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Toxicological safety and stability of the components of an irradiated Korean medicinal herb, Paeoniae Radix
ARTICLE IN PRESS
Radiation Physics and Chemistry 71 (2004) 115–119
Toxicological safety and stability of the components of an irradiated Korean medicinal herb, Paeoniae Radix Young-Beob Yua, Ill-Yun Jeongb, Hae-Ran Parkb, Heon Ohb, Uhee Jungb, Sung-Kee Job,* a
b
Korea Institute of Oriental Medicine, Yuseong, Daejeon 305-811, Republic of Korea Radiation Application Research Division, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600, Republic of Korea
Abstract As utilization of medicinal herbs in food and bio-industry increases, mass production and the supply of herbs with a high quality are required. As the use of fumigants and preservatives for herbs is being restricted, safe hygienic technologies are demanded. To consider the possibility of the application of irradiation technology for this purpose, the genotoxicological safety and stability of the active components of the g-irradiated Paeoniae Radix were studied. The herb was irradiated with g-rays at a practical dosage of 10 kGy, and then it was extracted with hot water. The genotoxicity of the extract of the irradiated herb was examined in two short-term in vitro tests: (1) Ames test in Salmonella typhimurium; (2) Micronucleus test in cultured Chinese hamster ovary (CHO) cells. The extract of the irradiated herb did not show mutagenicity in the Ames test of the Salmonella reverse mutation assay, and did not show cytogenetic toxicity in the culture of the CHO cells. HPLC chromatogram of paeoniflorin in the irradiated Paeoniae Radix was similar with that of the non-irradiated sample. The quantity of paeoniflorin did not change significantly with irradiation. These results suggest that g-irradiated Paeoniae Radix is toxicologically safe and chemically stable. r 2004 Elsevier Ltd. All rights reserved. Keywords: Paeoniae; Herb; Radiation; Toxicological; Chemical safety
1. Introduction The conventional method of decontamination was fumigation with gaseous ethylene oxide, which is now prohibited or being increasingly restricted in most advanced countries for health, environmental or occupational safety reasons (Uijl, 1992; Dickman, 1991). Food irradiation is increasingly recognized throughout the world as a means of reducing food losses due to microbial spoilage and insect damage. It can also reduce our reliance on the chemical fumigants and preservatives currently used by the food industry, and improve the hygienic quality of various foods and medicinal herbs *Corresponding author. Tel.: +82-42-868-8063; fax: +8242-868-8043. E-mail address: [email protected] (S.-K. Jo).
(Ahmed, 1991; WHO, 1992; Farkas, 1998). There are, however, still some debates about the safety of irradiated foods (Ehlermann, 2002; Tritsch, 2002), and therefore continuous scientific investigations should be performed to ensure the safety and stability of irradiated foods. The root of Paeonia lactiflora pallas (Paeoniae Radix) is one of the most important materials of Chinese crude drugs and has been used as a circulatory tonic and diuretic, and is prescribed for the treatment of abdominal pain. Paeoniflorin, main monoterpene glucoside of Paeoniae Radix, isolated by Ami et al. (1969) is well known as an active component in Paeoniae Radix and has been reported to have a hypotensive, vasodilative, and anti-oxytocic action. The purpose of the present study is to assess the genotoxicological safety and stability of the biological components of irradiated Paeoniae Radix to 10 kGy, by
0969-806X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2004.04.002
ARTICLE IN PRESS 116
Y.-B. Yu et al. / Radiation Physics and Chemistry 71 (2004) 115–119
comparing the results with those obtained from the untreated control samples.
number of revertant colonies was more than double that compared with the negative control and was dosedependent.
2. Materials and methods
2.3. Micronuclei test
2.1. Preparation of the extract for safety test
In the Chinese hamster ovary (CHO) cell culture, the induction of micronuclei by the water extract of the irradiated herb was examined by the cytokinesis-block method (Fenech and Morley, 1985) at dosages below 50% cell-growth inhibition. CHO cells were cultured in a RPMI 1640 medium with 10% FBS. Cells were exposed to the test substance in the absence or presence of the S9-mix for 6 h (Preston et al., 1981). After culture, medium was exchanged with fresh medium containing cytochalasin B. Cells were incubated for additional 18 h. After the incubation, the cells were treated with a hypotonic solution and stained with 10% Giemsa. 1000 cells per treatment were scored and the number of cells with micronucleus was determined.
Paeoniae Radix was collected at the cultivation area of Chonnam province in Korea. Voucher specimens of the Paeoniae Radix were deposited in the herbarium of the Oriental Medicine Resources Department of Sunchon National University. For g-irradiation, lots (100 g) of the samples were vacuum-packed in nylon bags and placed in PVC containers (25 cm 30 cm 3 cm). g-Irradiation was carried out in a cobalt-60 irradiator (AECL, IR-79, MDS Nordion International Co. Ltd., Canada) at room temperature (2070.5 C) and operated at a dose rate of 2 kGy/h. The applied dose level was 10 kGy. The absorbed dose was monitored with both free radical and ceric/cerous dosimeters. Non-irradiated and 10 kGy irradiated Paeoniae Radix was prepared by decocting the dried crude drug three times with boiling water (100 g of herb was extracted with 1000 ml of distilled water). The suspensions were spun at 10,000 g for 30 min and the extracts were dried with a speed vacuum. 2.2. Salmonella mutagenicity assay (Ames test) The Salmonella mutagenicity assay (Ames test) was performed according to the method of Maron and Ames (1983). S. typhimurium strains TA98, TA 100 and TA102 were obtained from Professor B.N. Ames (University of California, Berkely, California, USA). Each strain was tested for its genetic traits such as histidine requirement, deep rough (rfa) characteristic, UV sensitivity (uvrB mutation) and ampicillin- or tetracycline-resistance by the R-factor before use. S9 mix was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). The tester strain cultures were grown overnight in the Oxoid Nutrient Broth No. 2 (Oxoid Co. Ltd., Hampshire, England) with a density of 1–2 109 colony forming units (cfu/ml). The doses tested were 62, 185, 556, 1667 and 5000 mg/plate. Each dilution was performed with or without metabolic activation. For metabolic activation, preincubation was provided for 30 min. The positive controls were 4-nitro-o-phenylenediamine (NPD, 20 mg/plate), sodium azide (Na-Azide, 1.5 mg/plate), mitomycin C (MMC, 0.5 mg/plate) and 2-aminofluorene (2-AF, 10 mg/plate) and the negative control was distilled water (DW). The number of revertant colony was counted after 48 h of incubation at 37 C. According to Maron and Ames (1983), a mutagenic effect was acknowledged as positive when the
2.4. Isolation of paeoniflorin The dried and powdered Paeoniae Radix was refluxed with MeOH. The MeOH extract (95 g) was partitioned with H2O and Et2O. The Et2O fraction was subjected to chromatograph using SiO2 with CHCl3:MeOH(9:1) and CHCl3:MeOH(5:1). The CHCl3:MeOH(5:1) soluble fraction was also chromatographed on SiO2 with CHCl3:MeOH(4:1) as solvents to provide crude paeoniflorins and recrystalization in Et2O:MeOH (10:1). The NMR spectra were recorded with a Bruker DRX 300 NMR spectrometer containing TMS as an internal standard and the chemical shifts were given as d (ppm). 2.5. HPLC analysis of the water extract of irradiated Paeoniae Radix About 500 mg of Paeonae Radix was weighed accurately and sonicated in 50 ml of DW for 5 min. After filtration, residue was shaken vigorously in 10 ml of DW and centrifuged at 1000 rpm for 5 min. After repeating the extraction with DW once more, the aqueous fractions were combined and made up to 100 ml with DW. 10 ml of the aqueous solution injected to HPLC. HPLC was carried out with Shimadzu HPLC using Shim-Pack CLC-ODS(M) 25 column. To obtain the standard curve of paeoniflorin, standard paeoniflorin was accurately weighed and dissolved in methanol to provide various concentrations within the range of 100–500 mg/ml. The whole volume of the standard solution was 1 ml. Linearity of the responses was determined for five concentrations with three injections for each level. Calibration graphs were plotted after linear regression analysis of
ARTICLE IN PRESS Y.-B. Yu et al. / Radiation Physics and Chemistry 71 (2004) 115–119
the peak-area ratios with a concentration. The contents of paeoniflorin in the test samples were calculated using the regression parameters obtained from the standard curve.
3. Result and discussion 3.1. No mutagenicity of irradiated Paeoniae Radix The mutagenicity of the water extract of irradiated Paeoniae Radix was examined triplicatedly in Salmonella reversion assay at 5 dosages from 62 to 5000 mg/ plate. The number of revertant colonies of each strain in test group did not increased comparing with negative
117
control group, and the pattern of the revertant colony formation was similar to that of non-irradiated sample. The increase of the colony formation by irradiated sample was not appreciated in both direct non-activated and indirect activated tests (Table 1). From these results, any direct or indirect mutagen might be not formed in Paeoniae Radix by g-irradiation of 10 kGy. In previous studies, many kinds of food stuffs were examined to investigate possible toxic reaction. Most of the experiments produced no toxic effects in the Salmonella reversion assay. van Kooij et al. (1978) did not find any significant increase of mutation between irradiated and non-irradiated fresh vegetables. Hattori et al. (1979) and Farkas et al. (1981) also obtained the same results.
Table 1 Revertant colonies in the S. typhimurium reversion assay with water extract of Paeoniae Radix Test Material
Dose (mg/plate)
H2O
S9 mix
Number of revertant colonies(His+) per plate TA98
TA100
TA102
2379
17076
297758
Paeoniae Radix (0 kGy)
5000 1667 556 185 62
3374 2371 2774 1977 2472
166712 156711 15971 154711 167711
302720 335716 33978 310713 30772
Paeoniae Radix (10 kGy)
5000 1667 556 185 62
31711 3072 1871 1876 2072
15879 154711 14171 143720 154713
30877 322730 30479 29974 313713
2340792
+
2474 3173
22777 25776
309714 342716
5000 1667 556 185 62
+ + + + +
3173 2578 2573 32712 2071
315725 254711 25876 246710 235714
39473 382718 367712 351725 321716
5000 1667 556 185 62 10
+ + + + + +
2973 2471 26710 25711 2571 16417158
30176 22676 22971 247711 223725 909778
41176 37878 350735 329713 319711 531757
NPD Na-Azide MMC
20 1.5 0.5
H2O H2O Paeoniae Radix (0 kGy)
Paeoniae Radix (10 kGy)
2-AF
12297103 51897306
NPD (4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF (2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean 7S.D. of revertant colonies per plate in duplicate experiments except for negative control groups (triplicate).
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Table 2 Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of g-irradiated Paeoniae Radix Test material
Conc. (mg/ml)
S9 mix
Cells with n MNa 0
H2O Paeoniae Radix (0 kGy) Paeoniae Radix (10 kGy) MMC H2O Paeoniae Radix (0 kGy) Paeoniae Radix (10 kGy)
B(a)P
1
2
3
4
No. of MN
MN/1000 cellsb (Mean7S.D.)
500 150 50 500 150 50 0.1
2940 2925 2944 2943 2934 2951 2934 2712
56 68 47 50 60 44 57 247
4 7 5 7 6 5 2 33
0 0 0 0 0 0 2 8
0 0 0 0 0 0 0 0
64 82 57 64 72 54 67 337
21.375.0 27.377.8 19.073.6 21.372.5 24.0712.0 18.072.6 22.374.7 112.3716.1
500 150 50 500 150 50
+ + + + + + +
2944 2938 2934 2927 2951 2954 2957
53 54 59 66 48 45 43
2 8 7 3 1 1 0
1 0 0 1 0 0 0
0 0 0 0 0 0 0
60 70 73 75 50 47 43
20.074.9 23.372.5 24.374.5 25.074.6 16.772.1 15.772.5 14.372.1
+
2656
298
38
7
1
399
133.0720.5
0.1
MMC (Mitomycin C) and B(a)P (benzo(a)pyrene) were used as positive controls. a Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1000 binucleated cells were scored. b Number of MN/1000 binucleated cells in the triplicated experiments.
3.2. No induction of micronuclei in CHO cells by irradiated Paeoniae Radix The induction of micronuclei by the water extract of irradiated Paeoniae Radix was examined in cytokinesisblocked binucleated cells at dosages below 50% cellgrowth inhibition. The frequency of micronuclei in the test group was not significantly different from negative control group and similar to that of non-irradiated sample in both direct non-activated and indirect activated tests (Table 2). From these results, cytogenetic aberration in the nucleus division of the cell might not be induced by the extract of the irradiated Paeoniae Radix. 3.3. Stability of paeoniflorin in the irradiated Paeoniae Radix The paeoniflorin isolated and purified from Paeoniae Radix was analyzed using NMR (results not shown). The structure was confirmed by comparing the obtained data with that of a previous report (Lemmich, 1996). Quantitative HPLC analysis with UV detection was performed under conditions described in Table 3, and had a sufficient sensitivity for quantitative analysis of paeoniflorin in Paeoniae Radix. Paeoniflorin recrystalized in Et2O: MeOH (1:10) showed a purity of 97% in chromatogram by HPLC. The peak-area ratio of
Table 3 Analytical conditions for HPLC analysis of paeoniflorin Instrument System Pump Detector Column Mobile Phase Detection Flow rate (ml/min) Column Temp. ( C) Retention time(min)
Shimadzu HPLC CBM-10A LC-10AD SPD-10A Shim-Pack CLC-ODS(M)25 (4.6 250) Acetonitrile: H2O 20->100 gradient 230 nm 1 Room 7.982
paeoniflorin was proportional to its concentration from 100 to 500 mg/ml under the conditions described above. The results of the quantitative analysis of the paeoniflorin in the irradiated Paeoniae Radix are shown in Table 4 and a chromatogram is shown in Fig. 1. The results showed that the paeoniflorin contents (average of 3 replications) were 4.42% (CV=0.22%) in the irradiated sample and 4.59% (CV=0.45%) in the nonirradiated sample. The chromatogram of paeoniflorin from both the irradiated and non-irradiated samples was similar. Hence both samples showed a good agreement in the structure and the content of paeoniflorin, so it was found that paeoniflorin was not decomposed by the irradiation. Further tests of in vivo genotoxicity and
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Table 4 The content of the paeoniflorin in the g-irradiated Paeoniae Radix Irradiation
Sample (mg)
Paeoniflorin (mg)
Paeoniflorin(%) (n ¼ 3)
S.D.
CV(%)
0 kGy 10 kGy
500 500
22.95 22.10
4.59 4.42
0.01 0.02
0.22 0.45
References
Fig. 1. HPLC profiles of the water extracts of non-irradiated (0 kGy) and irradiated (10 kGy) Paenoiae Radix.
chronic toxicity and the analysis of other components are needed for public acceptance in the application of irradiation for the hygienic technology of the herbs.
Acknowledgements This project was supported by the Nuclear R&D Program from Ministry of Science and Technology of Korea.
Ahmed, M., 1991. Food irradiation, up-to-date status. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture IAEA 6626F, Vienna, November 27. Ami, N., Inaba, M., Watanabe, M., Shibata, S., 1969. Chemical studies on the oriental plant drugs. XXIII. Paeoniflorin, a glucoside of Chinese paeony root. Tetrahedron 25, 1825–1838. Dickman, S., 1991. Compromise eludes EC. Nature 349, 273. Ehlermann, D., 2002. Where is the proof? The science? Nutrition 18, 755–756. Farkas, J., 1998. Irradiation as a method for decontaminating food. Int. J. Food Microbiol. 44, 189–204. Farkas, J., Andrassy, E., Incze, K., 1981. Evaluation of possible mutagenicity of irradiated spices. Acta Aliment 10, 129. Fenech, M., Morley, A., 1985. Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36. Hattori, Y., Mori, M., Kaneko, F., Matsuyama, A., 1979. Mutagenicity tests of irradiated onions by Escherichia coli mutants in vitro. Mutat. Res. 60, 115–119. van Kooij, J.G., Leveling, H.B., Schubert, J., 1978. Application of the Ames mutagenicity test to food precessed by physical preservation methods. In: Food Preservation by Irradiation, Vol. 2. Proceedings of a Symposium, Wageningen, 21–25 November 1977. IAEA, Vienna, p. 63. Lemmich, J., 1996. Monoterpene and coumarin glucosides of Cnidium silaifolium. Phytochemistry 41, 1337–1340. Maron, D.M., Ames, B.N., 1983. Revised methods for Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Preston, R.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J.S., 1981. Mammalian in vivo and in vitro cytogenetic assays: a report of the U.S. EPA’s gene-tox program. Mutat. Res. 87, 143–188. Tritsch, G.L., 2002. Food irradiation: a risk not worth taking. Nutrition 18, 756–758. Uijl, C.H., 1992. Beating the bugs. Int. Food Ingredients 3, 9. WHO, 1992. Surveillance programme for control of foodborne infections and intoxications in Europe. Fifth Report 1985–1989. Institute of Veterinary Medicine––Robert von Ostertag Institute (FAO/WHO Collaboration Centre for Research and Training in Food Hygiene and Zoonosis), Berlin.
Radiation Physics and Chemistry 71 (2004) 115–119
Toxicological safety and stability of the components of an irradiated Korean medicinal herb, Paeoniae Radix Young-Beob Yua, Ill-Yun Jeongb, Hae-Ran Parkb, Heon Ohb, Uhee Jungb, Sung-Kee Job,* a
b
Korea Institute of Oriental Medicine, Yuseong, Daejeon 305-811, Republic of Korea Radiation Application Research Division, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600, Republic of Korea
Abstract As utilization of medicinal herbs in food and bio-industry increases, mass production and the supply of herbs with a high quality are required. As the use of fumigants and preservatives for herbs is being restricted, safe hygienic technologies are demanded. To consider the possibility of the application of irradiation technology for this purpose, the genotoxicological safety and stability of the active components of the g-irradiated Paeoniae Radix were studied. The herb was irradiated with g-rays at a practical dosage of 10 kGy, and then it was extracted with hot water. The genotoxicity of the extract of the irradiated herb was examined in two short-term in vitro tests: (1) Ames test in Salmonella typhimurium; (2) Micronucleus test in cultured Chinese hamster ovary (CHO) cells. The extract of the irradiated herb did not show mutagenicity in the Ames test of the Salmonella reverse mutation assay, and did not show cytogenetic toxicity in the culture of the CHO cells. HPLC chromatogram of paeoniflorin in the irradiated Paeoniae Radix was similar with that of the non-irradiated sample. The quantity of paeoniflorin did not change significantly with irradiation. These results suggest that g-irradiated Paeoniae Radix is toxicologically safe and chemically stable. r 2004 Elsevier Ltd. All rights reserved. Keywords: Paeoniae; Herb; Radiation; Toxicological; Chemical safety
1. Introduction The conventional method of decontamination was fumigation with gaseous ethylene oxide, which is now prohibited or being increasingly restricted in most advanced countries for health, environmental or occupational safety reasons (Uijl, 1992; Dickman, 1991). Food irradiation is increasingly recognized throughout the world as a means of reducing food losses due to microbial spoilage and insect damage. It can also reduce our reliance on the chemical fumigants and preservatives currently used by the food industry, and improve the hygienic quality of various foods and medicinal herbs *Corresponding author. Tel.: +82-42-868-8063; fax: +8242-868-8043. E-mail address: [email protected] (S.-K. Jo).
(Ahmed, 1991; WHO, 1992; Farkas, 1998). There are, however, still some debates about the safety of irradiated foods (Ehlermann, 2002; Tritsch, 2002), and therefore continuous scientific investigations should be performed to ensure the safety and stability of irradiated foods. The root of Paeonia lactiflora pallas (Paeoniae Radix) is one of the most important materials of Chinese crude drugs and has been used as a circulatory tonic and diuretic, and is prescribed for the treatment of abdominal pain. Paeoniflorin, main monoterpene glucoside of Paeoniae Radix, isolated by Ami et al. (1969) is well known as an active component in Paeoniae Radix and has been reported to have a hypotensive, vasodilative, and anti-oxytocic action. The purpose of the present study is to assess the genotoxicological safety and stability of the biological components of irradiated Paeoniae Radix to 10 kGy, by
0969-806X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2004.04.002
ARTICLE IN PRESS 116
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comparing the results with those obtained from the untreated control samples.
number of revertant colonies was more than double that compared with the negative control and was dosedependent.
2. Materials and methods
2.3. Micronuclei test
2.1. Preparation of the extract for safety test
In the Chinese hamster ovary (CHO) cell culture, the induction of micronuclei by the water extract of the irradiated herb was examined by the cytokinesis-block method (Fenech and Morley, 1985) at dosages below 50% cell-growth inhibition. CHO cells were cultured in a RPMI 1640 medium with 10% FBS. Cells were exposed to the test substance in the absence or presence of the S9-mix for 6 h (Preston et al., 1981). After culture, medium was exchanged with fresh medium containing cytochalasin B. Cells were incubated for additional 18 h. After the incubation, the cells were treated with a hypotonic solution and stained with 10% Giemsa. 1000 cells per treatment were scored and the number of cells with micronucleus was determined.
Paeoniae Radix was collected at the cultivation area of Chonnam province in Korea. Voucher specimens of the Paeoniae Radix were deposited in the herbarium of the Oriental Medicine Resources Department of Sunchon National University. For g-irradiation, lots (100 g) of the samples were vacuum-packed in nylon bags and placed in PVC containers (25 cm 30 cm 3 cm). g-Irradiation was carried out in a cobalt-60 irradiator (AECL, IR-79, MDS Nordion International Co. Ltd., Canada) at room temperature (2070.5 C) and operated at a dose rate of 2 kGy/h. The applied dose level was 10 kGy. The absorbed dose was monitored with both free radical and ceric/cerous dosimeters. Non-irradiated and 10 kGy irradiated Paeoniae Radix was prepared by decocting the dried crude drug three times with boiling water (100 g of herb was extracted with 1000 ml of distilled water). The suspensions were spun at 10,000 g for 30 min and the extracts were dried with a speed vacuum. 2.2. Salmonella mutagenicity assay (Ames test) The Salmonella mutagenicity assay (Ames test) was performed according to the method of Maron and Ames (1983). S. typhimurium strains TA98, TA 100 and TA102 were obtained from Professor B.N. Ames (University of California, Berkely, California, USA). Each strain was tested for its genetic traits such as histidine requirement, deep rough (rfa) characteristic, UV sensitivity (uvrB mutation) and ampicillin- or tetracycline-resistance by the R-factor before use. S9 mix was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). The tester strain cultures were grown overnight in the Oxoid Nutrient Broth No. 2 (Oxoid Co. Ltd., Hampshire, England) with a density of 1–2 109 colony forming units (cfu/ml). The doses tested were 62, 185, 556, 1667 and 5000 mg/plate. Each dilution was performed with or without metabolic activation. For metabolic activation, preincubation was provided for 30 min. The positive controls were 4-nitro-o-phenylenediamine (NPD, 20 mg/plate), sodium azide (Na-Azide, 1.5 mg/plate), mitomycin C (MMC, 0.5 mg/plate) and 2-aminofluorene (2-AF, 10 mg/plate) and the negative control was distilled water (DW). The number of revertant colony was counted after 48 h of incubation at 37 C. According to Maron and Ames (1983), a mutagenic effect was acknowledged as positive when the
2.4. Isolation of paeoniflorin The dried and powdered Paeoniae Radix was refluxed with MeOH. The MeOH extract (95 g) was partitioned with H2O and Et2O. The Et2O fraction was subjected to chromatograph using SiO2 with CHCl3:MeOH(9:1) and CHCl3:MeOH(5:1). The CHCl3:MeOH(5:1) soluble fraction was also chromatographed on SiO2 with CHCl3:MeOH(4:1) as solvents to provide crude paeoniflorins and recrystalization in Et2O:MeOH (10:1). The NMR spectra were recorded with a Bruker DRX 300 NMR spectrometer containing TMS as an internal standard and the chemical shifts were given as d (ppm). 2.5. HPLC analysis of the water extract of irradiated Paeoniae Radix About 500 mg of Paeonae Radix was weighed accurately and sonicated in 50 ml of DW for 5 min. After filtration, residue was shaken vigorously in 10 ml of DW and centrifuged at 1000 rpm for 5 min. After repeating the extraction with DW once more, the aqueous fractions were combined and made up to 100 ml with DW. 10 ml of the aqueous solution injected to HPLC. HPLC was carried out with Shimadzu HPLC using Shim-Pack CLC-ODS(M) 25 column. To obtain the standard curve of paeoniflorin, standard paeoniflorin was accurately weighed and dissolved in methanol to provide various concentrations within the range of 100–500 mg/ml. The whole volume of the standard solution was 1 ml. Linearity of the responses was determined for five concentrations with three injections for each level. Calibration graphs were plotted after linear regression analysis of
ARTICLE IN PRESS Y.-B. Yu et al. / Radiation Physics and Chemistry 71 (2004) 115–119
the peak-area ratios with a concentration. The contents of paeoniflorin in the test samples were calculated using the regression parameters obtained from the standard curve.
3. Result and discussion 3.1. No mutagenicity of irradiated Paeoniae Radix The mutagenicity of the water extract of irradiated Paeoniae Radix was examined triplicatedly in Salmonella reversion assay at 5 dosages from 62 to 5000 mg/ plate. The number of revertant colonies of each strain in test group did not increased comparing with negative
117
control group, and the pattern of the revertant colony formation was similar to that of non-irradiated sample. The increase of the colony formation by irradiated sample was not appreciated in both direct non-activated and indirect activated tests (Table 1). From these results, any direct or indirect mutagen might be not formed in Paeoniae Radix by g-irradiation of 10 kGy. In previous studies, many kinds of food stuffs were examined to investigate possible toxic reaction. Most of the experiments produced no toxic effects in the Salmonella reversion assay. van Kooij et al. (1978) did not find any significant increase of mutation between irradiated and non-irradiated fresh vegetables. Hattori et al. (1979) and Farkas et al. (1981) also obtained the same results.
Table 1 Revertant colonies in the S. typhimurium reversion assay with water extract of Paeoniae Radix Test Material
Dose (mg/plate)
H2O
S9 mix
Number of revertant colonies(His+) per plate TA98
TA100
TA102
2379
17076
297758
Paeoniae Radix (0 kGy)
5000 1667 556 185 62
3374 2371 2774 1977 2472
166712 156711 15971 154711 167711
302720 335716 33978 310713 30772
Paeoniae Radix (10 kGy)
5000 1667 556 185 62
31711 3072 1871 1876 2072
15879 154711 14171 143720 154713
30877 322730 30479 29974 313713
2340792
+
2474 3173
22777 25776
309714 342716
5000 1667 556 185 62
+ + + + +
3173 2578 2573 32712 2071
315725 254711 25876 246710 235714
39473 382718 367712 351725 321716
5000 1667 556 185 62 10
+ + + + + +
2973 2471 26710 25711 2571 16417158
30176 22676 22971 247711 223725 909778
41176 37878 350735 329713 319711 531757
NPD Na-Azide MMC
20 1.5 0.5
H2O H2O Paeoniae Radix (0 kGy)
Paeoniae Radix (10 kGy)
2-AF
12297103 51897306
NPD (4-nitro-o-phenylenediamine), Na-Azide(sodium azide), MMC(mitomycin C) and 2-AF (2-aminofluorene) were used as positive controls for the corresponding strains. Values represent mean 7S.D. of revertant colonies per plate in duplicate experiments except for negative control groups (triplicate).
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Table 2 Frequency of micronuclei(MN) in cytokinesis-blocked CHO cells following treatment with water extract of g-irradiated Paeoniae Radix Test material
Conc. (mg/ml)
S9 mix
Cells with n MNa 0
H2O Paeoniae Radix (0 kGy) Paeoniae Radix (10 kGy) MMC H2O Paeoniae Radix (0 kGy) Paeoniae Radix (10 kGy)
B(a)P
1
2
3
4
No. of MN
MN/1000 cellsb (Mean7S.D.)
500 150 50 500 150 50 0.1
2940 2925 2944 2943 2934 2951 2934 2712
56 68 47 50 60 44 57 247
4 7 5 7 6 5 2 33
0 0 0 0 0 0 2 8
0 0 0 0 0 0 0 0
64 82 57 64 72 54 67 337
21.375.0 27.377.8 19.073.6 21.372.5 24.0712.0 18.072.6 22.374.7 112.3716.1
500 150 50 500 150 50
+ + + + + + +
2944 2938 2934 2927 2951 2954 2957
53 54 59 66 48 45 43
2 8 7 3 1 1 0
1 0 0 1 0 0 0
0 0 0 0 0 0 0
60 70 73 75 50 47 43
20.074.9 23.372.5 24.374.5 25.074.6 16.772.1 15.772.5 14.372.1
+
2656
298
38
7
1
399
133.0720.5
0.1
MMC (Mitomycin C) and B(a)P (benzo(a)pyrene) were used as positive controls. a Total number of cytokinesis-blocked(CB) binucleated cells with n MN in the triplicated experiments which 1000 binucleated cells were scored. b Number of MN/1000 binucleated cells in the triplicated experiments.
3.2. No induction of micronuclei in CHO cells by irradiated Paeoniae Radix The induction of micronuclei by the water extract of irradiated Paeoniae Radix was examined in cytokinesisblocked binucleated cells at dosages below 50% cellgrowth inhibition. The frequency of micronuclei in the test group was not significantly different from negative control group and similar to that of non-irradiated sample in both direct non-activated and indirect activated tests (Table 2). From these results, cytogenetic aberration in the nucleus division of the cell might not be induced by the extract of the irradiated Paeoniae Radix. 3.3. Stability of paeoniflorin in the irradiated Paeoniae Radix The paeoniflorin isolated and purified from Paeoniae Radix was analyzed using NMR (results not shown). The structure was confirmed by comparing the obtained data with that of a previous report (Lemmich, 1996). Quantitative HPLC analysis with UV detection was performed under conditions described in Table 3, and had a sufficient sensitivity for quantitative analysis of paeoniflorin in Paeoniae Radix. Paeoniflorin recrystalized in Et2O: MeOH (1:10) showed a purity of 97% in chromatogram by HPLC. The peak-area ratio of
Table 3 Analytical conditions for HPLC analysis of paeoniflorin Instrument System Pump Detector Column Mobile Phase Detection Flow rate (ml/min) Column Temp. ( C) Retention time(min)
Shimadzu HPLC CBM-10A LC-10AD SPD-10A Shim-Pack CLC-ODS(M)25 (4.6 250) Acetonitrile: H2O 20->100 gradient 230 nm 1 Room 7.982
paeoniflorin was proportional to its concentration from 100 to 500 mg/ml under the conditions described above. The results of the quantitative analysis of the paeoniflorin in the irradiated Paeoniae Radix are shown in Table 4 and a chromatogram is shown in Fig. 1. The results showed that the paeoniflorin contents (average of 3 replications) were 4.42% (CV=0.22%) in the irradiated sample and 4.59% (CV=0.45%) in the nonirradiated sample. The chromatogram of paeoniflorin from both the irradiated and non-irradiated samples was similar. Hence both samples showed a good agreement in the structure and the content of paeoniflorin, so it was found that paeoniflorin was not decomposed by the irradiation. Further tests of in vivo genotoxicity and
ARTICLE IN PRESS Y.-B. Yu et al. / Radiation Physics and Chemistry 71 (2004) 115–119
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Table 4 The content of the paeoniflorin in the g-irradiated Paeoniae Radix Irradiation
Sample (mg)
Paeoniflorin (mg)
Paeoniflorin(%) (n ¼ 3)
S.D.
CV(%)
0 kGy 10 kGy
500 500
22.95 22.10
4.59 4.42
0.01 0.02
0.22 0.45
References
Fig. 1. HPLC profiles of the water extracts of non-irradiated (0 kGy) and irradiated (10 kGy) Paenoiae Radix.
chronic toxicity and the analysis of other components are needed for public acceptance in the application of irradiation for the hygienic technology of the herbs.
Acknowledgements This project was supported by the Nuclear R&D Program from Ministry of Science and Technology of Korea.
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