Phytochemical Analysis, In Vitro Anticholinesterase, Antioxidant Activity and In Vivo Nootropic Effect of Ferula ammoniacum (Dorema ammoniacum) D. Don. in Scopolamine-Induced Memory Impairment in Mice

Abstract

Background: Ferula ammoniacum (D. Don) is one of the endemic medicinal plants that is traditionally used to treat a number of diseases. Although the plant has been used to enhance memory, the investigational evidence supporting the nootropic effect was unsubstantial. Hence, the rationale for this study was to assess the potential beneficial effect of F. ammoniacum seed extracts on learning and memory in mice.

Methods: The powdered plant samples (aerial parts) were subjected to extraction ad fractionation. Among the extracts, crude and ethyl acetate extracts were screened for major phytochemicals through HPLC analysis. All the extracts were evaluated for the in vitro anticholinesterase (AChE and BChE) and antioxidant potentials. Among the extracts the active fraction was further assessed for improving learning and memory in mice using behavioural tests like Y-maze and novel object recognition test (NORT) using standard protocols. After behavioural tests, all the animals were sacrificed and brains tissues were assessed for the ex vivo anticholinesterase and antioxidant potentials.

Results: Phytochemicals like chlorogenic acid, quercetin, mandelic acid, phloroglucinol, hydroxy benzoic acid, malic acid, epigallocatechin gallate, ellagic acid, rutin, and pyrogallol were identified in crude methanolic extract (Fa.Met) and ethyl acetate fraction (Fa.EtAc) through HPLC. Fa.EtAc and Fa.Chf extracts more potently inhibited AChE and BChE with IC50 values of 40 and 43 µg/mL, and 41 and 42 µg/mL, respectively. Similarly highest free radical scavenging potential was exhibited by Fa.EtAc fraction against DPPH (IC50 = 100 µg/mL) and ABTS (IC50 = 120 µg/mL). The extract doses, 100 and 200 mg/kg body weight significantly (p < 0.01) improved the short-term memory by increasing the percent spontaneous alternation in the Y-maze test along with increasing discrimination index in the NORT that clearly indicated the enhancement in the recognition memory of mice.

Conclusion: The extracts more potently scavenged the tested free radicals, exhibited anticholinesterase activities, improved the learning abilities and reduced the memory impairment induced by scopolamine in mice model thus suggesting that these extracts could be effectively used for the management of oxidative stress, neurodegenerative diseases and memory loss.

Keywords: ABTS; Alzheimer’s disease; DPPH; Dorema ammoniacum (D. Don); Ferula ammoniacum (D. Don); HPLC; NORT; Y-Maze; cholinesterases (AChE and BChE).

Figure 1

HPLC chromatogram of Ferula ammoniacum (D. Don) aerial parts extract/fraction. (A) crude methanolic extract (Fa.Met) and (B) ethyl acetate fraction (Fa.EtAc).

Figure 2

Percent anticholinesterase and antioxidant potential of extract/fractions of Ferula ammoniacum (D. Don) aerial parts. {(A) Percent inhibition potential of extract/fractions for AChE and (B) BChE; (C) DPPH and (D) ABTS free radical. The data are expressed as Mean ± SEM, (n = 3). One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. Values are significantly different as compare to positive control, * p < 0.05, ** p < 0.01, *** p < 0.001}.

Figure 3

Effect of Fa.EtAc fraction of Ferula ammoniacum (D. Don) on mice in the behavioural Y-maze test. (A) Number of arm entries (B) Same arm returns (C) alternate arm returns (D) % Spontaneous alternation performance. The data are expressed as Mean ± SEM; each value corresponds to a mean of eight animals. One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. ** p < 0.01; comparison of DW + Sal^a (Normal control) vs. DW + Scop^b (Scopolamine treated), ^# p < 0.05 and ^## p < 0.01; comparison of (DW + Scop)^b vs. DZP + Scop^c (Donepezil treated)- and Fa.EtAc^c (50, 100 and 200 mg/kg)-treated groups), ns: values not significantly different in comparison to (DW + Scop)^b-treated group using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test.

Figure 4

Effect of Fa.EtAc fraction of Ferula ammoniacum (D. Don) on mice in behavioural NORT (A) Time spent in the sample phase (B) Time spent in the test phase (C) % Discrimination index were recorded in Fa.EtAc (50, 100 and 200 mg/kg)-treated groups versus scopolamine (Scop. 1 mg/kg)-treated group for assessment of recognition memory in mice model in behavioural NORT. The data are expressed as Mean ± SEM; each value corresponds to a mean of eight animals. One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. *** p < 0.001; comparison of DW + Sal^a (Normal control) vs. DW + Scop^b (Scopolamine-treated group), ^# p < 0.05 and ^## p < 0.01; comparison of (DW + Scop)^b vs. DZP + Scop^c (Donepezil treated) and Fa.EtAc^c (50, 100 and 200 mg/kg)-treated groups using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test.

Figure 5

Effect of Fa.EtAc fraction of Ferula ammoniacum (D. Don) on mice in behavioural NOL task (A) Percent exploration time between the objects A1 and A2 in familiar location in the sample phase (B) Percent exploration time between the objects A1 and A2 in novel location in the test phase. The data are expressed as Mean ± SEM; each value corresponds to a mean of eight animals. One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. *** p < 0.001, ^# p < 0.05, and ^## p < 0.01 and ^### p < 0.001; comparison of A1 (Object in familiar location) vs. A2 (Object in novel location).

Figure 6

Ex vivo % AChE activity in the (A) frontal cortex and (B) hippocampus of different animal groups in Y-maze behavioural test and (C,D) NORT. The data are expressed as Mean ± SEM; each value corresponds to a mean of eight animals. One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. ** p < 0.01; comparison of DW + Sal^a (Normal control) vs. DW + Scop^b (Scopolamine treated), ^# p < 0.05 and ^## p < 0.01; comparison of (DW + Scop)^b vs. DZP + Scop^c (Donepezil treated) and Fa.EtAc^c (50, 100 and 200 mg/kg)-treated groups), ns: values not significantly different in comparison to (DW + Scop)^b-treated group using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test.

Figure 7

Ex vivo % BChE activity in the (A) frontal cortex and (B) hippocampus of different animal in Y-maze behavioural task and (C,D) NORT. The data are expressed as Mean ± SEM; each value corresponds to a mean of eight animals. One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. ** p < 0.01; comparison of DW + Sal^a (Normal control) vs. DW + Scop^b (Scopolamine treated), ^# p < 0.05 and ^## p < 0.01; comparison of (DW + Scop)^b vs. DZP + Scop^c (Donepezil treated) and Fa.EtAc^c (50, 100 and 200 mg/kg)-treated groups), ns: values not significantly different in comparison to (DW + Scop)^b-treated group using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test.

Figure 8

Ex vivo %DPPH free radical scavenging effects in (A) frontal cortex and (B) hippocampus of different animal groups in Y-maze behavioural task and (C,D) NORT. The data are expressed as Mean ± SEM; each value corresponds to a mean of eight animals. One-way ANOVA followed by Dunnett’s post hoc multiple comparison test to determine the values of p. ** p < 0.01; comparison of DW + Sal^a (Normal control) vs. DW + Scop^b (Scopolamine treated), ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001; comparison of (DW + Scop)^b vs. DZP + Scop^c (Donepezil treated) and Fa.EtAc^c (50, 100 and 200 mg/kg)-treated groups), ns: values not significantly different in comparison to (DW + Scop)^b-treated group using one-way ANOVA followed by Dunnett’s post hoc multiple comparison test.

Figure 9

Mechanistic overview of Fa.EtAc as nootropic on the responsible factors involved in the aetiology of neurodegenerative diseases.