The extraction of antioxidant compounds from residue materials such as hulls, seed coats, peels, grape seeds, olive rape, and cocoa by product has been reported.

2004; 9(2): 92-97

INTRODUCTION

Rice is the principal cereal in Asia, some countries in Africa and Latin America. More than one million tons of rice hulls have been produced annually in South Korea after processing of rice. However, rice hulls are wasted or destined to undervalued uses. Cur- rently, agricultural and industrial residues are attrac-

tive sources of natural antioxidants.

The extraction of antioxidant compounds from residue materials such as hulls, seed coats, peels, grape seeds, olive rape, and cocoa by product has been reported.

In general, hulls play the major role in the defense of the plant seeds and together with bran fractions concentrate most tannin; therefore, the antioxidant activity of these fractions is higher than that of seeds.

Rice hull has been also reported to contain phenolic compounds Rice hull is known to contain an antioxidant defense system to protect rice seed from oxidative stress. The aim of this study was to evaluate the effect of methanol extract of rice hull on oxidative DNA damage induced by H

O

in human lymphocytes. Cells incubated in medium with 1 % DMSO (negative control) or with various concentration of rice hull extracts (12.5, 25, 50, 100μg/ml) for 30 min at 37

C were further treated with H

O

(200μM) as an oxidative stimulus for 5 min on ice. Oxidative damage was evaluated by Comet assay (single-cell gel electrophoresis) and quantified as % tail DNA.

Cell viability measured by trypan blue exclusion test was above 95% for all treatments.

An increased oxidative DNA damage by H

O

expressed by % tail DNA was significantly inhibited by pre-incubating with rice hull extracts in a dose dependent manner. When human lymphocytes were post-incubated with rice hull extract for 30 min after exposure to H

O

or incubated with rice hull and H

O

simultaneously, the protective ability of the rice hull was not changed. These results indicate that rice hull supplementation to human lymphocytes followed by H

O

treatment inhibited damage to cellular DNA, supporting a protective effect of rice hull against oxidative damage.

ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: Rice hull extract, DNA damage, Comet assay, Hydrogen peroxide,

Lymphocytes

Effect of Rice Hull Extracts on Oxidative DNA Damage in Human Lymphocytes

Kyung-Im Jeon

, Hae-Ryong Park

, Seung-Cheol Lee

and Eunju Park

1

Department of Food and Nutrition, and

Division of Food Science and Biotechnology, Kyungnam University, Masan 631-701, Korea

Corresponding author：Eunju Park, Department of Food and Nutrition, Kyungnam University, Masan 631-701, Korea. Tel:

+82-55-249-2218, Fax: +82-55-244-6504, E-mail: pej@kyungnam.ac.kr

Received：May 10, 2004, Accepted：June 14, 2004

such as isovitexin, phytic acid, vanillic acid, syringic acid, and ferulic acid, which showed strong antioxi- dant effects.

During last few decades, there has been much epidemiological research attention focused on plant phenolics due to their pronounced health benefits such as antioxidant, antimutagenic, anticarcinogenic, antia- therogenic, antiplatele aggregation etc.

In particular, phenolic compounds have been reported to associate with antioxidative action in biological systems, acting as scavengers of singlet oxygen and free radicals.

In addition, free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check only by the existence of multiple antioxidant and repair systems as well as the replacement of damaged lipids and proteins.

DNA is probably the most biologically significant target of oxidative attack, and it is widely thought that continuous oxidative damage to DNA is a significant contributor to the age-related develop- ment of the major cancers.

The objective of this work was to determine the antigenetoxic activity of rice hull extracts on oxidative DNA damage induced by H

O

in human lymphocytes in order to evaluate its potential as a natural antioxi- dant source for medicinal and food industry.

MATERIALS AND METHODS

1) Preparation of rice hull extracts

3 g of rice hulls were extracted with 100 ml of methanol for 1 h of incubation with shacking at room temperature and filtered through a Whatman No. 1 filter paper.

2) Preparation of human lymphocytes Blood samples were obtained from two healthy male volunteers (non-smokers, 24 and 25 years old, respectively). Five ml of fresh whole blood was added to 5 ml of phosphorous buffered saline (PBS) and layered onto 5 ml of Histopaque 1077. After centrifugation for 30 min at 400×g at room tempera-

ture, the lymphocytes were collected from the just above the boundary with the Histopaque 1077, washed with 5 ml PBS. Finally, they were freshly used for comet assay or resuspended in freezing medium (90%

fetal calf serum, 10% demethyl sulfoxide) at 6×10

cells/ml. The cells were frozen to -80

C using a Nalgene Cryo 1

C freezing container (Nalgene, Roche- ster, NY) and stored in liquid nitrogen. The cell were thawed rapidly prior to each experiment in a water bath at 37

C.

3) Treatment of rice hull extracts on human lymphocytes

Cells were incubated with rice hull extract in four different treatments (Fig. 1).

(1) Lymphocytes (2×10

cell/ml) were incubated with various concentration of rice hull extracts (12.5, 25, 50, 100μg/ml) for 30 min at 37

C in a dark incubator.

(2) Lymphocytes were incubated with various con- centrations of rice hull extracts (0, 12.5, 25, 50, 100μg/

ml) for 30 min at 37

C in a dark incubator and then were resuspended in PBS with 200μM H

O

for 5 min on ice.

(3) Lymphocytes were damaged oxidatively with 200μM H

O

for 5 min on ice and then incubated with rice hull extracts (0, 12.5, 25, 50, 100μg/ml) for 30 min at 37

C.

(4) Lymphocytes were incubated simultaneously with 200μM H

O

and rice hull extract

After each treatment, samples were centrifuged at 1,450 rpm for 5 min and washed with PBS. All the experiments were repeated twice with lymphocytes from each of two donors on the separate day.

4) Determination of DNA Damage (Comet assay)

The alkaline comet assay was conducted according

to Singh et al.

with little modification. The cell

suspension was mixed with 75μl of 0.5% low mel-

ting agarose (LMA), and added to the slides precoated

with 1.0% normal melting agarose. After solidifaction

Fig. 1. Treatment of rice hull extract and comet assay procedure.

1

Blood drawn Isolation of lymphocytes Sample treatments Rice hull (30')

Rice hull (30') H O (5') →

H O (5') Rice hull (30')

→

H O + Rice hull (5')

Cells with normal DNA Cells with damaged DNA

Suspend cells in agarose coat onto glass slide

Incubation in cell lysis solution

- nucleoids -

Incubate in alkaline electrophoresis solution subject to electrophoresis stain with EtBr/fluorescence microscopy

Negative control (DMSO 1%) Positive control (H O , 200

µM )

2 3 4

- + - +

of the agarose, slides were covered with another 75μl of 0.5% LMA, and then immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1%

sodium laurylasarcosine; 1% Triton X-100 and 10%

DMSO) for 1 h at 4

C. The slides were next placed into an electrophoresis tank containing 300 mM NaOH and 10 mM Na

EDTA (pH 13.0) for 40 min for DNA unwinding. For electrophoresis of the DNA, an electric current of 25 V/300 mA was applied for 20 min at 4

C. The slides were washed three times with a neutralizing buffer (0.4 M Tris, pH 7.5) for 5 min at 4

C, and then treated with ethanol for another 5 min before staining with 50μl of ethidium bromide (20μg/ml). Measurements were made by image analysis (Kinetic Imaging, Komet 5.0, U.K) and fluorescence microscope (LEICA DMLB, Germany), determining the percentage of fluorescence in the tail(tail intensity, TI; 50 cells from each of two replicate slides). Cell viability measured by trypan blue exclusion test was above 95% for all treatments.

5) Statistical Analysis

The mean values of the DNA damage (tail inten- sity) from each treatment were compared using one- way analysis of variance (ANOVA) followed by Dun-

can's test. p-value of less than 0.05 was considered significant.

RESULTS AND DISCUSSION

The genotoxic effects of H

O

and the protective ability of rice hull extract were assessed in normal human lymphocytes by comet assay. The comet assay is a sensitive and rapid method for the detection of DNA damage at the individual cell level

and speci- fically for detecting oxidative DNA strand breaks.

It is considered a useful tool for investigating issues related to oxidative stress in human lymphocytes.

The concentrations of rice hull extract (12.5∼100 μg/ml) used in this study were non-toxic and DMSO, the vehicle used throughout, had no effect on DNA strand breaks (Fig. 2).

Pretreatment of the cells for 30 min with rice hull extract significantly reduced the genotoxicity of hydrogen peroxide measured as DNA strand breaks (Fig. 3). The protective effect (inhibition rate) of rice hull extract increased as its concentration increased from 12.5 to 100μg/ml by 51 to 80% in rice hull

Fig. 2. The effect of supplementation in vitro with dif- ferent concentration of MeOH extract of rice hull in human lymphocytes. Values are mean with standard error of duplicate experiments with lymphocytes from each of two different donors. ns: not significant.

Fig. 3. The preventive effect of supplementation in

vitro with different concentration of methanol extract

of rice hull on 200 M H

O

-induced human lympho-

cytes DNA damage. Control: DMSO treated normal

control. Values are mean with standard error of dupli-

cate experiments with lymphocytes from each of two

different donors. Values not sharing the same letter are

significantly different from one another (p＜0.05).

extracts of H

O

treated positive control. Especially, the highest concentration (100μg/mL) of the rice hull extract was shown that there was no statistical dif- ference compared to DMSO-treated negative control.

This antigenotoxic effect of rice hull was the case when the rice hull and H

O

were treated simul- taneously on ice for 5 min (Fig. 4).

Hydrogen peroxide is believed to cause DNA strand breakage by generation of the hydroxyl radical (OH) close to the DNA molecule, via the Fenton reaction.

The possible mechanism by which rice hull extract inhibited oxidative DNA damage in human lympho- cytes can be ascribed to the chemical structure of the phenolic compound contained in rice hull, such as cinnamic acid and benzoic acid.

The phenolic com- pound in rice hull may work by providing hydrogen atoms from their phenolic hydroxyl groups to sca- venge hydroxyl radical generated from hydrogen pero- xide. Although the protective effect of cinnamic acid and benzoic acid, the main phenolic compound in rice hull extract, on hydrogen peroxide induced DNA damage was not reported so far, many other phenolic phytochemicals, such as quercetin, myricetin and

epigallocatechin gallate, work as antioxidants in this manner.

When human lymphocytes were post-incubated with rice hull extract for 30 min after exposure to hydrogen peroxide, the protective ability of the rice hull was not changed (Fig. 5). The damaged DNA by ROS can be repaired by DNA repair pathway and it is clear that individual variations in repair capability would have a bearing on cancer risk.

Collins et al. reported that when fresh isolated human lym- phocytes were incubated after hydrogen peroxide treat- ment, repair of strand breaks appears to be unusually slow.

In the present study, however, hydrogen peroxide induced DNA damage in human lympho- cytes was effectively repaired to almost similar level as DMSO treated negative control by post- treatment of rice hull extract for 30 min. Although the exact mechanism for DNA repair activity needs to be elucidated, the rice hull extract may contribute to stimulation of DNA repair.

These results indicate that rice hull supplementation to human lymphocytes could inhibit or repair H

O

induced damage to cellular DNA, supporting a protec- Fig. 4. The effect of supplementation in vitro with

different concentration of MeOH extract of rice hull and 200μM H

O

simultaneously in human lympho- cytes. Control: DMSO treated normal control. Values are mean with standard error of duplicate experiments with lymphocytes from each of two different donors.

Values not sharing the same letter are significantly different from one another (p＜0.05).

Fig. 5. The recovery effect of supplementation in vitro with different concentration of methanol extract of rice hull on 200μM H

O

-induced human lymphocytes DNA damage. Control: DMSO treated normal control. Values are mean with standard error of duplicate experiments with lymphocytes from each of two different donors.

Values not sharing the same letter are significantly

different from one another (p＜0.05).

tive effect of rice hull against oxidative damage.

ACKNOWLEDGEMENT

This study was supported by 2005 research fund of Kyungnam University, Korea.

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