한국방사선산업학회

INTRODUCTION

The chemical treatment has been widely permitted in food industry for improving storage effectiveness of many foods. Fumigation using ethylene oxide has been used as a general process in many countries for the sterilization and prolongation of storage periods. However, the application of this conventional technique to foods is being recently controlled due to the presence of chemical residues leading to human damage occasionally (Dickman 1991; Uijl 1992). Therefore, the use of naturally derived non-toxic

com-pounds and other alternative techniques have been studied for consumer’s safety.

Food irradiation is a simple and efficient technique to enhance the preservation and hygiene of foods because it can remove a wide variety of organic contaminants includ-ing harmful pathogenic microorganisms (Farkas 1998). Moreover, this technique possesses advantages in avoiding potentially harmful chemicals and irradiated foods are less transformed than heat-treated foods. Thus, the process has been endorsed by both WHO/FAO/IAEA/FDA groups and health authorities of many countries (Ahmed 1991; WHO 1992).

Artemisia capillaris Thunb has been used in traditional Oriental medicine as a choleretic, anti-inflammatory and diuretic agent in the treatment of epidemic hepatitis (Tang

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Functional and Chemical Stability of a Medicinal herb,

Artemisia capillaries, Following Gamma Sterilization

Radiation Research Center for Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeonbuk 580-185, Korea

Abstract -- The stability of functional and chemical properties of gamma-irradiated (10 kGy) Artemisia capillaris, a widely used herb in the traditional Oriental medicine, was investigated. Functional properties of the extracts of gamma-irradiated and non-irradiated A. capillaris were compared in antioxidant activities, such as 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical and superoxide anion radical scavenging, lipid peroxidation inhibition, and protection of lymphocyte and plasmid DNA. Their chemical properties were assessed by HPLC analysis, comparing with chlorogenic acid and caffeic acid, which were isolated from ethylacetate fraction as major compounds with strong antioxidant activities. No significant difference in functional properties between irradiated and non-irradiated A. capillaris was found in all antioxidant assays. Also HPLC analysis of ethyl acetate fractions of irradiated and non-irradiated A. capillaris revealed the preservation of chlorogenic acid (tR==3.124 min) and caffeic acid (tR==3.672 min), and showed

almost the same pattern in the general peaks. These results suggest that the chemical components and antioxidant properties of A. capillaris are not affected largely by gamma-ray irradiation. Therefore, this study may provide evidence that the irradiated herbs retain their potential functional properties.

Key words : Artemisia capillaris, Herb, Irradiation, Stability, Antioxidant

* Corresponding author: Sung-Kee Jo Tel. +82-63-570-3200, Fax. +82-63-570-3229. E-mail. [email protected]

and Eisenbrand 1992). Biologically active components such as capillarisin, chlorogenic acid, scopoletin and caffeic acid have been identified in A. capillaris (Wang et al. 2000; Sheu et al. 2001 ).

This study describes the stability of functional and che-mical properties of gamma-irradiated (10 kGy) A. capil-laris, thereby providing an evidence that gamma-irradiation is an efficient process for preservation of medicinal herbs.

MATERIAL AND METHODS

The herb, Artemisia capillaris, was purchased from the Kyoungdong Herb Market in Seoul, Korea. For gamma irradiation, lots (100 g) of the samples were vacuum-packed in nylon bags and placed in PVC containers (25 cm×30 cm ×3 cm). Gamma irradiation was carried out in a cobalt-60 irradiator (AECL, IR-79, MDS Nordion International Co. Ltd., Canada) at room temperature (20±0.5�C) and operat-ed at a dose rate of 2 kGy/hour. The applioperat-ed dose level was 10 kGy. The absorbed dose was monitored with both free radical and ceric/cerous dosimeters.

2. Preparation of water extract and isolation of active components

Gamma-irradiated and non-irradiated A. capillaris were extracted with boiling water (15 l of distilled water for 1.5 kg of herb) for 3 h. The water extracts were filtered and centrifuged at 10,000×g for 30 min to remove insoluble particles. The extracts were freeze-dried and stored at -20�C until use. To isolate the active antioxidant components of A. capillaris, the extract of non-irradiated herb was dissolved in water and further fractionated with ethylacetate and butanol successively, obtaining ethylacetate fraction, n-butanol fraction, and remaining water fraction. Based on antioxidant activities of solvent fractions, ethylacetate fraction was subjected to column chromatography for isolation of active antioxidant components of A. capillaris. Ethylacetate fraction was first subjected to MCI gel column chromatography with methanol : water (9 : 1) gradient and Sephadex LH-20 column chromatography with water : methanol (9 : 1) gradient. For the identification of isolated compounds, the 1_H-, 12_{C-NMR spectrometry and GC mass} spectrometry were performed.

3. Radical scavenging assay

1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scaveng-ing activity of the preparation was determined from the ab-sorbance at 517 nm as described previously (Gadow et al. 1997). Superoxide anion scavenging activity was determin-ed by superoxide dismutase-like activity assay with some modifications (Peskin and Winterbourn 2000). 7.5µl of sample was mixed with 75µl of 50 mM sodium phosphate buffer (pH 7.4) containing 1 mM ethelenediaminetetraacet-ic acid (EDTA), 0.5 mU xanthine oxidase, and 7.5µl of WST-8 solution (Dojindo Laboratories, Japan) in each well of 96-well microplates and, after storing at room tempera-ture for 1 min, 60µl of 2.5 mM xanthine solution was ad-ded. The absorbance change at 450 nm resulting from WST-8 oxidation by superoxide anion was monitored for 30 min. The superoxide anion scavenging activity was cal-culated as follows:

Scavenging activity (%) =

=(Acontrol-Asample)/(Acontrol-Ablank)×100

Acontrol: Absorbance with xanthine and xanthine oxidase, and without sample

Asample: Absorbance with xanthine and xanthine oxidase, and with sample

Ablank: Absorbance with xanthine oxidase, and without xanthine and sample

4. Lipid peroxidation assay

Lipid peroxidation assay was performed as described previously (Ohkawa et al. 1979). Briefly, malondialdehyde (MDA), the end product of lipid peroxidation, was produc-ed by Fe2++

/ascorbate system in the presence of rat liver mi-crosome. The amount of MDA was determined spectro-photometrically after thiobarbituric acid reaction. Lipid pe-roxidation inhibition was calculated as follows:

Inhibition (%)==(Acontrol-Asample)/(Acontrol-Ablank)×100 Acontrol: Absorbance with Fe2

+ +

/ascorbate and without sample Asample: Absorbance with Fe2

+ +

/ascorbate and with sample Ablank: Absorbance without Fe2

+ +

/ascorbate and sample

5. Single cell gel electrophoresis (Comet assay)

Lymphocytes were isolated from spleen of ICR mice. Lymphocytes were suspended in complete RPMI medium

with or without test samples and irradiated at a dose of 2 Gy. After irradiation, single cell gel electrophoresis was performed under an alkaline condition (Singh et al. 1988). After staining with ethidium bromide, the slides were observed using a fluorescent microscope and the results were analyzed with an image analysis system (Komet 4.0, Kinetic imaging, Ltd., Great Britain). The DNA damage was expressed as Tail Moment, where Tail Moment (TM) =

=(tail mean - head mean)×tail% DNA.

6. Plasmid DNA nick assay

Plasmid DNA nick assay was performed to assess the protective activities of samples against hydroxyl radical-induced DNA strand breaks. pUC18 plasmid (0.5µg) was mixed with 5µl of 50 mM Tris-HCl buffer (pH 7.4), 5µl of 3% H2O2, 5µl of 100µM FeCl2, and 5µl of sample. After incubation at 37�C for 30 min, the reaction mixture was mixed with 5µl of loading buffer and electrophoresed on a 1% agarose gel in TAE buffer. After staining the DNA in the gel with ethidium bromide, the plasmid bands were observed under UV light.

7. HPLC analysis of ethylacetate fraction

To confirm the stability of chemical properties of gam-ma-irradiated A. capillaris, qualitative HPLC analysis was performed with ethylacetate fraction of the A. capillaris extract. HPLC was performed with symmetry C18 column

(5µm, 4.6×250 mm) in a gradient of 0.1% phosphoric acid and acetonitrile at 0.5 ml/min flow rate, and peaks were detected with Waters 996 PDA detector (UV at 254 nm). Chlorogenic acid and caffeic acid were used as standard compounds.

RESULT AND DISCUSSION

gamma-irradiated A. capillaris

To examine the stability of antioxidant properties of gamma-irradiated A. capillaris extract, radical scavenging activities and DNA protection activities of the water extract and its solvent fractions were investigated. In DPPH radical scavenging assay, the water extract and its solvent fractions showed no difference between gamma-irradiated and non-irradiated A. capillaris (Fig. 1A). Superoxide anion sca-venging activity of the water extract and its water fraction showed a slight difference between irradiated and non-irradiated A. capillaris, but the activity of ethylacetate frac-tion and n-butanol fracfrac-tion was not different between irra-diated and non-irrairra-diated A. capillaris (Fig. 1B). The lipid peroxidation inhibition activity was slightly lower in the water extract of gamma-irradiated A. capillaris, and its n-butanol fraction, and water fraction, but the activity of its ethylacetate fraction was not different from non-irradiated A. capillaris (Fig. 1C). In DNA protection assay,

ethylace-0 20 40 60 80 100

Extract EtOAC BuOH Water

Concentration (50 µg ml-1) DPPH radical scavenging (%) Concentration (100 µg ml-1) 0 20 40 60 80 100

Extract EtOAC BuOH Water

Superoxideanion scavenging (%) 0 20 40 60 80 100

Extract EtOAC BuOH Water

Lipid peroxidation inhibition (%)

concentration (25 µg ml-1)

(A) (B) (C)

Fig. 1. Comparison of radical scavenging activities between each solvent fraction of gamma-irradiated and non-irradiated A. capillaris. The

water extract and its solvent fractions from gamma-irradiated (blank bars) and non-irradiated (filled bars) A. capillaris were tested for their DPPH radical scavenging activity (A), superoxide anion scavenging activity (B), and lipid peroxidation inhibitory activity (C). Extract: water extract; EtOAc: ethylacetate fraction; BuOH: n-butanol fraction, Water: water fraction. Data represent mean±SD.

tate fractions of irradiated and non-irradiated A. capillaris showed similar protective effects against Fenton reaction-mediated plasmid DNA nick formation (Fig. 2A) and

radiation-induced lymphocyte DNA damage (Fig. 2B). These results suggest that antioxidant properties of A. capillaris were not or little affected by gamma-irradiation.

2. Isolation and identification of active antioxidant components of A. capillaris

Based on its highest antioxidant activity among solvent fractions, ethylacetate fraction was subjected to the isola-tion of active antioxidant components of A. capillaris. Two active compounds were isolated by MCI gel column and Sephadex LH-20 column chromatography (Fig. 3) and were

Fig. 4. Comparative HPLC analysis of chemical components between irradiated and nonirradiated A. capillaris. (A) Ethylacetate fraction of

irradiated A. capillaris; (B) Ethylacetate fraction of nonirradiated A. capillaris; (C) caffeic acid and (D) chlorogenic acid as standards.

Absorbance (254nm) Minutes A B C D Minutes Minutes Minutes Au Au Au Au Supercoiled form Nicked form FeCl2 H2O2 Sample (100 µg ml-1) Irradiated A. capillaris non-irradiated A. capillaris Irradiated A. capillaris non-irradiated A. capillaris 0 1 2 3 4 5 T ail moment (TM) Lymphocyte Irradiation (2Gy) Sample (25 µg ml-1) (A) (B) - -- -- - --

-Fig. 2. Comparison of DNA protective effects between

ethylace-tate fractions of gamma-irradiated and non-irradiated A.

capillaris. (A) Fenton reaction-mediated pUC18 plasmid

nick formation assay; (B) Single cell gel electrophoresis (Comet assay) of gamma-irradiated mouse lymphocytes. Data represent mean±SD.

Artemisia capillaris (1.5 kg)

Extracted with hot water for 3h Extract (275g) 1. dissolved in water 2. ethylacetate fractionation 3. n-butanol fractionation Ethylacetate fraction (22 g) n-Butanol fraction (45 g) Water fraction (200 g) MCI gel column chromatography (methanol: water gradient)

Fraction ACE01 Fraction ACE02 Fraction ACE03 Sephadex LH-20

column chromatography (water: methanol gradient)

Sephadex LH-20 column chromatography (water: methanol gradient) Chlorogenic acid

(0.15 g)

Caffeic acid (0.3 g)

Fig. 3. Scheme for the isolation of chlorogenic acid and caffeic

identified as chlorogenic acid and caffeic acid by 1_H-, 12 C-NMR and GC mass spectrometry. These compounds show-ed high antioxidant activities to similar extents in DPPH radical and superoxide anion scavenging activity, lipid peroxidation inhibition, and DNA protection (Data not shown).

3. Stability of chemical properties of gamma-irradiated A. capillaris

HPLC analysis of ethylacetate fraction was performed to confirm the chemical stability of active components of A. capillaris following gamma-irradiation. Irradiated and non-irradiated A. capillaris showed almost same patterns of HPLC profile (Fig. 4A and 4B). The retention time and peak area of chlorogenic acid (tR==3.124 min) and caffeic acid (tR==3.672) were not changed by gamma-irradiation of A. carpillaris (Fig. 4). These results showed that gamma-irradiation did not alter the chemical components of A. capillaris.

ACKNOWLEDGMENTS

This project was supported by the Nuclear R&D Program from the Ministry of Science and Technology of Korea.

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Manuscript Received: July 25, 2007 Revision Accepted: August 14, 2007