Fitoterapia 77 (2006) 538–545www.elsevier.com/locate/fitote

Antimutagenic, antioxidant and antimicrobial properties ofMaytenus krukovii bark

Renato Bruni a,⁎, Damiano Rossi b, Mariavittoria Muzzoli c, Carlo Romagnoli d,Guglielmo Paganetto e, Elena Besco f, Fritz Choquecillo g, Katia Peralta g,

William Schmitt Lora h, Gianni Sacchetti c

a Dipartimento di Biologia Evolutiva e Funzionale, Parco Area delle Scienze 11, 43100 Parma, Italyb CATgroup — Centro Analisi Territoriali , via Provinciale 73, 44030 Copparo, Ferrara, Italyc Dipartimento delle Risorse Naturali e Culturali, C.so Porta Mare 2, 44100 Ferrara, Italy

d Dipartimento del Museo di Paleobiologia e dell'Orto Botanico, Università di Modena e Reggio Emilia,Viale dei Caduti in Guerra 127, 41100 Modena, Italy

e SISTA — Servizio Igiene Sicurezza e Tutela Ambientale, via Fossato di Mortara 17/19, 44100 Ferrara, Italyf Dipartimento di Scienze farmaceutiche, Via Fossato di Mortara 17/19, 44100 Ferrara, Italy

g Universidad Nacional Mayor de San Marcos, Facultad de Farmacia y Bioquimica, Av. Venezuela Cdra. 34, Lima, Peruh Instituto Peruano de Investigación Fitoterápica Andina (IPIFA), Av. El Rosario 512-526, Chaclacayo, Lima 08, Peru

Received 20 October 2005; accepted 22 June 2006Available online 6 July 2006

Abstract

The hydroalcoholic extract of Maytenus krukovii bark was investigated for its in vitro mutageno-protective activities by meansof the Ames Salmonella/microsome assay. The extract showed an inhibitory effect in both T98 and T100 strains against themutagenic activity of promutagen 2-aminoanthracene but was not protective against directly acting mutagens sodium azide and 2-nitrofluorene. When tested as a radical scavenger and antioxidant it produced a dose-dependent inhibition. The extract did not showsignificant antibacterial properties, and was weakly active against dermatophyte and phytopathogenic fungi, but inhibited thegrowth of phytopathogen Pithyum ultimum.© 2006 Elsevier B.V. All rights reserved.

Keywords: Maytenus krukovii; Antimutagenicity; Antioxidant activity; Antimicrobial activity

1. Introduction

Human cells are constantly exposed to reactive oxygen radicals generated by a number of biotic and abiotic factorssuch as irradiation, atmospheric and food pollutants or byproducts of metabolic processes. When the exposureoverwhelms endogenous preventive systems, cells are exposed to a potentially harmful load of oxidants, leading tovarious free-radical-induced noxious effects. These include, among others, oxidation of nucleic acids, proteins, lipidsand carbohydrates, which may subsequently determine mutagenesis and diseases related to DNA damage [1].

⁎ Corresponding author. Tel.: +39 521 906004; fax: +39 521 905403.E-mail address: bruni@biol.unipr.it (R. Bruni).

0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2006.06.009

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In particular, exposure to environmental mutagens is of great health concern and experimental data suggest arelationship between chemical mutagenesis and carcinogenesis [2]. Given that many mutagens act on the cell via itsactive metabolites or by generating free radicals, it is therefore suggested that supplementation of natural antioxidantscould ameliorate the harmful effects of oxidative processes in the living organism and both experimental and clinicaldata have been provided to prove this. Thus, in the constant quest for new therapeutic and commercial herbal products,several natural compounds and phytocomplexes have been tested for their ability to prevent oxidation and mutagenesis[3–5]. As a consequence, the market of healthy and herbal nutraceuticals constantly addresses its attention to new plantsources offering antioxidant or mutageno-protective efficacy and their use and positive image among the consumers isspreading.

Maytenus krukovii A.C. Sm (Maytenus chuchuhuasha Raymond–Hamet and Colas) (Celastraceae) is an amazoniancanopy tree indigenous to the tropical rainforests of Bolivia, Colombia, Ecuador and Peru [6,7]. Both for its botanicalcharacteristics and therapeutic use it appears strictly related to M. laevis and M. macrocarpa. It grows 30 m high andhas large leaves (10–30 cm) and small, white flowers. Its tough, heavy, reddish-brown bark is the main source of acrude drug locally called Chuchuhuasi, which hydroalcoholic extract is widely used in ethnopharmacology as a remedyfor arthritis, rheumatism and back pain [6,8]. Chuchuhuasi is also reported to be an astringent, antitumor, antiasthmatic,fertility-regulating agent and is used as well to treat stomach problems [6,8]. The entire Maytenus genus is char-acterized by an almost unique variety of phytochemicals, mostly triterpenes, flavonols, condensed tannins andsesquiterpene alkaloids with a very uncommon ansamacrolide-like skeleton ring [9–12]. M. krukovii makes noexception on this regard [13,14]. A number of Maytenus species have been reported for their antitumor, anti-inflammatory, antinociceptive activity and the presence of phenolic metabolites such as condensed tannins [6],flavonoids [15] and phenolic triterpenes [16,17] could justify the widespread popular use of some species asantiinflammatory and antiulcerogenic remedies and could also be related to some reports of antimutagenic activity [18].Condensed tannins and catechins are, in fact, known to be protective against various pathologies including cancer[19,20], thus the consumption of foods or herbal remedies rich in those compounds, e.g. Camelia sinensis infusions,has been proposed as a way to prevent some kinds of cancers [21]. Moreover, antimicrobic activity has been detected insome Maytenus species [22], but at present no data on this regard are available on M. krukovii.

Thus, the aim of this work was the assessment of antimutagenic, antioxidant and antimicrobial efficacy of M.krukovii bark hydroalcoholic extract, which is traditionally used in Andean folk medicine and commercially sold asnatural remedy.

2. Experimental

2.1. General

All chemicals and solvents were of analytical grade and supplied from Sigma-Aldrich (Steinheim, Germany). TA98,TA100 Salmonella typhimurium strains were purchased from Molecular Toxicology Inc. (Boone, NC, USA).

2.2. Plant

M. krukovii bark was identified by Dr. Fritz Choquecillo and Dr. Katia Peralta in the Herbarium of the NacionalMayor University de San Marcos, Lima. Vouchers of the crude drug were deposited in Dipartimento delle RisorseNaturali e Culturali of the University of Ferrara with code CHU1.

2.3. Extract preparation

The finely ground bark was extracted in 20% EtOH. The extract was filtered and freeze-dried. The resulting powderwas then re-dissolved in distilled water according to the corresponding assay conditions.

2.4. Metabolic activation system

Lyophilized post-mitochondrial supernatant S9 mix (Aroclor 1254-induced, Sprague–Dawley male rat liver in0.154 M KCl solution), commonly used for the activation of promutagens to mutagenic metabolites, was purchased

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from Molecular Toxicology, Inc. (Boone, NC, USA) and stored at −80 °C. Before its use, the S9 mix was filteredthrough a 0.45 μm Millipore disposable filter.

2.5. Mutagenicity/antimutagenicity of M. krukovii liophilized extract

The liophilized extract samples dissolved in dimethyl sulphoxide (DMSO) were tested with S. tiphymurium strainsTA98 and TA100 (100 μl/plate of a fresh overnight culture) with and without the addition of 0.5 ml of a 5% S9exogenous metabolic system, using plate incorporation assay [23]. The concentrations of the test samples used were 1,5, 10, 50, 100, 500, 1000 μg 0.1 ml−1 plate−1. The plate for negative control contained 100 μl of DMSO, with orwithout S9 mix. The positive control plates for the first contained 20 μg/plate of 2-aminoantracene and S9 mix or20 μg/plate of 2-nitrofluorene. The positive control plates for the latter contained 20 μg/plate of 2-AA and S9 mix or1 μg/plate of sodium azide. A sample was considered mutagenic when the observed number of colonies was at least 2-fold over the spontaneous level of revertants. The colonies were counted manually after 48 h of incubation at 37 °Cusing a Colony Counter 560 Suntex (Antibioticos, Italy).

The inhibitory effect ofM. krukovii extract (1, 5, 10, 50, 100, 500, 1000 μg 0.1 ml−1 plate−1) on mutagenic activityof direct acting mutagen 2-nitrofluorene (2 μg/plate) and sodium azide (1 μg/plate) was examined in plateincorporation assay, derived from mutagenicity test as described by Maron and Ames with some minor modifications,using tester strain TA98 and TA100 respectively. The inhibitory effect ofM. krukovii extract on the mutagenic activityof the indirectly acting mutagen 2-aminoanthracene (2 μg/plate) was examined in plate incorporation assay, using testerstrain TA98 and TA100 with S9 mix.

2.6. Statistical analysis

Data presented are the mean of 8 plates plus/minus Standard Deviation from two separate experiments eachperformed with quadruplicate plates. All data were analyzed for statistical significance and homogeneity of varianceusing Student's t-test and F-test respectively. All computations were made by employing the statistical software SPSSver. 10.0.

2.7. Toxicity of M. krukovii extract

In order to verify the toxicity of the analyzed samples on bacterial cells, a toxicity evaluation was performed [23]. Afresh 15-h culture was diluted 105 times to give a 1–2×104 bacteria/ml solution. The test samples at severalconcentrations (1, 5, 10, 50, 100, 500, 1000 μg 0.1 ml−1 plate−1) diluted in DMSO, mixed with 2 ml of molten topagar, were plated with 0.1 ml of the diluted culture. Minimal glucose agar plates were enriched with 10 μmol of L-

Fig. 1. Number of colonies/plate of S. typhimurium tester strain TA98 in the presence of liophilized extract of M. krukovii in minimal glucose agarplate enriched with L-histidine and biotin.

Fig. 2. Number of colonies/plate of S. typhimurium tester strain TA100 in the presence of liophilized extract of M. krukovii in minimal glucose agarplate enriched with L-histidine and biotin.

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histidine and 0.05 μmol of biotin by incorporating these nutrients into the soft agar overlay. Duplicate plates werepoured for each dose of the solution. The number of colonies was assessed after the plates were incubated at 37 °C for48 h and compared with that of control where no test samples were added (Figs. 1 and 2).

2.8. Determination of polyphenol concentration

Total polyphenol concentration was determined spectrophotometrically by means of the Folin–Ciocalteauphosphomolybdic–phosphotungstic acid reagents [24].

2.9. Scavenging and antioxidant activity

The antioxidant and radical scavenging activity of the Maytenus extract and reference compounds was assessed bythe DPPH (1,1-diphenyl-2-picrylhydrazyl) test and the β-carotene bleaching test as previously reported [25].Scavenging activity on oxygen radical absorbance capacity (ORAC) assay is based upon the early work of Ghiselli etal. [26] and Glazer [27], as developed further by Cao et al. [28]. Photochemiluminescence (PCL) assay was performedas described by Popov and Lewin [29–31] and involved the photochemical generation of superoxide O2U

- free radicalscombined with chemiluminescence detection. The assay is initiated by optical excitation of a photosensitizer, resultingin the generation of the superoxide radical anion.

Table 1Radical scavenging and antioxidant activity of M. krukovii hydroalcoholic extract

DPPH β-carotene

M. krukovii 38.38±0.21 586.85±1.21Gallic acid 4.98±0.09 1093.8±11.6Catechin 31.97±0.23 1791.34±9.7BHA 33.52±0.19 750.23±9.45BHT 40.31±0.29 1307.14±10.4Trolox 28.22±0.33 786.5±8.7GTE a 5.58±0.41 220.02±6.43GSEb 12.15±1.32 240.12±5.17PE c n.a. n.a.

n.a. = not active.a GTE, Green Tea polyphenol extract.b GSE, Grape Seed extract.c PE, Papaya extract.

Table 2Photochemiluminescence and ORAC values of M. krukovii hydroalcoholic extract

M. krukovii Vitamin E Ascorbic acid Trolox mmol/g±S.D. PE a GTEb GSE c

ORAC 4.497±0.113 – – – 0.121±0.08 6.479±0.061 4.033±0.077PCL-lypo 0.35±0.01 2.77±0.02 – 3.98±0.06 b1·10−3 1.33±0.01 1.66±0.03PCL-hydro 0.4±0.01 – 6.2±0.2 – b1·10−3 4.96±0.02 0.28±0.01

ORAC = oxygen radical absorbance capacity. PCL = Photochemiluminescence.a PE, Papaya extract.b GTE, Green Tea polyphenol extract.c GSE, Grape Seed extract.

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2.10. Antimicrobial activity

Biological activities were performed on different classes of microorganisms. For antibacterial assays, Gram (+)(Micrococcus luteus ATCC, Staphylococcus aureus ATCC, Bacillus subtilis ATCC, Enterococcus foecalis ATCC) andGram (−) (Klebsiella oxytoca ATCC, Escherichia coli ATCC, Pseudomonas aeruginosa ATCC, Proteus mirabilisATCC) bacteria. The culture media and conditions employed for ATCC strains were in accordance with American TypeCulture Collections protocols. The biological activity against these classes of microorganisms was determined byemploying the standard disks diffusion technique [32]. Antifungal properties were evaluated as described inRomagnoli and Sacchetti [33]. Trichophyton mentagrophytes (Robin) Blanchard strain no. 160.66 (CBS), Nannizziacajetani Ajello strain no. 3441 (IHME), Phythium ultimum Trow strain no. 58812 (ATCC), Magnaporthe grisea (T.T.Hebert) Yaegashi et Udagawa, strain no. 64413 (ATCC).

Each hydroalcoholic extract was aseptically added to the medium at 45 °C to obtain a final concentration of 50, 100,200 and 500 μg/ml.

3. Results and discussion

3.1. Antioxidant properties

In view of the differences among the test systems available, the results of a single assay can give only a suggestionon the protective potential of phytochemicals. Therefore, the use of more than one method is highly advisable. Amongthe methods that can be used for the evaluation of the antioxidant activity, few of them (TEAC, DPPH, PCL, ORAC)are useful to determine the activity of both hydrophilic and lipophilic species, thus ensuring a better comparison of theresults [34]. When screened for its antioxidant and radical scavenging properties, the hydroalcoholic extract of M.krukovii provided dose-dependent results on different assays. In particular Chuchuhuasi provided a radical scavengingcapacity comparable to that of most synthetic and natural reference compounds (Table 1). In particular, in the DPPHtest and in the β-carotene bleaching test the crude extract produced a dose-dependent inhibition (IC50: 38.38±0.21, and586.85±1.21 μg/ml, respectively). Moreover, IC50 of the bark extract were consistently better than those obtained from

Fig. 3. Mutagenic activity of 2-nitrofluorene on S. typhimurium tester strains TA98 and sodium azide on S. typhimurium tester strains TA100, in thepresence of liophilized extract of M. krukovii.

Fig. 4. Mutagenic activity of indirect mutagen 2-aminoanthracene on S. typhimurium tester strains TA98 and TA100, in the presence of liophilizedextract of M. krukovi.

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commercial Grape Seed and Green Tea polyphenol extracts. These data were confirmed by photochemiluminescence(PCL) where the extract showed a fair antioxidant capacity (Table 2), lower than that of Trolox and vitamin E buthigher than Grape Seed extract. The most relevant result was however obtained in the evaluation of oxygen radicalabsorbance capacity (ORAC), measuring a hydrogen atom transfer reaction mechanism, which is most relevant tohuman biology. Result in ORAC test was 4.497±0.113 mmol trolox/g, once again better or comparable than thoseobtained from commercial Grape Seed and Green Tea polyphenol extracts, which are marketed for their antioxidantproperties [35]. These data are of particular significance because the results of this assay easily correlate with thetherapeutical, nutriceutical and cosmeceutical potential of a given antioxidant and this method is accepted by theindustry to the point that some nutraceutical manufacturers are beginning to include ORAC values on product labels.

3.2. Mutagenic protection

When tested in the Ames Salmonella/microsome assay, no effect of increasing amounts of M. krukovii alcoholicextract was found on the activity of the directly acting mutagens 2-nitrofluorene and sodium azide (Fig. 3). However,the major result is that M. krukovii bark extract is able to induce an evident decrease on the mutagenicity of theindirectly acting mutagen 2-aminoanthracene, which acts as a genotoxic compound through a liver S9 fraction. Themutagenicity of 2-aminoanthracene was in both cases reduced by more than 90%. This is, to our knowledge, a novel

Table 3Antifungal activity of M. krukovii hydroalcoholic extract

Fungi M. krukovii hydroalcoholic extract a

(μl/ml) Growth %

T. mentagrophytes var. mentagrophytes 50 100100 89.4200 74.5500 87.2

N. cajetani 50 88.9100 91.9200 82.9500 80

P. ultimum 50 80.8100 65.6200 50500 32.4

M. grisea 50 91.8100 89.8200 87.7500 79.6

a Ketoconazole provided a 0% growth in all tested strains from 10 μl/ml.

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biological activity of a M. krukovii bark extract (Fig. 4). These results are consistently better than those reported byWeisburger et al. [20] according to the analogue antimutagenicity of green and black tea polyphenols, where at thesame concentration of extract per plate the reduction of the number of revertants was 60%.

The mechanism by which Chuchuhuasi extract inhibited the mutagenicity of 2-aminoanthracene is not known.However, some suggestions can be made on the basis of the present set of data. Since there is an evident difference inthe protective activity of M. krukovii bark against direct and indirect mutagens and since the great abundance offlavonoids, triterpene dimers and condensed tannins in the bark has been proved [13,14] it can be therefore suggestedthat these known antimutagens may interact synergically with some specific enzymes in the liver homogenates, whichare necessary for the activation of chemical mutagens. The polyphenolic abundance in the bark extract was alsoconfirmed by means of the total phenolic content, determined according to the Folin–Ciocalteu method and expressedas gallic acid equivalents (270 mg/g of extract).

Most of the polymeric tannins exerting an antimutagen activity on 2-aminoanthracene and other nitroaromaticindirect acting mutagens, can be classified as blocking agents since they inhibit the conversion of the latter to ultimatecarcinogens. Proanthocyanidins and flavonoids are known to form a complex with proteins and monooxygenasesmetabolizing xenobiotics, due to the fact that the formers can act as multidentate ligands and thus are able to bindsimultaneously more than one point to the protein surface [36,37]. The more the oligomeric nature, the more itsassociation produces an increased antimutagenic activity towards the inhibition of the enzyme mediated genotoxicityof indirect acting mutagens as 2-aminoanthracene [38]. As previously reported, this kind of action is likely to be thefinal result of many interactive effects, for which the whole phytocomplex [39], rather than a single chemicalconstituent, may be responsible. Therefore it could be suggested that M. krukovii interacts with the enzymes of the S9mix, thereby inhibiting the transformation of 2-aminoantracene into its active forms. A similar behaviour waspreviously noted [18] in M. illicifolia extract.

3.3. Antimicrobic properties

Regarding the antimicrobial properties of the extract, it must be noted that it was completely inactive at 1000 mg/mlagainst both Gram (+) and Gram (−) bacteria (data not shown), in accordance with previous reports regarding thesimilar specieM. macrocarpa [40]. The efficacy of the extract on fungal strains was weak, but not negligible (Table 3)and in particular it showed a inhibitory activity against the phytopathogenic fungus P. ultimum.

Since major attention has been recently devoted to natural antimutagenic and antioxidant factors that could lower therates of mutation by including them in dietary products, this activity is valuable towards an extension of the employ ofthis crude drug as new valuable ingredient for food and/or nutriceutical, cosmeceutical support in the promotion ofhealth, besides its consolidated ethnomedical use.

References

[1] Ames N, Shigenaga MK, Hagen TM. Proc Natl Acad Sci U S A 1993:7915.[2] Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Mol Cell Biochem 2004;266:37.[3] Ferguson LR, Philpott M, Karunasinghe N. Toxicology 2004;198:147.[4] Weiss JF, Landauer MR. Toxicology 2003;189:1.[5] Surh YJ. Food Chem Toxicol 2002;40:1091.[6] Gonzalez GJ, Delle Monache G, Delle Monache F, Marini-Bettolo BG. J Ethnopharmacol 1982;5:73.[7] Martinod P, Puredes A, Delle Monache F, Marini-Bettolo BG. Phytochemistry 1976;15:562.[8] Duke JA, Rodolfo V. Amazonian ethnobotanical dictionary. Boca Raton FL: CRC Press; 1994. p. 114.[9] Nakagawa H, Takaishi Y, Fujimoto Y, Duque C, Garzon C, Sato M, et al. J Nat Prod 2004;67:1919.[10] Shirota O, Tamemura T, Morita H, Takeya K, Itokawa H. J Nat Prod 1996;58:1072.[11] Kupchan SM, Komoda Y, Branfman AR, Sneden AT, Court WA, Thomas GJ, et al. J Org Chem 1977;42:2349.[12] Corsino J, Dulce HS, Zanoni MVB, Bolzani VS, Franco SC, Pereira AMS, et al. Phytochem Res 2003;17:913.[13] Shirota O, Sekita S, Satake M, Morita H, Takeya K, Itokawa H. Chem Pharm Bull 2004;52:1148.[14] Shirota O, Sekita S, Satake M, Morita H, Takeya K, Itokawa H. Heterocycles 2004;63:1891.[15] Leite JPV, Rastrelli L, Romussi G, Oliveira AB, Vilegas JHY, Vilegas W, et al. Agric Food Chem 2001;49:3796.[16] Itokawa H, Shirota O, Ikuta H, Morita H, Takeya K, Itaka Y. Phytochemistry 1991;30:3713.[17] Shirota O, Morita H, Takeya K, Itokawa H. J Nat Prod 1994;57:1675.[18] Horn RC, Ferrao-Vargas VM. Mutagenesis 2003;18:113.[19] De Bruyne T, Pieters L, Deelstra H, Vlietinck A. Biochem Syst Ecol 1999;27:445.

545R. Bruni et al. / Fitoterapia 77 (2006) 538–545

[20] Weisburger JH, Hara Y, Dolan L, Luo F, Pittman B, Zang E. Mutat Res 1996;371:57.[21] Bravo L. Nutr Rev 1998;56:317.[22] Moujir L, Gutiérrez-Navarro AM, González AG, Ravelo AG, Javler GL. Biochem Syst Ecol 1990;18:25.[23] Maron DM, Ames BN. Mutat Res 1983;113:173.[24] Singleton VL, Rossi JA. Am J Enol Vitic 1965;16:144.[25] Bruni R, Muzzoli M, Ballero M, Lio MC, Fantin G, Poli F, et al. Fitoterapia 2004;75:50.[26] Ghiselli A, Serafini M, Maiani G, Azzini E, Ferro-Luzzi A. Free Radic Biol Med 1995;18:29.[27] Glazer AN. Methods Enzymol 1990;186:161.[28] Cao G, Alessio HM, Cutler RG. Free Radic Biol Med 1993;14:303.[29] Popov I, Lewin G. Methods Enzymol 1999;300:437.[30] Popov I, Volker H, Lewin G. Redox Rep 2001;6:43.[31] Popov I, Lewin G. Free Radic Biol Med 1994;17:267.[32] Bruni R, Medici A, Andreotti E, Fantin C, Muzzoli M, Dehesa M, et al. Food Chem 2004;85:415.[33] Romagnoli C, Sacchetti G. In: Rai MK, Mares D, editors. Plant derived antimycotics. Binghamton NY: Harworth Press; 2003. p. 393.[34] Prior R, Wu X, Schaich K. J Agric Food Chem 2005;53:4290.[35] Rebabah TM, Hettiarachchy NS, Horax R. J Agric Food Chem 2004;52:5183.[36] Kloucek P, Polesny Z, Svobodova B, Vlkova E, Kokoska L. J Ethnopharmacol 2005;99:309.[37] Hodek P, Trefil P, Stiborova M. Chem Biol Interact 2002;139:1.[38] Naghabusan M, Amonkar AJ, Nair UJ, Santhanam U, Ammigan N, D'Souza AV, et al. Cancer Res Clin Onc 1988;114:177.[39] Gali HU, Perchellet EM, Klish DS, Johnson JM, Perchellet J. Int J Cancer 1992;51:425.[40] Bianchi A. Annali Istituto Superiore Sanità, vol. 35; 1999. p. 505.