ACHE BCHE PLANT EXTRACT.pdf

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RESEARCH ARTICLE

International Journal of Pharma and Bio Sciences

SCREENING OF TRADITIONAL INDIAN SPICES FOR INHIBITORY ACTIVITY OF

ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE ENZYMES

S. KUMAR*, BRIJESHLATA AND S.DIXIT

University School of Biotechnology, GGS Indraprastha University, Sector-16C, Dwarka, New Delhi-

110075

*Corresponding author

PHARMACOGNOSY

ABSTRACT

The Indian spices are known for their medicinal properties from ages apart from addingtaste to the Indian cuisines. In this study, traditional Indian spices such as Cuminum cyminum,Cinnamomum zeylanicu, Eletteria cardamom, Eugenia caryophyllus and Piper nigrum wereassessed for inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)enzymes. The assessment of cholinesterase enzyme inhibition was carried out by using acolorimetric method based on Ellmans reaction. Our findings demonstrate that aqueous andethanolic extract of these plants significantly inhibited AChE and BuChE enzymes. For aqueousextracts the maximum inhibitory activity was shown by Cuminum cyminum(66.660.005) % and(66.840.001) % for AChE and BuChE respectively. For ethanolic extracts, maximum inhibitoryactivity for AChE was observed for Eletteria cardamom i.e. 61.960.003% whereas for BuChEinhibition, Piper nigrum showed maximum inhibition i.e 73.260.005%. These results showed

that there could be great potential to search for novel usage of these medicinal plants for anti-cholinesterase compounds.

S. KUMARUniversity School of Biotechnology, GGS Indraprastha University, Sector-16C,

Dwarka, New Delhi-110075


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KEYWORDS

Acetylcholinesterase, butyrylcholinesterase, inhibition, Ellmans method, Alzheimers disease

INTRODUCTION

Acetylcholine (ACh) is one of the mostimportant neurotransmitters of central nervoussystem (CNS) associated with memory andcognition 1. A deficit of ACh levels in CNS leadsto conditions such as Alzheimers disease (AD).AD is the most common form of dementia, aprogressive neurologic disease of the brainthat leads to loss of mental ability severeenough to interfere with normal activities ofdaily living and decline in cognitive functionssuch as remembering, reasoning and planning.It affects parts of the brain that controlthought, memory, and language. It ischaracterized by nerve-cell loss, abnormaltangles and plaques within nerve cells anddeficiencies of several neurochemicals suchas acetylcholine (ACh) andbutyrylcholine(BuCh), which are essential forthe transmission of nerve messages. It waspostulated that blocking the enzymecholinesterase (ChE) induced hydrolysis ofACh and subsequent increase in AChconcentration in central synapses andenhancement of cholinergic functions providesthe symptomatic improvement to AD patient2,3,4,5. ChE inhibitors were developed toimprove the effectiveness of ACh byinhibiting its breakdown and increasing thelevels in the brain or by strengthening theway nerve cells responds to it. Increasedconcentrations of ACh in the brain leads to

increased communication between nerve cellsand may temporarily improve or stabilize thesymptoms of AD. These drugs appear towork best in the early and moderate stagesof AD 6,7. It has been further suggested thatdual inhibition of AChE and BuChE enzymesshould be one of the objectives in thetreatment of cognitive dysfunction associatedwith AD 8,9. Currently, most of the drugs

used for the treatment of AD are eitherAChE inhibitors like tacrine, physostigmineetc. or BuChE inhibitors astetrahydrofurobenzofuran cymserine (THFBFC),which have all been proven to improve thesituation of AD patients to some extent 10. Sofar, the four drugs that have been approvedby the Food and Drug Administration (FDA)to treat AD are tacrine , rivastigmine donepezil, and galanthamine, which all havesome success in slowing downneurodegeneration in AD patients

11. The

limitations of these drugs are their sideeffects such as aggression, depressiongastrointestinal disturbances andhepatotoxicity. Furthermore, these drugs areexpensive and require weekly bloodmonitoring

12. In view of the limitations of the

current drugs, there is an urgent need to lookfor new lead molecules from differentsources such as natural product, which canbe used to target these enzymes and helpsin alleviating the symptoms of AD. A varietyof plants has been reported to show ChEsinhibitory activity and may be relevant fortreatment of AD related to cholinergic deficit13, 14. Based on this hypothesis we screenedtraditional Indian spices such as Piper nigrum,Eletteria cardamom, Cinnamomum zeylanicum,Eugenia caryophyllus and Cuminum cyminumused in Indian cuisine which have a number

of medicinal properties and their use hasbeen advocated in traditional medicine forvarious problems like stomach disorders digestive problems, as CNS depressant andmany more. The present study evaluates theAChE and BuChE inhibitory activity of anaqueous and ethanolic extracts of thetraditional Indian spices in vitro by Ellmanmethod.


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MATERIALS AND METHODS

Chemicals: Acetylcholinesterase ( EC 3.1.1.7) from electric eel; butyrylcholinesterase ( EC3.1.1.8 ) from equine serum were purchasedfrom Sigma Aldrich, India ; acetylthiocholineiodide ( ATChI ); butyrylthiocholine iodide (BTChI ); 5, 5-dithio-bis-(2-nitrobenzoic acid)(DTNB); sodium bicarbonate were purchasedfrom Himedia Laboratories Pvt. Ltd., India .Phosphate buffer; and ethanol were obtainedfrom Sisco Research Laboratories Pvt. Ltd.,India.

Plant materials and extraction: All plantsamples namely Piper nigrum fruit (Voucherno.001) , Eletteria cardamom fruit (Voucherno.002), Cinnamomum zeylanicum bark(Voucher no.003), Eugenia caryophyllus flowerbud (Voucher no.004) , Cuminum cyminum fruit(Voucher no.005) were purchased from a localstore in Delhi, India . The plants wereauthenticated by a Botanist and voucherspecimens of these samples are stored in aherbarium at University School OfBiotechnology, Guru Gobind SinghIndraprasha University, Dwarka Sec- 16C, NewDelhi-110075.Fresh samples of the plant materials wereair dried at ambient room temperature andpowdered in a grinder. One gram of eachsample was weighed and extracted withdistilled water (1:25 w/v) and 90% ethanol. Thesample were boiled for 15-20 minutes andcooled. The samples were filtered usingmuslin cloth and the filtrate was lyophilized. Thelyophilized samples were collected and

stored at -200C until use.

Cholinesterase assay: An assessment ofcholinesterase inhibition was carried out inflat-bottom 96-well microtitre plates using thecolorimetric method of Ellman et al 15. Atypical run consisted of 5L of electric eelAChE solution, at final assay concentrationsof 0.03 U/mL; 200L of 0.1 M phosphate

buffer pH 7; 5L of DTNB at a finaconcentration of 0.3mM prepared in 0.1 Mphosphate buffer pH 7 with 0.12M of sodiumbicarbonate; and 5L of the test extract .The

reactants were mixed and pre incubated for15 minutes at 30C. The reaction wasinitiated by adding 5L of ATChI at a finaconcentration of 0.5mM. As a control theinhibitor solution was replaced with buffer. Althe reactions were carried out in triplicate(n=3). To monitor any non-enzymatichydrolysis in the reaction mixture two blanksfor each run were prepared in triplicate. Oneblank consisted of buffer replacing enzymeand a second blank had buffer replacing

substrate. Change in absorbance at 405 nmwas measured on a BioRad model 550(Ascent software version 2.24), 96-well platereader for a period of 6 min at 30 CSimilarly for butyrylcholinesterase assay sameprocedure was followed only difference beingAChE enzyme replaced by BuChE andATChI replaced by BTChI.

RESULT AND DISCUSSION

AChE and BChE inhibitory activities ofthe plant species used in this study wereevaluated and percentage inhibitions areshown in Table 1.

The present data revealed that althe extracts possessed potent AChE andBChE inhibitory activity at 100g/mconcentration. Among the plant screened, anaqueous extract of Cuminum cyminumshowed the maximum inhibition i.e66.660.005% for AChE and 66.840.001 %

for BuChE respectively. For ethanol extractmaximum AChE inhibition was shown byEletteria cardamom i.e. 61.960.003% andmaximum BuChE inhibition was shown byPiper nigrum i.e. 73.260.005%. The p value isless than 0.01 as compared to control (noenzyme inhibition) which is extremelysignificant result.


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Plants have been used as a source ofnew bioactive compounds for drug discoverysince ages and have many advantages inrelation to efficacy. Numerous medicinal

plants such as Centella asiatica, Nelumbonucifera, Myristica fragrans etc. have receivedmuch attention to improve cognitive functionagainst cognitive deficit condition included inAD condition

16. However the search for

potent long-acting anti-cholinesterase (AChEand BuChE) inhibitors is still ongoing. Basedon cholinergic hypothesis we screened someIndian traditional spices (listed in Table 1) fordual anti-cholinesterase activity against theAChE and BChE enzymes which are

considered to be related to the mechanismof memory dysfunction in AD.

Black pepper (Piper nigrum), is one ofthe oldest and most popular spices in theworld. It belongs to the family Piperaceae andis used in many Asian countries. It is usedin folk medicine for stomach disorders,digestive problems, and neuralgia and asCNS depressant 17. Its active constituent,piperine, has been reported to significantlyimprove memory impairment and

neurodegeneration in hippocampus. Moreover,piperine also demonstrated the neurotrophiceffect in hippocampus

18. The aqueous extract

of Piper nigrum showed an inhibitory activityof 52.250.002% for AChE enzyme and63.890.005% (p< 0.01) for BuChE enzyme.The ethanol extract from Piper nigrum fruitalso showed an inhibitory effect on AChEand BuChE with percentage inhibition of50.720.002 and 73.260.005 (p < 0.01)respectively.

Cinnamomum zeylanicum has beenused as a spice as well as traditionalmedicine for many centuries. It possessesmany unique medicinal properties such assugar control, anti-oxidant, anti-inflammatoryand antimicrobial activity 19. Cinnamon extracthas been found to have an inhibitory effecton tau aggregation related to Alzheimer'sdisease (AD). The extract can also promote

complete disassembly of recombinant taufilaments and cause substantial alteration ofthe morphology of paired-helical filamentsisolated from AD brain 20. In a recent study

orally administered cinnamon extract hasbeen found to reduce -amyloidoligomerization and correct cognitiveimpairment in AD animal models 21. In thisstudy an aqueous extract of C.zeylanicumshowed an inhibitory activity of 46.840.005% for AChE enzyme and 51.750.005% forBuChE enzyme. The ethanol extract showedpercentage inhibition of 40.830.005 and51.530.005 for AChE and BuChErespectively which is not as significant

compared to other extract.

Cumin (Cuminum cyminum) is anaromatic plant, belongs to Apiaceae family . Itis used to flavour foods and used in manymedicinal preparations. It has been used asan astringent, as carminative and eupepticas well as an analgesic agent

22. Cumin

extract has also been known to possessantistress, antioxidant and memory- enhancingactivity

23. An aqueous extract of Cuminum

cyminum fruit showed a strong inhibitory

activity for AChE i.e. 66.660.005% (p< 0.01)and 66.840.001% (p< 0.01) for BuChE. Anethanol extract of C.cyminum also showed astrong inhibitory effect with percentageinhibition of 61.910.005 (p < 0.01) and71.340.001 (p < 0.01) for AChE and BuChErespectively.

Eugenia caryophyllus (clove), belongingto the family Myrtaceae, has a number ofmedicinal properties. Clove has been reportedto possess a potent anti-oxidant activity in

vitro, which reduces the oxidative stress inthe body

24. Clove oil has been reported to

reverses learning and memory deficits inscopolamine treated mice 25. The aqueousextract of Eugenia caryophyllus has alsobeen found to possess AChE inhibitoryactivity in rats. It reduces the hydrolysis ofACh by AChE

26. The aqueous extract of

Eugenia caryophyllus flower bud showed


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inhibitory activity of 50.450.003 % for AChEenzyme and 59.090.006% for BuChEenzyme. The ethanol extract showedpercentage inhibition of 49.760.005 and

54.390.005 for AChE and BuChErespectively.

Eletteria cardamom belongs toZingiberaceae family.It has been traditionallyused as a culinary spice in foods. Thecognitive enhancing properties of this plant

might be related to its antioxidant activityrelevant to AD

27. In this study an aqueous

extract of Eletteria cardamom fruit showedinhibitory activity of 45.940.002 % for AChE

enzyme and 61.960.003% (p < 0.01) forBuChE enzyme. The ethanol extract of alsoshowed a strong inhibitory effect withpercentage inhibition of 61.990.001 (p < 0.01)and 66.110.001 (p < 0.01) for AChE andBuChE respectively.

Table 1

Anticholinesterase activity of plant extracts against AChE and BuChE

Sample Family Plant PartUsed

Inhibition (%)AChE aqueous

extract(100g/ml)

Inhibition (%)AChE

ethanol extract(100g/ml)

Inhibition (%)BChE aqueous

extract(100g/ml)

Inhibition (%)BChE ethanol

extract(100g/ml)

Cinnamomumzeylanicum

Lauraceae Bark 46.840.003 40.830.005 51.750.005 51.530.005

Cuminumcyminum

Umbellifereae Fruit 66.660.005* 61.910.005* 66.840.001* 71.340.001*

Eletteriacardamom

Zingiberaceae Fruit 45.940.002 61.960.003* 61.990.001* 66.110.001*

Eugenia

caryophyllus

Myrtaceae Flower bud 50.450.003 59.090.006 49.760.005 54.390.005

Piper nigrum PiperaceaeFruit

52.250.002 50.720.002 63.890.005 73.260.005*

Values are expressed as mean SDEV (n=3)*p < 0.01 compared to control (no enzyme inhibition).

CONCLUSION

An aqueous extract of Cuminumcyminum has more potent AChE inhibitory

activity as compared to Cinnamomumzeylanicum, Eletteria cardamom, Eugeniacaryophyllus and Piper nigrum. An ethanolicextract of Cuminum cyminum and Eletteriacardamom showed comparable AChEinhibitory activity and more potent thanCinnamomum zeylanicum, Eletteria cardamomand Piper nigrum. An aqueous extract ofCuminum cyminum, Eletteria cardamom and

Piper nigrum showed more potent BuChEactivity as compared to Cinnamomumzeylanicum and Eugenia caryophullus. Anethanolic extract of Cuminum cyminum,Eletteria cardamom and Piper nigrum showed

more potent inhibition of BuChE ascompared to Cinnamomum zeylanicum andEugenia caryophyllus. In the light of thesefindings, we conclude that most of the plantextracts screened herein showed significantChE inhibitory activity against both of theenzymes and they can be considered forfurther studies in the treatment of ADHowever, there is a need to isolate and


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characterize the compounds responsible forthe anticholinesterase activity for theireffective utilization in the treatment of

Alzheimer's disease and other stress relateddisorders. Studies in this direction arecurrently underway in our laboratory.

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