Evaluation of Tecoma stans flowers on Scopolamine induced Zebrafish model

RESEARCH POSTER PRESENTATION DESIGN © 2019
www.PosterPresentations.com
“EVALUATION OF NEUROPROTECTIVE PROPERTY OF TECOMASTANS FLOWER
EXTRACT IN SCOPALAMINE INDUCED ZEBRAFISH MODEL AMNESIA”
Sweety Gupta1,Prashanth Prjapathi1,Sandeep Chandakavate1,Nanjappaiah H M 1,V P Patil1, Akram Naikwadi2 &
Shivakumar H1
1. Dept. of Pharmacology, BLDEA’s SSM College of Pharmacy and Research Centre, Vijayapur, Karnataka, India.
2. Dept. of Pharmacology, Sri B M Medical College, Hospital and Research Centre, Vijayapur, Karnataka, India.

Introduction:
Alzheimer's disease (AD) is a progressive neurodegenerative disorder manifested by mental deterioration and cognitive impairment in older
adults, accounting for 50% to 60% of dementia cases.
The various animal models like rat, mice and fruit fly (Drosophila melanogaster) are employed for the screening of nootropic drugs1.
Zebrafish (Danio rerio) is a promising in vivo model for induction of AD as well as to screen drugs to combat AD2 . Previous researchers
reported that Zebrafish exhibits high sensitivity to all major classes of clinically effective neurotropic drugs in a similar fashion like humans.
It has been also demonstrated that brain disorders such as depression, autism, psychoses and cognitive deficits have been successfully
investigated in Zebrafish in a cheaper and more convenient manner than in rodents3.
The genus Tecoma comprising of 14 species of small trees or shrubs belonging to the family Bignoniaceae. Out of the 14 species 12
originate in America and 2 originate in Africa. All the plants in this genus possess a common name Trumpet bush. Tecoma stans is fast
growing ornamental plant which grows throughout India4 . Tecoma stans is also known as yellow bells, yellow elder, trumpet flower in
English and Piliya in Hindi. Tecoma stans is an important medicinal plant and leaves, bark and roots contains biologically active chemicals,
and extracts from these parts were used in traditional folk medicines5.
Tecoma stans, exhibited antitumor, antioxidant, antimicrobial, hypoglycemic, free radical scavenging, anti-inflammatory and
antidiabetic properties. In the traditional therapeutic method, different parts of Tecoma stans have been mentioned to be helpful in a
variety of diseases6.All parts of the plant, and especially it’s contained more amount of the compound alkaloids it’s called tecomine and
tecostamine7.
However, upon exhaustive literature review finds no report on neuroprotective property of the title plant till date. Hence, the present
study is an attempt to investigate the neuroprotective activity of Tecoma stans flower extract in scopolamine induced amnesia in zebrafish.

Methodology:
Collection of plant material-
The Tecoma stans flowers were collected in the month of November 2020 from BLDEA’s Campus Vijayapur, Karnataka after the
identification and authentication. The flowers of the plant were then dried under shade for the period of 10 days at room temperature.
Then the dried flowers were ground to coarse powder. The powder was preserved in air sealed polythene cover for further extraction.
Preparation of extract -The powdered material was extracted with methanol by Soxhlet extraction procedure till solvent turns to colorless.
Thereafter, the extract was concentrated using rotary flash evaporator. The yield of the extract was found to be 20.8%. The dried extract
was stored in refrigerator below 100C for further studies.

Methodology:
Phytochemical investigation on METSF-
Determination of flavonoid content in Tecoma stans flower extract using thin layer chromatography (TLC) 8,9
The TLC of METSF was performed on 10cm × 1.5cm precoated aluminium backed silica gel plate GF254 plates Merck. METSF
samples were spotted using thin capillary tubes on TLC plates. The spotted plate was placed in mobile phase of solvent
system (Ethyl acetate: Formic acid: Glacial Acetic Acid: Water 100:11:11:26 v/v/v/v solvent). The plate was placed in a
development chamber with a trial solvent. The solvent front was allowed to travel until about 1 cm from bottom to the top
end. The derivatization was done by using 1% Ethanolic aluminum chloride solution spraying method. The components were
visualized using UV lamp fluorescent at 254nm. Different spots were observed and its corresponding Rf values were
calculated.

Methodology:
Table- Physical parameters and normal values for laboratory Zebrafish 10

Methodology:
Acute oral toxicity study11,12
Principle The fishes were exposed to the test extract (METSF) for a period of 96 hours. Mortalities of Zebrafish were recorded at 24, 48, 72
and 96 hours and the concentration which killed 50 per cent of the fish (LC50) are determined where possible.
Method – Acute oral toxicity of METSF was tested in the Zebrafish (Danio rerio) as per the OECD guidelines 203 (modified, adopted June
18, 2019). The different concentrations (12.5, 25, 50, 100, 200 and 400 mg/L) of METSF were selected for performing the Oral toxicity in
Zebrafish. 7 healthy fishes were selected to each concentration and transferred to beakers containing different concentration of METSF
solution. The fish were observed after 24, 48, 72, and 96 hours. The mortalities were recorded for every 24 hours and fish were considered
dead if there was no visible movement and upon mechanical stimulation could produce no reaction13. LC50 was determined based on the
concentration of the METSF which killed 50% of fish.
Administration of drug14- In the present study, animals were divided into five groups (Group I - V) designated as normal Control,
Scopolamine control (100 µM/L), Standard and METSF (50, 100, and 200 mg/L) treated groups containing of seven Zebrafish. The three
different doses of METSF were administered by immersion to Zebrafish (Danio rerio) into 500 ml glass beaker for 1hour. Whereas
scopolamine and imipramine were administered by immersion into 500ml glass beaker for 30min.

Methodology:
Evaluation of Scopolamine induced amnesia activity
1. Novel Tank Test (NTT)
2. Y – Maze Test
1. Novel Tank Test (NTT) 15,16
Principle- NTT is a specific test widely used for investigation of memory improvement in Zebrafish. This apparatus is used observe the
behavioral parameters and cognitive impairments in animals. The novel tank test measures locomotor and anxiety in Zebrafish
and is highly sensitive both to anxiolytic and anxiogenic drugs114. In this trapezoidal tank of 1.5 L measured 15.2cm (height),
27.9cm (top), 22.5cm (Bottom), and 7.1cm (width) and divides into two equal horizontal portions by a line draw on the external
surface of tank walls.
Method- Zebrafish weighing 1 - 1.2 g were selected and divide into six groups of seven each.

Methodology:
Continue..
Group I served as normal control received water as vehicle, group II served as scopolamine control administered with scopolamine (100
µM/L), group III served as standard treated with standard drug imipramine (20 mg/L), groups IV, V and VI received different doses of
METSF.
The behavioral parameters observed are
1. Number of entries to the top
2. Number of entries to the bottom
3. Time spent in top T
4. ime spend in bottom
5. Total distance travel in tank (m)
6. Latency to enter the top

Methodology:
Y – Maze Test17,18
Principle-The response to novelty in Zebrafish can be assessed using the Y-maze test. The position in the Y-maze test is considered as an
index of memory. The apparatus consisted of a Y-maze glass aquarium (3 L) with three arms (25 X 8 X 15 cm). On the exterior walls of the
Y-maze, recognizable geometric shapes such as squares, circles and triangles are placed. The Y-maze arms are arbitrarily assigned: (I) the
start arm, where fish started to investigate (always open), (II) the novel arm, which is blocked during the first trial, but opened during the
second trial, and (III) the other arm (constantly open). The Y-maze centre (neutral zone) was not counted.
Method- Zebrafish weighing 1 - 1.2 gm were selected and divided into six groups of seven each.
Group I served as normal control received water as vehicle, group II served as
scopolamine control administered with scopolamine (100 µM/L), group III served as standard treated standard drug Donepezil (10mg/L),
whereas groups IV, V and VI received different doses of METSF.

Methodology:
Continue..
Group I served as normal control received water as vehicle, group II served as
scopolamine control administered with scopolamine (100 µM/L), group III served as standard treated standard drug Donepezil (10mg/L),
whereas groups IV, V and VI received different doses of METSF.
The behavioral parameters observed were
1. Time spent in each arm
2. Number of entries to new arm
3. Total distance travelled (m)
Biochemical Parameters Assay19
The following biochemical parameters were estimated using UV Spectroscopy
1. Acetylcholinesterase (AChE)
2. Superoxide dismutase (SOD)
3. Catalase (CAT)
4. Malondialdehyde (MDA)

Results and Discussion:

Conclusion:
The results of the current research work conclude that the methanolic extract of Tecoma stans flower exhibited neuroprotective effect
against scopolamine induced zebrafish model of amnesia.
Further scope However further study is necessary to isolate and active compounds responsible for this neuroprotective activity & to find out
exact mechanism of anti- amnesic effect.

References:
1. Rishitha N, Muthuraman A. Therapeutic evaluation of solid lipid nanoparticle of quercetin in pentylenetetrazole induced cognitive
impairment of Zebrafish. Life Sci. 2018; 199: 80 - 87.
2. Baser KHC.“Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils”.Current Pharmaceutical Design.
2008; 14(29): 3106 - 3119.
3. Jukic M, Politeo O, Maksimovic M, Milos M. “In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives
thymoquinone and thymohydroquinone”. Phytotherapy Research. 2007; 21(3): 259 - 261.
4. Anburaj G, Marimuthu M, Sobiyana P, Manikandan R. A Review on Tecoma stans. International journal of engineering research and
modern education. 2016; 1(1): 43 - 9.
5. Verma S. Phytochemical and pharmacological review study on Tecoma stans Linn. Journal of Medicinal Plants Studies. 2016; 4(5): 162 - 4.
6. Pelton J. A survey of the ecology of Tecoma stans. Butler University Botanical Studies. 1964; 53 - 88.
7. Lins AP, Felicio JD. Monoterpene alkaloids from Tecoma stans. Phytochemistry. 1993; 34(3): 876 - 8.
8. Lihua G, Tao W, Zhengtao W. TLC bioautography-guided isolation of antioxidants from fruit of Perilla frutescens var. acuta. LWT-Food Sci
Technol. 2009; 42: 131 – 136.
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Pharmacogn. Phytochem. Res. 2017; 9: 1051 -1059.
10. Zebrafish in biomedical research; biology, husbandry, diseases and research applications. american college of laboratory animal
Medicine series. 2020.
11. OECD (2019) guidelines for the testing of chemicals of effects on biotic systems. test guideline no. 203 fish, acute toxicity testing. 2019; 1
- 24.
12. Guidelines of CPCSEA for Experimentation on Fishes 2021.
13. Santos IVF, Duarte JL, Fernandes CP, Keita H, Amado JRR, VelazquezMoyado JA, et. al. Use of Zebrafish (Daniorerio) in experimental
models for biological assay with natural products. Afr. J. Pharm. Pharmacol. 2016; 10: 883 – 891.

References:
14. Capatin L, Boiangu RS, Dumitru G. Rosmarinusoffcinalis essential oil improves scopolamine-induced neurobehavioral changes via
restoration of Cholinergic function and brain antioxidant status in Zebrafish (Daniorerio). Antioxidants. 2020; 62: 1 – 14.
15. Dumitru G, El-Nashar HAS, Mostafa NM., et. al. “Agathisflavone isolated from Schinuspolygamus (Cav.) cabrera leaves prevents
scopolamine-induced memory impairment and brain oxidative stress in Zebrafish (Daniorerio). Phytomedicine. 2019; 58: 1 - 8.
16. Weller J, Budson A. Review current understanding of Alzheimer’s disease diagnosis and treatment. Research. 2014; 370(4): 311 – 321.
17. Tripathi KD. Essentials of medical pharmacology, 8th Ed, New Delhi; Jaypee Brothers. 2019; 517.
18. Cognato GD, Bortolotto JW, Blazina AR, Christoff RR, Lara DR, Vianna MR, Bonan CD. Y-Maze memory task in Zebrafish (Daniorerio): The
role of glutamatergic and cholinergic systems on the acquisition and consolidation periods. Neurobiology of learning and memory. 2012;
98(4): 321 - 328.
19. Batista FLA, Lima LMG, Abrante IA, Araújo JIF, Magalhães, EA, Lima DR, et.al. Anti-nociceptive activity of ethanolic extract of
Azadirachtaindica A. Juss (Neem, Meliaceae) fruit through opioid, glutamatergic and acid-sensitive ion pathways in adult Zebrafish (Danio
rerio). Biomed. Pharmacother. 2018; 108: 408 – 416.

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