EVALUATION OF LIPASE INHIBITORY ACTIVITY OF SWEET PEPPER
EXTRACTS
Puttacha Sornchan1,*, Kalyarat Kruawan2, Warangkana Srichamnong2, Somsri
Charoenkiatkul2, Uthaiwan Suttisansanee 2,#
1
Food science for nutrition, Institute of nutrition, Mahidol University, Thailand
2
Institute of nutrition, Mahidol University, Thailand
*e-mail: mayize.im@gmail.com, #e-mail: uthaiwan.sut@mahidol.ac.th
Abstract
Sweet pepper (or bell pepper) is a cultivar of Capsicum annuum species that produce
fruits in different colors, including red, yellow, orange and green. Many bioactive compounds
were found in sweet pepper such as beta-carotene, capsanthin, quercetin and luteolin, which
could promote health benefit. This study evaluated lipase inhibitory activity of different
colored (green and red) sweet pepper extracts. Freeze-dried sweet peppers were extracted
with hexane and 70% (v/v) aqueous ethanol using a Soxhlet extractor. Lipase inhibitory
activity was determined by a colorimetric microplate assay method using Ellman’s reagent.
Green and red sweet peppers extracted by hexane exhibited inhibition percentage of 82 and
49, respectively, while those extracted with 70% (v/v) aqueous ethanol exhibited inhibition
percentage of 51 and 64, respectively. The results suggested that green sweet pepper
extracted by hexane possessed significantly higher anti-lipase activity than red sweet pepper.
In the contrary, lipase inhibition of red sweet pepper extracted with 70% (v/v) aqueous
ethanol was slightly higher than that of green sweet pepper. These anti-lipase activities might
be the results of bioactive compounds such as capsaicinoids and phenolics, which abundantly
found in green pepper. Thus, consumption of green sweet pepper could possibly control
obesity through key enzyme, lipase, inhibition.
Keyword: sweet pepper, obesity, lipase, inhibitor
Introduction
Fruits and vegetables have been reported as sources of bioactive compounds with
effective antioxidant property that can promote potential health benefits against various
diseases such as atheroscelerosis, cancer, diabetes and cardiovascular disease (1). Obesity is a
major public health problem that is continuingly increased in Thailand. Obesity is associated
with many diseases and affects on body systems such as coronary heart disease, gallbladder
disease, cirrhosis, cancer, gout and stroke (2). Medicinal treatment of obesity is aimed to
decrease rate of fat accumulation in the body. By inactivating lipase, the enzyme that
hydrolyzes triglyceride into free fatty acids and glycerol, the absorption of fat could be
controlled. Orlistat is a medicine that used to treat obesity; however, side effects including
oily or fatty stools, gas with discharge, inability to control bowel movements, stomach pain,
nausea, vomiting and diarrhea are also reported. Thus, the natural compounds that can treat
obesity without causing side effects are of interest.
Sweet pepper or bell pepper is a cultivar of Capsicum annuum species, which produce
different fruit colors, including red, yellow, orange and green. Generally, these sweet peppers
are available in markets and consumed as fresh vegetables. The color of sweet peppers is the
major factor for consumer’s purchasing decisions. Sweet peppers are a rich source of
bioactive compounds such as beta-carotene, vitamin C and E, capsanthin, quercetin, luteolin,
flavonoids and phenolics (3, 4). Some of these bioactive compounds have been reported for
their biological functions against lipase (5). The different colored sweet peppers have been
reported to contain different nutrient compositions (6, 7), which, in turn, could alter health
promotion. Thus, this study was aimed to evaluate the lipase inhibitory activity of green
(immature) and red (mature) sweet pepper extracted from two solvents, hexane and 70% (v/v)
aqueous ethanol, using a Soxhlet extractor.
Methodology
Green and red sweet peppers were purchased from local markets in Bangkok and
Nakhon Pathom provinces (Thailand). Peppers were washed with deionized water, cut into
small pieces and removed the seeds before freeze-drying. The samples were then ground by a
grinder and stored at -20C until analysis.
Sweet pepper powder (5 g) was extracted under two solvents (400 mL), hexane and
70% (v/v) aqueous ethanol, using a Soxhlet extractor. The extractant was evaporated, and the
remaining was resuspended with minimal volume of 50% (v/v) dimethyl sulfoxide (DMSO).
The lipase inhibitory activity was determined by the colorimetric couple assays
utilizing the reaction of sulfhydryl product from lipase reaction and Ellman’s reagent (5,5'dithiobis(2-nitrobenzoic acid), DTNB). The assay consisted of Candida rugosa lipase (0.5
µg) in 50 mM Tris (pH 8.0) containing 0.1% (w/v) bovine serum albumin (BSA), 2,3dimercapto-1-propanol tributyrate (DMPTB, 0.05 mM) in 50 mM Tris (pH 7.2) containing 10
mM KCl and 1 mM EDTA, DTNB (0.8 mM) in 50 mM potassium phosphate buffer (KPB,
pH 7.0) and pepper extract. The reaction was monitored at a wavelength of 412 nm using a
microplate reader (BioTek Instruments, Inc., Winooski, VT) and Gen5 data analysis software.
The results were calculated as a percentage of inhibitory activity using the following
equation;
𝐵−𝑏
%inhibition = 100 x [1 − (
)],
𝐴−𝑎
where A is an initial velocity of the control reaction with lipase, a is an initial velocity of the
control reaction without lipase, B is an initial velocity of the enzyme reaction with extract and
b is an initial velocity of the reaction with extract but without lipase.
All data were expressed as mean of triplicate assays ± standard deviation (SD). One
way analysis of variance (ANOVA) and Tukey’s multiple comparison tests were performed
to determine the significant differences between values with p<0.05. All statistical analysis
was carried out using SPSS statistical analysis (version 17 for Windows, SPSS Inc., Chicago,
USA).
Results
The inhibition of lipase reaction could prevent fat absorption and reduce risk of
obesity. The anti-lipase property of sweet peppers was evaluated by couple assay utilizing the
reaction of sulfhydryl moiety, a free thiol product of lipase reaction and substrate DMPTB,
and Ellman’s reagent (DTNB), which acts as indicator. The free thiol would reduce disulfide
bond of DTNB, producing mixed disulfide product and yellow anion compound of 5-thio-2nitrobenzoic acid (TNB2-), which can be measured at a wavelength of 412 nm (Figure 1).
As results (Figure 2), it was found that green sweet pepper extracted with hexane
exhibited higher anti-lipase activity (82% inhibition) than red sweet pepper (49% inhibition)
under the same extraction condition. Opposite result was observed under 70% (v/v) aqueous
ethanol extraction, in which red sweet pepper exhibited slightly higher anti-lipase activity
(64% inhibition) than green sweet pepper (54% inhibition).
%inhibition of lipase
activities
Figure 1. The reaction of lipase enzyme with the substrate, 2,3-dimercapto-1-propanol tributyrate (DMPTB)
and the indicator, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).
90
80
70
60
50
40
30
20
10
0
a
b
a
b
Green pepper
Red pepper
Hexane
Solvent
70% (v/v) aqueous
ethanol
Figure 2. Lipase inhibitory activities of sweet peppers (green and red colors) extracted with hexane and
70% (v/v) aqueous ethanol. The same letter within same solvent extraction is not significantly different
with p<0.05 using one-way analysis of variance using the Tukey’s test.
Discussion and Conclusion
Peppers are potential sources of bioactive compounds that could function as antilipase agents. The content of bioactive compounds in pepper is influenced by various factors
such as climate, ripening stages and cultivar. The different colors of sweet peppers may be
due to different levels of bioactive compounds. The results showed that green sweet pepper
extracted by hexane exhibited higher anti-lipase activity than that of red pepper extracted
under the same condition. The opposite results, however, were observed under 70% (v/v)
aqueous ethanol extraction. Previous researches had reported the antioxidant activity of
different colored sweet peppers (4, 7, 8), while some suggested the relationship between
antioxidants and anti-lipase agents (9). Interestingly, green sweet pepper was found to contain
high quantity of some bioactive compounds such as beta-carotene, capsaicin and
dihydrocapsaicin that are highly dissolved in non-polar solvent. Red sweet pepper, on the
other hand, contains quercetin and luteonin, which are likely dissolved in polar solvent (8,
10). Thus, the content of those compounds may correlate to the extraction condition and
lipase inhibitory activity.
This research provided fundamental knowledge regarding the potential biological
functions of sweet peppers against obesity through inhibition of key enzyme, lipase. The
consumption of sweet pepper as daily vegetables could provide health benefits towards
supporting weight control and reducing risk of obesity. Besides, these results will be useful
for further production of functional food, nutraceutical or dietary supplement for prevention
of overweight and obesity.
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Acknowledgements: This research was performed at the Institute of Nutrition, Mahidol
University