Evaluation of the effect of Ferula asafoetida Linn. gum extract on learning and memory in Wistar rats.

OBJECTIVE: Memory loss is universal and is the first symptom to manifest in majority of the patients suffering from Alzheimer's disease. This study is designed to investigate the effect of Ferula asafoetida linn. (F. foetida) extract on learning and memory in rats.
MATERIALS AND METHODS: Learning and memory were evaluated using elevated plus maze and passive avoidance paradigm after the oral administration of two doses (200 mg/kg and 400 mg/kg) of F. foetida aqueous extract with rivastigmine as positive control. Brain cholinesterase activity, serum thiols and cholesterol were also estimated.
RESULTS: Extract produced significant improvement in memory score i.e. step through latency at 400 mg/kg dose in passive avoidance model (P< 0.05) and dose-dependent improvement of transfer latency in elevated plus maze model (P< 0.001). Dose-dependent inhibition of brain cholinesterase (P< 0.001) and significant improvement in antioxidant levels (P< 0.05) were also noted.
CONCLUSIONS: Memory enhancing potential of F. foetida can be attributed to acetylcholinesterase inhibiting and antioxidant properties. Hence, dietary usage of F. foetida is beneficial and can also be employed as an adjuvant to existing anti-dementia therapies.

Introduction

Dementia is a clinical syndrome characterized by a cluster of symptoms and signs manifested by difficulties in memory, disturbances in language, psychological and psychiatric changes, and impairments in routine activities. About 12 million people worldwide have dementia, and this is likely to increase by 2040 to 25 million. Alzheimer's disease (AD) accounts for nearly 50% of all cases of dementia. It affects about 6% of the population aged over 65 and increases in incidence with age.[1]

Cholinergic hypothesis of AD postulates that low synaptic levels of acetylcholine resulting from loss of cholinergic neurons in the nucleus basalis magnocellularis (NBM) leads to cognitive decline.[2] Apart from that, the distribution of brain acetylcholinesterase (AChE) is markedly altered with very little left in the normal axons and an intense activity in the neuritic plaques and neurofibrillary tangles.[3] This forms the basis for the symptomatic treatment of AD.

Oxidative stress increases with aging and may be related to AD and can be modified by diet. These observations imply the potentially important role of diet in the cause and prevention of AD and it has been a subject of extensive study.[4] There are reports of association of different types of dietary fats affecting the risk of AD.[5]

F. foetida (Family: Umbelliferae; common name:Asafoetida), a commonly used flavoring agent in Indian cuisine, is also known to age old system of medicine – Ayurveda. It is claimed to be a nervine stimulant.[6] It is claimed to have antispasmodic, hypotensive,[7] anticancer[8] and antidiabetic properties.[9] Oleoresin from the plant F. foetida commonly called as asafoetida is composed of volatile oil (4% to 20%), resin (40% to 60%), and gum (25%).[710] Glucuronic acid, galactose, arabinose and rhamnose have been isolated from the gum. Resin contains ferulic acid esters (60%), free ferulic acid (1.3%), coumarin derivatives – umbelliferone, foetidin and kamolonol and farnesiferoles A, B and C. Volatile oil has sulfur-containing compounds: disulfides, symmetric tri- and tetrasulfides.[11]

AChE inhibition by F. foetida has been demonstrated on snail nervous system and also on in-vitro assay.[12] Animal studies have shown that it also possesses antioxidant,[8] and cholesterol lowering properties.[13] To date, only AChE inhibitors and memantine have shown some demonstrable clinical efficacy in improving cognitive function in mild to moderate AD.[14] In view of these favorable properties, F. foetida aqueous extract was investigated for its effect on learning and memory using two experimental models – elevated plus maze (EPM) and passive avoidance (PA). Its effect on brain AChE activity, serum thiols and cholesterol was also assessed simultaneously.

Materials and Methods

Healthy male inbred albino rats of Wistar strain, weighing 200–300 g were used. The rats were maintained under a reverse photo cycle of 12 h day and 12 h night in temperature and humidity controlled environment with free access to food and water. All experiments were conducted between 9:00 am and 12:00 pm in a noise free environment. The study was approved by Institutional Animal Ethics Committee (Reg no. 94/99/CPCSEA).

Following drugs and chemicals were used: F. foetida gum (hard paste of light brown coloured, smells strongly, smeared with oily liquid), rivastigmine (Sun pharmaceuticals, Ind.ltd), DTNB (5’ 5’ dithio-2-nitrobenzoic acid), Sigma chemicals, St Louis, MO, USA, acetylthiocholine (ATC), Sigma chemicals, St Louis, MO, USA, reduced glutathione (GSH), sigma chemicals, St Louis, MO, USA, cholesterol kit (Aspen Laboratories Pvt. Ltd, Delhi), buffers and other reagents used were of analytical grade.

Method of Aqueous Extract Preparation

Hard paste of F. foetida of high purity was obtained from local ayurvedic dealer and its authenticity was confirmed by Dr. K Gopalkrishna Bhat, Professor of Botany, Poorna Prajna College Udupi. F. foetida(20 g) was soaked overnight in distilled water (100 ml) at room temperature and filtered for daily use.[7] Of the 20 g paste, 10 g was found to be insoluble in water. Thus, the final concentration of the extract was 100 mg/ml. The solution was stirred thoroughly before oral administration.

Acute Toxicity Study

The animals were grouped and fasted for 18 h prior to drug treatment. Extract was administered orally to group I to group VI in ascending and widely spaced doses viz. 10, 30, 100, 300, 1000 and 2000 mg/kg respectively. Rats were then observed periodically until 48 h for the signs of toxicity. Doses for the main study were selected on the basis of these results.

Experimental Protocol

Learning and memory was assessed with two behavioral paradigms viz. elevated plus maze and passive avoidance. Four study groups with eight animals in each were tested at the end of a treatment period of 15 days. Groups were as follows:

Group I (control): Equivolume of normal saline p.o.

Group II (200 mg): Aqueous extract of F. foetida 200 mg/kg p.o.

Group III (400 mg): Aqueous extract of F. foetida 400 mg/kg p.o.

Group IV (rivastigmine): Suspension of rivastigmine 5 mg/kg p.o.

Elevated plus maze

Utility of elevated plus maze in the assessment of learning and memory is well documented in literature.[15] The apparatus consists of two open arms (50 × 10 cm) and two closed arms (50 ×10 ×40 cm) extended from a central platform (10 ×10 cm). The maze is elevated to a height of 40 cm from the floor.

The experiment was performed in two stages. On day 1, each rat was placed at the end of an open arm facing away from the center. The time taken to enter any one of the closed arms was recorded as transfer latency (TL). All four legs inside the closed arm are counted as an entry. Cut off time allotted for each rat was 180 s. Those animals which did not enter the closed arms within cut off time were excluded from the study. Retention testing was conducted 24 h after the first trial (day 2) and transfer latency was recorded in a similar manner as mentioned before. Shortened transfer latency was considered as an index of improvement of memory.

Two Compartment Passive Avoidance Test

The apparatus consists of a square box with a grid floor (50 × 50 cm) and wooden walls of 35 cm height. This box was illuminated with a 100 W bulb placed 150 cm above the center of the compartment. In the center of one of the walls, there is an opening (6 × 6 cm) which can be opened or closed using a transparent plexy glass sliding door, which leads to a small dark compartment (15 × 15 cm).[16] This compartment is provided with an electrifiable grid floor which can be connected to a shock source and a removable ceiling.

The experiment was performed in three stages.[17] On the first day, the animal was placed at the center of the illuminated box facing away from the entrance to the small compartment. The door between the two compartments was kept open. The rat was allowed to explore the apparatus for 3 min and then returned to home cage. Twenty four hours later, the rat was placed in the illuminated chamber and time to enter the dark compartment was measured as step through latency (STL). Sliding door between the two compartments was closed as the rat entered dark chamber and unavoidable footshock (1.5 mA, 50 Hz, 2s) was delivered. The ceiling was opened and the rat was returned to home cage. Retention was tested after 24 h and STL was recorded. Cut off time allotted was 3 min. Increase in the STL was considered as an index of improvement of memory.

Collection of Samples

Soon after testing for retention of memory, blood was collected from retro-orbital vein under light ether anesthesia for estimation of serum cholesterol and protein thiols. The animals were then sacrificed. Immediately after decapitation, whole brain was carefully removed from the skull and kept in cold normal saline.

In vitro cholinesterase assay

The activity of brain AChE was measured according to Ellmans method with some modification.[1819] Rat brain was homogenized under 37°C in 0.1M KH2PO4 buffer (30 mg/ml rat brain wt/ml of buffer), at pH=8 and was kept frozen in an ice chest. The homogenate was then centrifuged at 3000 rpm for 10 min and the resultant cloudy supernatant was used for estimation of brain acetyl cholinesterase activity. A volume of 3 ml of phosphate buffer (pH=8) was added to the test tubes labeled as sample blank and test to which 200 μl of supernatant was added and the mixture was vortexed. Susequently DTNB was added to both the test tubes and incubated for 5 min at room temperature and absorbance was read at 412 nm using spectrophotometer and was set at zero absorbance when the enzyme activity had stopped increasing. Finally 20 μl of ATC was added to the test sample, and 20 μl of phosphate buffer (pH=8) to sample blank. Mixture was vortexed and absorbance was read at 412 nm for 10 min at 37°C at 1 min interval. The enzyme activity was calculated based on the changes in absorbance/min.

Total Protein Thiols

Serum total thiols were measured by spectrophotometric method using DTNB.[20] Na2HPO4 (900 μl of 0.2 M) containing 2 mM Na2 EDTA, 100 μl of serum and 20 μl of 10 mM DTNB in 0.2 M Na2HPO4 were taken in an Eppendorf tube and warmed to 37°C. The solution was mixed with a vortex mixer and the absorbance was measured at the end of 5 min at 412 nm. Simultaneously sample and reagent blanks were also prepared and their absorbance values were ascertained at 412 nm. Corrected absorbance value i.e. molar extinction coefficient 1600 M-1cm-1 was obtained from calibration curve produced using GSH dissolved in phosphate buffer saline (PBS). The absorbance of the sample and the reagent blanks were subtracted from the serum absorbance values. This value was then divided by molar extinction coefficient and the total thiol level was expressed as μmoles/L.

Serum Cholesterol Estimation

Total cholesterol was estimated by the ‘CHOD – POD (cholesterol oxidase–peroxidase) end point method’ using reagent kit (Aspen laboratories Pvt. Ltd).[21]

Statistical Analysis

The results are represented as mean ± SEM for expressing TL and STL, brain AChE activity, serum thiols and cholesterol level. One way ANOVA with Tukey post-hoc was applied wherever the data was homogenous; that means Levene's test for homogenity of variance showed insignificant results (P > 0.05). Wherever the data was non homogenous (Levene's test for homogenity of variance, P < 0.05), Welch ANOVA followed by Tamhane's T2 post hoc was applied. The analysis was carried out using SPSS version 14 for windows.

Results

No death or sign of toxicity was observed even at maximum dose of 2000 mg/kg body weight of F. foetida aqueous extract. Hence it was concluded to be safe. Thus, 1/10th of maximum safe dose i.e. 200 mg/kg was selected as the starting dose. A higher dose of 400 mg was also employed for further testing.

Effect on Transfer Latency Using Elevated Plus Maze

The rats treated with 200 and 400 mg/kg of F. foetida extract showed dose-dependent improvement in mean transfer latencies on acquisition day i.e. on day 1 indicating improvement in learning ability to some extent. However, it was not statistically significant [Table 1]. In retention performance i.e. on day 2, the mean latencies of the test groups were higher than that of control and the difference was statistically significant (P< 0.001). Although improvement of memory in F. foetida treated groups was not at par with rivastigmine group, it was qualitatively comparable.

Table 1

Effect of F. foetida extract on learning and memory

Effect on step through latency using passive avoidance paradigm

The results of acquisition and retention performance are depicted in Table 1. Mean STL on acquisition trial among the groups as compared to control was statistically insignificant. In retention performance, the mean STL of 200 mg, 400 mg and rivastigmine groups were 36.12, 60.12, 162.37 s respectively. Unlike in EPM model, significant improvement in memory was noted only at 400 mg/kg dose of F. foetida extracts compared to control (P< 0.001). However there was a highly significant (P< 0.001) improvement of memory in 400 mg group as compared to 200 mg in a same way as in EPM model. Rivastigmine showed highly significant improvement as compared to control (P < 0.001). Although memory improvement in 400 mg group was qualitatively comparable to rivastigmine group, it was not supported by statistical significance.

Effect on Acetylcholinesterase Enzyme Activity (Unit - Moles 0f Substrate Hydrolyzed × 10-6/min/g of Brain Tissue)

Extract-treated groups (200 and 400 mg) inhibited brain acetylcholinesterase enzyme significantly (P< 0.001) as compared to control. This inhibition was dose dependent and statistically significant (P < 0.05) in EPM model. This was also observed in the passive avoidance model (1.636 vs. 1.071) (P< 0.05). Effect on enzyme activity is depicted in Figures 1 and 2.

Figure 1

Effect of F. foetidaextract on acetylcholinesterase activity (EPM model)

Figure 2

Effect of F. foetida extract on acetylcholinesterase activity (PA model)

Effect on Serum Protein Thiols

F. foetida extract (400 mg/kg) consistently increased thiols (antioxidants) in serum as compared to control when measured on two different occasions (EPM and PA testing) and was statistically significant [Figures 3 and 4]. However, rivastigmine did not show any such increase in antioxidant level.

Figure 3

Effect of F. foetidaextract on serum thiols (EPM model)

Figure 4

Effects of F. foetidaextract on serum thiols (PA model)

Effect on Serum Cholesterol

Mean values of serum cholesterol in rats treated with F. foetida extract were similar to control [Table 2].

Table 2

Effect of different doses of F. foetida extract on serum cholesterol levels

Discussion

The management of age-related neurodegenerative diseases such as Alzheimer's disease is one of the greatest challenges faced by this century, owing to the complexity of its pathology and the availability of countable number of drugs which have limited benefit in long run. With increasing emphasis on dietary factors in recent times either in the prevention and/or progression of neurodegenerative diseases, it is worthwhile to take a note of the beneficial role of spices (turmeric, curry leaves, ginger, nutmeg etc) which are used generously in Indian dishes. This may partly explain the reason for the low incidence of AD in India (4.4 fold less in age group of 70- 79 yrs compared to their US counterparts).[22] Moreover, the cholinergic hypothesis of AD postulates that low synaptic levels of acetylcholine resulting from loss of cholinergic neurons in the nucleus basalis magnocellularis lead to cognitive decline.[2] Based on this, strategies for increasing synaptic levels of ACh have been widely explored in the development of antidementia drugs. One such strategy is blockade of synaptic degradation of ACh through the inhibition of acetylcholinesterase by donepezil, rivastigmine and galantamine and other agents that act by similar mechanisms. Reports reveal that F. foetida has AChE inhibiting property as evidenced by in-vitro assay and in vivo action on snail nervous system.[12] No study has been conducted so far to demonstrate similar effects in rats, which is in fact a useful property when it comes to the treatment of AD.

F. foetida/Asafoetida, commonly known as “hing,” is routinely added as a flavoring agent in Indian cuisine. The present study is undertaken to evaluate the effect of aqueous extract of F. foetida on learning and memory performance in rats using two models namely, two compartment passive avoidance test and elevated plus maze test and we simultaneously assessed its effect on brain AChE activity, serum cholesterol and total thiols.

The latency to enter the dark compartment (STL) and closed arm (TL) was taken as a parameter for assessment in experimental models. The rationale being, if the drug has positive effect on learning, it would be reflected as decrease in latency to enter dark compartment/closed arm. However, interpretation of TL/STL in retention trial is different in both the models, in that, decreased TL in EPM model is inferred as improvement in memory, whereas increased STL after shocking the animal in PA model is interpreted as memory enhancement.

Responses of two different doses of the extract (200 and 400 mg/kg) were compared against control and standard (rivastigmine) in both the models. Improvement was evident in extract treated and standard groups in acquisition trial, which suggests an enhancement of learning though it was not statistically significant.

In EPM model, retention trial, a dose-dependent improvement in memory was seen in extract-treated group as compared to control. In passive avoidance model, unlike in EPM model, the extract didn′t show any significant improvement in memory with lower dose (200 mg). However, higher dose (400mg) improved memory significantly compared to control group. This can be explained by a relatively high variability of this model; which could have been due to even a smallest error at some important stage of the experiment.[23] In both the models, even though the improvement in memory was qualitatively comparable to rivastigmine group, it was statistically insignificant.

Results of the study indicate that, the extract of F. foetida has memory enhancing effect as noted by changes in the TL/STL in retention trials. Inhibition of cholinestrase by F. foetida as reported earlier[12] was also demonstrated in our study and it was dose dependent. F. foetida exhibited a consistent enzyme inhibition activity when measured on two separate occasions. This can be taken as an indirect evidence of its ability to cross blood brain barrier effectively. However, the ability to inhibit AChE was found to be inferior to standard drug rivastigmine. These biochemical observations further strengthen our earlier findings of dose-dependent improvement in memory. This property could be attributed to the active principles ferulic acid and umbelliferone present in F. foetida.[12]

Oxidative stress has long been thought to play a major role in the pathogenesis of AD.[24] Certain plant products and diet rich in antioxidants are said to be neuroprotective and hence may have a role in improving cognition in aging and neurodegenerative diseases.[2225] F. foetida consistently increased total protein thiols (antioxidants) in serum at 400 mg dose. This finding is in agreement with earlier reports which demonstrated F. foetida's antioxidant potential and inhibition of lipid peroxidation in rats.[8] The protective effect of F. foetida extract may be partly attributed to its antioxidant property by virtue of which brain cells are subjected to less oxidative stress resulting in less brain damage and improved neuronal function, thereby enhancing memory. Besides, there are reports suggestive of inability of rivastigmine alone to improve total antioxidant status in rat brain although it improves learning performance, which may be solely due to AChE inhibition. In addition to that, rivastigmine attenuated the beneficial effect of selegeline on total antioxidant status when given as a combination therapy.[26] Since F. foetida produces AChE inhibition besides possessing antioxidant property, the combined effect may be advantageous over a drug that targets only one pathogenic mechanism.

Epidemiological studies have found positive correlations between hypercholesterolemia and development of AD.[27] In our study, F. foetida did not decrease serum cholesterol level suggesting no hypocholesterolemic effect. This is in contradiction with earlier reports of hypocholesterolemic effect of F. foetida wherein it was shown to increase fecal excretion of cholesterol and decrease cholesterol absorption in rats but there was no direct measurement of serum cholesterol as such.[6] However according to the current NIH state-of-the science conference consensus report, treatment with cholesterol lowering drugs, statins, may not offer any cognitive benefit.[28]

To the best of our knowledge, this is the first study of its kind where F. foetida has been evaluated for learning and memory enhancing potential in rats. It is evident from the above findings that the beneficial effects of F. foetida on memory can be attributed to facilitation of cholinergic transmission due to inhibition of AChE in rat brain, and partly due to its boosting effect on endogenous antioxidant system. Hence dietary usage of F. foetida would be beneficial and it can also be added as an adjuvant to existing therapies for the treatment of dementia. However, studies employing other memory models, with large sample size and characterization of active principles will be required to prove the benefits conclusively.