Botanical Studies (2006) 47: 231-237.

*

Corresponding author: E-mail:

wchou@tmu.edu.tw

; Fax:

886 (2) 2378-0134; Tel: 886 (2) 3736-1661 ext. 6160.

INTRODUCTION

Ya m ( Dioscorea species) is a member of the

monocotyledonous family Dioscoreaceae and is a staple

food in West Africa, Southeast Asia, and the Caribbean

(Akoruda, 1984). Yam is recognized as an herbal plant

since the tuber dried slices have freqently been used as

Chinese herbal medicines. The tuber storage proteins

of yam, dioscorin, exhibited carbonic anhydrase,

trypsin inhibitor activities (Hou et al., 1999a; Hou et

al., 2000), and both dehydroascorbate reductase and

monodehydroascorbate reductase activities (Hou et al.,

1999b). Chang et al. (2004) reported that Chinese yam (D.

alata cv. Tainong No. 2) feeding had antioxidant effects in

hyperhomocysteinemia rats. However, the component in

the freeze-dried powder of Chinese yam was responsible

for this activity was not clear. We reported previously that

the storage proteins (dioscorin) from Japanese yam (D.

batatas) exhibited scavenging activities against DPPH and

hydroxyl radicals (Hou et al., 2001). We also reported

that the crude and purified mucilages from Japanese yam

exhibited antioxidant activities (Lee et al., 2003). We

discovered this by testing anti-DPPH and anti-hydroxyl

radicals, reducing powers, and anti-lipid peroxidation

activities (Hou et al., 2002).

Liao et al. (2004) used near infrared Fourier transform

Raman spectroscopy to analyze the secondary structure of

dioscorin from three yam species [D. Japonica (Japanese

yam), D. alata L., and D. alata L. var. purpurea].

Although the dioscorin from three species has a similar

molecular mass, the amino acid compositions and the

secondary structure of dioscorin from D. alata L., and

D. alata L. var. purpurea were apparently different from

those of D. Japonica (Japanese yam). Dioscorin from

D. Japonica had lower contents of Cys, Ile, Lys, and

total essential amino acids than did the other two yam

species. The dioscorin from D. alata L., D. alata L. var.

purpurea, an d D. Japonica exhibited structures mostly

of the

α

-helix, antiparallel

β

-sheet, mixed

α

-helix, and

antiparallel

β

-sheet types, respectively. These differences

among yam species might result in different biological

activities. In this work we used dioscorin from two

different yam species, Chinese yam (D. alata cv. Tainong

No. 1, TN1) and Japanese yam (D. batatas Decne,

imported from Japan, JP), and compared their antioxidant

activity, using DPPH radical and hydroxyl radical

scavenging activity assay, reducing power test, anti-lipid

peroxidation test, DNA damage protection, and inhibition

of dihydrorhodamine 123 oxidation by peroxynitrite.

The results showed that dioscorins from two species

exhibited different scavenging activities even with heating

100oC for 5 min against DPPH and hydroxyl radicals.

The peptic hydrolysates of TN1 dioscorin were separated

BIOCHEMISTRY

Comparisons of in vitro antioxidant activities of storage

proteins in tuber of two Dioscorea species

Yuh-Hwa LIU

1

, Hong-Jen LIANG

2

, Huey-Chuan CHENG

3

, Yen-Wenn LIU

4

, and Wen-Chi HOU

4,

*

1

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

2

Department of Food Science, Yaunpei University of Science and Technology, Hsinchu 300, TAIWAN

3

Mackay Memorial Hospital, Taipei 104, and Mackay Medicine, Nursing and Management College, Taipei 112, TAIWAN

4

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, TAIWAN

(Received November 8, 2005; Accepted March 1, 2006)

Abstract.

Dioscorin was purified by DE-52 ion exchange chromatography from two yam species, Dioscorea

alata L. cv. Tainong 1 (TN1) and Dioscorea batatas Decne (imported from Japan, JP). By different in

vitro antioxidant tests, including DPPH radical and hydroxyl radical scavenging activity assay, a reducing

power test, an anti-lipid peroxidation test, DNA damage protection, and inhibition of dihydrorhodamine 123

oxidation by peroxynitrite, it was shown that dioscorins from the two species exhibited different scavenging

activities against DPPH and hydroxyl radicals, even after heating 100.C for 5 min. Dioscorins from TN1 were

hydrolyzed by pepsin for different durations and the peptic hydrolysates exhibited DPPH radical scavenging

activities. Peptic hydrolysates separated by Sephadex G-50 (F) gel filtration were tested for anti-DPPH radical

activity. Results showed that fractions of smaller molecular weight still have antioxidant activities.

Keywords: Antioxidant; Dioscorin; Peptic hydrolysates; Storage protein; Yam.

232

Botanical Studies, Vol. 47, 2006

by Sephadex G-50(F) column and were then analyzed

for anti-DPPH activity. It was found that the smaller

molecular weight fractions still had antioxidant activities.

MATERIALS AND METHODS

Material

Peroxynitrite was obtained from Calbiochem-

Novabiochem Co. (Darmstadt, Germany). Calf thymus

DNA (activated, 25 A

260

units) was purchased from

Amersham Biosciences (Uppsala, Sweden). Hydrogen

peroxide (33%) was from Wako Pure Chemical Industry

(Osaka, Japan). Reduced glutathione, dihydrorhodamine

123 (DHR 123), 1, 1-diphenyl-2-picrylhydrazyl (DPPH),

2-deoxy-ribose, and other chemicals and reagents were

purchased from Sigma Chemical Co. (St. Louis, MO,

USA).

Dioscorin extractions and purifications from

TN1 and JP yam

Tubers of fresh yams, D. alata L. cv. Tainong No. 1

(TN1) and D. batatas Decne (JP), were purchased from

Taipei Agricultural Products Marketing Co., Wanhua

District, Taipei. After washing and peeling, the tubers were

cut into strips for dioscorin extraction and purification.

After extraction and centrifugation, dioscorins were

purified from crude extracts successively by a DE-52 ion

exchange column according to the methods of Hou et al.

(2001). The concentrated dioscorin solution was dialyzed

against deionized water overnight and lyophilized for

further use.

DPPH radical scavenging activity of dioscorin

The DPPH radical scavenging activity of dioscorin

with or without heating at 100oC for 5 min and peptic

hydrolysates from TN1 and JP yams were measured

according to the method of Hou et al. (2001, 2002). Every

0.6 mL dioscorin solution (10, 20 and 40 nanomoles) was

added to 0.05 mL of 1 M Tris-HCl buffer (pH 7.9) and

then mixed with 0.6 mL of 50

μ

M DPPH in methanol for

20 min under light protection at room temperature. The

absorbance at 517 nm was measured. Deionized water was

used as a blank experiment. The scavenging activity of

DPPH radicals (%) was calculated following the equation:

(A517

blank

- A517

sample

) ÷ A517

blank

× 100%.

Scavenging activity of dioscorin against metal

ion-dependent hydroxyl radicals

The hydroxyl radical scavenging activity of dioscorin

with or without heating at 100oC for 5 min from TN1

and JP yams was determined by the deoxyribose method

(Halliwell et al., 1987). Every 0.5 ml sample containing

different amounts of dioscorin (0.357, 1.785, 3.57, 7.14,

and 17.85 nanomole) were added to 1.0 ml solution of

20 mM potassium phosphate buffer (pH 7.4), 2.8 mM

2-deoxy-ribose, 104

μ

M EDTA, 100

μ

M FeCl

3

, 100

μ

M

ascorbate, and 1 mM hydrogen peroxide. The mixtures

were incubated for 1 h at 37oC. After incubation, an equal

volume of 0.5% thiobarbituric acid in 10% trichloroacetic

acid was added and the mixtures were boiled at 100oC for

15 min. Deionized water was used as a blank experiment.

The absorbance at 532 nm was measured. The scavenging

activity of hydroxyl radicals (%) was calculated with the

equation: (A532

blank

- A532

sample

) ÷ A532

blank

× 100%.

The reducing power of dioscorin

The reducing power of different amounts (1.785, 3.57,

7.14, and 17.85 nmole) of dioscorin from TN1 and JP

yams in a 1.25 mL sample solution was measured by ferric

chloride-potassium ferricyanide methods (Yen and Chen,

1995) at a wavelength of 700 nm. Deionized water was

used as a blank experiment, and the reduced glutathione

(4.69, 9.37, 10.07, and 18.74 nmole) was used a positive

control. Increase of absorbance of the reaction mixture at

a wavelength of 700 nm (

.

A700 nm) indicates an increase

of reducing power.

Anti-linoleic acid peroxidation of dioscorin

The antiperoxidation activity of 5 μg (0.018 nmole)

dioscorin from TN1 and JP yams against 2.5 mL of 0.02

M linoleic acid emulsion at intervals after reactions at

37oC was measured by the thiocyanate method (Pham et

al., 2000). At intervals during incubation, a 0.1-mL aliquot

of the reaction mixture was mixed with 4.7 mL of 75%

ethanol, 0.1 mL of 30% ammonium thiocyanate, and 0.1

mL of 20 mM ferrous chloride in 3.5% hydrochloric acid.

Precisely 3 min after the addition of ferrous chloride to the

reaction mixture, the absorbance at a wavelength of 500

nm was determined. Deionized water was used in a control

experiment. Increase of absorbance at a wavelength of

500 nm indicates a decrease of antioxidant activity against

linoleic acid peroxidation.

Protecting hydroxyl radical-induced damages

of calf thymus DNA by TN1 dioscorin

The hydroxyl radical was generated by Fenton reaction

according to the method of Kohno et al. (1991). The 45

μ

L

reaction mixture included TN1 dioscorin (20, 50, 100, and

200

μ

g), 15

μ

L of calf thymus DNA, 18 mM FeSO

4

, and

60 mM hydroxygen peroxide at room temperature for 15

min or 30 min. Ten

μ

L of 10 mM EDTA was added to stop

the reaction. The only calf thymus DNA was used for the

blank test, and the control test was without TN1 dioscorin

additions. After agarose gel electrophoresis, the treated

DNA solutions were stained with ethidium bromide and

observed under UV light.

Protecting peroxynitrite-mediated DHR 123

oxidation by TN1 dioscorin

The protection against peroxynitrite-mediated DHR

123 oxidation was according to the methods of Kooy et

al. (1994). The total 175

μ

l reaction mixture included

different amounts of TN1 dioscorin (9, 12, and 16

μ

g),

10

μ

M DHR and 10

μ

l peroxynitrite in 50 mM phosphate

LIU et al. — Antioxidant activity of two species of yam tuber storage proteins

233

buffer (pH 7.4), containing 90 mM NaCl. After a 10-min

reaction, the fluorescent intensity was measured at the

excitation and emission wavelengths of 473 and 520 nm,

respectively, and at excitation and emission slit widths

of 2.5 nm and 3.0 nm, respectively. The control test was

without dioscorin additions.

Determination of the DPPH scavenging activity

of peptic hydrolysates of TN1 dioscorin

The 7 mg TN1 dioscorin was dissolved in 1 mL, 0.1 M

KCl-HCl buffer (pH 2.0). The 0.1 mL, 14 mg pepsin was

added for hydrolysis at 37oC for 8, 12, 24 and 32 h. After

hydrolysis, 0.5 mL of 0.5 M Tris-HCl buffer (pH 8.3) was

added and heated at 100oC for 5 min to stop hydrolysis.

The pepsin was heated before dioscorin hydrolysis for

zero hour reaction. Each dioscorin hydrolysate was used

for determination of DPPH scavenging activity.

Chromatograms of peptic hydrolysates of TN1

dioscorin on Sephadex G-50 column and their

DPPH scavenging activities

The peptic hydrolysates of TN1 dioscorin at 8, 12, 24

and 32 h were lyophilized and separated by Sephadex

G-50(F) chromatography (1×75 cm). The column was

eluted with 20 mM Tris-HCl buffer (pH 7.9). Flow rate

was 30 mL/h, and each fraction contained 2 mL. Each

fraction was determined at the absorbance of 210 nm for

peptide contents and for DPPH scavenging activity (570

nm).

Statistical analysis

Student’s t-test was used for comparisons between

control and each experimental test. A difference was

considered statistically significant when P< 0.05 (*) or P<

0.01 (**).

RESULTS AND DISCUSSION

Owing to the different amino acid compositions and the

secondary structures of dioscorin from Chinese yam and

Japanese yam (Liao et al., 2004), the antioxidant activity

of the dioscorin from two different yam species, Chinese

yam (D. alata cv. Tainong No. 1, TN1) and Japanese

yam (D. batatas Decne, imported from Japan, JP), was

compared. Our results show that dioscorin from Chinese

yam (TN1 yam) had higher antioxidant or scavenging

activities than did Japanese yam (JP yam) according to the

DPPH radical and hydroxyl radical scavenging activity

assay, reducing power test, anti-lipid peroxidation test.

Figure 1 shows the dioscorin scavenging activity

against DPPH radical from TN1 and JP with or without

heating at 100oC for 5 min. When DPPH radical was

scavenged, the color of the reaction mixture changed

from purple to yellow with the decrease of absorbance

at wavelength 517 nm. It was found that the dioscorin

from TN1 and JP exhibited dose-dependent DPPH radical

scavenging activity (Figure 1). Under the same 40 nmole

of dioscorin (28 kDa), the scavenging activity of JP

dioscorin decreased from 46% to 34% after heating at

100oC for 5 min. However, an activation of scavenging

activity (from 49% to 56%) was found in TN1 dioscorin.

Dioscorin from D. Japonica was found to have a content

of Cys roughly tenfold lower than the other two Taiwanese

yam species (Liao et al., 2004). The Cys in patatin (Liu

et al., 2003) and in sweet potato TIs (Hou et al., 2005)

was reported to contribute to DPPH scavenging activities.

It was proposed that the heating process could affect

JP dioscorin stability and reduce the DPPH scavenging

activity. However, it might also expose other inner Cys

residues in TN1 dioscorin and elevate TN1 dioscorin

scavenging activity.

Figure 2 shows the dioscorin scavenging activity

against hydroxyl radical from TN1 and JP with or without

heating at 100oC for 5 min. The dioscorin from TN1 and

JP was also found to exhibit dose-dependent hydroxyl

radical scavenging activity, even after heating at 100oC

for 5 min (Figure 2). Under the same 17.85 nmole of

dioscorin (28 kDa), the scavenging activity of TN1 (62%)

was higher than that of JP (48%). The heating processing

affected the hydroxyl radical scavenging activity in

dioscorins from two yam species. The higher hydroxyl

radical scavenging activity of the dioscorin from TN1

might be due partly to the different contents of Trp since

dioscorin from D. Japonica had less Trp (intensity ratio

of Raman spectra, I

878/759

) than the other two Taiwanese

yam species (Liao et al., 2004). The Trp in patatin (Liu

et al., 2003) and in sweet potato TIs (Hou et al., 2005)

was reported to contribute to hydroxyl radical scavenging

activities. Water spinach constituents were also reported to

have antioxidant activity (Huang et al., 2005). The heating

process could affect dioscorin stability and reduced

hydroxyl radical scavenging activity.

Figure 1. The scavenging activity against DPPH radical of

dioscorin from TN1 and J P yams with or without heating at

100oC for 5 min. Means of triplicates were measured. Deionized

water was used as a blank experiment. The scavenging activity

of DPPH radical (%) was calculated according to the following

equation: (A517

blank

- A517

sample

) ÷ A517

blank

× 100%.

234

Botanical Studies, Vol. 47, 2006

The reducing power of dioscorin from TN1 and JP

is shown in Figure 3. The reduced glutathione was used

as a positive control. TN1 dioscorin exhibited a dose-

dependent reducing power activity within the applied

concentrations (1.785, 3.57, 7.14, and 17.85 nmole) and

had higher activities than glutathione under the same

nmole concentration basis. The reducing power of TN1

dioscorin was about tenfold that of JP (Figure 3), which

was comparable to the reported ratio of Cys content in

each yam species (1.18±0.13 vs 0.13±0.07 mg/g protein,

Liao et al., 2004).

The products of lipid peroxidation (such as

malondialdehyde) could cause damage to proteins and

DNA (Esterbauer et al., 1991). The anti-lipid peroxidation

of 5 μg dioscorin from TN1 and JP is shown in Figure 4.

Both the dioscorin from TN1 and JP could retard linoleic

acid peroxidation during the intervals of 8 h at 37oC

compared to that of the control. Dioscorin from TN1

and JP were able to effect retardations of linoleic acid

peroxidation that were about 15.60 and 8.3-fold of control,

respectively, in the 12 h reaction.

Liao et al. (2004) reported the Cys content of dioscorin

in Chinese yam was about tenfold that of Japanese yam.

The secondary structures of dioscorin from D. alata L.

(Chinese yam) and D. Japonica (Japanese yam) were

mostly

α

-helix, a mixed

α

-helix type, and an antiparallel

β

-sheet type, respectively. From our present results,

under the same weight basis, dioscorin from Chinese yam

(TN1) had higher antioxidant or scavenging activities

than Japanese yam (JP yam) based on DPPH radical and

hydroxyl radical scavenging activity assay, a reducing

power test, and an anti-lipid peroxidation test. These

results might be attributable to the different amino acid

compositions and protein conformations. Therefore,

TN1 dioscorin was further studied in the following

Figure 3. The reducing power of dioscorin from TN1 and JP

yam (1.785, 3.57, 7.14, and 17.85 nmole). The glutathione (4.69,

9.37, 14.07, and 18.74 nmole) was used as a positive control.

Means of triplicate were determined at the absorbance of 700

nm.

Figure 5. The effects of TN1 dioscorin on the Fe

2+

-mediated

DNA oxidation. Lane B was native calf thymus DNA; lane C

was metal-mediated oxidized DNA; and lanes 1 to 4 were metal-

mediated oxidized DNA with 20, 50, 100, and 200

μ

g TN1

dioscorin additions. The reaction was stopped after 15 min or 30

min by adding 10 mM EDTA. After electrophoresis, the gel was

stained with ethidium bromide and observed under UV light.

Figu re 4. The effects of dioscorin from TN1 and JP yam on

the anti-linoleic acid peroxidation. Each of 5

μ

g dioscorin was

added to the emulsion and incubated at 37oC for 0, 2, 4, and 8 h.

At each time interval, 0.1 mL was picked and was determined

by thiocyanide method (A500 nm).

Figure 2. The scavenging activity against hydroxyl radical

of dioscorin from TN1 and JP yam with or without heating at

100oC for 5 min. Means of triplicates were measured. Deionized

water was used as a blank experiment. The absorbance at 532

nm was measured. The scavenging activity of hydroxyl radicals

(%) was calculated with the equation: (A532

blank

- A532

sample

) ÷

A532

blank

× 100%.

LIU et al. — Antioxidant activity of two species of yam tuber storage proteins

235

Figure 7. The DPPH scavenging activity (A) and chromatograms on Sephadex G-50(F) column (B) of peptic hydrolysates of TN1

dioscorin. TN1 dioscorin was hydrolyzed by pepsin at 37oC for 8, 12, 24 and 32 h. After hydrolysis, 0.5 mL of 0.5 M Tris-HCl buffer

(pH 8.3) was added and heated at 100oC for 5 min to stop hydrolysis. The pepsin was heated before dioscorin hydrolysis for zero

hour reaction. Each dioscorin hydrolysate was used for determinations of DPPH scavenging activity and was separated by Sephadex

G-50(F).

Figu re 6. The TN1 dioscorin (9, 12, and 16

μ

g) protected

peroxynitrite-mediated dihydrorhodamine 123 oxiadtion. The

total 175

μ

l reaction mixture included different amounts of

TN1 dioscorin, 10

μ

M DHR, and 10

μ

l peroxynitrite in 50 mM

phosphate buffer (pH 7.4) containing 90 mM NaCl. After 10 min

reaction, the fluorescent intensity was measured at the excitation

and emission wavelengths of 473 and 520 nm, res pectively,

and excitation and emission slit widths of 2.5 nm and 3.0 nm,

respectively. The control test was without dioscorins additions.

A difference between the control and the experimental test was

considered statistically significant when p<0.05 (*) or p<0.01

(**).

experiment of DNA damage protection, inhibition of

dihydrorhodamine 123 oxidation by peroxynitrite,

and DPPH scavenging activity of dioscorin peptic

hydroxylates.

Free radicals can damage macromolecules in DNA,

protein and the lipid cells in membranes (Halliwell, 1999).

Figure 5 shows TN1 dioscorin protected against hydroxyl

radical-induced calf thymus DNA damage in 15 or 30

min. The only calf thymus DNA was used for a blank

test (lane B), and the control test (lane C) was without

TN1 dioscorin additions. Compared to the blank test and

control test, it was found that the added TN1 dioscorin

above 50 μg (lane 2) could prevent hydroxyl radical-

induced calf thymus DNA damage in both 15 min or 30

min reactions.

Peroxynitrite is formed from a nearly diffusion-

limited reaction between nitric oxide and superoxide

anion and as an initiator of potentially harmful oxidation

reaction (Brannan et al., 2001). The results of Figure 6

demonstrate that the protective effect of peroxynitrite-

mediated DHR oxidation of TN1 dioscorin was dose-

dependent. Significant variation was observed among the

peroxynitrite, peroxnitrite + 12 μg TN1 dioscorin (p<

0.05), and peroxynitrite + 16 μg TN1 dioscorin (p<0.01).

236

Botanical Studies, Vol. 47, 2006

Figure 7(A) shows the DPPH scavenging activity

of TN1 dioscorin hydrolysates at different pepsin

hydrolysis times. As the results of Figure 7 make clear,

the scavenging activity against DPPH radical increased

from 7.1% (0 h) to about 21% (32 h). Figure 7(B) shows

the chromatograms of peptic dioscorin hydrolysates of 8,

12, 24 and 32 h on Sephadex G-50 chromatography. The

smaller peptides were found to increase with increasing

pepsin hydrolytic time and also exhibited DPPH radical

scavenging activities. The purifications of antioxidant

peptides will be investigated in the future.

Acknowledgments. The authors want to thank the

financial support (SKH-TMU-94-03) from Shin Kong Wu

Ho-Su Memorial Hospital, Taipei, Taiwan.

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兩品種山藥塊莖儲藏性蛋白½體外抗氧化活性之研究

劉玉華

1

　梁弘人

2

　鄭惠川

3

　劉燕雯

4

　侯文琪

4

1

新光醫院肝膽腸胃科

2

元培科學技術學院

3

馬偕醫院及馬偕醫護管理專科學校

4

臺北醫學大學生藥學研究所

　　兩品種山藥〈台農一號與日本山藥〉塊莖儲藏性蛋白½ dioscorin 經由 DE-52 離子交換層析法純

化。經由一系列體外抗氧化實驗，包括清除 DPPH 與氫氧自由基，還原能力，抗脂½過氧化能力，保

護去氧核糖核酸傷害能力，及抑制 peroxynitrite 氧化 dihydrorhodamine 123 能力。結果顯示，兩品種山

藥 dioscorin 具有不同的抗氧化能力，即使 100℃ 加熱五分鐘也仍具有不同的清除 DPPH 與氫氧自由基

的能力。以胃蛋白.水解台農一號 dioscorin 不同時間之水解產物也具有清除 DPPH 自由基的能力。以

Sephadex G-50 (F) ½濾層析進行分離並進行 DPPH 自由基清除實驗，結果顯示小分子水解產物也具有抗

氧化能力。

關鍵詞：抗氧化；儲藏性蛋白½；胃蛋白.水解物；山藥。