Nootropic Activity of Crataeva Nurvala Buch-Ham Against Scopolamine Induced Cognitive Impairment
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EXCLI Journal 2015;14:335-345 – ISSN 1611-2156 Received: August 16, 2014, accepted: January 21, 2015, published: February 27, 2015 Original article: NOOTROPIC ACTIVITY OF CRATAEVA NURVALA BUCH-HAM AGAINST SCOPOLAMINE INDUCED COGNITIVE IMPAIRMENT Atanu Bhattacharjee*1, Shastry Chakrakodi Shashidhara2, Santanu Saha1 NGSM Institute of Pharmaceutical Sciences, Deralakatte, Mangalore - 574 018, Karnataka, India 1 Department of Pharmacognosy 2 Department of Pharmacology * Corresponding author: Mr. Atanu Bhattacharjee, Tel: +91-8088741374, Fax: +91-824-2203992, E-mail: [email protected] http://dx.doi.org/10.17179/excli2014-541 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/). ABSTRACT Loss of cognition is one of the age related mental problems and a characteristic symptom of neurodegenerative disorders like Alzheimer’s. Crataeva nurvala Buch-Ham, a well explored traditional Indian medicinal plant of Westernghats, is routinely used as folkloric medicine to treat various ailments in particular urolithiasis and neu- rological disorders associated with cognitive dysfunction. The objective of the study was to evaluate the nootropic activity of Crataeva nurvala Buch-Ham stem bark in different learning and memory paradigm viz. El- evated plus maze and Y-maze against scopolamine induced cognitive impairment. Moreover, to elucidate possi- ble mechanism, we studied the influence of Crataeva nurvala ethanolic extract on central cholinergic activity via estimating the whole brain acetyl cholinesterase enzyme. Ethanolic extracts of Crataeva nurvala (100, 200 and 400 mg/kg body weight) were administered to adult Wistar rats for successive seven days and the acquisition, re- tention and retrieval of spatial recognition memory was determined against scopolamine (1 mg/kg, i.p.) induced amnesia through exteroceptive behavioral models viz. Elevated plus maze and Y-maze models. Further, whole brain acetyl cholinesterase enzyme was estimated through Ellman’s method. Pretreatment with Crataeva nurvala ethanolic extract significantly improved spatial learning and memory against scopolamine induced amnesia. Moreover, Crataeva nurvala extract decreased rat brain acetyl cholinesterase activity in a dose dependent man- ner and comparable to the standard drug Piracetam. The results indicate that ethanolic extract of Crataeva nur- vala might be a useful as nootropic agent to delay the onset and reduce the severity of symptoms associated with dementia and Alzheimer’s disease. The underlying mechanism of action of its nootropic potentiality might be at- tributed to its anticholinesterase property. Key words: Crataeva nurvala, nootropic, acetyl cholinesterase INTRODUCTION symptom of Alzheimer’s disease (Hanuman- thachar and Milind, 2006). The cholinergic Memory function is vulnerable to a va- hypothesis claims that decrease in cognitive riety of pathological condition including function in dementia is predominantly relat- many neuropsychiatric and neurodegenera- ed to a decrease in cholinergic neurotrans- tive diseases like Alzheimer’s disease mission. The cholinergic muscarinic antago- (Dinesh et al., 2004). Dementia, an age- nist like scopolamine is most widely used related mental problem is a characteristic drug to induce amnesia in experimental ani- 335 EXCLI Journal 2015;14:335-345 – ISSN 1611-2156 Received: August 16, 2014, accepted: January 21, 2015, published: February 27, 2015 mals (Kulkarni, 2013). Amnesic mild cogni- 04/2011) was preserved in the institutional tive impairment (through scopolamine ad- department. ministration) represents a transitional state between cognitive changes of normal aging Extraction and fractionation and the earliest clinical features of Alz- 500 gm coarsely powdered raw material heimer’s disease (Saxena et al., 2013). Noo- of C. nurvala stem bark was extracted by tropic drugs like Piracetam and cholinester- cold maceration with ethanol and concentrat- ase inhibitors are clinically used to improve ed through rotary flash evaporator at 40 °C learning and memory abilities, mood and be- under reduced pressure and stored in deep havior in those neurodegenerative diseases freezer at -20 °C (Parvin et al., 2011). The (Sibi and Delphia, 2013). But the resulting yield was found to be 17 % w/w. A portion side-effects associated with these synthetic of concentrated ethanolic extract was frac- drugs have made their utility limited (Hanu- tionated with different solvents with increas- manthachar and Milind, 2007). Therefore, it ing order of polarity (petroleum ether, chlo- is worthwhile to explore the utility of tradi- roform, ethyl acetate, n-butanol and metha- tional medicines in the treatment of various nol). A flow chat of detailed method of ex- neuropharmacological disorders associated traction and fractionation is given in Figure 1 with cognitive dysfunction as they are con- sidered to be safe and economical (Mura- Phytochemical analysis leedharannair et al., 2011). The crude fractions were subjected to Crataeva nurvala (C. nurvala) Buch- different qualitative phytochemical screening Ham (Family: Capparidaceae) commonly to identify the presence of various phytocon- known as Varuna, a well explored traditional stituents as described by Harborne (Harbone, Indian medicinal plant of Westernghats rou- 1998). The phytochemicals analyzed were tinely used to treat various ailments in par- alkaloids, phenols, flavonoids, saponins, ter- ticular urolithiasis and neurological disorders penoids, steroids, tannins, carbohydrates, (Shiddamallayya et al., 2010, Amod et al., proteins and coumarins. 2005). It is a medium sized branched decid- uous plant distributed throughout the river banks of southern India and other tropical, sub-tropical countries of the world, wild or cultivated (Rajesh et al., 2011). It requires dry, hot climate and shady places to grow ef- fectively (Patil et al., 2010). Hence, the pre- sent study was designed to evaluate the nootropic potentiality of C. nurvala through several in-vivo neuropharmacological assays and biochemical estimations. MATERIALS AND METHODS Figure 1: Schematic diagram of extraction and fractionation of C. nurvala Collection and authentication of plant material The stem bark of C. nurvala was collect- Screening procedure ed from the stream sides of Westernghat, In- dia and authenticated by Dr. K.V. Naga- Test for tannins and phenols lakshamma, Professor and Head, Department 5 ml of fraction was added to 2 ml of 5 % of Biotechnology (UG) of St. Aloysius Col- of alcoholic FeCl3 solution. Blue-black pre- lege, Mangalore, India. The herbarium cipitate indicated the presence of tannins and (voucher specimen no. NGSMIPS/Hb- phenols. 336 EXCLI Journal 2015;14:335-345 – ISSN 1611-2156 Received: August 16, 2014, accepted: January 21, 2015, published: February 27, 2015 Test for alkaloids obis (2-nitrobenzoic acid) (DTNB) were col- 2 ml of 2N HCl was added to 5 ml ex- lected from Sigma-Aldrich, Bangalore, In- tract and heated over water bath for 10 min. dia. All the toxic, standard and test drugs The cooled solution was filtered and a few (suspended in 0.6 % w/v of sodium CMC so- drops of Dragendorff’s reagent were added. lution) were administered in the morning Reddish-brown precipitate indicated the session i.e. 9 AM- 10 AM on each day. presence of alkaloid. Test for saponins Acute toxicity study About 1 g of dried powdered sample was The acute toxicity of ethanolic extract of boiled with 10 ml distilled water. Frothing C. nurvala stem bark was evaluated in fe- persistence indicated the presence of sapo- male Wistar rats as per OECD guidelines nins. 425 (Up and Down Procedure). A fixed dos- age study was adopted for acute toxicity Test for terpenoids study where the limit dose is 2000 mg/kg 5 ml of fraction was mixed with 2 ml of body weight of test animal. Clinical signs of chloroform and few drops concentrated toxicity, body weight changes, cage side pa- H2SO4 was carefully added to form a layer. rameters and mortality rate were observed Red ring indicated the terpenoids are present. every hour for the first 6 hours and every day Test for steroids for 7 days (Atanu et al., 2013a). 5 ml of fraction was mixed with 10 ml Experimental animals CHCl3 and 1 ml acetic anhydride and few Healthy young Wistar rats of either sex drops of concentrated H2SO4 were added. Green ring indicated the presence of steroids. aged between 8-12 week old and weighing between 150-200 g obtained from the institu- Test for flavonoids tional animal house were used for in-vivo To 5 ml of fraction few pieces of magnesium studies. The animals were housed in a room ribbon and few drops of concentrated HCl under standard environmental condition were carefully added. Red color indicated the (25 °C and 50-70 % relative humidity) of presence of flavonoids. 12/12 h light/dark cycle and fed with stand- Test for phlobatannins ard rat pellet and water ad libitum. Animals About 2 ml of fraction was boiled with were allowed to acclimatize for 7 days in la- 2 ml 1 % HCl. Deposition of a red color in- boratory conditions prior to experiment dicated the presence of phlobatannins. (Shirish and Shrikant, 2011). The experi- mental protocol was approved by the Institu- Test for amino acids (Ninhydrin test) tional Animal Ethics Committee (IAEC) un- 5-6 drops of ninhydrin reagent were added in der the IAEC no.: KSHEMA/AEC/34/2011. 5 ml of fraction and heated over boiling wa- ter bath for 5 min. Purple coloration indicat- Exteroceptive behavioral model ed the presence of amino acid. Elevated plus maze Test for proteins (Biuret test) To access the effect of C. nurvala on spa- 5-6 drops of 5 % NaOH and 5-7 drops of tial long term working memory of rats Ele- 1 % CuSO4 were added in 2 ml fraction. vated plus maze test was carried out (Dinesh Violet color indicated the presence of pro- and Varun, 1992). A typical Elevated plus teins. maze consists of two open (50x10 cm) and two close arms (50x10x40 cm) facing each Drugs and chemicals other with a central square (10x10 cm). The Scopolamine hydrobromide, piracetam, entire maze has been elevated to a height of sodium carboxy methyl cellulose (Sodium- 50 cm from the floor.