pg_0001

Botanical Studies (2007) 48: 133-140.

Corresponding author: Guan-Jhong Huang, E-mail:

gjhuang@mail.cmu.edu.tw; Yaw-Huei LIN, E-mail:

boyhlin@gate.sinica.edu.tw, Fax: 886-2-2782-7954, Tel:

886-2-2789-9590 ext. 321.

INTRODUCTION

It is commonly accepted that in a situation of oxidative

stress, reactive oxygen species such as superoxide (O

hydroxyl (OH·) and peroxyl (·OOH, ROO·) radicals are

generated. The reactive oxygen species play an important

role related to the degenerative or pathological processes

of various serious diseases, such as aging (Burns et al.,

2001), cancer, coronary heart disease, Alzheimer’s dis-

ease (Ames, 1983; Gey, 1990; Smith et al., 1996; Diaz

et al., 1997), neurodegenerative disorders, atheroscle-

rosis, cataracts, and inflammation (Aruoma, 1998). The

use of traditional medicine is widespread and plants still

present a large source of natural antioxidants that might

serve as leads for the development of novel drugs. Sev-

eral antiinflammatory, antinecrotic, neuroprotective, and

hepatoprotective drugs have recently been shown to have

an antioxidant and/or antiradical scavenging mechanism

as part of their activity (Perry et al., 1999; Repetto and

Llesuy, 2002). In the search for sources of natural anti-

oxidants and compounds with radical scavenging activity

during the last few years, some have been found, such

as whey proteins (Allen and Wrieden, 1982; Tong et al.,

2000), phenolic compounds (Rice-Evans et al., 1997), an-

thocyanin (Espin et al., 2000), echinacoside in Echinaceae

root (Hu and Kitts, 2000), water extract of roasted Cassia

tora (Yen and Chuang, 2000), both thioredoxin h protein

(Huang et al., 2004a) and mucilage (Huang et al., 2006a, b)

from sweet potato root.

Sporamin was the major storage protein in sweet potato

tuberous roots, first described by Maeshima et al. (Mae-

shima et al., 1985). It accounted for 60% to 80% of the to-

tal soluble protein in the sweet potato tuber. Expression of

Recombinant sporamin and its synthesized peptides

with antioxidant activities in vitro

Guan-Jhong HUANG

*, Hsien-Jung CHEN

, Yuan-Shiun CHANG

, Ming-Jyh SHEU

, and Yaw-

Huei LIN

Institute of Chinese Pharmaceutical Sciences, China Medical University, Taichung 404, Taiwan

Department of Horticulture, Chinese Culture University, Taipei 111, Taiwan

Department of Physiology, School of Medicine, China Medical University, Taichung 404, Taiwan

Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan

(Received October 17, 2006; Accepted November 29, 2006)

ABSTRACT.

Recombinant sporamin B overproduced in E. coli (M15) was purified by Ni

-chelated affinity

chromatography. The molecular mass of sporamin B is ca. 26 kDa as determined by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE). Total antioxidant status, 1,1-dipheny-2-picrylhydrazyl (DPPH)

staining, reducing power method, Fe

2＋

-chelating ability, ferric thiocyanate (FTC) method, and protecting

calf thymus DNA against hydroxyl radical-induced damage were studied. The sporamin B protein with a

concentration of 100 μg/mL exhibited highest activity (expressed as 4.21 ± 0.0078 mM Trolox equivalent

antioxidative value, TEAC) in total antioxidant status test. In the DPPH staining sporamin B appeared as

a white spot when the concentration was diluted to 25 mg sporamin B/mL (with an absolute amount of 75

μg). Like total antioxidant status, the reducing power, Fe

2＋

-chelating ability, FTC activity and protection

calf thymus DNA against hydroxyl radical-induced damage all showed that sporamin B polypeptides have

significant antioxidant activities. It was found that antioxidant activities of sporamin B increased from 19% (0 h)

to about 29% (24 h) after 24 h hydrolysis by pepsin. Smaller peptides increased with hydrolytic times. Eight

peptides for testing antioxidative activity were synthesized according to peptic hydrolysis simulation. The

obtained SNIP, VRL, SYCQ, GTEKC, RF, VKAGE, AH, KIEL showed IC

values of 8.36, 4.23, 0.206, 0.0884,

9.72, 14.9, 13.8 and 24.9 mM, respectively, when scavenging activity of DPPH radicals (%) was measured.

These findings mean that cysteine residue is most important in antiradical activities. It was suggested that

sporamin B might contribute to its antioxidant activities against hydroxyl and peroxyl radicals.

Keywords: Antioxidant activity; Recombinant protein; Gene expression; Sporamin; Sweet potato.

BIOChemISTRy

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Botanical Studies, Vol. 48, 2007

sporamin was shown to be mainly associated with tubers

(Hattori et al., 1990). Sporamin was found to strongly in-

hibit trypsin activity, and insect-defense capabilities were

confirmed in insect bioassays with transgenic tobacco (Yeh

et al., 1997). These showed that sporamin functions not

only as a storage protein for nutrient supply but also as a

factor against herbivore attacks. Sporamin genes belonged

to a large multigene family that was divided into subfami-

lies A and B. There was over 90% nucleotide homology

among intrasubfamily genes, and about 80% nucleotide

homology among inter-subfamily genes (Hattori et al.,

1989). The objectives of this work were to investigate the

antioxidant property of sporamin B from sweet potato in

comparison with chemical compounds such as butylated

hydroxytoluene (BHT), reduced glutathione or ascorbate

in a series of in vitro tests.

mATeRIALS AND meThODS

expression of sporamin B in

E. coli

Sporamin B (Gene Bank accession number: M16883)

was expressed in E. coli. The coding sequence was ampli-

fied from sporamin B cDNA using an oligonucleotide (5’

-GGATC CACAT GAAAG CCT TC G-3’), with a BamHI

site (underlined) at the putative initial Met residue, and an

oligonucleotide (5’-TCAGA AGCTT TGATC ACA-3’),

with a HindIII site at the 3’ end. The PCR fragment was

subcloned in pGEM T-easy vector. And the plasmid was

then digested with BamHI and HindIII and subcloned in

pQE-30 expression vector (QIAexpress expression system,

Qiagen). The resulting plasmid, termed pQE-sporamin

B, was introduced into E. coli (M15). Cultures of the

transformed E. coli (M15) overexpressed a protein of the

expected molecular mass, which was purified by affinity

chromatography in Ni-nitrilotriacetic acid (NTA) columns

(Qiagen), according to the manufacturer’s instructions.

Protein staining on 15% SDS-PAGe gels

Sporamin B was detected on 15% SDS-PAGE gels.

Samples treated with sample buffer and

-mercaptoethanol

(2-ME) with a final concentration of 14.4 mM were heated

at 100°C for 5 min before 15% SDS-PAGE.

measurement of total antioxidant status

Total antioxidant status of the sporamin B protein

was measured using the total antioxidant status assay

kit (Calbiochem

Corp) according to the manufacturer’s

instructions. The

assay relies on the antioxidant ability

of the protein to

inhibit oxidation of 2, 2’ azino-bis-[3-

ethylbenz-thiazoline-6-sulfonic

acid] (ABTS) to ABTS*

by metmyoglobin. The amount of ABTS*

produced

monitored by reading the absorbance at 600 nm. Under

these

reaction conditions, the antioxidant ability of

sporamin B protein decreases

the absorbance at 600 nm

in proportion

to its concentration. The final antioxidant

capacity of

sporamin B protein was calculated by the

following formula: Trolox equivalent value (mmol/L)=

[factor × (absorbance of blank-absorbance of sample)];

factor=[concentration of standard/(absorbance of blank-

absorbance

of standard)].

Rapid screening of antioxidant by dot-blot and

DPPH staining

An aliquot (3 μl) of each diluted sample of the sporamin

B was carefully loaded on a 20 cm × 20 cm TLC layer

(silica gel 60 F254; Merck) and allowed to dry (3 min).

Drops of each sample were loaded in order of decreasing

concentration along the row. The staining of the silica

plate was based on the procedure of Huang et al. (2005).

The sheet bearing the dry spots was placed upside down

for 10 s in a 0.4 mM DPPH solution. Then the excess of

solution was removed with a tissue paper and the layer

was dried with a hair-dryer blowing cold air. Stained silica

layer revealed a purple background with white spots at the

location where radical scavenger capacity presented. The

intensity of the white color depends upon the amount and

nature of radical scavenger present in the sample.

Scavenging activity against DPPh radical

The effect of sporamin B on the DPPH radical was

estimated according to the method of Huang et al. (2004b).

An aliquot of sporamin B (30 μL) was mixed with 100

mM Tris-HCl buffer (120 μL, pH 7.4) and then 150 μL

of the DPPH in ethanol with a final concentration of 250

μM was added. The mixture was shaken vigorously and

left to stand at room temperature for 20 min in the dark.

The absorbance at 517 nm of the reaction solution was

measured spectrophometrically. The percentage of DPPH

decolourization of the sample was calculated according to

the equation: % decolourization= [1- Abs

sample

/Abs

control

]×

100.

Determination of antioxidant activity by

reducing power measurement

The reducing powers of the sporamin B and glutathione

were determined according to the method of Yen and Chen

(1995). Sporamin B (0, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL)

or glutathione was mixed with an equal volume of 0.2 M

phosphate buffer, pH 6.6, and 1% potassium ferricyanide.

The mixture was incubated at 50°C for 20 min, during

which time ferricyanide was reduced to ferrocyanide.

Then an equal volume of 1% trichloroacetic acid was

added to the mixture, which was then centrifuged at 6,000

g for 10 min. The upper layer of the solution was mixed

with deionized water and 0.1% FeCl

at a radio of 1:1:2,

and the absorbance at 700 nm was measured to determine

the amount of ferric ferrocyanide (Prussian Blue) formed.

Increased absorbance of the reaction mixture indicated in-

creased reducing power of the sample.

Determination of antioxidant activity by Fe

＋

-chelating ability

The Fe

2＋

-chelating ability was determined according

to the method of Decker and Welch (1990). The Fe

2＋

was

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HUANG et al. — Sporamin and its peptides with antioxidant activities

135

monitored by measuring the formation of ferrous iron-fer-

rozine complex at 562 nm. Sporamin B (0, 0.4, 0.8, 1.2, 1.6

and 2 mg/mL) was mixed with 2 mM FeCl

and 5 mM fer-

rozine at a ratio of 10:1:2. The mixture was shaken and left

to stand at room temperature for 10 min. The absorbance

of the resulting solution at 562 nm was measured. The

lower the absorbance of the reaction mixture the higher

the Fe

2＋

-chelating ability. The capability of the sample to

chelate the ferrous iron was calculated using the following

equation:

Scavenging effect (%) = [1- Abs

sample

/ Abs

control

]×100

Determination of antioxidant activity by the

ferric thiocyanate (FTC) method

The FTC method was adapted from the method of

Osawa and Namiki (1981). Twenty mg/mL of samples

dissolved in 4 ml of 99.5% (w/v) ethanol were mixed with

linoleic acid (2.51%, v/v) in 99.5% (w/v) ethanol (4.1

ml), 0.05 M phosphate buffer pH 7.0 (8 ml) and deionized

water (3.9 ml) and kept in a screw-cap container at 40°C in

the dark. Then, to 0.1 ml of this solution was added 9.7 ml

of 75% (v/v) ethanol and 0.1 ml of 30% (w/v) ammonium

thiocyanate. Precisely 3 min after the addition of 0.1 ml

of 20 mM ferrous chloride in 3.5% (v/v) hydrochloric

acid to the reaction mixture, the absorbance at 500 nm

of the resulting red color [Fe (SCN)

, Fe

was formed

after linoleic acid peroxide was produced and Fenton

reaction occurred.] was measured every 24 h until the day

when the absorbance of the control reached the maximum

value. The inhibition of linoleic acid peroxidation was

calculated as (%) inhibition = 100 - [(absorbance increase

of the sample/absorbance increase of the control) × 100].

All tests were run in duplicate and analyses of all samples

were run in triplicate and averaged.

Protection of sporamin against hydroxyl

radical-induced calf thymus DNA damage

The hydroxyl radical was generated by Fenton reaction

according to the method of Kohno et al. (1991). The 15

μL reaction mixture containing sporamin B (0, 50, 100,

or 200 μg), 5 μL of calf thymus DNA (1 mg/mL), 18 mM

FeSO

, and 60 mM hydrogen peroxide were incubated at

room temperature for 15 min. Then 2 μL of 1 mM EDTA

was added to stop the reaction. Blank test contained

only calf thymus DNA and the control test contained all

reaction components except sporamin B. The treated DNA

solutions were subjected to agarose electrophoresis and

then stained with ethidium bromide and examined under

UV light.

Determination of the antioxidative activity of

sporamin B peptic hydrolysates

Six mg of sporamin B was dissolved in 1 mL of 0.1

M KCl buffer (pH 2.0). Then 0.1 mL (12 mg) of pepsin

was added at 37°C for 0 and 24 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°C for 5 min to stop enzyme

reaction. The pepsin was heated before sporamin B

hydrolysis for the 0 h reaction. Each of the 60 μL sporamin

B hydrolysates was used for determinations of the DPPH

antioxidative activities by spectrophotometry.

Chromatograms of peptic hydrolysates of

trypsin inhibitor on a Sephadex G-50 column

The unhydrolyzed sporamin B and peptic sporamin B

hydrolysates at 24 h were separated by Sephadex G-50

column chromatography (1 × 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 of which

the absorbance at 280 nm was then determined.

Statistical analysis

Means of triplicates were calculated. Student’s t test

was used for comparison between two treatments. All data

(expressed as percent of control value) were means ± SE.

A difference was considered to be statistically significant

when p < 0.05, p < 0.01 or p < 0.001.

ReSULTS and DISCUSSION

Purification of expressed sporamin B

Sporamin B cDNA clones from sweet potato storage

roots were isolated. Sporamin B was subcloned in a

pQE-30 expression vector in E. coli and sporamin B was

produced with a 6x His-tag at the N-terminus. SDS-PAGE

analysis of crude extracts from transformed E. coli (M15)

showed a high level of a polypeptide with the expected

molecular mass (ca. 26 kDa). This polypeptide was found

as a soluble protein in the supernatant (Figure 1, lane 2),

and was absent in protein extracts obtained from E. coli

transformed with pQE-30 vector (Figure 1, lane 1). The

expressed protein was purified from crude extracts by Ni

2＋

-chelate affinity chromatography, which yielded highly

purified His-tagged sporamin B (Figure 1, lane 3).

measurement of total antioxidant status

This was measured using the total antioxidant status

assay kit (Figure 2). Sporamin B protein exhibited a dose-

dependent total antioxidant activity within the applied

concentrations (0, 5, 10, 20, 40, 60, 80, and 100 μg/mL),

the highest at 100 μg/mL (expressed as 4.21 ± 0.008 mM

Trolox equivalent antioxidative value, TEAC). At 5 μg/

mL, sporamin B displayed the lowest total antioxidant

status (1.95 ± 0.002 mM TEAC).

Rapid screening of antioxidant by dot-blot and

DPPh staining

Antioxidant capacity of expressed sweet potato

sporamin B was eye-detected semi-quantitatively by a

rapid DPPH staining method in TLC. Each diluted sample

was applied as a dot on a TLC layer that was then stained

with DPPH solution (Figure 3). This method is typically

based on the inhibition of the accumulation of oxidized

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136

Botanical Studies, Vol. 48, 2007

products, since the generation of free radicals is inhibited

by the addition of antioxidants and scavenging the free

radicals shifts the end point. The appearance of white

color spot vs a purple background has a potential value

for the indirect evaluation of antioxidant capability of the

expressed sporamin B in the dot blots (Soler-Rivas et al.,

2000). Fast-reacted and strong intensites of white spots

appeared up to the dilutions of 25 mg sporamin B/mL (with

an absolute amount of 75 μg). The reduced glutathione

was used as a positive control.

measurement of reducing power

We investigated the Fe

-Fe

transformation in the

presence of the samples of sporamin B to measure its

reducing capacity. The reducing capacity of a compound

may serve as a significant indicator of its potential

antioxidant activity (Meir et al., 1995). The antioxidant

activity of putative antioxidants have been attributed

to various mechanisms, among them are prevention of

chain initiation, binding of transition metal ion catalysts,

decomposition of peroxides, prevention of continued

hydrogen abstraction, and radical scavenging (Diplock,

Figure 1. SDS-PAGE analysis of purified recombinant sweet po-

tato sporamin B. Crude extracts from E. coli (M15) transformed

with pQE30 (lane 1) or with pQE30-spormin B1 (lane 2) were

analyzed by 15% (w/v) SDS/PAGE with 10 μg protein applied

on each lane, and then the gel was stained with Coomassie blue

G-250. Molecular masses of standard proteins are indicated at

the left of the figure. His-tagged sporamin B was purified by

-chelated affinity chromatography (lane 3). The experiments

were done twice and a representative one is shown.

Figure 2. Total antioxidant activity of recombinant sporamin B

from sweet potato, as measured by the total antioxidant status

assay. Absorbance value represents average of triplicates of dif-

ferent samples analysed.

Figure 3. Dot blot assay of recombinant sporamin B from sweet

potato on a silica sheet stained with a DPPH solution in metha-

nol. Each 3 μl of sporamin B (100, 50, 25, 12.5, 6.25, and 3.125

mg/mL) was applied from left to right in sample row; while each

3 μl glutathione (10, 5, 2.5, 12.5, and 6.25 mg/mL) was applied

from left to right in control row.

Figure 4. Antioxidative activities of recombinant sporamin B

from sweet potato, as measured by the reducing power method.

Each absorbance value represents average of triplicates of dif-

ferent samples analysed. Results represent the means ± SE from

at least 3 separate experiments. *p < 0.05, **p < 0.01 and ***p

< 0.001 (unpaired t test) compared to sporamin unsupplemented

samples.

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HUANG et al. — Sporamin and its peptides with antioxidant activities

137

1997). The reducing power of sporamin B is shown in

Figure 4 with ascorbic acid served as a positive control.

The reducing power activity of sporamin B exhibited a

dose-dependence (significant at p<0.05) within the applied

concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL).

Measure of Fe

＋

-chelating ability

The metal chelating capacity of sporamin B and stan-

dard antioxidants was determined by assessing their ability

to compete with ferrozine for the ferrous ions. The Fe

2＋

-chelating ability of the sporamin B is shown in Figure 5.

EDTA was used as a positive control. The Fe

2＋

-chelat-

ing ability of sporamin B was lower than that of EDTA

and this difference was statistically significant (P<0.05).

Sporamin B at doses of 0.4, 0.8, 1.2, 1.6 and 2 mg/mL

exhibited 65.26, 70.15, 72.41, 74.61 and 76.03% iron

binding capacity, respectively. On the other hand, EDTA

at doses of 0.02, 0.1, 0.2, 0.3 and 0.4 mg/mL had 33.74,

78.16, 95.68, 96.68 and 96.69% chelating activity of iron,

respectively.

Ferric thiocyanate (FTC) method

Low-density lipoprotein (LDL) peroxidation has been

reported to contribute to atherosclerosis development

(Steinbrecher, 1987). Therefore, delay or prevention of

LDL peroxidation is an important function of antioxidants.

Figure 6 shows the time-course curve for the antioxidative

activity of the sporamin B from sweet potato, BHT and

O by the FTC method. The BHT was used as a positive

control, and H

O as a negative control. The results indicate

that sporamin B has antioxidative activity. Sporamin

B may act as a significant LDL peroxidation inhibitor

(P<0.05).

Protection against hydroxyl radical-induced calf

thymus DNA damage by sporamin B

Free radicals could damage macromolecules in cells,

such as DNA, protein, and lipids in membranes (Kohno

et al., 1991). Figure 7 shows that sporamin B protected

against hydroxyl radical-induced calf thymus DNA

damages. The blank contained calf thymus DNA only,

and the control contained all components except sporamin

B. Compared to the blank and control, it was found

that 100 μg sporamin B could protect against hydroxyl

radical induced calf thymus DNA damages during 15-min

reactions.

Determination of the antioxidative activity

of peptic sporamin B hydrolysates and their

peptide distributions

We used pepsin to hydrolyze sporamin B to mimic

the hydrolysis course during digestion in human’s (or

animal’s) stomach. Figure 8 shows the antioxidative

activity of peptic sporamin B hydrolysates and the

antioxidative activity (scavenging activity of DPPH

radicals, percent) of peptic sporamin B hydrolysates

collected at different pepsin hydrolysis times. From

the results, it was found that the antioxidative activity

increased from 19% (0 h) to about 29% (24 h). It was

found that smaller peptides increased with pepsin

hydrolytic time. The purifications of potential peptides

of antioxidative activity need further investigations. We

used synthetic peptides to measure antioxidative activity.

Synthetic peptides were designed by simulating pepsin

cutting sites of sporamin A and B gene products from

sweet potato (pH>2, http://expasy.nhri.org.tw/tools/

peptidecutter/). New peptides (Table 1) for antioxidative

activity, that is, SNIP, VRL, SYCQ, GTEKC, RF,

VKAGE, AH, KIEL were synthesized according to

simulation. IC

values of individual peptides were 8.36,

4.23, 0.206, 0.0884, 9.72, 14.9, 13.8 and 24.9 mM,

Figure 5. Antioxidative activities of recombinant sporamin B

from sweet potato, as measured by the Fe

2＋

-chelating ability

method. Each absorbance value represents average of triplicates

of different samples analysed. Results represent the means ± SE

from at least 3 separate experiments. *p < 0.05, **p < 0.01 and

***p < 0.001 (unpaired t test) compared to sporamin unsupple-

mented samples.

Figure 6. Inhibition of linoleic acid peroxidation by recombinant

sporamin B from sweet potato, as measured by the FTC method.

Each absorbance value represents average of triplicates of differ-

ent samples analysed.

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138

Botanical Studies, Vol. 48, 2007

respectively, when scavenging activity of DPPH radicals

(%) was measured. Cysteine residues with free-SH in

whey proteins (Allen and Wrieden, 1982; Tong et al.,

2000) were reported to have antioxidant activities. Our

results further indicate that cysteine residues (GTEKC

or SYCQ) in sweet potato sporamin B contribute to the

antiradical activities. The synthetic peptide, GTEKC, has

the highest antioxidant activity (IC

is 0.0884 mM) as

good as reduced glutathione (IC

is 0.0748 mM). Another

synthetic peptide, SYCQ, also has a good antioxidant

activity (IC

is 0.206 mM). These results demonstrated

that simulated synthetic peptides from peptic sporamin B

hydrolysates exhibited antioxidative activity.

In conclusion, the results from in vitro experiments,

including total antioxidant status assay (Figure 2), DPPH

staining (Figure 3), reducing power method (Figure 4),

2＋

-chelating ability (Figure 5), FTC method (Figure 6),

and hydroxyl radical-induced calf thymus DNA damage

(Figure 7), demonstrated that sporamin B in sweet potato

may have significant antioxidant activities. Sporamin B

may contribute significantly to change the redox states and

as a potent antioxidant against both hydroxyl and peroxyl

radicals when people consume sweet potato. The ex vivo

or in vivo antioxidant activity of sporamin B should be

performed in near further.

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Table 1. Sporamin B peptides with antioxidant activity.

Peptide

Scavenging activity of DPPH radicals (%),

(mM)

GSH (control)

0.0748

SNIP

8.36

VRL

4.23

SYCQ

0.206

GTEKC

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9.72

VKAGE

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KIEL

24.9

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