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SUPPLEMENTARY MATERIAL
Attenuating effect of bioactive coumarins from Convolvulus pluricaulis on scopolamine induced amnesia in mice

Jai Malik, Maninder Karan, Karan Vasisht*



University Institute of Pharmaceutical Sciences – UGC Centre of Advanced Study

Panjab University, Chandigarh-160014 (India)

Abstract

Convolvulus pluricaulis Chois. (Convolvulaceae) has been used in Ayurveda as Medhya Rasyana (nervine tonic) to treat various mental disorders. The present study was designed to isolate the bioactive compound(s) of this plant and to evaluate their effect against scopolamine induced amnesia. Column chromatography of the chloroform and ethyl-acetate fractions, led to the isolation of three coumarins identified as scopoletin, ayapanin and scopolin. All the three compounds at 2.5, 5, 10 and 15 mg/kg, p.o. were evaluated for memory enhancing activity against scopolamine induced amnesia using elevated plus maze (EPM) and step down (SD) paradigms. Effect on acetylcholinesterase activity in mice brain was also evaluated. Scopoletin and scopolin, in both the paradigms, significantly and dose dependently attenuated the scopolamine induced amnesic effect. Furthermore, these compounds at 10 and 15 mg/kg exhibited activity comparable to that of standard drug, donepezil. The compounds also exhibited significant acetylcholinesterase inhibitory activity.
Keywords: Scopoletin, Scopolin, Ayapanin, Shankhpushpi

1. Experimental

1.1. Plant material

Whole plant of CP was collected from Chandigarh, India, in the month of June 2006. The identity of each plant material was confirmed by Dr. S. Nautiyal, Forest Research Institute, Dehradun, Uttaranchal, India vide certificate number 765/2006-Bot/15-1. The plant material of CP was deposited for record in the Museum-cum-Herbarium of University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India, vide voucher specimen no. 1454.



1.2. Solvents and instruments

All the solvents (LR grade) and normal phase silica gel (60-120 mesh) for open column chromatography were procured from E. Merck (India) Ltd. Distilled water was used wherever mentioned. Borosilicate glass columns of appropriate capacity, depending on the quantity of material to be fractioned, were used to carry out column chromatography. The elution was done using suitable solvents and their combinations in an increasing order of polarity.

The 1H- and 13C-NMR were obtained using Bruker 400 MHz / 52 MM spectrophotometer (Bruker Instruments, Billerica, MA, USA) with tetramethylsilane as internal standard. EI-mass spectra were obtained on Vg-11-250J 70S mass spectrometer at 70ev using electron ionization source. IR spectroscopy was carried out using KBr disc on Perkin-Elmer 882 infrared spectrophotometer (Perkin Elmer, USA). UV spectroscopy was carried out, by dissolving the compound in suitable solvent, on Perkin-Elmer Lambda 15 UV spectrophotometer (Perkin Elmer, USA).

1.3. Animals

Laca mice weighing 25-30 g, of either sex, procured from the Central Animal House of Panjab University, Chandigarh were used in the present study. The animals were kept in polyacrylic cages and maintained under standard housing conditions with 12 h light/dark

reverse cycle. The food and water was made available ad libitum. Experiments were conducted between 9.00 and 15.00 h. All the animals were acclimatized to the laboratory conditions, prior to start of the experiment, 1 h everyday, for 5 days. The protocol was reviewed and approved by the Institutional Animal Ethics Committee, and the animal experiments were carried out in accordance with the Indian National Science Academy Guidelines for use and care of animals. The animals were divided into following different groups with six animals in each group.

Group I: Control group animals receiving vehicle

Group II: Animals receiving scopolamine (3 mg/kg, p.o.)

Group III: Animals treated with donepezil (1 mg/kg, p.o.) + socpolamine

Group IV- VII: Animals receiving 2.5-15 mg/kg, p.o. dose of scopoletin + socpolamine

Group VIII-XI: Animals receiving 2.5-15 mg/kg, p.o. dose of ayapanin + socpolamine

Group XII-XV: Animals receiving 2.5-15 mg/kg, p.o. dose of scopolin + socpolamine

Animals were acclimatized to laboratory environment daily for 1 h for 10 days, prior to the experiment. All the experimental protocols were approved by institutional animal ethical committee, Panjab University, Chandigarh, which complies with the international norms of Indian National Science Academy (INSA).



1.4. Extraction and isolation of bioactive compounds

The plant material was air-dried in shade and ground to a coarse powder. The powder (8 kg) of dried drug powder was macerated thrice for 48 h each with 80% aqueous methanol at room temperature in four batches of 2 kg each. The extract was filtered and volume was reduced to one fourth under reduced pressure. The concentrated extract was then suspended in 1 L of distilled water and partitioned successively for five times with 500 ml each of petroleum ether, chloroform, ethyl acetate and n-butanol. The partitioned extracts were filtered and dried under reduced pressure to get 70, 55, 45 and 150 g of the respective extracts.

A preliminary pharmacological study done by the authors, pertaining to memory enhancing activity using elevated plus maze model (the protocol used for screening the fractions was same as that is used in the present manuscript), revealed that amongst various partitioned extracts only chloroform and ethyl acetate fractions (100 and 75 mg/kg, p.o., respectively) showed significant memory enhancing activity (data not published). Following the results of preliminary study, the two active fractions viz. chloroform and ethyl acetate, were subjected to column chromatography using silica gel for isolation of the bioactive compound(s). Column chromatography of chloroform-soluble portion (50 g) led to the isolation of two compounds CP 1 (520 mg, 0.006% w/w) and CP 2 (80 mg, 0.001% w/w). Column chromatography of ethyl acetate-soluble portion (40 g) gave compounds CP 1 (340 mg, 0.004% w/w) and CP 3 (350 mg, 0.004% w/w). The total yield of compound 1 from both chloroform- and ethyl acetate-soluble portion was 860 mg (0.01% w/w).

1.5. Identification of the compounds

These compounds were identified, as scopoletin (1), ayapanin or herniarin (2) and scopolin (3), by the usual spectrometric techniques (UV, IR, 1H and 13C NMR and MS) and comparison with literature data (Ahmad and Mishra 1999, Lee et al. 2004, Thuong et al. 2005).



1.6. Dose preparation and drug treatment

All the doses of test compounds and standard drugs, scopolamine hydrobromide (Sigma Aldrich, USA) and donepezil (Esai Co. Ltd, Japan), were prepared in 1% of Tween 80 solution. The test compounds 1-3 (2.5–15 mg/kg) and donepezil (1 mg/kg) were administered orally for 14 days. Scopolamine (3 mg/kg, p.o.), was given 45 min after the administration of the dose of test/standard drug on day 10 and 13 (Das et al. 2002, Verloes et al. 1988). Training was carried out 30 min after the scopolamine administration.



1.7. Memory enhancing activity

Effect of the compounds on scopolamine induced memory impairment was assessed using elevated plus maze (EPM) test. The compounds that were active on EPM, were further evaluated on step down (SD) test.



1.7.1. Elevate plus maze test

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. 1990). On day 10, 30 min after the scopolamine administration, 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 i.e. day 11 (Retention test), at the same time period of the day. Decrease in the TL indicates the memory enhancing property of the test drug. The apparatus was cleaned using alcohol after trial of each animal to remove any olfactory traces.



1.7.2. Step down test

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 x 50 x 40 cm) with electrifiable grid floor, connected to an electric source to provide scramble foot shock. On day 13, 30 min after the scopolamine administration, each mouse was gently placed on a wooden platform (4 x 4 x 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 sec following which the mouse was shifted back to its cage immediately. Retention was tested after 24 h i.e. on day 14 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 sec. An increase in SDL indicate improved retention and memory of the animal.



1.8. Locomotor activity

In order to observe the effect of test drugs on the motor ability, each animal was tested for spontaneous locomotor activity on day 12. Each animal was observed over a period of 10 min in a square closed arena equipped with infrared light sensitive photocells using a digital actophotometer (INCO, India). As soon as the animal cross the light beam the photocell path gets obstructed and the recorder records a count. Decreased number of counts in comparison to control indicate a decreased locomotor activity of the animals.



1.9. Acetylcholinesterase activity

The activity of acetylcholinesterase (AChE) was determined in the brain of the animals according to Ellman's colorimetric method (Ellman et al. 1961). Briefly, the animals were sacrificed by cervical dislocation immediately after behavioral studies on day 14. The brain was isolated and homogenized with ice-cold 0.1  M phosphate buffer (pH 7.4) in a volume 10 times the weight of the tissue. The homogenate was centrifuged at


10,000×g for 15 min at 4̊ C, the supernatant was separated, and the aliquots were used for biochemical estimations. The assay was performed by adding requisite amount of supernatant from the tissue homogenate to a mixture of 3.0 ml of 0.1M phosphate buffer of pH 8.0 and 100 µL of 0.01 M DTNB (prepared in 0.1 M phosphate buffer of pH 7.0). Acetylthiocholine iodide, procured from Sigma Aldrich, USA (100 µL of 14.9 mM), was added to the mixture. The reaction mixture was vortexed for 3 sec and absorption was measured at 412 nm. The change in the absorption was recorded and the results were expressed in number of moles of acetylthiocholine iodide hydrolyzed/min/mg protein.

1.10. Protein estimation

The protein content was measured in all brain samples using bovine serum albumin (BSA; 1 mg/mL) as a standard (Lowry et al. 1951).



1.11. Statistical analysis

Results were expressed as mean ± SD. The inter group variation was measured by one way analysis of variance (ANOVA) followed by Tukey’s test. Statistical significance was considered at p < 0.05. The statistical analysis was done using Graph Prism Pad 5 statistical software.


References
Ahmad A, Mishra LN. 1999. Isolation of herniarin and other constituents from Matricaria chamomilla flowers. Pharm Biol. 35:121-125.

Das A, Shanker G, Nath C, Pal R, Singh S, Singh HK. 2002. A comparative study in rodents of standardized extracts of Bacopa monniera and Ginkgo biloba - Anticholinesterase and cognitive enhancing activities. Pharmacol, Biochem Behav. 73:893-900.

Ellman GL, Courtney KD, Andres VJ, Featherstone RM. 1961. A new rapid colorimetric determination of acetylcholineterase activity. Biochem Pharmacol. 7:88-95.

Itoh J, Nabeshima T, Kameyama T. 1990. Utility of an elevated plus-maze for the evaluation of memory in mice:effects of nootropics, scopolamine and electroconvulsive shock. Psychopharmacology. 101:27-33.

Kameyama T, Nabeshima T, Kozawa T. 1986. Step-down-type passive avoidance- and escape-learning method. Suitability for experimental amnesia models. J Pharmacol Methods. 16:39-52.

Lee JH, Ku CH, Baek N, Kim SH, Park HW, Kim DK. 2004. Phytochemical constituents from Diodia teres. Arch Pharm Res. 27:40-43.

Lowry OH, Roseburgh NJ, Farr AL, Randal RL. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem. 193:205-215.

Thuong PT, Na M, Su ND, Seong RS, Lee YM, Sok DE, Bae KH. 2005. Inhibitory effect of coumarins from Weigela subsessilis on low density lipoprotein oxidation. Chem Pharm Bull. 28:1095-1097.

Verloes R, Scotto AM, Gobert J, Wulfert E. 1988. Effect of nootropic drugs in scopolamine -induced amnesia. Psychopharmacology. 95:226-230.
 Table S1: Effect of scopoletin and scopolin at various dose levels on acetylcholinesterase activity.


Group

Dose

(mg / kg)

Acetylthiocholine iodide hydrolysed (µM) ± S.D.

Control

-

124.2 ± 10.2

Scopolamine

3

197.1 ± 9.3*b

Donepezil + Scopolamine

1

137.9 ± 8.3

Scopoletin + Scopolamine

2.5 + 3

5 + 3


10 +3

15 + 3


161.8 ± 9.7ab

148.7 ± 11.3ab

139.7 ± 8.3a

135.7 ± 7.7a



Scopolin + Scopolamine

2.5 + 3

5 + 3


10 +3

15 + 3


155.5 ± 12.8ab

147.8 ± 5.6ab

133.4 ± 6.3a

129.8 ± 5.2a



Values expressed as mean acetylcholine iodide hydrolysed ± S.D. (n = 6)

*P<0.05 vs control, aP<0.05 vs scopolamine, bP<0.05 vs donepezil



Results are compared by one way ANOVA followed by Tukey’s test.
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