Orient Pharm Exp Med

DOI 10.1007/s13596-013-0141-3

RESEARCH ARTICLE

Evaluation of anti-cholinesterase activity of the standardized

extract of Piper betel L. leaf
Manoj K. Dalai & Santanu Bhadra & Arun Bandyopadhyay &
Pulok K. Mukherjee

Received: 10 September 2013 / Accepted: 30 October 2013

# Institute of Korean Medicine, Kyung Hee University 2013

Abstract Piper betel L. (Piperaceae) is popularly known as

betel leaves. In Indian culture betel leaves play an important
role for the management of numerous diseases. The present
study was an attempt to evaluate the acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) inhibitory activity
of the standardized extract of P. betel leaf. After extraction the
P. betel leaf extract was standardized with the help of
hydroxychavicol and chlorogenic acid as bio- markers,
through HPLC. Further the anticholinesterase inhibitory activities of the standardized extract were evaluated by using 96well micro plate assay and thin layer chromatography bioassay detection methods. Results of the present study showed
that hydroxychavicol isolated from P. betel showed potential
enzyme inhibition activity than chlorogenic acid. Moreover,
the mixture of hydroxychavicol (HCH) and chlorogenic acid
(CGA) in 1:1 ratio have showed more potent cholinergic
activity than tested fraction and used bio marker compounds.
AChE and BChE inhibition (IC50) of HCH and CGA (1:1)
was found to be 21.230.33 g/mL and 45.551.89 g/mL
respectively. Outcome of the investigation exhibited that P.
betel can be a good lead as anti-cholinesterase agent and could
be explored further for its therapeutic potential for the management of Alzheimers disease (AD).
Keywords Piper betel . Acetylcholinesterase .
Butyrylcholinesterase . Hydroxychavicol . Chlorogenic
acid . Alzheimers disease

M. K. Dalai : S. Bhadra : P. K. Mukherjee (*)

Department of Pharmaceutical Technology, School of Natural
Product Studies, Jadavpur University, Kolkata 700 032, India
e-mail: naturalproductm@gmail.com
A. Bandyopadhyay
Indian Institute of Chemical Biology, Kolkata 700 032, India

Introduction
Application of the cholinesterase inhibitors are the clinically
recommended therapy for the management of Alzheimers
disease (AD), which is one of the major concerns in todays
society (Mukherjee et al. 2007a, b). Unfortunately, almost all
the present cholinesterase inhibitors available to treat AD have
displayed potential adverse effects such as gastric disturbance,
allergic reaction (Mukherjee et al. 2007a, b). Also those FDA
approved drugs failed to produce desired therapeutic potential.
Keeping the drawbacks of present anticholinesterases in mind
scientists, all over the world are bending towards the plant
resources for the development of better, novel and safe alternatives for these drugs (Mukherjee and Houghton 2009). It
was observed that, majority of the available cholinesterase
inhibitors are actually developed to inhibit acetyl cholinesterase (AChE). Whereas, recently it was reported that inhibition
of butyryl cholinesterase (BChE) is inevitable for the better
management of the AD (Bhadra et al. 2011).
Piper betel L (Piperaceae) is widely cultivated commercial
basis in the tropical moist region of all South Asian countries.
Five popularly known varieties of betel leaves are available in
the Indian subcontinent e.g. Bangla, Desasvari, Kapoori,
Meetha and Sanchi. These leaves are known for folk remedies
of boils, abscesses, conjunctivitis, constipation, headache, hysteria, itches, mastitis, mastoiditis, leucorrhoea, otorrhoea, ringworm, swelling of gum, rheumatism, abrasion, bad breath, cuts
and injuries (Norton 1998). In Indian culture, P. betel is used
primarily for welcoming guest, courtship and ancestral remembrance. Betel leaves are traditionally chewed as a mouth freshener and contains significant amounts of vitamins and minerals
(Guha 2007). The frequency of chewing of betel also varies
among local traditions of Asian people. While in different part
of Asian countries, peoples are usually taken Betel quid for
masticating. During mastication it produces euphoria, mild
stimulant and cholinergic effect (Norton 1998). Betel is

M. K. Dalai et al.

complex mixture of various natural occurring substances; includes leave from the P. betle L. vine, nut or fruit of A. catechu
L., slaked lime, paste of the bark from the A. catechu tree and
ingredient of culinary spices (Rai et al. 2005). Alkaloid compounds, arecoline from A. catechu have shown cholinergic
effect in dementia affected rat model (Saha et al. 2007), dual
activity of cholinomimetic and acetylcholinesterase (AChE)
inhibition in mice (Gilani et al. 2004). Betel leaves are attributed
with aromatic, digestive, stimulant, carminative properties
(Maity et al. 2012). Betel leaves are reported to possess a wide
range of biological and pharmacological activities including
cardio protection (Arya et al. 2010), hepato protection, antioxidant (Saravanan et al. 2006) and nootropic activities
(Vyawahare and Bodhankar 2007). Gilani et al. (2000) have
reported on the constituents of aqueous extract as well as ethyl
acetate fraction of P betle leaf possesses cholinergic effect.
Similarly, hydro alcoholic extract of P. betle leaf facilitates the
cholinergic effect in haloperidol induced catalepsy (Vyawahare
and Bodhankar 2007). An aqueous extract of P. betle leaf have
also exhibit AChE inhibition activity (Valenta et al. 2010).
Literature study reveals that P. betel leaves contains
phytoconstituents of estragole, catechols, terpenes, lominene,
cardinene, hydroxychavicol, eugenol, ascorbic acid and carotene (Prabu et al. 2012). Hydroxychavicol (HCH) and
chlorogenic acid (CGA) found from betel leaf showed potent
antioxidant, anti-inflammatory (Pin et al. 2010) and neuroprotective activities against cognitive deficit in animals (Pandey
and Bani 2010). From the above context it is clear that P. betel
leaves possess diverse biological activities and it can play a
pivotal role in search for better cholinesterase inhibitors, thus
the present study was undertaken to screen P. betel extracts for
AChE and BChE inhibitory activities for in search of novel
lead for better AD management.

Fig. 1 Hydroxychavicol (i) and Chlorogenic acid (ii)

procured from Sigma (Poole, UK). Methanol, n-butanol, ethyl

acetate, and all other solvents (HPLC reagent grade) were
purchased from Merck, India. All other solvents and reagents
used were of analytical grade.
Preparation of the extract and fractionation
Eight hundred grams of shade dried P. betel leaves were
crushed into coarse powder and extracted with methanol and
water mixture at 7:3 ratios (3 L), by cold maceration process
for 72 h. This process of extraction was repeated three more
times to get maximum yield. Extract was then filtered through
a nylon mesh and subsequently evaporated using rotary evaporator (EYELA, Tokyo, Japan) at a temperature not exceeding
45 C to obtain a dark brownish semisolid residue. The
semisolid extract was finally lyophilized to remove moisture
(yield 7.5 % w/w), which was stored in vacuum desiccators
for the study. The resultant hydroalcoholic extract was subsequently fractioned with n-butanol and ethyl acetate. The yields
of n-butanol and ethylacetate fractions were found to be 3.2
and 1.2 % (w/w) respectively.
RP-HPLC standardization P. betel leaves extracts

Materials and methods

Plant materials
P. betel leaves were purchased from local market in Kolkata,
West Bengal, India and authenticated by Dr. S. Rajan, field
botanist, medicinal plants collection unit, Ooty, Government
of India. A voucher specimen (SNPS-JU/2012/1052) has been
deposited in the herbarium of the School of Natural Product
Studies, Jadavpur University, Kolkata, India.
Chemical and reagents
Standard HCH and CGA (Fig. 1) were obtained from the
School of Natural Product Studies, Kolkata. AChE from bovine
erythrocytes, BChE from equine serum, acetyl thiocholine
iodide (ATCI), butyryl thiocholine iodide (BTCI), 5, 5dithiobis [2-nitrobenzoic acid] (DTNB) and galantamine was

RP-HPLC standardization of P. betel leaf extracts using HCH

and CGA as biomarker was done according to the previously
published protocol from our laboratory (Maity et al. 2012).
Briefly, HPLC system (Waters, Milford, MA, USA)
equipped with 600 quaternary HPLC pump, a Waters 2489
UVvis dual wavelength detector and a Rheodyne-7725i
injection valve (USA) with a sample loop of 20 L. A
Waters Spherisorb (Ireland) C18 column (2504.6 mm,
5 m particle size) was used as stationary phase. Binary
gradient elution process was carried out with optimized mobile phase of methanol (A), water acidified with 1 % glacial
acetic acid (B). The flow rate of the sample was 1 ml/min.
Standard solution of extract and biomarkers were prepared in
methanol solvent, which was filtered through 0.45 m ultra
membrane filters (Millipore, Germany), prior attempting
sampling. System operation conditions included: injection
volume, 20 l; ratio of gradient mobile phase for optimum
separation, 0.03 to 12.00 min. the ratio was 20:80 to 50:50

Anti-cholinesterase activity of Piper betel L.

and then 12.00 to 23.00 min the ratio was 50:50 to 20:80.
The absorbance was detected at wave length 280 nm.
The peak area was calculated with Empower 2 software.
The amount of HCH and CGA in the hydroalcoholic extract of
P. betel was measured through a calibration curve ranging
from 10 to 1000 g/mL.

Statistical analysis
Statistical analysis was performed using one way ANOVA.
The result was expressed as mean SEM. P 0.05 were
considered significant. The IC50 value was calculated between the percentages of inhibition of the enzyme versus
the concentrations.

Analysis of cholinesterase activity by bioautography method

Bio-autographic method for AChE and BChE inhibition was
carried out as per reported method (Kumar et al. 2010a, b;
Bhadra et al. 2012). Hydroalcoholic extract, n-butanol and
ethylacetate fractions, HCH and CGA (1 mg/ml) were spotted
over a stationary 2.5 mm silica gel TLC plate F254 (Merck,
Darmstadt, Germany). The chromatogram was developed by
using chloroform and hexane (3:7 v/v) as mobile phase.
Developed TLC plate was allowed to dry at ambient temperature. For AChE inhibition, the TLC plate was sprayed with
1 mM DTNB and 1 mM ATCI. Similarly for BChE inhibition,
the plate was sprayed with 1 mM DTNB and 1 mM BTCI.
TLC plates were allowed to dry for 5 min and then 3 U/mL of
enzyme solution (AChE/BChE) was sprayed on the respective
plates. A white spots appeared in yellow background of the
TLC plate was considered as true inhibition of the compounds. The spot was photo-documented within 15 min, as
the spot gets disappeared in 1520 min (Kumar et al. 2010a).
To validate the false-positive reaction, another TLC plate was
developed in the same way without using the extract in the
mixture. The white inhibitory spots appeared on the plate after
spraying of enzyme solution was considered as false positive
inhibition (Bhadra et al. 2012).
Analysis of cholinesterase inhibitory activity by micro plate
method
For the determination and estimation of AChE and BChE
inhibitory potential of hydro-alcoholic extracts, n-butanol and
ethylacetate fractions, HCH and CGA, 96 well microtiter plate
method was applied (Bhadra et al. 2012). Galantamine was
taken as the standard enzyme inhibitor in this method. The
activity of the cholinesterase was measured using a Bio Rad
96-well microplate reader (680 XR, USA). Each well of the
plate contained, 125 L of 3 mM, DTNB, 25 L of 15 mM
ATCI/BTCI (AChE/BChE inhibition), and 25 L of sample (at
various concentrations ranges between 5 and 100 g/mL) were
dissolved in phosphate buffer. The absorbance was detected at
405 nm for every 13 s up to 65 s. 25 L of 0.22 U/mL of AChE/
BChE solution in phosphate buffer were added into well and
the absorbance was again read at the intervals of 13 s for 104 s.
This procedure was repeated for six times. The % inhibition of
the enzyme activity was determined from plotted graph between absorbance. Inhibition curves were extrapolated to express the IC50 values for each compound.

Result
In the present study the standardization of the extracts of P.
betel leaves by HPLC analysis revealed that the extract contains a good amount of HCH (I) and CGA (II). Presence of
HCH and CGA in the extract was identified by comparison
with the retention time of reference HCH and CGA. The
retention time of the HCH and CGA were found to be
16.12 min and 13.43 min respectively. Content of HCH and
CGA in the hydro-alcoholic extract was calculated as 28.56 %
(w/w) and 0.40 % (w/w) respectively (Maity et al. 2012).
Cholinesterase potential of the hydro-alcoholic extract was
confirmed by TLC bioautography method using chloroform:
hexane (3:7 v/v) as the mobile phase. P. betel extract (1 mg/
mL) showed prominent inhibition spots (white spots) on the
TLC plate at the Rf of 0.75 and 0.68 which are similar to the Rf
of HCH and CGA respectively. The positive response of the
extract was justified by comparing with the false positive plates.
Further to establish the AChE and BChE inhibitory potential of
the betel leaves, the hydro-alcoholic extract and its fractions
(butanol and ethylacetate) and biomarker compound HCH and
CGA were analyzed by using 96 well-microtiter-plate assay,
using galantamine as standard cholinesterase inhibitors. The
results were expressed as IC50 values and shown in Table 1. It
was observed that HCH showed significant inhibitory activity
than CGA and other extracts and fractions. But they showed
synergistic activity when applied in 1:1 ratio. Order of the
inhibition activity of the extract, fractions and other
compounds were found to be as follows, HCH, galanthamine,
ethyl acetate fraction, CGA, hydro-alcoholic extract, butanol
fraction. Dose response relationships of the extract, fractions
and biomarkers are depicted in (Figs. 2 and 3).

Discussion
This study was an attempt to evaluate P. betel in search of
novel lead for cholinesterase inhibitors derived from natural
resources. Statistically, it was observed that BChE is capable
of compensating some function of AChE in the brain; particularly in progression of AD in the patients (Mukherjee 2002;
Bhadra et al. 2012), and the activity of BChE rises at which
concentration of AChE declines in the brain (Greig et al.
2001). The result of TLC bioautography and 96 well-

M. K. Dalai et al.
Table 1 IC50 values of the P betel hydroalcoholic extract, fraction, HCH,
CGA and galantamine against AChE and BChE (values are expressed as
Mean SEM, N =6)
Sample

AChE inhibition
IC50 value(g/mL)

BChE inhibition
IC50 value(g/mL)

Hydro-alcoholic extract
Butanol fraction
Ethyl acetate fraction
Chlorogenic acid isolated
from Piper betel
Hydroxychavicol isolated
from Piper betel
Hydroxychavicol +
Chlorogenic acid (1:1)
Galanthamine

90.230.14
126.620.21
63.510.19
70.870.42

126.481.241
160.031.01
91.241.22
121.761.61

29.930.26

69.301.44

21.230.33

45.551.89

42.560.54

55.341.56

The data were analyzed by one way ANOVA. Values are Mean SEM,
N =6 (N =Number of replicates)

Fig. 3 Dose response curve of P. betel extracts, fraction, HCH, CGA and
galantamine against BChE

HCH hydroxychavicol, CGA chlorogenic acid

microtiter plate assays were indicates that P. betel extract has

potential of AChE and BChE inhibition. Therefore both the
enzymes should be targeted to ameliorate the therapeutic
management of AD.
HCH and CGA, isolated from P. betel have showed significant AChE and BChE inhibitory activities than hydroalcoholic extract and its fractions. It was also observed that
the affinity of P. betel extract, its fractions and isolated compounds has more affinity towards the AChE than the BChE.
This indicates that compounds of the plant may play dual role
to inhibit cholinesterase enzymes. These types of biochemical
activities have been observed in plants which often protect the
plants from microbial infection (Schmeller et al. 1997). In due
of course of AD, the level of AChE and BChE in mammalian
brain also varies. When the concentration of AChE is decreases up to certain amount, at that time BChE could take

part in hydrolyzing both aceyl- and butryl-cholines in

mamlian tissue (Li et al. 2000).
It was reported that HCH and CGA are responsible for the
bioactivity of P. betel (Maity et al. 2012). In this present study
and from HPLC chromatogram it was observed that amount
phenolic compounds (HCH and CGA) are significant in the
hydro-alcoholic extract and well supports the above finding.
Further, Pandey and Bani (2010) have reported that HCH, a
phenolic compound derived from P. betel showed better protective effect against cognitive deficit in rat model. The result of
IC50 value of HCH (AChE, IC50: 29.930.26 g/mL and
BChE: IC50: 121.761.61 g/mL) of the present study has
indication of cholinesterase inhibition activity. The elevations
levels of BChE have been observed in the region of AD brain,
in contrast the AChE level decreases. In this case, BChE plays
an important role in hydrolysis of acetylcholine in the neuronal
junction, in contrast with decreased AChE levels in AD patient
brain region. Therefore, BChE is an important therapeutic agent
site to develop the cholinesterase inhibition (Arendt et al.
1992). The results of this present study indicate that the constituents of P. betel have capable to inhibit of both AChE and
BChE. The previous report on the P. betel and activity of HCH
and the present experimental outcome are in agreement to
justify that P. betel leaf extract, HCH and CGA can be explored
further for the development of novel lead as AChE and BChE
inhibitors in therapeutic of AD management.

Conclusion

Fig. 2 Dose response curve of P. betel extracts, fraction, HCH, CGA and
galantamine against AChE

This study showed that P. betel and HCH have potential

AChE and BChE inhibition activities. It can be concluded
that P. betel extract, HCH and CGA can be explored further
for the development of novel lead as AChE and BChE inhibitors in therapeutic management of AD.

Anti-cholinesterase activity of Piper betel L.

Acknowledgments We are gratefully acknowledging the financial support by the Department of Science and Technology, Government of India.
MKD also thank AICTE for providing QIP research scholarship award.
Conflict of interest The authors declare that they have no conflict of
interest with any matter.

References
Arendt T, Brckner MK, Lange M, Bigl V (1992) Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic developmenta study of molecular forms.
Neurochem Int 21:381396
Arya DS, Arora S, Malik S, Nepal S, Kumari S, Ojha S (2010) Effect of
Piper betel on cardiac function, marker enzymes, and oxidative
stress in isoproterenol-induced cardiotoxicity in rats. Toxicol Mech
Methods 20:564571
Bhadra S, Mukherjee PK, Kumar NS, Bandyopadhyay A (2011)
Anticholinesterase activity of standardized extract of Illicium verum
Hook. f. fruits. Fitoterapia 82:342346
Bhadra S, Mukherjee PK, Bandyopadhyay A (2012) Cholinesterase
inhibition activity of Marsilea quadrifolia Linn. an edible leafy
vegetable from West Bengal, India. Nat Prod Res 26:15191522
Gilani AH, Aziz N, Khurram IM, Rao ZA, Ali NK (2000) The presence
of cholinomimetic and calcium channel antagonist constituents in
Piper betle Linn. Phytother Res 14:436442
Gilani AH, Ghayur MN, Saify ZS, Ahmed SP, Choudhary MI, Khalid A
(2004) Presence of cholinomimetic and acetylcholinesterase inhibitory constituents in betel nut. Life Sci 75:23772389
Greig NH, Utsuki T, Yu Q, Zhu X, Holloway HW, Perry T, Lee B, Ingram
DK, Lahiri DK (2001) A new therapeutic target in Alzheimers
disease treatment: attention to butyrylcholinesterase. Curr Med Res
Opin 17:159165
Guha P (2007) Extraction of essential oil: an appropriate rural technology
for minimizing wastage of surplus betel leaves. Agr Mech Asia Afr
Lat Am 38:4750
Kumar NS, Mukherjee PK, Bhadra S, Saha BP (2010a) Acetylcholinesterase
enzyme inhibitory potential of standardized extract of Trigonella
foenum graecum L and its constituents. Phytomedicine 17:292295
Kumar NS, Mukherjee PK, Bhadra S, Saha BP, Pal BC (2010b)
Acetylcholinesterase inhibitory potential of a carbazole alkaloid,
mahanimbine, from Murraya koenigii. Phytother Res 24:629631
Li B, Stribley JA, Ticu A, Xie W, Schopfer LM, Hammond P, Brimijoin
S, Hinrichs SH, Lockridge O (2000) Abundant tissue

butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse. J Neurochem 75:13201331
Maity N, Nema NK, Sellamuthu MK, Sarkar BK, Mukherjee PK (2012)
Simultaneous estimation of hydroxychavicol and chlorogenic acid
from Piper betel L. through RP-HPLC. Nat Prod Res 26:13
Mukherjee PK (2002) Quality control on herbal drugs. Business Horizons,
New Delhi
Mukherjee PK, Houghton PJ (2009) The worldwide phenomenon of
increased use of herbal products: opportunities and threats. In:
Mukherjee PK, Houghton PJ (eds) Evaluation of herbal medicinal
products - perspectives of quality, safety and efficacy, 1st edn.
Pharmaceutical Press, London, pp 312
Mukherjee PK, Kumar V, Houghton PJ (2007a) Screening of Indian
medicinal plants for acetylcholinesterase inhibitory activity.
Phytother Res 21:11421145
Mukherjee PK, Kumar V, Mal M, Houghton PJ (2007b) In vitro acetylcholinesterase inhibitory activity of the essential oil from Acorus
Calamus and its main constituetns. Planta Med 73:283285
Norton SA (1998) Betel: consumption and consequences. J Am Acad
Dermatol 38:8188
Pandey A, Bani S (2010) Hydroxychavicol inhibits immune responses to
mitigate cognitive dysfunction in rats. J Neuroimmunol 226:4858
Pin KY, Chuah AL, Rashih AA, Mazura MP, Fadzureena J, Vimala S,
Rasadah MA (2010) Antioxidant and anti-inflammatory activities of
extracts of betel leaves (Piper betel) from solvents with different
polarities. J Trop For Sci 22:448455
Prabu SM, Muthumani M, Shagirtha K (2012) Protective effect of Piper
betel leaf extract against cadmium-induced oxidative stress and
hepatic dysfunction in rats. Saudi J Biol Sci 19:229239
Rai S, Mal M, Wahile A, Mukherjee PK (2005) Therapeutic potentials and
untoward effects of Piper betel. Orient Pharm Exp Med 5:272282
Saha I, Chatterji U, Chaudhuri-Sengupta S, Nag TC, Nag D, Banerjee S,
Maiti BR (2007) Ultrastructural and hormonal changes in the pinealtesticular axis following arecoline administration in rats. J Exp Zool
A Ecol Genet Physiol 307:187198
Saravanan R, Ramesh B, Pugalendi KV (2006) Effect of Piper betel on
hepatotoxicity and antioxidant defense in ethanol-treated rats. J
Herbs Spices Med Plants 12:6172
Schmeller T, Latz-Bruning B, Wink M (1997) Biochemical activities of
berberine, palmatine and sanguinarine mediating chemical defence
against microorganisms and herbivores. Phytochemistry 44:257
266
Valenta P, Gonc-alves Rui F, Belo C, Pinho PG, Andrade PB, Ferreres F
(2010) Improving the knowledge on Piper betle: targeted metabolite
analysis and effect on acetylcholinesterase. J Sep Sci 33:31683176
Vyawahare NS, Bodhankar SL (2007) Neuropharmacological profile of
Piper betel leaves extract in mice. Pharmacologyonline 2:146162