separately in accordance with the standard protocol. The animals were treated according to the guidelines of the Brazilian College of Animal Experimentation (COBEA), affiliated with the International Council for Laboratory Animal Science (ICLAS).

2.4 AChE inhibitory activity

The AChE activity was demonstrated as per the modified protocol of Rocha et al. (Rocha et al., 1993). In order to determine AChE activity, animals from each group were sacrificed by blowing their heads (without anaesthesia), and the cerebral tissues were quickly incised and kept on ice. The tissues were homogenised using Tris HCl buffer (50 mM; pH 7.5) and the homogenised product was centrifuged at 4000 × g for 10 min. After centrifugation, the supernatant was collected to pursue AChE activity.

The AChE activity was evaluated in a reaction mixture that contained 200 µL of acetylcholinesterase solution (0.415 U/mL prepared in 0.1 M phosphate buffer; pH 8.0), 100 µL of DTNB [5,5′-dithio-bis(2-nitrobenzoic)] acid solution, 500 µL of phosphate buffer (pH 7.0), and 50 µL of supernatant. The reaction mixture was incubated at room temperature for 20 min, following the addition of 100 µL of 0.05 M acetylcholine iodide solution as substrate. The absorbance was read at 412 nm using UV–Visible spectrophotometer (Shimadzu UV-1780) and the inhibitory activity (%) was estimated as: $$\mathit{AChE}activity\left(\%\right)=\left[\left(\mathit{Absorbanc}{e}_{\mathit{Control}}-Absorbanc{e}_{\mathit{Test}}\right)/Absorbanc{e}_{\mathit{Control}}\right]\times 100$$

2.5 Lipid peroxidation assessment

Since free radicals damage the brain tissues, cerebral tissues were used for thiobarbituric acid reactive substances (TBARS) assay. The lipid peroxidation assay was assessed by estimating TBARS production using the methodology of Puntel et al. (2007). Plant extracts fraction (100 µL), purified gallic acid, and quercetin were mixed separately into the freshly prepared 5 mM of sodium nitroprusside (SNP), Fe₂SO₄ (10 mM), and 2 mM of 3-nitropropionic acid (3-NPA). Pro-oxidants were used in order to induce oxidative stress which causes lipid peroxidation in the body. Following incubation time, acetate buffer, 8.1% SDS, and TBA was pipetted and a further incubation was done for 60 min at 100 °C. The reaction of TBA with malondialdehyde can be assessed with the production of light pink color. After cooling the reaction mixture, the absorbance was read at 532 nm.

2.6 Statistical analyses

The data was analysed statistically using one-way ANOVA, followed by DMRT (Duncan's Multiple Range Test) (for TBARS) and Bonferroni’s multiple comparison tests (for AChE activity). P value < 0.05 was considered significant. All the data included in this study was presented as mean ± standard error of mean (mean ± SEM).

3.1 AChE inhibitory activity

Results demonstrated that the aqueous extract fraction was unable to inhibit AChE activity at varied concentrations (Fig. 1A). On the other hand, ethyl acetate fraction significantly (P = 0.01) inhibited the enzyme in a concentration dependent manner in comparison to the control group (Fig. 1B). Gallic acid and quercetin showed higher inhibition of AChE activities than the ethyl acetate extract fractions (Fig. 1C and 1D). The IC₅₀ values for aqueous extract fractions, ethyl acetate extract fractions, gallic acid, and quercetin were estimated as 72.6 ± 2.72, 30.41 ± 0.56, 8.4 ± 0.22, and 6.82 ± 0.2 µg/mL, respectively (Fig. 2).

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Fig. 1

AChE inhibitory activity (%) of aqueous extract fractions (A), ethyl acetate extract fractions (B) of P. stewartii, gallic acid (C), and quercetin (D). Eserine was used as positive control. Data were a representative of mean ± SEM.

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Fig. 2

IC₅₀ values for aqueous extract fractions, ethyl acetate extract fractions, gallic acid, and quercetin.

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Acetylcholine is an important neurotransmitter distributed widely throughout the nervous system (Halder and Lal, 2021). Cholinesterase is classified into AChE and butyrylcholinesterase. AChE is a member of α/β hydrolases family. AChE causes the hydrolysis of acetylcholine, both in synapses and neuromuscular intersection, which results in nerve impulse termination (Asefy et al., 2021). It is reported that AChE is a good candidate for drug designing in AD treatment; as in AD there is a deficiency of acetylcholine (Zhou et al., 2021). After its transport to synapses, acetylcholine is hydrolysed into choline and acetate (Voet and Voet, 1995; Colović et al., 2013). Current strategy is to focus on the blockage of the AChE function in order to increase the quantity of acetylcholine. Most acetylcholinesterase drugs being used to treat AD are AChE inhibitors.

Several in vitro and in vivo studies have revealed the role of medicinal plants in treating neurological disorders (Ratheesh et al., 2017; Faheem et al., 2022). Suganthy et al. (2009) reported AChE inhibitory potential of Avicennia officinalis methanolic extract producing an IC₅₀ value of 1.24 ± 0.011 mg/mL and butyryl cholinesterase inhibition with an IC₅₀ value of 0.911 ± 0.007 mg/mL. Similarly, the ethanolic extract of Acacia catechu seeds exhibited promising AChE inhibition potential with an IC₅₀ value of 204.38 ± 2.54 μg/mL and recommended for the brain disorders management (Thangavelu and Ramasamy, 2015). In another study, the hydro-alcoholic extract of Hemidesmus indicus exerted significant AChE inhibitory activity with an IC₅₀ value of 28.4 ± 0.92 μg/mL (Kadiyala et al., 2014). Additionally, the aqueous and the methanolic extract of H. indicus roots also exhibited in vitro AChE inhibitory activity (Kundu and Mitra, 2013). In the present work, the ethyl acetate extract fractions of P. stewartii inhibited AChE activity significantly. Therefore, P. stewartii could be considered to assess its possibility as potent alternative candidate for designing effectual drug towards the treatment of AD.

3.2 Lipid peroxidation assessment

Results of TBARS levels induced by various pro-oxidant agents are illustrated in Table 1. In fact, the TBARS content measured the degree of peroxidation. Both the fractions were found to be protective against lipid peroxidation but ethyl acetate extract fraction reduced the TBARS levels significantly (P = 0.001) in comparison to the aqueous extract fraction. However, gallic acid and quercetin exhibited higher reductions of pro-oxidants than ethyl acetate and aqueous extract fractions. Findings of this context were found in consistence with the report of Tripathi et al. (1997). Rubiadin, (a dihydroxy anthraquinone) showed its remarkable anti-lipid peroxidation activity induced by FeSO₄ and t-butyl hydroperoxide in a concentration dependent manner. The inhibitory activity was estimated maximum in Fe⁺² induced lipid peroxidation. In this investigation, quercetin was found to be more potent as compared to the extracts and gallic acid. This might be due to the lipophilic characteristic of quercetin which enhanced its potency to block lipid peroxidation.