Download RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

6.0
BRIEF RESUME OF INTENDED WORK :
6.1 NEED FOR THE STUDY
Neurodegenerative disorders are relatively common in old age and represent a substantial
medical and social problem. It is estimated that there are currently about 6.29 % of population is
affected worldwide by neurological disorders. This figure is projected to 6.39 % in 2015 and 6.77
% in 2030. Presently available pharmacological treatment for neurodegenerative disorders are
symptomatic and do not alter the course or progression of the neurodegenerative disorders. These
treatments are less satisfactory and may lead to serious side effects such as in case of following
drugs1,2:
Levodopa
: dyskinesia and psychological effect
Amantadine : dizziness and insomnia, tacrine – hepatotoxicity, carbamazepin – severe mental
and motor disturbances,
Niluzole
: hepatic injury.
Therefore need new strategies to treat these disorders. Hence, the goal of present study is to
integrate allopathy system of medicine with Ayurvedic (herbal formulation) so as to demonstrate
the utility of such treatment strategy in preventing, retarding the progression of neurodegeneration
and enhancing the safety of the treatment. Hence it is hypothesized that poly herbal formulation
which is marketed in the name of Mentat containing many herbs having free radical scavenger
constituents; may protect the neurodegeneration due to oxidative stress. In addition Donezepil a
known acetyl-cholinesterase inhibitor used to treat neuro-degenrative diseases that act through
different mechanism. So, combination of these two may synergize the effect of each other and
enhance the efficacy of the treatment. In the present study three experimental models of
neurotoxicity will be adapted to assess the study hypothesis. They are:
i.
Mono sodium glutamate induced neuro toxicity in rats (which act by over
exciting
the glutamate receptors and release the free radicals19).
ii.
3nitro propionic acid induced neurotoxicity in rats (This also cause neuro toxicity
by generating free radicals20).
iii.
Bilateral carotid artery occlusion induced neuro toxicity in rats (This also causes
neurotoxicity by causing oxidative stress and reperfusion induced free radical
generation28) .
Mechanism responsible for neuronal degeneration in above mentioned experimental models are
excitotoxicity, oxidative stress and inflammation, hence controlling the glutamate release, NMDA
receptor antagonists, calcium channel blocker, free radical scavengers, anti-inflammatory and
estrogenic drugs may be beneficial in protecting neurons from damage5.
Hence the study drug Donezepil and polyherbal formulation Mentat act by two different
mechaninsm, which can prevent neurotoxicity, the combination of them may be highly useful.
Therefore the study is needed and justifiable.
6.2 REVIEW OF LITERATURE
Mentat syrup is manufactured by Himalaya herbal health care and it is the polyherbsal
formulation containing the following herbs and their concentrations6.
Brahmi (Bacopa monnieri)
288mg
Mandukaparani (Centella asiatica)
140mg
Ashvagandha (Withania somnifera)
104mg
Vishnukrantha (Evolvulus alsinoides)
104mg
Jatamansi (Nardostachys jatamansi)
104mg
Tagara (Valeriana wallichii Syn. V.jatamansi)
100mg
Vidanga (Embelia ribes)
100mg
Vatadha (Prunus amygdalus)
100mg
Vacha (Acorus calamus)
84mg
Haritaki (Terminalia chebula)
72mg
Amalaki (Emblica officinalis)
72mg
Guduchi (Tinospora cordifolia)
72mg
Jyotishmati (Celastrus paniculatus)
64mg
Shyonaka (Oroxylum indicum)
64mg
Kapikachchhu (Mucuna pruriens)
3.6mg
Ela (Elettaria cardamomum)
3.6mg
Arjuna (Terminalia arjuna)
3.6mg
Shatapushpa (Foeniculum vulgare)
3.6mg
Vidari (Ipomoea digitata)
3.6mg
Musali (Curculigo orchioides)
3.6mg
Sunthi (Zingiber officinale)
2.8mg
Vibhitaki (Terminalia belirica Syn. T.bellirica)
2.8mg
Jatiphalam (Myristica fragrans)
2.8mg
Lavanga (Syzygium aromaticum)
2.0mg
Few studies have been reported on this formula and several reports on the individual herbs are
available. However relevant reports are recorded here.
Ganesh et al.reported that the extract of mentat possesses concentration dependent NO
scavenging activity up to 250 μg/ml (67.21%), which was 1.22 fold higher than the equimolar
concentrations of G. biloba (54.92%)7. Bhattacharya reported that the mentat has anxiolytic
effect11. Preeti et al., reported that Mentat-200 restored locomotor activity in 3-NPA treated
animals12. Majumdar et al., reported that treatment with mentat syrup in children with simple
febrile convulsions is beneficial27.
Yamada et al., reported that the Brahmi enhanced immune function by increasing
immunoglobulin production21.
Ashwagandha is shown to possess anti-inflammatory, anticancer, antistress, Immunomodulatory
and adaptogenic properties. Further various protective effects have been described in endocrine,
central nervous system and cardiovascular system in both animal and human subjects. The root
extract significantly reduced the lipid peroxidation and increased superoxide dismutase (SOD) and
catalase activity, thus possessing a free radical scavenging property. The active constituents of
plant (Withaferin A, Sitoindosides VII- X) are reported to have antioxidant activity which may
contribute atleast in part to the reported antistress, immunomodulatory, cognition facilitating,
antiinflammatory and antiageing properties. WS is reported to modulate GABAergic [γ-aminobutyric acid (GABA)] or cholinergic neurotransmission accounts for various CNS related disorders.
Withaferin A has been recently reported to be inhibitory of angiogenesis and thus protective in
certain types of cancer. Two glycowithanolides (Sitoindoside IX or Sitoindoside X) were found to
possess antistress activity and augmented learning acquisition and memory relation in both young
and old rats.9
Bensimon et al., reported the effect of Centella asiatica (CA) extract in the prevention of
sporadic dementia of Alzheimer’s type using intracerebroventricular colchicines-induced rats.
Salient findings of this study are that pre- and postcolchicine treatment with CA improved
cognition, decreased malondialdehyde, and nitrite levels, restored decrease in GSH, increased
activities of glutathione-S-transferase, catalase, and SOD. This illustrates that central
administration of colchicine is characterized by progressive deterioration of learning and memory,
oxidative stress, and decrease in acetylcholine turnover.10
Indications Mentat syrup:
Pediatrics: Memory and learning disorders; Attention fluctuations, Concentration impairment,
Language and learning disability, Attention Deficit Hyperactivity Disorder (ADHD).
Adults: Behavioral disorders; Hyperkinetic states, Asocial behavior, Aggressive behavior, Anxiety
and stress- related anxiety disorders, Mental fatigue, As an adjuvant in Alzheimer’s and
Parkinson’s disease, As an adjuvant in epilepsy
Donepezil hydrochloride: (2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)4piperidinyl]methyl]-1H-inden-1-one) hydrochloride. is a piperidine derivative having a selective,
reversible and non-competitive acetylcholinesterase inhibitor with a relatively high central versus
peripheral specificity14. Neuroprotection by donepezil against glutamate excitotoxicity involves
stimulation of α7 nicotinic receptors and internalization of NMDA receptors15. Meunier et al.,
reported that donepezil is a potent antiamnesic and neuroprotective compound against the
neurodegeneration induced by excitotoxic insult, and its pharmacological actions as both an
acetylcholinesterase inhibitor and σ1 receptor agonist contribute to its marked efficacy. In
particular, the drug is a more potent postinsult protecting agent compared with more selective
cholinesterase inhibitors24.Takada et al., reported that donepezil protected cortical neurons against
glutamate Neurotoxicity via neuronal α4β2- and α7-nAChRs. In addition, donepezil prevented
glutamate-induced apoptosis. The present findings suggest that donepezil shows neuroprotective
action in addition to amelioration of cognitive deficits25.
Since, there are no reports on the Mentat and Donepezil, the study is planned with the following
objectives.
6.3 OBJECTIVES OF THE STUDY

To evaluate the neuroprotective activity of Mentat syrup and Donepezil against
Monosodium glutamate, 3-Nitropropionic acid and bilateral carotid occlusion induced
neurotoxicity in rats.

To study, neuroprotective activity of combination of Mentat syrup and Donepezil
hydrochloride treatment in Monosodium glutamate, 3-Nitropropionic acid and bilateral
carotid occlusion induced neurotoxicity in rats. .
MATERIALS AND METHODS
7.1 SOURCE OF DATA
Data will be generated by performing experiments on animals. The standard information from collected
from various journals, standard Text books in library of Govt. college of pharmacy, Indian
Institute of Science, RGUHS digital library and from various standard websites.
Web sites:
www.sciencedirect.com
www.pubmed.com
www.google.com
CHEMICALS
Monosodium glutamate, 3-Nitropropionic acid, ethanol, anesthetic ether, bovine serum
albumin, tetra ethoxy propane, glutathione reductase, glutathione, phenazine methosulphate,
nitroblue tetrazolinium etc.
ANIMALS
Adult rats weighing between 200-250 g of either sex will be used in the study. The animals will be
kept under standard conditions at ambient temp of 25± 20 C with 12hr light/ 12hr dark cycle, with
food and water. Animals will be acclimatized to laboratory condition before the test.
TOTAL ANIMALS REQUIRED
Total animals used in acute toxicity studies =12mice
Animals used in each group
= 06 rats
Total rats used
= 108 rats (6 groups for one model)
Total 108 wistar rats and 18 albino mice are required.
7.2 EXPERIMENTAL DESIGN
I Monosodium glutamate induced neurotoxicity model19 :
Total 30 rats of either sex will be randomly divided in to five groups of 6 rats each and treated for 7
days as fallows;
Group 1: Receives only vehicle (p.o.) for 7 days.
Group 2: Receives MSG 2 g/kg (i.p.) + normal saline (p.o.) for 7 days.
Group 3: Receives MSG 2 g/kg (i.p.) + mentat syrup (p.o.) for 7 days
Group 4: Receives MSG 2 g/kg (i.p.) + Donepezil hydrochloride (p.o.) for 7 days
Group 5: Receives normal saline (i.p) + mentat syrup and Donepezil hydrochloride (p.o) for 7 days.
One hour after the administration of MSG all animals will be treated with mentat and Donepezil
hydrochloride. The dose of MSG is selected based on previous literatures.19,26 During the drug
treatment rats will be observed for the behavioral changes for 50 minutes daily. On 8th day the rats
will be observed for General behavior, elevated plus maze test, rota rod test and despair swim test.
On 9th day rats will be sacrificed and brain will be isolated for estimation of glutathione, lipid
peroxidation, and histopathological study will be performed.
II 3-Nitropropionic acid induced neurotoxicity model20:
Total 30 rats of either sex will be randomly divided in to five groups of 6 each and treated as
fallows:
Group1 : Receives only vehicle alone for 7 days (p.o.) from 8th day onward vehicle and normal
saline (i.p.) in gap of 2 hours, daily up to 14 days.
Group 2 : Receives normal saline alone for 7 days (i.p) from 8th day onward saline followed
by 3-NP 10 mg/kg (i.p.) in gap of 2 hours, daily up to 14 days.
Group 3 : Pretreatment for 7 days with mentat syrup (p.o.)
from 8th day onward mentat syrup followed by 3-NP 10
mg/kg (i.p.) in gap of 2 hours, daily up to 14 days.
Group 4 : Pretreatment for 7 days with Donepezil hydrochloride (p.o.) from 8th day onward
Donepezil hydrochloride followed by 3-NP 10 mg/kg (i.p) in gap of 2 hours, daily up to
14
days.
Group 5 : Pretreatment for 7 days with mentat syrup and Donepezil hydrochloride (p.o.)
from 8th day onward mentat syrup and Donepezil hydrochloride followed by 3-NP
10 mg/kg (i.p) in gap of 2 hours, daily up to 14 days.
Mentat and Donepezil hydrochloride will be given 2 hour prior to 3-NP administration. The
dose of 3-NP is selected based on previous literature.22 During the drug treatment rats will be
observed for the behavioral changes for 50 minutes daily. On 14th day 4 hours after 3-NP
administration rats will be observed for General behavior, elevated plus maze test, rota rod test and
despair swim test. On 15th day rats will be sacrificed and brain will isolated for estimation of GSH,
lipidperoxidation, and histopathological study.
III. Bilateral carotid artery occlusion:
Total 48 rats of either sex will be randomly divided in to five groups of 6 each and treated as
fallows:
Group-I : Receives only vehicle orally. (n=8).
Group-II : Sham control (10 ml/kg), vehicle orally. (n=8).
Group-III : Normal saline (10 ml/kg,) oral, BCA occlusion for 30 min, followed by reperfusion
for 4 hr. (n=8).
Group-IV : mentat syrup , BCA occlusion for 30 min, followed by reperfusion
for 4 hr. (n=8).
Group-V : Donepezil hydrochloride, BCA occlusion for 30 min, followed by
reperfusion for 4 hr. (n=8).
Group-VI : mentat syrup and Donepezil hydrochloride, BCA occlusion for 30 min, followed by
reperfusion
for 4 hr. (n=8).
7.3 Parameters to be Evaluated:
I Measurement of body weight changes
II Behavioral models:
1 General behavior
3 Elevated Plus maze test
4 Rota rod test
III Brain tissue biochemical estimation:
1 Glutathione
2. Lipid peroxidation
IV Brain tissue histopathological evaluations
7.4 STATISTICAL ANALYSIS
The data obtained from experimentation will be subjected to one way analysis of variance
(ANOVA) with suitable post-hoc test
7.5 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS
TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS /ANIMALS? IF SO
PLEASE DESCRIBE BRIEFLY.
Yes, the above study requires investigation to be done on the Albino rats for the
determination of neurotoxicity.
7.6 HAS ANIMAL ETHICAL COMMITTEE CLEARANCE BEEN OBTAINED
FROM YOUR INSTITUTION IN CASE?
YES, The study has been referred to the ethical committee of the institution and clearance is
awaited. After obtaining from the IAEC the certificate will be submitted to the university.
8.0 REFERENCES
1. Hardman JG, Limbird LE, Goodman Gilman A. editors the pharmacological basis of
therapeutics. 2001; 549-66 10th ed. New York: McGraw-Hill.
2. Anonymous. Neurological disorders: public health challenges. Switzerland: World Health
Organization. 2006; 189.
3. Weil ZM, Norman GJ, DeVries AC, Nelson RJ. The injured nervous system: a Darwinian
perspective. Prog Neurobiol. 2008; 86:48-59.
4. Ahuja M, Bishnoi M, Copra K. Protective effect of minocycline, a semi synthetic second
generation Tetracycline against 3-nitropropionic acid induced neurotoxicity. Toxicology.
2008; 244:111-22.
5. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th ed. Edinburgh:
Churchil Livingstone. 2003; 490-3.
6. Composition of mentat syrup [online] December 27, 2012 available from URL:
http://members.tripod.com/kelab2020_p2020/mn001.htm
7. Jagetia GC, Rao SK, Baliga MS, Babu KS. The Evaluation of Nitric Oxide Scavenging
Activity of Mentat.2004;18: 561–565 .
8. Prabhu V, Karanth KS, Rao A effect of Nardostachys jatamansi on biogenic amines and
inhibitory amino acids in the rat brain, Planta Medica.1994; 60: 114.
9. Kulkarni Dhir Ashish. Withania somnifera: An Indian ginseng. Progress in
Neuropharmacology & Biological Psychiatry.2007.09.11; doi:10.1016/j.pnpbp.
10. Bensimon G, Chermat R. Microtubule disruption and cognitive defects: effect of colchicine
on learning behavior in rats. Pharmacology Biochemistry and Behavior.1991; vol.
38(1):141–145. View at Publisher · View at Google Scholar.
11. Battacharya SK. Behavioural studies on BR_16A(Mentat), A herbal psychotropic
formulation. Indian J. Exp. Bio. 1994: (32): 37-47
12. Preeti T, Patil PA, Suneel, Majagi I. locomotor activity in 3-NPA treated animals[online]
December 27, 2012 available from URL:
http://pharmacologyonline.silae.it/files/archives/2010/vol1/84.Patil.pdf
13. Donepezil [online] December 27, 2012 available from URL:
http://pubs.acs.org/doi/abs/10.1021/jm0109356
14. Donepezil chemistry [online] December 27, 2012 available from URL:
http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=3152
15. Shen H, Kihara T, Hongo H, Kem, Shimohama S, Kaike AA et al., Neuroprotection by
donepezil against glutamate excitotoxicity involves stimulation of α7 nicotinic receptors and
internalization of NMDA receptors Br. J. Pharmacol. 2010; 161(1): 127–139.
16. Kryger G, Silman I, Sossaman JL. Structure of acetylcholinesterase complexed with E2020
(Aricept): implications for the design of new anti-Alzheimer drugs. Structure. 1999; 7(3):297307.
17. Ki YS, Park EY, Lee HW, oh MS, Cho YW, Kwon YK. Donepezil, a potent
acetylcholinesterase inhibitor, induces caspase-dependent apoptosis in human promyelocytic
leukemia HL-60 cells. Biol Pharm Bull. 2010;33(6):1054-9.
18. Sugimoto H. Donepezil hydrochloride: a treatment drug for Alzheimer's disease. Chem Rec.
2001;1(1):63-73.
19. Ramanathan M, Sivakumar S, Anandvijayakumar PR, Saravanababu C,
Rathinavel PP. Neuroprotective evaluation of standardized extract of Centella
asiatica in monosodium glutamate treated rats. Indian J Exp Biol. 2007;45:425-31.
20. Szabo A, Andras P, Laszlo N. Effect of 3-nitropropionic acid in rats general toxicity and
functional Neurotoxicity Arh Hig Rada Toksikol. 2005; 56:297-302.
21. Yamada K, Hung P, Park TK, Park PJ. A comparison of the immunostimulatory effects of
The medicinal herbs Echinacea,Ashwagandha and Brahmi j. Ethnopharmacology. 2011; 137
(1): 231-235.
22. Kumar P, kumar A Effect of root extract of Withania somnifera in 3-Nitropropionic acid
induced Cognitive dysfunction and oxidative damage in rats. Int J Health Res. 2008; 1:13949.
23. Neuroprotective action of Donepezil[online] December 27, 2012 available from URL:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2962822
24. Meunier J, leni J, Maurice J. Antiamnesic and Neuroprotective Effects of Donepezil against
Learning Impairments Induced in Mice by Exposure to Carbon Monoxide Gas. J. Pharmacol
Exp. Therapeut. 2006 ; 317: 1307-1319.
25. Yuki Takada, Atsushi Yonezawa, Joshiaki Kume, Hiroshi Katsuki, Shuji Kaneka Nicotinic
Acetylcholine Receptor-Mediated Neuroprotection by Donepezil Against Glutamate
Neurotoxicity in Rat Cortical Neurons J. Pharmacology Exp. Therapeut. 2003; 306: 772-777.
26. Preethi K, Chandran, Kuttan R. Effect of Calendula officinalis flower extract on acute phase
Proteins , antioxidant defence mechanism and granuloma formation during thermal burns. J
Clin Biochem Nutr. 2008;43:58-64.
27. Majumdar, Ratna, Abhijeet Bagade, Marudula A, Phadke. Seizure Control and Pattern of
Behaviour in Children with Simple Febrile Convulsions – Effect of Mentat Syrup The Indian
Practitioner . 2001; 54 (10): 729.
28. Kim MS, Lee JI, Lee WY, Kim SE. Neuroprotective effect of Ginkgo biloba L. extract in
rat model of Parkinson’s disease. Phytother Res.2009;18:663-666.