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ORIGINAL RESEARCH
Inhibition of pancreatic lipase by berberine
and dihydroberberine: an investigation by docking simulation
and experimental validation
Mohammad Mohammad
•
Ihab M. Al-masri
•
Ala Issa
•
Ayman Khdair
•
Yasser Bustanji
Received: 7 June 2012 / Accepted: 24 August 2012 / Published online: 6 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Berberine (BBR) and dihydroberberine
(HBBR) were investigated as inhibitors of pancreatic lipase
in an attempt to explore their potential hypolipidemic
activities. The study included docking simulations and in
vitro enzymatic inhibition assays. At the molecular level,
docking simulations revealed several significant binding
interactions between the docked natural compounds and
the key amino acids in the binding pocket of the pancreatic
lipase enzyme. BBR had similar pattern of binding inter-
actions as HBBR; however, BBR has a permanent cationic
center which is suggested to have an adverse influence on
ligand–pancreatic lipase affinity. This trend is explainable
by the proposition that ionized ligands favor hydration
instead of docking into the binding site. This might explain
the lower inhibitory activity of BBR comparing to HBBR,
which appeared from their estimated IC
50
values. The
logarithmic regression of PL inhibition versus concentra-
tion revealed estimated IC
50
values of 106 and 8.0 lg/mL
for BBR and HBBR, respectively.
Keywords Berberine Dihydroberberine
Pancreatic lipase Obesity Docking simulations
Enzyme inhibition
Introduction
Obesity is one of the most common nutritional dilemmas in
the modern countries and is considered a potential risk
factor for the development of a plethora of devastating
diseases, including insulin resistance and type 2 diabetes,
lipid profile disorders, osteoarthritis, hyperuricemia,
malignancies, and cardiovascular diseases which includes
hypertension, coronary heart diseases, and stroke (Arbeeny,
2004;Cairns,2005; Gurevich-Panigrahi et al., 2009).
Many recent studies have placed obesity as one of the
greatest threats to global health in this millennium. Recent
reports showed that there are more than one billion over-
weight adults worldwide including at least 300 million
classified as clinically obese with increased risk of mor-
bidity and mortality (Arbeeny, 2004; Vega, 2001). Unfor-
tunately, these numbers are growing in alarming rates.
Obesity is considered as a metabolic disorder, which is
mainly caused by an imbalance between the energy intake
and expenditure. However, comprehensive understanding
of the molecular mechanisms that tightly regulate body
weight has afforded potential opportunities for therapeutic
managements and offered renewed hope for introducing
antiobesity drugs into the market (Bray and Tartaglia,
2000; Foster-Schubert and Cummings, 2006; Halford,
2006b).
Despite the surfeit of research data available on obesity,
it still remains alarming, unsolved medical and socioeco-
nomical problem (Cooke and Bloom, 2006; Halford,
2006a; Hofbauer, 2002). The relatively few medications
that are available in the market for obesity management
have minimal efficacy and are poorly tolerated. As such,
there is an urgent need for the development of new thera-
peutic agents to overcome obesity and its malicious clinical
consequences (Shi and Burn, 2004).
M. Mohammad (&) A. Issa Y. Bustanji
Faculty of Pharmacy, University of Jordan,
Amman 11942, Jordan
e-mail: mkmohammad@ju.edu.jo
I. M. Al-masri
Faculty of Pharmacy, Al-Azhar University,
Gaza, Gaza Strip, Palestine
A. Khdair
Faculty of Pharmacy, Applied Science University,
Amman, Jordan
123
Med Chem Res (2013) 22:2273–2278
DOI 10.1007/s00044-012-0221-9
MEDICINAL
CHEMISTR
Y
RESEARCH
Recently, new approaches for fighting obesity have
involved inhibition of dietary triglyceride absorption
through inhibition of pancreatic lipase (PL) which is being
the major source of excess calories (Ballinger and Peikin,
2002). PL (triacylgycerol acyl hydrolase), the primary
lipolytic enzyme synthesized and released in the pancreatic
juice by the pancreas, plays an essential role in the efficient
digestion and absorption of triglycerides from the gut.
PL is responsible for the hydrolysis and absorption of
50–70 % of total dietary fats.
X-ray crystallography has clarified the three-dimen-
sional structure of human PL. This enzyme is a monomeric
glycoprotein, composed of 449 amino acids, which is
divided into two main folding domains, the larger N-ter-
minal domain and a C-terminal domain (Vantilbeurgh
et al., 1992; Winkler et al., 1990). The N-terminal domain
is the catalytic domain to which substrate binds; while the
C-terminal domain binds to colipase, another enzyme that
acts as a required cofactor for activity (Vantilbeurgh et al.,
1992, Winkler et al., 1990).
Orlistat (Xenical
Ò
), widely prescribed for the manage-
ment of obesity, is the only FDA approved drug that acts
through inhibition of PL. Although Xenical
Ò
is one of the
best-selling drugs in the western countries, its use is
accompanied by uncomfortable intestinal adverse events
like oily stools, oily spotting, and flatulence (Drent et al.,
1995). Natural compounds identified from traditional
medicinal plants provide an enormous pool of PL inhibitors
that can possibly be turned into clinical products (Birari
and Bhutani, 2007; Bustanji et al., 2010, 2011a, b; Issa
et al., 2011).
Berberine (BBR) is an isoquinoline alkaloid found in
Hydrastis canadensis, Berberis, and Cortex phellodendri.It
has been comprehensively reviewed in the literature as an
attractive natural compound of broad potential medical
applications including hypolipidemic effects (Vuddanda
et al., 2010). BBR was found to ameliorate diabetes and
serum lipid profile by reducing serum cholesterol, triglyc-
erides, and LDL-cholesterol in animals and human (Bustanji
et al., 2006; Hu and Davies, 2010;Konget al., 2004;Lee
et al., 2006).
Consequently, current work was devoted to evaluate
BBR’s potential PL inhibitory activity as well as its bio-
logically available derivative dihydroberberine (HBBR).
Initially, we used computer-aided molecular docking of
BBR and HBBR into the binding pocket of PL to reach
preliminary conclusions about BBR/PL and HBBR/PL
binding energetics. Eventually, the docked active com-
pounds were tested in vitro against PL to evaluate their
inhibition potential.
Our findings help in understanding the mechanism of
action of BBR as hypolipidemic agent. It can also provide
new insights toward the binding interactions of BBR and
HBBR with PL which can lead to the screening and dis-
covery of new natural and safe PL inhibitors.
Materials and methods
Materials
Chemicals and reagents used in these experiments were
reagent grade and obtained from the following sources:
porcine PL type II, Tris–HCl buffer, orlistat; BBR, and
p-nitrophenyl butyrate (Sigma, USA). HBBR (PhytoLab
GmbH, Germany).
Docking experiment
Docking simulations and settings were employed as pre-
viously described in Bustanji et al. (2011a). Briefly, the
chemical structure of BBR and HBBR (Fig. 1) were sket-
ched in Chemdraw Ultra (7.01) and saved in MDL molfile
format. Subsequently, an ensemble of energetically acces-
sible conformers were created using OMEGA2 software
(OMEGA, 2006). OMEGA builds initial models of struc-
tures by assembling fragment templates along sigma bonds.
Once an initial model of a structure is built, OMEGA
generates additional models by enumerating ring confor-
mations and invertible nitrogen atoms. The produced
conformers are saved in SD format.
The 3D coordinates of PL were recovered from the Pro-
tein Data Bank (PDB code: 1LPB, resolution; 2.46 (A
˚
)
(Egloff et al., 1995). Hydrogen atoms were inserted to the
protein using the DS visualizer templates for protein resi-
dues. The docking study was performed in the presence of
crystallographically explicit water molecules. The tested
compounds were docked into the binding site of PL using
FRED software (FRED,
2006). The ligand conformers and
protein structure are treated as rigid items during the docking
process. FRED’s docking plan is to comprehensively score
all possible positions of each ligand in the binding pocket
(FRED, 2006). The conformational ensemble of the BBR
N
O
O
H
3
CO
OCH
3
N
O
O
H
3
CO
OCH
3
Berberine Dihydroberberine
Fig. 1 Two-dimensional structure of berberine (BBR) and dihydrob-
erberine (HBBR)
2274 Med Chem Res (2013) 22:2273–2278
123
and HBBR generated using OMEGA software was used as
input in the FRED software. For further details about FRED
parameters which were employed in docking simulations
see reference (Bustanji et al., 2011a).
Preparation of compounds and extract for in vitro assay
BBR or HBBR were initially dissolved in sufficient vol-
umes of DMSO to give five stock solutions with an esca-
lating concentration range of about 5–250 lg/mL.
Thereafter, 20 lL of each stock solution was accurately
transferred to the reaction mixture to produce BBR or
HBBR final concentration range of 0.1–5 lg/mL.
PL inhibition assay
The PL activity was determined using a colorimetric assay
approach as previously described, in which, the release of
p-nitrophenol (PNPB) will be measured spectrophotomet-
rically at 410 nm (Bustanji et al., 2011a). Briefly, 50 mg
(2,000 unit) of crude porcine PL type II (Sigma, USA, EC
3.1.1.3) was suspended in 10 mL of Tris–HCl buffer
(2.5 mmol, pH 7.4 with 2.5 mmol NaCl) and mixed well
using a stirrer for 15 min. Thereafter, the prepared solution
was centrifuged at 1,5009g for 10 min and the clear
supernatant was collected. The final enzyme concentration
was about 200 unit/mL.
BBR and HBBR solutions were preincubated with
0.10 mL of PL solution in Tris–HCl buffer for 5 min at
37 °C. After that, the PNPB substrate (10 mM in acetoni-
trile, 5 lL) was added to obtain a final volume of 1 mL of
the reaction mixture. The absorbance was spectrophoto-
metrically measured at 410 nm for at least 5 time points:
1–5 min. The release of PNPB is estimated as the incre-
ment increase in absorbance against blank. The percentage
of residual activity of PL was determined for BBR and
HBBR by comparing the lipase activity in the presence and
the absence of the tested inhibitors. Orlistat was used as a
positive standard inhibitor and all experiments were repe-
ated thrice.
Results and discussion
Lipid metabolism is cunningly balanced to maintain
homeostasis (Foster-Schubert and Cummings, 2006;
Mukherjee, 2003). When the balance is misplaced, obesity
or dyslipidemia appears ending with a range of serious
metabolic diseases. One of the most important strategies to
control obesity is to develop inhibitors of PL.
Phytochemicals identified from traditional medicinal
plants provide exciting opportunity for the development of
novel antiobesity therapeutics. As a part of our screening
project for biologically active antiobesity agents from
natural resources, BBR and HBBR have been investigated
for their antilipase activities. Several studies, however,
have reported the hypolipidemic activity of BBR alkaloid
(Kong et al., 2004). BBR was found to decrease weight
gain and food intake in mice. Moreover, serum glucose,
triglyceride, and total cholesterol levels were reduced
accompanied with a down-regulation of PPARc expression
in mice fed a high fat diet (Hu and Davies, 2010). Like-
wise, BBR improved lipid profile in obese mice by its
effect on central and peripheral AMP-dependent protein
kinase activities (Kim et al., 2009). Furthermore, oral
administration of BBR in hypercholesterolemic patients
ameliorated lipid profile and reduced serum triglycerides
and LDL-cholesterol with a suggested mechanism of
stimulation of LDL-receptors expression (Kong et al.,
2004).
Our investigation started by evaluating the possibility of
BBR/HBBR-PL binding via computer-aided molecular
modeling technique. Accordingly, both BBR and HBBR
were docked into the binding cleft of PL (PDB code:
1LPB). Generally, docking process consists of two stages:
(i) prediction of the conformation and pose of the bioactive
ligand into the binding pocket, and (ii) evaluation of the
rigidity of target-ligand interactions (scoring). The final
docked conformations are chosen according to their scores.
The docking experiments were performed utilizing the
docking capabilities of the software engine FRED (Fred).
However, an optimal set of parameters for the docking
experiments should be provided to FRED. Therefore, to
identify the optimal docking configuration and scoring
function for PL, the ligand CllP (Fig. 2) was first extracted
from PL crystallographic structure (PDB code: 1LPB)
(Egloff et al., 1995) and then redocked again into the same
protein (self-docking) via a variety of docking conditions.
Chemgauss2 was found to yield the closest model to the
crystallographic structure as shown in Fig. 2.
The preliminary docking study has postulated that BBR
and HBBR might have PL inhibitory activity and this has
encouraged further investigation to evaluate the antilipase
activities of the two alkaloids. Accordingly, BBR and
HBBR were used for in vitro activity bioassay. The enzy-
matic reaction progression was monitored through the
release of PNPB. The in vitro activity was expressed as the
concentration of natural alkaloids that could inhibit PL
activity by 50 % (IC
50
).
Figure 3 shows clearly the dose-dependent relation
between the BBR/HBBR concentrations and the degree of
PL inhibition. However, HBBR showed higher inhibitory
effect (Fig. 3b) than that of BBR (Fig. 3a) as indicated by
Med Chem Res (2013) 22:2273–2278 2275
123
relatively lower IC
50
value of HBBR. The logarithmic
regression of PL inhibition versus concentration revealed
estimated IC
50
values of 106 and 8.0 lg/mL for BBR and
HBBR, respectively (Table 1). Our assay was validated
using orlistat as a positive control with IC
50
equal to
0.21 lg/mL (see Table 1).
On the molecular level, docking simulations have
revealed several significant binding interactions between the
docked natural compounds and the PL (Fig. 4). Comparison
of the docked poses of the BBR and HBBR (Fig. 4b, c) with
the co-crystallized ligand within the binding site of PL
(Fig. 4a) illustrates similarities in their binding profiles.
Both have potential hydrophobic interactions with the key
amino acids Phe-215, Phe-77, and Tyr-114. However, the
dioxymethylene substituted-aromatic ring of BBR and
HBBR is well fitted in the hydrophobic pocket which sug-
gests the existence of van der Waals’ aromatic stacking
attraction between them. Moreover, the two natural com-
pounds having the oxygen of the 1,3-dioxolane ring moiety
seem to be tightly hydrogen-bonded to the hydroxyl and
imidazole NH groups of Ser-152 and His-263, respectively,
albeit at closer proximities in the HBBR (Fig. 4). These
multiple strong hydrogen bonds with Ser-152 and His-263
stabilizes the ligand–protein complex and contributes to the
relatively good affinity of the two alkaloids. Similarly, the
co-crystallized ligand has analogous interactions with Ser-
152 and His-263 (Fig. 4a). In the structure of PL, His-263,
Asp-176, and Ser-152 form a triad representing the lipolytic
site. Furthermore, enzymatic activity has shown to be
diminished after chemical modification of Ser-152 indicat-
ing its essential role for the catalytic activity (Winkler et al.,
1990). Therefore, it is predictable that compounds that
strongly bind to the catalytic triad, especially Ser-152, could
inhibit the lipolytic activity. Although BBR has similar
pattern of binding interactions as HBBR (Fig. 4), it has a
permanent cationic center which may have an adverse effect
on the ligand–PL affinity. This trend is explainable by the
proposition that ionized ligands favor hydration instead of
docking into the binding site, particularly if they are misa-
ligned with their corresponding counterparts in the binding
pocket. A similar analysis was recently used to explain the
general nonspecific enhancement in ligand–receptor affinity
concomitant to increases in ligand lipophilicity (Manly et al.,
2008). This may help to explain the lower affinity of BBR
comparing to HBBR. In our study, the reported significant
decrease in serum cholesterol, triglycerides, LDL-choles-
terol, and weight gain can be attributed, at least partially, to
its inhibitory action of PL. This hypothesis can be supported
by the initial docking studies and in vitro inhibitory assay.
Fig. 2 a Comparison between the docked pose (green) of the ligand
CllP as produced by docking simulation and the crystallographic
structure of this ligand (red) within the binding pocket of PL. b The
solvent accessible surface area of the binding site of PL (1LPB) and
the co-crystallized inhibitor (CllP) [18] (Color figure online)
B
Dihydroberberine
y = 9.5701Ln(x) + 29.991
R
2
= 0.9184
0
20
40
60
80
100
% of Inhibition
A
Berberine
y = 5.0283Ln(x) + 26.526
R
2
= 0.8732
0
20
40
60
80
100
0.01 0.1 1 10
Concentration (µg/mL)
% of Inhibition
0.01 0.1 1 10
Concentration (µg/mL)
Fig. 3 The inhibitory effect of
escalating concentrations of
BBR (a) and HBBR (b) on the
PL activity
Table 1 IC
50
values for tested compounds estimated from logarith-
mic regression of PL inhibition versus concentration
Compound IC
50
(lg/mL)
Berberine (BBR) 106.0
Dihydroberberine (HBBR) 8.0
Orlistat 0.21
2276 Med Chem Res (2013) 22:2273–2278
123
Conclusion
The results of this study clearly proved, through experi-
mental testing and theoretical docking simulations, that
HBBR and BBR have substantial PL inhibition activities.
This PL inhibitory effect can be used to explain, at least
partially, the reported use of BBR as an effective hypo-
lipidemic and fat-mass reduction activities. In conclusion,
this work is considered a step toward the discovery of
new natural and safe hypolipidemic/antiobesity PL
inhibitors.
Acknowledgments This project was sponsored by the Deanship of
Scientific Research at the University of Jordan. The authors wish to
thank the Deanship of Scientific Research at the University of Jordan
for their generous funds. The authors would also like to thank the
OpenEye Scientific Software for providing us with a free license for
FRED software (FRED, version 2.1.5).
References
Arbeeny CM (2004) Addressing the unmet medical need for safe and
effective weight loss therapies. Obes Res 12:1191–1196
Ballinger A, Peikin SR (2002) Orlistat: its current status as an anti-
obesity drug. Eur J Pharmacol 440:109–117
Birari RB, Bhutani KK (2007) Pancreatic lipase inhibitors from
natural sources: unexplored potential. Drug Discov Today 12:
879–889
Bray GA, Tartaglia LA (2000) Medicinal strategies in the treatment
of obesity. Nature 404:672–677
Bustanji Y, Taha MO, Yousef AM, Al-Bakri AG (2006) Berberine
potently inhibits protein tyrosine phosphatase 1B: investigation
by docking simulation and experimental validation. J Enzym
Inhib Med Chem 21:163–171
Bustanji Y, Issa A, Mohammad M, Hudaib M, Tawah K, Alkhatib H,
Almasri I, Al-Khalidi B (2010) Inhibition of hormone sensitive
lipase and pancreatic lipase by Rosmarinus officinalis extract and
selected phenolic constituents. J Med Plants Res 4:2235–2242
Bustanji Y, Al-Masri IM, Mohammad M, Hudaib M, Tawaha K,
Tarazi H, AlKhatib HS (2011a) Pancreatic lipase inhibition
Fig. 4 a Detailed view of the co-crystallized structure (CllP) and the
corresponding interacting amino acids within the binding site of PL
(PDB code: 1LPB). b Detailed view of the docked BBR structure and
the corresponding interacting amino acid moieties within the binding
site of PL. c Detailed view of the docked HBBR-PL interactions
Med Chem Res (2013) 22:2273–2278 2277
123
activity of trilactone terpenes of Ginkgo biloba. J Enzym Inhib
Med Chem 26:453–459
Bustanji Y, Mohammad M, Hudaib M, Tawaha K, Al-Masri IM,
AlKhatib HS, Issa A, Alali F (2011b) Screening of some
medicinal plants for their pancreatic lipase inhibitory potential
Jordan. J Pharm Sci 4:81–88
Cairns E (2005) Obesity: the fat lady sings? Drug Discov Today
10:305–307
Cooke D, Bloom S (2006) The obesity pipeline: current strategies in
the development of anti-obesity drugs. Nat Rev Drug Discov
5:919–931
Drent ML, Larsson I, Williamolsson T, Quaade F, Czubayko F,
Vonbergmann K, Strobel W, Sjostrom L, Vanderveen EA (1995)
Orlistat (Ro-18-0647), a lipase inhibitor, in the treatment of
human obesity—a multiple-dose Study. Int J Obes 19:221–226
Egloff MP, Marguet F, Buono G, Verger R, Cambillau C, Vantilbeurgh
H (1995) The 2.46-Angstrom resolution structure of the pancreatic
lipase-colipase complex inhibited by a C-11 alkyl phosphonate.
Biochemistry 34:2751–2762
Foster-Schubert KE, Cummings DE (2006) Emerging therapeutic
strategies for obesity. Endocr Rev 27:779–793
FRED VUM (2006) OpenEye Scientific Software(www.eyesopen.com),
Boston
Gurevich-Panigrahi T, Panigrahi S, Wiechec E, Los M (2009)
Obesity: pathophysiology and clinical management. Curr Med
Chem 16:506–521
Halford JCG (2006a) Obesity drugs in clinical development. Curr
Opin Investig D 7:312–318
Halford JCG (2006b) Pharmacotherapy for obesity. Appetite 46:6–10
Hofbauer K (2002) Molecular pathways to obesity. Int J Obes
26:S18–S27
Hu YS, Davies GE (2010) Berberine inhibits adipogenesis in high-fat
diet-induced obesity mice. Fitoterapia 81:358–366
Issa A, Mohammad M, Hudaib M, Tawah K, Abu Rjai T, Oran S,
Bustanji Y (2011) A potential role of Lavandula angustifolia in
the management of diabetic dyslipidemia. J Med Plants Res
5:3876–3882
Kim WS, Lee YS, Cha SH, Jeong HW, Choe SS, Lee MR, Oh GT,
Park HS, Lee KU, Lane MD, Kim JB (2009) Berberine improves
lipid dysregulation in obesity by controlling central and periph-
eral AMPK activity. Am J Physiol–Endocrinol Metab 296:E812–
E819
Kong WJ, Wei J, Abidi P, Lin MH, Inaba S, Li C, Wang YL, Wang
ZZ, Si SY, Pan HN, Wang SK, Wu JD, Wang Y, Li ZR, Liu JW,
Jiang JD (2004) Berberine is a novel cholesterol-lowering drug
working through a unique mechanism distinct from statins. Nat
Med 10:1344–1351
Lee YS, Kim WS, Kim KH, Yoon MJ, Cho HJ, Shen Y, Ye JM, Lee
CH, Oh WK, Kim CT, Hohnen-Behrens C, Gosby A, Kraegen
EW, James DE, Kim JB (2006) Berberine, a natural plant
product, activates AMP-activated protein kinase with beneficial
metabolic effects in diabetic and insulin-resistant states. Diabetes
55:2256–2264
Manly CJ, Chandrasekhar J, Ochterski JW, Hammer JD, Warfield BB
(2008) Strategies and tactics for optimizing the Hit-to-Lead
process and beyond—a computational chemistry perspective.
Drug Discov Today 13:99–109
Mukherjee M (2003) Human digestive and metabolic lipases—a brief
review. J Mol Catal B Enzym 22:369–376
OMEGA VUM (2006) OpenEye Scientific Software Inc., Santa Fe,
July, 2006
Shi YG, Burn P (2004) Lipid metabolic enzymes: emerging drug
targets for the treatment of obesity. Nat Rev Drug Discov
3:695–710
Vantilbeurgh H, Sarda L, Verger R, Cambillau C (1992) Structure of
the pancreatic lipase procolipase complex. Nature 359:159–162
Vega GL (2001) Obesity, the metabolic syndrome, and cardiovascular
disease. Am Heart J 142:1108–1116
Vuddanda PR, Chakraborty S, Singh S (2010) Berberine: a potential
phytochemical with multispectrum therapeutic activities. Expert
Opin Inv Drug 19:1297–1307
Winkler FK, Darcy A, Hunziker W (1990) Structure of human
pancreatic lipase. Nature 343:771–774
2278 Med Chem Res (2013) 22:2273–2278
123
