Development of a bioautographic method for the detection of lipase inhibitors

Article abstract of DOI:10.1016/j.bbrc.2014.10.030

An autobiographic method based on the thin layer chromatogram was developed by using the chemical system that comprises p-Nitrophenyl butyrate and bromothymol blue for detecting the lipase inhibitor. Lipase inhibitory zones were visualized as blue spots a

Full text of DOI:10.1016/j.bbrc.2014.10.030

Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Development of a bioautographic method for the detection of lipase
inhibitors
⇑
Venkata Krishna Bayineni, Sukrutha Suresh, Gurmeet Singh, Ravi-Kumar Kadeppagari
Food Science and Technology Division, Centre for Emerging Technologies, Jain University, Jain Global Campus, Jakkasandra, Kanakapura Main Road, Ramanagara Dist.,
Karnataka 562112, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 October 2014
Available online xxxx
An autobiographic method based on the thin layer chromatogram was developed by using the chemical
system that comprises p-Nitrophenyl butyrate and bromothymol blue for detecting the lipase inhibitor.
Lipase inhibitory zones were visualized as blue spots against the greenish yellow background. This
method could able to detect the well known lipase inhibitor, orlistat up to the concentration of 1 ng
which is better than the earlier method. This method could also able to detect the lipase inhibition
activities from the un-explored species of Streptomyces. The developed method can be used not only
for the screening of unknown samples for the lipase inhibitors but also for the purification of the lipase
inhibitors from the unknown samples.
Keywords:
Autobiographic method
p-Nitrophenyl butyrate
Bromothymol blue
Lipase inhibitor
Orlistat
Ó 2014 Elsevier Inc. All rights reserved.
Streptomyces
1. Introduction
and glucosidase inhibitors [5]. Recently, TLC based bioautographic
method was developed for the detection of lipase inhibitors from
Lipase (EC 3.1.1.3) is required for the digestion of triglycerides
and inhibition of this enzyme reduces the absorption of fat and
leads to the loss of body weight. Recent approaches for the treat-
ment of obesity focused on the inhibition of dietary triglyceride
absorption via pancreatic lipase inhibition [1,2]. Lipase inhibitors
will also be helpful in treating atherosclerosis. Lipase inhibitors
are mainly reported from plant and microbial sources [2].
However, till now orlistat is the only pancreatic lipase inhibitor
that was approved clinically for the treatment of obesity [3]. But,
recently orlistat was reported to show the adverse side effects like
acute kidney injury and oxalate nephropathy [4]. Hence, there is a
requirement for the lipase inhibitors that has no or reduced side
effects.
Camellia sinensis and Rosmarinus officinalis [9]. This method was
developed by using naphthyl acetate and fast blue salt B. Recently,
lipase inhibition activity was assayed spectrophotometrically by
using p-Nitrophenyl butyrate as a substrate and this method is
faster [10]. However, this substrate was not explored for the
development of bioautographic method. In the present study,
p-Nitrophenyl butyrate was used along with bromothymol blue,
a pH indicator for the development of bioautographic method for
the detection of lipase inhibitors. This method could able to
localize the orlistat and this method was used to detect the lipase
inhibitors from the cultures of un-explored species of Streptomyces.
2. Materials and methods
Usually, people prefer therapeutic molecules from the natural
resources over synthetic ones due to their reduced side effects.
However, the screening of natural resources for lipase inhibitors
is cumbersome and there is a need for the quick and effective
method. The chromatography based autobiographic methods were
reported to determine the activity in situ and quickly provide the
information on the presence and localization of activity in the
complex plant matrices [5]. Autobiographic methods were used
for the detection of antifungal activity initially [6] and later used
for the determination of acetylcholine esterase inhibitors [7,8]
2.1. Microbial cultures and reagents
Cultures of Streptomyces coelicolor, Streptomyces tendae,
Streptomyces aurantiacus and Streptomyces albaduncus were
obtained from microbial type culture collection, Chandigarh, India
and maintained on the agar slants containing the medium 1 (0.4%
yeast extract, 1% malt extract and 0.4% glucose; pH 7.2) for S. coe-
licolor and medium 2 (0.4% glucose, 0.4% yeast extract, 0.4% meat
extract and 0.2% CaCO3; pH 7.2) for the other cultures. They were
grown as submerged cultures in the medium containing glucose
(0.5%), glycerol (2%), soya bean meal (2%), yeast extract (0.5%)
and NaCl (0.3%) for the production of lipase inhibitor at 28 °C
and 150 RPM for 5–7 days. Orlistat, porcine pancreatic lipase and
⇑
Corresponding author. Fax: +91 80 2757 7211.
E-mail address: k.ravikumar@jainuniversity.ac.in (R.-K. Kadeppagari).
http://dx.doi.org/10.1016/j.bbrc.2014.10.030
0006-291X/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: V.K. Bayineni et al., Development of a bioautographic method for the detection of lipase inhibitors, Biochem. Biophys.
Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.10.030

2
V.K. Bayineni et al. / Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx
p-Nitrophenyl butyrate (PNPB) were obtained from Sigma.
Bromothymol blue and methanol were purchased from Nice
Chemicals Pvt. Ltd., India. Chloroform and benzene were obtained
from Spectrochem, India. Silica gel 60 F254 plates (Merck) were
used for carrying out TLC.
2.2. Bioautographic assay
Orlistat was dissolved into water (pH 7.7) at the concentration
of 120 mg/ml and 10
for the loading on the TLC plate. Cultures of Streptomyces were
centrifuged after the growth and 20 l of the supernatant was
ll of appropriately diluted sample was used
l
loaded on to the plate after adjusting the pH to 7.7. Solvent system
containing chloroform and methanol (90:10) was used as mobile
phase for the separation of orlistat and the one containing benzene
and methanol (60:40) was used for the separation of supernatants
of Streptomyces cultures. The TLC plate was air dried till the solvent
was evaporated completely after the separation of samples. Then
the plate was sprayed with the porcine pancreatic lipase enzyme
dissolved (10 mg/ml) in the water (pH 7.7) and dried at room
temperature. Later the plate was incubated in the humidified
chamber at 37 °C for 1 h in order to allow the enzyme–inhibitor
interaction to take place. Then the plate was air dried and sprayed
1
2
3
4
5
Fig. 1. Bioautographic thin layer chromatogram performed using different concen-
trations of orlistat. Lanes 1, 2, 3 and 4 spotted with 100, 10, 1 and 0.1 ng of orlistat,
respectively. Lane 5 was spotted with distilled water.
the reagents used in this assay was adjusted to 7.7. Hence,
wherever the lipase acts it must show up as green and the spots
where there is no lipase action must be visualized as blue when
the bromothymol blue was sprayed. To test this possibility on
the TLC plate we have spotted the different concentrations of well
known lipase inhibitor, the orlistat on the plate and samples were
separated by using the solvent system that contains chloroform
and methanol and the plate was developed by using lipase, PNPB
and bromothymol blue as described in Section 2. After the bioauto-
graphic assay the presence of blue spots against the greenish
yellow background was observed in the lanes of TLC plate (Fig. 1)
spotted with orlistat. However blue spot was not appeared when
0.1 ng (Fig. 1, lane 4) of orlistat was used suggesting the limit of
detection by this method and it could able to detect the orlistat
up to 1 ng (Fig. 1, lane 3). The sensitivity of this method is better
than the autobiographic assay based on the chemical system
containing naphthyl acetate and fast blue salt B for the detection
of orlistat [9]. Yellow color in the background is due to the
p-Nitrophenol that formed after the enzymatic reaction. The Rf
with PNPB (200 lM) prepared in the water (pH 7.7). After air
drying, the plate was incubated at 37 °C for 30 min for allowing
the enzyme and substrate reaction to take place. Then the plate
was air dried and sprayed with bromothymol blue solution to
visualize the lipase inhibitor as blue spot against the greenish
yellow background.
2.3. Lipase inhibition assay
The blue spot on the TLC plate was scrapped and suspended in
the water sufficient to submerge the silica gel for 3–4 h and silica
was separated by centrifuging for 3 min at 10,000 RPM. The super-
natant was separated and extracted with equal volume of ethyl
acetate by shaking at 150 rpm and 30 °C for 60 min. Then the mix-
ture was centrifuged at 10,000 RPM for 30 min and the organic
layer was collected and dried at 40 °C. Afterwards, the sample
was dissolved into the minimum volume of DMSO and the 40 ll
of it was used for assaying the lipase inhibition activity. Pancreatic
lipase corresponding to the 20 units was pre-incubated with the
extract of the blue spot for 60 min at 37 °C before adding the sub-
strate. The percentage of residual activity of the lipase was deter-
mined by the spectrophotometric assay described below. Here,
PNPB (200 lM) was employed as the lipase substrate and the reac-
tion was maintained at 37 °C for 30 min and the release of p-Nitro-
phenol was measured at 410 nm. The enzyme activity was
detected as the
l moles of p-Nitrophenol released per minute. Orli-
stat was used as a positive control in the assay and the % of enzyme
inhibition was calculated with reference to the control that has sol-
vent instead of extract [10].
3. Results and discussion
In order to screen un-explored species of Streptomyces for the
lipase inhibitors an autobiographic method based on the TLC was
developed. The principle behind this assay is lipase leads to the
formation of p-Nitrophenol and butyric acid after acting upon
p-Nitrophenyl butyrate and the released butyric acid will bring
down the pH of the reaction mixture. Hence, the addition of pH
indicator must indicate this change by means of color change.
We have selected bromothymol blue since this indicator will be
in blue color when the pH was above 7.6 and its color will be
changed to light green in the pH range of 6.0–7.6. The pH of all
1
2
3
4
Fig. 2. Bioautographic thin layer chromatogram performed using culture superna-
tants of different Streptomyces spp. Twenty micro liters of supernatants of S.
coelicolor (lane 1), S. tendae (lane 2), S. aurantiacus (lane 3) and S. albaduncus (lane 4)
were spotted. Arrows point the blue spots used for assaying the lipase inhibition
activity. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
Please cite this article in press as: V.K. Bayineni et al., Development of a bioautographic method for the detection of lipase inhibitors, Biochem. Biophys.
Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.10.030

V.K. Bayineni et al. / Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx
3
Table 1
Conflict of interest
Lipase inhibition activities corresponding to the blue or greenish yellow regions of
bioautographic TLC.
Authors declare no conflict of interest.
S.
No.
Source of extract
Lipase inhibition
(%)
Acknowledgments
1
2
3
4
5
6
Blue spot from the lane 3 of Fig. 1
Greenish yellow region from the lane 5 of Fig. 1
Blue spot from the lane 1 of Fig. 2
Blue spot from the lane 2 of Fig. 2
Blue spot from the lane 3 of Fig. 2
76
0
66
66
63
0
Authors thank Dr. Chenraj Roychand, President, Jain University
Trust and Dr. Krishna Venkatesh, Director, Centre for Emerging
Technologies, Jain University for the research facilities.
Greenish yellow region from the lane 4 of Fig. 2
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This assay was applied to screen the cultures of un-explored
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described under Section 2. The cultures of S. coelicolor, S. tendae
and S. aurantiacus have shown the lipase inhibitory blue zones
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all the extracts from the blue zones, whereas no lipase inhibition
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Please cite this article in press as: V.K. Bayineni et al., Development of a bioautographic method for the detection of lipase inhibitors, Biochem. Biophys.
Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.10.030