INTRODUCTION:

Alzheimer’s disease (AD) is gradual degeneration of neuron is common form of dementia. Atrophy of cholinergic neurons of Alzheimer’s disease patient is associated with impairment in learning ability and decline in cognitive functions¹. At present 30 million people survive with dementia, and 65.7 million persons may affected by 2050 globally^2,3.

The main histopathological characteristics of Alzheimer’s disease includes: The extracellular deposits of insoluble amyloid β peptide (Aβ ) as a result of enzymatic cleavage of amyoid precursor protein (APP), and the intracellular neurofibrallary tangles of hyperphosphorylated tau proteins.^4,5 It is well known that cholinergic neuronal system plays an important role in cognitive process.⁶ Currently available treatment for AD is symptomatic use of acetylcholine esterase inhibitors (AchEI) and N-methyl-D asparate receptor antagonist memantine such treatment is short term not effective.⁷Due to resulting side effect of these agents, they have limited their use.⁸ Currently there is no satisfactory therapeutic regimen available for the management of cognitive dysfunction. India has rich heritage in herbal medicine. Demand of herbal medicines in the treatment of Alzheimer’s disease is increased day by day.⁹ Ayurveda an ancient, traditional science of medicine classify rasayana (rejuventor) are drug that promotes, intellect, memory, immunity, longevity, strength of sense and improve strength of digestive system.¹⁰ Medhya rasayana is special class of rasayana which improve memory and intellect.¹¹ Sesbania grandiflora Linn (Fabaceae), commonly known as Agati is a widely available plant. The plant is known as Agastya in Sanskrit and is used as Medhya in Dravyaguna Vijnyan.^12,13 During ripening period fruits are sweet and invigorator of memory and in Auyrveda it has been reported that fruit brightens the intellect.¹⁴The present study is based on hypothesis that fruits of Sesbania grandiflora can show cognition enhancing activity.

MATERIAL AND METHOD:

Plant material:

The fruits of Sesbania grandiflora were collected from local market of karad, Maharashtra. It was identified and authentificated by Priyanka A. Ingle, Scientiest B, Botanical Survey of India, Western Regional Center, Pune. Herbarium of Sesbania grandiflora was deposited at Botanical Survey of India, Western Regional Center, Pune and voucher specimen (196 dated) was deposited in Herbarium.

Extraction process:

The fruits of Sesbania grandiflora were dried in shade for 15 days. The shed dried powder of fruits of Sesbania grandiflora were reduced to coarse powder and sieved. The powder was subjected to successive hot continuous extraction (soxhlet apparatus) with petether (60-80), benzene, chloroform, acetone, ethanol to about 40cycles per batch for 10 batches. The extraction was continued until the solvent in the thimble become clear then few drops were collected in a test tube during the completion of cycle (during siphoning) chemical test of that solvent was performed. Each time before extracting with next solvent the powdered material was dried at room temperature.

Phytochemical Screening:

Preliminary phytochemical extract was done by standard method of Khandelwal to identify active principle like carbohydrate, alkaloids, flavonoids, steroids, saponin, proteins, glycosides, tannins and polyphenol.

Laboratory animals:

The experiments were carried out using Swiss albino mice weighing (25-30gm) were used in present study. The animal had free access to food and water and they were housed in natural 12h light dark cycle. The animals were acclimazed for at least 5 days to the laboratory conditions before behavioral experiment. Experimental protocol was presented in the IAEC meeting held on 22 January 2018. The approval for use of lab animals (Reference no. IAEC/ABCO/09/2017-18) was taken from the Institutional Animal Ethics Committee (IAEC), Appasaheb Birnale College of Pharmacy, Sangali. The experimental procedure were performed in accordance with guideline recommended by Control and Supervision of Experiments on Animals (CPCSEA).

Acute oral toxicity study:

The acute oral toxicity study of extract was carried out as per the guidelines set by Organization for Economic Cooperation and Development (OECD), received draft guidelines, received from Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Experimental protocol:

Mice were divided into eight groups (n=6 animals/group)

Group I: Normal control mice received saline.

Group II: Negative control mice received scopolamine (0.4mg/kg,i.p)

Group III: Standard control mice received piracetam (200mg/kg,p.o)

Group IV: pretreated with petether extract of fruits of Sesbania grandiflora (PESG ,200mg/kg, p.o.) and scopolamine (0.4mg/kg,i.p)

Group V: pretreated with Benzene extract of fruits of Sesbania grandiflora of (BESG, 200mg/kg p.o).and scopolamine (0.4mg/kg,i.p).

Group VI: pretreated with Chloroform extract of fruits of Sesbania grandiflora of (CESG ,200mg/kg p.o.) and scopolamine (0.4mg/kg,i.p).

Group VII: pretreated with Acetone extract of fruits of Sesbania grandiflora of (AESG, 200mg/kg p.o.) and scopolamine (0.4mg/kg,i.p)

Group VIII: pretreated with Ethanol extract of fruits of Sesbania grandiflora of (EESG ,200mg/kg p.o.) and scopolamine (0.4mg/kg,i.p)

Elevated plus maze¹⁵:

The elevated plus maze served as the exteroceptive behavioral model (wherein the stimulus existed outside the body) to evaluate learning and memory in mice. The apparatus consisted of two open arms (16cm × 5cm) and two covered arms (16cm × 5cm × 12cm). The arms ex-tended from a central platform (5cm × 5cm), and maze was elevated to a height of 25cm from the floor. On the first day, each mouse was placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) was taken as the time taken by the mouse to move into any one of the covered arms with all its four legs. TL was recorded on the first day. If the animal did not enter into one of the covered arms within 90 sec., it was gently pushed into one of the two covered arms and the TL was assigned as 90 sec. The mouse was allowed to explore the maze for 10 sec and then returned to its home cage. Retention of this learned task (memory) was examined 24h after the first day trial (i.e.9^thday, 24 h after last dose). Significant reduction in TL value of retention indicated improvement in memory. (Joshi and Parle, 2006)

The TL was expressed as inflexion ratio using given formula.

Where

L₁ is the TL on day 8^th and

L_O is the TL on the 9^th day

Morris water maze¹⁶:

Morris water maze tasks were performed to evaluate spatial learning and memory MWM consist of a circular pool (150cm in diameter, 45cm in height) filled to a depth of 30cm with water maintained at 25◦C. The water was made opaque with non-toxic white colored dye. The tank was divided into four equal quadrants with the help of two threads, fixed at right angle to each other on the rim of the pool. In the center of 1st quadrant was a removable escape platform below the water level. For each animal, the location of invisible platform was placed at the center of one quadrant and remained there throughout training. The rats must memorize the platform location in relation to various environmental cues because there was nothing to directly show the location of the escape platform in and outside the pool. Therefore, the placement of the water tank and platform were the same in all acquisition trials.Each animal was subjected to four consecutive trials each day with a gap of 5 min for four consecutive days (starting from 15^th day of drug administration to 18^th day), during which they were allowed to escape on to the hidden platform and to remain there for 20 s. If the mouse failed to find the platform within 120 s, it was guided gently onto the platform and allowed to remain there for 20 s. Escape latency time (ELT) was defined as the time taken by the animal to locate the hidden platform. ELT was noted as an index of learning. On fifth day (i.e. 19^th day of drug administration), the platform was removed. Mouse was placed in water maze and allowed to explore the maze for 120 s. Each mouse was subjected to four such trials and each trial was started from a different quadrant. Mean time spent in all the three quadrants i.e. Q1, Q2 and Q3 was recorded and the time spent in the target quadrant (TSTQ) in search of the missing platform provided as an index of retrieval. Care was taken not to disturb the relative location of water maze with respect to other objects in the laboratory.

Estimation of Brain Acetylcholinesterase¹⁷:

Immediately after the experiment mice were sacrified by cervical decapitation with light anaesthesia and whole brain was carefully removed. The brain AChE activity was estimated by Ellman's method. The esterase activity is measured by providing an artificial substrate, acetylthiocholinelodide (ACI). Thiocholine released because of the cleavage of ACI by AChE is allowed to react with the -SH reagent 5,5'- dithiobis-(2-nitrobenzoic acid) (DTNB), which is reduced to thionitrobenzoic acid, a yellow coloured anion with an absorption maxima at 412nm. The rate of moles substrate hydrolyzed per min per gram of tissue was calculated.

Estimation of Malondiadehyde (MDA)¹⁸:

The content of MDA was determined as per the method described by Ohkawa et al; 1979 The assessment of MDA level from tissue homogenate measured by method of malondialdehyde (MDA) is formed mainly from the peroxidation of PUFAs. MDA is a TBA reacting substance (TBARs) and the product formed between the reaction of MDA and TBA is extracted into an organic layer and estimated at 532nm.

Estimation of Glutathione (GSH)¹⁹:

Reduced glutathione was measured by its reaction with 5,5 -dithiobis (2-nitrobenzoic acid) (DTNB) to give a yellow colored 5-thio-2-nitrobenzoic acid (TNB) compound that absorbs at 412nm. The produced disulfide is reduced by NADPH in the presence of glutathione reductase. GSH level was measured by Ellman’s method. Value was expressed as nmole/min/mg protein.

Statistical analysis:

The result of study were expressed as ±S.E.M. Data was analyzed by using one way analysis of variance test (ANOVA) followed by Dunnett’s test for multiply comparisons. Statistical significance was set at p<0.05.

RESULT:

Effect of different extracts of fruits of Sesbania grandiflora on EPM:

EPM was used to assess acquisition and retention of memory process. Retentional trial showed significant increase in transfer latency in scopolamine treated group indicated that cognitive dysfunction in mice. Pretreatment of BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significantly decrease transfer latency (TL) and increase in inflexion ratio as compared to scopolamine treated group. (Fig.1)

Fig 1: Effect of sesbania grandiflora fruits extract on Transfer Latency using Elevated plus maze.

All values are mean±SEM, ***P < 0.001, **P < 0.01, *P < 0.05versus scopolamine group.

Fig 2: Effect of sesbania grandiflora fruits extract on Inflexion ratio using Elevated plus maze.

All values are mean±SEM, ***P < 0.001, **P < 0.01, *P < 0.05versus scopolamine group.

Effect of different extracts of Sesbania grandiflora on MWM:

Scopolamine treated mice showed significant increase in escape latency time (ELT) indicate acquisition and decrease in time spend in target quadrant (TSTQ) indicate retention in MWM. Pretreatment of BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant decrease in ELT and increase in TSTQ indicated protection and retrivel of amnesia induced by scopolamine and improve the memory. Pretreatment of PESG did not produce any significant improvement in memory score of mice. (Fig 3)

Fig 3: Effect of sesbania grandiflora fruits extract on Escape Latency and time spend in Target Quadrant using Morris Water Maize. All values are mean±SEM, ***P < 0.001, **P < 0.01, *P < 0.05versus scopolamine group, ###P < 0.001, ##P < 0.01, #P < 0.05versus control group.

Effect of different extracts of Sesbania grandiflora on AchE:

Scopolamine treated mice showed significant increase in AchE. Pretreatment of piracetam and BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant decrease in brain AchE level as compared to scopolamine treated group. No significant change was observed in pet ether extract of Sesbania grandiflora. (Fig. 4)

Fig 4: Effect of sesbania grandiflora fruits extract on Brain acetylcholinesterase (AchE)

All values are mean±SEM, ***P < 0.001, **P < 0.01, *P < 0.05versus scopolamine group.

Effect of different extracts of Sesbania grandiflora on MDA:

Scopolamine treated mice showed significant increase (p< 0.01) in oxidative stress which is determined by the level of MDA as compared to control group. Pretreatment of BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant decrease in MDA level as compared to scopolamine treated group. Treatment with piracetam significantly reduce the brain MDA level as compared to control. Pretreatment of PESG did not produce any significant change in MDA level. (Fig .5)

Fig 5: Effect of sesbania grandiflora fruits extract on malondialdehyde (MDA). All values are mean±SEM, ***P < 0.001, **P < 0.01, *P < 0.05versus scopolamine group, ###P < 0.001, ##P < 0.01, #P < 0.05versus control group

Effect of different extracts of Sesbania grandiflora on GSH:

Brain GSH level was significantly decreased (P < 0.01) in scopolamine treated groups as compared to normal control. Pretreatment of piracetam and BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant increase in brain GSH level as compared to scopolamine treated group.

Effect of pet ether extract on brain GSH level was not significant as compared. to scopolamine treated group.

However, BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora significantly protected brain from scopolamine induced decrease in brain GSH level. Standard drug pirace effect of tam significantly attenuated the Sesbania grandiflora on glutathione level. (fig 6)

Fig 6: Effect of sesbania grandiflora fruits extract on GSH. All values are mean±SEM, ***P < 0.001, **P < 0.01, **P < 0.05versus scopolamine group

DISCUSSION:

Present study investigated the different extracts of fruits of Sesbania grandiflora PESG, BESG, CESG, AESG, EESG on scopolamine induced cognitive dysfunction in mice using behavioural and biochemical paradigm. The scopolamine induced amnesia is interoceptive behavioural paradigm is used as primary screening test for Anti alzhiemers drugs.²⁰ Non selective centrally acting muscarinic cholinergic antagonist scopolamine produce reversible impairement in attention level of individuals and total distruption of learning and memory capacity and subsequent causes short term and long term memories, increased oxidative stress and acetylcholineesterase in mice brain.^21,22Blockade of cholinergic neuronal system with decline in various cholinergic parameters such as Choline acetyltransferase and cholineasterase activity, Cholineacetyltransferase mRNA and acetylcholine receptors were observed²³. Ach plays crucial role in basic nerve transmission, concentration, memory and learning. The result of present study exposed that pretreatment of piracetam and BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant decrease in brain AchE level as compared to scopolamine treated group. EPM was used to assess acquisition and retention of memory process. Pretreatment of BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significantly decrease in transfer latency (TL) and increase in inflexion ratio as compared to scopolamine treated group. Pretreatment of BESG, CESG, AESG, EESG extracts of fruits of Sesbania grandiflora for fifteen successive days prevented memory loss of mice as indicated by decrease in ELT and increase in TSTQ using MWM. Brain is vulruneable to damage from free radicals caused by oxidative process due to excessive consumption of oxygen²⁴. Oxidative stress in one of the primary factor responsible for the degeneration of neurons^25.Ample number of studies reported that aggregation of product of ROS/RNS damage in alzhiemer disease²⁶. TBARS is formed as a result of lipid molecule react with oxygen free radicals. TBARS measure MDA which is end product of lipid peroxidation²⁷. Pretreatment of BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant decrease in MDA level as compared to scopolamine treated group. Treatment with piracetam significantly reduce the brain MDA level as compared to control. Pretreatment of PESG did not produce any significant change in MDA level. Scopolamine treated mice increases level of MDA represent index of invivo lipid peroxidation. GSH is found in cell and act as natural defence system protect the brain cells from damaging effect of reactive oxygen species. GSH is endogenous antioxidant play important role in maintaining balance between generation of reactive oxygen species and antioxidative defence system.²⁸ In thse present study Pretreatment of BESG, CESG, AESG, EESG extract of fruits of Sesbania grandiflora showed significant increase in GSH level as compared to scopolamine treated group.

CONCLUSION:

The above findings conclude that, pretreatment with different extracts of fruits of Sesbania grandiflora pet ether extract (PESG), benzene extract (BESG), chloroform extract (CESG), acetone extract (AESG) and ethanolic extract (EESG) demonstrated a significant reversal of cognition impairment in mice by inhibition of AchE enzyme, decrease in MDA level, increase in GSH level in mice brain, possesses cognition enhancing property in scopolamine induced amnesia in mice.

ACKNOWLEDGMENT:

Authors are thankful to Management of Shree Santkrupa Shikshan Sanstha’s Ghogaon, and Department of Pharmacology, Aappasaheb Birnale College of Pharmacy, Sangali for providing constant support and facilities for completion of this work.

CONFLICT OF INTEREST:

None declared.

REFRENCES:

1. Mahboubi M. Melissa officinalis and rosmaric acid in management of memory functions and Alzheimer disease. Asian Pacific Journal of Tropical Biomedicine2019; 9(2): 47-52.

2. Pattanashetti LA, Taranalli AD, Parvatrao V, Malabade RH, Kumar D. Evaluation of neuroprotective effect of quercetin with donepezil in scopolamine –induced amnesia in rats. Indian Journal of Pharmacology 2017;49:60-4.

3. Surabhi, Singh BK. Alzheimer’s disease: A comprehensive review. International Journal of Pharmaceutical Sciences and Research 2019; 10(3) : 993-1000.

4. Liu Z, Zhang A, Sun H, Han Y, Kong L, Wang X. Two decades of new drug discovery and development for Alzheimer’s disease. Royal Society of Chemistry. 2017;7: 6046-6058.

5. Mani A, Chaudhary A, Maurya P, Yadav B, Singh S. Current therapeutic targets for Alzheimer’s. disease. Journal of Biomedicine 2018;3:74-84.

6. Hopper S, Udawela M, Scarr E, Dean B. Allosteric modulation of cholinergic system: Potential approach to treating cognitive deficits of schizophrenia. World Journal of Pharmacology s2016;(5)1:32-43.

7. Kucca K, Soukup O, Maresova P: Current approaches against Alzheimer’s disease in clinical trials. Journal of Brazilian. Chemical Science 2016;27: 641-649.

8. Joshi H, Parle M: Zingiber officinale: Evaluation of its nootropic effect in mice. African Journal of Traditional Complementary and Alternative Medicines 2006;3:64-74.

9. Andrade C, Monteiro I, Hedge RP, Chandra JS. Investigation of the possible role of Shankapushpi in the attenuation of ECT induced amenestic deficits. Indian Journal of Psychiatry 2012;54:166-71.

10. Neelam, Patekar R, Choudhary A, Soni G, Dwivedi K: Concept of medhya rasayana in aurveda: An Overview. International Journal of Research in Ayurveda Pharm 2017;8 :78-81.

11. Chaudhari K, Murthy A. R: Effect of Rasayana on Mental health-a review study. International Journal of Ayurveda and Alternative Medicine. 2014; 2:1-7.

12. Kirtikar B. D., Basu B. D. Indian Medicinal Plants.1998; 2735.

13. Nadkarni A.K. Nadkarni K.M. Indian material medica.1982;3, 52-54

14. Deshpande A. P. Javalgekar R. R. Rande S., Dravyaguna Vijnyan,2013;820-821.

15. Joshi H, Parle M: Zingiber officinale: Evaluation of its nootropic effect in mice. African Journal of Traditional Complementary and Alternative Medicines 2006;3:64-74.

16. Morris R. Spatial localization dose not depend on the presence of local cues. Learning and Motivation 1981;12: 239-260

17. Ellman G, Courtney KD, Andres VJ. Feather-Stone RM. Anew and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol1961; 7:88-95

18. Ohkawa H, Ohishi N, Yagi K, Assay of lipid peroxidase tissue by thiobarbituri acid reaction. Anal Biochem 1979; 95:351-358.

19. Ellman G, Courtney KD, Andres VJ. Feather-Stone RM. Anew and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol1961; 7:88-95

20. Jawaid T, Shakya A, Siddiqui H, Kamal M. Antiamnesic activity of leaves of Bambusa arunndinacea in rats. An International Journal of Advances in Pharmaceutical Sciences. 2013;4(5):919-932.

21. Malabade R, Taranalli A. Cassia tora A potential cognition enhancer in rats with experimentally induced amnesia 2015;7(4):455-461

22. Young K, Jeong E, Lee K, Kim S, Sung S. Cognitive –enhancing and antioxidant activities of iridoid glycosides from Scrophularia buergeriana in scopolamine- treated mice.

23. Rao N, Pujar B, Nimba S, Shanta Kumar S, Satyanarayana S. Nootropic activity of tuber extract of Pueraria tuberose (Roxb) 2008;46:591-598.

24. Uddin S, Nasruah, Hossain S Evaluation of nootropic activity of Persicaria flaccida on cognitive performance, brain antioxidant markers and acetylcholinesterase activity in rats; implication for the management of Alzheimer’s Disease. American Journal of Psychiatry and Neuroscience 2016;4(2):26-35.

25. Muralidharan P, Balamurugan G, Venu Bapu. Cerebroprotective effect of Glycyrrhiza glabra Linn.root extract on hypoxic rats. Bangladesh J Pharmacology 2009; 4:60-64.

26. Khare P, Chaudhary S, Singh L, Yadav G, Verma S. Evaluation of Nootropic activity of Cressa cretica in scopolamine –induced memory impairment in mice. International Journal of Pharmacology and Toxicology 2014;2(2):24-29.

27. Parle M, Kaura S. Anti-Alzheimer Potential of Green Moong Bean 2016;37(2): 178-182.

28. Uddin S, Nasruah, Hossain S Evaluation of nootropic activity of Persicaria flaccida on cognitive performance, brain antioxidant markers and acetylcholinesterase activity in rats; implication for the management of Alzheimer’s Disease. American Journal of Psychiatry and Neuroscience 2016;4(2):26-35.

Received on 11.01.2020 Modified on 24.02.2020

Research J. Pharm. and Tech. 2020; 13(11):5057-5062.

DOI: 10.5958/0974-360X.2020.00886.0