Research Article

Phytochemical evaluation and pharmacological screening of nootropic activity of Eclipta alba Maimunna khathun*1, Habeeba Sulthana2, Jyoti Sahu3, Kouser Unnisa4, Hafsa Siddiqua5

ABSTRACT

Objective: The present study focuses on the evaluation of nootropic activity using passive avoidance paradigm test model (step-down method) in white albino mice using ethanolic extracts of Eclipta alba aerial parts. Materials and Methods: The study was comprised of primary phytochemical constituents screening of extracts using standard methods such as alkaloids, glycosides, steroids, flavonoids, tannins, saponins, and terpenoids. The nootropic activity is analyzed by step-down method using nine white albino mice (120-150 g) at doses of 100 mg in 0.52 ml saline solution (oral administration) based on body weight. Results: Results showed that the phytochemical constituents present in plants increase the nootropic activity as there was a significant increase in step-down latency compared to untreated and standard. Conclusion: It was concluded from the present study finding that the ethanolic extract of plant has a potential nootropic activity.

KEY WORDS: Dementia, Eclipta alba, Nootropic, Piracetam

INTRODUCTION Pyramidal cells of the cortex and subcortex are affected.[6] Dementia is a mental disorder characterized by loss of cerebral activity sufficiently severe that interferes Nootropics, also referred as smart drugs, memory with one’s profession or social activities. It is several enhancers, neuro enhancers, cognitive enhancers, types and invariously destruction of memory. The and intelligence boosters, are drugs supplements, major cause of dementia is Alzheimer disease which nutraceuticals, and functional foods that eventually is progressive neurodegenerative disorders associated improve mental function.[7] Herbal plants are used with loss of neurons in distinct areas of the brain.[1,2] as medicines from ancient times, and it is already In recent decades, it has become one of the principle proved that it has lower side effects, and due pillars of a branch of science called cognitive to this reason, nowadays, it is attracting many neuroscience and interdisciplinary link between researchers to synthesize a new chemical entity cognitive and neurosciences.[3] using plant extracts, and for this reason, presently, it is widely used. We also focus on one of the herbal The central cholinergic pathway plays an important plants, i.e., Eclipta alba which contain various role in learning and memory process. Centrally acting phytochemical constituents in their extracts such muscarinic drugs (e.g., scopolamine) impair learning as alkaloids, anthracene glycosides, anthraquinones and memory both in animals and human beings.[4,5] It is associated with deposition of beta amyloid protein and carotenoids’, Coumadin’s, flavonoids, gallic in brain tissue and abnormal phosphorylation of tannins, saponins, triterpenoids, and volatile oils intracellular tau-protein, causing abnormalities of which are responsible for various pharmacological [8] microtubule assembly collapse of the cytoskeleton. activity. Plant extract of E. alba (100 mg/kg) had been orally Access this article online administered to the albino mice taken as a test group and piracetam (3.12 mg/kg approximately as per mice Website: jprsolutions.info ISSN: 0974-6943 body weight) referred to standard group for 3 days.

1-3Department of Pharmacology, 4Department of Pharmaceutical Chemistry, 5Department of Pharmaceutics Azad College of Pharmacy, Moinabad, Telangana, India

*Corresponding author: Maimunna Khathun, Azad College of Pharmacy, Moinabad, Telangana, India. E-mail: [email protected]

Received on: 15-06-2017; Revised on: 28-07-2017; Accepted on: 14-08-2017

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The nootropic activity of test group if compared in albino mice, in accordance with the OECD with the standard group it was shown better activity guidelines (423).[10] Acute toxicity studies in compared to standard. ethanolic extract of plant were performed in mice in each group (6 animals in each group) by following MATERIALS AND METHODS orally administering graded doses of 100 mg/kg, 200 mg/ kg, 1000 mg/kg and 2000 mg/kg, it was observed Plant Materials for 2 weeks. It was found that the ethanolic extract Plant E. alba was collected commercially from has produced significant toxicity at the dose of 2000 cultivator during the winter season. Mainly, the mg/kg as 2 animal of this group was died. Thus the aerial part of the plant was used, and it was washed extract was highly tolerable below 2000 mg/ kg for with normal tap water so that the stick dirt particles ethanolic extract. were removed, and finally, it was dried in shade. It was placed to size reduction by mechanical grinder Shock Device Delivering Scramble Foot Shock and makes it into a fine powder. About 50 g of dry Step-down method powder was taken in a Soxhlet apparatus, and first, it was extracted with 250 ml ethanol for about 8 Mice of either sex are used. A rectangular box days at 15-20°C, and further, it was extracted with (50 × 50 cm) with electrifiable grid floor and 35 cm 250 ml distilled water after the collection of marc. fits over the block. The grid floor is connected to a The marc left after successive extraction was taken shock device which delivers scramble foot shocks. out and dried it separately under room temperature The animals are divided into 3 groups, and each to get a dry mass, i.e., free of solvent. The both final group contains 10 animals and goes through different obtained extract was weighed, packed in a paper paradigm consists of three phases. The groups are as bags, and stored in airtight container at cool place follows: until use. • Group-I: Control group (effect of normal saline on Phytochemical Analysis mice) Preliminary phytochemical studies of ethanolic • Group-II: Test group (effect of E. alba 100 mg/kg extract of aerial parts of E. alba was performed on mice) for major classes of constituents such as alkaloids, • Group-III: Standard group (piracetam 3.12 mg/kg). glycosides, anthraquinones, steroids, Coumadin’s, The three paradigm phases are as follows: flavonoids, tannins, saponins, terpenoids, and volatile • Familiarization: The animal is placed on the oils.[9] platform, release after raising the cylinder and Preparation of Dosage Form the latency to descend is measured. After 10 s of exploration, it is returned to the home cage. For in vivo studies, both standard drugs Piracetam (3.12 mg/kg approximately as per mice body weight) Learning: Immediately after the animal has descend and the concentrated ethanolic extracts of Eclipta alba. from the platform, an unavoidable foot shock is were suspended in distilled water (100 mg/kg) and applied (foot shock: 50 Hz; 1.5 mA; 1 s), and the orally administered. The freshly prepared solution of animal is return to the home cage. both standard and test drug was used in experiment. • Retention test: 24 h after the learning trial, Test doses were selected on the basis of acute toxicity the animal is again placed on the platform, test on mice. and the step-down latency (SDL) is measured (Figure 1).[11] Animals Swiss albino mice (120-150 g) of either sex had been obtained from Shadan Institute of Medical Science, Hyderabad (11/CEAD/SES/2015) and housed 5 animals per cage made up of polypropylene, habituated at laboratory conditions for 2 weeks before experiment procedures.

The animals were fed with standard pellets and water ad libitum.

Acute Toxicity Study and Dose Selection The dose limits were selected on the basis of previously performed oral acute toxicity studies Figure 1: Step-down latency test

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RESULTS AND DISCUSSION time it is clear that test dose showing better retention time. Safety Study Result Indications recorded during acute toxicity studies Table 6 is representing the average retention time between untreated, standard and test dose and the By the acute toxicity test, it was found that the ethanolic average is taken from table 5 clearly mention that extract was non-toxic up to 2000 mg/kg. Hence, by standard and test dose showing higher learning considering the test, the dose was taken 100 mg/kg. time. The preliminary phytochemical test indicated that the extract contains alkaloids, anthracene glycosides, Table 7 is representing the comparison of time taken anthraquinones and carotenoids, Coumadin’s, for familiarization, learning, and retention time and it flavonoids, gallic tannins, saponins, triterpenoids, and was concluded from the table that time taken for step volatile oils. During observation studies, convulsions, down latency in test dose had shown remarkably more tremors, diarrhea, salivation, lethargy, sleep, and coma compare to untreated and standard. had been identified. Table 1: Familiarization (3‑30 s) day‑0 Step-down Method Result Control Standard group (s) Test group (s) Graph 1 showing differences between untreated, group (s) piracetam E. alba standard, and test, it was clearly seen from graph 4 9 4 that the standard and test dose showing similar 5 8 5 6 7 6 familiarization. 5 5 7 4 4 8 Graph 2 showing difference between learning time of 6 6 9 untreated, standard and test, It was clearly seen from 7 6 5 the graph that the test dose showing higher learning 9 5 6 8 7 7 time in the day 1. 6 4 4 Graph 3 representing average retention time of untreated, standard, and test groups and it shows that Table 2: Comparison of average familiarization time the test dose has higher retention time compare to of untreated group, standard group, and test group standard on day 2. Groups SDL (s) Untreated 6.0 Table 1 is representing the familiarization time Standard 6.1 between untreated, standard and test dose and from Test 6.1 the above time it is clear that standard and test dose SDL: Step‑down latency showing similar familiarization time. Table 3: Learning time –day 1 Table 2 is representing the average familiarization time between untreated, standard and test dose and Untreated (s) SDL Standard (s) Test drug (s) step‑down step‑down the average is taken from Table 1, it is clear that the latency latency standard and test dose showing similar familiarization 70 68 65 time. 66 69 70 65 65 75 Graph 4 showing that in each three phases of paradigm 67 67 67 the test dose is giving remarkable potent result 68 70 72 69 66 66 compare to standard. 64 65 64 67 64 63 Table 3 is representing the learning time between 63 68 69 untreated, standard and test dose and from the above 64 65 68 time it is clear that test dose showing better learning SDL: Step‑down latency time.

Table 4 is representing the average learning time Table 4: Comparison of average learning time of between untreated, standard and test dose and the untreated, standard and test groups average is taken from table 3 clearly mention that Groups SDL (s) standard and test dose showing higher learning time. Untreated 66.3 Standard 66.7 Table 5 is representing the retention time between Test 67.9 untreated, standard and test dose and from the above SDL: Step‑down latency

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Table 5: Retention time: Day‑2 Untreated group (s) Standard Test group (s) SDL group (s) SDL SDL 2 min 26 s 2 min 7 s 2 min 45 s 2 min 45 s 3 min 2 min 50 s 5 min 3 min 45 s 3 min 4 min 4 min 3 min 45 s 4 min 45 s 4 min 30 s 4 min 10 s 3 min 4 min 45 s 4 min 45 s 3 min 45 s 5 min 5 min Graph 1: Step down latency (Day-0 familiarization time) 4 min 2 min 3 min 47 s 4 min 45 s 2 min 45 s 2 min 45 s 2 min 2 min 50 s 2 min 50 s SDL: Step‑down latency

Table 6: Comparison of average retention time of untreated, standard, and test groups Groups SDL (s) Untreated 201.7 Standard 213.7 Test 218.6

Graph 2: Step down latency (Day-1 learning time) Table 7: Comparison of time taken for familiarization, learning, and retention time Groups Day‑0 (s) Day‑1 (s) Day‑2 (s) Untreated 6 66.3 201.7 Standard 6.1 66.7 213.7 Test 6.1 67.9 218.6

method was used for observing neurological behavior during research, and it is one of the most extensively used for screening of drugs affecting Graph 3: Step down latency (Day-2 retention time) learning and memory. This method is fast and highly reliable, reproducible, quick, and safe from safety studies.

The ethanolic extract has shown greatly significant results in all parameters, i.e., familiarization, learning, and retention time of nootropic activity compared to control and standard. Extract shown that there was a significant increase in SDL compared to untreated and standard and it can be efficiently use.

Graph 4: Comparison of day 0, day 1 and day 2 ACKNOWLEGMENT The author would like to express my gratitude to CONCLUSION all those who gave me the possibility to complete The aim of the present study was to investigate the this work, specially my family who always give me nootropic activity of ethanolic extract of E. alba support and encouraging me at every moment. I am plant using step-down method in Swiss albino mice. deeply indebted my guide. I have furthermore thanks The nootropic activity was conducted to evaluate to my friends. the phytochemical constituents of unexplored herbal plant which is evidence of their claimed REFERENCES pharmacological properties. The nootropic activity 1. American psychiatric association. Diagnostic and Statistical is due to phytochemicals present in the plant such Manual of Mental Disorders. Washington DC, USA: American as triterpenoids, flavonoids, saponins, alkaloids, Psychiatric Association; 1994. tannins, glycosides, and steroids. During toxicity test, 2. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of alzheimer’s disease: Report there are no signs of changes in animal neurological of the NINCDS-ADRDA work group under the auspices behavior up to the dose 2000 mg/kg. Step-down of department of health and human services task force on

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