Nootropic, anxiolytic and CNS-depressant studies on different plant sources of shankhpushpi

Abstract

Context: Shankhpushpi, a well-known drug in Ayurveda, is extensively used for different central nervous system (CNS) effects especially memory enhancement. Different plants are used under the name shankhpushpi in different regions of India, leading to an uncertainty regarding its true source. Plants commonly used under the name shankhpushpi are: Convolvulus pluricaulis Chois., Evolvulus alsinoides Linn., both from Convolvulaceae, and Clitoria ternatea Linn. (Leguminosae).

Objective: To find out the true source of shankhpushpi by evaluating and comparing memory-enhancing activity of the three above mentioned plants. Anxiolytic, antidepressant and CNS-depressant activities of these three plants were also compared and evaluated.

Materials and methods: The nootropic activity of the aqueous methanol extract of each plant was tested using elevated plus-maze (EPM) and step-down models. Anxiolytic, antidepressant and CNS-depressant studies were evaluated using EPM, Porsolt’s swim despair and actophotometer models, respectively.

Results: C. pluricaulis extract (CPE) at a dose of 100 mg/kg, p.o. showed maximum nootropic and anxiolytic activity (p < 0.001). E. alsinoides extract (EAE) and C. ternatea extract (CTE) showed maximum memory-enhancing and anxiolytic activity (p < 0.001) at 200 and 100 mg/kg, respectively. Amongst the three plants, EAE and CTE showed significant (p < 0.05), while CPE did not exhibit any antidepressant activity. All the three plants showed CNS-depressant action at higher dose levels.

Discussion and conclusions: The above results showed all the three plants possess nootropic, anxiolytic and CNS-depressant activity. The results of memory-enhancing activity suggest C. pluricaulis to be used as true source of shankhpushpi.

Keywords::

• Ayurveda
• Convolvulus pluricaulis
• Clitoria ternatea
• Evolvulus alsinoides

Introduction

Epidemiological transitions have led to the change in the global burden of illness. Several factors have contributed to this change, including improvements in maternal and child health, increasing age of population, and newly recognized disorders of the nervous system. It is now evident that neurologic disorders have emerged as priority health problems worldwide. This is reflected in the Global Burden of Disease Study, jointly published by the World Health Organization and other groups. The proportionate share of the total global burden of disease resulting from neuropsychiatric disorders is projected to rise to 14.7% by 2020. Although neurologic and psychiatric disorders comprise only 1.4% of all deaths, they account for a remarkable 28% of all age groups living with a disability (Menken et al., 2000). In the developing countries, changing lifestyle, poverty and aging are considered as three main determinants for the increase in the burden of mental disorders (Brundtland, 2001). The rising average life expectancy is increasing the number of elderly patients suffering from geriatric diseases particularly dementia which is characterized by impaired memory and other cognitive disabilities (Davis, 2002). It is estimated that within the next 50 years approximately 30% of the population will be aged 65 years or older, and six million of those between 75 and 84 years of age, will exhibit some form of Alzheimer’s disease (AD) symptoms (Bradford & Gupta, 2005).

According to Ayurveda, the traditional Indian System of Medicine, AD is an imbalance of vata, pitta and kapha. Medhya Rasyanas (drugs which act as nervine tonic) such as shankhpushpi, Brhami [Bacopa moniera Wettst. (Scrophulariaceae)], Vacha [Acorus calamus Linn. (Acoraceae)] and Jyotishmati [Celastrus paniculatus Willd. (Celastraceae)] are beneficial in cognitive disorders (Joshi & Parle, 2006). ‘Charak Samhita’ has described shankhpushpi as one of the best Medhya rasayana or brain tonic (Sharma, 2001; Chunekar & Pandey, 2002). It has been used traditionally for its memory-enhancing, anticonvulsant, antianxiety and sedative properties. However, different plants are being used under the name shankhpushpi in different parts of India. The plants commonly used under the name shankhpushpi in different regions of India are Convolvulus pluricaulis Chois. (syn. Convolvulus prostratus, Convolvulus microphyllus) and Evolvulus alsinoides Linn. (Convolvulaceae) and Clitoria ternatea Linn. (Leguminosae) (Pillai, 1976; Aulakh et al., 1988; Handa & Chakaraborti, 1989). Despite being a very popular drug, there has been no authentic information available as to which plant is the true source of shankhpushpi. In one report, a survey of crude drug samples of shankhpushpi from various pharmaceutical companies showed that nine samples were of C. pluricaulis, one sample consisted of E. alsinoides, one was mixture of these two, and others were comprised of some different plants (Singh & Vishwanathan, 2001). In an attempt to find out the true source of shankhpushpi, and to validate its traditional uses, the above said three plants were studied for their nootropic activity. These plants were also studied and compared on the basis of their effect on anxiety, depression and spontaneous locomotor activity.

Materials and methods

Plant material

Whole plant of C. pluricaulis and E. alsinoides were collected from different places in Chandigarh, Punjab and Khurampur, District Saharanpur, Uttar Pradesh, India in June 2008, respectively. Roots of C. ternatea were procured from Tirunelveli, Tamil Nadu, India in August 2008. The identity of each plant material was confirmed by Dr. S. Nautiyal, Forest Research Institute, Dehradun, Uttaranchal, India with certificate number 765/2008-Bot/15-1. The three plants, C. pluricaulis, C. ternatea and E. alsinoides, respectively, were deposited for record in the Museum-cum-Herbarium of University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India, with voucher specimen nos. 1454, 1455 and 1456.

Animals

Laca mice weighing 25–30 g, of either sex were procured from the Central Animal House of Panjab University, Chandigarh. The animals were allowed standard laboratory feed and water ad libitum under standard laboratory conditions. For each study, animals were divided into different groups, viz, control, test groups of different doses, and standard group. Animals were acclimatized to laboratory environment daily for 1 h for 10 days, prior to the experiment. All the experimental protocols were approved by animal ethical committee, Panjab University, Chandigarh, which complies with the international norms of Indian National Science Academy.

Extraction of plant material and phytochemical screening

The plant material was dried in shade and ground to a coarse powder. Powder of each drug (1 kg) was macerated three time for 48 h each with 80% aqueous methanol at room temperature. The extracts after filtration were dried under reduced pressure. The aqueous methanol extract of each plant was then subjected to phytochemical screening (Evans, 2002).

Dose preparation and drugs used

All the doses were prepared by suspending the extracts and standard drugs in 3% v/v aqueous solution of Tween 80. Each plant extract was evaluated for nootropic, anxiolytic and antidepressant studies at four oral dose levels of 50, 100, 200 and 400 mg/kg. To evaluate their effect on the spontaneous locomotor activity they were tested at dose levels of 100, 200, 400 and 600 mg/kg. Piracetam (Uni-UCB, Mumbai, India), diazepam and imipramine (Sigma, New Delhi, India), and scopolamine hydrobromide (Buscpon-Boehringer Ingelheim, Germany) were used in this study. Solvents used in this study were of analytical grade.

Drug treatment

The animals of control and test group were given vehicle (3% aqueous solution of Tween 80) and different doses of plant extracts, respectively. The dosing was done for 30 days for studying the effect on memory. Piracetam, a standard memory enhancer, was administered orally at a dose of 100 mg/kg for 30 days (Une et al., 2001). The last dose was administered 45 min before the training on day 30. Scopolamine hydrobromide (3 mg/kg, p.o.), a standard amnesic drug, was administered 30 min before training (Verloes et al., 1988; Das et al., 2002). This dosing protocol was used for assessing memory-enhancing/nootropic activity in mice. The remaining activities were evaluated by using single dose of the vehicle, standard drugs and different test extracts administered orally, 45 min before the test. Diazepam, at doses of 2 and 10 mg/kg (Ali et al., 1995; Shri et al., 2010), was used as standard anxiolytic and CNS-depressant, respectively. Imipramine (12.5 mg/kg, p.o.) was used as a standard antidepressant drug (Kalariya et al., 2010).

Nootropic/memory-enhancing activity

Nootropic or memory-enhancing activity was assessed using elevated plus-maze (EPM) and step-down passive avoidance models.

EPM model

An EPM consisting of two open and two closed arms, placed at a height of 50 cm, was employed for memory testing in mice (Itoh et al., 1991). On day 30, each mouse was placed at the end of the open arm facing away from the central platform and enclosed arms. Time taken by the mice to enter the enclosed arm, with its all the four paws inside, was recorded as transfer latency (TL). TL was again recorded after 24 h (retention test), at the same time period of the day. Decrease in the TL indicates the memory-enhancing property of the test drug.

Step-down passive avoidance model

Passive avoidance behavior based on negative reinforcement was used to examine the memory-enhancing property (Kameyama et al., 1986). The apparatus consisted of a rectangular box (50 × 50 × 40 cm) with electrifiable grid floor, connected to an electric source to provide scramble foot shock. On day 30, 45 min after the last dose administration, each mouse was gently placed on a wooden platform (4 × 4 × 4 cm) in the centre of the box. The time taken by the mouse to step-down was recorded as step-down latency (SDL). As soon as mouse steps down with all its four paws on the grid, an electric shock of 0.5 mA was delivered for 5 s following which the mouse was returned to its cage immediately. Retention was tested after 24 h in a similar manner, except that the electric shocks were not applied to the grid floor. SDL was recorded, with an upper cut-off time of 300 s.

Anxiolytic activity

Anxiolytic activity was evaluated using EPM model (Pellow & File, 1986). An EPM consisted of two open arms (16 × 5 cm) and two closed arms (16 × 5 × 12 cm) having an open roof and similar arms faced each other. The maze was elevated to a height of 50 cm from ground level. The animal was placed in the centre of the plus-maze with its head facing open arm. During the next 5-min observation, the behavior of the animal was recorded as the number of entries and time spent in open arms. An entry into an arm was recorded only when the animal with all its four paws, crossed the line marking the entry of an arm.

Antidepressant activity

Antidepressant activity was evaluated using Porsolt’s swim despair test (Dhingra & Sharma, 2005). The mouse was forced to swim in a glass jar (25 × 12 × 25 cm) containing water to a height of 15 cm at room temperature (22 ± 1°C). After an initial 2-min period of vigorous activity to escape, the animal assumed a typical immobile posture (ceased to struggle and made minimal limb movements just sufficient to keep the in head above the water level). More often, animals continuously struggle in bouts interspersed by a period of immobility. The total immobility was noted for the next 4 min during a 6-min test session. To remove bias, the investigator recording the immobility period, was unaware of the treatment schedule. The decrease in the immobility period in comparison to the control group animals indicated antidepressant activity.

Spontaneous locomotor/CNS-depressant activity

Effect on locomotor activity was measured in an actophotometer (Ganachari et al., 2004), with slight modification in the method. The apparatus consisted of a square chamber with grid floor, fitted with six light beams on the side walls near to floor connected to photocells on opposite walls. Whenever a light beam was obstructed by the movement of animal, it produced a count, which was automatically recorded on the digital counter. The units of the activity are arbitrary and based on beam obstructions resulting from the movement of animal. The locomotor activity was measured for a period of 5 min beginning 45 min after the administration of various doses of the plant extracts.

Statistical analysis

Results were expressed as mean ± SD. The inter group variation was measured by one-way analysis of variance followed by Tukey’s test. Statistical significance was considered at p < 0.05, 0.01 and 0.001. The statistical analysis was done using Jandel Sigma statistical software version 2.0 (Jandel Corporation, San Rafel, California, USA).

Results

Phytochemical screening

The yield (w/w) of 80% aqueous methanol extract of C. pluricaulis (CPE), E. alsinoides (EAE) and C. ternatea (CTE) was 10, 10 and 11%, respectively. The results for the phytochemical screening in Table 1 clearly showed that all the three plants showed similar profiles. Flavonoids, steroids and carbohydrates were present, while anthraquinone and cardiac glycosides were absent, in all the three plants, respectively. Further, CPE showed absence of alkaloids while tannins were absent in CTE.

Table 1.  Phytochemical screening of Convolvulus pluricaulis, Evolvulus alsinoides and Clitoria ternatea.

Phytochemical group	Screening result
	C. pluricaulis	E. alsinoides	C. ternatea
Alkaloids	−	+	+
Carbohydrates	+	+	+
Triterpenoids/steroids	+	+	+
Flavonoids	+	+	+
Anthraquinone glycosides	−	−	−
Cardiac glycosides	−	−	−
Tannins and phenolics	+	+	−

+Indicates presence of a phytoconstituent.

−Indicates absence of a phytoconstituent.

Memory-enhancing/nootropic activity

The three most commonly used plants under the name shankhpushpi were compared for the memory-enhancing activity in order to determine the true source of the drug. The memory-enhancing property of the aqueous methanol extracts of the three plants was evaluated at doses of 50, 100, 200 and 400 mg/kg, using EPM and step-down passive avoidance model.

In EPM model, all the three plants possess memory-enhancing activity (Figure 1–3). CPE showed memory-enhancing activity at all the tested dose levels (100 mg/kg, p < 0.01; 50, 200 and 400 mg/kg, p < 0.05) when compared with control group (Figure 1). EAE treated animals showed significant results at 200 (p < 0.01) and 400 mg/kg (p < 0.05) when compared with vehicle-treated animals (Figure 2). CTE showed activity at 100 (p < 0.01) and 200 mg/kg (p < 0.05) in comparison to control group (Figure 3). CPE and CTE (100 mg/kg, p < 0.01) and EAE (200 mg/kg, p < 0.01) showed maximum activity when compared to vehicle-treated group. EAE at dose of 50 and 100 mg/kg, and CTE at 50 and 400 mg/kg did not produce significant (p < 0.05) memory-enhancing effect. Among the three plants, maximum activity (reduction in TL when compared to their training-day latency) was shown by CPE (70%) followed by EAE (66%) and CTE (65%). The control group animals showed 42, 43 and 43% reduction in TL, respectively, with respect to their training-day latency.

Figure 1.  Effect of hydromethanol extract of Convulvulus pluricaulis (CPE) at various dose levels on the transfer latency of mice in elevated plus-maze. Ordinates express mean transfer latency, in seconds. *p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus piracetam; ^bp < 0.05 versus scopolamine. Results are compared by one-way analysis of variance followed by Tukey’s test (n = 5 per group). PIR, piracetam (100 mg/kg, p.o.); SCP, scopolamine (3 mg/kg, p.o.).

Figure 2.  Effect of hydromethanol extract of Evolvulus alsinoides (EAE) at various dose levels on the transfer latency of mice in elevated plus-maze. Ordinates express mean transfer latency, in seconds. *p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus piracetam; ^bp < 0.05 versus scopolamine. Results are compared by one-way analysis of variance followed by Tukey’s test (n = 5 per group). PIR, piracetam (100 mg/kg, p.o.); SCP, scopolamine (3 mg/kg, p.o.).

Figure 3.  Effect of hydromethanol extract of Clitoria ternatea (CTE) at various dose levels on the transfer latency of mice in elevated plus-maze. Ordinates express mean transfer latency, in seconds. *p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus piracetam; ^bp < 0.05 versus scopolamine. Results are compared by one-way analysis of variance followed by Tukey’s test (n = 5 per group). PIR, piracetam (100 mg/kg, p.o.); SCP, scopolamine (3 mg/kg, p.o.).

In the step-down passive avoidance model, CPE at dose levels of 100 and 200 mg/kg profoundly increased the SDL. The results at 100 mg/kg were comparable to piracetam and were significantly different (p < 0.001) from vehicle-treated groups (Table 2). Furthermore, CPE also showed significant effects (p < 0.001 and p < 0.01) at the higher dose levels of 200 and 400 mg/kg, respectively, in comparison to control. EAE at doses of 200 and 400 mg/kg (Table 3) and CTE at doses of 100 and 200 mg/kg showed significant activity (p < 0.001) in comparison to the control group of animals, but was not as much as that of piracetam.

Table 2.  Effect of 80% aqueous methanol extract of Convolvulus pluricualis (CPE) on the step-down latency.

Group	Dose (mg/kg)	Mean step-down latency (s)^n ± SD
		On day 30	On day 31
Control/vehicle	–	4.0 ± 1.26	53.3 ± 6.21
Piracetam	100	5.0 ± 1.67	233.1 ± 33.87***
Scopolamine	3	5.0 ± 1.78	24.3 ± 8.50*^a
CPE	50	5.0 ± 1.78	47.0 ± 7.87^ab
	100	5.8 ± 2.31	215.3 ± 22.96***^b
	200	4.5 ± 1.37	198.8 ± 20.54***^ab
	400	6.6 ± 3.20	112.5 ± 12.34**^ab

Values are expressed as mean ± SD (n = 6).

*p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 vs piracetam; ^bp < 0.05 versus scopolamine.

Analysis of variance followed by Tukey’s test.

Table 3.  Effect of 80% aqueous methanol extract of Evolvulus alsinoides (EAE) on the step-down latency.

Group	Dose (mg/kg)	Mean step-down latency (s)^n ± SD
		On day 30	On day 31
Control/vehicle	–	4.1 ± 1.16	58.5 ± 5.39
Piracetam	100	6.5 ± 3.2	251.8 ± 27.56***
Scopolamine	3	5.0 ± 1.78	24.3 ± 8.50*^a
EAE	50	5.6 ± 1.63	63.6 ± 5.39^ab
	100	3.83 ± 0.98	60.5 ± 5.85^ab
	200	4.3 ± 1.21	214.1 ± 21.92***^ab
	400	5.1 ± 3.20	211.0 ± 16.76***^ab

Values are expressed as mean ± SD (n = 6).

*p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus piracetam; ^bp < 0.05 versus scopolamine.

Analysis of variance followed by Tukey’s test.

Table 4.  Effect of 80% aqueous methanol extract of Clitoria ternatea (CTE) on the step-down latency.

Group	Dose (mg/kg)	Mean step-down latency (s)^n ± SD
		On day 30	On day 31
Control/vehicle	–	5.3 ± 2.25	57.8 ± 6.11
Piracetam	100	4.8 ± 1.72	241.6 ± 26.90***
Scopolamine	3	5.0 ± 1.78	24.3 ± 8.50*^a
CTE	50	4.8 ± 1.47	57.5 ± 5.46^ab
	100	6.0 ± 2.09	199.1 ± 10.74***^ab
	200	5.5 ± 1.87	147.8 ± 12.70***^ab
	400	6.3 ± 2.80	68.8 ± 7.05^ab

Values are expressed as mean ± SD (n = 6).

*p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus piracetam; ^bp < 0.05 versus scopolamine.

Analysis of variance followed by Tukey’s test.

Anxiolytic activity

All the three extracts (CPE, EAE and CTE) exhibited anxiolytic activity. Among the extracts of the three plants, only CPE showed significant anxiolytic activity (Table 5) at all tested dose levels (50, 100, 200 and 400 mg/kg) when compared to the control group in terms of time spent in open arms. EAE (100, 200 and 400 mg/kg) and CTE (100 and 200 mg/kg) showed anxiolytic activity when compared with the vehicle-treated group. Both CPE and CTE (100 mg/kg) showed significant activity (p < 0.001) in terms of time spent by the animals in open arms (22.1 and 17.6 s, respectively), in comparison to control group (3.6 s). E. alsinoides (200 mg/kg) also showed a significant activity (20.4 s in open arm, p < 0.001) in comparison to the vehicle-treated group (Table 5). Furthermore, the results of CPE (100 mg/kg) and EAE (200 mg/kg) were comparable to diazepam (standard drug). The animals of the control group showed a preference for the closed arms with an average of only 1.4 entries in open arms. Animals treated with CPE (100 mg/kg), EAE (200 mg/kg) and CTE (100 mg/kg) showed an average of 9.4, 10.6 and 9.6 entries, respectively, in the open arms.

Table 5.  Anxiolytic activity of 80% aqueous methanol extract of Convolvulus pluricaulis, Evolvulus alsinoides and Clitoria ternatea.

Group	Dose (mg/kg)	^nMean number of entries in open arm ± SD	^nMean time spent in open arms (s) ± SD
Control	–	1.4 ± 0.5^a	3.6 ± 1.1
Diazepam	2	9.2 ± 1.9***	22.6 ± 3.7***
CPE	50	3.6 ± 1.5*^a	9.3 ± 1.5**^a
	100	9.4 ± 2.6***	22.1 ± 4.3***
	200	2.2 ± 1.4^a	13.8 ± 2.3**^a
	400	1.0 ± 0.4^a	7.6 ± 1.4*^a
EAE	50	2.4 ± 1.6^a	4.4 ± 2.3^a
	100	4.2 ± 0.8**^a	7.2 ± 1.4*^a
	200	10.6 ± 1.9***	20.4 ± 2.5***
	400	4.4 ± 2.0**^a	10.0 ± 1.5**^a
CTE	50	2.8 ± 1.4^a	5.8 ± 1.3^a
	100	9.6 ± 2.8***	17.6 ± 4.2***^a
	200	4.8 ± 2.4**^a	8.8 ± 1.9**^a
	400	1.6 ± 1.1^a	2.8 ± 1.9^a

Values are expressed as mean ± SD (n = 5).

*p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus diazepam.

Analysis of variance followed by Tukey’s test.

CPE: 80% aqueous methanol extract of Convolvulus pluricaulis; CTE: 80% aqueous methanol extract of Clitoria ternatea; EAE: 80% aqueous methanol extract of Evolvulus alsinoides.

Antidepressant activity

The antidepressant activity was evaluated using the forced swimming despair test in mice. Imipramine, 12.5 mg/kg, p.o., was used as a standard antidepressant. Imipramine reduced the average immobility time (Figure 4) to 95 s producing a 37% fall in immobility period with respect to control animals which remain immobile for 151 s. CTE (50, 100 and 200 mg/kg) showed antidepressant activity (p < 0.05) in terms of a decrease in the immobility time period (114, 120.3 and 122.5 s, respectively) in comparison to control group animals (151 s). EAE also showed significant activity (p < 0.05) at only the two-dose levels of 50 and 100 mg/kg (123.6 and 129.5 s of immobility, respectively) in comparison to the control group. The activity of these two plants extracts decrease with the increase in the dose as indicated by an increase in immobility time. EAE showed an increase in immobility time to 163 s (7.9% increase) with the 400 mg/kg dose in comparison to control animals. CPE showed no activity (Figure 4) at any of the four dose levels. Moreover, rather than decreasing the immobility time, it increased the immobility time to 156, 167 and 191 s at 100, 200 and 400 mg/kg dose levels, respectively (showing 3.3, 11 and 26% increase, respectively), in comparison to control.

Figure 4.  Effect of hydromethanol extract of Convulvulus pluricaulis (CPE), Evolvulus alsinoides (EAE) and Clitoria ternatea (CTE) on the immobility of mice. Ordinates express mean immobility time, in seconds. *p < 0.05 versus control; ^ap < 0.05 versus imipramine. Results are compared by one-way analysis of variance followed by Tukey’s test (n = 5 per group). Imipra, imipramine (12.5 mg/kg, p.o.).

Spontaneous locomotor/CNS-depressant activity

A slight decrease in memory-enhancing, anxiolytic and antidepressant activities of all the three plants pointed towards possible CNS-depressant activity at higher doses. So, in order to evaluate CNS-depressant activity of CPE, EAE and CTE, a test of locomotor activity of mice was performed in an actophotometer. The extracts of all the three plants were tested at 100–600 mg/kg against a standard drug, diazepam. The results in Figure 5 clearly showed that all the three plants possessed a dose-dependent, significant CNS-depressant activity at higher dose levels of 400 and 600 mg/kg. Animals treated with CPE showed significant activity (p < 0.05 and p < 0.001) at 400 and 600 mg/kg, respectively. CPE at these two-dose levels showed an average 27 and 45% decrease in the locomotor activity (decrease in number of counts), respectively, in comparison to control animals. EAE at doses of 400 mg/kg (p < 0.05) and 600 mg/kg (p < 0.01) also showed a significant decrease in locomotor activity in comparison to vehicle-treated animals. The locomotor activity of the animals was also decrease when treated with CTE (400 and 600 mg/kg) when compared with control group animals (p < 0.05). Furthermore, none of these two extracts (EAE and CTE) did not show as much activiy as that of diazepam. The results confirmed that C. pluricaulis possesses maximum CNS-depressant activity at a dose of 600 mg/kg. Only C. pluricaulis (600 mg/kg) showed activity (45% reduction) comparable to diazepam (52% reduction in the locomotor activity). The depressant action of all the three plants at higher dose levels gave a plausible explanation of reduced memory-enhancing, anxiolytic and antidepressant activity of these plants at higher doses.

Figure 5.  Effect of hydromethanol extract of Convulvulus pluricaulis (CPE), Evolvulus alsinoides (EAE) and Clitoria ternatea (CTE) on the locomotor activity of mice. Ordinates express mean number of counts. *p < 0.05, **p < 0.01, ***p < 0.001 versus control; ^ap < 0.05 versus diazepam. Results are compared by one-way analysis of variance followed by Tukey’s test (n = 5 per group). Diaz, diazepam (10 mg/kg, p.o.).

Discussion

Shankhpushi has been used since ages as a brain tonic and memory enhancer in Ayurveda. The controversy regarding the true source of the drug has led to the limited use of this drug by the herbal drug industry. The results of the present investigation have shown that among the three plants, C. pluricaulis has maximum memory-enhancing activity in both the models. Moreover, the effect of all the three plants was not dose-dependent and a slight decrease in the activity was observed with the increase in the dose. The results of the present study and the description of the drug in the traditional texts (Sharma, 2001; Chunekar & Pandey, 2002) support the use of C. pluricaulis as the true source of shankhpushpi. Recently, the alcohol, ethyl acetate and aqueous extracts of C. pluricaulis showed significant memory-enhancing activity in rodents when tested using Cook and Weidley’s pole climbing apparatus, active and passive avoidance models (Nahata et al., 2008). Furthermore, these extracts significantly reversed the amnesia induced by scopolamine. Review of literature has shown that cholinergic system, especially acetylcholine, plays an important role in memory functions both in humans and rodents (Levin & Simon, 1998; Hasselmo, 2006; Román & Kalaria, 2006). The drug which reverses the scopolamine induced amnesia might have some effects on the cholinergic system, mainly on the levels of acetylcholine in brain. Moreover, scopolamine is a muscarinic antagonist, and it has been reported that blockade/or decreased muscarinic activity causes excess phosphorylation of tau proteins leading to the formation of β-amyloid in the brain leading to memory impairment (Francis et al., 1999). Cholinergic or muscarinic agonists have been found to prevent the formation of β-amyloids through the GSK-3 enzyme pathway and improve memory (Forlenza et al., 2000). Therefore, it is hypothesized that the C. pluricaulis may enhance memory by two different pathways: (1) by affecting acetylcholine levels in brain either by increasing its synthesis or by inhibiting the acetylcholinesterase enzyme, and/or (2) by acting as muscarinic agonist which further reduce the β-amyloid formation.

All the three plants showed a significant anxiolytic activity in comparison to control. CPE (100 mg/kg) and EAE (200 mg/kg) showed activity comparable to diazepam. CTE was the least active among the three (Table 5). A similar pattern of decrease in the anxiolytic activity at doses higher than showing maximum activity was observed in all the three plants. This decrease in the activity at higher dose levels could be due to the CNS-depressant activity of the plants. Recently, C. pluricaulis and E. alsinoides were compared for anxiolytic activity and a similar pattern of activity was also observed (Nahata et al., 2009). It was quite evident from the results in Figure 4 CTE and EAE showed a very weak antidepressant effect at lower doses. All the three plants showed a dose-dependent response but, rather than showing an increase, a decrease in the activity was observed. The decrease in the activity with increase in dose was indicated by the increase in the immobility time period of the animals. These results further suggest evaluating the antidepressant activity of the three plants at lower dose levels.

The decrease in the above three activities at higher dose levels strongly suggested the CNS-depressant activity of the three plants. It is well known that depression leads to the decrease in locomotor activity of the animals (Nyeem et al., 2006). Therefore, the CNS-depressant activity of the three plants was evaluated in terms of locomotor activity. The results in Figure 5 clearly indicated that extract of C. pluricaulis showed best CNS-depressant activity followed by E. alsinoides and C. ternatea. The CNS-depressant property of the three plants at higher dose levels gave a possible explanation for the decrease in the activities at higher dose levels.

Conclusion

In conclusion, all the three plants possess memory-enhancing, anxiolytic and CNS-depressant activity with C. pluricaulis showing the maximum activity. E. alsinoides and C. ternatea showed significant antidepressant activity but a lower dose levels. C. pluricaulis did not show any antidepressant activity but a significant depressant activity was observed at higher doses. Further, the decrease in nootropic and anxiolytic activities of the three plants at higher dose levels can be attributed to their depressant effect. The results of memory-enhancing activity suggest that C. pluricaulis should be used as the true source of shankhpushpi while the other two plants can be used as its substitutes.

The findings validate the traditional use of shankhpushpi for its memory-enhancing property and provide great relevance for its use in the prevention and therapies of memory disorders, especially AD. Further investigations are in progress to isolate, identify and describe the bioactive constituent(s) of the plant (C. pluricaulis) and their possible mechanisms of action.

Acknowledgement

The Research Fellowship awarded to JM by the University Grants Commission, New Delhi, India is gratefully acknowledged.

Declaration of interest

The authors report no declarations of interest.