Botanical Studies (2007) 48: 141-146.

*

Corresponding author: E-mail: wchou@tmu.edu.tw; Fax:

886 (2) 2378-0134.

INTRODUCTION

Free radical-mediated reactions have involved in

degenerative or pathological processes such as aging

(Harman, 1995), cancer, coronary heart disease and

Alzheimer¡¦s disease (Ames, 1983; Smith et al., 1996; Diaz

et al., 1997). Meanwhile there are many epidemiological

results revealing an association between a diet rich in

fresh fruit and vegetable and a decrease in the risk of

cardiovascular diseases and certain forms of cancer

in humans (Salah et al., 1995). Several reports were

concerned natural compounds in fruit and vegetable for

their antioxidant activities, such as phenolic compounds

(Rice-Evans et al., 1997), anthocyanin (Espin et al., 2000),

echinacoside in Echinaceae root (Hu and Kitts, 2000),

methanolic and hot-water extracts of Liriope spicata L.

(Hou et al., 2004), the storage proteins of sweet potato root

(Hou et al., 2001a), yam tuber (Hou et al., 2001b), potato

tuber (Liu et al., 2003b) and yam mucilages (Hou et al.,

2002; Lin et al., 2005).

There are several classes of pharmacological agents

which have been used in the treatment of hypertension

(Mark and Davis, 2000), one class of anti-hypertensive

drugs known as angiotensin I converting enzyme (ACE)

inhibitors (i.e. peptidase inhibitors) has a low incidence

of adverse side-effects and are the preferred class of anti-

hypertensive agents when treating patients with concurrent

secondary diseases (Fotherby and Panayiotou, 1999). ACE

(EC 3.4.15.1) is a dipeptide-liberating exopeptidase, which

has been classically associated with the renin-angiotensin

system regulating peripheral blood pressure (Mullally

et al., 1996). The potent ACE inhibitors were frequently

derived from food proteins (Ariyoshi, 1993; Hsu et al.,

2002). However, pomegranate juice (Aviram and Dornfeld,

2001), flavan-3-ols and procyanidins (Actis-Goretta et

al., 2003), yam mucilages (Lee et al., 2003) and tannins

(Liu et al., 2003a) were reported to have ACE inhibitory

activity.

Alginic acids, extracted from brown seaweeds or

Phaeophyceae, are unbranched high molecular-weight

polymers containing two types of uronic acid residues

of

£]

-(1

¡÷

4)-linked

D

-mannuronic acid and

£\

-(1

¡÷

4)-linked

L

-guluronic acid acid. Its derivatives have wide

Methanol-soluble, £]-elimination products from

preparations of alginic acid hydroxamate exhibited

DPPH scavenging and angiotensin converting enzyme

inhibitory activities

Yuh-Hwa LIU

1

, Mao-Te CHUAnG

2

, and Wen-Chi HOU

3,

*

1

Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan

2

St. Martin De Porres Hospital, Chiayi 600, Taiwan

3

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan

(Received June 2, 2006; Accepted August 30, 2006)

AbstrAct.

The methanol-soluble sugar hydroxamic acid derivatives obtained during the preparation

of alginic acid hydroxamates (AAH) were separated by BioSil-ODS HPLC column (10¡Ñ250 mm) in

acetonitrite: 0.05% trifluoroacetic acid, 10:90 (V/V). The absorbance at 235 nm was set for monitoring

£]

-elimination products. Each fraction was collected and assayed for hydroxamic acid contents, 1, 1-diphenyl-

2-picrylhydrazyl (DPPH) scavenging activity, and inhibitory activity against angiotensin converting enzyme

(ACE). The fraction with higher hydroxamic acid content was further separated by Sephadex G-15 column.

Fractions containing both ACE inhibitory activity and DPPH scavenging activity overlapped with fractions

containing high levels of hydroxamic acid derivatives. The chromatogram showed little tailing when judged by

hydroxamic-acid content but significant tailing when judged by biological activity. Each fraction from above

was further separated by silica-TLC in acetonitrite: distilled water, 95:5 (V/V). It was found that a methanol

soluble fraction prepared from alginic acid hydroxamates (AAH) exhibited DPPH scavenging activity.

Keywords: Alginic acid hydroxamates; Angiotensin converting enzyme (ACE); DPPH;

£]

-elimination.

BIOCHeMISTRy

142

Botanical Studies, Vol. 48, 2007

applications in different industry (Anderson et al., 1991;

Taqieddin and Amiji, 2004). We recently reported that

monohydroxamates of aspartic acid and glutamic acid

exhibit antioxidant and angiotensin converting enzyme

inhibitory activities (Liu et al., 2004), and the pectin

hydroxamic acids exhibited both semicarbazide-sensitive

amine oxidase and ACE inhibitory activities (Hou et al.,

2003), and antioxidant activities (Yang et al., 2004). In

our recent report, the methyl ester of alginic acid was used

to react alkaline hydroxylamine in methanol to produce

alginic acid hydroxamates (AAH), which was proven to

exhibit antioxidant and semicarbazide-sensitive amine

oxidase inhibitory activities (Liu et al., 2006). However,

the filtrates of AAH reaction solution by a G3 glass

filter after being adjusted to neutral pH exhibited potent

antioxidant activities. In this report, the small molecule

of methanol-soluble alginic acid hydroxamate (MSAAH)

was further separated by BioSil-ODS HPLC column and

Sephadex G-15 column. Each fraction was analyzed for its

DPPH scavenging and ACE inhibitory activities.

MATeRIALS AND MeTHODS

Materials

ACE (I unit, rabbit lung) was purchased from Fluka

Chemie GmbH (Switzerland); Alginic acid (low viscosity,

A-2158), 1, 1-diphenyl-2-picrylhydrazyl (DPPH), n-(3-

[2-furyl] acryloyl)-Phe-Gly-Gly (FAPGG), and other

chemicals and reagents were from Sigma Chemical Co.

(St. Louis, MO, USA). Silica gel 60 F

254

were purchased

from E. Merck Inc. (Darmstadt, Germany). Sephadex

G-15 powder was purchased from Amersham Biosciences

(Uppsala, Sweden).

Preparation of small molecules of methanol-

soluble alginic acid hydroxamate (MSAAH)

The preparation of AAH was previously reported (Liu

et al., 2006). In brief, the 8 g of methyl ester of alginic

acid suspended in 500 ml methanol were stirred at room

temperature for 20 h with a mixed solution (insoluble salt

was removed by filtration) containing 13 g of potassium

hydroxide in 50 ml methanol and 12 g of hydroxylamine-

HCl in 150 ml methanol. The filtrates from AAH reaction

medium by a G3 glass filter after being adjusted to neutral

pH were collected and dried by the rotary evaporator.

The salt in dried powder was removed through filtration

process by being dissolved in methanol repeatedly to

get the small molecules of methanol-soluble alginic acid

hydroxamate (MSAAH).

Separation of methanol-soluble alginic acid

hydroxamate (MSAAH)

Chromatographic separation of MSAAH (dissolved

in distilled water) was carried out in the Hitachi (Japan)

HPLC system equipped with a photodiode array detector

(L-2450) and a BioSil-ODS HPLC column (10¡Ñ250

mm). The MSAAH was separated isocratically with a

mobile phase consisting of a mixture of acetonitrite:

0.05% trifluoroacetic acid, 10:90 (V/V). The flow

rate was 1 ml/min and each fraction contained 1 ml.

The absorbance at 235 nm was set for monitoring

£]

-elimination products. Each fraction was collected and

assayed for hydroxamic acid contents, DPPH scavenging

activity, and ACE inhibitory activity. The fraction with the

highest hydroxamic acid content was further separated by

Sephadex G-15 column (1¡Ñ75 cm) and eluted by distilled

water. The flow rate was 0.5 ml/min and 1 ml was saved

for 50 fractions. Each fraction was collected and assayed

for hydroxamic acid contents, DPPH scavenging activity,

and ACE inhibitory activity.

Determination of hydroxamic acid contents

The hydroxamic acid contents were determined by

acidic ferric chloride solution (Soloway and Lipschitz,

1952) with some modifications as followed. Each 0.2

ml of separated fraction was mixed with 0.3 ml of 4 n

HCl and 0.5 ml of 10% ferric chloride in 0.1 n HCl.

The absorbance at 540 nm was determined after 10 min

standing, and the acetohydroxamic acid was used to plot

the standard curve and was expressed as

£g

mole nHOH

equivalent/ml.

Scavenging activity against DPPH radicals

The scavenging activity against DPPH radical was

measured according to the method of Hou et al. (2001a, b)

and Lee et al. (2006). Each 10 £gl of sample solution was

added to 0.1 ml of 1 M Tris-HCl buffer (pH 7.9), and then

mixed with 500 £gM DPPH in methanol for 20 min under

light protection at room temperature. Means of triplicates

were measured. The solvent system in each separation

process was used as a blank experiment. The scavenging

activity of DPPH radicals (%) was calculated with the

equation: (ĉ A517

blank

- ĉ A517

sample

)

¡Ò

ĉ A517

blank

¡Ñ

100%.

Determination of ACe inhibitory activity

The ACE inhibitory activity was measured according to

the method of Holmquist et al. (1979) and Lee et al. (2003)

with some modifications. The 15 £gl (15 mU) commercial

ACE (1 U/mL, rabbit lung) were mixed with 200 £gl

of sample solution and then 1 ml of 0.5 mM FAPGG

[dissolved in 50 mM Tris-HCl buffer (pH 7.5) containing

0.3 M naCl] was added. The decreased absorbance at

345 nm (ĉA

inhibitor

) was recorded during 5 min at room

temperature. The solvent system in each separation process

was used instead of sample solution for blank experiments

(ĉA

blank

). The ACE activity was expressed as ĉA 345 nm

and the ACE inhibition (%) was calculated as followed:

[1 - (ĉA

inhibitor

¡Ò

ĉA

control

)] ¡Ñ 100%. Means of triplicates

were determined.

Qualitative determination of sugar, hydroxamic

acid, and DPPH scavenging activity by TLC

Each fraction (fractions 34 to 45) separated by

LIU et al. ¡X Methanol-soluble sugar hydroxamic acid derivatives

143

the Sephadex G-15 column was further qualitative

determination of total sugar, hydroxamic acid, and DPPH

scavenging activity by TLC. The 10 £gl of each fraction

was spotted on a silica gel 60 F

254

TLC plate and was

separated by acetonitrite: distilled water, 95:5 (V/V). For

sugar staining, the plate was dipped in cerium molybdate

solution (in 10% sulfuric acid), and then heated at hot

plate (Ren et al., 2002), the white zones against blue

background showed the position of sugar. For hydroxamic

acid staining, the acidic ferric chloride solution in

methanol was used (Soloway and Lipschitz, 1952), and

the reddish color against the yellow background showed

the position of hydroxamic acid. For DPPH scavenging

activity, the plate was dipped in 500 £gM DPPH solution (in

methanol) and then was protected from light for a suitable

time, the white zones against purple background showed

the position of anti-DPPH radicals.

ReSULTS AND DISCUSSION

In our recent report, the methyl ester of alginic acid

was used to react alkaline hydroxylamine in methanol to

produce alginic acid hydroxamates (AAH), which was

proven to exhibit antioxidant and semicarbazide-sensitive

amine oxidase inhibitory activities (Liu et al., 2006). It

was found that the AAH exhibited a smaller molecular

size than the original materials identified by gel filtration.

It was proposed that the £]-elimination might be occurred

(Sajjaanatakul et al., 1989) during alkaline hydroxylamine

reaction and resulted in polymer breakdown and produced

a smaller molecule. In this report, the filtrates from AAH

reaction medium, adjusted to neutral pH, were collected

and dried by the rotary evaporator to get methanol-soluble

alginic acid hydroxamates (MSAAH) that were further

separated by BioSil-ODS HPLC column and Sephadex

G-15 column. It was found that small molecule of

MSAAH exhibited DPPH scavenging and ACE inhibitory

activities.

Separations of methanol-soluble alginic acid

hydroxamate (MSAAH) by BioSil-ODS HPLC

column

The suitable amounts of MSAAH (dissolved in distilled

water) were separated by a BioSil-ODS HPLC column

isocratically with a mobile phase consisting of a mixture of

acetonitrite: 0.05% trifluoroacetic acid, 10:90 (V/V). The

absorbance at 235 nm was set for monitoring £]-elimination

products (Figure 1). From the result of Figure 1, it was

found that the separation was complete within 10 min,

and the major peaks were found between 3 to 5 min. This

chromatographic process (within 10 min) was repeated

several times to collect the available amounts of separated

fractions for further biological assays.

Properties and biological activities of separated

MSAAH

Figure 2 showed (A) the absorbance at 235 nm; (B)

hydroxamic acid contents (expressed as £gmole equiv./

ml); (C) ACE inhibition (%, 200 £gl); and (D) scavenging

activity against 500 £gM DPPH radical (%, 10 £gl) of the

separated MSAAH. From the results of Figure 2(A), it

was found that the fractions 4 and 5 exhibited the major

absorbance at 235 nm. The double bond in the position of

C4 and C5 was characterized to have major absorbance

at 235 nm (Deng et al., 2006). From the results of Figure

2(B), it was found that the fraction 5 contained the highest

amounts of hydroxamic acid moiety (3.18 £gmole NHOH

equivalent/ml). The fraction 4 contained 0.2415 £gmole

nHOH equivalent/ml. Each fraction was used to test

the ACE inhibitory (Figure 2C) and DPPH scavenging

(Figure 2D) activities. The fractions 4 and 5 were found

to have 79.44% and 70%, respectively, ACE inhibitory

activities (Figure 2C, 200 £gl). In our previous report, the

hydroxamates of L-aspartic acid £]-hydroxamate (AAH)

Retention time (min)

1000

800

600

400

200

0

0 2 4 6 8 10 12 14 16 18

Figu re 1. The HP LC chromatogram of BioSil-ODS column

(10¡Ñ250 mm) for the separation of m ethanol-soluble alginic

acid hydroxamates (MS AAH). T he MS AAH was s epa rated

isocratically with a mobile phas e consisting of a mixture of

acet onit rit e: 0.05% tri fl uoroace tic ac id, 10:90 (V/V). The

flow rate was 1 ml/min and each fraction contained 1 ml. The

abs orbance at 235 nm was s et for monitoring

£]

-elimination

products.

Figure 2. Properties and biological activities of the separated

MSAAH by BioSil-ODS HPLC column. (A) The absorbance

at 235 nm; (B) hydroxamic acid contents (expressed as £gmole

equiv/ml); (C) ACE inhibition (%, 200 £gl); and (D) scavenging

activity against 500 £gM DPPH radical (%, 10 £gl). The separation

method was described at Figure 1.

144

Botanical Studies, Vol. 48, 2007

and L-glutamic acid £^-hydroxamate (GAH) showed

dose-dependent ACE inhibitory activities, and the IC

50

was 4.92 mM and 6.56 mM, respectively, for AAH and

GAH (Liu et al., 2004). The pectin hydroxamic acids

exhibited ACE inhibitory activities (Hou et al., 2003),

however, the ACE inhibitory activity was less active as

MSAAH did. It was also found that fractions 4 and 5

were exhibited 68.84% and 70.17%, respectively, DPPH

(500 £gM) scavenging activities (Figure 2D, 10 £gl). The

hydroxamates of L-aspartic acid £]-hydroxamate (AAH)

and L-glutamic acid £^-hydroxamate (GAH) exhibited

scavenging activities against DPPH radicals, and the IC

50

for AAH and GAH against DPPH (60 £gM) was 36 £gM

and 48 £gM, respectively (Liu et al., 2004). The IC

50

of

scavenging activity against DPPH (60 £gM) were 1.51,

5.43 and 5.63 mg/mL for DE94T4, DE65T4, DE25T4,

respectively (Yang et al., 2004). The fraction with the

highest hydroxamic acid content (fraction 5) was chosen

for further separation.

Separation of methanol-soluble alginic acid

hydroxamate (MSAAH) by Sephadex G-15

column

The fraction 5 was further separated by Sephadex G-15

column and eluted by distilled water. The hydroxamic acid

contents, ACE inhibition, and scavenging activity against

DPPH radical were assayed in each fraction (Figure 3).

It was found that both ACE inhibitory (Figure 3A) and

DPPH scavenging activities (Figure 3B) were overlapped

with fraction containing hydroxamic acid moiety (Figures

3A and 3B). The available fractionation range of Sephadex

G-15 was below 1500 Da. The methylglucopyranoside

(MW 194.2) was eluted in the fraction 35. However, the

hydroxamic acid fraction was eluted in a broader manner

(fractions 34 to 41), and the tailing phenomenon in the

biological activity assay was found (Figures 3A and 3B).

Qualitative determination of sugar, hydroxamic

acid, and DPPH scavenging activity on the TLC

The fractions 34 to 45 in the Sephadex G-15 were

further analyzed to qualitatively determine the total sugar,

hydroxamic acid, and DPPH scavenging activity on the

TLC which was separated by acetonitrite: distilled water,

95:5 (V/V). From the results of Figure 4A, there were

Figure 4. Thin-layer chromatography for the determination of sugar (A), hydroxamic acid (B), and DPPH scavenging activity (C) in

the fractions 34 to 45 that was separated by the Sephadex G-15 column. The 10 £gl of each fraction was spotted on a silica gel 60 F

254

TLC plate and was separated by acetonitrite: distilled water, 95:5 (V/V). The arrow indicated the position of sugar-hydroxamic acid

derivatives with DPPH scavenging activities.

Figure 3. The Sephadex G-15 chromatograms of the fraction 5

of the MSAAH that was separated by BioSil-ODS column. (A)

The hydroxamic acid contents (£gmole equiv/50 £gl), and ACE

inhibition (%, 50 £gl); (B) The scavenging activity against DPPH

radical (%, 10 £gl) were assayed in each fraction of Sephadex

G-15 column. The column (1¡Ñ75 cm) was eluted by distilled

water. The flow rate was 0.5 ml/min and 1 ml was saved for 50

fractions.

LIU et al. ¡X Methanol-soluble sugar hydroxamic acid derivatives

145

several white zones in each fraction which indicated the

positions of sugar. It meant that each fraction contained

sugar mixtures. From the results of Figure 4B, there was

only one position (arrow indicated) in fractions 35 to 41

with reddish color after spraying the acidic ferric chloride.

It meant that in the sugar mixtures (Figure 4A) only one

was belonged to hydroxamic acid derivatives (Figure 4B,

arrow indicated), and which exhibited the potent DPPH

scavenging activities (Figure 4C, arrow indicated).

In conclusion, the small molecule of methanol-

soluble AAH exhibited both DPPH scavenging and ACE

inhibitory activities in the Sephadex G-15 column (Figure

3). From the results of TLC qualitative assay, it was

found that only one sugar was belonged to hydroxamic

acid derivatives (Figure 4B, arrow indicated), and

which exhibited the potent DPPH scavenging activities

(Figure 4C, arrow indicated). The isolation and structure

identification of AAH with potent biological activity will

be investigated further.

Acknowledgments. The authors want to thank the

financial support (SKH-TMU-95-06) from Shin Kong Wu

Ho-Su Memorial Hospital, Taipei, Taiwan.

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