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In This Article
 »  Abstract
 » Introduction
 »  Materials and Me...
 » Results
 » Discussion
 » Conclusion
 »  References
 »  Article Figures
 »  Article Tables

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 Table of Contents    
RESEARCH ARTICLE
Year : 2018  |  Volume : 50  |  Issue : 1  |  Page : 30-38
 

Zanthoxylum alatum ameliorates scopolamine-induced amnesia in rats: Behavioral, biochemical, and molecular evidence


1 Department of Pharmacology and Toxicology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India
2 School of Biological Sciences, National Institute of Science Education and Research, HBNI, Bhimpur-Padanpur, Jatni, Khurda, Odisha, India
3 Department of Veterinary Pathology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India
4 Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India
5 Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India
6 Department of Veterinary Public Health, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India

Date of Submission13-Jul-2017
Date of Acceptance05-Mar-2018
Date of Web Publication30-Apr-2018

Correspondence Address:
Beenita Saikia
Department of Pharmacology and Toxicology, College of Veterinary Science, Khanapara, Guwahati - 781 022, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijp.IJP_417_17

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 » Abstract 


OBJECTIVE: Hydroethanolic extract of Zanthoxylum alatum seeds (HEZA) in scopolamine-induced amnesia was investigated for memory enhancing activity.
MATERIALS AND METHODS: Radial arm maze (RAM) test was performed to evaluate the behavioral activity. Rats were treated with HEZA (50, 100, and 200 mg/kg, p. o.) and tacrine (3 mg/kg. i. p.) for 14 days. Scopolamine (0.4 mg/kg) was injected i. p. into rats after 45 min of drug administration on the 14th day. The messenger RNA (mRNA)/protein profile of few markers (acetylcholinesterase [AChE], heme oxygenase-1 [HO-1], nuclear factor-kappa B [NFκB], nuclear factor erythroid 2–related factor 2 [Nrf2], protein phosphatase 2A[PP2A], Tau, brain-derived neurotrophic factor [BDNF], tropomyosin-related kinase B [TrkB], Bcl-2-associated X protein [Bax], and Caspase-3) were also measured by polymerase chain reaction (PCR) and immunoblotting assay. Brain cytokines (tumor necrosis factor alpha [TNF-α], interleukin [IL]-1 β, and IL-10) in hippocampus were evaluated using commercially available enzyme-linked immunosorbent assay kits.
RESULTS: HEZA exhibited anti-amnesic activity as indicated by a significant reduction in the working memory error and reference memory error in RAM. Pretreatment with HEZA significantly down-regulated the expression of AChE, NFκB, Tau, Bax, and Caspase-3 with simultaneous up-regulation of Nrf2, HO-1, PP2A, BDNF, and TrkB genes in the hippocampal tissues similar to tacrine when compared with scopolamine-treated rats. Pretreatment with HEZA attenuated scopolamine-induced elevation of TNF-α, IL-1 β, levels in hippocampus and reversed diminished IL-10 concentrations towards normal levels in the brain.
CONCLUSION: Zanthoxylum alatum seeds could probably counteract amnesia. Since its use is mainly reported as a stimulant and tonic, this novel activity could be a boon for the scientists to explore more in this direction.


Keywords: Amnesia, rats, scopolamine, Zanthoxylum alatum


How to cite this article:
Saikia B, Barua CC, Sarma J, Haloi P, Tamuli SM, Kalita DJ, Purkayastha A, Barua AG. Zanthoxylum alatum ameliorates scopolamine-induced amnesia in rats: Behavioral, biochemical, and molecular evidence. Indian J Pharmacol 2018;50:30-8

How to cite this URL:
Saikia B, Barua CC, Sarma J, Haloi P, Tamuli SM, Kalita DJ, Purkayastha A, Barua AG. Zanthoxylum alatum ameliorates scopolamine-induced amnesia in rats: Behavioral, biochemical, and molecular evidence. Indian J Pharmacol [serial online] 2018 [cited 2019 Jul 20];50:30-8. Available from: http://www.ijp-online.com/text.asp?2018/50/1/30/231477





 » Introduction Top


Herbs have been used since ancient times in the treatment of various diseases including cognitive disorders, such as Alzheimer's disease (AD). Therefore, ethnopharmacological screening of plants may provide useful leads in the discovery of new drugs for AD therapy. AD is a progressive neurodegenerative disorder, associated with dysregulation of several biogenic pathways due to genetic, epigenetic, and environmental causes.[1] The main symptoms of the AD include difficulty in several areas of mental function including language, memory, perception, emotional behavior, personality, and cognitive skills. Once a person with Alzheimer's disease has reached the severe stages of the disease, they become immobile and totally disabled.[2] Although there is no cure for Alzheimer's disease, treatment options are available to help manage the patient's progression of the disease, behavioral problems, sleep disturbances, and confusion.

Zanthoxylum alatum (ZA) Roxb. (Rutaceace) is an important small medicinal xerophyte, tree or shrub, grows up to 6 m in height with dense foliage and armed branched flattened prickles, which comprises about 150 genera.[3] Dried fruit of ZA contains an aroma, which is present in pericarp shell of brown fruit wall.[4] Seeds of ZA are rounded and shining black,[5] and have a bitter taste.[4] In India, ZA has been reported from the warmer valleys of the Himalaya from Jammu and Kashmir to Assam and Khasi in the Eastern Ghats in Orissa and Andhra Pradesh and the lesser Himalayan regions in the North-Eastern part of India, i.e., Naga Hills, Meghalaya, Mizoram, and Manipur.[6] ZA is locally known as Tejphal (Hindi), Tejowati (Sanskrit), Mukthrubi (Manipur), and Timur (Nepal).[7] Common names are Indian Prickly Ash, Nepal pepper or toothache tree. In indigenous system of medicine, the bark, fruits, and seeds of ZA are used as carminative, stomachic, and anthelmintic.[7] The fruit and seeds are used for fever and dyspepsia as aromatic tonic. Fruits extract is useful in roundworm infestation. In Nepal folk medicine, it is used in cold and cough, tonsillitis, headache, fever, vertigo, diarrhea, and dysentery.[8] Ethanolic extract of ZA possesses antioxidant,[9] and anti-inflammatory activity.[10] Essential oil of ZA has antispasmodic, antimicrobial, cytotoxic, and phytotoxic properties.[5] Essential oils of ZA seeds contain various alkaloids, flavonoids, flavonol glycosides, lignins, phenolics, sterols, terpenoids, fatty acids, alkanoic acids, amino acids, and number of other compounds have also been identified and isolated in good quantity.[11] Ethanol and n-hexane extract of ZA contains different phytochemicals such as alkaloids, sterols, saponins, tannins, phenols, and flavonoids.[12]

Scopolamine is a muscarinic receptor antagonist and is able to cause a transient disruption of memory in experimental animals by increasing the acetylcholinesterase (AChE) activity, leads to decrease in the endogenous acetylcholine content at cholinergic synapses, as a result, weakened neurotransmission and thereby memory loss.[13],[14] Hence, the protective role and molecular mechanisms of hydroethanolic extract of ZA seeds (HEZA), in scopolamine-induced impaired memory were undertaken in the present study.


 » Materials and Methods Top


Drugs and reagents

The following drugs/chemicals were procured from different sources. Scopolamine hydrobromide, tacrine hydrochloride (9-Amino-1, 2, 3, 4-tetrahydro-acridine hydrochloride hydrate), and 3,3′, 5, 5′-tetramethylbenzidine (TMB) were obtained from Sigma-Aldrich, USA. Enzyme-linked immunosorbent assay (ELISA) Kit (RayBio ®, USA), TRIzol ® reagent (Invitrogen, USA), RIPA lysis buffer (Amresco, USA and Canada), and cDNA synthesis kit (Thermo Scientific, EU, Lithuania). All other chemicals and reagents used in the present work were of analytical grades.

Plant material

The fresh seeds of ZA were collected from local market of Arunachal Pradesh in August 2014. It was identified and authenticated by Dr. Iswar Chandra Barua, Principal Scientist, Department of Agronomy, Assam Agricultural University, Jorhat, Assam and a voucher specimen (5109 dated 25.09.2014) was deposited in the Herbarium.

Preparation of hydroethanolic extract

Cleaned seeds of ZA were shade dried, powdered mechanically, weighed, and stored in airtight container. Afterward, 250 g of powdered material was soaked in 700 ml ethanol, and 300 ml distilled water (70:30) for 72 h in a beaker and mixture was stirred every 18 h using a sterile glass rod. The filtrate was obtained three times with the help of Whatman filter paper No. 1, and the solvent was removed by rotary evaporator (BUCHI, R-210, Labortechnik AG, Meierseggstrasse, Switzerland) under reduced pressure leaving a dark brown residue. It was stored in airtight container at 4°C until use. The recovery percentage with respect to dry powder was found to be 19.43% w/w.

Phytochemical analysis

Qualitative phytochemical analysis of HEZA was done by the standard method of Harborne [15] to identify the different active constituents (alkaloid, glycoside, flavonoids, terpenoids, tannin, and saponin) present in the extract.

Acute toxicity studies

The acute toxicity studies of HEZA were performed according to Organization of Economic Corporation Development Guidelines No. 425 using albino mice of either sex (20–30 g). The extract was administered orally at 2000 mg/kg to a group of mice (n = 3) and the percentage mortality, if any, was recorded. The animals were kept under observation for next 14 days for mortality or gross abnormality with the given doses. Based on the acute toxicity study, 50, 100, and 200 mg/kg oral dose were selected for the study.

Animals

Wistar albino male rats (150–200 g) were selected for the study and housed in polypropylene cages with clean bedding materials and safe drinking water with 12 h light-dark cycle. They were fed with rodent pellet diet and drinking water ad libitum. Experimental procedures were performed in accordance with the guidelines recommended by Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. All the protocols were approved by Institutional Animal Ethical Committee (IAEC) of College of Veterinary Sciences, Assam Agricultural University with registration number (770/ac/CPCSEA/FVSc, AAU/IAEC/16-17/452 dated 30.07.2016).

Treatment schedule

The rats were divided into six groups (n = 6–10) – Group I: Normal control received vehicle, p. o.(Tween 80 and saline), Group II: Negative control, received scopolamine 0.4 mg/kg, i. p., Group III: Standard control, received tacrine 3 mg/kg, i. p. + scopolamine, 0.4 mg/kg i. p., Group IV: HEZA 50 mg/kg, p. o. + scopolamine 0.4 mg/kg i. p., Group V: HEZA 100 mg/kg, p. o. + scopolamine 0.4 mg/kg i. p., and Group VI: HEZA 200 mg/kg p. o. + scopolamine 0.4 mg/kg i. p. The control, tacrine, and HEZA-treated groups were given respective treatment once daily for 14 consecutive days. On the 14th day, scopolamine (0.4 mg/kg) was injected i. p. into rats after 45 min of drug administration. One hour after scopolamine challenge, the behavioral test was performed. After behavioral measurements, the rats were sacrificed, and brains were quickly dissected out. Then, the hippocampus was isolated and stored at −80°C until detection.

Behavioral assessments

Radial arm maze (RAM) test was measured to test spatial working memory in rats.[16] The RAM consisted of an octagonal central platform with eight equally spaced radial arms (Stoelting and Co. USA). The 1st, 3rd, 5th, and 7th arms were baited while the 2nd, 4th, 6th, and 8th arms were unbaited. Baited and unbaited arms were fixed throughout the experiment. Rats were previously kept for 48 h fasting to achieve a convenient motivational level. Food-deprived rats were expected to search for specific arms with rewards and subsequently register and retain the memory of each entered arm where food was present. The experimental rats were placed in the central starting platform of the RAM in the position facing towards the 1st arm. Each rat was allowed to freely explore and consume food rewards for 10 min. An entry was recorded every time the rat placed all four paws into the initial part of the arm. The movements of the animals were monitored via a digital video tracking system (ANY-maze ). The first entry into never-baited arms was scored as a reference memory error (RME) and re-entry into arms where the food reward had already been eaten was scored as a working memory error (WME).

Reverse transcriptase polymerase chain reaction analysis

Expression of AChE, heme oxygenase-1 (HO-1), nuclear factor-kappa B (NFκB), nuclear factor erythroid 2-related factor 2 (Nrf2), protein phosphatase 2A (PP2A), and Tau in the hippocampus of rats exposed to RAM was studied by reverse transcriptase polymerase chain reaction (RT-PCR). The total RNA was isolated using TRIzol (Ambion). The messenger RNA (mRNA) was reverse transcribed by RevertAid First Strand cDNA synthesis kit (Thermo Scientific) with random hexamer primers as per protocol of the kit. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control for the experiment with a concentration of 10 pmol. Amplification was carried out in a thermal cycler (Applied Biosystems-Veriti-Thermal cycler). Polymerase chain reaction (PCR) condition included pre denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, variable annealing for different genes for 45 s [Table 1], and extension at 72°C for 45 min followed by final extension at 72°C for 10 min. The PCR product was visualized in 1.5% agarose gel in 1X Tris-acetate-ethylenediaminetetraacetic acid. The gel picture was then analyzed by Image Lab software 5.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Table 1: Oligonucleotide primer sequences for target genes used in reverse transcriptase polymerase chain reaction

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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis

Hippocampal tissue samples (30 mg approximately) were homogenized in 5 ml of chilled lysis buffer (RIPA Buffer, Amresco, USA) and centrifuged at 23,000×g for 20 min at 4°C. The protein estimation of the supernatants was carried out by Bradford reagent (Himedia) with Bovine serum albumin (BSA) as the standard. Fifty micrograms of total protein was loaded on each lane of 10% gel, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Hoefer Midi Gel apparatus (Harvard Apparatus, Holliston, MA). Gels were electrophoresed at 150 V and the fractionated proteins were transferred to polyvinylidene-difluoride membranes (Pierce Biotechnology, Rockford, IL, USA) using semi-dry blotting apparatus (Hoefer). The membranes were then blocked using 10 ml of cold blocking buffer containing 3% BSA in Tris-buffered saline with 0.1% tween 20 (TBST) for 1 h at room temperature, and incubated overnight (4°C) with 5 ml of 1% BSA in TBST containing anti-serum rabbit/mouse polyclonal IgG (Santa Cruz Biotechnology, Inc.) against brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrKB), Bcl-2-associated X protein (Bax), and Caspase-3 in 1: 500 dilution. After overnight incubation, blots were washed 3 times with TBST for 15 min and incubated with goat anti-rabbit/goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. After washing with cold TBST, the color reaction on the membrane was obtained by treating the membranes with commercially available UltraTMB blotting buffer. The membranes were scanned, and band intensities were quantified using Image J software (NIH, Bethesda, MD, USA).

Estimation of inflammatory cytokines

Hippocampal tissue samples (10% w/v) were homogenized with ice-cold phosphate buffer solution (pH 8) and centrifuged at 10,000 rpm for 15 min, and the supernatant was used for ELISA estimations. The levels of inflammatory cytokines such as interleukin-1 β (IL-1 β), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10) in the brain tissue were estimated using commercially available cytokine ELISA kits (Ray Biotech, USA) according to the manufacturer's instructions.

Statistical analysis

Results were expressed as mean ± standard error of the mean statistical analysis was performed by one way analysis of variance followed by post hoc Tukey's multiple range tests, using GraphPad Prism software version 5.0 (Inc. 7825 Fay Avenue, Suite 230 La Jolla, CA 92037 USA). Results were considered statistically significant when P < 0.05.


 » Results Top


Phytochemical analysis

The phytochemical analysis of HEZA was found to be positive for the presence of tannins and terpenoids by ferric chloride and Salkowski's test.

Acute toxicity study

No mortality or significant changes in behavior and body weight was observed following oral administration of HEZA with the highest dose 2000 mg/kg, p. o. It was safe up to 2000 mg/kg body weight, orally in the animals. Hence, 50, 100, and 200 mg/kg body weight oral doses were used in the study.

Effect of hydroethanolic extract of Zanthoxylum alatum on scopolamine-induced memory impairment in radial arm maze test

In this test, the WME was significantly higher in scopolamine-treated group (P < 0.001), when compared with the control group, indicating memory impairment. There was significant (P < 0.001) reduction of WME in those groups, pretreated with HEZA at 50, 100, and 200 mg/kg, oral doses, and tacrine, when compared to scopolamine-treated group [Figure 1]a. The incidence of RME was significantly (P < 0.001) lower in tacrine pretreated group, as compared to scopolamine treated group. Similarly, pretreatment with HEZA, showed significant reduction of RME at 50 (P < 0.001), 100 (P < 0.05), and 200 mg/kg (P < 0.001) dose, respectively, [Figure 1]b when compared to scopolamine-treated group.
Figure 1: Effect of hydroethanolic extract of Zanthoxylum alatum pretreatment on scopolamine-induced amnesia in behavioral study by radial arm maze model in rat showing (a) working memory errors and (b) reference memory errors. Values are expressed as mean ± standard error of the mean (n = 6). *P < 0.05 compared with normal control group; #P < 0.05 compared with scopolamine treated group

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Effects of hydroethanolic extract of Zanthoxylum alatum on acetylcholinesterase, nuclear factor-kappa B, Tau, nuclear factor erythroid 2–related factor 2, heme oxygenase-1, and protein phosphatase 2A gene expression

The RT-PCR analysis of AChE, NFκB, Tau, Nrf2, HO-1, and PP2A gene expression were represented in [Figure 2]a, the mRNA expression levels of AChE, NFκB, and Tau were significantly (P < 0.05, P < 0.01, P < 0.001) upregulated in the hippocampal tissues of scopolamine-treated rats respectively, compared to the normal control group. However, pretreatment with HEZA significantly down-regulated the mRNA expression of AChE (P < 0.05) with 200 mg/kg HEZA [Figure 2]b, NFκB (P < 0.05), with 200 mg/kg, HEZA [Figure 2]c, and Tau (HEZA, 100, 200 mg/kg; P < 0.001) [Figure 2]d genes in the hippocampal tissues of rat, similar to that of standard drug tacrine as compared to the scopolamine treated rats. On the other hand, mRNA expression levels of Nrf2 (P < 0.01), HO-1 (P < 0.01), and PP2A (P < 0.001) were significantly down-regulated in the hippocampal tissues of scopolamine treated rats compared to the control group. Pretreatment with HEZA (100 and 200 mg/kg) significantly up regulated the mRNA expression of Nrf2 ( 100 mg/kg; P < 0.05, 200 mg/kg; P < 0.01) [Figure 2]e, HO-1 ( 100 and 200 mg/kg; P < 0.05) [Figure 2]f, and PP2A (50 mg/kg; P < 0.05, 100 mg/kg; P < 0.001 and 200 mg/kg; P < 0.001) [Figure 2]g genes in the hippocampal tissues of rats similar to tacrine, when compared with scopolamine treated rats.
Figure 2: Effect of hydroethanolic extract of Zanthoxylum alatum pretreatment on scopolamine-induced amnesia on acetylcholinesterase, nuclear factor-kappa B, Tau, nuclear factor erythroid 2-related factor 2, heme oxygenase-1, and protein phosphatase 2A messenger RNA expression in the hippocampus of the rat. (a) Lane 1: DNA marker, lane 2–7: normal control, scopolamine, tacrine, hydroethanolic extract of Zanthoxylum alatum 50, hydroethanolic extract of Zanthoxylum alatum 100, hydroethanolic extract of Zanthoxylum alatum 200. (b-g) Quantitative expressing messenger RNA level was assessed using Image J software and is expressed as percentage fold change when compared with the internal control (glyceraldehyde 3-phosphate dehydrogenase). Values are expressed as mean ± standard error of the mean (n = 6). *P < 0.05 compared with normal control group; #P < 0.05 compared with scopolamine treated group

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Immunoblotting assay of brain-derived neurotrophic factor, tropomyosin-related kinase B, Bcl-2-associated X protein, and Caspase-3

The immunoblotting assay of BDNF, TrkB, Bax, and Caspase-3 showed [Figure 3]a, significant (P < 0.001) downregulation of TrkB and BDNF protein in the hippocampal tissue of scopolamine-treated rats compared to control group. However, pretreatment with HEZA significantly upregulated BDNF (50,100 and 200 mg/kg; P < 0.05, P < 0.001, P < 0.001) [Figure 3]b, and TrkB (100 and 200 mg/kg; P < 0.001) [Figure 3]c, protein expression in the hippocampal tissues of rats similar to tacrine (P < 0.001), as compared to scopolamine treated rats. On the other hand, significant (P < 0.001) upregulation in the expression of Bax and caspase-3 in the hippocampal tissue of scopolamine-treated rats compared to the control group were observed. Pretreatment with HEZA significantly downregulated Bax (100 and 200 mg/kg; P < 0.01, P < 0.001) [Figure 3]d, and Caspase-3 (100 and 200 mg/kg; P < 0.01) [Figure 3]e protein in the hippocampal tissues of rats similar to that of standard drug, tacrine (P < 0.001), when compared with scopolamine-treated rats.
Figure 3: Effect of hydroethanolic extract of Zanthoxylum alatum pretreatment on scopolamine-induced amnesia on brain-derived neurotrophic factor, tropomyosin-related kinase B, tropomyosin-related kinase B and Caspase-3 proteins expression in rat hippocampus. (a) Lane 1: DNA marker, lane 2–7: Normal control, scopolamine, tacrine, hydroethanolic extract of Zanthoxylum alatum 50, hydroethanolic extract of Zanthoxylum alatum 100, hydroethanolic extract of Zanthoxylum alatum 200. (b-e) Quantitative expressing messenger RNA level was assessed using Image J software and is expressed as percentage fold change when compared with the internal control (β actin). Values are expressed as mean ± standard error of the mean (n = 6). *P < 0.05 compared with normal control group; #P < 0.05 compared with scopolamine treated group

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Effect of hydroethanolic extract of Zanthoxylum alatum on pro and anti-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1 β, and interleukin-10)

Proinflammatory cytokines, (TNF-α and IL-1 β) levels were significantly (P < 0.001) increased in scopolamine treated rats when compared with the control group. However, the standard drug, tacrine (P < 0.01) and the test drug HEZA at 100, and 200 mg/kg (P < 0.01) decreased TNF-α level significantly [Figure 4]a when compared with scopolamine treated group. Likewise, the standard drug, tacrine (P < 0.001), and the test drug HEZA at 100, and 200 mg/kg (P < 0.001) decreased IL-1 β [Figure 4]b levels when compared with scopolamine-treated group. On the other hand, anti-inflammatory cytokine, i.e., IL-10 level was significantly (P < 0.05) decreased in scopolamine treated group, in comparison to the control group animals. This significant fall in the IL-10 level was ameliorated by preventive treatment with the test drug HEZA at 200 mg/kg (P < 0.05) [Figure 4]c.
Figure 4: Effect of hydroethanolic extract of Zanthoxylum alatum pretreatment on scopolamine-induced amnesia on pro- and anti-inflammatory cytokines (a) tumor necrosis factor alpha, (b) interleukin-1β, (c) interleukin-10 in rat hippocampus. Values are expressed as mean ± standard error of the mean (n = 6). *P < 0.05 compared with normal control group; #P < 0.05 compared with scopolamine-treated group

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 » Discussion Top


Memory formation is a very complex process which involves various neurotransmitters and multiple neuronal pathways.[17] Deprived memory, lower retention, and slow recall are common problems in today's stressful and competitive world. Alzheimer's disease is a neurodegenerative disorder characterized by aphasia, apraxia, agnosia with a progressive loss of memory and cognition.[18] In the present study, HEZA showed significant protection from loss of memory and cognition impairment in scopolamine-induced cognitive dysfunction in rats RAM model.

The main approach of symptomatic treatment of mild AD is through improving the cholinergic deficit via the inhibition of AchE.[13] Scopolamine induces amnesia through increased calcium influx followed by oxidative stress which in turn increases the activity of AchE.[19] In our study, scopolamine-induced increase AchE expression was attenuated by pretreatment with HEZA and tacrine. Therefore, it is possible that memory-enhancing the activity of HEZA extract is also mediated by AchE inhibition.

Nrf2 is an upstream transcription factor modulating Phase II enzyme activity, which interacts with the antioxidant response element (ARE) in the nucleus to induce ARE-dependent gene expression. During oxidative stress, Nrf2 translocates into the nucleus to induce the expression of HO-1.[20] which plays an essential role in maintaining cellular redox homeostasis against reactive oxygen species (ROS) generation and oxidative stress.[21] Pretreatment with HEZA and tacrine led to significant increase in the Nrf2 level along with HO-1 gene expression and decreased oxidative stress and strengthen endogenous antioxidant. Therefore, we speculated that HEZA exhibited antioxidant activity through up-regulating Nrf2-mediated HO-1 expression.

Under oxidative stress, ROS may initiate and exaggerate the inflammatory response due to their capability to stimulate and regulate the inflammatory signaling cascades genes like NFκ B and pro inflammatory cytokines.[22] In our study, scopolamine-treated rats showed increased NFκ B activity and also proinflammatory cytokines, i.e., TNFα and IL-1β and attenuated IL-10 level. Pretreatment with HEZA and tacrine downregulated NFκB activity and its downstream proinflammatory cytokines TNFα and IL-1β. This decrease in neuroinflammation with HEZA and tacrine may be due to inhibition of NFκB expression.

The balance between the phosphorylation and dephosphorylation of Tau is regulated by many kinds of proteinases, such as PP2A. It had been demonstrated that during oxidative stress, endogenous PP2A is specifically and reversibly inhibited.[23] In our study, scopolamine treated rats showed significantly decreased PP2A activity/mRNA expression and up-regulated Tau mRNA expression in hippocampus regions. Pretreatment with HEZA and tacrine restored PP2A activity/mRNA expression, and finally decreased Tau hyperphosphorylation that may prevent neurofibrillary tangle formation and deposition. Our findings are supported by Dwivedi et al.[24] where Bacopa monniera attenuated okadaic acid-induced Tau hyperphosphorylation in rats through restoring of PP2A activity.

During oxidative stress, neuroinflammation, and Tau phosphorylation, excessive-free radical attacked membrane phospholipids, result in mitochondrial membrane potential loss and release of apoptosis-inducing factors which activate apoptosis cascades.[25] Apoptosis in excess is related to cellular degeneration by oxidative stress, frequently associated with aging and pathogenesis of neurodegenerative conditions.[26] Expression of caspase-3 is a frequently activated death protease catalyzing the specific cleavage of many key cellular proteins. In our study, scopolamine-induced apoptotic neuronal cell death by increasing Bax and Caspase-3 (apoptotic proteins) activity. Preventive treatment with HEZA and tacrine lead to decreased proapoptotic Bax and Caspase-3 activity indicating neuroprotection. HEZA protected rat's neuronal injury modulating the apoptotic regulatory proteins and enzymes Caspase-3 in scopolamine-induced Caspase-3 activation and neuronal apoptosis.

BDNF is an important regulator of synaptogenesis and synaptic plasticity mechanisms involving learning and memory formations.[27] Decrease in the expression of BDNF during stress leads to loss of normal plasticity, damage, and neurons. BDNF also have an antioxidant role.[28] A number of studies have demonstrated that BDNF may reverse memory deficits, making it an attractive candidate for selective enhancement of failing memories in disease models.[29] Studies have also shown increased BDNF mRNA or protein levels following memory enhancement treatments.[30] Interestingly, in the current study, we found a decrease in BDNF and TrkB levels following scopolamine administration which was significantly reverted by HEZA.


 » Conclusion Top


Our study demonstrated that HEZA probably exhibits multiple pathways for cognition enhancement in scopolamine-induced amnesia in rats. The mechanism by which HEZA performs anti-amnesic activity could be through inhibition of AChE activity, antioxidants effect, inhibition of neuroinflammation, modulating the apoptotic regulatory proteins and increased BDNF expression. So far, anti-amnestic potential of seeds of ZA was not reported; therefore, this new activity may lure the scientists to direct their attention toward an important paradigm. Further study is ongoing to isolate compounds responsible for memory enhancing the property.

Acknowledgment

The authors express their sincere thanks to the Director of Research (Vety), AAU, Khanapara for providing facility to carry out this work, and also thankful to taxonomist Dr. Iswar Chandra Barua, Principal Scientist, Department of Agronomy, AAU, Jorhat for identifying the plant.

Financial support and sponsorship

DRDO, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

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