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DOI 10.1007/s13596-013-0141-3
RESEARCH ARTICLE
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.
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.
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
Conclusion
Fig. 2 Dose response curve of P. betel extracts, fraction, HCH, CGA and
galantamine against AChE
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