EFFECT OF SOLVENTS ON BUTYRYLCHOLINESTERASE INHIBITORY
ACTIVITIES OF SWEET PEPPER EXTRACTS
Kantamanee Thuphairo1,*, Kalyarat Kruawan2, Warangkana Srichamnong2, Somsri
Charoenkiatkul2 and Uthaiwan Suttisansanee2,#
1
Food Science for Nutrition, Institute of Nutrition, Mahidol University, Thailand
2
Institute of Nutrition, Mahidol University, Thailand
*e-mail: kantamanee.thp@student.mahidol.ac.th, #e-mail: uthaiwan.sut@mahidol.ac.th
Abstract
Sweet pepper (Capsicum annuum) consists of many colors such as green, red, yellow
and orange. These colors are results of different phytochemicals resided in pepper coats.
Besides its colors, sweet peppers that extracted under solvents with various polarities may
possess different health benefits. Butyrylcholinesterase (BChE) is one of the enzymes in
cholinergic hypothesis that predicts the occurrence of Alzheimer’s disease (AD). Thus,
inactivation of BChE by plant extract is alternative pathway to prevent AD. This research
aimed to investigate the effect of solvents on colored sweet peppers regarding their BChE
inhibitory activities. Sweet peppers (immature green and mature red peppers) were extracted
by hexane and 70% (v/v) aqueous ethanol using a Soxhlet extractor. Anti-BChE activities
were measured spectrophotometrically at a wavelength of 412 nm using a 96-well microplate
reader. As results, it was found that green pepper extracted with hexane exhibited twice
higher BChE inhibitory activities (~10% inhibition) than that of red pepper. On the other
hand, red pepper extracted with 70% (v/v) aqueous ethanol exhibit twice higher anti-BChE
activities (~40% inhibition) than that of green pepper. These results suggested that hexane is
a suitable solvent to extract anti-BChE agents from green pepper, while red pepper preferred
aqueous ethanol. This research will be benefit for development of extraction method for antiBChE agents from sweet peppers, which, in turn, would support green medicine as safe and
economically effective source for preventing AD.
Keywords: sweet pepper, butyrylcholinesterase (BChE), inhibitor, solvent extraction
Introduction
Alzheimer’s disease (AD) is the type of dementia, which frequently occurs in elderly
individuals. The cerebral neurotransmitters, acetylcholine and butyrylcholine, are normally
degraded by two cholinesterase enzymes, acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE), causing AD occurrence. Therefore, inhibition of these
enzymes may be an alternative pathway for AD treatment (1-3). The later enzyme, BChE,
possesses higher substrate specificity, thus maybe a better choice for accepting a wider
inhibitor range.
Pepper in the genus Capsicum of family Solanaceae is an important ingredient in Thai
food as a source of spicy favor. Peppers are reported to contain significant bioactive
compounds such as vitamin C, vitamin E, provitamin A, carotenoids, phenolics and
flavonoids (4). These compounds could prevent many diseases related to free radical
oxidation, cardiovascular disease, cancer, diabetes and neurodegenerative diseases (1, 5).
Pepper is found in several varieties with C. annuum being a less spicy species. Sweet peppers
(C. annuum) that could be consumed as fresh vegetables are present in various fruit colors
such as green (immature) and red (mature) (6). Previous researches had reported that different
colored peppers consist of different nutrient compositions such as the content of vitamin C
and total phenolics (7). Carotenoids (including capsanthin, capsorubin and capsanthin 5,6epoxide) and flavonoids are main pigment compounds in red pepper (7, 8), while the color of
green pepper is from chlorophyll and carotenoids typical of the chloroplast (9). In addition,
green peppers are often harvested in immature state, while color change indicates maturity of
the peppers (red color). Pepper maturity may affect the content of phytonutrients (9). Besides
the color of peppers, polarities of solvent may affect phytochemicals and their health benefit
properties. For example, lipophilic carotenoids could be extracted in non-polar solvents,
while hydrophilic flavonoids are better dissolved in polar solvents.
Therefore, the aim of this research was to investigate BChE inhibitory activities of
sweet peppers in both immature (green) and mature (red) stages regarding their extraction
solvents (non-polar hexane and polar aqueous ethanol).
Methodology
Sweet peppers including green pepper and red pepper were purchased from the local
markets in Bangkok and Nakhon Pathom provinces, Thailand. The peppers were prepared by
removing seed and cutting into small pieces before being freeze-dried using a lyophilizer.
The dried samples were then homogenized using a kitchen blender and stored at -20 oC for
further analysis.
Green and red peppers (5 g) were extracted with solvents (400 mL) including hexane
and 70% (v/v) aqueous ethanol using a Soxhlet extractor. The extracts were evaporated and
redissolved in 50% (v/v) dimethyl sulfoxide (DMSO). The BChE inhibitory activity was
determined using BChE (50 ng), butyrylthiocholine chloride (BTCh, 0.1 mM), 5,5’-dithiobis(2-nitrobenzoic)acid (DTNB, 0.8 mM) and pepper extract (17.3 mg/mL). The enzyme
reaction was spectrophotometrically measured at a wavelength of 412 nm using a 96-well
microplate reader (BioTek Instruments, Inc., Winooski, VT) with a Gen5 data analysis
software. The inhibitory activity was determined as % inhibition. The percentage of
inhibition is 100 x (1 – ((B–b)/(A–a))), where A is an initial velocity of the control reaction
with enzyme, a is an initial velocity of the control reaction without enzyme, B is an initial
velocity of the enzyme reaction with extract and b is an initial velocity of the reaction with
extract but without enzyme. Significant differences between means (p < 0.05) were detected
by t-test using the SPSS 16.0 statistical package (version 16.0, SPSS Inc., IL, USA).
Results
The BChE inhibitory activities of green pepper and red pepper extracted with solvents
including hexane and 70% (v/v) aqueous ethanol were determined as shown in Figure 1. The
substrate, BTCh, is hydrolyzed by enzyme BChE to produce thiocholine, which is
subsequently reacted with Ellman’s reagent, DTNB, to form a yellow 5-thio-2-nitrobenzoate
anion. This final product possesses yellow color that can be measured at a wavelength of 412
nm. However, under the presence of inhibitor, the production of thiocholine is reduced as a
result of interference between inhibitor and enzyme. Therefore, BChE inhibitory activity of
pepper extracts was calculated from the reduction of 5-thio-2-nitrobenzoate anion formation
and reported as percentage of inhibition.
The results suggested that green and red peppers extracted with aqueous ethanol could
significantly inactivate BChE with greater inhibitory activities (20-40% inhibition) than those
extracted with hexane (5-10% inhibition). By using hexane extraction, green pepper exhibited
higher anti-BChE activity (10% inhibition) than red pepper (5% inhibition). However, red
pepper exhibited higher anti-BChE activity (40% inhibition) than that of green pepper (20%
inhibition) under aqueous ethanol extraction.
A
Substrate
Thiocholines
Enzyme-Substrate Complex
BChE
DTNB
Measured at wavelength of 412 nm
B
5-thio-2-nitrobenzoate anion(Yellow)
Inhibitor
Enzyme-Inhibitor Complex
BChE
Figure 1. The scheme showed the reaction of BChE enzyme under (A) general condition and (B) the presence
of inhibitor using BTCh as substrate and DTNB as indicator. The reaction could be monitored at the wavelength
of 412 nm (yellow color).
%inhibition of BChE activities
45
b,**
40
35
30
a,**
25
20
15
10
5
Green pepper
Red pepper
a,*
b,*
0
Hexane
70% (v/v) aqueous ethanol
Solvent
Figure 2. The BChE inhibitory activities of green and red sweet peppers extracted with different solvents,
hexane and 70% (v/v) aqueous ethanol. The different letters (a and b) and symbols (* and **) within same
solvent extraction condition and colored pepper, respectively, are significantly different with p<0.05 using
t-test.
Discussion and Conclusion
Hexane extract of green pepper exhibited significantly higher BChE inhibitory
activities than that of red pepper. These results are corresponded to the previous research,
which indicated that green pepper contained the high level of β-carotene (7) and capsaicin
(10) that is highly soluble in lipophilic solvent, hexane (11). Thus, β-carotene and capsaicin
may be the key phytochemical that cause higher BChE inhibitory activities in green pepper
than in red pepper. On the other hand, red pepper extract in 70% (v/v) aqueous ethanol was
found to exhibit significantly higher BChE inhibitory activities than those of green pepper.
Previous research found that extraction of red pepper by methanol (hydrophilic solvent) cause
higher quantity of flavonoids such as quercetin and luteolin than in green pepper (4, 7).
Therefore, higher anti-BChE activities in aqueous ethanol extracted red pepper might be a
result of these bioactive compounds. In addition, these results were corresponded to our
preliminary data on the total phenolic compounds (TPC) and antioxidant capacity as being
detected using 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, ferric reducing
antioxidant power (FRAP) assay and oxygen radical absorbance capacity (ORAC) assay,
which indicated that green pepper extracted with hexane and red pepper extract in 70% (v/v)
aqueous ethanol possessed higher anti-oxidative properties than hexane extracted red pepper
and aqueous ethanol extracted green pepper, respectively. Thus, it could be suggested that
some anti-oxidative agents could possibly inactivate BChE reaction. It was previously
reported that oxidative stress is another cause of pathological changes in AD (1), thus
bioactive compounds with anti-oxidative properties may prevent AD occurrence through
antioxidant function as well as inhibition of cholinergic pathway.
From these results, it could be concluded that the color of sweet pepper and polarities
of solvent are significant factors that affect BChE inhibitory activities. These properties may
be relevant to the bioactive compounds, which yield different color pigments and polarities.
This research will be benefit for development of extraction method for anti-BChE agents
from sweet peppers, which in turn would support green medicine as safe and economically
effective source for preventing AD.
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Acknowledgements: This research was performed at the Institute of Nutrition, Mahidol
University.