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Is the yin–yang nature of Chinese herbal medicine equivalent to antioxidation–oxidation?
Journal of Ethnopharmacology 108 (2006) 361–366
Is the yin–yang nature of Chinese herbal medicine equivalent to antioxidation–oxidation? Yim-Tong Szeto a,b,∗ , Iris F.F. Benzie b a
School of Health Sciences, Macao Polytechnic Institute, Alameda Dr. Carlos D’Assump¸ca˜ o, No. 335-341 Edif. Centro Hotline, 5◦ Andar, Macao b Department of Health Technology & Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China Received 21 January 2006; received in revised form 17 May 2006; accepted 20 May 2006 Available online 23 June 2006
Abstract It has been suggested that yin–yang theory described in traditional Chinese medicine is somewhat equivalent to the modern theory of antioxidant–oxidant balance. Some yin-tonic Chinese herbal medicines possess antioxidant properties. In this context, the DNA protective effect of 12 yin-tonic and 13 yang-tonic herbs were tested using the single cell gel electrophoresis (comet) assay. Lymphocytes from three healthy subjects were pre-incubated with aqueous herb extract, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide. Results using this ex vivo cellular assay showed protection by some herbs. Seven out of 12 yin-tonic Chinese herbs demonstrated decreased DNA damage after treatment while 10 out of 13 yang-tonic herbs showed protection. Among 25 herbs tested, rhizome of Ligusticum sinensis Oliv. and aerial part of Artemisia annua L. demonstrated greatest DNA protective effect. Results indicated that the yin nature of herbs may not be necessarily associated with superior antioxidative effect to yang-tonic herbs, at least in terms of DNA protection against oxidant challenge. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Antioxidant; Comet assay; Traditional Chinese medicine; Herb; DNA; Oxidative stress
1. Introduction Traditional Chinese herbal medicine (TCM) has been used for thousands of years and attention is being paid increasingly by the western world for the more widespread utilization of this ancient method of treatment. However, the ideology underlying traditional Chinese medicine is quite different from that of the modern/western world, which makes the global application or marketing of TCM limited. Seeking to widen the usage of TCM, some scientists investigate the therapeutic effects of TCM and try to align these with the concepts of modern medicine, validating the potential beneficial effects of different types of herbs. It has been proposed that the yin–yang relationship, which includes the concepts of balance, harmony and homeostatis, is actually the antioxidant–oxidant balance concept described in modern medicine (Ou et al., 2003; Van Dyke et al., 1998). One of these studies correlated the antioxidant potential (by oxy-
∗
Corresponding author. Tel.: +852 92892854; fax: +852 753159. E-mail addresses: [email protected], [email protected] (Y.-T. Szeto). 0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.05.033
gen radical absorbance capacity) of a range of Chinese herbs and compared the antioxidant power of yin-tonic and yangtonic herbs. It was suggested that yin-tonic herbs were herbs rich in antioxidants, e.g. polyphenolics, which contributed the yin-tonic or antioxidative effect (Ou et al., 2003). In this context, assessment of a more specific antioxidative effect, such as DNA protection against oxidant challenge, of yin and yang type herbs would be of interest. A useful method of assessing DNA damage is the single cell gel electrophoresis, or ‘comet’ assay, which detects DNA strand breaks in individual cells (Collins et al., 1997). In brief, relaxed DNA containing strand breaks is pulled towards the anode during electrophoresis, forming a ‘comet’ tail. The level of DNA in the tail is related to the degree of DNA damage (Collins et al., 1997; Szeto and Benzie, 2002). This assay is useful, versatile and in controlled experiments allows direct comparison of ex vivo effects, and has been used successfully in biomonitoring and antioxidant studies (Panayiotidis and Collins, 1997; Noroozi et al., 1998; Szeto et al., 2002; Szeto and Benzie, 2002; Szeto et al., 2005). The aim of the current study was to use this cellular assay to assess ex vivo DNA protective effects of selected Chinese herbs to investigate whether DNA protective activity of Yin-
362
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tonic herbs is greater than that of Yang-tonic herbs. The 12 yintonic and 13 yang-tonic Chinese herbal medicines tested by Ou et al. (2003) were studied for the sake of comparison.
2.4. Treatments For herbal treatment and oxidant challenge of lymphocytes, 1 ml of PBS [with or without herbal extract at between
2. Materials and methods 2.1. Chemicals The highest purity grades of the following reagents or materials were purchased: Fetal bovine serum (FBS) was from HyClone, Logan, Utah; RPMI 1640 without phenol red, Histopaque 1077, dimethyl sulfoxide (DMSO), type VII low gelling point agarose, phosphate buffered saline (PBS) tablets, hydrogen peroxide solution, Triton X-100, disodium ethylenediaminetetraacetic acid dihydrate, and sodium chloride, were from Sigma, St. Louis, MO, USA; agarose 3:1 was from Amresco, Solon, OH, USA; hydrochloric acid and sodium hydroxide were from Merck, Darmstadt, Germany. Tris was from BioBasic Inc., Markham, Ontario, Canada; ethidium bromide was from Promega, Madison, WI, USA. 2.2. Herbs All herbal medicines were from Wai Yuen Tong (retail) Ltd., Hong Kong. Herbs were identified by the Chinese Medicine Practioner and Associate Professor, Dr. Ken GU, School of Health Sciences, Macao Polytechnic Institute. Voucher specimens were deposited in the School of Health Sciences, Macao Polytechnic Institute. For the preparation of aqueous herbal extracts, herbs were cut into small pieces and 2 g of each herb was added to 200 ml boiling deionized water (NANOpure Diamond, Barnstead/Thermolyne, Dubuque, IA, USA) and boiled for 30 min. The extract was filtered through Whatman no. 1 filter paper and the volume of filtrate was made up to 200 ml with NANOpure water (i.e. final concentration 1% (w/v), known as initial extract). Aqueous extracts of herbs were kept at 4 ◦ C until used. Herb extracts were further diluted in PBS to 10−2 , 10−3 , 10−4 , 10−5 , 10−6 % (w/v), before testing. 2.3. Cells Lymphocytes from three healthy subjects (two males, one female) were harvested from venous blood following the procedure described previously (Szeto and Benzie, 2002). Ten ml of blood was added to 10 ml RPMI medium in a 50 ml culture tube. Blood and medium were mixed gently before layering with 20 ml Histopaque 1077. Tubes were spun at 700 g for 30 min at 20 ◦ C. Lymphocytes were retrieved from just above the boundary between the RPMI and Histopaque layers, and added to 20 ml RPMI medium in the other culture tube. The centrifugation step was repeated for 30 min at 4 ◦ C and as much supernatant as possible was removed from the pelleted lymphocytes. Five ml of cold freezing medium (4.5 ml FBS + 0.5 ml DMSO) was added to the lymphocytes. Cells were gently mixed and aliquoted into microtubes and frozen slowly to −65 ◦ C for storage. Cells from each individual were tested, in separate experiments, with each test herb within 3 months of cryopreservation.
Fig. 1. Representative comet images showing different levels of damage in visual scoring: (a) grade 0 (undamaged DNA); (b) grade 1; (c) grade 2; (d) grade 3; (e) grade 4 (severe damage).
Y.-T. Szeto, I.F.F. Benzie / Journal of Ethnopharmacology 108 (2006) 361–366
1 × 10−2 % and 1 × 10−6 % (w/v)] was added to the tube containing washed lymphocytes and gently mixed. The cell suspension was incubated at 37 ◦ C for 30 min. The tubes were then centrifuged at 200 × g for 5 min at room temperature, the supernatant was discarded, cells were washed once with 1 ml cold PBS and the centrifugation step was repeated. Herbal extracttreated lymphocytes were stressed in the tube with 1 ml of 60 M cold hydrogen peroxide in PBS for 5 min. The cell suspension was then washed by centrifugation. Finally, the comet assay was performed following the procedure of Szeto and Benzie (2002). 2.5. Scoring One hundred cells were scored per treatment in each of three independent experiments, each series using cells from a different subject, i.e. 300 cells for each herb at each dose tested. Analysis of comets was performed using a fluorescence microscope (Olympus CH30, Tokyo, Japan, fitted with DMG-2 filter set) by manual scoring (grades 0–4, where grade 4 signified the greatest damage, see Fig. 1). 2.6. Statistical analysis The unpaired t-test was used to investigate differences in score between cells with or without pre-treatment with various concentrations of extract. Prism 4.0 (GraphPad Software, San Diego, CA, USA) was used.
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3. Results None of the herbs tested induced DNA strand breaks between 1 × 10−2 % and 1 × 10−6 % (w/v). Ten of the 13 yang-tonic and 7 of the 12 yin-tonic herbs were protective against oxidant challenge-induced DNA damage (Table 1). There was no apparent relationship between the antioxidative capacity (assessed as ORAC value) and the DNA protective effect. Among those herbs offering DNA protective effect, rhizome of Ligusticum sinensis Oliv., a yang-tonic herb, demonstrated the highest ability to enhance resistance of cellular DNA to oxidative challenge, with the effect seen over a wide range of concentrations (1 × 10−2 to 1 × 10−6 % (w/v)) (Fig. 2b). A yin-tonic herb, aerial part of Artemisia annua L. also demonstrated effective DNA protection from 1 × 10−2 to 1 × 10−6 % (w/v) (Fig. 2a). The comet assay results of individual herbs and dilutions are shown in Table 2. 4. Discussion In this study, results showed that Ligusticum sinensis Oliv. (Rhizoma ligustici) and Artemisia annua L. (Herba artemisiae annuae) possess strong DNA protective effect against oxidative stress (see Szeto and Benzie, 2002 on other DNA protective agents for comparison purpose). Rhizome of Ligusticum sinensis Oliv. is an analgesic. In TCM theory, this herb ‘dissipates wind and dispels dampness’ (Xu and Wang, 2002). It can also be used
Table 1 A comparison of DNA protective effect and in vitro antioxidant level of yin- and yang-tonic Chinese herbs DNA protection (herb in any dilution showing significant decrease in DNA damage after oxidant challenge)
ORAC values (mol TE/g) (source: Ou et al., 2003)
Yin-tonic herbs Bark of Phellodendron chinense Schneid Fruit of Forsythia suspensa (Thunb.) Vahl. Root bark of Paeonia suffruticosa Andr. Artemisia scoparia Waldst. et Kit. Flower bud of Chrysanthemum morifolium RAMAT. Flower bud of Lonicera japonica Thunb. Aerial part of Artemisia annua L. Rhizome of Coptis chinensis Franch. Root of Salvia miltiorrhiza Bge. Rhizome of Belamcanda chinensis (L.) DC. Root of Scutellaria baicalensis Georgi Root of Rheum palmatum LINN
− − + − + + + + − − + +
433 450 473 521 525 760 841 855 1248 1672 1684 1939
Yang-tonic herbs Processed root of Aconitum carmichaeli Debx. Root of Saposhnikovia divaricata (Turcz.) Schischk. Root of Codonopsis tangshen Oliv. Root of Angelica dahurica (Fisc.) Benth. et Hook. f. Rhizome of Cibotium barometz (L.) J. Sm. Ripe fruit of Alpinia oxyphylla Miq. Ripe seed of Cuscuta chinensis Lam. Root of Dipsacus asperoides C. Y. Cheng et T. M. Ai Cinnamomum cassia Presl Asarum sieboldii Miq. Fleshy stem with scale of Cistanche deserticola YC Ma. Rhizome of Ligusticum sinensis Oliv. Curculigo orchioides Gaertn.
− + + + − + − + + + + + +
16 42 47 83 112 113 174 193 214 235 235 240 243
364 Table 2 Effect of pre-treatment with herbal extracts on DNA damage in oxidatively challenged cells in the comet assay; results are expressed as % of DNA damage seen in challenged cells without pre-treatment with the herb Dilution of initial extract (1%, w/v)
%DNA damage after H2 O2 treatment compared to cells without pre-treatment with herb of interest: results are mean (S.D.), n = 3 10−6 %
10−5 %
10−4 %
10−3 %
10−2 %
Yin-tonic herbs Bark of Phellodendron chinense Schneid Fruit of Forsythia suspensa (Thunb.) Vahl. Root bark of Paeonia suffruticosa Andr. Artemisia scoparia Waldst. et Kit. Flower bud of Chrysanthemum morifolium RAMAT. Flower bud of Lonicera japonica Thunb. Aerial part of Artemisia annua L. Rhizome of Coptis chinensis Franch. Root of Salvia miltiorrhiza Bge. Rhizome of Belamcanda chinensis (L.) DC. Root of Scutellaria baicalensis Georgi Root of Rheum palmatum LINN
0.039 0.058 0.114 0.097 0.274 0.302 0.147 0.245 0.525 0.132 0.128 0.659
93 (10) 88 (10) 90 (10) 97 (21) 98 (9) 81 (16) 81 (21) 87 (3)** 90 (19) 109 (6) 89 (13) 102 (16)
83 (20) 100 (14) 90 (14) 104 (7) 91 (2)** 80 (24) 86 (2)*** 75 (15)* 85 (10) 100 (11) 96 (16) 103 (13)
90 (5) 93 (9) 91 (11)** 95 (10) 96 (3) 87 (8)* 71 (12)* 88 (9) 97 (14) 104 (22) 89 (12) 100 (2)
110 (11) 95 (9) 92 (9) 105 (4) 99 (14) 88 (8) 77 (4)*** 96 (18) 91 (25) 95 (15) 96 (7) 99 (5)
93 (13) 92 (25) 91 (10) 101 (14) 93 (12) 69 (22) 72 (8)** 71 (22) 98 (5) 84 (20) 87 (8)* 84 (7)*
Yang-tonic herbs Processed root of Aconitum carmichaeli Debx. Root of Saposhnikovia divaricata (Turcz.) Schischk. Root of Codonopsis tangshen Oliv. Root of Angelica dahurica (Fisc.) Benth. et Hook. f. Rhizome of Cibotium barometz (L.) J. Sm. Ripe fruit of Alpinia oxyphylla Miq. Ripe seed of Cuscuta chinensis Lam. Root of Dipsacus asperoides C. Y. Cheng et T. M. Ai Cinnamomum cassia Presl Asarum sieboldii Miq. Fleshy stem with scale of Cistanche deserticola YC Ma. Rhizome of Ligusticum sinensis Oliv. Curculigo orchioides Gaertn.
0.110 0.194 0.212 0.129 0.279 0.124 0.109 0.449 0.024 0.042 0.485 0.145 0.093
81 (13) 87 (16) 73 (23) 90 (1)*** 81 (13) 81 (5)** 97 (4) 90 (23) 92 (3)* 72 (7)*** 92 (8) 88 (2)*** 89 (6)
88 (10) 88 (3)** 88 (20) 79 (10)* 88 (19) 87 (15) 94 (11) 93 (20) 91 (12) 86 (8)* 89 (5)* 82 (6)** 84 (6)**
85 (23) 84 (15) 75 (9)** 88 (9) 94 (10) 64 (31) 91 (8) 74 (13)* 87 (22) 85 (14) 84 (18) 64 (18)* 80 (14)
* p < 0.05; ** p < 0.01; *** p < 0.001,
compared to control cells, unpaired t-test.
91 (10) 87 (5)** 89 (14) 67 (24) 92 (31) 75 (15)* 98 (10) 84 (23) 94 (14) 85 (28) 99 (7) 86 (1)*** 78 (8)*
76 (21) 88 (15) 93 (16) 76 (15) 92 (19) 95 (9) 100 (15) 91 (11) 90 (19) 93 (9) 102 (17) 81 (7)* 86 (12)
Y.-T. Szeto, I.F.F. Benzie / Journal of Ethnopharmacology 108 (2006) 361–366
Soluble content in initial extract (g/100 ml)
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medicine. None of the herbs tested induced DNA damage at any of the concentrations tested. The mechanism of protection and the active ingredients of the herbs are not clear, but the effect did not seem to be related to their measured antioxidant capacity as assessed using the ORAC radical scavenging method. Further, yin-tonic herbs, which have been suggested to be antioxidative, did not in general show superior DNA protective effects to yang-tonic herbs (Ou et al., 2003). Results do not support the proposal that yin–yang equates to the antioxidation–oxidation concept, at least, in terms of DNA protection against oxidant challenge. Acknowledgments The authors wish to thank the Macao Polytechnic Institute and The Hong Kong Polytechnic University for supporting this work and Miss Chan Hong for her technical assistance. References
Fig. 2. Effect of aqueous extracts of (a) Artemisia apiacea Hance and (b) Ligusticum sinensis Oliv. pretreatment on DNA of human lymphocytes using the comet assay. Results shown expressed as % of DNA damage in control cells and are mean + 1S.D. of data obtained in three separate experiments. Cells were pre-incubated in different dilutions of herb extract followed by H2 O2 challenge (60 M); * p < 0.05; ** p < 0.01; *** p < 0.001, compared to control cells, unpaired t-test.
for treating cardiovascular disorders (Rhyu et al., 2004; Zhu et al., 2004). It has been reported that Rhizoma ligustici contains antioxidants such as -pinene, ␣-terpinene and terpine-4-ol (Xu and Wang, 2002) which showed hydroxyl radical scavenging effect or lipid peroxidation inhibition (Burits and Bucar, 2000; Candan et al., 2003; Foti and Ingold, 2003). Herba artemisiae annuae also exhibited a strong DNA protective effect. Herba artemisiae annuae is the dried aerial part of Artemisia annua L. Its actions include ‘removing summerheat and relieve fever due to Yin deficiency’ (Xu and Wang, 2002), and it is used in the treatment of malaria (Mueller et al., 2004), as justified by its content of artemisinin. The ingredients of this herb include sesquiterpenes, flavones and volatile oil and coumarin (Xu and Wang, 2002), some of which possess antioxidant activity (Fylaktakidou et al., 2004). 5. Conclusions In conclusion, our results using an ex vivo cellular assay indicate that DNA of human lymphocytes is protected from oxidant challenge by several yin and yang-type herbs used in Chinese
Burits, M., Bucar, F., 2000. Antioxidant activity of Nigella sativa essential oil. Phytotherapy Research 14, 323–328. Candan, F., Unlu, M., Tepe, B., Daferera, D., Polissiou, M., Sokmen, A., Akpulat, H.A., 2003. Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. Millefolium Afan. (Asteraceae). Journal of Ethnopharmacology 87, 215–220. Collins, A.R., Dusinska, M., Franklin, M., Somorovska, M., Petrovska, H., Duthie, S., Fillion, L., Panayiotidis, M., Raslova, K., Vaughan, N., 1997. Comet assay in human biomonitoring studies: Reliability, validation, and applications. Environmental & Molecular Mutagenesis 30, 139– 146. Foti, M.C., Ingold, K.U., 2003. Mechanism of inhibition of lipid peroxidation by γ-terpinene, an unusual and potentially useful hydrocarbon antioxidant. Journal of Agricultural and Food Chemistry 51, 2758–2765. Fylaktakidou, K.C., Hadjipavlou-Litina, D.J., Litinas, K.E., Nicolaides, D.N., 2004. Natural and synthetic coumarin derivatives with antiinflammatory/antioxidant activities. Current Pharmaceutical Design 10, 3813–3833. Mueller, M.S., Runyambo, N., Wagner, I., Borrmann, S., Dietz, K., Heide, L., 2004. Randomized controlled trial of a traditional preparation of Artemisia annua L. (Annual Wormwood) in the treatment of malaria. Transactions of the Royal Society of Tropical Medicine & Hygiene 98, 318– 321. Noroozi, M., Angerson, W.J., Lean, M.E.J., 1998. Effects of flavonoids and vitamin C on oxidative damage to human lymphocytes. American Journal of Clinical Nutrition 67, 1210–1218. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., 2003. When east meet west: the relationship between yin–yang and antioxidation–oxidation. FASEB Journal 17, 127–129. Panayiotidis, M., Collins, A.R., 1997. Ex vivo assessment of lymphocyte antioxidant status using comet assay. Free Radical Research 27, 533– 537. Rhyu, M.R., Kim, E.Y., Kim, B., 2004. Nitric oxide-mediated vasorelaxation by Rhizoma Ligustici wallichii in isolated rat thoracic aorta. Phytomedicine 11, 51–55. Szeto, Y.T., Benzie, I.F.F., Collins, A.R., Choi, S.W., Cheng, C.Y., Yow, C.M.N., Tse, M.M.Y., 2005. A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutrition studies. Mutation Research 578, 371–381. Szeto, Y.T., Benzie, I.F.F., 2002. Effects of dietary antioxidants on human DNA ex vivo. Free Radical Research 36, 113–118. Szeto, Y.T., Collins, A.R., Benzie, I.F.F., 2002. Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure. Mutation Research 500, 31–38.
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Van Dyke, K., Sacks, M., Qazi, N., 1998. A new screening method to detect water-soluble antioxidants: acetaminophen (Tylenol) and other phenols react as antioxidants and destroy peroxynitrite-based luminol-dependent chemiluminescence. Journal of Bioluminescence & Chemiluminescence 13, 339–348.
Xu, L., Wang, W., 2002. Chinese Materia Medica: Combinations and Applications. St. Albans, Herts: Donica. Zhu, Y.Z., Huang, S.H., Tan, B.K., Sun, J., Whiteman, M., Zhu, Y.C., 2004. Antioxidants in Chinese herbal medicines: a biochemical perspective. Natural Product Reports 21, 478–489.
Is the yin–yang nature of Chinese herbal medicine equivalent to antioxidation–oxidation? Yim-Tong Szeto a,b,∗ , Iris F.F. Benzie b a
School of Health Sciences, Macao Polytechnic Institute, Alameda Dr. Carlos D’Assump¸ca˜ o, No. 335-341 Edif. Centro Hotline, 5◦ Andar, Macao b Department of Health Technology & Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China Received 21 January 2006; received in revised form 17 May 2006; accepted 20 May 2006 Available online 23 June 2006
Abstract It has been suggested that yin–yang theory described in traditional Chinese medicine is somewhat equivalent to the modern theory of antioxidant–oxidant balance. Some yin-tonic Chinese herbal medicines possess antioxidant properties. In this context, the DNA protective effect of 12 yin-tonic and 13 yang-tonic herbs were tested using the single cell gel electrophoresis (comet) assay. Lymphocytes from three healthy subjects were pre-incubated with aqueous herb extract, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide. Results using this ex vivo cellular assay showed protection by some herbs. Seven out of 12 yin-tonic Chinese herbs demonstrated decreased DNA damage after treatment while 10 out of 13 yang-tonic herbs showed protection. Among 25 herbs tested, rhizome of Ligusticum sinensis Oliv. and aerial part of Artemisia annua L. demonstrated greatest DNA protective effect. Results indicated that the yin nature of herbs may not be necessarily associated with superior antioxidative effect to yang-tonic herbs, at least in terms of DNA protection against oxidant challenge. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Antioxidant; Comet assay; Traditional Chinese medicine; Herb; DNA; Oxidative stress
1. Introduction Traditional Chinese herbal medicine (TCM) has been used for thousands of years and attention is being paid increasingly by the western world for the more widespread utilization of this ancient method of treatment. However, the ideology underlying traditional Chinese medicine is quite different from that of the modern/western world, which makes the global application or marketing of TCM limited. Seeking to widen the usage of TCM, some scientists investigate the therapeutic effects of TCM and try to align these with the concepts of modern medicine, validating the potential beneficial effects of different types of herbs. It has been proposed that the yin–yang relationship, which includes the concepts of balance, harmony and homeostatis, is actually the antioxidant–oxidant balance concept described in modern medicine (Ou et al., 2003; Van Dyke et al., 1998). One of these studies correlated the antioxidant potential (by oxy-
∗
Corresponding author. Tel.: +852 92892854; fax: +852 753159. E-mail addresses: [email protected], [email protected] (Y.-T. Szeto). 0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.05.033
gen radical absorbance capacity) of a range of Chinese herbs and compared the antioxidant power of yin-tonic and yangtonic herbs. It was suggested that yin-tonic herbs were herbs rich in antioxidants, e.g. polyphenolics, which contributed the yin-tonic or antioxidative effect (Ou et al., 2003). In this context, assessment of a more specific antioxidative effect, such as DNA protection against oxidant challenge, of yin and yang type herbs would be of interest. A useful method of assessing DNA damage is the single cell gel electrophoresis, or ‘comet’ assay, which detects DNA strand breaks in individual cells (Collins et al., 1997). In brief, relaxed DNA containing strand breaks is pulled towards the anode during electrophoresis, forming a ‘comet’ tail. The level of DNA in the tail is related to the degree of DNA damage (Collins et al., 1997; Szeto and Benzie, 2002). This assay is useful, versatile and in controlled experiments allows direct comparison of ex vivo effects, and has been used successfully in biomonitoring and antioxidant studies (Panayiotidis and Collins, 1997; Noroozi et al., 1998; Szeto et al., 2002; Szeto and Benzie, 2002; Szeto et al., 2005). The aim of the current study was to use this cellular assay to assess ex vivo DNA protective effects of selected Chinese herbs to investigate whether DNA protective activity of Yin-
362
Y.-T. Szeto, I.F.F. Benzie / Journal of Ethnopharmacology 108 (2006) 361–366
tonic herbs is greater than that of Yang-tonic herbs. The 12 yintonic and 13 yang-tonic Chinese herbal medicines tested by Ou et al. (2003) were studied for the sake of comparison.
2.4. Treatments For herbal treatment and oxidant challenge of lymphocytes, 1 ml of PBS [with or without herbal extract at between
2. Materials and methods 2.1. Chemicals The highest purity grades of the following reagents or materials were purchased: Fetal bovine serum (FBS) was from HyClone, Logan, Utah; RPMI 1640 without phenol red, Histopaque 1077, dimethyl sulfoxide (DMSO), type VII low gelling point agarose, phosphate buffered saline (PBS) tablets, hydrogen peroxide solution, Triton X-100, disodium ethylenediaminetetraacetic acid dihydrate, and sodium chloride, were from Sigma, St. Louis, MO, USA; agarose 3:1 was from Amresco, Solon, OH, USA; hydrochloric acid and sodium hydroxide were from Merck, Darmstadt, Germany. Tris was from BioBasic Inc., Markham, Ontario, Canada; ethidium bromide was from Promega, Madison, WI, USA. 2.2. Herbs All herbal medicines were from Wai Yuen Tong (retail) Ltd., Hong Kong. Herbs were identified by the Chinese Medicine Practioner and Associate Professor, Dr. Ken GU, School of Health Sciences, Macao Polytechnic Institute. Voucher specimens were deposited in the School of Health Sciences, Macao Polytechnic Institute. For the preparation of aqueous herbal extracts, herbs were cut into small pieces and 2 g of each herb was added to 200 ml boiling deionized water (NANOpure Diamond, Barnstead/Thermolyne, Dubuque, IA, USA) and boiled for 30 min. The extract was filtered through Whatman no. 1 filter paper and the volume of filtrate was made up to 200 ml with NANOpure water (i.e. final concentration 1% (w/v), known as initial extract). Aqueous extracts of herbs were kept at 4 ◦ C until used. Herb extracts were further diluted in PBS to 10−2 , 10−3 , 10−4 , 10−5 , 10−6 % (w/v), before testing. 2.3. Cells Lymphocytes from three healthy subjects (two males, one female) were harvested from venous blood following the procedure described previously (Szeto and Benzie, 2002). Ten ml of blood was added to 10 ml RPMI medium in a 50 ml culture tube. Blood and medium were mixed gently before layering with 20 ml Histopaque 1077. Tubes were spun at 700 g for 30 min at 20 ◦ C. Lymphocytes were retrieved from just above the boundary between the RPMI and Histopaque layers, and added to 20 ml RPMI medium in the other culture tube. The centrifugation step was repeated for 30 min at 4 ◦ C and as much supernatant as possible was removed from the pelleted lymphocytes. Five ml of cold freezing medium (4.5 ml FBS + 0.5 ml DMSO) was added to the lymphocytes. Cells were gently mixed and aliquoted into microtubes and frozen slowly to −65 ◦ C for storage. Cells from each individual were tested, in separate experiments, with each test herb within 3 months of cryopreservation.
Fig. 1. Representative comet images showing different levels of damage in visual scoring: (a) grade 0 (undamaged DNA); (b) grade 1; (c) grade 2; (d) grade 3; (e) grade 4 (severe damage).
Y.-T. Szeto, I.F.F. Benzie / Journal of Ethnopharmacology 108 (2006) 361–366
1 × 10−2 % and 1 × 10−6 % (w/v)] was added to the tube containing washed lymphocytes and gently mixed. The cell suspension was incubated at 37 ◦ C for 30 min. The tubes were then centrifuged at 200 × g for 5 min at room temperature, the supernatant was discarded, cells were washed once with 1 ml cold PBS and the centrifugation step was repeated. Herbal extracttreated lymphocytes were stressed in the tube with 1 ml of 60 M cold hydrogen peroxide in PBS for 5 min. The cell suspension was then washed by centrifugation. Finally, the comet assay was performed following the procedure of Szeto and Benzie (2002). 2.5. Scoring One hundred cells were scored per treatment in each of three independent experiments, each series using cells from a different subject, i.e. 300 cells for each herb at each dose tested. Analysis of comets was performed using a fluorescence microscope (Olympus CH30, Tokyo, Japan, fitted with DMG-2 filter set) by manual scoring (grades 0–4, where grade 4 signified the greatest damage, see Fig. 1). 2.6. Statistical analysis The unpaired t-test was used to investigate differences in score between cells with or without pre-treatment with various concentrations of extract. Prism 4.0 (GraphPad Software, San Diego, CA, USA) was used.
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3. Results None of the herbs tested induced DNA strand breaks between 1 × 10−2 % and 1 × 10−6 % (w/v). Ten of the 13 yang-tonic and 7 of the 12 yin-tonic herbs were protective against oxidant challenge-induced DNA damage (Table 1). There was no apparent relationship between the antioxidative capacity (assessed as ORAC value) and the DNA protective effect. Among those herbs offering DNA protective effect, rhizome of Ligusticum sinensis Oliv., a yang-tonic herb, demonstrated the highest ability to enhance resistance of cellular DNA to oxidative challenge, with the effect seen over a wide range of concentrations (1 × 10−2 to 1 × 10−6 % (w/v)) (Fig. 2b). A yin-tonic herb, aerial part of Artemisia annua L. also demonstrated effective DNA protection from 1 × 10−2 to 1 × 10−6 % (w/v) (Fig. 2a). The comet assay results of individual herbs and dilutions are shown in Table 2. 4. Discussion In this study, results showed that Ligusticum sinensis Oliv. (Rhizoma ligustici) and Artemisia annua L. (Herba artemisiae annuae) possess strong DNA protective effect against oxidative stress (see Szeto and Benzie, 2002 on other DNA protective agents for comparison purpose). Rhizome of Ligusticum sinensis Oliv. is an analgesic. In TCM theory, this herb ‘dissipates wind and dispels dampness’ (Xu and Wang, 2002). It can also be used
Table 1 A comparison of DNA protective effect and in vitro antioxidant level of yin- and yang-tonic Chinese herbs DNA protection (herb in any dilution showing significant decrease in DNA damage after oxidant challenge)
ORAC values (mol TE/g) (source: Ou et al., 2003)
Yin-tonic herbs Bark of Phellodendron chinense Schneid Fruit of Forsythia suspensa (Thunb.) Vahl. Root bark of Paeonia suffruticosa Andr. Artemisia scoparia Waldst. et Kit. Flower bud of Chrysanthemum morifolium RAMAT. Flower bud of Lonicera japonica Thunb. Aerial part of Artemisia annua L. Rhizome of Coptis chinensis Franch. Root of Salvia miltiorrhiza Bge. Rhizome of Belamcanda chinensis (L.) DC. Root of Scutellaria baicalensis Georgi Root of Rheum palmatum LINN
− − + − + + + + − − + +
433 450 473 521 525 760 841 855 1248 1672 1684 1939
Yang-tonic herbs Processed root of Aconitum carmichaeli Debx. Root of Saposhnikovia divaricata (Turcz.) Schischk. Root of Codonopsis tangshen Oliv. Root of Angelica dahurica (Fisc.) Benth. et Hook. f. Rhizome of Cibotium barometz (L.) J. Sm. Ripe fruit of Alpinia oxyphylla Miq. Ripe seed of Cuscuta chinensis Lam. Root of Dipsacus asperoides C. Y. Cheng et T. M. Ai Cinnamomum cassia Presl Asarum sieboldii Miq. Fleshy stem with scale of Cistanche deserticola YC Ma. Rhizome of Ligusticum sinensis Oliv. Curculigo orchioides Gaertn.
− + + + − + − + + + + + +
16 42 47 83 112 113 174 193 214 235 235 240 243
364 Table 2 Effect of pre-treatment with herbal extracts on DNA damage in oxidatively challenged cells in the comet assay; results are expressed as % of DNA damage seen in challenged cells without pre-treatment with the herb Dilution of initial extract (1%, w/v)
%DNA damage after H2 O2 treatment compared to cells without pre-treatment with herb of interest: results are mean (S.D.), n = 3 10−6 %
10−5 %
10−4 %
10−3 %
10−2 %
Yin-tonic herbs Bark of Phellodendron chinense Schneid Fruit of Forsythia suspensa (Thunb.) Vahl. Root bark of Paeonia suffruticosa Andr. Artemisia scoparia Waldst. et Kit. Flower bud of Chrysanthemum morifolium RAMAT. Flower bud of Lonicera japonica Thunb. Aerial part of Artemisia annua L. Rhizome of Coptis chinensis Franch. Root of Salvia miltiorrhiza Bge. Rhizome of Belamcanda chinensis (L.) DC. Root of Scutellaria baicalensis Georgi Root of Rheum palmatum LINN
0.039 0.058 0.114 0.097 0.274 0.302 0.147 0.245 0.525 0.132 0.128 0.659
93 (10) 88 (10) 90 (10) 97 (21) 98 (9) 81 (16) 81 (21) 87 (3)** 90 (19) 109 (6) 89 (13) 102 (16)
83 (20) 100 (14) 90 (14) 104 (7) 91 (2)** 80 (24) 86 (2)*** 75 (15)* 85 (10) 100 (11) 96 (16) 103 (13)
90 (5) 93 (9) 91 (11)** 95 (10) 96 (3) 87 (8)* 71 (12)* 88 (9) 97 (14) 104 (22) 89 (12) 100 (2)
110 (11) 95 (9) 92 (9) 105 (4) 99 (14) 88 (8) 77 (4)*** 96 (18) 91 (25) 95 (15) 96 (7) 99 (5)
93 (13) 92 (25) 91 (10) 101 (14) 93 (12) 69 (22) 72 (8)** 71 (22) 98 (5) 84 (20) 87 (8)* 84 (7)*
Yang-tonic herbs Processed root of Aconitum carmichaeli Debx. Root of Saposhnikovia divaricata (Turcz.) Schischk. Root of Codonopsis tangshen Oliv. Root of Angelica dahurica (Fisc.) Benth. et Hook. f. Rhizome of Cibotium barometz (L.) J. Sm. Ripe fruit of Alpinia oxyphylla Miq. Ripe seed of Cuscuta chinensis Lam. Root of Dipsacus asperoides C. Y. Cheng et T. M. Ai Cinnamomum cassia Presl Asarum sieboldii Miq. Fleshy stem with scale of Cistanche deserticola YC Ma. Rhizome of Ligusticum sinensis Oliv. Curculigo orchioides Gaertn.
0.110 0.194 0.212 0.129 0.279 0.124 0.109 0.449 0.024 0.042 0.485 0.145 0.093
81 (13) 87 (16) 73 (23) 90 (1)*** 81 (13) 81 (5)** 97 (4) 90 (23) 92 (3)* 72 (7)*** 92 (8) 88 (2)*** 89 (6)
88 (10) 88 (3)** 88 (20) 79 (10)* 88 (19) 87 (15) 94 (11) 93 (20) 91 (12) 86 (8)* 89 (5)* 82 (6)** 84 (6)**
85 (23) 84 (15) 75 (9)** 88 (9) 94 (10) 64 (31) 91 (8) 74 (13)* 87 (22) 85 (14) 84 (18) 64 (18)* 80 (14)
* p < 0.05; ** p < 0.01; *** p < 0.001,
compared to control cells, unpaired t-test.
91 (10) 87 (5)** 89 (14) 67 (24) 92 (31) 75 (15)* 98 (10) 84 (23) 94 (14) 85 (28) 99 (7) 86 (1)*** 78 (8)*
76 (21) 88 (15) 93 (16) 76 (15) 92 (19) 95 (9) 100 (15) 91 (11) 90 (19) 93 (9) 102 (17) 81 (7)* 86 (12)
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Soluble content in initial extract (g/100 ml)
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medicine. None of the herbs tested induced DNA damage at any of the concentrations tested. The mechanism of protection and the active ingredients of the herbs are not clear, but the effect did not seem to be related to their measured antioxidant capacity as assessed using the ORAC radical scavenging method. Further, yin-tonic herbs, which have been suggested to be antioxidative, did not in general show superior DNA protective effects to yang-tonic herbs (Ou et al., 2003). Results do not support the proposal that yin–yang equates to the antioxidation–oxidation concept, at least, in terms of DNA protection against oxidant challenge. Acknowledgments The authors wish to thank the Macao Polytechnic Institute and The Hong Kong Polytechnic University for supporting this work and Miss Chan Hong for her technical assistance. References
Fig. 2. Effect of aqueous extracts of (a) Artemisia apiacea Hance and (b) Ligusticum sinensis Oliv. pretreatment on DNA of human lymphocytes using the comet assay. Results shown expressed as % of DNA damage in control cells and are mean + 1S.D. of data obtained in three separate experiments. Cells were pre-incubated in different dilutions of herb extract followed by H2 O2 challenge (60 M); * p < 0.05; ** p < 0.01; *** p < 0.001, compared to control cells, unpaired t-test.
for treating cardiovascular disorders (Rhyu et al., 2004; Zhu et al., 2004). It has been reported that Rhizoma ligustici contains antioxidants such as -pinene, ␣-terpinene and terpine-4-ol (Xu and Wang, 2002) which showed hydroxyl radical scavenging effect or lipid peroxidation inhibition (Burits and Bucar, 2000; Candan et al., 2003; Foti and Ingold, 2003). Herba artemisiae annuae also exhibited a strong DNA protective effect. Herba artemisiae annuae is the dried aerial part of Artemisia annua L. Its actions include ‘removing summerheat and relieve fever due to Yin deficiency’ (Xu and Wang, 2002), and it is used in the treatment of malaria (Mueller et al., 2004), as justified by its content of artemisinin. The ingredients of this herb include sesquiterpenes, flavones and volatile oil and coumarin (Xu and Wang, 2002), some of which possess antioxidant activity (Fylaktakidou et al., 2004). 5. Conclusions In conclusion, our results using an ex vivo cellular assay indicate that DNA of human lymphocytes is protected from oxidant challenge by several yin and yang-type herbs used in Chinese
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