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LWT 40 (2007) 955–962

Antioxidant activities of five different mulberry cultivars in Korea

Song-Hwan Baea, Hyung-Joo Suhb,

aDepartment of Food and Biotechnology, Anseong-si 456-749, KoreabDepartment of Food and Nutrition, College of Health Sciences, Korea University, 1 Jeongneung-dong, Sungbuk-ku, Seoul 136-703, Korea

Received 20 February 2006; received in revised form 2 June 2006; accepted 29 June 2006

Abstract

Five major mulberry [Pachungsipyung (M-1), Whazosipmunja (M-2), Suwonnosang (M-3), Jasan (M-4) and Mocksang (M-5)]cultivated in Korea were assessed for their polyphenolic composition using spectrophotometric methodology, and tested for antioxidant

potential by some different assays. The total polyphenol (TP) was found from 2235 to 2570 mg/g gallic acid equivalents, total anthocyanin

(TA) content to vary from 1229 to 2057 mg/g, coloured anthocyanins (CA) from 126 to 190 mg/g, and total flavanol (TF) from 16.4 to

65.4mg/g catechin equivalents except Mocksang (M-5). The ethanolic extract from mulberry fruit shows a rapid and concentration-

dependent increase of antioxidant activity. Especially, the antioxidant activities of M-2 and M-4 are higher than those of the others in a

hemoglobin-induced linoleic acid system. The reducing power compared with BHT was observed to high value in M-2, M-3 and M-4

extracts. Effectiveness in reducing powers was in a descending order of M-44M-24M-34M-14M-1. The DPPH-scavenging ability of

the ethanolic extract from mulberry fruit was 60.0% at 200 and 212 mg of M-2 and M-4, respectively. M-2 and M-4 also showed sharply

increase of hydroxyl scavenging ability with concentration of the extracts. IC 50 values in scavenging abilities on hydroxyl radicals were

30mg and in a descending order of M-54M-34M-14M-44M-2. Superoxide anion-sacavenging activities of M-2, M-3 and M-4

showed 17.1, 14.5 and 14.8 SOD unit/mg, respectively.

r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Mulberry; Morus alba L.; Anthocyanin; Cyanidin-3-glucoside; Cyanidin-3-rutinoside; Antioxidant; Radical scavenging activity

1. Introduction

There is great interest in determining the role of

phytonutrients in promoting improved health and in

reducing cancer, cardiovascular disease, and the effects of

aging. It is widely believed that antioxidant phytonutrients

can inhibit the propagation of free radical reactions that

may ultimately lead to the development of diseases,

especially those which are aging related. Analysis in several

laboratories shows that many fruits and vegetables havestrong antioxidant capacities, and that this capacity is due

primarily to nonvitamin C phytochemicals (Wang, Cao, &

Prior, 1997; Prior et al., 1998).

Flavonoids are a large group of natural phenolic

compounds, consisting mainly of flavonols, flavanols and

anthocyanidins. Among these flavonoids, the water-soluble

glycosides and acylglycosides of anthocyanins are an

important group of natural antioxidants (van Acker

et al., 1996; Wang, Cao, & Prior, 1997; Tsuda, Horio,

Kitoh, & Osawa, 1999). While in vitro antioxidant

activities of anthocyanins are superior to vitamin E (Wang,

Nair, Strasburg, Booren, & Gray, 1999b), little is known

regarding comparable in vivo capacity or the bioavail-

ability of these compounds. Anthocyanin variations lend to

a complexity that has made these compounds, as a group,

difficult to study with regard to bioavailability.

Anthocyanins are becoming increasingly important notonly as food colorants, but also as antioxidants. Antho-

cyanins are reported to have some therapeutic benefits

including vasoprotective and antiinflammatory properties

(Lietti, Cristoni, & Picci, 1976), anticancer and chemopro-

tective properties (Karaivanova, Drenska, & Ovcharov,

1990), as well as antineoplastic properties (Kamei et al.,

1995). Anthocyanins are therefore, considered to contri-

bute significantly to the beneficial effects of consuming

fruits and vegetables (Wang, Cao, & Prior, 1997). There is

a rising demand for natural sources of food colorants with

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0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.lwt.2006.06.007

Corresponding author. Tel.: +82 2 9402853; fax: +82 2 9417825.

E-mail address: [email protected] (H.-J. Suh).

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nutraceutical benefits (Boyd, 2000) and alternative sources

of natural anthocyanins are becoming increasingly im-

portant.

Mulberry fruit is a traditional Chinese edible fruit that is

used effectively in folk medicines to treat fever, protect liver

from damage, strengthen the joints, facilitate discharge of

urine and lower blood pressure. Recently, it has gained animportant position in the local soft drink market, although

its biological and pharmacological effects are still poorly

defined. Important constituents of mulberry fruits are the

anthocyanins (Gerasopoulos & Stavorulakis, 1997).

Particularly for the mulberry varieties cultivated in

Korea, the anthocyanin composition and contents have

not been examined in detail. The investigation presented

herein was undertaken for anthocyanin composition and

content and further comparison of the antioxidant capacity

of five major mulberry cultivars.

2. Materials and methods

2.1. Preparation of mulberry fruit extract

Mulberry fruits (Morus alba L.) of the cultivars,

Pachungsipyung (M-1), Whazosipmunja (M-2), Suwonno-

sang (M-3), Jasan (M-4) and Mocksang (M-5), were

obtained from National Institute of Agricultural Science

and Technology, Suwon, Korea. Fruits were selected

according to the uniformity of shape and colour. The

fruits were then stored at 20 1C for further studies.

According to Lee and Wicker (1991), mulberry fruit

(50 g) was homogenized and extracted in 100 ml of 70%

ethanol for 4 h at room temperature. The extract wasfiltered through Whatman No. 41 paper and rinsed with

50 ml of ethanol. Extraction of the residue was repeated

using the same conditions. The two filtrates of ethanol were

combined and evaporated under vacuum at 40 1C to obtain

dry extract. The extracts were were placed in a plastic

bottle and then stored at 20 1C until used.

2.2. Determination of total polyphenols (TP)

Total polyphenol (TP) content was determined using the

Folin-Ciocalteu method (Waterman & Mole, 1994), adapted

to a microscale. In a 1.5-ml Eppendorf tube, 0.79 ml distilled

water, 0.01ml mulberry extract appropriately diluted, and

0.05 ml Folin-Ciocalteu reagent was added and mixed. After

exactly 1 min, 0.15 ml of sodium carbonate (20 g/100 ml) was

added, and the mixture was mixed and allowed to stand at

room temperature in obscurity, for 120 min. The absorbance

was read at 750 nm, and the total polyphenol concentration

was calculated from a calibration curve (r2 ¼ 0:9990), using

gallic acid as standard (50–800 mg/l).

2.3. Determination of total and coloured anthocyanins

Measurements were performed using well-established

spectrophotometric methodology (Somers & Evans, 1977;

Zoecklein, Fugelsang, Gump, & Nury, 1990). Analytically,

mulberry extract was placed in a 0.2-cm path length quartz

cuvette, and the absorbance was measured at 520 nm

(A520). Following this, 0.02 ml of a sodium metabisulphite

(20 g/100 ml) solution was added, the sample was mixed

well, and after 1 min the absorbance was read at 520 nm

ASO2

520 . A 12% ethanolic solution was used as blank. Allmeasurements were corrected to a 1.0-cm path length.

Further, extract (0.02 ml) was mixed with 0.98 ml of 1 mol/l

of HCl solution in a 1.5-ml eppendorf tube, mixed, and

allowed to stand for 180 min at room temperature. The

absorbance was read at 520 nm (AHCI520 ), using a 1.0-cm path

length cuvette. For the blank, 0.02 ml of a 12% ethanolic

solution was used instead of extract. The concentration of

total anthocyanins (TA) and coloured (ionized) anthocya-

nins (CA) was expressed as malvidin-3-glucoside and

calculated as follows:

TA ðmg=gÞ ¼ 20 ½AHCI520 ð5=3Þ ASO2

520

final extract volume ðmlÞ

=homogenate weight ðgÞ;

CA ðmg=gÞ ¼ 20 ½A520 ASO2

520

final extract volume ðmlÞ

=homogenate weight ðgÞ:

2.4. Determination of total flavanols (TF)

The total flavanol (TF) content was estimated using

the p-dimethylaminocinnamaldehyde (DMACA) method

(Li, Tanner, & Larkin, 1996; McMorrough, Madigan, &Smyth, 1996). This method has a great advantage over the

widely used vanillin assay, since there is no interference by

anthocyanins. Further, it provides higher sensitivity and

specificity (Li, Tanner, & Larkin, 1996). Extract (0.2 ml),

diluted 1:100 with MeOH, was introduced into a 1.5-ml

Eppendorf tube and added 1 ml DMACA solution (0.1%

in 1 mol/l of HCl in MeOH). The mixture was vortexed and

allowed to react at room temperature for 10 min. Follow-

ing this, the absorbance at 640 nm was read against blank

prepared similarly without DMACA. The concentration of

TF was estimated from a calibration curve, constructed by

plotting known solutions of catechin (1–16 mg/l) against

A640 (r2 ¼ 0:9987).

2.5. Quantitative analysis of anthocyanins

Quantitative analysis of anthocyanins for the mulberry

extracts was performed by HPLC (Shimadzu LC-6A)

equipped with a diode array detector (Shimadzu SPD-

M10Avp) on a Waters C18 (4.6 250 mm) column at 40 1C

with a flow rate of 1 ml/min, monitoring at 530 nm (Ando

et al., 2000). The solvent system employed was a linear

gradient elution for 40 min from 20% to 85% solvent B

(1.5% H3PO4, 20% HOAc, 25% MeCN in H2O) in solvent

A (1.5% H3PO4 in H2O).

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2.6. Antioxidant activity in a hemoglobin-induced linoleic

acid system

The antioxidant activity of mulberry extract was

determined by a modified photometry assay (Kuo, Yeh,

& Pan, 1999). Reaction mixtures (200 ml) containing 10ml

extracts (10–400mg), 1 mmol/l of linoleic acid emulsion,40 mmol/l of phosphate buffer (pH 6.5), and 0.0016%

hemoglobin, were incubated at 37 1C for 45 min. After the

incubation, 2.5 ml of 0.6% HCl in ethanol was added to

stop the lipid peroxidation. The amount of peroxide value

was measured in triplicate using the thiocyanate method by

reading the absorbance at 480 nm after colouring with

100ml of 0.02 mol/l of FeCl2 and 50ml of ammonium

thiocyanate (30 g/100 ml). a-Tocopherol was used as

positive control.

2.7. Reducing power

The mulberry extract (20–800 mg) were mixed with 200 ml

of 0.2 mol/l of phosphate buffer, pH 6.5 and 200ml of

1 g/100 ml of potassium ferricyanide, and, then, incubated

at 50 1C for 20 min. Two hundred and fifty microlitres of

trichloroacetic acid (10 g/100 ml) was added to the mixture

and centrifuged at 3000g for 10 min at room temperature.

The resulting supernatant was taken and mixed with 500 ml

of H2O and 100 ml of ferric chloride (0.1 g/100 ml), and,

then, incubated at 37 1C for 10 min. The absorbance at

700nm was measured. Increased absorbance indicated

increased reducing power (Oyaizu, 1986).

2.8. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) scavengingeffect

Reactions were performed in 1.25ml of methanol

containing 0.5 mmol/l freshly made DPPH and 10–1200mg

of extract. Reaction mixtures were incubated at 37 1C for

30min, and the absorbance at 517 nm was measured

(Schimada, Fujikawa, Yahara, & Nakamura, 1992).

2.9. Scavenging effect on hydroxyl radicals

Reaction mixtures containing 2–40 mg extract, 0.02 mol/l

phosphate buffer (pH 7.4), 2 mmol/l of H2

O2

, 0.05 mmol/l

of ferric chloride, 0.05 mmol/l of ascorbate, 6 mmol/l of

deoxyribose, and 0.05 mmol/l of EDTA were incubated at

37 1C for 30 min. The degree of deoxyribose oxidation was

analysed as thiobarbituric acid-reactive material (Halliwell,

Gutteridge, & Arurma, 1987).

2.10. Superoxide anion radical-scavenging activities

0.1 ml of aqueous superoxide dismutase (SOD) standard

solutions (5, 10, 25, 50, 100 units/ml) and various amount

of the extract were separately added to a 1.0 ml mixture of

0.4 mmol/l xanthine and 0.24 mmol/l nitro blue tetrazolium

chloride (NBT) in 0.1 mol/l phosphate buffer (pH 8.0).

A 1.0 ml of xanthine oxidase (0.049 units/ml), diluted in

0.1 mol/l phosphate buffer (pH 8.0), was added and the

mixture was incubated in a water-bath at 37 1C for 20 min.

The reaction was terminated by adding 2.0 ml of an

aqueous solution of 69 mmol/l sodium dodecylsulphate

(SDS), and the absorbance of NBT was measured at

560 nm. The superoxide radical-scavenging activity of thedry weight was calculated as SOD equivalents (units/mg)

from the SOD standard curve and IC50 value.

In all cases analyses were performed in triplicate, unless

elsewhere specified, and values averaged. The standard

deviation (SD) was also calculated. All data were analysed

by one-way analysis of variance and Duncan’s multiple

range tests using the Statistical Analysis System (SAS).

Results were considered significant at P o0.05.

3. Results

3.1. Polyphenol composition of mulberry ethanol extract

We examined the polyphenol composition of the ethanol

extracts from various mulberry samples as shown in

Table 1. Using 60% ethanol as extractant, the yields

were in a descending order of M-1 (19.9%)4M-4

(18.8%)4M-2 (14.7%)4M-3 (14.5)4M-5 (9.6%) (Data

was not shown). The total phenol contents in M-1, M-2,

M-3 and M-4 were found from 2235 to 2570, respectively,

as mg gallic acid/g dry sample, but M-5 (Mocksang) was

in very low content compared with others. The total

anthocyanin content in mulberry extracts (M-1–M-4),

examined by spectrophotometric assay was varied from

1230 to 2057 as mg cyanidin-3-glucoside/g dry sampleexcept M-5. The contents of well-known cyanidin-3-gluco-

side and cyanidin-3-rutinoside of anthocyanins were

analysed by HPLC system and the results showed from

106 to 185 mg/100 g of dry weight except for M-5.

Total flavonoid content varied from 5.6 to 65.4 mg/g of

dry weight and the exception was M-4 (Jasan) variety,

where we observed twice the amount of the total flavonoid,

65.4mg/g, compared to other cultivars.

3.2. Antioxidant activity and reducing power of mulberry

ethanol extract

The antioxidant activities of ethanol extract of mulberry

cultivars were determined by the method of Kuo, Yeh, and

Pan (1999) using the hemoglobin-induced linoleic acid

system. This method could evaluate the results with only

1 h for oxidation time. Generally antioxidant assays with

linoleic acid need more auto-oxidation for 5–6 days. The

ethanolic extract from mulberry fruit showed moderate

inhibitory ability on lipid oxidation (23.7–47.6%) at 76 mg

and high inhibitory ability (52.7–73.3%) at 255 mg (Fig. 1).

However, the ethanolic extract from mulberry fruit shows a

rapid and concentration-dependent increase of antioxidant

activity. Especially, the antioxidant activities of M-2 and

M-4 are higher than those of the others. However, the

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inhibitory abilities were 69.2% at 62mg and 79.1% at

100mm for a-tocopherol.

The antioxidant activities of natural components may

have a reciprocal correlation with their reducing powers.

The reducing powers of the ethanol extracts of mulberry

samples and BHT were determined in this study (Fig. 2).

Reducing powers of the ethanolic extracts increased in

three patterns with increased concentrations, i.e., a fast

increase to 0.75 at 122.1 and 122.5 mg of M-2 and M-4,

respectively, intermediate increase to 0.75 at 160.3mg of

M-3 and 230.2 mg of M-1 and a slow increase to 0.75 at

456.7 mg of M-5 (Fig. 2). However, BHA showed an

excellent reducing power of 0.94 at 28mg. The reducing

power compared with BHT was observed to high value in

M-2, M-3 and M-4 extracts. The reducing power in M-5

showed the lowest level among the extracts. The high value

of reducing power indicated some compounds in mulberryextract were both electron donors could react with free

radicals to convert them into more stable products and to

terminate radical chain reactions.

3.3. Radical-scavenging activity of mulberry extracts

It is well known that antioxidants can seize the free-

radical chain of oxidation and form stable free radicals,

which would not initiate or propagate further oxidation.

DPPH has been used extensively as free radical to evaluate

reducing substances. The scavenging ability of the etha-

nolic extract from mulberry fruit was 60.0% at 200 mg and

212mg of M-2 and M-4, respectively, whereas the extracts

scavenged 60.0% of DPPH radicals at 363 and 683 mg of

M-1 and M-5, respectively (Fig. 3). At 100 mg, a-tocopherol

showed excellent scavenging abilities of 84.0%.

The mulberry fruit extracts were also evaluated for their

ability to scavenge hydroxy radical and/or to chelate iron

using the deoxyribose degradation assay. As shown in

Fig. 4, all the samples found to have the ability to scavenge

hydroxyl radicals at concentrations of 10 ml, with a similar

profile. Especially, M-2 and M-4 showed sharply increase

of scavenging ability with concentration of the extracts.

Results showed that mulberry extracts inhibited the

formation of hydroxyl radicals. The deoxyribose assay

was employed to verify whether the samples were able to

protect this carbohydrate from oxidation provoked by

OH. Our results revealed that the extract and fractions

significantly inhibited deoxyribose degradation. Com-

pounds with the most hydroxyl groups apparently exert

the greatest antioxidant activity in aqueous systems. The

antioxidant effect demonstrated by these phenolic com-

pounds in the deoxyribose assay may also be due to their

capacity to chelate transition metals.

In the xanthine/xanthine oxidase system of superoxide

anion-scavenging activity the mulberry extracts were

effective in inhibition of NBT reduction by O2. Five

samples showed superoxide anion-scavenging activity,

which was demonstrated with the respective data (SOD

unit/mg): M-1 (10.7), M-2 (17.1), M-3 (14.5), M-4 (14.8)

and M-5 (8.4). Moreover, this effect was more evident with

M-2 and M-5 was less effective than the samples. At the

ARTICLE IN PRESS

Mulberry fruit extract (µg)

0 100 200 300 400

I n h i b i t o r y a b i l i t y ( % )

0

20

40

60

80

100

Fig. 1. Antioxidant activities of mulberry fruit extracts against linoleic

acid peroxidation induced by hemoglobin. Each value is expressed as

mean7standard deviation (n ¼ 3). Values with different superscripts are

significantly different, P o 0.05. J: Pachungsipyung (M-1), &: Whazo-

sipmunja (M-2), &: Suwonnosang (M-3), &: Jasan (M-4), &: Mocksang

(M-5), : positive control (a-tocopherol).

Table 1

Polyphenol composition of mulberry extract

Mulberry fruit extract TP (mg/g) TA (mg/g) CA (mg/g) Cyanidin-3-glucoside (mg/g) Cyanidin-3-rutinoside (mg/g) TF (mg/g)

M-1 2235.7778.2b 1229.3721.4d 134.2710.4b 847.1711.2c 259.3715.3d 16.471.8c

M-2 2570.4757.1a 2057.3718.5a 190.5711.2a 1364.9710.4a 486.7718.5a 34.572.2b

M-3 2494.6734.9a 1599.3721.0c 126.477.4b 1091.6719.6b 347.7712.4c 37.272.3b

M-4 2532.5735.1a 1664.1711.9b 134.173.0b 1077.3711.2b 420.5720.1b 65.474.0a

M-5 959.9723.1c 137.377.7e 10.270.8c 93.2 77.4d 30.673.8e 5.670.2d

Values (as dry basis) represent means of triplicate determination ( n ¼ 3)7SD. Values in a column with different superscripts are significantly different,

P o 0.05.

Total phenols (TP) expressed as gallic acid equivalents.

Total anthocyanins (TA) expressed as malvidin-3-glucoside equivalents.

Coloured (ionized) anthocyanins (CA) as expressed as malvidin-3-glucoside equivalents.

Total flavanols (TF) expressed as catechin equivalents. M-1: Pachungsipyung, M-2: Whazosipmunja, M-3: Suwonnosang, M-4: Jasan, M-5: Mocksang.

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IC50 value level, M-2 was more potent; the M-3 and M-4

exhibited the same potency (Table 2).

3.4. IC 50 values in antioxidant properties

The antioxidant properties assayed herein were summar-

ized in Table 3 except for scavenging ability on superoxide

radicals, and the results were normalized by computing the

concentration of ethanolic extracts at which the effect was

50% or the absorbance was 0.5 and expressed as IC50

values (mg extract) for comparison. Effectiveness in

antioxidant properties inversely correlated with IC50 value.

With regard to IC50 values in inhibitory ability on lipid

oxidation, the extracts of M-2 and M-4 were better than

those of the others. Effectiveness in reducing powers was in

a descending order of M-44M-24M-34M-14M-1.

Scavenging abilities on DPPH radicals were excellent for

ethanolic extract of M-2 and IC50

values of M-2 (138.4 mg)

was significantly lower than those of the others (P o0.05).

IC50 values in scavenging abilities on hydroxyl radicals

were 30mg and in a descending order of M-54M-34

M-14M-44M-2. Scavenging abilities on hydroxyl radi-

cals were also excellent for the extract of M-2 and IC50

values of M-2 (14.7 mg) was also significantly lower than

those of the others (P o0.05).

4. Discussion

Mulberries are a good source of sugars, acids and

anthocyanin pigments, which are important constituents of

juices, beverages and wines. Recently, mulberry fruits have

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0 200 400 600 800 1000 1200 1400

S c a v e n g i n g

a b i l i t y o n D P P H

r a d i c a l s (

% o

f i n h i b i t i o n )

0

20

40

60

80

100

Fig. 3. Scavenging effects of mulberry fruit extracts on DPPH radicals.

Each value is expressed as mean7standard deviation (n ¼ 3). Values with

different superscripts are significantly different, P o 0.05. J: Pachungsi-

pyung (M-1), &: Whazosipmunja (M-2), &: Suwonnosang (M-3), &:

Jasan (M-4), & Mocksang (M-5), : positive control (a-tocopherol).

Mulberry fruit extract (mg)

0 10 20 30 40

S c a v e n i n g a b i l i t y

o f h y d r o l x y

r a d i c a l ( % o f i n

h i b i t i o n )

15

30

45

60

75

90

Fig. 4. Scavenging effects of mulberry extracts on hydroxyl radicals.

DMSO (10mg/ml) was used as a positive control. Each value is expressed

as mean7standard deviation (n ¼ 3). Values with different superscriptsare significantly different, P o 0.05. J: Pachungsipyung (M-1), &:

Whazosipmunja (M-2), &: Suwonnosang (M-3), &: Jasan (M-4), &:

Mocksang (M-5), : positive control (DMSO).


0 200 400 600 800

A b s o r b a n c e a t 7 0 0 n m

0.0

0.5

1.0

1.5

2.0

Fig. 2. Reducing powers of mulberry fruit extracts. BHT (1.0mg/ml) was

used as a positive control. Each value is expressed as mean7standard

deviation (n ¼ 3). Values with different superscripts are significantlydifferent, P o 0.05. J: Pachungsipyung (M-1), &: Whazosipmunja (M-2),

&: Suwonnosang (M-3), &: Jasan (M-4), &: Mocksang (M-5), :

positive control (BHT).

Table 2

Superoxide anion radical scavenging activities of mulberry fruit extracts

Mulberry fruit extract Unit/mg solid IC50 (mg)

M-1 10.771.2c 0.09470.011b

M-2 17.170.7a 0.05970.002a

M-3 14.570.4d 0.06970.002c

M-4 14.870.3b 0.06870.001c

M-5 8.470.2d 0.11970.003a

Each value is expressed as mean7standard deviation (n ¼ 3). Means with

different letters within a row at a specific IC50 are significantly different

(P o0.05).

M-1: Pachungsipyung, M-2: Whazosipmunja, M-3: Suwonnosang, M-4:

Jasan, M-5: Mocksang.

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been reported to have several biological actions such as

antidiabetic (Asano et al., 2001), antioxidantive (Kim,

Park, & Lee, 1998), antiinflammtory (Kim, Park, & Lee,

1998) and antihyperlipidemic (Kim, Kim, Ryu, Lee, &

Moon, 2001) activities. These biological activities were due

to polyphenol components including anthocyanins in

mulberry fruits. Anthocyanin pigments are of prominent

importance in mulberry fruits because of their dual role;

first they constitute an integral part of the sensory

attributes because their levels, various forms and deriva-

tives pertain directly to the colouration of the final product;

second, they have been claimed to possess diverse

biological properties and therefore are considered as

secondary metabolites with potential nutritional value.

Table 1 shows the contents of phenolic compounds in

five mulberry cultivars. Similar levels of TP were found in

the cultivars of M-2 and M-4. Especially, M-2 had a highercontent of TA (2057.3 mg/g) compared to that of M-4

(1664.1 mg/g). Kim, Bang, Lee, Seuk, and Sung (1999)

showed that several mulberries and a wild variety

contained relatively high levels of total anthocyanin

(2.45–3.14 mg/g). In contrast, Park, Jung, and Ko (1997)

reported that total anthocyanin contents of matured

mulberry ranged form 0.19 to 3.29 mg/g. Thus anthocyanin

contents of mulberry fruits can be somewhat variable,

depending on cultivar and maturation. M-2 cultivar also

showed high level of cyanidin-3-glucoside and cyanidin-3-

rutinoside compared to the others. Lee and Wicker (1991)

reported that the two major anthocyanin contents (cyani-

din-3-glucoside and cyanidin-3-rutinoside) varied from

30.9 to 924.9 mg/100 g dried weight of several mulberries

cultivar. On the other hand, two major anthocyanin

contents of our samples are in contrast regular with their

result generally.

Cyanidins are considered the widest spread anthocyanin

in the plant kingdom. They are largely distributed in the

human diet through crops, beans, fruits, vegetables and red

wines, suggesting that we daily ingest significant amounts

of these compounds from plant-based diets. Cyanidin-3-

glycoside, also known as kuromanin, is probably the most

notorious and investigated among cyanidin-glycosides.

One of the most important dietary sources of cyanidin-3-

glycoside is surely represented by pigmented oranges,

named Moro, Sanguinello and Tarocco, typically growing

in Sicily (Italy) (Amorini et al., 2001) as well as in Florida

(USA) (Lee, 2002).

The ethanolic extract from mulberry fruit showed

moderate inhibitory ability on lipid oxidation (23.7–47.6%)

at 76mg and high inhibitory ability (52.7–73.3%) at 255 mg

(Fig. 1). The antioxidant activities of M-2 and M-4 are higher

than those of the others in hemoglobin-induced linoleic acid

system (Fig. 1). The reducing power compared with BHT

was observed to high value in M-2, M-3 and M-4 extracts

(Fig. 2). IC50 values in reducing power of M-2 and M-4 were

46.9 and 54.3mg, respectively (Table 3). M-2 and M-4

cultivar showed a high level of TP and cyanidin-3-glucoside.

M-4 cultivar showed higher level of TF than the others.

Anthocyanins are considered very good antioxidant

agents, their high activity being attributed to their peculiarstructure, namely the oxonium ion in the C ring (van Acker

et al., 1996). The antioxidant functions of anthocyanins

have been ascribed to the aglycone moiety, and this was

demonstrated for cyanidin and some of its glycosides

(Wang et al., 1999a), but the number of sugar residues at

the 3-position (Wang et al., 1999a), the oxidation state of

the C ring (Lapidot, Harel, Akiri, Granit, & Kanner, 1999),

the hydroxylation and methylation pattern (Wang, Cao, &

Prior, 1997; Espin, Soler-Rivas, Witchers, & Garcia-

Viguera, 2000), as well as the acylation by phenolic acids

(Degenhardt, Knapp, & Winterhalter, 2000) are considered

crucial factors for the expression of antioxidant effects. In

some small fruits (Moyer, Hummer, Finn, Frei, &

Wrolstad, 2002), raspberries (Mullen et al., 2002), sweet

potatoes (Oki, Masuda, Furuta, Nishiba, & Suda, 2002),

and caneberries (Wada & Ou, 2002), the antioxidant

capacity has been correlated to a significant degree with

anthocyanin content, indicating that anthocyanins may

govern to a certain extent the antioxidant capacity of

several plant tissues.

M-4 cultivar, containing slightly lower level of cyanid-

ing-3-glucoside than M-2, was similar antioxidant activ-

ities. This was due to high level of TF content. In grapes,

fractions containing cyanidin-3-glucoside were the least

efficient in inhibiting LDL oxidation, whereas the highest

ARTICLE IN PRESS

Table 3

IC50 values of ethanolic extracts from mulberry fruit in antioxidant properties

Mulberry fruit extract M-1 M-2 M-3 M-4 M-5

Inhibitory ability on lipid oxidation (mg) 226.473.6ab 138.475.8c 212.378.2b 146.476.2c 229.373.9a

Reducing power (mg) 111.7710.2b 46.973.1d 54.374.7c 46.372.1d 192.176.8a

Scavenging ability on DPPH radicals (mg) 436.3712.7b 225.9715.9d 412.9718.1b 305.4714.5c 537.679.8a

Scavenging ability on hydroxyl radicals (mg) 21.970.4c 14.770.5d 25.871.2b 20.370.8c 29.571.1a

IC50 value: the effective concentration at which lipid oxidation was inhibited by 50%; the absorbance was 0.5 for reducing power; 1,1-diphenyl-2-

picrylhydrazyl (DPPH) radicals and hydroxyl radicals were scavenged by 50%. IC 50 value was obtained by interpolation from regression analysis.

Each value is expressed as mean7standard deviation (n ¼ 3). Means with different letters within a row at a specific IC50 are significantly different

(P o0.05).

M-1: Pachungsipyung, M-2: Whazosipmunja, M-3: Suwonnosang, M-4: Jasan, M-5: Mocksang.

S.-H. Bae, H.-J. Suh / LWT 40 (2007) 955–962960

8/13/2019 1-s2.0-S0023643806001873-main


activity was seen with fractions containing flavanols

(Teissedre, Frankel, Waterhouse, Peleg, & German, 1996).

It has been revealed that active oxygen species such as

OH and O2 — , are thought to be agents that cause

oxidative damage, and much attention has been focused

on active oxygen scavenging agents such as a-tocopherol

and natural phenolics like flavonoids and tannins inpreventing cell damage. Fig. 3, 4 and Table 2 show the

radical scavenging activity of the mulberry fruit extract.

Two cultivar (M-2 and M-4) had a similar radical

scavenging activity, however, M-2 cultivar showed

a slightly higher scavenging ability than M-4 (Table 3).

M-2 and M-4 showed a high level of TP contents.

Especially, M-2 showed a higher level of TA content than

that of M-4, but M-4 showed a higher level of TF content

than that of M-2.

Tsuda, Shiga, Ohshima, Kawakishi, and Osawa (1996)

reported that three anthocyanins, Pelargonidin-3-gluco-

side, Cyanidin-3-glucoside and Delphinidin-glucoside, had

the same lC50 values for OH scavenging activity (not

significant). In general, the flavylium cation form of the

anthocyanins is stable in the acidic condition, but the

structure changes in neutral and alkaline conditions and

breaks down (Brouillard, 1988). When anthocyanins

scavenge active oxygen or lipid hydroperoxide radicals,

the structure also would be broken, and the radicals may be

scavenged by the reaction products and show antioxidative

activity.

In this study, the antioxidant activities of mulberry

ethanol extract varied with the five test models. Our results

revealed that M-2 and M-4 (rich in anthocyanin com-

pounds) showed strong antioxidant activities in all fiveassays. On the other hand M-5 which the phenolic

compounds is low expressed most low antioxidant activ-

ities.

Although additional data are needed to better under-

stand this antioxidant activity, the present study confirms

that ethanol extract of mulberry represent a significant

source of phenolic (especially anthocyanic) antioxidants.

Mulberry fruits have recently been received much

attention as potential sources of functional foods due to

several biological and pharmacological effects of antho-

cyanins (Tsuda, Horio, Kitoh, & Osawa, 1999) and

flavonoids (Havsteen, 1983; Solimani, 1997; ). We think

that research about the isolation and identification of

phenolic compounds existed mulberry fruits (especially

M-2 and M-4) and determination of their biological

activity in vivo must be processed continuously.

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