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Nootropic and Free Radical Scavenging Activity of Plumbago zeylanica Linn. By Different In-Vivo & In-Vitro Methods

Department of Pharmacology, Faculty of Pharmacy, Oriental University Indore-India

Article Info:

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Article History:

Received 02 April 2022

Reviewed 29 May 2022

Accepted 05 June 2022

Published 15 June 2022

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Cite this article as:

Chandanwala SA, Jain NL, Singh N, Nootropic and Free Radical Scavenging Activity of Plumbago zeylanica Linn. By Different In-Vivo & In-Vitro Methods, International Journal of Medical Sciences & Pharma Research, 2022; 8(2):12-18

DOI: http://dx.doi.org/10.22270/ijmspr.v8i2.31

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*Address for Correspondence:

Sakina Akbarali Chandanwala, Department of Pharmacology, Faculty of Pharmacy, Oriental University Indore-India

Abstract

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AIM- The aim of the present investigation is to evaluate Plumbago zeylanica L. (Plumbaginaceae) for evaluation of memory enhancing activity. MATERIAL & METHODS- All the plant materials were dried under shade and subjected to coarse powder for extraction process. Accurately weighed quantity of roots of Plumbago zeylanica Linn. extracted using petroleum ether, chloroform, methanol, butanol and finally water by soxhlet apparatus for 72 h. Qualitative chemical tests of chloroform, methanol, butanol and water extracts were subjected to various chemical tests to detect various Phytoconstituents. Briefly, 6.0 mg ß-carotene was dissolved in 10 ml of chloroform, and then 1 ml of solution pipette to glass filled of 20 mg linoleic acid. 5 ml of mixture then pipette to reaction tube filled of extract in a range of concentration, mixed homogenously. Briefly, 1 mL of 0.3 mM of DPPH solution was added to 1 mL each of the test solutions, and was incubated in the dark at room temperature for 30 min. Escape latency (EL) was recorded 120 min after drug administration from 11th day to 14th day. On 15th day, time spent in target quadrant (TSTQ) was noted 120 min after the drug administration. In case of animals administered with physostigmine, EL and TSTQ was noted after 30 min of drug administration. 0.4 ml of brain homogenate was added into a test tube containing 2.6 ml of phosphate buffer. 5,5-dithiobis-2-nitrobenzoic acid reagent (0.1 ml) was added to the above mixture and absorbance was noted at 412 nm. RESULTS- The preliminary phytochemical analysis revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino acids, fat, oils, steroids, terpenoids, glycosides, alkaloids, tannins and other phenolics compounds. Vitamin E as standard was used in this assay and 84% inhibition was found to be at 30 minutes. Chloroform extract also showed 70% inhibition at 30 minutes which was reduced to 45% at the time of 120 minutes. Hydroxyl radical scavenging ability calculated as IC₅₀ reveals that chloroform; methanol, butanol and water extracts have IC₅₀ values of 47 μg/mL, 100μg/mL, 200 μg/mL and 150μg/mL, respectively. Among all the extracts, chloroform extract showed a highly significant effect on EL and TSTQ. Chloroform extracts significantly decreased (P<0.001) EL and significantly increased TSTQ as compared to vehicle treated control. CONCLUSION- The deterioration and dysfunction of cortical cholinergic neurons is closely associated with cognitive deficits of Alzheimer’s disease.

Keywords: Alzheimer’s disease, Vitamin E, DPPH solution, Plumbago zeylanica Linn., Plumbaginaceae, EL and TSTQ

The mean age at commencement of AD is around 75 years of age and the overall occurrence is about 1% in most urbanized nations. If AD prevalence is divided into dissimilar age bands then we see a rapid augment from 1% in the 60-64 year age group to greater than 25% in individuals over 85 years of age. The National Institute of Health predicts, if the in progress trend continues, there will be more than 8.5 million AD patients by the year 2013 in USA alone (Joshi & Parle, 2006).

Formation of memory is the most complex procedure and involves multiple neuronal pathways and neurotransmitters. It is well recognized that the cholinergic neuronal system plays an important responsibility in learning and memory in humans and animals. This is the remedial rational at the back the use of nootropic agents such as Piracetam, various Piracetam analogues like oxiracetam, aniracetam, and metrifonate. However, the adverse effects connected with these agents have limited their use (Kriti et al., 2010).

Nootropic are agents that augment the cognitive skills, and amnestics are agents that disrupt the learning and memory process (Kshirsagar, 2011). Nootropic agents such as Piracetam, Aniracetam, and cholinesterase inhibitors like Donepezil are presently used for humanizing memory, mood and behavior (Joshi & Parle, 2006). The cholinergic neural system theater an important role in learning and memory in humans and animals.

The Plumbago zeylanica L. roots (Plumbaginaceae) is included in “Rasayana”- an Ayurvedic ancient system of medicine for rejuvenation. It also contains Plumbagin as active constituent which is an alkaloid which may act as nervine tonic. Hence, by keeping in view, Plumbago zeylanica L. (Plumbaginaceae) are selected for evaluation of memory enhancing activity.

The roots of Plumbago zeylanica Linn. were purchased from the local market. The roots were taxonomically identified by Dr. Gynendra Tiwari, Senior Scientist at Horticulture College. The herbarium sheets were submitted in Department of Pharmacognosy, under voucher specimen.

All the plant materials were dried under shade and subjected to coarse powder for extraction process. Accurately weighed quantity of roots of Plumbago zeylanica Linn. extracted using petroleum ether, chloroform, methanol, butanol and finally water by soxhlet apparatus for 72 h. The extracts were dried completely under reduced pressure. After drying, the respective extracts were weighed and percentage yield was determined (Kumar et al., 2009).

Qualitative chemical tests of chloroform, methanol, butanol and water extracts were subjected to various chemical tests to detect various Phytoconstituents (Kokate, 2003; Khandelwal, 2006).

This assay is based on the capacity of β-carotene oxidative bleaching in β-caroten/linoleic acid mixture with and without the addition of different extract of both plants, the method described by Kikuchi and Kitamura (1987) with a slight modification. Briefly, 6.0 mg ß-carotene was dissolved in 10 ml of chloroform, and then 1 ml of solution pipette to glass filled of 20 mg linoleic acid. 5 ml of mixture then pipette to reaction tube filled of extract in a range of concentration, mixed homogenously. Sample absorptions were conducted before and after incubation at 50^oC for 30, 60, and 120 minutes.

β-carotene bleaching inhibition percentage was calculated by the following formula:

One mL of the reaction mixture contained 100 μL of 2.8 mM 2-deoxyribose (dissolved in phosphate buffer (10 mM), pH 7.4), 500 μL solution of various concentrations of the extract (500n1000 μg/mL), 200 μL of 200 μM FeCl3 and 1.04 μM EDTA (1:1 v/v), 100 μL of H2O2 (1.0 mM) and 100 μL of ascorbic acid (1.0 mM). After incubation time of 1 hour at 37^OC, the amount of deoxyribose degradation was measured by TBA reaction (Badmus et al., 2011; Halliwell et al., 1987).

When DPPH reacts with an antioxidant compound which can donate hydrogen, it is reduced. The modification in color from deep violet to golden/light yellow can be measured at 518 nm. Briefly, 1 mL of 0.3 mM of DPPH solution was added to 1 mL each of the test solutions, and was incubated in the dark at room temperature for 30 min (Badmus et al., 2011). The absorbance values were read at 518 nm, and converted into percentage antioxidant activity, using the below mentioned formula:

Wistar albino mice of either sex between 1 and 2 months of age weighing 25-35 g were used which were procured from the central animal house, India. The animals were allowed free to access commercial rat pallet diet (Lipton India Ltd, Mumbai, India) and water ad libitum. All the laboratory conditions and animals were maintained as per CPCSEA guidelines throughout the experiments. The study designs were approved by the Institutional Animal Ethical Committee.

The acute oral toxicity studies were carried out according to the guidelines set by the Organization for Economic Co-operation and Development (OECD), revised draft guideline 423.

One tenth and one fifth of the lethal dose was taken as effective dose (therapeutic dose) and cut off value was selected as 200 and 400 mg/kg to evaluate the dose dependent action for the evaluation (OECD guidelines, 2001).

The procedure and parameters for testing learning and memory of mice using Morris water maze were followed as reported earlier (Domange et al., 2013; Parle & Singh, 2007).

Animals were divided in ten groups and six animals were placed in each group. Groups 1 served as control and group 2 as standard drug (physostigmine, 0.1 mg/kg i.p.) treated. Group 3 to 10 were treated by different extracts (chloroform, methanol, butanol and water) in a dose of 200 and 400 mg/kg, respectively were administered for 15 successive days. Escape latency (EL) was recorded 120 min after drug administration from 11th day to 14th day. On 15th day, time spent in target quadrant (TSTQ) was noted 120 min after the drug administration. In case of animals administered with physostigmine, EL and TSTQ was noted after 30 min of drug administration.

Group-3 Animals were treated by chloroform extract of P.zeylanica in dose of 200 mg/kg

Group-4 Animals were treated by chloroform extract of P.zeylanica in dose of 400 mg/kg

Group-5 Animals were treated by methanolic extract of P.zeylanica in dose of 200 mg/kg

Group-6 Animals were treated by methanolic extract of P.zeylanica in dose of 400 mg/kg

Group-7 Animals were treated by butanolic extract of P.zeylanica in dose of 200 mg/kg

Group-8 Animals were treated by butanolic extract of P.zeylanica in dose of 400 mg/kg

Group-9 Animals were treated by water extract of P.zeylanica in dose of 200 mg/kg

Group-10 Animals were treated by water extract of P.zeylanica in dose of 400 mg/kg

The procedure and parameters for testing learning and memory of mice using Morris water maze were followed as reported earlier (Domange et al., 2013; Morris, 1984; Parle & Singh, 2007).

To assess plus maze test, the mouse was individually positioned on the central platform facing towards open arm. The percentage of time spent (duration) in open arms and frequency of open arm entries were counted for a period of 5 min. All precaution was taken to ensure that no external stimuli, other than the height of plus maze could invoke anxiety in the animals. The percentage of time spent in the open arms and number of open arm entries were calculated using the formulas [100 × open/ (open + enclosed)] and (100 × open / total entries), respectively.

After 1 h of oral administration of vehicle, diazepam and different extracts of P. zeylanica (chloroform, methanol, butanol and water) were assessed for memory enhances or behavior studies using elevated plus-maze test. Test extracts of both plants were used in a dose of 200 & 400 mg/kg.

After 15^th day using Morris water maze, the animals were sacrificed on 16^th day by cervical dislocation. Whole brain was carefully removed from the animals. The fresh whole brain was weighed first and then homogenized in 10 volumes of 0.1 M phosphate buffer (pH 8) using a glass homogenizer. The homogenate was centrifuged at 3000 rpm for 10 min at 4°C using refrigerated centrifuge (Remi, Mumbai). The resultant cloudy supernatant liquid was used for the estimation of brain acetyl cholinesterase activity (Ellman et al., 1961).

0.4 ml of brain homogenate was added into a test tube containing 2.6 ml of phosphate buffer. 5,5-dithiobis-2-nitrobenzoic acid reagent (0.1 ml) was added to the above mixture and absorbance was noted at 412 nm. Then 0.02 ml of acetylcholine iodide solution was added and again absorbance was noted 15 min thereafter. Change in absorbance per minute was calculated (Ellman et al., 1961).

The values are expressed in mean ± SEM. The results were analyzed by using one way analysis of variance (ANOVA) followed by Dunnet’s "t” test to determine the statistical significance. p< 0.05 was chosen as the level of significance.

The preliminary phytochemical analysis revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino acids, fat, oils, steroids, terpenoids, glycosides, alkaloids, tannins and other phenolics compounds.

In this activity, we found that chloroform extract among all extracts showed best inhibitory activity. Then methanolic extract showed higher activity as compared to butanolic and water extracts. Vitamin E as standard was used in this assay and 84% inhibition was found to be at 30 minutes. Chloroform extract also showed 70% inhibition at 30 minutes which was reduced to 45% at the time of 120 minutes.

Hydroxyl radical scavenging ability calculated as IC₅₀ reveals that chloroform; methanol, butanol and water extracts have IC₅₀ values of 47 μg/mL, 100μg/mL, 200 μg/mL and 150μg/mL, respectively. Since chloroform extract had very lowest IC₅₀as compared to other extracts so it was considered as best extract for the further evaluation.

Table 2: Effect of different extracts on Hydroxyl radical scavenging activity

DPPH scavenging ability calculated as IC50 from Tables shows that chloroform extract has IC₅₀ of 36 μg/mL followed by ascorbic acid (3.2 μg/mL), gallic acid (3.5μg/mL), methanol (40μg/mL), butanolic (47μg/mL) and water (45μg/mL) extracts. The result revealed that chloroform extract had the highest DPPH scavenging ability.

Table 3: Effect of different extracts on Percentage DPPH scavenging activities

Pharmacological Evaluation of Different Extracts for Memory Enhancing Activity

Different extracts and Physostigmine (0.1mg/ kg, i.p.) administered for 15 successive days significantly decreased EL of mice from 11^th to 14^th day and increased TSTQ by mice on 15^th day as compared to the control, thus showed significant improvement of learning and memory. Among all the extracts, chloroform extract showed a highly significant effect on EL and TSTQ. Chloroform extracts significantly decreased (P<0.001) EL and significantly increased TSTQ as compared to vehicle treated control.

Table 5: Effect of different extract on escape latency (EL) of mice using Morris Water Maze

Values are expressed as mean±SEM, n=6 in each group; * p <0.05, compared to disease control ** p<0.01, compared to disease control. *** p <0.001, compared to disease control

Table No 6: Effect of different extract on time spent in target quadrant (TSTQ) of Morris Water Maze

Values are expressed as mean±SEM, n=6 in each group; * p <0.05, compared to disease control ** p<0.01, compared to disease control. *** p <0.001, compared to disease control

Table No 7: Effect of different extracts on brain Acetyl cholinesterase activity of mice

Values are expressed as mean±SEM, n=6 in each group; * p <0.05, compared to disease control ** p<0.01, compared to disease control. *** p <0.001, compared to disease control

Oral administration of chloroform extract of P. zeylanica in 200 and 400 mg/kg, respectively exhibited significant (P < 0.01) increase in the percentage of number of open arm entries and time spent in open arm whereas, in the closed arm number of entries and time spent was significantly (P < 0.01) reduced as compared to vehicle-treated group.

Values are expressed as mean±SEM, n=6 in each group; * p <0.05, compared to disease control ** p<0.01, compared to disease control. *** p <0.001, compared to disease control

Lipid peroxidation is an accumulated effect of reactive oxygen species (ROS), which leads to worsening of biological systems (Dzingiral et al., 2007). Oxygen radicals and other reactive kind are generated in biological systems either as by-products of oxygen reduction or by xenobiotic catabolism (Chance et al., 1979). These ROS such as superoxide anion (O2-),hydroxyl radicals (OH.), nitric oxide (NO) and peroxy radical (ROO.) are unbalanced and can attack key biomolecules such as lipids, proteins and nucleic acids (Halliwell & Gutteridge, 1999).

β-carotene bleaching inhibition method was deliberate based on the ability of an antioxidant to slow down orange color reduction of β-carotene due to the oxidation occurred in linoleic acid/β-carotene mixture (Kulisic et al., 2004; Wang et al., 2010). β-carotene is very responsive to free radical formed by linoleic acid oxidation (Fukumoto & Mazza, 2000; Apak et al.,2007).

In case of P. zeylanica, we found that chloroform extract among all extracts showed best inhibitory activity. Then methanolic extract showed higher activity as compared to butanolic and water extracts. Vitamin E as standard was used in this assay and 84% inhibition was found to be at 30 minutes. Chloroform extract also showed 70% inhibition at 30 minutes which was reduced to 45% at the time of 120 minutes.

Hydroxyl radicals are well thought-out to be one of the rapid initiators of lipid peroxidation process, abstracting hydrogen atoms from polyunsaturated fatty acid, which brings about peroxidic reactions of membrane lipids (Kitada et al., 1979) and also, from each of the carbon atom of the sugar moiety of DNA causing oxidative smash up to DNA. These effects have been concerned in mutagenesis, carcinogenesis and aging (Halliwell & Gutteridge, 1999). Ferric-EDTA incubated with H2O2 and ascorbic acid at pH 7.4, produces hydroxyl radicals and was detected by their aptitude to degrade 2 deoxyribose into fragments, on heating with TBA at low pH forming a pink chromogen (Aruoma et al., 1989).

In case of P. zeylanica, Hydroxyl radical scavenging ability calculated as that chloroform, methanol, butanol and water extracts have IC₅₀ values of 47 μg/mL, 100μg/mL, 200 μg/mL and 150μg/mL, respectively. These results implied that chloroform and methanol extracts have the highest OH^ͦradical scavenging abilities as compared to butanolic and water extracts.

DPPH is a free radical constant at room temperature, and produces a purple color solution in methanol. It is condensed in the presence of antioxidant molecule, giving rise to a yellowish methanol solution. In case of P. zeylanica, DPPH scavenging ability calculated as IC50 shows that chloroform extract has IC₅₀ of 36 μg/mL followed by ascorbic acid (3.2 μg/mL), gallic acid (3.5μg/mL), methanol (40μg/mL), butanolic (47μg/mL) and water (45μg/mL) extracts. The result revealed that chloroform extract had the highest DPPH scavenging ability.

In our study of Plumbago zeylanica Linn.Learning and memory are associated with escape latency and time spent in target quadrant. Decline of EL and augment of TSTQ by mice in Morris water maze indicates improvement of learning and memory and vice versa. Among all the extracts, chloroform extract showed a highly significant effect on EL and TSTQ. Chloroform extracts significantly decreased (P<0.001) EL and significantly increased TSTQ as compared to vehicle treated control.

In our study, all the extracts in a dose of 200 & 400 mg/kg were administered for 15 successive days significantly improved learning and memory of mice. Memory improving effects of extracts were comparable to physostigmine. Chloroform extract of both plants significantly decreased EL during training and it significantly increased TSTQ during retrieval, indicating improvement of learning and memory.

Acetylcholine is measured as the most important neurotransmitter involved in the regulation of cognitive functions (Hasselmo, 2006). Selective defeat of cholinergic neurons or decreased synthesis of acetylcholine was reported to be an attribute feature of neurodegenerative disorder. Drugs that increases the overall quantity of acetylcholine was considered as memory enhancing drug (Deutsch & Rocklin, 1967).

The deterioration and dysfunction of cortical cholinergic neurons is closely associated with cognitive deficits of Alzheimer’s disease (Bartus et al.,1982; Coyle et al., 1983). Thus, the drugs which enhance cholinergic function can be used for treatment of dementia intimately related to Alzheimer’s disease.

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S. No.	Concentration (µg/mL)	Chloroform (%)	Methanol (%)	Butanol (%)	Water (%)
	50	53.78±2.33	48.22±2.44	40.22±2.44	48.38±2.34
	100	57.21±3.21	50.24±3.34	42.21±3.56	49.11±3.11
	150	65.78±3.45	55.44±3.78	46.28±3.31	50.78±3.51
	200	74.34±3.66	58.34±3.99	50.56±3.55	53.21±3.87
	250	82.76±3.55	62.31±3.51	53.76±3.48	60.99±3.53

S. No.	Concentration (µg/mL)	Chloroform (%)	Methanol (%)	Butanol (%)	Water (%)
	10	28.23±2.11	22.55±2.12	25.01±2.42	26.33±2.31
	20	33.11±3.32	26.26±3.15	28.11±3.22	29.11±2.14
	30	41.78±3.45	36.14±3.78	32.28±3.11	33.12±2.51
	40	55.37±3.55	50.44±3.33	42.56±3.24	45.31±3.44
	50	62.76±3.12	52.31±2.11	53.11±3.22	54.17±2.66

S. No.	Concentration (µg/mL)	Vitamin C (%)	Gallic Acid (%)
	2	47.98±2.33	43.66±3.56
	3	49.22±3.67	48.77±3.77
	4	62.33±3.28	55.22±3.11
	5	70.38±3.78	60.78±3.59

Treatment Schedule	Time spent (sec) in target quadrant (15^th day)
Control	45.42 ± 2.23
Physostigmine, 0.1 mg	98.21±2.15***
Chloroform Extract, 200 mg/kg	99.31±2.45***
Chloroform Extract, 400 mg/kg	101.45±2.98***
Methanol Extract, 200 mg/kg	58.35±1.26*
Methanol Extract, 400 mg/kg	60.21±1.78*
Butanolic Extract, 200 mg/kg	62.42±2.87**
Butanolic Extract, 400 mg/kg	65.36±2.64**
Water Extract, 200 mg/kg	63.89±1.74**
Water Extract, 400 mg/kg	65.68±1.64**

Treatment Schedule	Acetyl cholinesterase activity (mol/l per min × 10-6/g of tissue)
Control	0.061 ± 0.010
Physostigmine, 0.1 mg	0.019±0.002***
Chloroform Extract, 200 mg/kg	0.021±0.005***
Chloroform Extract, 400 mg/kg	0.020±0.008***
Methanol Extract, 200 mg/kg	0.050±0.006*
Methanol Extract, 400 mg/kg	0.046±0.006*
Butanolic Extract, 200 mg/kg	0.045±0.007**
Butanolic Extract, 400 mg/kg	0.042±0.005**
Water Extract, 200 mg/kg	0.041±0.004**
Water Extract, 400 mg/kg	0.038±0.002**

S. No.	Treatments	No of Entries		Time Spent (Sec)
S. No.	Treatments	Open Arm	Closed Arm	Open Arm	Closed Arm
	Vehicle	6.2 ± 1.2	22.2 ± 2.4	25.7 ± 3.8	198.3 ± 4.8
	Diazepam	12.5 ± 1.1***	14.2 ± 1.3***	37.7 ± 2.7**	140.2 ± 4.6
	Chloroform extract, 200 mg/kg	11.3 ± 1.4**	10.2 ± 1.8**	44.2 ± 3.8**	132.7 ± 3.6**
	Chloroform extract, 400 mg/kg	11.6 ± 1.6 **	11.3 ± 1.6**	50.2 ± 3.1**	148.5 ± 3.9**
	Methanolic extract, 200 mg/kg	8.4 ± 1.1 *	11.5 ± 1.8**	30.3 ± 3.8*	111.6 ± 4.2**
	Methanolic extract, 400 mg/kg	9.2 ± 1.3 *	11.6 ± 1.1**	35.2 ± 3.4 *	118.7 ± 4.6**
	Butanolic extract, 200 mg/kg	8.5 ± 1.2 *	11.2 ± 1.4**	44.3 ± 3.4**	126.5 ± 3.6**
	Butanolic extract, 400 mg/kg	9.2 ± 1.2 *	11.4 ± 1.2**	47.9 ± 3.3 **	130.3 ± 5.9**
	Water extract, 200 mg/kg	10.9 ± 1.7 *	11.2 ± 1.8**	48.5 ± 3.2**	136.8 ± 4.6**
	Water extract, 400 mg/kg	11.2 ± 1.1 **	11.6 ± 1.5**	49.3 ± 3.6 **	140.9 ± 4.9**