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Antimutagenic, antioxidant and antimicrobial properties of Maytenus krukovii bark
Fitoterapia 77 (2006) 538 – 545 www.elsevier.com/locate/fitote
Antimutagenic, antioxidant and antimicrobial properties of Maytenus 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, Italy b CATgroup — Centro Analisi Territoriali , via Provinciale 73, 44030 Copparo, Ferrara, Italy c 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, Italy f 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, Peru h Instituto Peruano de Investigación Fitoterápica Andina (IPIFA), Av. El Rosario 512-526, Chaclacayo, Lima 08, Peru Received 20 October 2005; accepted 22 June 2006 Available online 6 July 2006
Abstract The hydroalcoholic extract of Maytenus krukovii bark was investigated for its in vitro mutageno-protective activities by means of the Ames Salmonella/microsome assay. The extract showed an inhibitory effect in both T98 and T100 strains against the mutagenic activity of promutagen 2-aminoanthracene but was not protective against directly acting mutagens sodium azide and 2nitrofluorene. When tested as a radical scavenger and antioxidant it produced a dose-dependent inhibition. The extract did not show significant antibacterial properties, and was weakly active against dermatophyte and phytopathogenic fungi, but inhibited the growth 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 factors such as irradiation, atmospheric and food pollutants or byproducts of metabolic processes. When the exposure overwhelms endogenous preventive systems, cells are exposed to a potentially harmful load of oxidants, leading to various free-radical-induced noxious effects. These include, among others, oxidation of nucleic acids, proteins, lipids and 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: [email protected] (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 a relationship between chemical mutagenesis and carcinogenesis [2]. Given that many mutagens act on the cell via its active metabolites or by generating free radicals, it is therefore suggested that supplementation of natural antioxidants could ameliorate the harmful effects of oxidative processes in the living organism and both experimental and clinical data 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 plant sources offering antioxidant or mutageno-protective efficacy and their use and positive image among the consumers is spreading. Maytenus krukovii A.C. Sm (Maytenus chuchuhuasha Raymond–Hamet and Colas) (Celastraceae) is an amazonian canopy tree indigenous to the tropical rainforests of Bolivia, Colombia, Ecuador and Peru [6,7]. Both for its botanical characteristics and therapeutic use it appears strictly related to M. laevis and M. macrocarpa. It grows 30 m high and has large leaves (10–30 cm) and small, white flowers. Its tough, heavy, reddish-brown bark is the main source of a crude drug locally called Chuchuhuasi, which hydroalcoholic extract is widely used in ethnopharmacology as a remedy for 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 characterized by an almost unique variety of phytochemicals, mostly triterpenes, flavonols, condensed tannins and sesquiterpene alkaloids with a very uncommon ansamacrolide-like skeleton ring [9–12]. M. krukovii makes no exception on this regard [13,14]. A number of Maytenus species have been reported for their antitumor, antiinflammatory, 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 as antiinflammatory 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 in some 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 as natural 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 Nacional Mayor University de San Marcos, Lima. Vouchers of the crude drug were deposited in Dipartimento delle Risorse Naturali 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 powder was 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 in 0.154 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 filtered through 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 strains TA98 and TA100 (100 μl/plate of a fresh overnight culture) with and without the addition of 0.5 ml of a 5% S9 exogenous 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 or without S9 mix. The positive control plates for the first contained 20 μg/plate of 2-aminoantracene and S9 mix or 20 μg/plate of 2-nitrofluorene. The positive control plates for the latter contained 20 μg/plate of 2-AA and S9 mix or 1 μg/plate of sodium azide. A sample was considered mutagenic when the observed number of colonies was at least 2fold over the spontaneous level of revertants. The colonies were counted manually after 48 h of incubation at 37 °C using a Colony Counter 560 Suntex (Antibioticos, Italy). The inhibitory effect of M. krukovii extract (1, 5, 10, 50, 100, 500, 1000 μg 0.1 ml− 1 plate− 1) on mutagenic activity of direct acting mutagen 2-nitrofluorene (2 μg/plate) and sodium azide (1 μg/plate) was examined in plate incorporation 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 of M. krukovii extract on the mutagenic activity of the indirectly acting mutagen 2-aminoanthracene (2 μg/plate) was examined in plate incorporation assay, using tester strain 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 each performed with quadruplicate plates. All data were analyzed for statistical significance and homogeneity of variance using Student's t-test and F-test respectively. All computations were made by employing the statistical software SPSS ver. 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]. A fresh 15-h culture was diluted 105 times to give a 1–2 × 104 bacteria/ml solution. The test samples at several concentrations (1, 5, 10, 50, 100, 500, 1000 μg 0.1 ml− 1 plate− 1) diluted in DMSO, mixed with 2 ml of molten top agar, 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 agar plate enriched with L-histidine and biotin.
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Fig. 2. Number of colonies/plate of S. typhimurium tester strain TA100 in the presence of liophilized extract of M. krukovii in minimal glucose agar plate enriched with L-histidine and biotin.
histidine and 0.05 μmol of biotin by incorporating these nutrients into the soft agar overlay. Duplicate plates were poured for each dose of the solution. The number of colonies was assessed after the plates were incubated at 37 °C for 48 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–Ciocalteau phosphomolybdic–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 by the 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 et al. [26] and Glazer [27], as developed further by Cao et al. [28]. Photochemiluminescence (PCL) assay was performed as described by Popov and Lewin [29–31] and involved the photochemical generation of superoxide O2U- free radicals combined with chemiluminescence detection. The assay is initiated by optical excitation of a photosensitizer, resulting in the generation of the superoxide radical anion.
Table 1 Radical scavenging and antioxidant activity of M. krukovii hydroalcoholic extract
M. krukovii Gallic acid Catechin BHA BHT Trolox GTE a GSE b PE c n.a. = not active. a GTE, Green Tea polyphenol extract. b GSE, Grape Seed extract. c PE, Papaya extract.
DPPH
β-carotene
38.38 ± 0.21 4.98 ± 0.09 31.97 ± 0.23 33.52 ± 0.19 40.31 ± 0.29 28.22 ± 0.33 5.58 ± 0.41 12.15 ± 1.32 n.a.
586.85 ± 1.21 1093.8 ± 11.6 1791.34 ± 9.7 750.23 ± 9.45 1307.14 ± 10.4 786.5 ± 8.7 220.02 ± 6.43 240.12 ± 5.17 n.a.
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Table 2 Photochemiluminescence and ORAC values of M. krukovii hydroalcoholic extract
ORAC PCL-lypo PCL-hydro
M. krukovii
Vitamin E
Ascorbic acid
Trolox mmol/g ± S.D.
PE a
GTE b
GSE c
4.497 ± 0.113 0.35 ± 0.01 0.4 ± 0.01
– 2.77 ± 0.02 –
– – 6.2 ± 0.2
– 3.98 ± 0.06 –
0.121 ± 0.08 b1 · 10−3 b1 · 10−3
6.479 ± 0.061 1.33 ± 0.01 4.96 ± 0.02
4.033 ± 0.077 1.66 ± 0.03 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.
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) and Gram (−) (Klebsiella oxytoca ATCC, Escherichia coli ATCC, Pseudomonas aeruginosa ATCC, Proteus mirabilis ATCC) bacteria. The culture media and conditions employed for ATCC strains were in accordance with American Type Culture Collections protocols. The biological activity against these classes of microorganisms was determined by employing the standard disks diffusion technique [32]. Antifungal properties were evaluated as described in Romagnoli and Sacchetti [33]. Trichophyton mentagrophytes (Robin) Blanchard strain no. 160.66 (CBS), Nannizzia cajetani 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 suggestion on the protective potential of phytochemicals. Therefore, the use of more than one method is highly advisable. Among the 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 the results [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 scavenging capacity comparable to that of most synthetic and natural reference compounds (Table 1). In particular, in the DPPH test and in the β-carotene bleaching test the crude extract produced a dose-dependent inhibition (IC50: 38.38 ± 0.21, and 586.85 ± 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 the presence of liophilized extract of M. krukovii.
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Fig. 4. Mutagenic activity of indirect mutagen 2-aminoanthracene on S. typhimurium tester strains TA98 and TA100, in the presence of liophilized extract of M. krukovi.
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 but higher than Grape Seed extract. The most relevant result was however obtained in the evaluation of oxygen radical absorbance capacity (ORAC), measuring a hydrogen atom transfer reaction mechanism, which is most relevant to human biology. Result in ORAC test was 4.497 ± 0.113 mmol trolox/g, once again better or comparable than those obtained from commercial Grape Seed and Green Tea polyphenol extracts, which are marketed for their antioxidant properties [35]. These data are of particular significance because the results of this assay easily correlate with the therapeutical, nutriceutical and cosmeceutical potential of a given antioxidant and this method is accepted by the industry 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 alcoholic extract 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 the indirectly acting mutagen 2-aminoanthracene, which acts as a genotoxic compound through a liver S9 fraction. The mutagenicity of 2-aminoanthracene was in both cases reduced by more than 90%. This is, to our knowledge, a novel
Table 3 Antifungal activity of M. krukovii hydroalcoholic extract Fungi
T. mentagrophytes var. mentagrophytes
N. cajetani
P. ultimum
M. grisea
a
Ketoconazole provided a 0% growth in all tested strains from 10 μl/ml.
M. krukovii hydroalcoholic extract a (μl/ml)
Growth %
50 100 200 500 50 100 200 500 50 100 200 500 50 100 200 500
100 89.4 74.5 87.2 88.9 91.9 82.9 80 80.8 65.6 50 32.4 91.8 89.8 87.7 79.6
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biological activity of a M. krukovii bark extract (Fig. 4). These results are consistently better than those reported by Weisburger et al. [20] according to the analogue antimutagenicity of green and black tea polyphenols, where at the same 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 in the protective activity of M. krukovii bark against direct and indirect mutagens and since the great abundance of flavonoids, triterpene dimers and condensed tannins in the bark has been proved [13,14] it can be therefore suggested that these known antimutagens may interact synergically with some specific enzymes in the liver homogenates, which are necessary for the activation of chemical mutagens. The polyphenolic abundance in the bark extract was also confirmed by means of the total phenolic content, determined according to the Folin–Ciocalteu method and expressed as gallic acid equivalents (270 mg/g of extract). Most of the polymeric tannins exerting an antimutagen activity on 2-aminoanthracene and other nitroaromatic indirect acting mutagens, can be classified as blocking agents since they inhibit the conversion of the latter to ultimate carcinogens. Proanthocyanidins and flavonoids are known to form a complex with proteins and monooxygenases metabolizing xenobiotics, due to the fact that the formers can act as multidentate ligands and thus are able to bind simultaneously more than one point to the protein surface [36,37]. The more the oligomeric nature, the more its association produces an increased antimutagenic activity towards the inhibition of the enzyme mediated genotoxicity of indirect acting mutagens as 2-aminoanthracene [38]. As previously reported, this kind of action is likely to be the final result of many interactive effects, for which the whole phytocomplex [39], rather than a single chemical constituent, may be responsible. Therefore it could be suggested that M. krukovii interacts with the enzymes of the S9 mix, thereby inhibiting the transformation of 2-aminoantracene into its active forms. A similar behaviour was previously 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/ml against both Gram (+) and Gram (−) bacteria (data not shown), in accordance with previous reports regarding the similar specie M. 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 the rates of mutation by including them in dietary products, this activity is valuable towards an extension of the employ of this crude drug as new valuable ingredient for food and/or nutriceutical, cosmeceutical support in the promotion of health, besides its consolidated ethnomedical use. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
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Antimutagenic, antioxidant and antimicrobial properties of Maytenus 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, Italy b CATgroup — Centro Analisi Territoriali , via Provinciale 73, 44030 Copparo, Ferrara, Italy c 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, Italy f 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, Peru h Instituto Peruano de Investigación Fitoterápica Andina (IPIFA), Av. El Rosario 512-526, Chaclacayo, Lima 08, Peru Received 20 October 2005; accepted 22 June 2006 Available online 6 July 2006
Abstract The hydroalcoholic extract of Maytenus krukovii bark was investigated for its in vitro mutageno-protective activities by means of the Ames Salmonella/microsome assay. The extract showed an inhibitory effect in both T98 and T100 strains against the mutagenic activity of promutagen 2-aminoanthracene but was not protective against directly acting mutagens sodium azide and 2nitrofluorene. When tested as a radical scavenger and antioxidant it produced a dose-dependent inhibition. The extract did not show significant antibacterial properties, and was weakly active against dermatophyte and phytopathogenic fungi, but inhibited the growth 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 factors such as irradiation, atmospheric and food pollutants or byproducts of metabolic processes. When the exposure overwhelms endogenous preventive systems, cells are exposed to a potentially harmful load of oxidants, leading to various free-radical-induced noxious effects. These include, among others, oxidation of nucleic acids, proteins, lipids and 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: [email protected] (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 a relationship between chemical mutagenesis and carcinogenesis [2]. Given that many mutagens act on the cell via its active metabolites or by generating free radicals, it is therefore suggested that supplementation of natural antioxidants could ameliorate the harmful effects of oxidative processes in the living organism and both experimental and clinical data 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 plant sources offering antioxidant or mutageno-protective efficacy and their use and positive image among the consumers is spreading. Maytenus krukovii A.C. Sm (Maytenus chuchuhuasha Raymond–Hamet and Colas) (Celastraceae) is an amazonian canopy tree indigenous to the tropical rainforests of Bolivia, Colombia, Ecuador and Peru [6,7]. Both for its botanical characteristics and therapeutic use it appears strictly related to M. laevis and M. macrocarpa. It grows 30 m high and has large leaves (10–30 cm) and small, white flowers. Its tough, heavy, reddish-brown bark is the main source of a crude drug locally called Chuchuhuasi, which hydroalcoholic extract is widely used in ethnopharmacology as a remedy for 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 characterized by an almost unique variety of phytochemicals, mostly triterpenes, flavonols, condensed tannins and sesquiterpene alkaloids with a very uncommon ansamacrolide-like skeleton ring [9–12]. M. krukovii makes no exception on this regard [13,14]. A number of Maytenus species have been reported for their antitumor, antiinflammatory, 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 as antiinflammatory 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 in some 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 as natural 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 Nacional Mayor University de San Marcos, Lima. Vouchers of the crude drug were deposited in Dipartimento delle Risorse Naturali 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 powder was 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 in 0.154 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 filtered through 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 strains TA98 and TA100 (100 μl/plate of a fresh overnight culture) with and without the addition of 0.5 ml of a 5% S9 exogenous 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 or without S9 mix. The positive control plates for the first contained 20 μg/plate of 2-aminoantracene and S9 mix or 20 μg/plate of 2-nitrofluorene. The positive control plates for the latter contained 20 μg/plate of 2-AA and S9 mix or 1 μg/plate of sodium azide. A sample was considered mutagenic when the observed number of colonies was at least 2fold over the spontaneous level of revertants. The colonies were counted manually after 48 h of incubation at 37 °C using a Colony Counter 560 Suntex (Antibioticos, Italy). The inhibitory effect of M. krukovii extract (1, 5, 10, 50, 100, 500, 1000 μg 0.1 ml− 1 plate− 1) on mutagenic activity of direct acting mutagen 2-nitrofluorene (2 μg/plate) and sodium azide (1 μg/plate) was examined in plate incorporation 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 of M. krukovii extract on the mutagenic activity of the indirectly acting mutagen 2-aminoanthracene (2 μg/plate) was examined in plate incorporation assay, using tester strain 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 each performed with quadruplicate plates. All data were analyzed for statistical significance and homogeneity of variance using Student's t-test and F-test respectively. All computations were made by employing the statistical software SPSS ver. 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]. A fresh 15-h culture was diluted 105 times to give a 1–2 × 104 bacteria/ml solution. The test samples at several concentrations (1, 5, 10, 50, 100, 500, 1000 μg 0.1 ml− 1 plate− 1) diluted in DMSO, mixed with 2 ml of molten top agar, 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 agar plate enriched with L-histidine and biotin.
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Fig. 2. Number of colonies/plate of S. typhimurium tester strain TA100 in the presence of liophilized extract of M. krukovii in minimal glucose agar plate enriched with L-histidine and biotin.
histidine and 0.05 μmol of biotin by incorporating these nutrients into the soft agar overlay. Duplicate plates were poured for each dose of the solution. The number of colonies was assessed after the plates were incubated at 37 °C for 48 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–Ciocalteau phosphomolybdic–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 by the 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 et al. [26] and Glazer [27], as developed further by Cao et al. [28]. Photochemiluminescence (PCL) assay was performed as described by Popov and Lewin [29–31] and involved the photochemical generation of superoxide O2U- free radicals combined with chemiluminescence detection. The assay is initiated by optical excitation of a photosensitizer, resulting in the generation of the superoxide radical anion.
Table 1 Radical scavenging and antioxidant activity of M. krukovii hydroalcoholic extract
M. krukovii Gallic acid Catechin BHA BHT Trolox GTE a GSE b PE c n.a. = not active. a GTE, Green Tea polyphenol extract. b GSE, Grape Seed extract. c PE, Papaya extract.
DPPH
β-carotene
38.38 ± 0.21 4.98 ± 0.09 31.97 ± 0.23 33.52 ± 0.19 40.31 ± 0.29 28.22 ± 0.33 5.58 ± 0.41 12.15 ± 1.32 n.a.
586.85 ± 1.21 1093.8 ± 11.6 1791.34 ± 9.7 750.23 ± 9.45 1307.14 ± 10.4 786.5 ± 8.7 220.02 ± 6.43 240.12 ± 5.17 n.a.
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Table 2 Photochemiluminescence and ORAC values of M. krukovii hydroalcoholic extract
ORAC PCL-lypo PCL-hydro
M. krukovii
Vitamin E
Ascorbic acid
Trolox mmol/g ± S.D.
PE a
GTE b
GSE c
4.497 ± 0.113 0.35 ± 0.01 0.4 ± 0.01
– 2.77 ± 0.02 –
– – 6.2 ± 0.2
– 3.98 ± 0.06 –
0.121 ± 0.08 b1 · 10−3 b1 · 10−3
6.479 ± 0.061 1.33 ± 0.01 4.96 ± 0.02
4.033 ± 0.077 1.66 ± 0.03 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.
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) and Gram (−) (Klebsiella oxytoca ATCC, Escherichia coli ATCC, Pseudomonas aeruginosa ATCC, Proteus mirabilis ATCC) bacteria. The culture media and conditions employed for ATCC strains were in accordance with American Type Culture Collections protocols. The biological activity against these classes of microorganisms was determined by employing the standard disks diffusion technique [32]. Antifungal properties were evaluated as described in Romagnoli and Sacchetti [33]. Trichophyton mentagrophytes (Robin) Blanchard strain no. 160.66 (CBS), Nannizzia cajetani 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 suggestion on the protective potential of phytochemicals. Therefore, the use of more than one method is highly advisable. Among the 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 the results [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 scavenging capacity comparable to that of most synthetic and natural reference compounds (Table 1). In particular, in the DPPH test and in the β-carotene bleaching test the crude extract produced a dose-dependent inhibition (IC50: 38.38 ± 0.21, and 586.85 ± 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 the presence of liophilized extract of M. krukovii.
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Fig. 4. Mutagenic activity of indirect mutagen 2-aminoanthracene on S. typhimurium tester strains TA98 and TA100, in the presence of liophilized extract of M. krukovi.
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 but higher than Grape Seed extract. The most relevant result was however obtained in the evaluation of oxygen radical absorbance capacity (ORAC), measuring a hydrogen atom transfer reaction mechanism, which is most relevant to human biology. Result in ORAC test was 4.497 ± 0.113 mmol trolox/g, once again better or comparable than those obtained from commercial Grape Seed and Green Tea polyphenol extracts, which are marketed for their antioxidant properties [35]. These data are of particular significance because the results of this assay easily correlate with the therapeutical, nutriceutical and cosmeceutical potential of a given antioxidant and this method is accepted by the industry 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 alcoholic extract 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 the indirectly acting mutagen 2-aminoanthracene, which acts as a genotoxic compound through a liver S9 fraction. The mutagenicity of 2-aminoanthracene was in both cases reduced by more than 90%. This is, to our knowledge, a novel
Table 3 Antifungal activity of M. krukovii hydroalcoholic extract Fungi
T. mentagrophytes var. mentagrophytes
N. cajetani
P. ultimum
M. grisea
a
Ketoconazole provided a 0% growth in all tested strains from 10 μl/ml.
M. krukovii hydroalcoholic extract a (μl/ml)
Growth %
50 100 200 500 50 100 200 500 50 100 200 500 50 100 200 500
100 89.4 74.5 87.2 88.9 91.9 82.9 80 80.8 65.6 50 32.4 91.8 89.8 87.7 79.6
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biological activity of a M. krukovii bark extract (Fig. 4). These results are consistently better than those reported by Weisburger et al. [20] according to the analogue antimutagenicity of green and black tea polyphenols, where at the same 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 in the protective activity of M. krukovii bark against direct and indirect mutagens and since the great abundance of flavonoids, triterpene dimers and condensed tannins in the bark has been proved [13,14] it can be therefore suggested that these known antimutagens may interact synergically with some specific enzymes in the liver homogenates, which are necessary for the activation of chemical mutagens. The polyphenolic abundance in the bark extract was also confirmed by means of the total phenolic content, determined according to the Folin–Ciocalteu method and expressed as gallic acid equivalents (270 mg/g of extract). Most of the polymeric tannins exerting an antimutagen activity on 2-aminoanthracene and other nitroaromatic indirect acting mutagens, can be classified as blocking agents since they inhibit the conversion of the latter to ultimate carcinogens. Proanthocyanidins and flavonoids are known to form a complex with proteins and monooxygenases metabolizing xenobiotics, due to the fact that the formers can act as multidentate ligands and thus are able to bind simultaneously more than one point to the protein surface [36,37]. The more the oligomeric nature, the more its association produces an increased antimutagenic activity towards the inhibition of the enzyme mediated genotoxicity of indirect acting mutagens as 2-aminoanthracene [38]. As previously reported, this kind of action is likely to be the final result of many interactive effects, for which the whole phytocomplex [39], rather than a single chemical constituent, may be responsible. Therefore it could be suggested that M. krukovii interacts with the enzymes of the S9 mix, thereby inhibiting the transformation of 2-aminoantracene into its active forms. A similar behaviour was previously 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/ml against both Gram (+) and Gram (−) bacteria (data not shown), in accordance with previous reports regarding the similar specie M. 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 the rates of mutation by including them in dietary products, this activity is valuable towards an extension of the employ of this crude drug as new valuable ingredient for food and/or nutriceutical, cosmeceutical support in the promotion of health, besides its consolidated ethnomedical use. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
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