Survey, Isolation and Purification of

Bioactive Peptides with Anti-

hypertensive Activity from Sweet
Potato (Ipomoea batatas) and
Winged Bean [Psophocarpus
tetragonolobus (L) D.C.]
Dr. Mary Ann O. Torio
Prof. Mark Rickard N. Angelia
Institute of Chemistry
College of Arts and Sciences
University of the Philippines Los Banos
• Study 1: Screening of Biologically Active
Peptides from Sweet Potato (Ipomoea batatas)
and Winged Bean [Psophocarpus
tetragonolobus (L) D.C.]
• Study 2: Purification and Biochemical
Characterization in Terms of Anti-hypertensive
Activity of Biologically Active Peptides from
Sweet Potato (Ipomea batatas) and Winged
Bean [Psophocarpus tetragonolobus (L) D.C.]
 Introduction
• In the turn of the century, there has been
widespread interest in the roles that smaller
proteins play in many physiological processes
and their possible roles of treating human
diseases.
• These small proteins exhibiting biological
activities are called bioactive peptides.
• Bioactive peptides are specific protein
fragments that give a positive impact on body
functions or conditions and ultimately
improve health.
• Peptides with angiotensin I-converting
enzyme (ACE) inhibition activity are commonly
found in milk proteins, egg, chicken meat,
sardines, tuna and soybean.
• Recent studies have shown that there is a
rapid increase in the stroke mortality and
prevalence of hypertension.
• It is also alarming that occurrence of
hypertension is at relatively younger age (20-
25 years old) and at lower levels of body mass
index.
• This research aimed to identify bioactive
peptides present in the chosen plant samples
and determine its properties.
• The information that will be obtained will
allow further tailoring of their properties using
biochemistry and molecular biology
techniques.
 ISOLATION AND PURIFICATION OF THE MAJOR STORAGE
PROTEIN FROM SWEET POTATO (Ipomea batatas) WITH
BIOACTIVE PEPTIDES EXHIBITING ANGIOTENSIN CONVERTING
ENZYME INHIBITORY ACTIVITY
• Sweet potato, Ipomea batatas, is a crop plant
that can be found throughout tropical and
warm temperate regions wherever there is
sufficient water to support its growth.
• Sweet potato is fourth among other foods as
an important alternative of carbohydrate
taking after rice, corn, and cassava.
• The major storage protein in tuberous roots of
sweet potato is sporamin.
• It was reported that sporamin comprises 60%
to 80% of the total soluble proteins in sweet
potato tubers.
• Earlier characterization studies of sporamin
described that its molecular weight is
apparently 25 kDa.
 Methodology
• Sample Preparation
• Extraction of Proteins
• Purification of Proteins
–Gel Filtration Chromatography
–Ammonium Sulfate Precipitation
–Dialysis
• Protein Characterization
– Sodium Dodecyl Sulfate – Polyacrylamide
Gel Electrophoresis (SDS-PAGE)
– Protein Content Determination
– Pepsin and Thermolysin Digestion
– Densitometric Analysis
– Spectrophotometric Assay of Angiotensin-
Converting Enzyme (ACE) Activity
 Results and Discussion
• In Silico Analysis
1 mkaltfalfl alslyllpnp ahsrfnpirl ptthepasse
tpvldingde vraggnyymv
61 saiwgagggg lrlahldmms kcasdvivsp ndldngdpit
itpatadpes tvvmastyqt
121 frfniatnkl cvnnvnwgiq hdsasgqyfl kagefvsdns
nqfkievvda nlnfykltyc
181 qfgsdkcynv grfhdplmrt trlalsnspf vfvikptdv

The sporamin sequence from the locus AAL55800 with DBSource, locus
AF289060 accession AF289060.1. The highlighted peptides are found to be
ACE inhibitors (http://www.uwm.edu.pl/biochemical).
 PROTEIN ISOLATION AND
PURIFICATION

The SDS-PAGE profile of the crude

SDS- PAGE profile of crude extract of
total soluble proteins of sweet potato.
Lane MW – molecular weight marker;
lane 1 – crude extract
extract reveals two major proteins, a
25 kDa (Peak B) that is 75.94% of
the total proteins while the 55 kDa
(Peak A) is 17.21%
The protein contents of
the crude extract and the
dialysate were found to be
0.752 mg/mL and 0.415
mg/mL, respectively.
 PEPSIN DIGESTION

•The percent remaining

proteins after one hour and
24 hour digestion of the 25
kDa polypeptide are
36.34% and 28.72%,
respectively .
• Pepsin completely
hydrolyzed the 25 kDa
SDS- PAGE profile of peptic hydrolysates protein after 1 hour.
after 0 min (lane 0), 3 min (lane 3), 5 min
(lane 5), 10 min (lane 10), 15 min (lane
15), 20 min (lane 20), 30 min (lane 30), 60
min (lane 60), and 24 hrs (lane 24);
molecular weight marker (lane MW).
1mkalt|fal|flalsl|yllpnpahsr|fnpirlptthepassetpvldingd
eraggn|y|ymv
61
sai|wgagggglrlahldmmskcasdvivspndldngdpititpata
dpestvvmastyqt
121
|fr|fniatnklcvnnvn|wgiqhdsasgq|y|flkage|fvsdnsnq|fk
ievvdanln|f|yklt|yc
181 q|fgsdkc|ynvgr|fhdplmrttrlalsnsp|f vfvikptdv
 THERMOLYSIN DIGESTION

•SDS-PAGE reveals
that the major proteins
of sweet potato were
completely digested
within 0-3 min of
reaction.

SDS- PAGE profile of thermolysin

hydrolysates after 0 min (lane 0), 3 min (lane
3), 5 min (lane 5), 10 min (lane 10), 15 min
(lane 15), 20 min (lane 20), 30 min (lane 30),
60 min (lane 60), and 24 hrs (lane 24);
molecular weight marker (lane MW)
1mka|lt|fal|f|lalsl|yllpnpahsr|fnp|ir|lptthepassetp|v|ld|ing
de|vraggn|y|ymv
61sai|wgagggg|lr|lah|ldmmskcasd|v|i|vspnd|ldngdp|it|it
patadpest|v|vmast|yqt
121 |fr|fn|iatnk|lc|vnn|vn|wg|iqhdsasgq|y|f|l
kage|fvsdnsnq|fk|ie|v|vdan|ln|f|yk|lt|yc
181 q|fgsdkc|yn|vgr|fhdp|lmrttr|la|lsnsp|f|vf|v|ikptd|v
ANGIOTENSIN CONVERTING ENZYME
INHIBITION ASSAY
SAMPLE % INHIBITION REMAINING ACE IC50
ACE ACTIVITY INHIBITORY (mg/mL)
(units) ACTIVITY
(units)
24-hr peptic 0.420 1.32 x10-3 3.00 x 10-6 0.252
hydrolysate

1-hr 72.7 1.65 x 10-4 1.17 x 10-3 0.150

thermolysin
hydrolysate

24-hr 83.2 4.66 x 10-5 1.28 x 10-3 0.121

thermolysin
hydrolysate
ISOLATION AND PARTIAL PURIFICATION OF THE MAJOR
STORAGE PROTEIN FROM WINGED BEAN [Psophocarpus
tetragonolobus (L) D.C.] WITH BIOACTIVE PEPTIDES
EXHIBITING ANGIOTENSIN CONVERTING
ENZYME INHIBITORY ACTIVITY
• Winged bean [Psophocarpus tetragonolobus (L)
D.C.] is a legume plant that grows in hot humid
equatorial countries like the Philippines.
• The common name of winged bean in the
Philippines is “sigarilyas”.
• Its seeds contain about 29.8 to 39% protein
whereas the tubers have 12.5 to 15%.
• Protein content of the winged bean tubers is 2 to
4 times higher than potatoes and 8 times than
cassavas.
 METHODOLOGY
• Sample Preparation
• Protein Extraction
• Purification of Proteins
– Ammonium Sulfate Fractionation
– Hydrophobic Interaction Chromatography
– Gel Filtration Chromatography
• Protein Characterization
– Sodium Dodecyl Sulfate – Polyacrylamide
Gel Electrophoresis (SDS-PAGE)
– Protein Content Determination
– Pepsin and Thermolysin Digestion
– Densitometric Analysis
– Spectrophotometric Assay of Angiotensin-
Converting Enzyme (ACE) Activity
 RESULTS AND DISCUSSION
• In Silico Analysis

ADDPVYDAEGNKLVNRGKYTIVSFSDGAGIDVVATGNEN
PEDPLSIVKSTRNIMYATSISSEDKTPPQPRNILENMRLKI
NFATDPHKGDVWSVVDFQPDGQQLKLAGRYPNQVKGA
FTIQKGSNTPRTYKLLFCPVGSPCKNIGISTDPEGKKRLVV
SYQSDPLVVKFHRHEPE

Amino acid sequence of winged bean albumin-1 (WBA-1) with the peptides (in
red letters) known to exhibit anti-hypertensive activity (Kortt, 1989)
• In Silico Analysis
KTISFNFNQFHQNEEQLKLQRDARISSNGVLQLTKVVNG
VPKWQSTGRALYAKPVQIWDSTTGNVASFETRFSFSIPQ
PFPRPTPADGLVFFIAPLNTITGPGGGYHLIYNKLGEDDNI
FVVEGNEFDTFRNTWDPQIPHIGIDVNSVISTKTVPFTLD
NGQIANVVIKYDASTKILHVVLVFPSSGTIYTIAQLVNVQE
SVNVGFSAATGDPSGKQRNATETHDILSWSFSASLPGTN

Amino acid sequence of winged bean basic agglutinin (WBA) with the
peptides (in red letters) known to exhibit anti-hypertensive activity (Puri and
Surolia, 1994).
PROTEIN ISOLATION AND PURIFICATION

SDS-PAGE protein profiles of (MW) molecular weight marker specifically

BenchMark Protein Ladder, (A) and (E) are crude extracts, (B) supernatant after
35% (NH4)2SO4 precipitation, (C) precipitate dissolve in buffer after 80%
(NH4)2SO4 precipitatiom from HIC pooled fractions, (D) supernatant after 80%
(NH4)2SO4 precipitation, and (F) 11th fraction from gel chromatography.
 • The protein band having
the highest protein
concentration which is
17.34% is the low
molecular weight protein
which was partially isolated.
•The percent concentration
of the high molecular
Densitometric analysis of the crude weight protein is only
extract using NIH Scion Image 14.24%.
 PROTEIN DIGESTION
SDS-PAGE of the digested
samples. (MW) Molecular
Weight Marker; (A) Crude
extract; (F) Fraction #11 of gel
chromatography; (G) 1:8 v/v
enzyme to protein digest after
one hour and (H) 24 hours
digestion time.
• The purified sample was
digested using thermolysin.
• The purified sample was
partially digested after one
hour and almost completely
hydrolyzed after 24 hours.
• The protein content of the
crude extract was found to be
4.10 mg/mL while protein
content after gel filtration is
2.30 mg/mL.
ANGIOTENSIN CONVERTING ENZYME
INHIBITION ASSAY
SAMPLE PERCENT REMAINING INHIBITORY IC50
INHIBITION ACE ACTIVITY (mg/mL)
ACTIVITY
(%) (units)
(units)
1-hr 88.7 1.51 x 10-4 1.18 x 10-3 0.126
digestion
24-hr 92.5 1.01 x 10-4 1.23 x 10-3 0.118
digestion
 SUMMARY AND CONCLUSION
• The percent inhibition of thermolysin
hydrolysates of sweet potato at 1-hr and 24-hr
digestion time are 72.7 and 82.3, repectively.
• The percent inhibition of thermolysin
hydrolysates of winged bean at 1-hr and 24-hr
digestion time are 88.7 and 92.5, respectively.
• Highest inhibition was achieved using
thermolysin digests for both sample.
• Thermolysin digests from both samples
exhibited ACE inhibitory activity.
• Thus, both samples could serve as possible
source of bioactive peptides with anti-
hypertensive activity.
 ACKNOWLEDGMENTS
Biochemistry Laboratory (IPB) headed by Dr.
Roberta N. Garcia
Biochemistry and Agricultural Chemistry Division
(IC) headed by Dr. Veronica C. Sabularse
OVCRE headed by Dr. Enrico Supangco
`