AD individuals exhibit deterioration in mental functions rendering them incapacitated to perform normal daily activities. However, evidence shows that AD can also afflict young individuals as early as 40 years of age (Sugimoto et al., 2002). Neuritic plaques (consisting of a core of β- amyloid aggregates covered by dead neurons, microglia and apolipoprotein E) and neurofibrillary tangles are the major pathological hallmarks of an Alzheimer brain (Selkoe, 2001). Cholinergic drugs such as Donepezil^® improve learning, memory and attention. The non-cognitive aspects of dementia however are linked to serotonin and dopamine rather than acetylcholine because these neurotransmitter systems most probably influence mood, emotional balance and psychosis (Zhang et al., 2004).

There has been a steady rise in the number of patients suffering from Alzheimer’s disease (AD) all over the world. There are around 30 million patients suffering from Alzheimer’s disease all over the world, out of which United States of America alone has around 4.5 million patients (Hebert et al., 2003), despite the severity and high prevalence of this disease. Therefore, neurobiologists all over the world are looking for new directions and alternative strategies for managing this disease of senior citizens. In India AD patents are estimated to be less than 3 million (Shaji, 2005). These prevalence figures however, point out that the number of patients suffering from AD are considerably small in India when compared to USA.

Nootropics are a class of psychotropic agents with selective facilitatory effect on integrative functions of the central nervous system, particularly on intellectual performance, learning capability and memory (Plan et al., 1996; Reddy et al., 1998). Piracetam, the first representation of a class of nootropic agents, has been shown to improve memory deficits in geriatric individuals. Repeated injections of piracetam had improved learning abilities and memory capacities of laboratory animalss(Bhattacharya et al., 1993).

Acetylcholine is considered the most important neurotransmitter involved in the regulation of cognitive functions. Cognitive dysfunction has been shown to be associated with reduced cholinergic transmission and the facilitation of central cholinergic transmission with improved memory (Bhattacharya et al., 1993; Finucane et al., 2006). Selective loss of cholinergic neurons and decrease in cholinacetyl transferase activity was reported to be a characteristic feature of senile dementia of the Alzheimer’s type (Agnolli et al., 1983; Foster, 2006). There are extensive evidences linking the central cholinergic system to memory (Fodale et al., 2006; Nordberg, 2006; Ghelardini et al., 1998; Peng et al., 1997; Olney, 1990).

Anticholinesterases such as Metrifonate (Ringman et al., 1999), Physostigmine, Tacarine, Donepezil (Sugimoto et al., 2002), Huperzine-A (Bai et al., 2000), Rivastigmine (Potkin et al., 2001), Galanthamine (Sramek et al., 2000) and Eptastigmine (Braida et al., 2000) have all been shown to reverse amnesia produced by disruption of cholinergic system. Enzyme choline acetyltransferase is involved in the synthesis of acetylcholine and acetylcholinesterase is involved in the degradation of acetylcholine. In the present study,

Plant extracts of Zingiber officinale (Joshi et al., 2006), Nardostachys jatamansi (Joshi et al., 2006a), Foeniculum vulgare (Joshi et al., 2006b), Hibiscus sabdariffa (Joshi et al., 2006), Ocimum sanctum (Joshi et al., 2006), and Desmodium gangeticum (Joshi et al., 2006), Piper nigrum (Joshi et al., 2006), Glycyrrhiza glabara (Parle et al.,2003, 2004) have been found to posses nootropic effects and they had significantly lowered the whole brain AChE activity thereby elevating acetylcholine levels in the brain.

MEMORHIS is a poly-herbal formulation comprising of the herbal ingredients and pharmaceutical adjuvants. The plant extracts used for formulating this preparation were selected since they had exhibited very promising cognition improving effects in mice. This suspension was prepared in our research laboratory using plant extracts of N. jatamansi, O. sanctum, A. racemosus, P. nigrum, M. elengi, P. niruri, G. glabra. Other ingredients of the preparations were ascorbic acid, cardamom oil, methyl paraben, propyl paraben, propylene glycol, sodium carboxy methyl cellulose and purified water.

METHODS:

Preparation of poly-herbal formulation-MEMORHIS

MEMORHIS suspension was prepared using lyophilized extracts of N. jatamansi, O. sanctum, A. racemosus, P. nigrum, M. elengi, P. niruri, G. glabra, and other ingredients were ascorbic acid, cardamom oil, methyl paraben, propyl paraben, propylene glycol, sodium carboxy methyl cellulose and purified water. Each 5 ml of MEMORHIS contained lyophilized extracts of N. jatamansi (20 mg), O. sanctum (20 mg), A. racemosus (20 mg), P. nigrum (5 mg), M. elengi (20 mg), P. niruri (10 mg), G. glabra (10 mg), ascorbic acid (5 mg) and cardamom oil (Q.S.).

Acute toxicity studies

Acute toxicity studies were performed according to OECD/OCDE guidelines (Ecobichon, 1997). Male Swiss mice selected by random sampling technique were employed in this study. The animals were fasted for 4 h with free access to water only. All the plant extracts were administered orally at a dose of 5 mg/kg initially. During the first four hours after the drug administration, the animals were observed for gross behavioral changes if any for 7 days. The parameters such as hyperactivity, grooming, convulsions, sedation, hypothermia and mortality were observed. If mortality was observed in two out of three animals, then the dose administered was considered as toxic dose. However, if the mortality was observed in only one animal out of three animals then the same dose was repeated again to confirm the toxic effect. If no mortality was observed, then only higher doses of test drugs were employed for further toxicity studies.

Laboratory models for testing memory

Elevated plus Maze

The elevated plus maze served as the exteroceptive behavioral model (wherein the stimulus existed outside the body) to evaluate learning and memory in mice. The apparatus consisted of two open arms (16 cm x 5 cm) and two covered arms (16 cm x 5 cm x 12 cm). The arms extended from a central platform (5 cm x 5 cm), and maze was elevated to a height of 25 cm from the floor. On the first day, each mouse was placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was taken as the time taken by the mouse to move into one of the covered arms with all its four legs. TL was recorded on the first day. If the animals did not enter into one of the covered arms within 90 sec., it was gently pushed into one of the two covered arms and the TL was assigned as 90 sec. The mouse was allowed to explore the maze for additional 10 sec and then returned to its home cage. Memory retention was examined 24 h after the first day trial i.e. on the second day (Parle et al., 2004a; Itoh et al., 1990).

Passive shock avoidance paradigm

Passive avoidance behavior based on negative reinforcement was used to examine the long-term memory. The apparatus consisted of a box (27 X 27 X 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 X 7 X 1.7 cm) in the center of the grid floor. The box was illuminated with a 15 W bulb during the experimental period. Electric shock (20V AC) was delivered to the grid floor. Training was carried out in two similar sessions. Each mouse was gently placed on the wooden platform set in the center of the grid floor. When the mouse stepped down from the wooden platform set in the center of the grid floor and placed all its paws on the grid floor, shocks wee delivered for 15 sec and the step down latency (SDL) was recorded (Parle et al., 2004). SDL was defined as the time taken by the mouse to step down from wooden platform to grid floor with its entire paw on the grid floor. Animalss showing SDL in the range (2-15 sec) during the first test were used for the second session and the retention test. The second-session was carried out 90 min after the first test. When the animalss stepped down before 60 sec, electric shocks were delivered for 15 sec. During the second test, animalss were removed from shock free zone, if they did not step down for a period of 60 sec. Retention was tested after 24 h in a similar manner, except that the electric shocks were not applied to the grid floor (Parle et al., 2003).

Estimation of brain acetyl cholinesterase (AChE) activity

The animals were euthanized by cervical dislocation carefully to avoid any injuries to the tissue. The whole brain AChE activity was measured using the Ellman method (Ellman et al., 1961). This was measured on the basis of the formation of yellow color due to the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylcholine iodide in the presence of tissue cholinesterase was measured using a spectrophotometer. The sample was first treated with 5, 5’-dithionitrobenzoic acid (DTNB) and the optical density (OD) of the yellow color compound formed during the reaction at 412 nm every minute for a period of three minutes was measured. Protein estimation was done using Folin’s method. AChE activity was calculated using the following formula:

R= d O.D. X Volume of Assay (3 ml)

E X mg of protein

Where,

R= rate of enzyme activity in ‘n’ mole of acetylcholine iodide hydrolyzed / min / mg protein

d O.D.= Change in absorbance / minute

E = Extinction coefficient = 13600 / M / cm

Statistical analysis

All the results were expressed as mean ± standard error (SEM). The data was analyzed using one-way ANOVA followed by Tukey Kramer’s test. P values <0.05 were considered as statistically significant.

RESULTS:

Acute toxicity studies: All the doses (5, 50, 250, 500 and 2000 mg/kg, p.o.) of MEMORHIS (MEM) did not produce any mortality even with the highest dose (2000 mg/kg, p.o.) employed. Three submaximal doses (50, 100 and 250 mg/kg, p.o.) were selected for further psychopharmacological and biochemical studies.

Effect on transfer latency using elevated plus maze: MEMORHIS (50, 100 and 200 mg/kg, p.o.) showed dose-dependent reduction in TL of 8^th day and 9^th day, indicating remarkable improvement in learning ability and memory of the young and aged mice as compared to respective control groups (Fig. 1). Diazepam (1 mg/kg, i.p.) and scopolamine (0.4 mg/kg, i.p.) significantly increased (P < 0.01) the TL of 9^th day indicating impairment in memory (amnesia). MEMORHIS (100 and 250 mg/kg, p.o.) successfully (P< 0.001) reversed the amnesia induced by both diazepam and scopolamine (Fig. 2).

Effect on step down latency using passive avoidance paradigm.

MEMORHIS (50, 100 and 250 mg/kg, p.o.) administered to young and aged mice for consecutive 8 days, showed dose-dependent increase in SDL values as compared to respective control groups (Fig. 3). MEMORHIS (50, 100 and 250 mg/kg, p.o.) also exhibited reversal of amnesia induced by diazepam and scopolamine in young mice (Fig. 4).

Effect on brain cholinesterase activity.

MEMORHIS (50, 100 and 250 mg/kg, p.o.) showed a remarkable reduction in the brain acetyl cholinesterase activity in young and aged mice, as compared to respective control groups. Whereas, phenytoin (12 mg/kg, p.o.) significantly (P< 0.01) increased the acetyl cholinesterase activity. Piracetam (200 mg/kg, i.p.) was the standard nootropic agent employed (Fig. 5).

DISCUSSION:

Memory function is vulnerable to a variety of pathologic processes including neurodegenerative diseases, strokes, tumors, head trauma, hypoxia, cardiac surgery, malnutrition, attention deficit disorder, depression, anxiety, the side effects of medication, and normal ageing (Newman et al., 2001; Mesulam, 2000). As such, memory impairment is commonly seen by physicians in multiple disciplines including neurology, psychiatry, medicine, and surgery. Memory loss is often the most disabling feature of many disorders, impairing the normal daily activities of the patients and profoundly affecting their families.

The ancient Ayurvedic physicians had understood the delicate cellular mechanisms of the body and the deterioration of the functional efficiency of the body tissues. These Ayurvedic scientists had thus developed certain dietary and therapeutic measures to delay the ageing process, while rejuvenating functional dynamics of the body organs. This revitalization and rejuvenation is known as the ‘rasayana chikitsa’ (rejuvenation therapy) (Govindarajan et al., 2005). Rasayana drugs act inside the human body by modulating the nuero-endocrino-immune systems and have been found to be a rich source of antioxidants (Brahma et al., 2003). Brahmi rasayana, Trikatu churna were reported to exhibit significant decrease in AChE activity in whole brain homogenates of mice, indicating their anti-cholinesterase potential (Joshi et al., 2006). They had also reversed diazepam, scopolamine and ageing-induced impairment in learning and memory in mice (Joshi et al., 2005).

Glycyrrhiza glabra and ascorbic acid were proved to be memory enhancers in earlier studies (Parle et al., 2004; 2003) from our laboratory. MEMORHIS successfully reversed scopolamine, diazepam or ageing-induced amnesia, when administered for successive 8 days. Piracetam, the established nootropic agent was used in the present study for comparison because, it improves memory as a net result of several protective actions such as increased resistance to adverse conditions, brain protection against physical and chemical injuries and enhancement of reserve energy stores. Piracetam also increased choline uptake in cholinergic nerve endings, thereby facilitating cholinergic transmission in brain (Wu et al., 1994; Parson et al., 1993). Piracetam elevated the density of frontal cortex acetylcholine receptors by 30-40%, restoring the levels of acetylcholine in the brain (Balaraman et al., 2002).

MEMORHIS exhibited highly significant anticholinesterase activity in both and young and aged mice. Thus, it is possible that enhanced cholinergic transmission resulting from increased acetylcholine synthesis in brain due to abundant availability of choline and reduction of brain cholinesterase activity in young and aged mice may explain the memory improving effect exhibited by MEMORHIS. Hence, MEMORHIS can be of enormous use in the preliminary management of early symptoms of cognitive dysfunctions such as Alzheimer’s disease and dementia. Further investigations using human volunteers are warranted for further confirmation of nootropic potential. The possible involvement of other neurotransmitters like glutamate, GABA, catecholamines, serotonin etc. in the pathogenesis of cognitive disorders.

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Received on 20.01.2012

Modified on02.02.2012

Accepted on 12.02.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(2): March-April 2012, 97-103