Neuroprotective Property of Tecoma Stans Flower Extract in Scopolamine Induced Amnesia and Chlorpromazine Induced Rigidity in Laboratory Animals

Introduction

The Alzheimer's and Parkinson's disease (PD) are associated with slow death of neurons resulting in loss of sensory and cognitive functions.¹ Pathological processes such as oxidative stress, apoptosis, and mitochondrial dysfunction lead to neuronal deterioration in Alzheimer’s disease (AD).² The research reports documented that lipid peroxidation may cause destruction of cholinergic neurons in AD³ and dopaminergic neurons in PD.⁴ The low antioxidant activity of the brain tissue makes it liable to oxidative damage because the brain contains high levels of polyunsaturated fatty acids and is more sensitive to peroxidation reactions.⁵ Clinically, PD may cause slowness of movement, muscle rigidity, and rest tremor.⁶ The PD can be treated with levodopa and carbidopa which act by reversing the symptoms of PD. However, long-term usage of these drugs produces side effects such as hallucinations, convulsions, anxiety and transient dizziness.⁷

The use of herbal drugs in the present scenario is increasing daily because allopathic drugs have more side effects. Also, the presence of chemical constituents in herbal drugs have contributed significantly to the development of new drugs.⁸

Tecoma stans Linn belongs to the family Bignoniaceae originating in India and is commonly known as ‘yellow bells’ and ‘trumpet flower’. It is an important medicinal plant exhibiting anticancer,⁹ antioxidant,¹⁰ antimicrobial,¹¹ anti-proliferative¹⁰ properties. Each of the part of the plant has been used in treating a variety of diseases in traditional medicinal system. Different parts of plant contain alkaloids namely tecomine and tecostamine.¹² In traditional system of practice, the flower and bark were used for their anti-proliferative, wound healing, cytotoxic, antimicrobial, antifungal properties and for treating various cancers.

However, the neuroprotective efficacy of flowers of this plant with respect to learning, loss of memory, motor coordination and muscle grip strength in animal models resembling to Alzheimer’s disease and Parkinson’s syndrome has not been validated scientifically till date. Hence, the present research was designed.

Materials and Methods

Authentication of plant material by the Botanist The flower of Tecoma stans was identified and authenticated by Mrs. Gangambika Biradar, Professor, Botany Dept., KCP Science College, Vijayapur. Later required quantities of Tecoma stans flowers were obtained from the BLDE University garden, Vijayapur.

Extraction of plant material

For this study, the collected flowers were shade dried and grounded to coarse powder. The coarse powder was then extracted with methanol by Soxhlet’s extraction method. Later, using rotary flash evaporator, the extract was concentrated. The yield of the obtained extract was 20.8%.

Preliminary phytochemical screening¹³

The screening of phytochemicals present in crude extract was done by reported methods mentioned in Practical Pharmacognosy by Kandelwal.

Institutional Animals Ethics Committee (IAEC) clearance

The research work was approved by IAEC before initiation of the experiment bearing approval number IAEC No.: BLDE/BPC/2019-20/645, dated 21.09.2019.

Acute toxicity study¹⁴

The guideline No.423 of Organisation for Economic Co-operation and Development (OECD) was adapted for acute toxicity study of methanolic extract of Tecoma stans flowers (METSF) in female albino mice weighing 20-30g. The METSF at a dose of 2000 mg/kg i.p. did not cause any mortality. Hence, 2500 mg/kg was taken as LD50 cutoff value.

Evaluation of plant extract for neuroprotective property in experimental animals

The neuroprotective activity of METSF was evaluated against various experimental models in animals as mentioned under:

1. Motor coordination test using Rotarod Apparatus (RA)

2. Memory improving activity using Elevated Plus Maze (EPM)

3. Learning and memory study using Morris Water Maze (MWM)

Motor coordination test by rotarod apparatus^15,16

Rats were allocated into six groups of six animals each.

G - 1: Vehicle 1% gum acacia

G - 2: Chlorpromazine 3 mg/kg, i.p. for a period of 21 days

G - 3: Carbidopa + Levodopa (1:10 ratio) (10 mg/kg,i.p.).

G - 4: METSF 100 mg/kg orally 21 days

G - 5: METSF 250 mg/kg orally 21 days

G - 6: METSF 500 mg/kg orally 21 days

Chlorpromazine 3 mg/kg, i.p. was administered 30 minutes before the administration of standard and METSF for a period of 21 days.

The muscle grip strength was recorded on 0 day, 7th day, 14th day and 21st day using rotarod test. After the 21st day, the brains were removed from sacrificed animals and weighed. The lipid peroxidation (LPO), glutathione (GSH), nitrites and total protein were measured in the brain tissue homogenate.

Assessment of brain LPO¹⁷

The LPO content was determined as per the method described by Ohkawa H et al. 

Assessment of brain GSH¹⁸

The brain content of GSH was estimated by a procedure described by Moron MS et al.

Assessment of brain nitrites¹⁹

The brain content of nitrites was estimated using a procedure described by Lidija R et al.

Estimation of brain total protein²⁰

Lowry method was applied for the measurement of protein content of brain.

Memory improving activity in EPM^19-21

Mice were allocated into six groups comprising of six animals in each group.

G 1: Normal saline for 10 days

G 2: Scopolamine (SCP) 10 days

G 3: SCP and Donepezil for 10 days

G 4: SCP and METSF for 10 days

G 5: SCP and METSF for 10 days

G 6: SCP and METSF for 10 days

Dose: SCP 0.3 mg/kg, i.p., Donepezil 1 mg/kg, p.o., METSF G 4 100, G 5 250 and G 6 500 mg/kg, p.o., respectively. 

Behavioral study

EPM serves as the exteroceptive behavioral model to evaluate memory acquisition and retention. On day 1, each mouse was assigned at the end of an open arm, facing away from the central platform. Transfer latency (TL) was documented on the first day (i.e. 10th day of drug administration) for each animal. If the animal did not enter into one of the closed arms within 90 seconds, it was gently pushed into one of the two closed arms and TL was assigned as 90 seconds. The mouse was allowed to explore the maze for another two minutes and then returned to its home cage. Retention of this learned-task (memory) was examined 24 h (11th day) after the first day trial.

Memory retention was calculated after 24 hrs of acquisition trial (on the day 1) as inflation ratio using the following formula:

Inflation ratio (IR) = L1- L0 / L0

Where, L0 is initial LT on day 1 (i.e. 10th day of drug administration) in seconds and L1 is the LT after 24 hrs (11th day) of acquisition trial (11 days).

Estimation of Acetylcholinesterase²²

After behavioral assessment, the mice were sacrificed, brain was isolated and weight was recorded. Then the brain tissue was homogenized in phosphate buffer and was centrifuged at 3000 rpm for 10 min. Later the supernatant was separated and used for the estimation of acetylcholinesterase in brain tissue using Ellman et al. method.

Scopolamine employed amnesia in MWM test

Mice were allocated into six groups containing six animals each.

G 1: Normal saline only

G 2: Scopolamine (SCP) 0.3 mg/kg, i.p.

G 3: SCP + Donepezil

G 4: SCP + METSF

G 5: SCP + METSF

G 6: SCP + METSF

Dose: SCP 0.3 mg/kg, i.p., Donepezil 1 mg/kg, p.o., METSF G 4 100, G 5 250 and G 6 500 mg/kg, p.o., respectively

After one week of training period (first seven days), from 8th day to 21st day, the G 2 animals received SCP, i.p., G 3 animals received SCP + Donepezil, G 4 to G 6 received SCP and different doses of METSF. Later learning and memory was evaluated using the MWM test. On 7th day, the results were documented which the mice were placed into the maze 180° from the platform. The time taken by the mice to reach the platform was recorded as initial acquisition latency (IAL). On 14th and 21st day, the retention transfer latency (RTL) was recorded. The actual trial was conducted 30 min after the treatment. On 21st day after recording of RTL, the mice were sacrificed and brains were isolated. The isolated brains were further homogenized in phosphate buffer at pH 7.4. The homogenate obtained was centrifuged for 15 min. The supernatant was used for the estimation of brain acetylcholinesterase.²² 

Lipid peroxidation (LPO-MDA)¹⁷

The LPO content was determined as per the method described by Ohkawa H et al.

Statistical analysis

The data obtained from the research were subjected to statistical analysis using one-way ANOVA test followed by Turkey Kramer multiple comparison test.

Results

Phytochemical screening

The METSF showed the presence of carbohydrates, alkaloids, tannins, flavonoids, saponins and phenolic compounds.

Acute toxicity

The METSF did not cause mortality of the animals at a dose of 2000 mg/kg. Hence, 2500 mg/kg was taken as LD50 cutoff value. Screening doses chosen for the activity were:

125 mg/kg - 1/20th of 2500 mg/kg b.w.

250 mg/kg - 1/10th of 2500 mg/kg b.w.

500 mg/kg - 1/5th of 2500 mg/kg b.w.

METSF effect on muscle rigidity in rotarod test

Muscle rigidity was determined in the rotarod apparatus. The positive control animals showed decreased mean fall off time on 0 day, 7th day, 14th day and 21st day when compared to normal control animals. The rats treated with combination of Carbidopa + Levodopa showed a significant increase in muscle grip activity on the 0 day, 7th day, 14th day and 21st day when compared to G 2 animals, whereas pretreatment with METSF at different doses showed a significant increase in the muscle grip activity on 0 day, 7th day, 14th day and 21st day as compared to G 2 animals. The results are presented in Table 1. 

METSF effect on brain LPO and Nitrites in rotarod test

The G 2 animals treated with CPZ showed increas in LPO and nitrite levels in brain (nM/mg protein) as compared to G 1 animals. Pretreatment with standard and test extract at different doses showed a significant reduction in levels of LPO in brain and nitrite levels when compared to G 2 animals. The results are represented in Table 2.

METSF effect on brain GSH and Total protein in rotarod test

The animals administered with CPZ (G 2) showed a substantial decrease in GSH and total protein levels in brain (nM/mg protein) as compared to G 1 animals. Pretreatment with Carbidopa + Levodopa and METSF at different doses showed a substantial increase in levels of GSH and total protein when compared to G 2 animals. The results are indicated in Table 3.

METSF effect on memory performance in EPM

The administration of SCP increased TL in animals, representing its amnesia as compared to G 1 animals. The administration of standard Donepezil and METSF of various doses for 10 days did not affect TL of mice on 10th day (learning) as matched to the G 1 animals. But on 11th day, Donepezil and METSF of various doses significantly reversed (decreased the TL) SCP induced memory impairment in mice as matched to G 2 animals. The results are tabulated in Table 4. 

METSF effect on brain Acetylcholinesterase in EPM

The augmented level of brain Acetylcholinesterase was seen in SCP treated animals as compared to G 1 animals. Treatment with different doses of METSF and Donepezil produced a decrease in brain Acetylcholinesterase as matched to G 2 animals. The results are indicated in Table 5.

METSF effect on SCP induced impairment of learning and memory in MWM test

The SCP administered mice exhibited significant increase in the Escape Latency Time (ELT) as compared to G 1 animals. However, the treatment with various doses of METSF and Donepezil was found to decrease the ELT compared to G 2 animals. The results are shown in the Table 6. 

Effect of METSF on brain Acetylcholinesterase

Administration of SCP caused increased levels of brain Acetylcholinesterase when matched to G 1 mice. The brain Acetylcholinesterase level was found to decrease in standard and METSF treated groups as matched with the SCP administered group. The results are indicated in Table 7.

METSF effects on brain MDA

SCP treated animals suggestively increased the brain MDA levels matched to the G 1 animals, indicating increased oxidative stress. The administration of standard and different doses of METSF significantly restored the brain MDA level as compared with G 2 animals. The results are presented in Table 7.

Discussion

This research work aimed to evaluate the neuroprotective efficacy of METSF in cholopromazine induced muscle rigidity using rotarod apparatus and SCP produced memory alterations using EPM and MWM in investigational animals.

Chlorpromazine is an antipsychotic drug discovered after World War II. Administration of Chlorpromazine blocks dopamine D₂ receptors in the brain, the mechanism believed to relieve the positive symptoms of schizophrenia. The blockade of nigrostriatal dopamine receptors causes movement disorders, collectively known as extrapyramidal side effects.¹⁸

In the present research work, the administration of Chlorpromazine in the experimental animals caused muscle rigidity and increased levels of oxidative stress in brain, which may result in altered levels of LPO, GSH, nitrites and total protein. The muscle grip strength was determined by rotarod apparatus in which the treatment with METSF at different doses and standard drug increased the mean fall off time. The increased levels of LPO and nitrates and decreased levels of GSH and total protein in brain are considered to be an indication for neuronal damage and oxidative stress. The administration of different doses of METSF and standard drug reversed the chlorpromazine induced changes which suggest the protective effect of METSF by preventing the oxidative stress, exhibiting potent anti-oxidant property.

Elevated plus maze is used as exteroceptive behavioral model to evaluate acquisition and retention of memory. The administration of SCP in experimental animals causes detrimental effects on short term memory, memory acquisition, learning and psychomotor speed. SCP produced amnesia has been projected as a model for dementia where reduced cholinergic function is the suspected cause. The dysfunctions are reversible by physostigmine which suggests that the memory impairment is specifically related to reduced cholinergic transmission caused by SCP.

Administration of different doses of METSF and Donepezil decreases the transfer latency in experimental animals indicating that plant extract is exhibiting the anti-amnestic activity. The anti-amnestic activity of different doses of METSF may be due to the facilitation of cholinergic transmission.

Acetylcholine, an important neurotransmitter helps in the regulation of cognitive functions. Acetylcholinesterase (AChE) is an enzyme of brain cholinergic system that hydrolyses the neurotransmitter acetylcholine to choline and acetate in the synaptic cleft.²³ The evidences have shown reduced activity of AChE in several brain disorders. The loss of cholinergic neurons in brain is the characteristic feature of dementia. Administration of SCP in experimental animals caused increased levels of AChE whereas treatment with METSF at different doses and Donepezil reduced the AChE levels, indicating improvement in memory due to the potent drug METSF.  

It has been documented that SCP alters short memory, acquisition of new knowledge and increases AChE activity and oxidative stress in the brain.¹⁹ Scopolamine significantly increased the levels of MDA, a marker of cellular degeneration and AChE. Administration of METSF at different doses restored the MDA levels and AChE compared to G 1 animals. 

Conclusion

The neuroprotective activity of METSF could be due to the inhibition of brain acetylcholinesterase activity and antioxidant property.

Conflicts of Interest

Nil

Acknowledgement

Authors are grateful to Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru for providing the financial assistance to carry out the research work and to the Management BLDE Association, Vijayapur, Principal and HOD, Department of Pharmacology, BLDEA’s SSM College of Pharmacy and Research Centre, Vijayapur for providing the necessary facilities to carry out the research work.