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.

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.

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).

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.

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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