Botanical Studies (2006) 47: 397-402.
*
Corresponding author: E-mail: boyhlin@gate.sinica.edu.
tw; Fax: +886-2-2782-7954; Tel: +886-2-2789-9590.
INTRODUCTION
Many bioactive peptides have common structural
properties that include a relatively short length of peptide
residues (e.g. 2-9 amino acids), possessing hydrophobic
amino acid residues in addition to proline, lysine or
arginine groups. Bioactive peptides are among the many
functional components identified in foods. These are
small protein fragments that have biological effects once
they are released during gastrointestinal digestion in
the organism or by previous in vitro protein hydrolysis.
Bioactive peptides with immunostimulating (Parker
et al., 1984; Fiat et al., 1993), opioid (Zioudrou et al.,
1979), antithrombotic (Scarborough et al., 1991), caseino-
phosphopeptic (Maubois and Leonil, 1989; Fox and
Mulvihill, 1993), bactericidal (Bellamy et al., 1993),
antioxidant or angiotensin-converting enzyme inhibitor
(Ehlers and Riordan, 1989; Ariyoshi, 1993; Huang et al.,
2004a) functions have been the focus of research in recent
years.
ACE (peptidyldipeptide hydrolyase EC 3.4.15.1) is
a glycoprotein and a dipeptide-liberating exopeptidase
classically associated with the renin-angiotensin system
regulating peripheral blood pressure (Mullally et al.,
1996). ACE removes a dipeptide from the C-terminus of
angiotensin I to form angiotensin II, a very hypertensive
compound. Several endogenous peptides, such as
enkephalins,
£]
-endorphin, and substance P, were
reported to be competitive substrates and inhibitors
of ACE (Mullally et al., 1996). Several food-derived
peptides also inhibited ACE, including £\-lactalbumin and
£]
-lactoglobulin (Pihlanto-Leppala et al., 1998), casein
(Maruyama et al., 1987), zein (Yano et al., 1996), and
gelatin (Chen et al., 1999; Kim et al., 2001). Several
antioxidant peptides (reduced glutathione and carnosine-
related peptides) (Hou et al., 2003) and synthetic peptides
also exhibited ACE I activities (Chen et al., 2003).
The major components of plant mucilage are pectins.
Pectins are largely acidic polysaccharides that form
gels in the extracellular matrix and are present in all
cell walls. The two most common pectins found in
dicotyledonous plants are polygalacturonic acid (PGA)
and rhamnogalacturonan I (RG I) (Brett and Waldron,
1990; Carpita and Gibeaut, 1993; Cosgrove, 1997).
PGA
is an unbranched chain of £\-1,4-linked galacturonic acid
(GalUA) residues, while RG I is a highly substituted,
branched polysaccharide with a backbone of alternating
£\-1,4-linked GalUA and £\-1,2-linked rhamnose (Brett
and Waldron, 1990).
There are reports concerning the
BIOChemISTRy
Sweet potato (Ipomoea batatas
(L.) Lam. ¡¥Tainong 57¡¦)
storage root mucilage exhibited angiotensin converting
enzyme inhibitory activity in vitro
Dong-Jiann HUANG
1
, Wen-Chi HOU
2
, Hsien-Jung CHEN
3
, and Yaw-Huei LIN
1,
*
1
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, TAIWAN
2
Graduate Institute of Pharmacognosy Science, Taipei Medical University, Taipei 110, TAIWAN
3
Department of Horticulture, Chinese Culture University, Taipei 111, TAIWAN
(Received February 21, 2006; Accepted April 11, 2006)
ABSTRACT.
Sweet potato (Ipomoea batatas (L.) Lam. ¡¥Tainong 57¡¦) storage root mucilage was extracted
(crude mucilage) and further purified by SDS and heating treatment (purified mucilage). Purified mucilage
treated with 2-mercaptoethanol moved as a single band in SDS-PAGE. This purified mucilage was active to
inhibit angiotensin converting enzyme (ACE) as shown by spectrophotometric method in a dose-dependent
manner (28.7 to 59.8% ACE inhibition, respectively, by 50 to 400 £gg/mL mucilage) using (N-(3-[2-furyl]
acryloyl)-Phe-Gly-Gly) (FAPGG) as a substrate.
The concentration of mucilage required for 50% inhibition
(IC
50
) of ACE activity was 364.5 £gg/mL while that of captopril was 10 nM
(8.68 £gg/mL).
The commercial
polysaccharide pectin (50 to 400 £gg/mL) showed no inhibitory activity against ACE. When using fluorescent
silica TLC to detect FAPGG and FAP, the results also showed that mucilage inhibited ACE. The mucilage
showed mixed type inhibition against ACE, and the Michaelis constant in the presence of mucilage was 12
mM. We suggest that consumption of sweet potato storage root mucilage may benefit people¡¦s health.
Keywords: Angiotensin converting enzyme (ACE); Mucilage; Sweet potato.
pg_0002
398
Botanical Studies, Vol. 47, 2006
physiological activities of pectins on the interactions
between fibroblast growth factors and receptors (Liu
et al., 2001), on the modulation of lung colonization of
B16-F1 melanoma cell (Platt and Raz, 1992), and on the
inhibition of human cancer cell growth and metastasis
in nude mice (Nangia-Makker et al., 2002). Pectin diets
could also reduce the incidence of colon cancer in rats
(Hardman and Cameron, 1995). Yam (Dioscorea batatas,
Dioscoreaceae) is a major tuber crop and its mucilages are
mainly composed of mannan-protein macromolecules with
antioxidant activities and angiotensin converting enzyme
inhibitory activities (Tsai, and Tai, 1984; Hou et al., 2001;
Hou et al., 2002; Hou et al., 2003, Lee et al., 2003). The
mucilage from sweet potato storage roots has antioxidant
activities against both hydroxyl and peroxyl radicals
(Huang et al., 2005).
No report concerning mucilage of sweet potato on
the ACE activities is presently available. In this work we
report for the first time that purified mucilage from sweet
potato displayed ACE inhibitor activity in comparison
with captopril serving as a positive control and commercial
pectin as a negative control in a series of in vitro tests.
mATeRIALS AND meThODS
materials
Tris, electrophoretic reagents, and silica gel 60
F254 were purchased from E. Merck Inc. (Darmstadt,
Germany); Captopril was purchased from Calbiochem
Co. (CA, USA); Seeblue prestained markers for SDS-
PAGE including myosin (250 kDa), BSA (98 kDa),
glutamic dehydrogenase (64 kDa), alcohol dehydrogenase
(50 kDa), carbonic anhydrase (36 kDa), myoglobin (30
kDa), and lysozyme (16 kDa) were from Invitrogen
(Groningen, The Netherlands); FAPGG, ACE (I unit/mL,
rabbit lung), nonylamine, pectin (from citrus fruit, degree
of esterification 94%), coomassie brilliant blue R-250, and
other chemicals and reagents were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
mucilage extraction and purification
Mucilage extraction and purification were done
according to the method of Hou et al. (2002). Fresh
sweet potato (Ipomoea batatas (L.) Lam. ¡¥Tainong 57¡¦)
storage roots were purchased from a local market. After
cleaned with water, the storage roots were cut into strips
for crude mucilage extraction. Sweet potato strips were
homogenized with two volumes (w/v) of 50 mM Tris-
HCl buffer (pH 8.3) containing 1% vitamin C. After
centrifugation at 14,000 xg for 30 min, the supernatants
were mixed with isopropanol to a final concentration of 70
%, and stirred quickly at 4¢XC overnight. The precipitates
were filtered and dehydrated with 100% isopropanol,
then rinsed with acetone. After drying at 40¢XC in an
oven, the crude mucilage was ground and collected for
further purification by both SDS and heating procedures.
About 1.0 g crude mucilage powder was dissolved in
200 mL distilled water and kept in a 50¢XC water bath.
Forty mL of 5% SDS solution (dissolved in 45% ethanol)
were added to the crude mucilage solution. The mixture
was kept with gentle stirring at 50¢XC for 30 min, then
at room temperature for another 2 h. After that, the
mucilage solution was placed in an ice bath to quickly
lower the temperature in order to precipitate the SDS-
protein complex. After centrifugation at 14,000 xg at 0
¢XC for 30 min, the supernatants were precipitated with
isopropanol and dried at 40¢XC in an oven as described
earlier. The semi-purified mucilage was again ground,
dissolved, and then heated in boiling water for 20 min.
After centrifugation at 14,000 xg at 0¢XC for 30 min,
the supernatants were mixed with isopropanol to a final
concentration of 70%. The purified mucilage was filtered,
dehydrated, rinsed with acetone, dried, and then collected
for further uses.
Protein and PAS stainings on 10% SDS-PAGe
gels
Samples were mixed with sample buffer, namely
60 mM Tris-HCl buffer (pH 6.8) containing 2% SDS,
25% glycerol, and 0.1% bromophenol blue with
2-mercaptoethanol, and subjected to electrophoresis
according to the method of Laemmli (1970). Coomassie
brilliant blue G-250 was used for protein staining (Huang
et al., 2004b). Periodic acid-Schiff (PAS) staining was
used for oligosaccharide staining. After electrophoresis
the gel was placed in fixative (7.5% acetic acid), shaken
gently for 60 min. To oxidize the oligosaccharides the gel
was treated with 0.2% periodic acid for 45 min at 4¢XC.
The gel was washed with distilled water and stained with
Schiff ¡¦s reagent in dark at 4¢XC (Deepak et al., 2003).
Determination of ACe inhibitory activity by
spectrophotometry
The ACE inhibitory activity was measured according
to the method of Holmquist et al. (1979) with some
modifications. Four microliters (4 microunits) of
commercial ACE (1 unit/mL, rabbit lung; Sigma Chemical
Co.) was mixed with 50 £gL of different amounts of
mucilage and commercial pectin (50, 100, 200 and
400 £gg), and then 200 £gL of 5 ¡Ñ 10
-4
M N-[3-(2-furyl)
acryloyl]-Phe-Gly-Gly [FAPGG, dissolved in 50 mM Tris-
HCl buffer (pH 7.5) containing 0.3 M NaCl] was added.
The decreased absorbance at 345 nm (£G A inhibitor) was
recorded during 5 min at room temperature. Deionized
water was used instead of sample solution for blank
experiments (£GA blank). Captopril (molecular mass 217.3
Da) was used as a positive control for ACE inhibitor
(1.25, 2.5, 5, 10, 20, 40, and 80 nM). The ACE activity
was expressed as £GA 345 nm, and the ACE inhibition
(percent) was calculated as follows: [1 - (£GA inhibitor /£G A
control)] ¡Ñ 100. Means of triplicates were determined. The
50% inhibition (IC50) of ACE activity was calculated as
the concentrations of samples that inhibited 50% of ACE
activity under these conditions.
pg_0003
HUANG et al. ¡X Mucilage with angiotensin converting enzyme inhibitory activity
399
Determination of ACe inhibitory activity by TLC
The ACE inhibitory activity of mucilage was
determined by the TLC method (Holmquist et al., 1979).
The reactions between mucilage and ACE or pectin and
ACE were according to the methods of Anzenbacherova
et al. (2001) with some modifications. Each 100 £gL
of mucilage and pectin (250 £gg) was premixed with
15 microunits ACE for 1 min, and then 200 £gL of 5
¡Ñ 1 0
-4
M FAPGG was added and allowed to react at
room temperature for 10 min. Then 800 £gL of methanol
was added to stop the reaction. The blank experiment
contained FAPGG only; in the control experiment, ACE
reacted with FAPGG under the same conditions. Each was
dried under reduced pressure and redissolved with 400
£gL of methanol, and 50 £gL was spotted on a silica gel 60
F254. The FAPGG and FAP (ACE hydrolyzed products)
were separated by TLC in butanol - acetic acid-water,
4:1:1 (v/v/v), and observed under UV light.
Determination of the kinetic properties of ACe
inhibition by mucilage
The kinetic properties of ACE (4 mU) without or with
purified mucilage (200 £gg) in total volume of 250 £gL were
determined using different concentrations of FAPGG as
substrates (0.1 mM to 0.5 mM). The Michaelis constants
Km (without mucilage) and Km¡¦ (with 200 £gg/mL
mucilage) were calculated from Lineweaver-Burk plots.
ReSULTS AND DISCUSSION
extraction and purification of mucilage from
sweet potato storage roots
The crude mucilage was obtained by isopropanol
precipitation and the yield was about 1.5%. For further
purification, it was first treated by SDS, which binds to
proteins, and the complexes formed were removed by
centrifugation. The semi-purified mucilage was then
heated in boiling water for 20 min. The total recovery
of purified mucilage after the two purification steps was
about 24% that of the crude one. After drying at 40¢XC
in an oven, the purified mucilage was ground and then
collected for further uses (Figure 1).
Determination of ACe inhibitory activity of
mucilage by spectrophotometry
The purified mucilage was used for determinations of
ACE inhibitory activity. Figure 2 shows the dose effect of
mucilage (50, 100, 200, and 400 £gg) on the ACE activity
(£G A 345 nm). Each line shows the time course of mucilage
effect in 5 min. Compared with the ACE only (control), it
was found that the higher the amount of mucilage added,
the lower the £GA 345 nm found during 300 sec ACE
inhibitory reactions (Figure 2).
effects of mucilage, pectin and captopril on
ACe activity by spectrophotometry
From Figure 2, it was found that mucilage exhibited
ACE inhibitory activity. It was interesting to know
whether commercial pectin also exhibited the ACE
inhibitory activity. Figure 3A shows the effects of
mucilage (50, 100, 200, and 400 £gg), commercial pectin
(50, 100, 200, and 400 £gg) and captopril (Figure 3B;
1.25, 2.5, 5, 10, 20, 40 and 80 nM) on ACE activity. It
was found that commercial pectin showed less ACE
inhibitory activity (less than 10% inhibition) and no
dose-dependent inhibition patterns. However, mucilage
exhibited dose-dependent ACE inhibitory activities
(50~400 £gg respectively, 23.4~53.2% inhibitions). From
calculations, the 50% inhibition (IC
50
) of ACE activity
was 364.5 £gg/mL mucilage compared to that of 10 nM
(8.68 £gg/mL) for captopril, which was similar to the report
Figure 1. The protein (A) and PAS (B) stainings of the crude
(lane 1) and purified (lane 2) mucilage from sweet potato storage
root on SDS-PAGE gels after 2-mercaptoethanol treatment. The
gel system contained 2.5 cm, 4% stacking gel and 4.5 cm, 15%
separating gel. M indicates the Seeblue
TM
prestained markers
of SDS-PAGE. The mucilage of 10 £gg was loaded in each well.
Arrow indicates the position of the mucilage.
Figure 2. Dosage effect of the mucilage (50, 100, 200, and 400
£gg) from sweet potato storage root on the ACE activity (.A 345
nm).
pg_0004
400
Botanical Studies, Vol. 47, 2006
(7 nM) of Pihlanto-Leppala et al. (1998). And the IC
50
of
yam mucilage in inhibiting ACE activity was 256.2 £gg/mL
(Lee et al., 2003). Both pectin and purified mucilage were
macromolecules, but only the purified mucilage showed
special dose-dependent ACE inhibitory activity. In the
literature, the protein hydrolysates were used as sources
for purification of peptides as ACE inhibitors
(Maruyama
et al., 1987; Mullally et al., 1996; Yano et al., 1996; Chen
et al., 1999; Kim et al., 2001; Chen et al., 2003). From
calculations, the IC
50
of mucilage for inhibiting ACE
activity was 364.5 £gg/mL, which was close to those of the
synthetic peptides £\-lactorphin (YGLF, 322.7 £gg/mL)
and
£]-lactoglobulin hydrolysates (GLDIQK, 391 £gg/mL).
Determinations of ACe inhibitory Activity of
mucilage by TLC
The FAPGG and FAP (product of ACE catalyzed
hydrolysis reaction) were separated by TLC using water
saturated 1-butanol:acetic acid:water, 4:1:1 (V/V/V)
as developing solvents according to the methods of
Holmquist et al. (1979). Figure 4 shows the qualitative
results of TLC chromatograms of a silica gel 60 F254
showing the effects of 250 £gg of commercial pectin
(lane 3) or mucilage (lane 4) on 15 microunits of ACE.
When compared to the control test (lane 2), it was found
that mucilage (lane 4) inhibited ACE activity (i.e. less
FAP produced) as observed under UV light. However,
similar FAP productions were found between the control
test (lane 2) and commercial pectin (lane 3). This
result demonstrated again that mucilage exhibited ACE
inhibitory activity.
Determination of the kinetic properties of ACe
inhibition by mucilage
The Lineweaver-Burk plots of ACE (4 mU) without
or with purified mucilage (200 £gg/mL) with different
concentrations of FAPGG are shown in Figure 5. The
results indicated that purified mucilage acted as a mixed
type inhibitor with respect to the substrate (FAPGG).
Without the purified mucilage, the calculated Km was
1 mM FAPGG for ACE. In the presence of purified
mucilage (200 £gg/ mL), the calculated Km¡¦ was 12 mM.
In conclusion, the mucilage exhibited dose-dependent
ACE inhibitory activity and acted as a mixed type
inhibitor with respect to the substrate (FAPGG). In the
yam without the purified mucilage, the calculated Km
was 0.255 mM FAPGG for ACE and in the presence of
purified mucilage, the calculated Km¡¦ was 0.3304 mM.
The purified mucilage exhibited dose-dependent ACE
inhibitory activity and acted as a mixed type inhibitor
with respect to the substrate (FAPGG) in the yam too. The
Lineweaver-Burk plots of ACE inhibitory activity were
just the same for purified mucilage of both sweet potato
and yam (Lee et al., 2003).
Figure 3. The effects of the purified mucilage, commercial
pe ct in, a nd c apt opri l on ACE ac tivi ty as det erm in ed b y
spectrophotometry. (A) Purified mucilage (50, 100, 200, and
400 £gg/mL) or commercial pectin (50, 100, 200, and 400 £gg/
mL); (B) Captopril (1.25, 2.5, 5, 10, 20, 40, and 80 nM). The
inhibition of ACE (%) was calculated according to the equation
[(1-(
.
A inhibitor) ¡Ò
.
A control)] ¡Ñ 100 %.
Figu re 4. The TLC chromatogram s of a silica gel 60 F254
showing the effects of the mucilage from sweet potato storage
root or commercial pectin on ACE activity. Lane 1, blank test
(FAPGG only); lane 2, control test (ACE reacted with FAPGG
to produce FAP); lane 3, control test plus 250 £gg commercial
pectin; lane 4, control test plus 250 £gg mucilage. Each solution
was dried under reduced pressure and redissolved with 400 £gL
methanol. Each 50 £gL was spotted on a silica gel 60 F254. The
FAPGG and FAP were separated by water saturated 1-butanol :
acetic acid : water, 4:1:1 (V/V/ V). Arrow indicated the position
of FAP.
pg_0005
HUANG et al. ¡X Mucilage with angiotensin converting enzyme inhibitory activity
401
In conclusion, the purified mucilage of sweet potato
storage roots exhibited dose-dependent ACE inhibitory
activity in vitro. The mucilage acted as a mixed type
inhibitor toward ACE with an IC
50
of 364.5 £gg/mL,
which is less than those of several peptides acting as
ACE inhibitors. It might be a potential ingredient for
hypertension control and deserves further investigations.
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