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 Table of Contents  
Year : 2019  |  Volume : 9  |  Issue : 2  |  Page : 116-123

Pharmacological evaluation of Hibiscus abelmoschus against scopolamine-induced amnesia and cognitive impairment in mice

Department of Pharmacology, School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh, India

Date of Web Publication8-May-2019

Correspondence Address:
Dharamveer Panjwani
Department of Pharmacology, School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/AIHB.AIHB_3_19

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Objective: Progressive loss of memory has a prominent role in devastating neurodegenerative diseases such as amnesia, dementia and Alzheimer's disease. The present study was, thus, designed to investigate the potential of Hibiscus abelmoschus (H. abelmoschus) in scopolamine-induced amnesic Swiss Albino mice. Materials and Methods: Phytochemical and acute toxicity studies of H. abelmoschus ethanolic extract (HAEE) were performed. Memory loss was induced by scopolamine (1 mg/kg, i.p), a muscarinic antagonist, and evaluated by using Morris water maze (MWM), elevated plus maze (EPM) and modified passive avoidance tests on Swiss Albino mice. Besides this, locomotor activity, serum biochemical parameters such as total cholesterol and triglyceride levels were evaluated. Lipid peroxidation (LPO), glutathione (GSH) level and catalase (CAT) activity were also checked. HAEE at a dose of 200 and 400 mg/kg body weight administered orally to the animals. Donepezil hydrochloride (1 mg/kg) was taken as standard. Results: Pre-treatment of mice with HAEE significantly reversed scopolamine-induced memory impairments, as evidenced by a significant decrease in escape latency in the MWM task, transfer latency in EPM task and an increase in step-down latency in the modified passive avoidance task. HAEE also exhibited a remarkable cholesterol and triglyceride-lowering property in the present study. Moreover, treatment with HAEE (200 and 400 mg/kg, p.o) to scopolamine-induced rats significantly decreased the LPO and increased the GSH and catalase levels. Conclusion: The memory restorative effects of H. abelmoschus in amnesia may involve its multiple functions including antioxidative and lipid-lowering properties and the presence of phytoconstituents. Thus, H. abelmoschus may act as memory enhancer and may also be useful as a supportive adjuvant in the treatment of impaired memory functions.

Keywords: Amnesia, catalase, glutathione, Hibiscus abelmoschus, scopolamine

How to cite this article:
Nigam A, Kulshreshtha M, Panjwani D. Pharmacological evaluation of Hibiscus abelmoschus against scopolamine-induced amnesia and cognitive impairment in mice. Adv Hum Biol 2019;9:116-23

How to cite this URL:
Nigam A, Kulshreshtha M, Panjwani D. Pharmacological evaluation of Hibiscus abelmoschus against scopolamine-induced amnesia and cognitive impairment in mice. Adv Hum Biol [serial online] 2019 [cited 2022 Sep 27];9:116-23. Available from: https://www.aihbonline.com/text.asp?2019/9/2/116/257812

  Introduction Top

Memory is a vital function of brain, includes basically three main stages, namely, acquisition, consolidation and retrieval. Learning a task is acquisition; process through which the memory stabilises is consolidation and bringing back of the learned task is retrieval.[1] If there is no learning, there can be no memory later.[2] Part of memory which comes to be known through perception, reasoning, intuition and knowledge is cognition which involves a plethora of functions such as attention, perception, memory coding, retention, recall, decision-making along with reasoning, problem-solving, imaging, planning and executing actions.[3] Decline in memory functions is supposed to be related to various etiological factors such as increasing age, production of free radicals, up and downs of emotions, reduction in cholinergic firing in brain, elevated oxidative stress, hypercholesterolaemia and neuroinflammatory reactions, which causes occurrence of amnesia, dementia to more threatening conditions such as schizophrenia and Alzheimer's disease (AD).[4],[5],[6]

The estimated global dementia prevalence in people aged over 60 is approximate 3.9% with regional prevalence being 1.6% in Africa, 3.9% in Eastern Europe, 4.0% in China, 4.6% in Latin America, 5.4% in Western Europe and 6.4% in North America. There will be an exponential increase in age-related neurodegenerative patients, as estimated by United Nations which depicts its range from 25.5 million in 2000 to an approximately 114 million in 2050.[7] Furthermore, in India, the patients of AD and other dementias are raising their counts and are expected to become thrice as now by 2050.

The cholinergic system plays an important role in learning and memory.[8] The neurotransmitter acetylcholine has important recognition functions such as memory,[9] and it is synthesised in certain neurons by the enzyme choline acetyltransferase from choline to acetyl-CoA. Acetylcholinesterase (AChE) is an enzyme that hydrolyses acetylcholine into choline and acetate.[10] A loss of cholinergic neurons and reduced choline acetyltransferase activity in the cerebral cortex and hippocampus is consistent with findings in AD.[11],[12],[13]

Since ages, drugs and natural remedies have been prescribed to enhance memories in people. The Indian System of Medicine Ayurveda has a treasury of such memory enhancing drugs, which are today popular all over the world due to their proven effective qualities. The herbs acting on the brain are called as Nootropic herbs (Nootropic is derived from Greek and means acting on the mind) and their isolated constituents referred to as smart drugs. Memory enhancer herbs enhance the memory and increase blood circulation in the brain.[14]

H. abelmoschus (syn: Abelmoschus moschatus), belonging to Family Malvaceae, is a native of India and grows in the tropical subtropical and hilly regions of country.[15] Seeds of this herb are commonly named 'Musk daana.' Phytochemical studies have identified the presence of a wide variety of constituents such as flavonoids like myrecetin, quercetin, along with glycosides and β-sitosterol. It was mentioned in traditional Indian medicine as a useful therapy for treating nervous debility, hysteria and other nervous disorders and for the treatment of intestinal complaints, constipation, dyspepsia and gonorrhoea. It acts as cardiac tonic, aphrodisiac, diuretic and antispasmodic.[16],[17] Seeds and leaves of H. abelmoschus are found to possess various pharmacological properties, such as larvicidal, antioxidant, antimicrobial, aphrodisiac, skin protective, wound healing, antilithiatic activity and sexual activity for the treatment of impotency.[18],[19],[20],[21],[22],[23]

The present research was thus, carried out to evaluate the effect of ethanolic extract of seed of H. abelmoschus on learning and memory activity at different doses against scopolamine-induced amnesia in mice.

  Materials and Methods Top

Plant material

Seeds of H. abelmoschus, were purchased from N. N. Aushdhalaya, Lucknow and authenticated by Dr. RR Singh, ex-HOD, Department of Botany, Lucknow University, Lucknow, UP, India, and a voucher specimen was deposited (Ref. no. Bot/Q/2323).

Preparation of the extract

The seeds of H. abelmoschus were cleaned, dried and coarsely powdered. A weighed quantity of the coarse powder was taken and extracted with 70% ethanol in a soxhlet apparatus. The extract was concentrated to dryness with the help of rotary evaporator and finally, air-dried thoroughly to remove all traces of the solvent. The H. abelmoschus ethanolic extract (HAEE) was kept for further analysis. The yield of the HAEE was found to be 11.5%.[24]

Experimental animals

Swiss Albino mice (25–35 g, either sex) were used in this study after Institutional Animal Ethics Committee approval (IAEC No. BBDNIIT/IAEC/022/2014). The mice were allowed free access to food and water ad libitum. Animals were kept in polyacrylic cage in groups of six/cage and maintained under standard housing conditions (temperature: 24°C–27°C and humidity: 60%–65%) with 12 h light and dark cycle. Animals were acclimatized for at least 7 days to the laboratory conditions before behavioural experiments. Experiments were carried out between 900 h and 1600 h to avoid experimental deviations due to diurnal variations.

Drugs and chemicals

Scopolamine hydrobromide (Sigma-Aldrich, USA), 5,5'-dithiobis-(2-nitrobenzoic acid), (Ellman's reagent), trichloroacetic acid and thiobarbituric acid (TBA) were purchased from Sigma-Aldrich (Bangalore, India) Dithiobis-nitrobezoic acid (SD Fine-Chem Limited), Donepezil (as a gift sample from Vasudha Pharma, Hyderabad), all other chemicals and reagents used for the study were of analytical grade.

Grouping and treatment protocol

Learning and memory were assessed with three behavioural models, namely, Morris water maze (MWM), elevated plus maze (EPM) and modified passive avoidance test. Animals were randomly divided into five groups of six animals each. The grouping for pharmacological screening models was as follows:

  • Group 1: Positive control (PC) mice received only vehicle
  • Group 2: Negative control (NC) mice received vehicle and scopolamine (1 mg/kg, i.p)
  • Group 3: Standard drug (STD), donepezil (1 mg/kg, i. p.) and scopolamine (1 mg/kg, i.p)
  • Group 4: Lower dose of HAEE (T1) (HAEE 200 mg/kg, p. o) and scopolamine
  • Group 5: Higher dose of HAEE (T2) (HAEE 400 mg/kg, p. o) and scopolamine.

These animals were dosed once daily with the respective drugs. Donepezil and scopolamine hydrochloride were dissolved in distilled water and administered intraperitonealy, while HAEE was administered orally after get dissolved in distilled water.[25]

Acute toxicity study

Mice were kept on overnight fasting and water was withdrawn 3–4 before administration of test compound. HAEE was administered orally in increasing doses of 50, 100, 500, 1000, 2000 and 4000 mg/kg body weight. Immediately after dosing, the mice were observed continuously for 4 h for symptoms of toxicity such as motor activity, tremors, convulsions, tonic extension, muscle spasm, loss of righting reflex, ataxia, sedation, hypnosis, lacrimation, diarrhoea, salivation and writhing. Mice were then kept under observation up to 72 h for any mortality.

Memory models

Induction of amnesia

Amnesia was induced in mice by using scopolamine at a dose of 1 mg/kg, i.p only once during the model. It was given 30 min before retention trial in MWM, and 30 min before acquisition trial in EPM. In modified passive avoidance, it was injected to animals 30 min before acquisition trial.

Morris water maze

The maze was a white circular pool (100 cm diameter and 50 cm height) with a featureless inner face filled with water (20°C ± 1°C) upto a depth of 30 cm. The treatment was allotted to animals for 14 days. Scopolamine was injected on day 14 1 h after all treatments. The animals were trained for maze task from day 10th to 13th four times per day at a intertrial gap of 5 minutes. Mice failed to find the platform within the allotted period (120 s, initial learning period; 180 s, scopolamine-induced amnesia trials) were placed on the platform for 30 s. On day 14, water made opaque by adding milk and animals were tested three times with each of the three trials being performed between 30 and 45 min following scopolamine injection. Escape latency, number of crossing and residence time were evaluated as parameters for testing of spatial learning.[4],[26]

Elevated plus maze

Elevated plus maze served as the exteroceptive behavioural model to evaluate long-term memory in mice. Grouping and dosing schedule were same as MWM test. Maze consisted of two open (16 × 5) and two closed arms (16 × 5 × 20). All treatments were given for 14 days. On the 14th day, after 60 min of administration of treatments, scopolamine (1 mg/kg, i.p) was injected to all groups except Group 1. After 30 min of scopolamine injection, animals were placed individually at the end of either of the open arm, facing away from the centre and the time taken by the animal to move from open-to-closed arm, i.e., transfer latency (TL) was noted for the acquisition trial. TL was defined as the time (in seconds) taken by the animals to move from the open arm into one of the covered arms with all its four legs.[27] If the mice were unable to reach the closed arm within allotted period, that is, 180 s, it was placed into one of the closed arms for 15 s, so as to explore it and the TL was taken as 180 s.[28] After 24 h of exposure, the procedure was again repeated and TL was recorded as the parameter for memory on the day of retention.

Modified passive avoidance test

Passive avoidance apparatus is based on negative reinforcement was used to examine long-term memory. The apparatus consisted of a rectangular box (27 cm × 27 cm × 27 cm) having three walls of wood and one wall of Plexiglas featuring a grid floor (3-mm stainless steel rods set 8 mm apart), with a wooden platform (10 cm × 7 cm × 1.7 cm) at the centre of the grid floor an electric shock was delivered to the grid floor. The box was illuminated with a 15 W bulb during the experimental period. All treatments were given to the animal for 14 days. On the 14th day, scopolamine was injected to the animals after 60 min of drug administration. After 30 min of scopolamine injection each mice were placed on a wooden platform set in the centre of the grid floor. When the mice stepped down and placed its paw on the grid floor, shock (foot shock: 50 Hz; 1.5 mA; 1 s) was delivered and the step-down latency (SDL) was recorded. SDL is defined as the time taken by the mice to step down and place all four paws on the grid floor. Mice showing SDL in the range of 2–15 s were taken for the acquisition and the retention tasks. The acquisition task was carried out 90 min after the training session. During the acquisition test, animals were removed from the shock-free zone if they did not step down for a period of 60 s. Retention was tested after 24 h in a similar manner, except with an upper cut-off time of 180 s.[29],[30],[31]

Locomotor activity

The locomotor activity was studied using actophotometer, which operates on photoelectric cells are connected in circuit with a counter when the beam of light falling on photocell is cut off by the animal, a count is recorded. The animals were placed individually in the activity cage for 5 min and the activity was monitored. The photocell count was noted and decrease or increase in locomotor activity was calculated.[30],[32]

Biochemical estimation

Biochemical tests were conducted 24 h after the last behavioural test, that is, MWM. Following the completion of experiment on the 14th day, blood was collected from retro-orbital plexus under mild ether anaesthesia. The collected blood was centrifuged to separate serum for estimation of serum cholesterol and triglyceride levels. After collection of blood, the animals were sacrificed by decapitation. Brains were removed and rinsed with ice-cold isotonic saline. Brains were then homogenised with ice cold 0.1 mmol/l phosphate buffer (pH 7.4). The homogenates (10% w/v) were then centrifuged at 10,000 g for 15 min and the supernatant so formed was used for the biochemical estimations.[30]

Measurement of lipid peroxidation

The extent of lipid peroxidation (LPO) in the brain was determined quantitatively by performing the method as described by Ohkawa et al.[32] The amount of malondialdehyde (MDA) was measured by reaction with TBA at 532 nm using spectrophotometer.[33]

Catalase activity

Catalase activity was assessed by the method of Beer and Sizer, based on the ability of catalase to oxidise hydrogen peroxide. The change in absorbance was recorded for 3 min.[34]

Glutathione levels

Glutathione (GSH) level was measured as non-protein thiols based on the protocol developed by Ellman (1959). The homogenate was precipitated in cooled trichloroacetic acid 10% and centrifuged at 15,000 g for 2 min, and the supernatant was incubated with 5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB in a 1 M phosphate buffer, pH 7.0. Absorbances were measured at 412 nm. A standard curve of reduced GSH was used to calculate the GSH levels.[35]

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey's post-hoc test. Values are expressed as mean ± standard error of the mean (SEM).

  Results and Discussions Top

Preliminary phytochemical analysis

The preliminary phytochemical studies were performed for testing different phytochemical constituents present in HAEE. The observations showed the presence of alkaloids, flavonoids, glycosides, tannins and the absence of saponins [Table 1].
Table 1: Phytochemical analysis of Hibiscus abelmoschus ethanolic extract

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Acute toxicity studies

In acute toxicity study, HAEE did not show any mortality in mice. Even at this higher dose, i.e., 4000 mg/kg, there were no gross behavioural changes such as skin, fur condition, eyes colour, motor activity, convulsions, tonic extension, loss of righting reflex, ataxia, sedation, hypnosis and writhing were observed, and 200 and 400 mg/kg doses were used for the evaluation of different activities.

Morris water maze

The effect of HAEE (200 and 400 mg/kg, p.o) on spatial long-term memory was evaluated using the MWM task. Daily administration of both the doses of HAEE and STD for 14 days significantly decreased latency to find hidden platform when compared to NC group, indicating their effect on improvement in learning and memory in retention trial. Scopolamine increased the latency to find hidden platform, but at the end of the experiment, escape latency was decreased in HAEE-treated groups at the dose of 400 mg/kg. Donepezil showed maximum reduction in escape latency. However, there were no significant differences in number of crossings and residence time between negative control (NC) and HAEE-treated groups during training and hidden platform trial [Table 2] and [Table 3].
Table 2: Effect of Hibiscus abelmoschus ethanolic extract on escape latency (s) of mice using Morris water maze

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Table 3: Effects of extract on number of crossing and residence time of mice using Morris water maze

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Elevated plus maze

The effect of the vehicle, scopolamine control, HAEE (200 and 400 mg/kg) and donepezil (1 mg/kg) were evaluated at the end of day 14. The scopolamine control group showed a significant increase in TL values on the acquisition as well as on the retention days as compared with vehicle control rats, indicating impairment in learning and memory. In the retention trial on day 15, the result analysis revealed significant differences in TL in EPM performance between HAEE (200 and 400 mg/kg) and NC group animals (***P < 0.001). The results were comparable with donepezil (1 mg/kg). TL of 2nd day (day 15) reflected retention of learned task or memory [Table 4].
Table 4: Effect of extract on transfer latency of mice using elevated plus maze

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Modified passive avoidance

In the modified passive avoidance test, scopolamine decreased SDL on the acquisition and retention trial training, indicating impairment of memory. SDL of the 2nd day (day 15 of drug treatment) reflected the long-term memory of animals. There was a statistically significant increase (***P < 0.001) in SDL after the administration of HAEE (200 and 400 mg/kg po) for 14 days as compared with the NC group in learning and memory of mice. However, donepezil showed maximum increase in SDL [Table 5].
Table 5: Effect of extract on step-down latency of mice in modified passive avoidance test

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Locomotor activity

The HHAE at doses of 200 and 400 mg/kg p.o did not produce any significant reduction in locomotor activity as compared to the control animals receiving only the vehicle. Furthermore, the scopolamine control group and the donepezil-treated group failed to produce any significant effect on locomotor activity as compared to the control. This result indicates that the scopolamine injection did not affect the general locomotor activities of the mice, but gave rise to learning disabilities in them [Table 6].
Table 6: Effect of Hibiscus abelmoschus ethanolic extract on locomotor activity in scopolamine-induced amnesia in mice

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Effect of Hibiscus abelmoschus ethanolic extract on biochemical parameters

HAEE at the doses of 200 and 400 mg/kg showed marked reduction in serum total cholesterol and a significant reduction in triglyceride levels. The dose of 200 mg/kg showed more reduction in cholesterol level as compared to test group 2 of 400 mg/kg. Donepezil showed reduction in blood cholesterol level of mice similar to HAEE (200 mg/kg) [Table 7].
Table 7: Effect of Hibiscus abelmoschus ethanolic extract on serum total cholesterol and triglyceride level in mice

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Effects of Hibiscus abelmoschus ethanolic extract on malondialdehyde levels and antioxidant enzyme activities

The analysis of MDA levels using one-way ANOVA showed a difference among the five groups. Between-groups comparisons indicated stress exposure significantly increased MDA levels in NC group compared to PC group. HAEE treatment caused a significant decrease in the MDA levels when compared to the NC group. Furthermore, there was a significant decrease in MDA levels of standard group as compared to NC group. Furthermore, one-way ANOVA on catalase and GSH levels revealed a significant difference in five groups. Between-groups comparisons indicated stress exposure significantly decreased catalase and GSH levels in NC group compared to PC group. HAEE treatment at a dose of 400 mg/kg caused a significant increase in the catalase and GSH levels when compared to the NC group. Furthermore, there was a significant increase in catalase and GSH levels of the standard group as compared to NC group.

Oxidative parameters

In the restraint stress-induced model, catalase, GSH and LPO decreased and increased, respectively, in stressed group compared with the unstressed group. HAEE increased catalase, GSH level and decreased LPO as compared to NC group. Similarly, donepezil treatment also increased catalase, GSH level and depleted LPO compared with stressed group [Table 8].
Table 8: Effects of Hibiscus abelmoschus ethanolic extract on oxidative stress parameters in mice brain

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  Discussion Top

Herbal extracts with multiple active constituents have emerged as preferred prophylactics owing to their broad spectrum curative potential and minimum negative effects when compared to synthetic molecules. H. abelmoschus seeds have been recommended in traditional Indian Medicine System for the treatment of nervous debility, disorders and hysteria, but no research has been done yet. The present study, thus, demonstrates beneficial effect of HAEE on scopolamine-induced amnesia as assessed by behavioural test using MWM, EPM, modified passive avoidance test and biochemical parameters such as total serum cholesterol and triglyceride levels.

Scopolamine interferes with memory and cognitive function in human beings and experimental animals by blocking muscarinic receptors[36] along with the impairment of long-term potentiation and the central cholinergic system.[4] Its interference in acetylcholine transmission in the central nervous system proved it as a useful experimental animal model to screen for drugs with potential therapeutic value in dementia. It is reported that memory impairment in the scopolamine (1 mg/kg, i.p)-induced animal model is associated with the increased oxidative stress within rat and mice brain. We treated mice with scopolamine and corroborated that it increased EL in MWM, TL in EPM and reduced SDL in passive avoidance, which would validate our paradigm as an effective measurement of learning and memory in mice.[37]

Donepezil is a reversible and highly selective acetylcholinesterase inhibitor with a long plasma half-life of approximately 70 h that is approved for the enhancement of memory function in AD. The results of MWM and EPM task had shown significant effectiveness in observed parameters when compared with NC group. The main mechanism of action through which it influences cognition and function is presumed to be the inhibition of AChE enzyme in the brain along with the prevention of free radical-mediated neuroinflammation, upregulation of ChAT, which is known to be responsible for the synthesis of ACh or enhancement of release of ACh through nicotinic receptors.[38],[39],[40],[41],[42]

The MWM learning task is used to assess hippocampal-dependent spatial learning ability.[4] In the present study, the results obtained from a MWM test in all groups during the training phase showed normal learning profiles as indicated by decreases of escape latencies from day 1 to 4. A decrease in day 4, escape latencies (ELT) of control animals during on-going acquisition trials denoted normal acquisition of memory and an increase in time spent in target quadrant, in search of the missing platform during retrieval trial indicated, retrieval of memory[43] which was shown by HAEE treatment. The accurate directionality of their escape behaviour (and other measures of performance) provides evidence that rats escape by learning the spatial position of the platform relative to distal cues.[24]

The EPM paradigm (TL) was employed to deduce spatial long-term memory of animals.[44] Decrease in TL on the 2nd day (i.e., 24 h after the first trial) indicated improvement of memory.[45] It showed retention or consolidation of memory, whereas acquisition of memory-related action of drugs was checked by treating the animals before 1st day. There was also improvement in cognition of animals as indicated by quick migration of mice after exposure to open arm.[46]

The step-down type of passive avoidance task has been used to examine the long-term memory based on negative reinforcement.[44] In case of the passive avoidance paradigm, the SDL was increased on the administration of HAEE[47] which was also recorded before to determine memory disturbance produced by the cholinergic antagonist scopolamine.[45] With 200 mg/kg dose, the increase in number of avoidances and prolongation of SDL (mice remained on the platform for longer time) was higher than 400 mg/kg, but not significantly different than standard.

Epidemiological studies have found positive correlations between hypercholesterolemia and development of AD.[48] In our study, H. abelmoschus decreased the serum cholesterol level as compared to NC. Cholesterol turnover appears to play a crucial role in the deposition and clearance of amyloid peptide in the brain, and in high risk of developing AD,[49] and drugs, that inhibit cholesterol synthesis lower amyloid-beta (Aβ) in these cases.[26] HAEE at both the doses significantly lower the serum cholesterol and triglyceride level which showed its hypolipidaemic nature.

There is a growing body of evidence in the literature that indicates oxidative stress as a crucial factor for several disorders including dementia, furthermore, data indicate that oxidative stress is one of the earliest events in pathogenesis of memory impairment.[50] Oxidative stress is thought to be important in the progression of AD and is temporally linked to the development of plaques and neurofibrillary tangles.[51] The LPO is well-known elements to play an essential role in oxidative stress balance.[50] It is demonstrated increased oxidation of lipids, proteins and deoxyribonucleic acid, alterations in mitochondrial function and a possible role of Aβ and its precursor protein in oxidative reactions in experimental models of AD. Scopolamine-induced TBA reactive substance activity represents an important marker for LPO in the frontal cortex and hippocampus. Because oxidative stress contributes significantly to the perturbation of calcium homeostasis and subsequent apoptosis, as seen in AD patients.[4] There is considerable evidence that scopolamine leads to oxidative stress in rat leading to cognitive impairment.[50]

LPO may enhance due to depletion of GSH content in the brain, which is often considered as the first-line of defence of the cell by this endogenous antioxidant against oxidative stress. Evidence has been presented that the neuronal defence against H2O2, which is the most toxic molecule to the brain, is mediated primarily by the GSH system. GSH is a tripeptide, an endogenous antioxidant found in all animal cells in variable amounts and is a very accurate indicator of oxidative stress. MDA is an indicator of LPO.[25] In the present study, scopolamine administration increased (1.21 ± 0.17), the MDA level when compared to PC (0.50 ± 0.07) mice. Donepezil appreciably decreased (P < 0.001) (0.03 ± 0.04) the MDA level when compared to scopolamine-administered mice. HAEE (200 mg/kg, p.o) administration did not produce any considerable changes (P > 0.05) in MDA level when compared to scopolamine-administered mice. HAEE administered mice at the dose of 400 mg/kg, p. o appreciably decreased (0.70 ± 0.03) in MDA level when compared to NC group.[25]

Catalase is the major enzymatic defence for aerobic cells combating the toxic effect of superoxide radicles.[51] Catalase has the capability to detoxify H2O2 radicals. Release of H2O2 promotes the formation of numerous other oxidant species that greatly contributes for oxidative stress leading to the pathogenesis of AD. The present study shows that scopolamine treatment decreased brain catalase activity and pre-treatment with EEAS (400 mg/kg, p.o) remarkably restored catalase activities. Improvement of catalase activity was observed in standard and HAEE-treated groups compared to NC.[29]

GSH is a premiere cellular antioxidant. It exists in reduced (GSH) and oxidized (glutathione disulfide [GSSG]) states within our cells. In the reduced state, the thiol group of cysteine are able to quench free radicals by donating a reducing equivalent (H++ e-). This includes quenching various reactive oxygen species that are generated during the use of oxygen by our cells in aerobic metabolism. In donating an electron, GSH itself becomes reactive but readily reacts with another reactive GSH to form GSSG. This is possible due to the normally high concentration of GSH in cells (up to 5 mM in the liver). GSH can be regenerated from GSSG by the enzyme GSH reductase.[52]

The phytochemical studies showed the presence of high amounts of flavonoids[53] and polyphenols which are known to possess antioxidative, free radical scavenging and LPO inhibition activity.[17] Flavonol myricetin (3, 5, 7, 3′,4′,5′-hexahydroxy flavone), the active principle of A. moschatus, has proven activity of antioxidative and cytoprotective.[54] One more flavonoid, i.e., quercetin which is also identified in plant through studies[25] has been demonstrated to improve cerebral blood flow along with preventing memory impairment, oxidative stress, altered brain energy metabolism and cholinergic dysfunction in mice.[55] HAEE exhibited significant activity against scopolamine-induced amnesia, probably due to the presence of high amount of flavonoids and polyphenols. These antioxidant, free radical scavenging and LPO inhibitory activity are probably the basic mechanism of plant for anti-amnesic activity. LPO inhibitory activity of A. moschatus extracts may be the probable mechanism of hypolipidaemic activity.[56]

  Conclusion Top

Our results suggested that HAEE pre-treatment prevents scopolamine-induced impairment of memory in MWM, EPM and modified passive avoidance test. Plant was also effective in improving the lipid profile of animal, although the cellular and molecular mechanisms that underlie the action of HAEE were not fully understood. Thus, it can be concluded that plant has the potential to be considered as a therapeutic agent and it would be beneficial to explore it.


The authors would like to express their hearty thanks to Dr. Sunita Garg, NISCAIR, New Delhi for the authentication of plant material.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]

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