Enhancement of pancreatic lipase inhibitory activity of curcumin by radiolytic transformation

Article abstract of DOI:10.1016/j.bmcl.2010.12.122

The naturally occurring yellow dietary diarylheptanoid curcumin (1) was converted by γ-ray to two new γ-lactones, curculactones A (2) and B (3), as well as four known transformates, erythro-1-(3-methoxy-4-hydroxy-phenyl) -propan-1,2-diol (4), threo-1-(3-methoxy-4-hydroxy-phenyl)-propan-1,2-diol (5), vanillic acid (6), and vanillin (7). The structures of the two new γ-lactone derivatives were elucidated on the basis of spectroscopic methods. The steroisomeric phenylpropanoids 4 and 5 exhibited significantly enhanced inhibitory activity against pancreatic lipase when compared to parent curcumin.

Full text of DOI:10.1016/j.bmcl.2010.12.122

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1512–1514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enhancement of pancreatic lipase inhibitory activity of curcumin
by radiolytic transformation
a,
⇑
b
c
d,
⇑
Tae Hoon Kim , Jae Kyung Kim , Hideyuki Ito , Cheorun Jo
a
Department of Herbal Medicinal Pharmacology, Daegu Haany University, Gyeongsan 712-715, Republic of Korea
b
c
Radiation Food Science and Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea
Division of Pharmaceutical Sciences, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan
Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea
d
a r t i c l e i n f o
a b s t r a c t
Article history:
The naturally occurring yellow dietary diarylheptanoid curcumin (1) was converted by c-ray to two new
Received 4 November 2010
Accepted 25 December 2010
Available online 4 January 2011
c
-lactones, curculactones A (2) and B (3), as well as four known transformates, erythro-1-(3-methoxy-4-
hydroxy-phenyl)-propan-1,2-diol (4), threo-1-(3-methoxy-4-hydroxy-phenyl)-propan-1,2-diol (5),
vanillic acid (6), and vanillin (7). The structures of the two new -lactone derivatives were elucidated
c
on the basis of spectroscopic methods. The steroisomeric phenylpropanoids 4 and 5 exhibited signifi-
cantly enhanced inhibitory activity against pancreatic lipase when compared to parent curcumin.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Radiolytic transformation
Curcumin
c
-Lactones
Phenylpropanoids
Pancreatic lipase
Curcumin is the main yellow bioactive constituent isolated
from Curcuma longa. It has been shown to possess a wide spectrum
of biological properties such as anti-inflammatory, anti-carcino-
genic, anti-mutagenic, anti-coagulant, anti-diabetic, and antioxi-
research to identify new enzyme inhibitors derived from several
1
3
medicinal plants.
Recently, several papers have demonstrated that
has potential in modifying flavonoids to have higher bioactivity.
c
-irradiation
14
1
dant activities. Recent studies have demonstrated that some
However, research relating to radiolytic transformation of natu-
rally occurring compounds is still very limited. As part of our
continuing search for novel bioactive compounds, we herein report
demethoxy derivatives of curcumin also exhibit promising cancer
chemopreventive and antioxidative effects as well as effects on
2
–4
cancer cell lines.
However, curcumin is unstable under neutral
the radiolytic transformation of curcumin into new c-lactones as
and basic pH conditions due to a highly reactive b-diketone moiety
and has limited clinical efficacy due to its low bioavailability under
physiological conditions.5 Therefore, many studies have been at-
tempted for the structural modification of curcumin to enhance
well as related derivatives that exhibited significantly improved
pancreatic lipase inhibitory activity than that of their parent
compound.
,6
Methanol solution containing curcumin (1) was subjected to
7–9
15
its bioactivities and bioavailability.
radiolytic transformation by c-irradiation, and their degraded
Obesity is caused by an imbalance between energy intake and
expenditure and is widely recognized as a major public health
problem. Obesity can result in hypertension, hyperlipidemia,
products were detected by HPLC analysis. Chromatographic separa-
1
6
tion
yielded two new compounds, curculactone A (2) and
curculactone B (3), along with previously known erythro-1-(3-meth-
10
17
arteriosclerosis, and type II diabetes. Pancreatic lipase is a key
enzyme for triglyceride absorption in the small intestine. This
enzyme is secreted from the pancreas and hydrolyzes triglycerides
into glycerol and fatty acids.11 Therefore, pancreatic lipase inhibi-
tors are considered to be a valuable therapeutic reagent for treat-
oxy-4-hydroxy-phenyl)-propan-1,2-diol (4),
threo-1-(3-meth-
1
8
19
oxy-4-hydroxy-phenyl)-propan-1,2-diol (5), vanillic acid (6),
and vanillin (7). The known compounds were identified by compar-
ison of their physicochemical and spectroscopic data with those of
authentic samples and literature values (Fig. 1).
1
2
20
D
ing diet-induced obesity in humans. The success of orlistat,
which is a specific pancreatic lipase inhibitor, has prompted
Compound 2 was obtained as a colorless oil, ½
aꢀ
ꢁ7.0° (MeOH).
The characteristic absorbances at 229 and 281 nm in the UV spec-
trum suggested the presence of an aromatic group. The molecular
formula C12
H
14
+
O
4
for 2 was determined from an ion peak at m/z
2
22.0890 [M] in the HREIMS and NMR data described below.
⇑
Corresponding authors. Tel.: +82 53 819 1371; fax: +82 53 819 1272 (T.H.K.);
tel.: +82 42 821 5774; fax: +82 42 825 9754 (C.J.).
1
The H NMR spectrum of 2 (Table 1) showed ABX-type aromatic
0
E-mail addresses: skyey7@dhu.ac.kr (T.H. Kim), cheorun@cnu.ac.kr (C. Jo).
signals at d
H
6.81 (1H, d, J = 8.4 Hz, H-5 ), 6.77 (1H, d, J = 1.8 Hz,
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.122

T. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1512–1514
1513
5
6
O
OCH
3
R
O
OCH3
O
3
OCH
3
HO
4
OH
O
OCH3
R1
HO
1
'
2
'
2'
1
'
OH
1
3
R1
2
OCH
3
R2
OH
COSY
HMBC
OH
R2
OH
: R = OH, R2 = H, 4,5-erythro
1
2
3
4: 1,2-erythro
5: 1,2-threo
6
: R = COOH
7: R = CHO
: R = H, R2 = OH, 4,5-threo
1
1
2
2: R = OH, R = H
: R = H, R = OH
4
1
2
3
Figure 1. Structures of radiolytic degradation products 2–7 of curcumin.
Figure 2. Selected COSY and key HMBC correlations of 2–4.
Table 1
1H and 13C NMR chemical shifts of compounds 2 and 3a
O
H
O
H
H
CH3
Hβ
Hβ
Position
2
3
O
O
3
5
3
5
b
b
4
4
d
H
(J in Hz)
d
C
, mult.
d
H
(J in Hz)
d
C
, mult.
Hα
H CH3
OCH3
Hα
H
H
2
—
179.8
35.9
—
178.5
38.5
H
1'
H
1'
2
'
2'
3
3
4
5
6
1
2
3
4
5
6
a
b
3.00 (dd, 17.4, 8.4)
2.84 (dd, 17.4, 6.0)
3.79 (dd, 14.4, 6.0)
4.98 (quint, 6.0)
1.04 (d, 6.6)
—
6.77 (d, 1.8)
—
—
2.90 (dd, 9.6, 1.2)
OCH3
45.7
82.2
16.8
130.8
112.8
149.1
147.1
116.4
121.4
56.5
3.22 (m)
4.57 (dd, 9.6, 6.0)
1.41 (d, 6.0)
—
6.96 (s)
—
6.81 (s)
—
6.81 (s)
3.90 (s)
50.7
85.1
19.1
131.2
112.1
149.4
116.5
147.2
121.1
56.5
H
HO
OH
H
2
3
0
0
0
0
0
0
Figure 3. Selected NOESY correlations of 2 and 3.
6.81 (d, 8.4)
6.66 (dd, 8.4, 1.8)
3.88 (s)
techniques showed the presence of a unique skeleton20 with the
same molecular formula as in 2. The relative configuration of 3
was also determined by NOESY correlations of H-2 /H-5 , H-4/H-6
OCH
3
0
0
a
1H NMR measured at 600 MHz, 13C NMR measured at 150 MHz; obtained in
3
OD with TMS as internal standard. Assignments based on HMQC and HMBC
0
(
CH
3
), 3b, and H-6 /H-3
a. Consequently, the structure of compound
CD
2
3
NMR spectra.
3 was assigned to curculactone B, which is unknown in the
b
J values (Hz) are given in parentheses.
literature.
2
0
Compound 4 was obtained as a colorless oil, ½
a
ꢀ
ꢁ10.0°
D
(
MeOH). The HRESMS of 4 had a molecular ion peak at m/z
0
0
+
H-2 ), and 6.66 (1H, dd, J = 8.4, 1.8 Hz, H-6 ), indicating the pres-
ence of a 1,3,4-trisubstituted benzene ring in 2. The presence of a
198.0889 [M] , which is consistent with the molecular formula
. The 1D H NMR and H–1H COSY spectra of 4 indicated
the presence of aromatic protons at d
1
1
C
10
H
14
O
4
0
c
-lactone moiety was also revealed in the 1H NMR spectrum in
H
6.97 (1H, d, J = 1.8 Hz, H-2 ),
0
0
the well-defined aliphatic region, which shows signals from one
oxygenated methine proton at d 4.98 (1H, quint, J = 6.0 Hz, H-5),
one benzylic methine proton at d 3.79 (1H, dd, J = 14.4, 6.0 Hz,
H-4), one methylene group at d 3.00 (1H, dd, J = 17.4, 8.4 Hz, H-
6.77 (1H, dd, J = 7.8, 1.8 Hz, H-6 ), and 6.73 (1H, dd, J = 7.8 Hz, H-6 ),
H
as well as two oxymethine protons at d
1) and 3.82 (1H, dq, J = 6.6, 5.4 Hz, H-2), a methyl proton at d
(3H, d, J = 6.6 Hz, H-3), and a methoxyl group at d 3.84 (3H, s). In
addition to the methoxyl carbon signal, nine skeletal carbon signals
H
4.38 (1H, d, J = 5.4 Hz, H-
H
H
1.11
H
H
3
1
a), 2.84 (1H, dd, J = 17.4, 6.0 Hz, H-3b), one methyl group at d
H
1
3
.04 (3H, d, J = 6.6 Hz, H-6), and one methoxyl group at d
H
3.88
appeared in the C NMR spectrum. These spectral features suggest
(
3H, s). This partial structure was supported by the appearance of
that 4 was a penylpropanoid with two hydroxyl groups in its pro-
pane moiety. The methoxyl group was located at C-3 , based on
1
3
24
0
C NMR resonances (Table 1), which were further supported by
1
0
cross peaks of H-4/H-3, -5,and H-5/H-6 in the H–1H COSY as well
as long-range correlations of H-4/C-1 , -2, -2 , -3, -5, -6, -6 , H-5/C-
1
the HMBC and NOESY (d
H
3.84/H-2 ) correlations (Figs. 2 and 3).
0
0
0
25
The erythro relationship between H-1 and H-2 was inferred from
their coupling constant (J1,2 = 5.4 Hz) and verified by key NOESY
0
3
, 3, -4, -6, and CH -6/C-4, -5 in the HMBC spectrum. The position
0
of the methoxyl group was confirmed unambiguously by HMBC
correlations between H-1 and H-2, 2 . Compound 4 was previously
0
0
17
(
OCH
3
/C-3 ) and NOESY (OCH
3
/H-2 ) experiments (Figs. 2 and 3).
reported as a synthetic compound without assigning the NMR
1
13
Comparison of the H and C NMR spectra of 2 with previously
data. Thus, compound 4 was assigned to erythro-1-(3-methoxy-4-
known descurainolide A20 readily identified that this new com-
pound possesses the same structure, except for the absence of a
methoxyl group at the C-3 position. The relative stereostructures
of the 4 and 5-positions in the
by NOESY spectrum (Fig. 3). The spectrum of 3 displayed correla-
tions between H-2 /H-6 (CH
ing an erythro relationship between the phenyl moiety and the
methyl group. Thus, the structure of curculactone A (2) was as-
signed as shown.
hydroxy-phenyl)-propan-1,2-diol.
26
The compounds isolated from irradiated curcumin solution
were evaluated for their ability to inhibit pancreatic lipase activ-
0
2
7
c
-lactone moiety were characterized
ity using orlistat as a positive control (Table 2). Among the tested
compounds, some derivatives possessed significantly higher pan-
creatic lipase inhibitory activity than that of their parent curcumin
(1). Two phenylpropanoid-type byproducts, erythro-1-(3-meth-
oxy-4-hydroxy-phenyl)-propan-1,2-diol (4) and threo-1-(3-meth-
oxy-4-hydroxy-phenyl)-propan-1,2-diol (5), exhibited the most
potent pancreatic lipase inhibitory activities in a dose-dependent
0
0
3
), H-4/H-5, 3b, and H-6 /H-3a, indicat-
2
1
2
D
0
The HREIMS of compound 3, ½
pseudomolecular ion peak at m/z 222.0891 [M] , indicating that
the molecular formula (C12 ) was the same as that of 2. Most
of the NMR signals in 3 were nearly identical to those of 2, except
for the different AB
splitting patterns22 of the aromatic signals at
6.96 (1H, s) and 6.81 (2H, s) in 3. These proton signals along
with the corresponding carbon resonances assigned by 2D NMR
aꢀ
ꢁ24.5° (MeOH), showed a
+
manner with IC50 values of 9.1 ± 0.2 and 12.1 ± 0.3
lM, respec-
H
14
O
4
tively. On the other hand, the unusual -lactone derivatives
c
curculactones A (2) and B (3) displayed lower inhibitory effects
than those of 4 and 5 with IC50 values of 231.5 ± 2.9 and
2
d
H
65.3 ± 2.2
lM, respectively. These results suggest that the stereo-
chemistry at the
c-lactone moiety may have influenced pancreatic

1
514
T. H. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1512–1514
Table 2
Pancreatic lipase inhibitory activity of compounds 1–7
8. Fuchs, J. R.; Pandit, B.; Bhasin, D.; Etter, J. P.; Regan, N.; Abdelhamid, D.; Li, C.;
Lin, J.; Li, P. K. Bioorg. Med. Chem. Lett. 2009, 19, 2065.
9.
Zhang, Q.; Fu, Y.; Wang, H. W.; Gong, T.; Qin, Y.; Zhang, Z. R. Chin. Chem. Lett.
2008, 19, 281.
Compound
IC50 value (lM)
1
1
1
0. Cooke, D.; Bloom, S. Nat. Rev. Drug Disc. 2006, 5, 919.
1. Lowe, M. E. Gastroenterology 1994, 107, 1524.
2. Hill, J. O.; Hauptman, J.; Anderson, J. W.; Fujioka, K.; O’Neil, P. M.; Smith, D. K.;
Zavoral, J. H. Am. J. Clin. Nutr. 1999, 69, 1108.
3. (a) Ninomiya, K.; Matsuda, H.; Shimoda, H.; Nishida, N.; Kasajima, N.; Yoshino,
T.; Morikawa, T.; Yoshikawa, M. Bioorg. Med. Chem. Lett. 2004, 14, 1943; (b)
Yoshikawa, M.; Shimoda, H.; Nishida, N.; Takada, M.; Matsuda, H. J. Nutr. 2002,
132, 1819; (c) Nakai, M.; Fukui, Y.; Asami, S.; Toyoda-Ono, Y.; Iwashita, T.;
Shibata, H.; Mitsunaga, T.; Hashimoto, F.; Kiso, Y. J. Agric. Food. Chem. 2005, 53,
1
2
3
4
5
6
7
>250
231.5 ± 2.9
65.3 ± 2.2
12.1 ± 0.3
9.1 ± 0.2
>250
1
74.4 ± 2.1
0.6 ± 0.2
Orlistata
4593.
a
Used as positive control.
1
4. (a) Marfak, A.; Trouillas, P.; Allais, D. P.; Calliste, C. A.; Cook-Moreau, J.;
Duroux, J. L. Radiat. Res. 2003, 160, 355; (b) Marfak, A.; Trouillas, P.; Allais,
D. P.; Champavier, Y.; Calliste, C. A.; Duroux, J. L. J. Agric. Food. Chem. 2002,
5
3
0, 4827; (c) Jung, H. J.; Park, H. R.; Jo, S. K. Radiat. Phys. Chem. 2009, 78,
86.
5. Irradiation was carried out at ambient temperature, using
lipase inhibition. Two simpler phenolic compounds among the
obtained compounds, vanillic acid (6) and vanillin (7), did not
show significantly improved inhibitory activity when compared
to the other tested compounds. During the last decade, synthetic
modification of curcumin to enhance its bioactivity has been
1
a
cobalt-60
irradiator (point source AECL, IR-79, MDS Nordion International Co. Ltd,
Ottawa, ON, Canada) in the Advanced Radiation Technology Institute, Korea
Atomic Energy Research Institute (Jeongup, Korea). The source strength was
approximately 320 kCi, with dose rate at the location of the sample of 10 kGy/
h. Dosimetry was performed using 5 mm diameter alanine dosimeters (Bruker
Instruments, Rheinstetten, Germany). The dosimeters were calibrated against
an International Standard Set by the International Atomic Energy Agency
2
8,29
performed.
Apart from the previously reported biological
improvement of modified curcumin, this is the first report on the
isolation and evaluation of the biological activities of curcumin
(
Vienna, Austria). Sample solution (2 g curcumin in 200 mL MeOH) in chapped
byproducts produced by radiolytic degradation using
The results of this study established that curcumin (1) was con-
verted into small amounts of two new compounds, curculactones A
c
-ray.
vials were irradiated with 30 kGy (absorbed dose). The irradiated methanolic
solution was immediately evaporated to remove the solvent and lyophilized.
6. The dried methanolic solution was directly subjected to column
1
chromatography over
a YMC GEL ODS AQ 120-50S column (1.1 cm
(
2) and B (3), as well as four previously known compounds,
erythro-1-(3-methoxy-4-hydroxy-phenyl)-propan-1,2-diol (4),
threo-1-(3-methoxy-4-hydroxy-phenyl)-propan-1,2-diol (5), vanil-
lic acid (6), and vanillin (7) by -irradiation. Their structures,
i.d. ꢂ 37 cm) with aqueous MeOH, to yield pure compounds 2 (1.8 mg, t
R
1
6.3 min), 3 (4.3 mg, t
R
20.5 min), 4 (2.2 mg, t
R R
2.9 min), 5 (1.6 mg, t 3.4 min),
6
(1.5 mg, t 6.9 min), and 7 (3.6 mg, t
R
R
8.7 min). HPLC analysis was carried out
on a YMC-Pack ODS A-302 column (4.6 mm i.d. ꢂ 150 mm; YMC Co., Ltd) and
c
was developed at 40 °C with 10 mM H PO /10 mM KH PO /MeCN (4.3:4.3:1.4,
3
4
2
4
including four stereochemically pure compounds (2–5), were
established on the basis of spectroscopic data interpretation.
Compounds 2–5 were evaluated for their inhibitory activities
against pancreatic lipase, and compounds 3 and 4 were the most
potent molecules with much lower IC50 values than that of original
curcumin or the other products tested. In addition, it can be con-
flow rate: 1.0/min, detection: 280 nm).
1
7. Peng, K.; Chen, F.; She, X.; Yang, C.; Cui, Y.; Pan, X. Tetrahedron Lett. 2005, 46,
217.
8. Ma, J.; Jin, X.; Yang, L.; Liu, Z. L. Phytochemistry 2004, 65, 1137.
1
1
19. He, X.; Liu, R. H. J. Agric. Food. Chem. 2006, 54, 7069.
2
2
0. Sun, K.; Li, X.; Wang, J.; Sha, Y. Chem. Pharm. Bull. 2004, 52, 1483.
20
1. Curculactone A (2): colorless oil, ½
aꢀ
ꢁ7.0° (c 0.5, MeOH); UV kmax MeOH nm
13 +
D
1
(
log
HREIMS m/z 222.0890 [M] (calcd for C12
2. Okada, Y.; Ishimaru, A.; Suzuki, R.; Okuyama, T. J. Nat. Prod. 2004, 67, 103.
3. Curculactone B (3): colorless oil, ½
e): 229 (3.71), 281 (1.70); H and C NMR, see Table 1; EIMS m/z 222 [M] ,
+
cluded that
c-irradiation of curcumin may be a favorable method
14 4
H O , 222.0892).
2
2
for modifying structure and enhancing bioactivity. Further
isolation and biological evaluation of non-phenolic constituents
are currently in progress.
2
0
a
ꢀ
ꢁ24.5° (c 0.2, MeOH); UV kmax MeOH nm
D
1
1
3
+
(
log
e): 229 (3.73), 281 (1.72); H and C NMR, see Table 1; EIMS m/z 222 [M] ,
+
HREIMS m/z 222.0891 [M] (calcd for C12
14 4
H O , 222.0892).
2
2
2
4. Delazar, A.; Biglari, F.; nazemiyeh, H.; Talebpour, A. H.; Nahar, L.; Sarker, S. D.
Phytochemistry 2006, 67, 2176.
5. Balboul, B. A. A. A.; Ahmed, A. A.; Otsuka, H.; Adams, A. D. Phytochemistry 1996,
Acknowledgments
4
2, 1191.
2
0
6. Erythro-1-(3-methoxy-4-hydroxy-phenyl)-propan-1,2-diol (4): colorless oil, ½
a
ꢀ
The research discussed herein was supported by the Basic Sci-
ence Research Program through the National Research Foundation
of Korea (NRF), which is funded by the Ministry of Education, Sci-
ence and Technology (2010-0022783).
D
1
ꢁ10.0° (c 0.1, MeOH); UV kmax MeOH nm (log
NMR (CD OD, 600 MHz): d 6.97 (1H, d, J = 1.8 Hz, H-2 ), 6.77 (1H, dd, J = 7.8,
1.8 Hz, H-6 ), 6.73 (1H, d, J = 7.8 Hz, H-5 ), 4.38 (1H, d, J = 5.4 Hz, H-1), 3.84
e): 229 (3.00), 280 (1.35); H
0
3
H
0
0
0
(3H, s, MeO-3 ), 3.82 (1H, dq, J = 6.6, 5.4 Hz, H-2), 1.11 (3H, d, J = 6.6 Hz, H-3);
13
0
0
0
C NMR (CD OD, 150 MHz): 148.7 (C-3 ), 146.8 (C-4 ), 134.8 (C-1 ), 120.9 (C-
3
0
0 0 0
), 115.6 (C-5 ), 111.6 (C-2 ), 79.0 (C-1), 72.4 (C-2), 59.3 (C-3 MeO), 18.5 (C-
+ +
6
Supplementary data
3); EIMS m/z 198 [M] , HREIMS m/z 198.0893 [M] (calcd for C10
14 4
H O ,
198.0892).
2
7. Assay of pancreatic lipase activity: The ability of the compounds to inhibit
porcine pancreatic lipase was evaluated using the previously reported method
with a minor modification (Kim, J. H.; Kim, H. J.; Park, H. W.; Youn, S. H.; Choi,
D. Y.; Shin, C. S. FEMS Microbiol. Lett. 2007, 276, 93.). Briefly, an enzyme buffer
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.122.
was prepared by the addition of 30
pancreatic lipase (Sigma, St. Louis, MO) in 10 mM MOPS (morpholine-
propanesulphonic acid) and 1 mM EDTA, pH 6.8] to 850 L of Tris buffer
(100 mM Tris–HC1 and 5 mM CaCl , pH 7.0). Then, 100 L of the compounds at
the test concentration or orlistat (Roche, Basel, Switzerland) was mixed with
880 L of the enzyme-buffer, and incubated for 15 min at 37 °C, with 20 L of
the substrate solution [10 mM p-NPB (p-nitrophenylbutyrate) in dimethyl
formamide] added and the enzymatic reactions allowed to proceed for 15 min
at 37 °C. The pancreatic lipase activity was determined by measuring the
hydrolysis of p-NPB to p-nitrophenol at 405 nm using an ELISA reader (Tecan,
Infinite F200, Austria). Inhibition of the lipase activity was expressed as the
percentage decrease in the OD when porcine pancreatic lipase was incubated
with the test compounds.
lL (10 units) of a solution of porcine
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1
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.
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2
2
2
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