Anti-Amnesic effect of methanolic extract of Tagetes erecta flower heads on Aluminium Induced Cognitive Impairment in Albino Mice

 

Dr. M. Ganga Raju*, Srilakshmi S

Department of Pharmacology, Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad, Telangana State.

*Corresponding Author E-mail: mgrpharma@gmail.com

 

ABSTRACT:

Objective: Anti-amnesic activity of methanolic extract of Tagetes erecta flower heads were carried out using various in-vitro and in-vivo models.  Methods: Preliminary Phytochemical screening of Tagetes erecta extract was carried out by using standard tests. Acute toxicity studies were performed as per OECD-425 guidelines.

In-vivo anti-amnesic activity was evaluated in aluminium chloride induced amnesia (chronic) using Cook’s pole climbing apparatus and elevated plus maze. Various biochemical parameters like anticholinesterase (AChE) using Ellman’s assay and thiobarbituric acid reactive species (TBARS) were also estimated. Histopathological studies of brain were carried out in AlCl3 induced amnesic model. Results: The acute toxicity studies revealed that the extract was safe upto 2000 mg/kg, bd.wt. The METE at two doses levels 200 and 400 mg/kg, bd. wt reversed the memory deficit induced by aluminium in mice models. METE significantly (p<0.05) reduced the brain AChE activity and TBARS level. Conclusion: The presence of active constituents like flavonoids, terpenoids, steroids and phenols in methanolic extract of flower heads of Tagetes erecta might be responsible for its anti-amnesic and anti-cholinesterase activity.

 

KEYWORDS: Tagetes erecta, anti-amnesic, AChE inhibition, TBARS, aluminium chloride

 

 


INTRODUCTION:

Dementia is an umbrella term that covers different types of clinical syndromes such as Alzheimer’s disease (AD), vascular dementia, dementia with Lewy bodies, frontotemporal dementia (FTD) and Parkinson’s disease etc1. Alzheimer’s disease is associated with brain shrinkage and localised loss of neurons, mainly in the hippocampus and basal forebrain. The loss of cholinergic neurons in the hippocampus and frontal cortex is a feature of the disease, and is thought to underlie the cognitive deficit and loss of short-term memory that occur in AD.

 

 

The brain is especially vulnerable to damage from oxidative stress because of its high oxygen consumption rate, abundant lipid content, and relative paucity of antioxidant enzymes compared to other organs. In neurons, oxidation can result in numerous problems, including up regulation of proinflammatory cytokines and irreparable DNA damage. Oxidative stress is thought to be important early in AD progression because it is temporally linked to the development of plaques and NFTs.

 

Tagetes is a genus of annual or perennial, mostly herbaceous plants in the sunflower family (Asteraceae). Flowers of Tagetes erecta Linn used traditionally from ancient times and are used in folk medicine to cure various types of diseases. The flower are used to cure fever, epileptic fits according to Ayurveda, They are said to purify blood and flower juice is given as a remedy in cold, rheumatism and bronchitis2, 3.

 

Aluminium metal is abundantly present in the earth’s crust. Aluminium has been implicated in aging-related changes and neurodegenerative diseases. Aluminium promotes the formation of amyloid-β protein plaques4. Aluminium causes disturbances in cholinergic neurotransmission and disrupts the cognitive behaviour of animals by increasing the stress levels. The elevations in AChE are either direct result of neurotoxic effect of metals or due to increased lipid peroxidation. Aluminium causes marked oxidative damage by increasing the redox active iron concentration in the brain mainly via the Fenton reaction5. Thus, the present work focused on evaluating the Anti-amnesic effect of Tagetes erecta against aluminium induced cognitive impairment.

 

2. MATERIALS AND METHODS:

2.1 Preparation of extract:

The powdered crude material (200 g) was extracted with methanol by Soxhlation. At the end of the extraction process extract obtained was filtered and evaporated to solid mass. The extract was preserved in desiccators to remove remaining moisture, if present, and finally stored in air tight containers for further use.

 

2.2 Identification of phytochemical constituents:

Phytochemical screening of Tagetes erecta extract was carried out by using standard tests.

 

2.3 Acute toxicity testing:

Acute toxicity study was carried out in order to check the toxic effects of methanolic extract of Tagetes erecta flowers. Acute toxicity studies were carried out as per the OECD 425 guidelines.

 

2.4 Experimental animals and diets:

Adult Swiss albino mice (20-25 g) were used for the present study. They were kept in polypropylene cages at 25 ± 2° C, with relative humidity 45-55% under 12 h light and dark cycles. All the animals were acclimatized to the laboratory conditions for a week before use. They were fed with standard animal feed and water ad libitum. All the pharmacological experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) with (Reg. No. 1175/PO/ERe/S/08/CPCSEA).

 

2.5 In vivo methods for evaluation of Anti- amnesic activity:      

The in vivo evaluation of anti-amnesic activity of the methanolic extract of Tagetes erecta flower heads was evaluated in Aluminium chloride induced (chronic) amnesic model using Cook’s pole climbing apparatus & Elevated plus Maze.

 

1. Aluminium chloride induced (chronic) amnesic model:

30 Healthy Swiss albino mice of either sex weighing 20-25 g were selected and these animals were divided into 5 groups (6 in each group) as follows6.

 

Study design for Aluminium chloride induced (chronic) amnesic model

S. No

Groups

Treatment

1

Group – I

Saline

2

Group – II

Disease control received AlCl3 (100 mg/kg bd.wt, p.o) for 1-42 days

3

Group – III

AlCl3 (100 mg/kg, p.o) for 1-41 days + METE (200 mg/kg bd.wt, p.o) for 21-41 days

4

Group – IV

AlCl3 (100 mg/kg, p.o) for 41 days + METE (400 mg/kg bd.wt, p.o) for 21-41 days

5

Group – V

AlCl3 (100 mg/kg, p.o) for 41 days + Donepezil (1mg/kg bd.wt, p.o) for 21-41 days

 

a. Cook’s pole climbing method:

The basic operational mode of this method is that following an auditory warning stimulus, the animal learns to avoid the foot shock delivered through the cage floor by jumping to pole. This method has long been accepted as a reliable technique to evaluate learning and memory in experimental animals. The training and testing of mice was conducted in 25 X 25 X 40 cm chamber that is enclosed in a dimly light, sound attenuating box. The mice had learned to jump on a pole to avoid foot shock. A tone 50 Hz was used as a conditional stimulus and foot shock of 1 mA - 2 mA was the unconditioned stimuli. In the training procedure, the animal was initially allowed to adopt in the chamber for 1 min. This was followed, in succession by conditioned and unconditioned stimuli for a period of 10 sec each. The trial ended either after the animal responded by jumping on the pole or after of 10 seconds each. The trial ended either after the animal responded by jumping on the pole or after 30 secs, which ever was earlier the animal was given such trial every day for 10 days, mice were initially trained to escape the foot shock by climbing on the pole, i.e. the shock free zone and only those mice, which could climb the pole and escape the foot shock were included in the study. Retention of the memory of the painful stimuli established in learning procedure was tested before and after drug treatment. It was quantified as the percentage of animals avoiding shock by jumping on the pole 7.

 

b. Elevated Plus Maze:

EPM is extensively used to examine cognition in rodents. The Plus maze apparatus is based on the innate aversion of rodents to open and high space. The apparatus has a central platform of 5 cms connected to two open arms (15 cms × 5 cms) and two closed arms (15 cms × 5 cms × 12 cms), bisecting each other. The maze was raised to a height of 25 cms from the ground. On 20th day, 30 min after METE administration, each mouse was gently placed at the distal end of an open arm, facing away from central platform. The time taken by the mouse to enter either of the closed arms with all four paws from open arm is designated as transfer latency time (TLT). If the mice do not enter either of the closed arms within 90 secs, it is gently pushed into one of the two closed arms and the TLT was assigned as 90 secs. The mouse was allowed to explore the maze for 10 s and then was returned to its home cage. TLT was again recorded after 24 h (retention test) and on the 42nd day. Decline in TLT indicates memory enhancing effect of extract 1.

 

2.6 In vitro AChE inhibition assay:

The prepared extract were screened for AChE inhibitory activity using method of Ellman et al (1961)8.

 

2.7 Histopathological studies:

One rat each from five groups was sacrificed by decapitation, the brain was isolated by clearing its extraneous connections and stored in 10 % formalin solution; these specimens were used for histopathological studies.

2.8 Statistical analysis:

Graph Pad prism 7 software and MS excel was used for statistical analysis of data. All the results were expressed as mean±standard error of mean (SEM), analyzed for ANOVA and Dunnett’s t test (Multiple). Differences between test groups and standard group were considered significant at p<0.05, p<0.01 levels.

 

3. RESULTS:

3.1 Preliminary phytochemical analysis:

The preliminary phytochemical investigation of methanolic extract of Tagetes erecta flower heads showed the presence of terpenoids, flavonoids, phenols, steroids and alkaloids.

 

3.2 Acute Toxicity Studies:

The methanolic extract of Tagetes erecta did not exhibit any signs of toxicity and mortality even upto 2000 mg/kg. bd.wt. All animals were safe even after 14 days of observation. The doses selected for the present study are 200 and 400 mg/kg bdwt

 


 

3.3 Aluminium chloride induced amnesic model:

TABLE. 1: Effect of METE on passive avoidance time using cook’s pole climbing apparatus in Aluminium chloride induced amnesic model.

Groups

Treatment

Time taken to climb the pole (secs)

20th day

21st day

42nd day

I

Normal Control

21.83± 0.30

21.16 ± 0.79

19.16 ±0.83

II

Negative control (AlCl3 100 mg/kg)

34.5± 0.67** A

36.3± 0.68** A

42.5 ± 0.42** A

III

METE (200 mg/kg)

28.83±1.24**A, a

27.5± 0.84** A, a

25.5± 0.56** A, a

IV

METE (400 mg/kg)

26.5± 0.84** a

25.66 ± 0.33** B. a

23.83±0.47** B, a

V

Donepezil (1mg/kg)

25 ± 0.36* a

23.83 ± 0.30* b

21.66 ± 0.33* A

Values were expressed as mean ± SEM (n=3). Statistical analysis was performed by using ANOVA followed by Dunnet’s t-test by comparing with control, negative control & standard. Significant values are expressed as control group (**p<0.01), negative control (a=p<0.01) & standard (A=p<0.01, p<0.05)

 

B. Evaluation of METE on transfer latency time using elevated plus maze in in Aluminium chloride induced amnesic model.

 

TABLE.2: Effect of METE on Transfer latency time using elevated plus maze

Groups

Treatment

Transfer latency time (secs)

20thday

21stday

42ndday

I

Normal Control

20.83± 0.47

20.16 ± 0.47

18.16 ±0.49

II

Negative control (AlCl3 100 mg/kg)

33.36± 0.71** A

35.33± 0.66** A, a

46.33 ± 0.71** A, a

III

METE (200 mg/kg)

29.66±1.22** B, a

28.0± 0. 93** A, a

25.83 ± 0.87** A, a

IV

METE (400 mg/kg)

28.00± 1.12** a

26.1 ± 0.47** A, a

23.66 ± 0.33** B, a

V

Donepezil (1mg/kg)

25.66 ± 0.88** a

23.1 ± 0.47** A

21.3± 0.33** A

Values were expressed as mean ± SEM (n=6). Statistical analysis was performed by using ANOVA followed by Dunnet’s t-test by comparing with control, negative control & standard. Significant values are expressed as control group (**p<0.01), negative control (a=p<0.01) & standard (A=p<0.01, B = p<0.05).

 


Biochemical Estimations:

Effect of METE on AChE activity and oxidative stress (TBARS) in brain in aluminium chloride model

TABLE.3: Effect of METE on AChE activity and oxidative stress (TBARS) in brain in aluminium chloride model

Groups

AChE activity (nM/min/mg of protein)

TBARS (nM/ mg of protein)

Control

0.60 ± 0.008

7.17± 0.02

Negative Control

1.31 ± 0.02** A

16.09± 0.04** A

METE (200 mg/kg)

0.89± 0.017**, A, a

10.68±0.02**, A, a

METE (400 mg/kg)

0.83 ± 0.012**,B, a

9.35±0.01**,A, a

Donepezil (1mg/kg)

0.75 ± 0.012**,a

8.91±0.01**,A

Values were expressed as mean ± SEM (n=3). Statistical analysis was performed by using ANOVA followed by Dunnet’s t-test by comparing with control, negative control & standard. Significant values are expressed as control group (**p<0.01), negative control (a=p<0.01) & standard (A=p<0.01, p<0.05).


3.4 In vitro AChEInhibition:

TABLE. 4: AChE inhibition of methanolic extract of Tageteserectaflower heads

S. No

Compounds

Concentrations (µg/mL)

% Inhibition

IC50Values

1.

METE

10

20

30

40

50

9.78±1.61

21.84±2.26

46.04 ±1.69     57.49±1.17      77.7±1.42

34.5

2.

Donepezil

10

20

30

40

50

13.05±0.86

23.84±0.86

43.78±0.84

64.17±0.56

84.31±1.13

31.5

 

Since AD pathogenesis is potentially attributed to acetylcholine decline and Aβ formation, the anti-amnesic effects of METE was evaluated by inhibition of AChE (Jung et al., 2010). METE exhibited AChE-inhibitory activity with IC50 values of 34.5 μg/ml and donepezil exhibited the AChE inhibition with an IC50 value 31.5 μg/ml, respectively. These results indicate that METE may reverse aluminium chloride induced cognitive impairment through inhibition of cholinesterase enzyme as implicated in Alzheimer's disease.

 

Histopathology Studies:

Histopathology study of hippocampus region of mice brain in aluminium chloride induced chronic amnesic model. The pathological changes were viewed under light microscope after staining with haematoxylin and eosin.

 

 

Fig. 1.Control group:

 

Control animal showed five to six layers of compactly arranged small pyramidal cells with vesicular nuclei in CA1 region of Hippocampus

 

 

Fig. 2.Aluminium chloride (100 mg/kg bd. wt)

Two to three layers of shrinked pyramidal cells of CA1 region of hippocampus with irregular arrangement and most of the nucleus appears pale in colour was observed group II mice brain.

 

 

Fig. 3: METE (200 mg/kg, bd. wt)

 

Increased layer of pyramidal cells with dark prominent nucleus appears in CA1 region of hippocampus. Arrangement of neurons is still improper in group III mice brain.

 

 

Fig. 4: METE (400 mg/kg bd. wt)

 

Four to five layers of pyramidal cells with regular arrangement at deeper layer was observed in CA1 region of hippocampus in group IV micebrain.

 

 

Fig. 5: Donepezil (1 mg/kg bd. wt)

 

Five to six layers of regularly arranged pyramidal cells with prominent nucleus appearing in CA1 region of hippocampus in group V mice brain.

 

4. DISCUSSION:

In the present study methanolic extract of Tagetes erecta flower heads was evaluated for anti-amnesic activity by using aluminium induced amnesic animal models and various behavioural parameters like passive avoidance and transfer latency time were evaluated. Apart from the above parameters, the biochemical parameters like AChE levels and TBARS levels are also estimated in brains of mice in aluminium induced amnesic model.

 

Aluminium causes disturbances in cholinergic neurotransmission and disrupts the cognitive behaviour of animals by increasing the stress levels. The elevations in AChE are either direct result of neurotoxic effect of metals or due to increased lipid peroxidation. Aluminium causes marked oxidative damage by increasing the redox active iron concentration in the brain mainly via the Fenton reaction 5. Thus, reduced cholinergic activity due to the neurodegeneration in cortical regions is one of the consequences of ailment.

 

During the study chronic exposure to AlCl3 results in memory impairment indicated by the increase in latency and passive avoidance time and increased AChE and TBARS levels in brain. But at the end of the study the cognitive functioning is found to be improved by decreased latency and passive avoidance time in the animals treated with extract and the standard drug. The biochemical parameters such as AChE and TBARS levels were also reduced which indicates acetylcholinesterase inhibition and antioxidant potential which can be the possible mechanism to reduce amnesia.

 

Histopathological results clearly indicated the improvement in number of layers of pyramid cells with vesicular nuclei in CA1region of hippocampus in animal treated with extract and standard drug Donepezil.

 

The various phytochemicals identified in the methanolic extract of Tagetes erecta flower heads are phenolics like (syringic acid and gallic acid), terpenoids like (β- amyrin and erythrodiol), flavonoids (quercetin, kaemferol), steroids (β- sitisterol, stigma sterol), caretonoids (lutein and zexanthin) and tocopherol (α and β tocopherol)9.   Improvement in cognitive function might be due to the presence of phytochemical constituents which may act through inhibiting AChE. AD has great impact on the personal and social life of human beings and no doubt cholinesterase inhibitors offer great help in the effective management and treatment of AD.

 

5. ACKNOWLEDGEMENTS:

The authors are grateful to the Principal and the Management of the Gokaraju Rangaraju College of Pharmacy, for the constant support and encouragement during the course of the work.

 

6. CONFLICT OF INTEREST:

The authors have no conflict of interest.

 

7. REFERENCES:

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2.     Nadakarni KM. The Indian MateriaMedica. Popular prakashan. Bombay. 1954.

3.     Kadam PV, Bhingare CL, Sumbe RB, Nikam RY, Patil MJ. Pharmacocognostic, Physicochemical and Phytochemical investigation of Tagetes erecta Linn flowers (Asteraceae). Journal of Biological and Scientific Opinion. 2013; 1(1): 21- 4.

4.     Kawahara M, Kato M, Kuroda Y. Effects of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of beta-amyloid protein. Brain Research Bulletin. 2001; 55:211–217

5.     Rani A, Neha, Sodhi R and Kaur A. Protective effect of a calcium channel blocker “diltiazem” on aluminum chloride-induced dementia in mice. Naunyn-Schmiedeberg's Archives of Pharmacology. 2015;388(11):1151-1161.

6.     Thippeswamy A, Rafiq M, Viswantha G, Kavya K, Anturlikar S and Patki P. Evaluation of Bacopa monnierafor its Synergistic Activity with Rivastigmine in Reversing Aluminum-Induced Memory Loss and Learning Deficit in Rats. Journal of Acupuncture and Meridian Studies. 2013;6(4):208-213.

7.     Kannan R, Sivaraman D, Muralidharan P and Deepak  venkataraman N. Neuroprotective effect of hydroalcoholic extract of Areca catechu Linn on β-amyloid (25-35) induced cognitive dysfunction in mice. International journal of Research in Ayurveda & Pharmacy. 2013;4(5):747-753.

8.     Ellman G, Courtney K, Andres V and Featherstone R. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology. 1961;7(2):88-95.

9.     MH Kumar, YK Gupta. Antioxidant property of Celastrus paniculatus Willd. A possible mechanism in enhancing cognition Phytomedicine. 2002;9 (4) : 302-311.

 

 

 

Received on 14.08.2018          Modified on 25.09.2018

Accepted on 11.10.2018  ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 299-303.

DOI: 10.5958/0975-4385.2018.00048.1