Mutation Research, 282 (1992) 93-98 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

93

MUTLET 0663

Glycyrrh&a glabra extract

as an e f f e c t o r o f i n t e r c e p t i o n

in Escherichia coli K12 + Simon Kuo a, Delbert M. Shankel a, Hanumaiah Telikepalli b and Lester A. Mitscher b a Departments of Microbiology and o Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045 (USA) (Received 26 August 1991) (Revision received 24 January 1992) (Accepted 3 February 1992)

Keywords: Interception; Glycyrrhiza glabra; Antimutagenesis; Glutathione; Flavonoids; Antioxidants

Summary Glycyrrhiza glabra polar lipid extract contains a number of flavonoids and related chemical compounds. Studies on the effectiveness of Glycyrrhiza glabra polar lipid extract in intercepting reactive molecules generated from the illumination of the photosensitizers rose bengal and phenosafranin indicate that it is effective in preventing cytotoxicity against E. coli K12 + in a dose-related fashion using illuminated rose bengal. Since only a modest scavenging of singlet oxygen generated from phenosafranin is observed, the effects of the extracts are less related to singlet oxygen-mediated oxidation of substrate (type II reactions) than non-singlet oxygen-mediated oxidation of substrate (type I reactions). Elevated levels of glutathione observed in exponentially growing cells of E. coli K12 were also observed.

Naturally-derived antioxidants form an important and ubiquitous group of substances. Plants characteristically produce a variety of antioxidants as chemoprotectants against molecular damage from active oxygen species. These substances may also serve as protection against other electrophilic substances which may be potentially damaging. Phenolics compose the major class of plant-derived antioxidants. They have been implicated in the inhibition of 4-nitroquinoline 1-oxide and N-methyloN'-nitro-N-nitrosoguanidine mutaCorrespondence: Dr. Simon Kuo, Department of Microbiology, The University of Kansas, Lawrence, KS 66045, USA. Abbreviations: DTNB, 5,5'-dithiobis(2-nitrobenzoic RNO, N, N-dimet hyl-4-nitrosoaniline.

acid);

genesis in Escherichia coli (Jain et al., 1989; Shimoi et al., 1986); induction of glutathione-Stransferases and increased levels of glutathione (Batzinger et al., 1978; Benson et al., 1979; Kuo et al., 1989). Among phenolic compounds, the flavonoids are perhaps the most important group. The average daily human intake of flavonoids is about 1 g (Jongen and Dorgelo, 1986). The range of protection afforded by these compounds, in addition to their activity as antioxidants, includes chelation of metal ions (Brown, 1980); enhancing the breakdown of epoxides (Yagi et al., 1988); antimutagenesis in Salmonella against aflatoxin B1 (Francis, 1989) and MNNG (Bhattacharya, 1989); inhibition of nitrosation reactions by depleting nitrite (Stich and Rosin, 1984). Mutagenic activity has also been observed (Jongen and

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Dorgelo, 1986; MacGregor and Wilson, 1986). It has been conjectured that mutagenesis by flavonoids involves the oxidation of adjacent hydroxyls to o-quinones (Hartman and Shankel, 1990). Thus lack of adjacent hydroxyls could mean that a compound is innocuous yet antimutagenic. G. glabra is the licorice plant, and has a history of consumption for the past 6000 years (Mitscher, 1986). It is consumed in a variety of herbal medicines, primarily in the Far East, as a flavoring, and as a dietary supplement. Previous studies have shown that extracts of the plant exhibit antimutagenic activity in several test systems (see below). The polar lipid fraction of G. glabra is markedly purified relative to crude extracts and contains a number of phenolic constituents, including flavonoids, which do not possess a 1,2-dihydroxybenzene (catechol) moiety. Components identified in the polar lipid fraction include: glabridin, glabrol, glabrene, foromonetin, phaseollinisoflavan, hispaglabridin A and hispaglabridin B (Mitscher et al., 1986). Using Escherichia coli GW5352, a strain which carries a T n l 0 insertion in the ada locus, Mitscher et al. (1986) demonstrated that the number of revertants to auxotrophy was reduced when cells were preincubated with G. glabra crude extract, and that survival of ABl157, which does not carry the transposon insert, was increased relative to GW5352 when pre-incubated with G. glabra. They also demonstrated that the number of EMS-induced revertants to His + in Salmonella typhimurium TA100 and TA1535 decreased when pretreated with G. glabra extracts at varying concentrations, and that they are protective in preventing growth inhibition by EMS in the Bacillus subtilis Rec assay (Mitscher et al., 1986). We have endeavored to ascertain the properties of G. glabra in intercepting type I and II products of rose bengal illumination as well as its ability to scavenge singlet oxygen generated by the illumination of phenosafranin. Materials and methods

Strains, media and chemicals E. coil K12 + strains were kindly provided by Dr. Philip Hartman of The Johns Hopkins Uni-

versity. Rose bengal, N,N-dimethyl-4-nitrosoaniline, 2,5-bis(hydroxymethyl)-furan, phenosafranin, 5,5'-dithiobis(2-nitrobenzoic acid), N,N-dimethyl4-nitrosoaniline and 1-chloro-2,4-dinitrobenzene, were purchased from The Sigma Chemical Corporation, St. Louis, MO. Bacterial strains were stored on LB or NB agar slants at 4°C. Prior to each experiment, the strains were inoculated into 10-250 ml of M9 minimal medium supplemented with 0.2% dextrose, and grown overnight on a gyrotory water bath shaker at 37°C.

Growth assays 0.1 ml of an overnight culture grown to an O.D. of 0.4 at 600 nm was used to inoculate 10 ml duplicate cultures of M9 media supplemented with 0.2% glucose. G. glabra polar lipid fraction was delivered in a total volume of 0.1 ml (for a total of 0.1% final DMSO concentration). The cultures were grown with vigorous shaking on a gyrotory water bath for 24 h. Absorbance readings were taken at the indicated time intervals. 0.1-ml aliquots of random cultures were taken at various timepoints, diluted and plated to verify cell numbers. Photodynamic inactivation of E. coli The procedure used was essentially that of Dahl et al. (1988a). Fresh cultures of E. coli K12 were grown up overnight in M9 media supplemented with 0.2% glucose at 37°C with shaking to an O.D. of 0.30 at 600 nm. The cultures were diluted with 0.9% NaCI to approximately 105 C F U / m l . Rose bengal salts were dissolved at 20 × final concentration in DMSO. Illumination mixtures contained 0.05 M NaC1, 1 mM sodium phosphate (pH 7.0), 5% DMSO, and 104 C F U / m l E. coli. Glycyrrhiza glabra extract was added at the indicated concentrations in a total volume of 0.01 ml DMSO. Illumination mixtures were preincubated for a total of 120 min at 37°C in complete darkness. Preincubation alleviates the lag time for illuminated rose bengal inactivation of gram negative organisms due to lipopolysaccharide hindering of photosensitizer penetration (Dahl, 1988a). Illuminations were performed with 3 ml of mixture in 35 × 10 mm petri plates at 4°C using a 150 W G E tungsten filament lamp with a

95 light intensity of 1.1 m E / m 2 / s e c . A 10-cm column of water was used to filter out infrared and ultraviolet radiation. At 15-min time intervals 0.1 ml of the illumination mixture was plated on nutrient agar. Plates were kept at 4°C in complete darkness during the course of the assay. Subsequently all the plates were incubated for 24 h in complete darkness and survivors scored using a Dynatech Laboratories AutoCount automated colony counter.

Scavenging of singlet oxygen Light-dependent bleaching of N,N-dimethyl4-nitrosoaniline (RNO)~" in the presence of phenosafranin was used as an indicator of the efficiency of G. glabra extract in trapping singlet oxygen. The procedure used followed that of H a r t m a n et al. (1987). G. glabra extract dissolved in D M S O was added to a reaction mixture containing a total of 9 × 10 -6 M phenosafranin and 1.8× 10 -5 M N,N-dimethyl-4-nitrosoaniline in 0.05 M phosphate buffer p H 7.5. The final concentration of D M S O in all cases was 0.1%. The reaction mixtures were poured into 2 ml spect r o p h o t o m e t e r cuvettes and placed under a 150-W G E miser spotlamp with a light intensity of 1.1 m E / m Z / s e c suspended from the surface on which the cuvettes were placed. The experiment was performed at room temperature. Infrared radiation was absorbed using a 5-cm column of 2% CuSO 4 solution. The experiment was performed in darkness except for spotlamp illumination. Optical densities at 440 nm were followed using a Beckman DU-40 series spectrophotometer. Resuits are the averages of at least three experiments performed in duplicate.

equal volumes of phosphate buffer (0.1 M, p H 7.0), centrifuged and the supernatant discarded. The cells were then resuspended in 5% TCA, or 5% 5-sulfosalicylic acid. 0.70 ml of a N A D P H / buffer solution (phosphate 0.125 m o l e s / l ; E D T A 6.3 mmoles/1; N A D P H , 0.3 m m o l e s / l ) was placed into a cuvette. 0.1 ml of 5,5'-dithiobis(2-nitrobenzoic acid) solution (phosphate 0.125 m o l e s / l ; E D T A 6.3 mmoles/1, D T N B 6 mmoles/1) w a s added followed by the sample in distilled water (for a total sample volume of 200/~1). The cuvette was capped and mixed thoroughly, then placed in a water bath for 4 min until its t e m p e r a t u r e was stable. Glutathione reductase solution was then added (5 /~1), the contents of the cuvette mixed and the absorbance at 412 nm monitored for approximately 20 min. The amount of reduced glutathione was determined by comparison to a standard curve constructed using various amounts of glutathione diluted in 5% 5-sulfosalicylic acid in place of the cell-derived sample. Results

and discussion

To determine the effects of G. glabra extract on growth of E. coil K12, cells were grown in the presence of varying concentrations of the extract and growth monitored over a 24-h period (Fig. 1). It was observed that no significant growth effects 1.2-

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Assay of total reduced glutathione The total amount of reduced glutathione was measured using the glutathione reductase recycling assay described by Tietze (1969) and following the modifications of Anderson (1985). E. coli K12 was grown in M9 medium to exponential phase at 37°C on a gyrotory water bath (approximately 8 - 1 0 h). G. glabra extract was then added to the cultures at the indicated concentrations and they were incubated for varying lengths of time. 7 × 109 cells of E. coli K12 were then spun down. The cells were washed twice with

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Fig. 1. Growth of Escherichia coli K12+ in the presence and absence of Glycyrrhiza glabra polar lipid extract. A, 200 p~g/ml extract dissolved in DMSO; [2, 0 ~g/ml extract. Values are the averages of at least three duplicate experiments, p < 0.05.

96

occurred. Doses of 100 ~ g / m l , 10 ~zg/ml, 1 /~g/ml, 0.1 / x g / m l and 0.01 p~g/ml of the extract and D M S O alone yielded essentially the same curve. Slight variations in growth pattern observed at 12 h with 100 ~xg/ml of G. glabra and at 24 h with 200 ~ g / m l of extract were not significant. This confirms the extract's low toxicity reported previously (Mitscher et al., 1986). To determine the efficacy of the compound as an interceptor, cellular inactivation assays of E. coli K12 incubated with illuminated rose bengal were conducted. The presence of increasing concentrations of G. glabra polar lipid fraction resuited in increased survival of cells in a dose-related fashion (Fig. 2). Carnosine, used as a positive control, has previously been shown to be an effective scavenger of singlet oxygen (Dahl et al., 1987, 1988b; H a r t m a n and Shankel, 1990). Since type I (non-singlet oxygen-mediated) as well as type II (singlet oxygen-mediated) oxidation of substrate are possible in this assay, the phenosafranin/N,N-dimethyl-4-nitrosoaniline assay for singlet oxygen scavenging was performed to determine which scavenging pathway is most important.

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Time (mins.) Fig. 2. Percent survival of E. coli K12 in the presence of illuminated rose bengal with and without added G. glabra polar lipid extract. The illumination mixtures contained the following unless otherwise indicated: 0.05 M NaC1, 1 mM sodium phosphate (pH 7.0), 5% DMSO, and 104 CFU/ml E. coli. I~1, 0 ~g/ml extract; [] 200 ,~g/ml extract; B, 100 /zg/ml extract; B, 10 ~.g/ml extract; El, 5 mM carnosine. Values are the averages of at least three duplicate experiments, p < 0.05.

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Time (rains.) Fig. 3. Bleaching of N,N-dimethyl-4-nitrosoaniline (RNO) by illuminated rose bengal with and without added G. glabra polar lipid extract. The illumination mixtures contained the following unless otherwise noted: 9 × 10 6 M phenosafranin, 1.8× 10 -5 M N,N-dimethyl-4-nitrosoaniline in 0.05 M phosphate buffer (pH 7.5). m, R N O + p h e n o s a f r a n i n alone; /~, 200 ~ g / m l e x t r a c t + R N O alone; A, 10 ~ g / m l extract; ©, 100 ~zg/ml extract; t2, 200 ~zg/ml extract; e, i m M carnosine; × , 5 m M carnosine. Values are the averages of at least three triplicate experiments, p < 0.03.

G. glabra extract was observed to be modestly effective in mediating the bleaching of N,N-dimethyl-4-nitrosoaniline in the presence of the illuminated photosensitizer phenosafranin (Fig. 3). The latter compound in this system does not react with the substrate ( R N O ) itself, thus interception of the singlet oxygen generated from illumination of photosensitizer by the components of the G. glabra fraction is implicated. The extract was observed to be effective at a concentration of 200 tzg/ml, although effects were not nearly as vigorous as observed with carnosine, which has previously been shown to be an effective scavenger of singlet oxygen (Dahl, 1988b). Assays of reduced glutathione were performed to determine what contribution this endogenous scavenging system might have in the prevention of cytotoxicity. It was observed that there was, in exponentially-growing cells of E. coli K12, approximately 20% difference in glutathione levels in cells incubated in the presence of 100 / z g / m l of G. glabra extract compared to cells grown in

97 120

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Time (mins.) Fig. 4. Amounts of reduced glutathione in E. coli K12 grown in the presence and absence of G. glabra polar lipid extract. ©, 100 /~g/ml extract; [3, 10 /~g/ml extract; *, 0 /xg/ml extract. Values are the averages of at least three triplicate experiments and are per 1.4× 109 cells, p < 0.05. the a b s e n c e of the polar lipid fraction or incub a t e d with 10 / x g / m l of the fraction after 120 m i n of i n c u b a t i o n (Fig. 4). G i v e n the e v i d e n c e that the G. glabra polar lipid fraction is effective in p r e v e n t i n g cellular cytotoxicity from i l l u m i n a t e d rose bengal, this result may be i n t e r p r e t e d as s t e m m i n g either from scavenging of the singlet oxygen g e n e r a t e d , or as a n i n h i b i t i o n of direct i n t e r a c t i o n of the photosensitizer with cellular c o m p o n e n t s which lead to toxicity, or as a n i n h i b i t i o n of secondary p r o d u c t s g e n e r a t e d from i n t e r a c t i o n of i n t r a c e l l u l a r substances with the photosensitizer. As the polar lipid fraction exhibits low effectiveness in scave n g i n g singlet oxygen in the R N O / p h e n o s a f r a n i n assay, the latter two possibilities seem likely. T h e o b s e r v a t i o n of i n c r e a s e d g l u t a t h i o n e levels is interesting, t h o u g h u n i m p o r t a n t in the p r e v e n t i o n of cytotoxicity observed here since a significant increase in g l u t a t h i o n e level is only observed in cells grown with 100 / x g / m l of G. glabra p o l a r lipid fraction after 120 m i n of i n c u b a t i o n .

References Anderson, M.E. (1985) Determination of glutathione and glutathione disulfide in biological samples, Meth. Enzymol., 113, 548-555.

Batzinger, R.P., S.L. Ou and E. Bueding (1978) Antimutagenic effects of 2(3)-tert.-butyl-4-hydroxyanisole and of antimicrobial agents, Cancer Res., 38, 4478-4485. Benson, A.M., Y.N. Cha, E. Bueding, H.S. Heine and P. Talalay (1978) Elevation of extrahepatic glutathione Stransferase and epoxide hydratase activities by 2(3)-tert.butyl-4-hydroxyanisole,Cancer Res., 39, 2971-2977. Bhattacharya, R.A. (1989) Modulation of mutagenicity by plant flavonoids, Environ. Mol. Mutagen., 14(Suppl 15), 21. Brown, J.P. (1980) A review of the genetic effects of naturally occurring flavonoids, anthraquinones and related compounds, Mutation Res., 75, 243-277. Dahl, Thomas A., Robert W. Midden and P.E. Hartman (1987) Pure singlet oxygen toxicity for bacteria, Photochem. Photobiol., 46, 345-352. Dahl, T.A., W.R. Midden and D.C. Neckers (1988a) Comparison of photodynamic action of rose bengal in gram-positive and gram-negative bacteria, Photochem. Photobiol., 48, 607-612. Dahl, T.A., R.W. Midden and P.A. Hartman (1988b) Some prevalent biomolecules as defenses against singlet oxygen damage, Photochem. Photobiol., 47, 357-362. Francis, A.R., T.K. Shetty and R.K. Bhattacharya (1989) Modifying role of dietary factors on the mutagenicity of afiatoxin BI: in vitro effect of plant flavonoids, Mutation Res., 22, 393-401. Hartman, P.E., and D.M. Shankel (1990) Antimutagens and anticarcinogens: A survey of putative interceptor molecules, Environ. Mol, Mt~tagen., 15, 145-182. Hartman, P.E., Z. Hartman artd K.T. Ault (1990) Scavenging of singlet molecular oxygcrl by imidazole compounds: High and sustained activitie~ of carboxy terminal histidine dipeptides and excepti0rlal activity of imidazole-4-acetic acid, Photochem. Photobiol., 51, 59-66. Jain, A.K., K. Shimoi, Y. Nakamura, T. Kada, Y. Hara and I. Tomita (1989) Crude tea extracts decrease the mutagenic activity of N-methyl-N'nitro-N-nitrosoguanidine in vitro and in intragastric tract of rats, Mutation Res., 210, 1-8. Jongen, W.M.F., and F.O. Dorgelo (1986) Naturally occurring carcinogens and modulating factors in food of plant origin, Neth. J. Agric. Sci., 34, 395-404. Kuo, S., and D.M. Shankel (1989) Antimutagenic effects of glutathione S-transferase induction in E. coli, Environ. Mol. Mutagen., 14(Suppl. 15), 109. MacGregor, J.T., and R.E. Wilson (1988) F!avone mutagenicity in Salmonella typhimurium: Dependence on the pKM101 plasmid and excision repair capacity, Environ. Mol. Mutagen., 11,315-322. Mitscher, L.A., S. Drake, S.R. Gollapudi, J.A. Harris and D.M. Shankel (1986) Isolation and identification of higher plant agents active in antimutagenic assay systems: Glycyrrhiza glabra, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis Mechanisms, Plenum, New York, pp. 153-165. Shimoi, K., Y. Nakamura, I. Tomita and T. Kada (1985) Bio-antimutagenic effects of tannic acid on UV and chem-

98 ically induced mutagenesis in Escherichia coli B/r, Mutation Res., 173, 239-244. Stich, H.F., and M.P. Rosin (1984) Naturally occurring phenolics as antimutagenic and anticarcinogenic agents, Adv. Exp. Med. Biol., 177, 1-29. Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione, Anal. Biochem., 27, 502-522. Yagi, H., J.M. Sayer, D.M. Jerina, A.W. Wood, M.T. Huang,

R.L. Chang and A.H. Conney (1988) Inhibition of the biological activity of benzo[a]pyrene diol epoxide by naturally occurring small molecules, in: Y. Kuroda, D.M. Shankel and Y. Shirasu (Eds.), Abstracts, 2nd International Conference on Mechanisms of Antimutagenesis and Anticarcinogenesis, Plenum, New York, 36 pp.

Communicated by H. Hayatsu

Glycyrrhiza glabra extract as an effector of interception in Escherichia coli K12+.

Glycyrrhiza glabra polar lipid extract contains a number of flavonoids and related chemical compounds. Studies on the effectiveness of Glycyrrhiza gla...
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