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Evaluation of the in vitro and in vivo genotoxicity of magnolia bark extract
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Regulatory Toxicology and Pharmacology 49 (2007) 154–159 www.elsevier.com/locate/yrtph
Evaluation of the in vitro and in vivo genotoxicity of magnolia bark extract Ning Li a, Yan Song a, Wenzhong Zhang a, Wei Wang a, Junshi Chen a, Andrea W. Wong b, Ashley Roberts b,* a
b
Department of Toxicological Safety Assessment, National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing, China Cantox Health Sciences International, 2233 Argentia Road, Suite 308, Mississauga, Ont., Canada L5N 2X7 Received 20 April 2007 Available online 4 July 2007
Abstract Magnolia bark extract (MBE) is an extract of the dried stem, root, or branch bark of magnolia trees that has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. To study the genotoxic potential of MBE, a bacterial reverse mutation assay and an in vivo micronucleus test were conducted. Compositional analysis of the test substance revealed that MBE contains 94% magnolol and 1.5% honokiol. MBE exerted no mutagenic activity in various bacterial strains of Salmonella typhimurium and in Escherichia coli WP2 uvrA, either in the absence or presence of metabolic activation at all doses tested. In the micronucleus test, various doses of MBE did not affect the proportions of immature to total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of Swiss albino mice. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro bacterial reverse mutation assay and the in vivo micronucleus test, and support the safety of MBE for dietary consumption. Ó 2007 Published by Elsevier Inc. Keywords: Magnolia bark extract; MBE; Safety; Genotoxicity; Ames assay; Mutagenicity; Micronuclei; Micronucleus test
1. Introduction Magnolia bark extract (MBE) is produced primarily from the dried stem, root, or branch bark of Magnoliae officinalis, and less commonly from Magnoliae obovata (Chang and But, 1986). MBE is used as a constituent of dietary supplements and topically applied cosmetic products. Magnolia bark has historically been used for many years as a component of traditional Chinese medicines and Japanese remedies for the treatment of depression, anxiety, nervous disorders, gastrointestinal disturbances, asthma, and stroke, as well as to relieve headaches and alleviate muscular pain or fever (Hattori et al., 1986; Tsai
*
Corresponding author. Fax: +1 905 542 1011. E-mail address: [email protected] (A. Roberts).
0273-2300/$ - see front matter Ó 2007 Published by Elsevier Inc. doi:10.1016/j.yrtph.2007.06.005
et al., 1995; Ogata et al., 1997; Sarker, 1997; Hsieh et al., 1998; Maruyama et al., 1998). The two main active compounds in magnolia bark have been identified as the neolignans magnolol and honokiol (Fujita et al., 1972; Zhao et al., 1991; Hsieh et al., 1998; Bang et al., 2000). These compounds are found at levels ranging from 40-90% in commercially available MBE preparations. Magnolol, and to a lesser extent honokiol, have been shown to inhibit tumor metastasis in vitro and in vivo (Nagase et al., 2001; Ikeda et al., 2003), promote apoptosis in tumor cells (Ikeda and Nagase, 2002), and inhibit skin tumor promotion (Konoshima et al., 1991). Animal studies indicate that magnolia bark and its extracts are of low oral toxicity, with an oral LD50 value for MBE of >50 g/kg body weight (Yang and Chen, 1997), and a lack of effects, with the exception of decreased alanine aminotransferase and creatinine levels and
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increased blood urea nitrogen levels, following administration of a magnolia bark preparation (5 or 10 g/kg body weight) to male rats for 14 days by esophageal intubation (Yang and Chen 1997, 1998). Studies investigating the efficacy of magnolia bark and MBE for improving cough reflex, functional dyspepsia symptoms, menopausal symptoms, and for weight management (Iwasaki et al., 2002; Oikawa et al., 2005; Garrison and Chambliss, 2006; Mucci et al., 2006), as well as for the treatment of sleep and panic disorders (Hisinaga et al., 2002; Mantani et al., 2002) also demonstrate the lack of adverse effects and the safety of these preparations. Although MBE and magnolia bark are components of currently marketed dietary supplements and cosmetics, the potential of these substances to cause genotoxicity has not previously been investigated. In the present study, the in vitro mutagenic potential of MBE was evaluated in a bacterial reverse mutation test, while the ability of MBE to induce cytogenetic damage was assessed in a mouse bone marrow erythrocyte micronucleus test. 2. Materials and methods1,2 2.1. Compositional analysis Compositional analysis of MBE was conducted by high performance liquid chromatography (HPLC), as previously described (Tsai and Chen, 1992). An Agilent 1100 Series system equipped with a variable UV detector was used with a Waters Nova Pack C-18 column. The column temperature was set at 35 °C with a mobile phase flow rate of 1.0 ml/min (CH3CN/H2O, 50/50 volume ratio).
2.2. Bacterial reverse mutation test The histidine-requiring Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and the tryptophan-requiring Escherichia coli (E. coli) mutant WP2 uvrA (WOOJUNG BSC Inc., Korea) were cultured in a nutrient broth at 37 °C with shaking. In a cytopreliminary toxicity study, all strains were tested using three plates per dose (ranging from 18.75 to 5000 lg/plate) of MBE to determine an appropriate range of concentrations for the mutagenicity study. The preliminary study was conducted without metabolic activation. Cultures were assessed by microscopic evaluation for a reduced rate of spontaneously occurring colonies and a visible thinning of the bacterial lawn. Based on 1
Studies were conducted to the standards of the United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations, 21 Code of Federal Regulations (CFR) Part 58, with the Organization for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice (1997), and with any applicable amendments. The Ames study was conducted in compliance with OECD Guideline for Testing of Chemicals-471, Bacterial Reverse Mutation Test , 1997 and FDA Redbook 2000 Toxicological Principles for the Safety Assessment of Food Ingredients, 2000 IV.C.1.a. Bacterial Reverse Mutation Test. The Micronucleus study was conducted in accordance with the OECD Guideline for Testing of Chemicals, Mammalian Erythrocyte Micronucleus Test, 1997, and FDA Redbook, 2000, Toxicological Principles for the Safety Assessment of Food Ingredients, In Vivo Mammalian Erythrocyte Micronucleus Test. 2 Studies were conducted at the Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention (CDC), Beijing, China.
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the results of this experiment (discussed in Section 3.1), a range of concentrations from 18.75 to 300 lg/plate were selected for the mutagenicity study. The Ames assay (plate incorporation method) was performed with or without metabolic activation (S-9 obtained from Aroclor 1254-induced rat livers) (Ames et al., 1975; Green and Muriel, 1976; Maron and Ames, 1983). The nutrient broth culture of the bacterial strain (pH 7.4, containing per L of medium: 10 g peptone, 5 g beef extract, 5 g NaCl, 2.6 g K2HPO4Æ3H20, 1 L distilled water) cultured overnight (0.1 ml), 0.1 ml test substance dissolved in DMSO, 0.5 ml S-9 mix or PBS buffer (pH 7.4) were added to 2 ml of molten top agar (containing 0.6% agar, 0.5% NaCl and 10 ml of 0.5 mM histidine–biotin solution for the S. typhimurium strains or 2.5 ml of 0.5 mM tryptophan for the E. coli strain) maintained at approximately 45 °C. The ingredients were mixed and poured onto a minimal agar Petri culture dish (1.5% agar, 40% glucose, and phosphate buffer). Following incubation at approximately 37 °C for 48–72 h, the number of his+ and trp+ revertants were counted. The test sample was assayed in triplicate at 5 concentrations (18.75, 37.5, 75, 150 and 300 lg/plate), and two independent experiments were performed for each bacterial strain. Negative (DMSO only) and positive control experiments were also conducted. The positive control mutagens are listed in Table 1. The test substance was considered to be mutagenic if either: (a) a twofold or greater increase in the mean number of revertants per plate was observed compared to the mean number of revertants per plate in the appropriate negative control in at least one of the tester strains, in the absence of cytotoxicity; or (b) a dose-related increase in the mean number of revertants per plate, compared to that of the appropriate negative control, was observed in at least 2–3 concentrations of the test substance and in at least one bacterial strain, in the absence of cytotoxicity (Maron and Ames, 1983; U.S. FDA, 2000).
2.3. Micronucleus test Male and female Swiss Albino (CD-1) mice were obtained from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) at 7–9 weeks of age, subjected to a general physical examination upon receipt, and acclimatized for 7 days. Animals were housed in cages (5 animals per sex per cage) and provided food (Vital River Laboratory Animal Technology Co. Ltd.) and water (tap water) ad libitum. The housing facility was designed to maintain appropriate environmental conditions (23 ± 2 °C, 12-h light/dark cycle, 30–70% relative humidity, and a ventilation frequency of 10–15 times/hour). In order to assess the toxicity of MBE in mice and to select dose levels for the micronucleus test, a dose range-finding study was conducted in 5 male and 5 female mice. Animals were administered 2500 mg MBE/kg body weight (bw) suspended in 0.5% aqueous carboxymethyl cellulose by oral gavage, and observed for 14 days. Based on the results of the dose range-finding study (discussed in Section 3.2), 2500 mg/kg bw was selected as the high dose for the micronucleus test, while 1250 and 625 mg/kg bw were selected as mid- and lowdose levels. Animals were randomly allocated into one of the following five groups (5/sex/group): negative control (vehicle) group, 625 mg/kg bw (low-dose group), 1250 mg/kg bw (mid-dose group), 2500 mg/kg bw (high-dose group), and a positive control (40 mg cyclophosphamide/kg bw) group. Group assignments are outlined in Table 2. Animals were administered vehicle, positive control, or various doses of MBE twice at an interval of 24 h by oral gavage. Following dosing, animals were examined regularly for mortality and clinical signs of toxicity until sacrifice. Mice were euthanized by carbon dioxide asphyxiation 24 or 48 h after the last treatment administered (all doses). For the cyclophosphamidetreated group, mice were euthanized at the 24-h time point only. Both femors were removed and the bones were cleaned of muscle tissue. The proximal ends of the femora were opened and the bone marrow was flushed into a 5 ml centrifuge tube containing 3 ml fetal bovine serum. The mixture was centrifuged for 5 min at 1000 rpm and the resulting supernatant was discarded. Following thorough mixing, one drop of the sediment was smeared onto a clean slide and air-dried. Slides were briefly
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Table 1 Positive control substances used in the bacterial reverse mutation test Strain
( ) S-9 mix
Concentration (lg/plate)
(+) S-9 mix
Concentration (lg/plate)
S. typhimurium TA98 S. typhimurium TA100 S. typhimurium TA1535 S. typhimurium TA1537 E. coli WP2 uvrA
Furylfuramide (AF2) Furylfuramide (AF2) Sodium azide (SA, NaN3) 9-Aminoacridine (9-AA) Furylfuramide (AF2)
0.1 0.01 0.5 80 0.02
Benzo[a]pyrene (B[a]P) Benzo[a]pyrene (B[a]P) 2-Aminoanthracene (2-AA) 2-Aminoanthracene (2-AA) 2-Aminoanthracene (2-AA)
5 5 2 2 10
Table 2 Animal group assignments in the in vivo micronucleus test Group
Dose level (mg/kg bw)
Concentration of MBE (%)
Sampling time (h postadministration)
1 2 3 4 5 1 2 3 4
0 625 1250 2500 40 0 625 1250 2500
0 2.08 4.16 8.33 0.2 0 2.08 4.16 8.33
24 24 24 24 24 48 48 48 48
(Negative control) (Low-dose) (Mid-dose) (High-dose) (Positive control)* (Negative control) (Low-dose) (Mid-dose) (High-dose)
Abbreviations: bw, body weight; h, hours. * Positive control = cyclophosphamide (40 mg/kg body weight).
flamed, then fixed by immersion in 95% methanol for 10 min and stained in ordinary staining jars with Giemsa Working Solution for 15 min. Stained slides were treated with xylene, cover-slipped, and analyzed microscopically. All slides were coded to ensure that that the evaluation was blinded. The proportion of immature (polychromatic, PCE) erythrocytes to total [immature + mature (normochromatic, NCE)] erythrocytes was determined for each animal by analyzing 200 erythrocytes (Odagiri et al., 1997; OECD, 1997). For a valid test, the proportion of immature to total (PCE + NCE) erythrocytes was required not to be less than 20% of the negative control value. To determine the presence of micronuclei 2000 PCEs were analyzed per animal. Both biological and statistical significance were considered for evaluation of a positive response. The test substance was considered positive for a mutagenic response if there was a statistically significant, dose-related increase in the number of micronucleated PCEs for at least one of the time points, compared to the concurrent negative control group.
2.4. Statistical analysis For the micronucleus test, homogenous data were analyzed using a One-Way Analysis of Variance (ANOVA), and the significance of intergroup differences were analyzed using Duncan’s test. Heterogeneous data were analyzed using the Kruskal–Wallis test, and the significance of intergroup differences between the control and treated groups was assessed using Dunn’s test. All statistical tests were performed at the p < 0.05 level of significance.
3. Results 3.1. Compositional analysis HPLC analysis revealed that MBE contains 94% magnolol and 1.5% honokiol (Fig. 1), indicating that the test
Fig. 1. Compositional analysis of magnolia bark extract.
substance used in the genotoxicity assays contains high amounts of the major active components of MBE. 3.2. Bacterial reverse mutation test In the preliminary cytotoxicity and range-finding study, evidence of cytotoxicity (reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn) was observed at concentrations of P75 lg/plate in S. typhimurium TA 1535 and TA 1537, P150 lg/plate in S. typhimurium TA 98, and P300 lg/plate in S. typhimurium TA100 and E. coli WP2uvrA. Based on these results, 300 lg/plate was selected as the highest dose for all strains in the bacterial reverse mutation test. A range of 5 concentrations from 18.75 to 300 lg/plate were tested. In the mutagenicity study, MBE did not increase the mean number of revertants per plate of any S. typhimurium strain or the E. coli strain with or without metabolic activation in comparison to the spontaneous reversion rate in the negative control (Table 3). The mean number of revertant colonies of the negative control was within the historical range typically reported in the literature (Mortelmans et al., 1986; Gee et al., 1994). The positive control mutagens induced increases in revertant colonies (Table 4), confirming the validity of the assay. A reduction in the background bacterial lawn was observed at 300 lg/plate
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Table 3 Bacterial reverse mutation test results Concentration (lg/plate)
Number of revertants (number of colonies/plate) S. typhimurium TA98
0 (Exp. 1) 0 (Exp. 2) 18.5 (Exp. 1) 18.5 (Exp. 2) 37.5 (Exp. 1) 37.5 (Exp. 2) 75 (Exp. 1) 75 (Exp. 2) 150 (Exp. 1) 150 (Exp. 2) 300 (Exp. 1) 300 (Exp. 2)
E. coli TA100
TA1535
TA1537
WP2uvrA
( ) S-9
(+) S-9
( ) S-9
(+) S-9
( ) S-9
(+) S-9
( ) S-9
(+) S-9
( ) S-9
(+) S-9
27 ± 2.6 31 ± 5.2 33 ± 8.2 26 ± 3.2 30 ± 5.6 28 ± 4.6 25 ± 6.0 23 ± 7.9 15 ± 4.6 19 ± 1.5 0±0 0±0
32 ± 4.4 37 ± 9.5 39 ± 4.9 35 ± 5.9 33 ± 3.6 39 ± 6.1 35 ± 10.0 32 ± 4.6 31 ± 6.7 33 ± 5.3 18 ± 1.7 15 ± 4.7
112 ± 5.5 115 ± 7.5 126 ± 30.0 146 ± 13.9 137 ± 19.4 139 ± 10.1 133 ± 13.6 145 ± 18.7 128 ± 3.6 139 ± 7.5 74 ± 9.0 108 ± 6.0
108 ± 5.8 135 ± 20.1 147 ± 12.7 135 ± 18.5 141 ± 12.7 146 ± 14.2 135 ± 7.0 151 ± 12.5 119 ± 7.8 132 ± 25.3 130 ± 3.6 131 ± 12.3
12 ± 4.7 16 ± 5.7 11 ± 2.9 19 ± 2.9 10 ± 1.0 17 ± 1.7 8 ± 1.7 7 ± 0.6 0±0 0±0 0±0 0±0
13 ± 4.6 13 ± 1.7 14 ± 2.5 11 ± 2.0 13 ± 0.0 14 ± 4.6 10 ± 4.6 14 ± 4.6 8 ± 2.1 8 ± 3.2 0±0 0±0
9 ± 1.2 10 ± 3.5 9 ± 3.5 9 ± 1.2 7 ± 1.5 13 ± 7.0 5 ± 1.7 4 ± 0.6 0±0 0±0 0±0 0±0
10 ± 2.0 15 ± 3.2 12 ± 1.0 13 ± 6.0 9 ± 3.2 13 ± 1.0 9 ± 3.8 13 ± 3.1 3 ± 1.5 8 ± 2.6 0±0 0±0
46 ± 6.0 31 ±1.5 50 ± 9.8 34 ± 2.6 46 ± 5.0 31 ± 3.0 46 ± 4.6 39 ± 1.0 29 ± 4.9 36 ± 6.1 21 ± 3.1 22 ± 3.2
51 ± 5.3 34 ± 3.5 56 ± 3.6 44 ± 0.6 55 ± 7.5 36 ± 1.5 57 ± 2.6 37 ± 4.0 50 ± 7.1 35 ± 2.6 27 ± 4.2 35 ± 1.2
Abbreviation: Exp., Experiment. All values presented as means ± standard deviation.
Table 4 Bacterial reverse mutation test results (Positive controls) Strain (S. typhimurium unless otherwise noted)
Positive control
Mean ± SD Experiment 1
Experiment 2
( ) S-9 activation TA98 TA100 TA1535 TA1537 E. coli WP2 uvrA
AF2 AF2 SA 9-AA AF2
667 ± 10.1 866 ± 92.5 582 ± 178.0 535 ± 43.0 305 ± 4.4
523 ± 17.7 574 ± 16.6 479 ± 30.4 472 ± 109.7 291 ± 8.3
(+) S-9 activation TA98 TA100 TA1535 TA1537 E. coli WP2 uvrA
B[a]P B[a]P 2-AA 2-AA 2-AA
144 ± 24.2 523 ± 11.8 352 ± 97.7 427 ± 32.2 673 ± 59.5
146 ± 19.7 584 ± 25.2 307 ± 32.3 404 ± 15.5 741 ± 43.5
Abbreviations: AF2, furylfuramide; SA, sodium azide; 9-AA, 9-aminoacridine; B[a]P, benzo[a]pyrene; 2-AA, 2-aminoanthracene.
in S. typhimurium strains TA98 and TA100 and E. coli WP2 uvrA. The number of revertants could not be counted for S. typhimurium strains TA1535 and TA1537 at concentrations of 150 and 300 lg/plate due to excessive cytotoxicity, but a lack of increase in the mean number of revertants per plate compared to negative control was observed for the remaining three concentrations, the highest of which produced evidence of cytotoxicity. 3.3. Micronucleus test In the dose range-finding study, no mortalities and no clinical signs of toxicity were observed in animals given 2500 mg MBE/kg body weight. In the micronucleus test, no mortalities were recorded, and gross necropsy of the animals revealed no macroscopic findings. The criterion for a valid test was met.
Table 5 Proportion of polychromatic to total erythrocytes in the in vivo micronucleus test Sex
MBE dose level (mg/kg bw)
24 h
48 h
No. of PCE (mean ± SD)
PCE/ (PCE + NCE) (%)
No. of PCE (mean ± SD)
PCE/(PCE + NCE) (%)
Female
0 625 1250 2500 Positive control
110.2 ± 9.8 108.2 ± 8.2 109.4 ± 4.4 110.0 ± 9.3 102.1 ± 6.9
55.1 54.1 54.7 55.0 51.1
111.0 ± 6.7 107.8 ± 3.8 109.6 ± 5.0 109.2 ± 5.5 —
55.5 53.9 54.8 54.6 —
Male
0 625 1250 2500 Positive control
109.2 ± 8.8 112.2 ± 8.5 106.0 ± 9.5 108.6 ± 6.7 99.8 ± 3.5
54.7 56.1 53.0 54.3 49.9
109.0 ± 5.5 107.6 ± 8.8 108.2 ± 7.7 107.2 ± 8.5 —
54.5 53.8 54.1 53.6 —
Abbreviations: bw, body weight; MBE, magnolia bark extract; NCE, normochromatic erythrocyte; No., number; PCE, polychromatic erythrocyte; SD, standard deviation. Positive control = cyclophosphamide (40 mg/kg body weight).
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Table 6 Number of micronucleated polychromatic erythrocytes in the in vivo micronucleus test Sex
MBE dose level (mg/kg bw)
24 h
48 h
No. of MNPCE (mean ± SD)
MNPCE (%)
No. of MNPCE (mean ± SD)
MNPCE (%)
Female
0 625 1250 2500 Positive control
3.8 ± 1.9 3.6 ± 1.1 4.2 ± 1.8 4.6 ± 2.1 23.2 ± 2.9*
1.9 1.8 2.1 2.3 11.6*
4.8 ± 2.2 4.0 ± 1.0 4.2 ± 1.3 3.8 ± 0.8 —
2.4 2.0 2.1 1.9 —
Male
0 625 1250 2500 Positive control
4.4 ± 1.1 4.0 ± 1.2 4.2 ± 1.5 4.2 ± 1.6 24.6 ± 3.8*
2.2 2.0 2.1 2.1 12.3*
4.4 ± 1.8 4.2 ± 1.3 4.6 ± 1.1 4.4 ± 1.3 —
2.2 2.1 2.3 2.2 —
Abbreviations: bw, body weight; MBE, magnolia bark extract; MNPCE, micronucleated polychromatic erythrocyte; No., number; PCE, polychromatice erythrocyte; SD, standard deviation. Positive control = cyclophosphamide (40 mg/kg body weight). * Significantly different compared to negative control (P < 0.05).
The proportion of immature to total erythrocytes was not affected by MBE administration (Table 5). No statistically significant increase in the number of micronucleated PCEs was observed in any of the MBE-treated groups compared to the negative control group at either time point (Table 6). The positive control substance (cyclophosphamide) induced a marked and statistically significant increase in the number of PCEs with micronuclei but did not affect the proportion of immature to total erythrocytes. 4. Discussion The results of the genotoxicity tests demonstrate that MBE, consisting of 94% magnolol and 1.5% honokiol, is not mutagenic under the experimental conditions for S. typhimurium TA98, TA100, TA1535 and TA1537 and for E. coli WP2 uvrA, and that MBE does not affect the proportions of PCEs to total erythrocytes or increase the number of micronucleated PCEs in Swiss albino mice. All genotoxicity assays were conducted in replicate using multiple doses that were determined in preliminary dose range-finding experiments. No evidence of mutagenicity was observed at any concentration tested, including those that caused cytotoxicity. The bacterial reverse mutation tests were conducted with or without S-9 activation to determine whether MBE could be converted to mutagenic metabolites following bioactivation by microsomal enzymes. Appropriate positive and negative controls were included for both genotoxicity tests and produced the expected results, demonstrating the validity of the assays. Although there are no other known published studies on the genotoxic potential of MBE, it has been reported that magnolol inhibits UV-induced mutations (Fujita and Taira, 1994), as well as the mutagenic activity induced by several indirect mutagens (Saito et al., 2006). These reports demonstrate that magnolol has anti-mutagenic activity, and support the findings of the current study.
In summary, the potential genotoxic effects of MBE were assessed in vitro and in vivo. MBE, containing over 90% of the two main active compounds magnolol and honokiol, showed no mutagenic activity in the Ames assay and did not induce micronuclei in the immature erythrocytes of Swiss albino mice. These data indicate that MBE does not have genotoxic potential, and support the safety of MBE for dietary consumption. References Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens with the Salmonella/mammalian microsome mutagenicity test. Mutat. Res. 31, 347–364. Bang, K.H., Kim, Y.K., Min, B.S., Na, M.K., Rhee, Y.H., Lee, J.P., Bae, K.H., 2000. Antifungal activity of magnolol and honokiol. Arch. Pharm. Res. 23, 46–49. Chang, H.-M., But, P.P.-H. (Eds.), 1986. Houpo Magnolia officinalis. Pharmacology and Applications of Chinese Materia-Medica, Vol. 2. Wiley, Philadelphia, pp. 878–880. Fujita, S., Taira, J., 1994. Biphenyl compounds are hydroxyl radical scavengers: their effective inhibition for UV-induced mutation in Salmonella typhimurium TA102. Free Radic. Biol. Med. 17, 273–277. Fujita, M., Itokawa, H., Sashida, Y., 1972. Honokiol, a new phenolic compound isolated from the bark of Magnolia obobvata Thunb. Chem. Pharm. Bull. 20, 212–213. Garrison, R., Chambliss, W.G., 2006. Effect of a proprietary Magnolia and Phellodendron extract on weight management: a pilot, doubleblind, placebo-controlled clinical trial. Altern. Ther. Health Med. 12, 50–54. Gee, P., Maron, D.M., Ames, B.N., 1994. Detection and classification of mutagens: a set of base-specific {{{ Salmonella}}} tester strains. Proc. Natl. Acad. Sci. USA 91 (24), 11606–11610. Green, M.H.L., Muriel, W.J., 1976. Mutagenicity testing using TRP+ reversion in Escherichia coli. Mutat. Res. 38, 3–32. Hattori, M., Endo, Y., Takebe, S., Kobashi, K., Fukasaku, N., Namba, T., 1986. Metabolism of magnolol from magnoliae cortex. II. Absorption, metabolism and excretion of [ring-(14)C]magnolol in rats. Chem. Pharm. Bull. 34, 158–167. Hisinaga, A., Itoh, T., Hasegawa, Y., Emore, K., Kita, T., Okabe, A., Kurachi, M., 2002. A case of sleep choking syndrome improved by the Kampo extract of Hange-koboku-to. Psychiatry Clin. Neurosci. 56, 237–325.
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Odagiri, Y., Uchida, H., Shibazaki, S., 1997. Interindividual variation in cytogenetic response to x-ray and colchicine measured with the cytokinesis-block micronucleus assay. Mutat. Res. 381, 1–13. OECD, 1997. Mammalian erythrocyte micronucleus test, in: OECD Guideline for the Testing of Chemicals, Section 474, Organisation for Economic Co-operation and Development (OECD), Paris, revised 21st July, 1997. Ogata, M., Hoshi, M., Shimotohno, K., Urano, S., Endo, T., 1997. Antioxidant activity of magnolol, honokiol, and related phenolic compounds. J. Am. Oil Chem. Soc. 74, 557–562. Oikawa, T., Ito, G., Koyama, H., Hanawa, T., 2005. Prokinetic effect of a Kampo medicien, Hange-koboku-to (Banxia-houpo-tang), on patients with functional dyspepsia. Phytomedicine 12, 730–734. Saito, J., Sakai, Y., Nagase, H., 2006. In vitro anti-mutagenic effect of magnolol against direct and indirect mutagens. Mutat. Res. 609, 68–73. Sarker, S.D., 1997. Biological activity of magnolol: a review. Fitoterapia 68, 3–8. Tsai, T.T., Chen, C.F., 1992. Identification and determination of honokiol and magnolol from Magnolia officinalis by high-performance liquid chromatography with photodiode-array UV detection. J. Chromatogr. 598, 143–169. Tsai, T.H., Chou, C.J., Chen, C.F., 1995. Glucuronidation of magnolol assessed using HPLC/fluorescence. Planta Med. 61, 491–492. U.S. FDA, 2000. Bacterial reverse mutation test, in: Redbook 2000: Toxicological Principles for the Safety Assessment of Food Ingredients. U.S. Food and Drug Administration (U.S. FDA), Center for Food Safety and Nutrition (CFSAN), College Park, MD, available from: http://www.cfsan.fda.gov/redbook/redivc1a.html. Yang, H.-Y., Chen, C.-F., 1997. Subacute toxicity of 15 commonly used Chinese drugs (II). Yao Wu Shi Pin Fen Xi 5, 355–379. Yang, H.-Y., Chen, C.-F., 1998. Pharmacology and toxicology of herbal medicine: Subacute toxicity of commonly used Chinese drugs. J. Toxicol. Sci. 23 (Suppl. 2), 229–233. Zhao, Z., Tang, X.H.U.M., Sashida, Y., 1991. Pharmacognostical studies on the Magnolia bark (3). Determination of magnolol and honokiol in ‘‘Hou po’’(cortex magnoliae) prepared from the bark of different age. Shoyakugaku Zasshi 45, 145–147.
Regulatory Toxicology and Pharmacology 49 (2007) 154–159 www.elsevier.com/locate/yrtph
Evaluation of the in vitro and in vivo genotoxicity of magnolia bark extract Ning Li a, Yan Song a, Wenzhong Zhang a, Wei Wang a, Junshi Chen a, Andrea W. Wong b, Ashley Roberts b,* a
b
Department of Toxicological Safety Assessment, National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing, China Cantox Health Sciences International, 2233 Argentia Road, Suite 308, Mississauga, Ont., Canada L5N 2X7 Received 20 April 2007 Available online 4 July 2007
Abstract Magnolia bark extract (MBE) is an extract of the dried stem, root, or branch bark of magnolia trees that has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. To study the genotoxic potential of MBE, a bacterial reverse mutation assay and an in vivo micronucleus test were conducted. Compositional analysis of the test substance revealed that MBE contains 94% magnolol and 1.5% honokiol. MBE exerted no mutagenic activity in various bacterial strains of Salmonella typhimurium and in Escherichia coli WP2 uvrA, either in the absence or presence of metabolic activation at all doses tested. In the micronucleus test, various doses of MBE did not affect the proportions of immature to total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of Swiss albino mice. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro bacterial reverse mutation assay and the in vivo micronucleus test, and support the safety of MBE for dietary consumption. Ó 2007 Published by Elsevier Inc. Keywords: Magnolia bark extract; MBE; Safety; Genotoxicity; Ames assay; Mutagenicity; Micronuclei; Micronucleus test
1. Introduction Magnolia bark extract (MBE) is produced primarily from the dried stem, root, or branch bark of Magnoliae officinalis, and less commonly from Magnoliae obovata (Chang and But, 1986). MBE is used as a constituent of dietary supplements and topically applied cosmetic products. Magnolia bark has historically been used for many years as a component of traditional Chinese medicines and Japanese remedies for the treatment of depression, anxiety, nervous disorders, gastrointestinal disturbances, asthma, and stroke, as well as to relieve headaches and alleviate muscular pain or fever (Hattori et al., 1986; Tsai
*
Corresponding author. Fax: +1 905 542 1011. E-mail address: [email protected] (A. Roberts).
0273-2300/$ - see front matter Ó 2007 Published by Elsevier Inc. doi:10.1016/j.yrtph.2007.06.005
et al., 1995; Ogata et al., 1997; Sarker, 1997; Hsieh et al., 1998; Maruyama et al., 1998). The two main active compounds in magnolia bark have been identified as the neolignans magnolol and honokiol (Fujita et al., 1972; Zhao et al., 1991; Hsieh et al., 1998; Bang et al., 2000). These compounds are found at levels ranging from 40-90% in commercially available MBE preparations. Magnolol, and to a lesser extent honokiol, have been shown to inhibit tumor metastasis in vitro and in vivo (Nagase et al., 2001; Ikeda et al., 2003), promote apoptosis in tumor cells (Ikeda and Nagase, 2002), and inhibit skin tumor promotion (Konoshima et al., 1991). Animal studies indicate that magnolia bark and its extracts are of low oral toxicity, with an oral LD50 value for MBE of >50 g/kg body weight (Yang and Chen, 1997), and a lack of effects, with the exception of decreased alanine aminotransferase and creatinine levels and
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increased blood urea nitrogen levels, following administration of a magnolia bark preparation (5 or 10 g/kg body weight) to male rats for 14 days by esophageal intubation (Yang and Chen 1997, 1998). Studies investigating the efficacy of magnolia bark and MBE for improving cough reflex, functional dyspepsia symptoms, menopausal symptoms, and for weight management (Iwasaki et al., 2002; Oikawa et al., 2005; Garrison and Chambliss, 2006; Mucci et al., 2006), as well as for the treatment of sleep and panic disorders (Hisinaga et al., 2002; Mantani et al., 2002) also demonstrate the lack of adverse effects and the safety of these preparations. Although MBE and magnolia bark are components of currently marketed dietary supplements and cosmetics, the potential of these substances to cause genotoxicity has not previously been investigated. In the present study, the in vitro mutagenic potential of MBE was evaluated in a bacterial reverse mutation test, while the ability of MBE to induce cytogenetic damage was assessed in a mouse bone marrow erythrocyte micronucleus test. 2. Materials and methods1,2 2.1. Compositional analysis Compositional analysis of MBE was conducted by high performance liquid chromatography (HPLC), as previously described (Tsai and Chen, 1992). An Agilent 1100 Series system equipped with a variable UV detector was used with a Waters Nova Pack C-18 column. The column temperature was set at 35 °C with a mobile phase flow rate of 1.0 ml/min (CH3CN/H2O, 50/50 volume ratio).
2.2. Bacterial reverse mutation test The histidine-requiring Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and the tryptophan-requiring Escherichia coli (E. coli) mutant WP2 uvrA (WOOJUNG BSC Inc., Korea) were cultured in a nutrient broth at 37 °C with shaking. In a cytopreliminary toxicity study, all strains were tested using three plates per dose (ranging from 18.75 to 5000 lg/plate) of MBE to determine an appropriate range of concentrations for the mutagenicity study. The preliminary study was conducted without metabolic activation. Cultures were assessed by microscopic evaluation for a reduced rate of spontaneously occurring colonies and a visible thinning of the bacterial lawn. Based on 1
Studies were conducted to the standards of the United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations, 21 Code of Federal Regulations (CFR) Part 58, with the Organization for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice (1997), and with any applicable amendments. The Ames study was conducted in compliance with OECD Guideline for Testing of Chemicals-471, Bacterial Reverse Mutation Test , 1997 and FDA Redbook 2000 Toxicological Principles for the Safety Assessment of Food Ingredients, 2000 IV.C.1.a. Bacterial Reverse Mutation Test. The Micronucleus study was conducted in accordance with the OECD Guideline for Testing of Chemicals, Mammalian Erythrocyte Micronucleus Test, 1997, and FDA Redbook, 2000, Toxicological Principles for the Safety Assessment of Food Ingredients, In Vivo Mammalian Erythrocyte Micronucleus Test. 2 Studies were conducted at the Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention (CDC), Beijing, China.
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the results of this experiment (discussed in Section 3.1), a range of concentrations from 18.75 to 300 lg/plate were selected for the mutagenicity study. The Ames assay (plate incorporation method) was performed with or without metabolic activation (S-9 obtained from Aroclor 1254-induced rat livers) (Ames et al., 1975; Green and Muriel, 1976; Maron and Ames, 1983). The nutrient broth culture of the bacterial strain (pH 7.4, containing per L of medium: 10 g peptone, 5 g beef extract, 5 g NaCl, 2.6 g K2HPO4Æ3H20, 1 L distilled water) cultured overnight (0.1 ml), 0.1 ml test substance dissolved in DMSO, 0.5 ml S-9 mix or PBS buffer (pH 7.4) were added to 2 ml of molten top agar (containing 0.6% agar, 0.5% NaCl and 10 ml of 0.5 mM histidine–biotin solution for the S. typhimurium strains or 2.5 ml of 0.5 mM tryptophan for the E. coli strain) maintained at approximately 45 °C. The ingredients were mixed and poured onto a minimal agar Petri culture dish (1.5% agar, 40% glucose, and phosphate buffer). Following incubation at approximately 37 °C for 48–72 h, the number of his+ and trp+ revertants were counted. The test sample was assayed in triplicate at 5 concentrations (18.75, 37.5, 75, 150 and 300 lg/plate), and two independent experiments were performed for each bacterial strain. Negative (DMSO only) and positive control experiments were also conducted. The positive control mutagens are listed in Table 1. The test substance was considered to be mutagenic if either: (a) a twofold or greater increase in the mean number of revertants per plate was observed compared to the mean number of revertants per plate in the appropriate negative control in at least one of the tester strains, in the absence of cytotoxicity; or (b) a dose-related increase in the mean number of revertants per plate, compared to that of the appropriate negative control, was observed in at least 2–3 concentrations of the test substance and in at least one bacterial strain, in the absence of cytotoxicity (Maron and Ames, 1983; U.S. FDA, 2000).
2.3. Micronucleus test Male and female Swiss Albino (CD-1) mice were obtained from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) at 7–9 weeks of age, subjected to a general physical examination upon receipt, and acclimatized for 7 days. Animals were housed in cages (5 animals per sex per cage) and provided food (Vital River Laboratory Animal Technology Co. Ltd.) and water (tap water) ad libitum. The housing facility was designed to maintain appropriate environmental conditions (23 ± 2 °C, 12-h light/dark cycle, 30–70% relative humidity, and a ventilation frequency of 10–15 times/hour). In order to assess the toxicity of MBE in mice and to select dose levels for the micronucleus test, a dose range-finding study was conducted in 5 male and 5 female mice. Animals were administered 2500 mg MBE/kg body weight (bw) suspended in 0.5% aqueous carboxymethyl cellulose by oral gavage, and observed for 14 days. Based on the results of the dose range-finding study (discussed in Section 3.2), 2500 mg/kg bw was selected as the high dose for the micronucleus test, while 1250 and 625 mg/kg bw were selected as mid- and lowdose levels. Animals were randomly allocated into one of the following five groups (5/sex/group): negative control (vehicle) group, 625 mg/kg bw (low-dose group), 1250 mg/kg bw (mid-dose group), 2500 mg/kg bw (high-dose group), and a positive control (40 mg cyclophosphamide/kg bw) group. Group assignments are outlined in Table 2. Animals were administered vehicle, positive control, or various doses of MBE twice at an interval of 24 h by oral gavage. Following dosing, animals were examined regularly for mortality and clinical signs of toxicity until sacrifice. Mice were euthanized by carbon dioxide asphyxiation 24 or 48 h after the last treatment administered (all doses). For the cyclophosphamidetreated group, mice were euthanized at the 24-h time point only. Both femors were removed and the bones were cleaned of muscle tissue. The proximal ends of the femora were opened and the bone marrow was flushed into a 5 ml centrifuge tube containing 3 ml fetal bovine serum. The mixture was centrifuged for 5 min at 1000 rpm and the resulting supernatant was discarded. Following thorough mixing, one drop of the sediment was smeared onto a clean slide and air-dried. Slides were briefly
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Table 1 Positive control substances used in the bacterial reverse mutation test Strain
( ) S-9 mix
Concentration (lg/plate)
(+) S-9 mix
Concentration (lg/plate)
S. typhimurium TA98 S. typhimurium TA100 S. typhimurium TA1535 S. typhimurium TA1537 E. coli WP2 uvrA
Furylfuramide (AF2) Furylfuramide (AF2) Sodium azide (SA, NaN3) 9-Aminoacridine (9-AA) Furylfuramide (AF2)
0.1 0.01 0.5 80 0.02
Benzo[a]pyrene (B[a]P) Benzo[a]pyrene (B[a]P) 2-Aminoanthracene (2-AA) 2-Aminoanthracene (2-AA) 2-Aminoanthracene (2-AA)
5 5 2 2 10
Table 2 Animal group assignments in the in vivo micronucleus test Group
Dose level (mg/kg bw)
Concentration of MBE (%)
Sampling time (h postadministration)
1 2 3 4 5 1 2 3 4
0 625 1250 2500 40 0 625 1250 2500
0 2.08 4.16 8.33 0.2 0 2.08 4.16 8.33
24 24 24 24 24 48 48 48 48
(Negative control) (Low-dose) (Mid-dose) (High-dose) (Positive control)* (Negative control) (Low-dose) (Mid-dose) (High-dose)
Abbreviations: bw, body weight; h, hours. * Positive control = cyclophosphamide (40 mg/kg body weight).
flamed, then fixed by immersion in 95% methanol for 10 min and stained in ordinary staining jars with Giemsa Working Solution for 15 min. Stained slides were treated with xylene, cover-slipped, and analyzed microscopically. All slides were coded to ensure that that the evaluation was blinded. The proportion of immature (polychromatic, PCE) erythrocytes to total [immature + mature (normochromatic, NCE)] erythrocytes was determined for each animal by analyzing 200 erythrocytes (Odagiri et al., 1997; OECD, 1997). For a valid test, the proportion of immature to total (PCE + NCE) erythrocytes was required not to be less than 20% of the negative control value. To determine the presence of micronuclei 2000 PCEs were analyzed per animal. Both biological and statistical significance were considered for evaluation of a positive response. The test substance was considered positive for a mutagenic response if there was a statistically significant, dose-related increase in the number of micronucleated PCEs for at least one of the time points, compared to the concurrent negative control group.
2.4. Statistical analysis For the micronucleus test, homogenous data were analyzed using a One-Way Analysis of Variance (ANOVA), and the significance of intergroup differences were analyzed using Duncan’s test. Heterogeneous data were analyzed using the Kruskal–Wallis test, and the significance of intergroup differences between the control and treated groups was assessed using Dunn’s test. All statistical tests were performed at the p < 0.05 level of significance.
3. Results 3.1. Compositional analysis HPLC analysis revealed that MBE contains 94% magnolol and 1.5% honokiol (Fig. 1), indicating that the test
Fig. 1. Compositional analysis of magnolia bark extract.
substance used in the genotoxicity assays contains high amounts of the major active components of MBE. 3.2. Bacterial reverse mutation test In the preliminary cytotoxicity and range-finding study, evidence of cytotoxicity (reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn) was observed at concentrations of P75 lg/plate in S. typhimurium TA 1535 and TA 1537, P150 lg/plate in S. typhimurium TA 98, and P300 lg/plate in S. typhimurium TA100 and E. coli WP2uvrA. Based on these results, 300 lg/plate was selected as the highest dose for all strains in the bacterial reverse mutation test. A range of 5 concentrations from 18.75 to 300 lg/plate were tested. In the mutagenicity study, MBE did not increase the mean number of revertants per plate of any S. typhimurium strain or the E. coli strain with or without metabolic activation in comparison to the spontaneous reversion rate in the negative control (Table 3). The mean number of revertant colonies of the negative control was within the historical range typically reported in the literature (Mortelmans et al., 1986; Gee et al., 1994). The positive control mutagens induced increases in revertant colonies (Table 4), confirming the validity of the assay. A reduction in the background bacterial lawn was observed at 300 lg/plate
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Table 3 Bacterial reverse mutation test results Concentration (lg/plate)
Number of revertants (number of colonies/plate) S. typhimurium TA98
0 (Exp. 1) 0 (Exp. 2) 18.5 (Exp. 1) 18.5 (Exp. 2) 37.5 (Exp. 1) 37.5 (Exp. 2) 75 (Exp. 1) 75 (Exp. 2) 150 (Exp. 1) 150 (Exp. 2) 300 (Exp. 1) 300 (Exp. 2)
E. coli TA100
TA1535
TA1537
WP2uvrA
( ) S-9
(+) S-9
( ) S-9
(+) S-9
( ) S-9
(+) S-9
( ) S-9
(+) S-9
( ) S-9
(+) S-9
27 ± 2.6 31 ± 5.2 33 ± 8.2 26 ± 3.2 30 ± 5.6 28 ± 4.6 25 ± 6.0 23 ± 7.9 15 ± 4.6 19 ± 1.5 0±0 0±0
32 ± 4.4 37 ± 9.5 39 ± 4.9 35 ± 5.9 33 ± 3.6 39 ± 6.1 35 ± 10.0 32 ± 4.6 31 ± 6.7 33 ± 5.3 18 ± 1.7 15 ± 4.7
112 ± 5.5 115 ± 7.5 126 ± 30.0 146 ± 13.9 137 ± 19.4 139 ± 10.1 133 ± 13.6 145 ± 18.7 128 ± 3.6 139 ± 7.5 74 ± 9.0 108 ± 6.0
108 ± 5.8 135 ± 20.1 147 ± 12.7 135 ± 18.5 141 ± 12.7 146 ± 14.2 135 ± 7.0 151 ± 12.5 119 ± 7.8 132 ± 25.3 130 ± 3.6 131 ± 12.3
12 ± 4.7 16 ± 5.7 11 ± 2.9 19 ± 2.9 10 ± 1.0 17 ± 1.7 8 ± 1.7 7 ± 0.6 0±0 0±0 0±0 0±0
13 ± 4.6 13 ± 1.7 14 ± 2.5 11 ± 2.0 13 ± 0.0 14 ± 4.6 10 ± 4.6 14 ± 4.6 8 ± 2.1 8 ± 3.2 0±0 0±0
9 ± 1.2 10 ± 3.5 9 ± 3.5 9 ± 1.2 7 ± 1.5 13 ± 7.0 5 ± 1.7 4 ± 0.6 0±0 0±0 0±0 0±0
10 ± 2.0 15 ± 3.2 12 ± 1.0 13 ± 6.0 9 ± 3.2 13 ± 1.0 9 ± 3.8 13 ± 3.1 3 ± 1.5 8 ± 2.6 0±0 0±0
46 ± 6.0 31 ±1.5 50 ± 9.8 34 ± 2.6 46 ± 5.0 31 ± 3.0 46 ± 4.6 39 ± 1.0 29 ± 4.9 36 ± 6.1 21 ± 3.1 22 ± 3.2
51 ± 5.3 34 ± 3.5 56 ± 3.6 44 ± 0.6 55 ± 7.5 36 ± 1.5 57 ± 2.6 37 ± 4.0 50 ± 7.1 35 ± 2.6 27 ± 4.2 35 ± 1.2
Abbreviation: Exp., Experiment. All values presented as means ± standard deviation.
Table 4 Bacterial reverse mutation test results (Positive controls) Strain (S. typhimurium unless otherwise noted)
Positive control
Mean ± SD Experiment 1
Experiment 2
( ) S-9 activation TA98 TA100 TA1535 TA1537 E. coli WP2 uvrA
AF2 AF2 SA 9-AA AF2
667 ± 10.1 866 ± 92.5 582 ± 178.0 535 ± 43.0 305 ± 4.4
523 ± 17.7 574 ± 16.6 479 ± 30.4 472 ± 109.7 291 ± 8.3
(+) S-9 activation TA98 TA100 TA1535 TA1537 E. coli WP2 uvrA
B[a]P B[a]P 2-AA 2-AA 2-AA
144 ± 24.2 523 ± 11.8 352 ± 97.7 427 ± 32.2 673 ± 59.5
146 ± 19.7 584 ± 25.2 307 ± 32.3 404 ± 15.5 741 ± 43.5
Abbreviations: AF2, furylfuramide; SA, sodium azide; 9-AA, 9-aminoacridine; B[a]P, benzo[a]pyrene; 2-AA, 2-aminoanthracene.
in S. typhimurium strains TA98 and TA100 and E. coli WP2 uvrA. The number of revertants could not be counted for S. typhimurium strains TA1535 and TA1537 at concentrations of 150 and 300 lg/plate due to excessive cytotoxicity, but a lack of increase in the mean number of revertants per plate compared to negative control was observed for the remaining three concentrations, the highest of which produced evidence of cytotoxicity. 3.3. Micronucleus test In the dose range-finding study, no mortalities and no clinical signs of toxicity were observed in animals given 2500 mg MBE/kg body weight. In the micronucleus test, no mortalities were recorded, and gross necropsy of the animals revealed no macroscopic findings. The criterion for a valid test was met.
Table 5 Proportion of polychromatic to total erythrocytes in the in vivo micronucleus test Sex
MBE dose level (mg/kg bw)
24 h
48 h
No. of PCE (mean ± SD)
PCE/ (PCE + NCE) (%)
No. of PCE (mean ± SD)
PCE/(PCE + NCE) (%)
Female
0 625 1250 2500 Positive control
110.2 ± 9.8 108.2 ± 8.2 109.4 ± 4.4 110.0 ± 9.3 102.1 ± 6.9
55.1 54.1 54.7 55.0 51.1
111.0 ± 6.7 107.8 ± 3.8 109.6 ± 5.0 109.2 ± 5.5 —
55.5 53.9 54.8 54.6 —
Male
0 625 1250 2500 Positive control
109.2 ± 8.8 112.2 ± 8.5 106.0 ± 9.5 108.6 ± 6.7 99.8 ± 3.5
54.7 56.1 53.0 54.3 49.9
109.0 ± 5.5 107.6 ± 8.8 108.2 ± 7.7 107.2 ± 8.5 —
54.5 53.8 54.1 53.6 —
Abbreviations: bw, body weight; MBE, magnolia bark extract; NCE, normochromatic erythrocyte; No., number; PCE, polychromatic erythrocyte; SD, standard deviation. Positive control = cyclophosphamide (40 mg/kg body weight).
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Table 6 Number of micronucleated polychromatic erythrocytes in the in vivo micronucleus test Sex
MBE dose level (mg/kg bw)
24 h
48 h
No. of MNPCE (mean ± SD)
MNPCE (%)
No. of MNPCE (mean ± SD)
MNPCE (%)
Female
0 625 1250 2500 Positive control
3.8 ± 1.9 3.6 ± 1.1 4.2 ± 1.8 4.6 ± 2.1 23.2 ± 2.9*
1.9 1.8 2.1 2.3 11.6*
4.8 ± 2.2 4.0 ± 1.0 4.2 ± 1.3 3.8 ± 0.8 —
2.4 2.0 2.1 1.9 —
Male
0 625 1250 2500 Positive control
4.4 ± 1.1 4.0 ± 1.2 4.2 ± 1.5 4.2 ± 1.6 24.6 ± 3.8*
2.2 2.0 2.1 2.1 12.3*
4.4 ± 1.8 4.2 ± 1.3 4.6 ± 1.1 4.4 ± 1.3 —
2.2 2.1 2.3 2.2 —
Abbreviations: bw, body weight; MBE, magnolia bark extract; MNPCE, micronucleated polychromatic erythrocyte; No., number; PCE, polychromatice erythrocyte; SD, standard deviation. Positive control = cyclophosphamide (40 mg/kg body weight). * Significantly different compared to negative control (P < 0.05).
The proportion of immature to total erythrocytes was not affected by MBE administration (Table 5). No statistically significant increase in the number of micronucleated PCEs was observed in any of the MBE-treated groups compared to the negative control group at either time point (Table 6). The positive control substance (cyclophosphamide) induced a marked and statistically significant increase in the number of PCEs with micronuclei but did not affect the proportion of immature to total erythrocytes. 4. Discussion The results of the genotoxicity tests demonstrate that MBE, consisting of 94% magnolol and 1.5% honokiol, is not mutagenic under the experimental conditions for S. typhimurium TA98, TA100, TA1535 and TA1537 and for E. coli WP2 uvrA, and that MBE does not affect the proportions of PCEs to total erythrocytes or increase the number of micronucleated PCEs in Swiss albino mice. All genotoxicity assays were conducted in replicate using multiple doses that were determined in preliminary dose range-finding experiments. No evidence of mutagenicity was observed at any concentration tested, including those that caused cytotoxicity. The bacterial reverse mutation tests were conducted with or without S-9 activation to determine whether MBE could be converted to mutagenic metabolites following bioactivation by microsomal enzymes. Appropriate positive and negative controls were included for both genotoxicity tests and produced the expected results, demonstrating the validity of the assays. Although there are no other known published studies on the genotoxic potential of MBE, it has been reported that magnolol inhibits UV-induced mutations (Fujita and Taira, 1994), as well as the mutagenic activity induced by several indirect mutagens (Saito et al., 2006). These reports demonstrate that magnolol has anti-mutagenic activity, and support the findings of the current study.
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