Botanical Studies (2008) 49: 101-108.
*
Corresponding author: E-mail: boyhlin@gate.sinica.edu.tw;
Fax: 886-2-2782-7954; Tel: 886-2-2789-9590 ext. 321.
BiochemiStry
Sweet potato storage root trypsin inhibitor and their
peptic hydrolysates exhibited angiotensin converting
enzyme inhibitory activity in vitro
Guan-Jhong HUANG
1
, Yu-Ling HO
2
, Hsien-Jung CHEN
3
, Yuan-Shiun CHANG
1
, Shyh-Shyun
HUANG
1
, Hsin-Jung HUNG
1
, and Yaw-Huei LIN
4,*
1
Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 404, Taiwan
2
Department of Nursing, Hung Kuang University, Taichung, 433, Taiwan
3
Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan
4
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan
(Received September 11, 2007; Accepted October 26, 2007)
ABStrAct.
Trypsin inhibitor (TI), the root storage protein of sweet potato, was shown by
spectrophotometric methods to inhibit angiotensin converting enzyme (ACE) in a dose-dependent manner
(50-200 £gg/mL, with 31.9-53.2% inhibition) using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as a
substrate. The 50% inhibition (IC
50
) of ACE activity required 187.96ƒn £gg/mL TI compared to 10 nM (868 ng/
mL) of Captopril. The use of TLC also showed TI as ACE inhibitor. TI acted as a mixed type inhibitor against
ACE using FAPGG as a substrate. When 200 £gg/mL TI were added, Vmax and Km were, respectively 0.013
£GA/min and 0.715 mM while without TI they were 0.027 £GA/min and 0.419 mM. Pepsin was used for TI
hydrolysis for different times. ACE inhibitory activity was found to increase from 34% to about 83% after 24
h of hydrolysis. The results suggested that when small peptides increased by pepsin hydrolysis of the TI ACE
inhibitory capacity also increased up to 24 h and then decreased, it may be due to the disappearance of some
conformational requirements. Ten peptides¡Xnamely HDHM, LR, SNIP, VRL, TYCQ, GTEKC, RF, VKAGE,
AH and KIEL¡Xwere synthesized based on the simulated pepsin digestion of trypsin inhibitor and then tested
for ACE inhibitory activity. IC
50
values of individual peptides were 276.2, 746.4, 228.3, 208.6, 2.3, 275.8,
392.2, 141.56, 523.5 and 849.7 £gM, suggesting that TYCQ might represent the main active site for the ACE
inhibition. TI and its hydrolysates might be good for control of hypertension and other diseases when people
consume sweet potato tuberous roots.
Keywords: Angiotensin converting enzyme (ACE); Trypsin inhibitor; Pepsin; Sweet potato.
iNtroDUctioN
Many bioactive peptides have common structural
properties that include a relatively short peptide residue
length (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, 1991),
caseino-phosphopeptic (Maubois
and Leonil, 1989),
bactericidal (Bellamy et al., 1993),
antioxidant or angiotensin-converting enzyme inhibitor
(Ehlers and Riordan, 1989) functions have been the
research focus 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.
Several food-derived peptides from £\-lactoalbumin,
£]-lactoglobulin (Pihlanto-Leppala et al., 1998),
casein
(Maruyama et al., 1987), zein, mucilage (Huang et al.,
2006), and azein (Yano et al., 1996) also inhibited ACE.
Several antioxidant peptides (reduced glutathione and
carnosine-related peptides) (Hou et al., 2003) and synthetic
peptides also exhibited ACE inhibitor activities (Chen et
al., 2003).
pg_0002
102
Botanical Studies, Vol. 49, 2008
Protease inhibitors in plants may be important in
regulating and controlling endogenous proteases and
in acting as protective agents against insect and/or
microbial proteases (Ryan, 1973; Ryan, 1989).
Sohonie
and Bhandarker (Sohonie and Bhandarker, 1954)
reported
for the first time the presence of trypsin inhibitor (TI) in
sweet potato (SP). Later, it was indicated that TI activities
in SP are positively correlated with concentrations of
water-soluble protein (Lin and Chen, 1980), and that they
increase in response to drought (Lin, 1989). Polyamines,
including cadaverine, spermidine and spermine, were
bound covalently to SPTI, which might participate in
regulating the growth and developmental processes of
SP (Hou and Lin, 1997). TIs in SP storage roots account
for about 60% of total water-soluble proteins and could
be recognized as storage proteins (Lin, 1989). Matsuoka
et al. (Matsuoka et al., 1990) identified sporamin as the
major storage protein in SP root, accounting for 80% of
the total proteins there. A dramatic decrease of the amount
of sporamin to 2% of the original value was found during
sprouting. Lin (Lin, 1993; Huang et al., 2007) considered
sporamin as one form of TIs in SP, a finding confirmed
later by Yeh et al. (1997).
In our previous report, TI exhibited both
dehydroascorbate reductase and monodehydroascorbate
reductase activities, and 33 kDa TI exhibited antioxidant
activities against different radicals (Hou et al., 2001;
Huang et al., 2007). In this work we report for the first
time that TI exhibited dose-dependent ACE inhibitory
activity when Captopril was used as a positive control.
Commercial bovine serum albumin (BSA), which was
frequently found in the literature as the peptide resource of
ACE inhibitors, was chosen for comparison. The K
i
values
of trypsin inhibitor against ACE were calculated. We also
used pepsin to hydrolyze TI for different times, and the
changes of ACE inhibitory activity were determined. The
IC
50
of ACE inhibitory activities by synthetic peptides was
also determined.
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 (1 unit, rabbit lung);
coomassie brilliant blue G-250; peptide (GL Biochem,
China), and other chemicals and reagents were purchased
from Sigma Chemical Co. (St. Louis, MO, USA).
Plant materials
Fresh storage roots of sweet potato (Ipomoea batatas
(L.) Lam. ¡¥Tainong 57¡¦) were purchased from a local
market.
Purification of sweet potato trypsin inhibitor
Sweet potato storage roots were washed and peeled, and
then were cut into strips and extracted with distilled water.
The crude extracts were loaded directly onto a trypsin
Sepharose-4B affinity column. The adsorbed TI was eluted
by pH changes with 0.2 M KCl (pH 2.0) according to
Huang et al. (2005).
Protein staining and activity staining of trypsin
inhibitor on 15% denaturing polyacrylamide
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 or without
2-mercaptoethanol. Coomassie brilliant blue G-250 was
used for protein staining (Huang et al., 2004). For sweet
potato TI activity staining, the gel was stained according
to the method of Hou et al. (2002).
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, rabbit lung) was mixed with 50
£gL of different amounts of trypsin inhibitor or BSA (50,
100, and 200 £gg/mL), and then 200 £gL of 0.5 mM N-[3-
(2-f uryl) acryloyl]-Phe-Gly-G ly [FAPGG, dissolved in
50 mM Tris-HCl buffer (pH 7.5) containing 0.3 M NaCl]
was added. The decreased absorbance at 345 nm (£GA
inhibitor) was recorded during 5 min at room temperature.
Deionized water was used instead of sample solution for
blank experiments (£GA control). 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 percent
inhibition was calculated as follows: [1 - (£G A inhibitor /
£GA control)] ¡Ñ 100. Means of triplicates were determined.
The 50% inhibition (IC
50
) of ACE activity was defined as
the concentrations of samples that inhibited 50% of ACE
activity under experimental conditions.
Determination of Ace inhibitory activity by tLc
The ACE inhibitory activity of TI was determined
by TLC method (Holmquist et al., 1979). The reactions
between TI and ACE or BSA and ACE were according to
the method of Anzenbacherova et al. (2001) with some
modifications. Each 100 £gL of TI and BSA (225 £gg/mL)
was premixed with 15 microunits ACE for 1 min, and then
200 £gL of 0.5 mM 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, and in the control experiment,
ACE reacted with FAPGG under the same conditions.
pg_0003
HUANG et al. ¡X Trypsin inhibitor with angiotensin converting enzyme inhibitory activity
103
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
product) were separated by TLC in 1-butanol-acetic acid-
water, 4:1:1 (v/v/v) and observed under UV light.
Determination of the kinetic properties of Ace
inhibition by trypsin inhibitor
The kinetic properties of ACE (4 mU) without or with
purified TI (200 £gg/mL) in a total volume of 250 £gL were
determined using different concentrations of FAPGG
as substrate (0.1 mM to 0.5 mM). The Km (without TI)
and Km¡¦ (with TI) were calculated from Lineweaver-
Burk plots, where Km¡¦ was the Michaelis constant in the
presence of 200 £gg/mL TI.
Determination of the Ace inhibitory activity of
peptic hydrolysates of trypsin inhibitor
Six mg of TI were dissolved in 1 mL of 0.1 M KCl
buffer (pH 2.0). Then 0.1 mL of 12 mg of pepsin was
added at 37¢XC for 8, 12, 24, and 32 h. After hydrolysis,
0.5 mL of 0.5 M Tris-HCl buffer (pH 8.3) was added, and
the solution was heated at 100¢XC for 5 min to stop enzyme
reaction. The pepsin was heated before TI hydrolysis
for the 0 h reaction. Each of the 60 £gL TI hydrolysates
was used for determinations of ACE inhibition by
spectrophotometry.
chromatograms of peptic hydrolysates of
trypsin inhibitor on a Sephadex G-50 column
The unhydrolyzed TI and peptic TI hydrolysates at 24 h
were separated by Sephadex G-50 chromatography (1 ¡Ñ 60
cm). The column was eluted with 20 mM Tris-HCl buffer
(pH 7.9). The flow rate was 30 mL/h, and each fraction
contained 2 mL, the absorbance of which was determined
at 280 nm.
reSULtS and DiScUSSioN
extraction and purification of trypsin inhibitor
from sweet potato storage root
TI was purified from sweet potato storage root
according to the method of Huang et al. (2005) Figure
1 shows protein staining (lane 1 and lane 3) and activity
staining (lane 2 and lane 4) of purified TI without (lane 1
and lane 2) or with (lane 3 and lane 4) 2-mercaptoethanol
treatment on a 15% SDS-PAGE gel.
Determination of Ace inhibitory activity of
trypsin inhibitor by spectrophotometry
The purified TI was used for determinations of ACE
inhibitory activity. Figure 2 shows time course of the effect
of the different amounts of TI (0, 50, 100, and 200 £gg/
mL) on ACE activity (£GA 345 nm). Compared with ACE
only (control), it was found that the higher the amount of
TI added, the lower the £GA 345 nm was during the 300-s
reaction period. Results of Figure 2 shows that purified TI
could inhibit ACE activity in a dose-dependent manner.
effects of trypsin inhibitor, BSA, and captopril
on Ace activity shown by spectrophotometry
We wanted also to know whether BSA also exhibited
ACE inhibitory activity. Figure 3A shows the effects of TI
(0, 50, 100, 200 £gg/mL), BSA (0, 50, 100, 200 £gg/mL),
or Captopril (Figure 3B; 0, 1.25, 2.5, 5, 10, 20, 40 and
80 nM; corresponding to 0, 108.5, 217, 434, 868, 1,736,
3,472 and 6,844 ng/mL, respectively) on ACE activity.
BSA displayed less ACE inhibitory activity (less than 15%
inhibition) and without dose-dependent inhibition patterns.
However, TI exhibited dose-dependent ACE inhibitory
Figure 1. The protein (lane 1 and lane 3) and activity (lane 2
and lane 4) stainings of the trypsin inhibitor from sweet potato
storage root on SDS -PAGE gels without (lane 1 and lane 2)
or with (lane 3 and lane 4) 2-mercaptoethanol. The gel system
contained 2.5 cm, 4% stacking gel and 4.5 cm, 15 % separating
gel. M indicates the S eeblue
TM
prestained markers of S DS-
PAGE. Ten micrograms of trypsin inhibitor were loaded in each
well.
Figure 2. Inhibitory activity of different amounts of trypsin
inhibitor (0, 50, 100 and 200 £gg/mL) from sweet potato storage
root on the ACE activity (.A 345 nm).
pg_0004
104
Botanical Studies, Vol. 49, 2008
activity (50~200 £gg/mL giving, respectively, 31.9 ~ 53.2%
inhibition). From calculations, the 50% inhibition (IC
50
) of
TI against ACE activity was 187.96 £gg/mL, compared to
10 nM (868 ng/mL) for Captopril, which was similar to the
7 nM reported by Pihlanto-Leppala et al. (1998). The IC
50
of yam dioscorin was 250 £gg/mL (Hsu et al., 2002). Both
BSA and purified TI are proteins, but only the purified TI
showed specific dose-dependent ACE inhibitory activity.
In the literature, the protein hydrolysates are used as
sources to purify peptides like ACE inhibitors (Mullally
et al., 1996; Maruyama et al., 1987).
By our calculations,
the IC
50
of TI against ACE activity was 187.96 £gg/mL,
which was smaller than that of the synthetic peptide
£\-lactorphin (YGLF, 322.7 £gg/mL). Several identified
peptide fragments exhibited much lower IC
50
values than
our purified TI: for example, Tyr-Pro of whey proteins,
8.1 £gg/mL (Yamamoto et al., 1999) and HHL of soybean
proteins, 2.2 £gg/mL (Shin et al., 2001). Conversely, several
identified peptide fragments exhibited IC
50
values much
higher than our purified TI: for example, hydrolysates of
whey proteins (£\-lactalbumin and £]-lactoglobulin) with
IC
50
values between 345-1,733 £gg/mL (Pihlanto-Leppala,
et al., 2000); LAHKAL of £\-lactalbumin hydrolysates, 406
£gg/mL; GLDIQK of £]-lactoglobulin hydrolysates, 391 £gg/
mL; and VAGTWY of £]-lactoglobulin hydrolysates, 1,171
£gg/mL.
Determinations of Ace inhibitory activity of
trypsin inhibitor 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 method of Holmquist
et al. (1979). Figure 4 shows the TLC chromatograms
of a silica gel 60 F254 and the qualitative results of the
effects of 225 £gg/mL of commercial BSA (lane 3) or
TI (lane 4) on 15 microunits of ACE. Compared to the
control test (lane 2), TI (lane 4) was found to inhibit the
ACE reaction with less FAP production observable under
UV light. However, the control test (lane 2) and BSA
(lane 3) produced similar amounts of FAP. These results
demonstrated again that TI exhibited ACE inhibitory
activity.
Determination of the kinetic properties of Ace
inhibition by trypsin inhibitor
The Lineweaver-Burk plots of ACE (4 mU) without
or with purified TI (200 £gg/mL) under different
concentrations of FAPGG are shown in Figure 5. The
Figure 3. The effects of trypsin inhibitor, albumin, and Captopril
on ACE activity determined by s pectrophotometry. Trypsin
inhibitor (0, 50, 100 and 200 £gg/mL) or bovine serum albumin
(0, 50, 100 and 200 £gg/mL) was used. The inhibition of ACE (%)
was calculated according to the equation [1-(.A inhibitor ¡Ò .A
control)] ¡Ñ 100%.
Figu re 4. The TLC chromatograms of a silica gel 60 F254
s howing the effec ts of tryps in inhibitor from s wee t pota to
storage root or bovine serum albumin on ACE activity. Lane
1, blank test (FAPGG only); lane 2, control test (ACE reacted
with FAPGG to produce FAP); lane 3, 225 £gg/mL bovine serum
albumin added; lane 4, 225 £gg/mL trypsin inhibitor added. 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). Arrows indicated
the positions of both FAP and FAPGG.
pg_0005
HUANG et al. ¡X Trypsin inhibitor with angiotensin converting enzyme inhibitory activity
105
results indicated that purified TI acted as a mixed type
inhibitor against ACE using FAPGG as a substrate.
When 200 £gg/mL TI were added, Vmax and Km were,
respectively, 0.013 £GA/min and 0.715 mM; while without
TI they were 0.027 £GA/min and 0.419 mM. In conclusion,
TI exhibited dose-dependent ACE inhibitory activity
and acted as a mixed type inhibitor with respect to the
substrate (FAPGG). A similar finding was reported with
the calculated Km as 0.255 mM FAPGG for ACE, and in
the presence of purified dioscorin, the calculated Km¡¦ was
0.3304 mM (Hsu et al., 2002).
Determination of the Ace inhibitory activity of
peptic trypsin inhibitor hydrolysates and their
peptide distributions
Pepsin is frequently used for protein hydrolysis to
purify potential ACE inhibitory peptides (Pihlanto-Leppala
et al., 2000). Therefore, we used pepsin to hydrolyze
TI. Figure 6 shows the ACE inhibitory activity (£GA 345
nm) of peptic TI hydrolysates. Figure 6A shows the ACE
inhibition (percent) of peptic TI hydrolysates collected at
different pepsin hydrolysis times. The results (Figure 6A)
show ACE inhibitory activity increasing from 34% (0 h)
to about 83% (24 h). Figure 6B shows the chromatograms
of unhydrolyzed TI and peptic TI hydrolysates (24 h) on
Sephadex G-50 chromatography. It was found that smaller
peptides increased with increasing pepsin hydrolytic time.
The ACE inhibitor activities of peptic TI hydrolysates
decreased after 36 h of hydrolysis (Figure 6A), suggesting
that some proper conformational requirements got lost
thereafter.
Lin (Lin, 1993) considered sporamin as one form of
TI in sweet potato, a finding confirmed later by Yeh et
al. (1997). So we used synthetic peptides to measure
ACE inhibitor activity by the sporamin A and B gene
sequences. Kohmura et al. (1989) synthesized some
peptide fragments of human £]-casein and found that
the length of those peptides had an influence on the
ACE inhibitory activity. Namely, peptides composed of
3-10 amino acids with proline on the C-terminal were
necessary for ACE inhibitors (Kohmura et al., 1990).
Thus
the peptide Leu-Arg-Pro from food protein hydrolysates
has been reported to be the most potent natural ACE
inhibitor, with an IC
50
value of 0.27
or 1.0 £gM. Byun et al.
(1980) studied the ACE inhibitory activity of a series of
dipeptides and indicated that tryptophan, tyrosine, proline,
or phenylalanine at the C-terminal and branched-chain
aliphatic amino acid at the N-terminal were suitable for a
peptide binding to ACE (Byun and Kim, 2002).
Synthetic peptides were designed by simulating the
pepsin cutting sites of sporamin A (accession number:
P14715) and B (accession number: P14716) gene products
from sweet potato (pH >2, http://expasy.nhri.org.tw/tools/
peptidecutter/). Ten new inhibitory peptides (Table 1) for
ACE¡X HDHM, LR, SNIP, VRL, TYCQ, GTEKC, RF,
VKAGE, AH and KIEL¡Xwere synthesized according to
simulation. IC
50
values of individual peptides were 276.2,
Figure 5. The Lineweaver-Burk plots of ACE (4 mU) without
or with trypsin inhibitor (200 £gg/mL) from sweet potato storage
root using different concentrations of FAPGG (0.1 to 0.5 mM).
F igure 6. ACE inhibitory activity of peptic hydrolysates of
sweet potato trypsin inhibitor. The plot shows the ACE inhibition
(%) of peptic trypsin inhibitor hydrolysates obtained at different
peps in hydrolys is tim es (A). The proteins and the inhibition
of ACE (%) were shown (B). The inhibition of ACE (%) was
calculated according to the equation [1-(.A inhibitor ¡Ò .A
control)] ¡Ñ 100%.
pg_0006
106
Botanical Studies, Vol. 49, 2008
746.4, 228.3, 208.6, 2.3, 275.8, 392.2, 141.56, 523.5 and
849.7 £gM, respectively. These results demonstrated that
simulated synthetic peptides from peptic TI hydrolysates
exhibited ACE inhibitory activities. In addition, some of
these synthetic peptides also had antioxidantive activity
(Huang et al., 2004, 2007). Our work suggests: (1) TYCQ
might represent the main active site of the ACE inhibition,
and (2) there are marked structural similarities for
peptides with antihypertensive, immunomodulatory, and
antioxidant activities, and these may be used as criteria
for selecting or designing multifunctional ingredients of
functional foods to control cardiovascular diseases.
In summary, trypsin inhibitor purified from sweet
potato storage roots exhibited dose-dependent ACE
inhibitory activity. TI acted as a mixed type inhibitor
toward ACE with an IC
50
of 187.96 £gg/mL. Its peptic
hydrolysates also showed ACE inhibitory activities. Some
peptides derived from food proteins were demonstrated
to have antihypertensive activities against spontaneously
hypertensive rats (Fujita et al., 2000; Yoshii et al., 2001).
The potential for hypertension control when people
consume sweet potato deserves further investigation.
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Hou, W.C., Y.C. Chen, H.J. Chen, Y.H. Lin, L.L. Yang, and
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Hou, W.C., D.J. Huang, and Y.H. Lin. 2002. An aspartic type
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Table 1. Trypsin inhibitor (sporamin A and B) peptides with
ACE inhibitor activity.
IC
50
(
£g
M)
Sporamin A peptides
523.5
37
AH
38
523.5
127
KIEL
130
849.7
141
TYCQ
144
2.3
157
HDHM
160
276.2
Sporamin B peptides
37
VRL
39
208.6
83
RF
84
392.2
121
VKAGE
125
141.5
144
GTEKC
148
275.8
167
SNIP
170
228.3
Sporamin A and B peptides
35
RL
36
or
34
RL
35
746.4
85
RF
86
or
83
RF
84
392.2
Note: The sequences of sporamin A and B were retrieved from
the NCBI (National Center for Biotechnology Information,
http://www.ncbi.nlm.nih.gov) with the following accession
numbers: sporamin A: ABB97547 and sporamin B: P14716,
neither of which includes pre-pro-sequence.
pg_0007
HUANG et al. ¡X Trypsin inhibitor with angiotensin converting enzyme inhibitory activity
107
Huang, D.J. C.D. Lin, H.J. Chen, and Y.H. Lin. 2004.
Antioxidant and antiproliferative activities of sweet potato
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