Abstracts
In these work the in vitro antioxidant activity and the in vivo genotoxicity of M. dasyclada was compared to the reference species M. aquifolium and M. ilicifolia. M. dasyclada showed in vitro antioxidant activity comparable to M. aquifolium but lower than M. ilicifolia, being that a inverse Pearson correlation between DPPH IC50 values and total phenolic content was detected (–0.932). The carbonyl content of M. dasyclada and M. aquifolium extracts promoted a similar increase in protein oxidation in vivo, while M. ilicifolia no altered the carbonyl levels. The comet assay demonstrated that the three analyzed species promoted a low and similar level of genotoxicity; which is compatible with DNA damage induced by other medicinal plants and is partially recovered by a co-treatment with vitamin C. The data showed M. dasyclada as antioxidant activity in vitro, and that its genotoxic and pro-oxidant effects in vivo are comparable to the Maytenus reference species.
Maytenus ilicifolia ; M. aquifolium ; M. dasyclada ; comet assay; antioxidant activity; phenolic pompounds
No presente trabalho a atividade antioxidante in vitro e a genotoxicidade in vivo de M. dasyclada foi comparada com as espécies de referência M.aquifolium e M. ilicifolia. M. dasyclada mostrou atividade antioxidante in vitro comparável a de M. aquifolium mas inferior a M. ilicifolia, sendo que foi detectada uma correlação de Pearson inversa entre os valores de IC50 por DPPH e o conteúdo fenólico total (–0,932). Em relação ao teor de carbonila, os extratos de M. dasyclada e M. aquifolium promoveram um aumento semelhante na oxidação das proteínas in vivo, ao passo que Maytenus ilicifolia não alterou os níveis de carbonila. O ensaio do cometa demonstrou que as três espécies analisadas promoveram um nível baixo e semelhante de genotoxicidade, o que é compatível com os danos no DNA induzidos por outras plantas medicinais e é parcialmente recuperada por um co-tratamento com a vitamina C. Os dados mostraram M. dasyclada com atividade antioxidante in vitro, e que os seus efeitos genotóxicos e pró-oxidantes in vivo são comparáveis às espécies de referência de Maytenus.
Maytenus ilicifolia ; M. aquifolium ; M. dasyclada ; teste cometa; atividade antioxidante; substâncias fenólicas
1 Introduction
Maytenus aquifolium Mart. (synonym M. aquifolia) and
M. ilicifolia Mart. ex Reissek (synonym M. muelleri)
(Celastraceae) are widely employed in Brazilian popular medicine, being known as
‘espinheira-santa’ or ‘cancarosa’. Both are native plants, with natural occurrence in South of
Brazil and are used in form of teas for stomach and ulcer illness treatment (Mariot and Barbieri, 2007Mariot, MP. and Barbieri, RL., 2007. Metabólitos secundários e propriedades
medicinais da espinheira-santa (Maytenus ilicifolia Mart. ex Reiss. e Mart.).
Maytenus aquifoliumRevista Brasileira de Plantas Medicinais, vol. 9, no. 3,
p. 89-99.). Souza-Formigoni et al. (1991)Souza-Formigoni, ML., Oliveira, MG., Monteiro, MG., Silveira-Filho, NG., Braz,
S. and Carlini, EA., 1991. Antiulcerogenic effects of two Maytenus species in laboratory
animals. Journal of Ethnopharmacology, vol. 34, no. 1, p. 21-27.
http://dx.doi.org/10.1016/0378-8741(91)90185-G. PMid:1753784
http://dx.doi.org/10.1016/0378-8741(91)9...
have demonstrated the antiulcerogenic property of these
two species, which seems to be closely related to the presence of two classes of substances,
namely, phenols and triterpenes (Mossi et al.,
2009Mossi, AJ., Mazutti, M., Paroul, N., Corazza, ML., Dariva, C., Cansian, RL. and
OLIVEIRA, JV., 2009. Chemical variation of tannins and triterpenes in Brazilian populations of
Maytenus ilicifolia Mart. Ex Reiss. Brazilian Journal of Biology, vol. 69, no. 2, p. 339-345.
http://dx.doi.org/10.1590/S1519-69842009000200015.
http://dx.doi.org/10.1590/S1519-69842009...
).
This increasing interest by natural antioxidants is related to their low toxicity as compared
to synthetic antioxidants (Soares et al., 2008Soares, M., Welter, L., Gonzaga, L., Lima, A., Macini-Filho, J. and Fett, R.,
2008. Evaluation of antioxidant activity and identification of phenolic acids present in the
pomace of Gala variety apples. Ciência e Tecnologia deAlimentos, vol. 28, no. 3, p.
727-732.). In
these sense, investigations about the presence of new antioxidants in plants emerges as a
natural alternative against the deleterious effects of reactive oxygen species (ROS) and free
radicals (FR) (Galvão et al., 2008Galvão, EL., Silva, DCF., Silva, JO., MOREIRA, AVB. and SOUSA, EMBD., 2008.
Evaluation of the antioxidant potential and sub-critical extraction of linseed oil. Ciência e
Tecnologia de Alimentos, vol. 28, no. 3, p. 551-557.). Several works have
already shown the antioxidant activity of M. aquifolium and M.
ilicifolia (Corsino et al., 2003Corsino, J., Silva, DHS., Zanoni, MVB., Bolzani, VS., França, SC., Pereira, AM.
and Furlan, M., 2003. Antioxidant flavan-3-ols and flavonol glycosides from Maytenus
aquifolium. Phytotherapy research : PTR, vol. 17, no. 8, p. 913-916.
http://dx.doi.org/10.1002/ptr.1249. PMid:13680823
http://dx.doi.org/10.1002/ptr.1249...
; Vellosa et al., 2007Vellosa, JCR., Barbosa, VF., Khalil, NM., Santos, VAFFM., Furlan, M., Brunetti,
IL. and Oliveira, OMMF., 2007. Profile of action over free radicals and reactive oxygen
species. Maytenus aquifoliumBrazilian Journal of Pharmaceutical Sciences, vol.
43, no. 3, p. 447-453.; Mariot
and Barbieri, 2007Mariot, MP. and Barbieri, RL., 2007. Metabólitos secundários e propriedades
medicinais da espinheira-santa (Maytenus ilicifolia Mart. ex Reiss. e Mart.).
Maytenus aquifoliumRevista Brasileira de Plantas Medicinais, vol. 9, no. 3,
p. 89-99.; Pessuto et al., 2009Pessuto, MB., Costa, IC., Souza, AB., Nicoli, FM., Mello, JCP., Petereit, F. and
Luftmann, H., 2009. Antioxidant activity of extracts and condensed tannins from leaves of
.Maytenus ilicifolia Mart. ex ReissQuimica Nova, vol. 32, no. 2, p. 412-416.
http://dx.doi.org/10.1590/S0100-40422009000200027.
http://dx.doi.org/10.1590/S0100-40422009...
), which
probably is due to the action of polyphenols and flavonoids as the free radicals scavengers. The
pharmacological properties of M. ilicifolia were reviewed in literature (Santos-Oliveira, 2009Santos-Oliveira, R., Coulaud-Cunha, S. and Colaço, W., 2009. Review of Mart. Ex
Reissek, Celastraceae. Contribution to the studies of pharmacological properties.
Maytenus ilicifoliaBrazilian Journal of Pharmacognosy, vol. 19, no. 2b, p.
650-659.).
The production of ROS and FR can occur as consequence of exposition to toxic agents
(radiation, antibiotics, several types of environmental pollutants, among others) or as result
of normal metabolism in the living cells (Halliwell and
Gutteridge, 2007Halliwell, B. and Gutteridge, JMC., 2007. Free Radicals in biology and medicine.
4th ed. New York: Oxford University Press Inc.). In situations where the ROS and FR production exceeds the defense
capacity of the organism, a condition known as oxidative stress can occurs and provoke damages
to important biomolecules, including DNA, proteins and membrane lipids, compromising cell
general functions (Vargas et al., 1991Vargas, VMF., Guidobono, RR. and Henriques, JAP., 1991. Genotoxicity of plant
extracts. Memorias do Instituto Oswaldo Cruz, vol. 86, no. 2, supplement 2, p. 67-70.
http://dx.doi.org/10.1590/S0074-02761991000600017. PMid:1842016
http://dx.doi.org/10.1590/S0074-02761991...
).
Despite of the increasing in the use of medicinal plants in the last decade, the most of them was not sufficiently studied in relation to the quality, safety, efficiency patterns or even in relation to their cytotoxic and mutagenic potential (Bagatini et al., 2007BAGATINI, MD., SILVA, ACF. and TEDESCO, SB., 2007. The use of test as a bioindicator of genotoxicity of medicinal plants infusions. Allium cepaBrazilian Journal of Pharmacognosy, vol. 17, p. 444-447.). Currently, the application of M. ilicifolia as medicinal plant is allowed in Brazil, so that their register and commercialization in the phytomedicine don't requires the application of efficiency and safety tests (Brasil, 2011Brasil. Agência Nacional de Vigilância Sanitária – ANVISA,2011.Formulário de Fitoterápicos da Farmacopeia Brasileira. Brasília: ANVISA.).
However, Maytenus dasyclada have been popularly used as teas without knowledge of their therapeutic properties and their toxic and mutagenic potential. There is no literature that describes the antioxidant activity of the specie in vivo. There is only one manuscript in the literature about chemical characterization and in vitro biological properties of this specie (Schwanz et al., 2013Schwanz, M., Dresch, RR., Manfredini, V. and Henriques, AT., 2013. Antioxidative effects of Mart. (Celastraceae). Maytenus dasycladaInternational Journal of Pharma and Bio Sciences, vol. 4, no. 3, p. 957-969.). These work identified the presence of quercetin and kaempherol in ethyl acetate and ethanolic extracts of M. dasyclada, being that both showed antioxidant and antigenotoxic activity in vitro. However, there is no literature describing biological properties of M. dasyclada in vivo.
Viewing expands the knowledge about Maytenus genus and their economic and/or medicinal potential, the objective of this study was compare the in vitro antioxidant activity and the in vivo genotoxicity of M. dasyclada with the reference species M. aquifolium and M. ilicifolia.
2 Material and Methods
The leaves of Maytenus species were collected in the northern Rio Grande do Sul, Brazil. The exsicatas were identified and deposited in the Herbarium Padre Balduino Rambo of the URI University, Erechim, RS, Brazil. The exsicatas were identified by the numbers HPBR 11,512 (M. dasyclada), HPBR 11,508 (M. aquifolium) and HPBR 11,502 (M. ilicifolia) by Dr. Altemir J. Mossi.
The fresh aerial parts of the plant were air-dried at temperature to 30-35°C for 5 days, after being broken into small pieces. The extract was obtained by water infusion with 20 g of the leaves to 100mL of distilled water for 15minutes at 70°C (20% w/v). After cooling and filtration, the extract was frozen and concentrated by lyophilization.
The antioxidant activity of Maytenus spp. was evaluated in vitro by the DPPH
method. Briefly, the absorbance of free radical 2.2-diphenyl-1-picryl-hydrazil alone (DPPH
control) or in the presence of plant extracts (0.01, 0.025, 0.05, 0.075, 0.1, 0.25 and 0.5
mg.mL–1) was measured in 515 nm (Miranda and Fraga,
2006Miranda, ALP. and FRAGA, CAM., 2006. Atividade sequestradora de radical livre
determinação do potencial antioxidante de substâncias bioativas. In MONGE, A. and GANELLIN, CR.
(Eds.). Practical Studies for Medicinal Chemistry. IUPAC. p. 1-14.Available from:
<http://www.iupac.org/fileadmin/user_upload/publications/cd/Medicinal-Chemistry/Practica-I-5.pdf>.
Access in: 10 Mar. 2015.
http://www.iupac.org/fileadmin/user_uplo...
), with ethanol used as solvent and blank. The scavenging activity of extracts upon
DPPH radical was calculated as percentage of antioxidant activity (AA%), using the Equation 1:
The concentration of extract able to scavenge 50% of DPPH radical (IC50) was calculated through regression analysis.
The content of total polyphenolics (TP) present in the extracts was estimated by a colorimetric assay at 691 nm in according with Glasl (1983)Glasl, H., 1983. ZurPhotometrie in der drogenstandardisierung -3. Gehaltsbestimmung von gerbstoffdrogen. Deutsche Apotheker-Zeitung, vol. 123, p. 1979-1987.. The percentages of total phenolics were determined in triplicate. Results were expressed as milligrams of galic acid equivalents by grams of extract (mg EAG.g–1) and were calculated using the Equation 2:
where TP = total polyphenolics (%); Abs = absorbance of each sample; M = mass (g) of extracts.
The correlation between phenolic content and antioxidant activity in three Maytenus species was performed in Excel.
This study was conducted in accordance with Federal Law N° 11,794 and was approved by the Research Ethics Committee of URI University (RS, Brazil) upon register numbers 077/TCA/09. Male Wistar rats (Rattus norvegicus Bakenhouff) two months old, weighing 120 ± 5 g, were obtained from Laboratory of Animal Experiments of URI University (RS, Brazil). The animals were kept in photoperiod of 12 h of light and dark, minimal noise (< 20 db), ambient temperature (22 ± 2 °C) and had free access to food and water.
For experimental procedures, the animals were distributed randomly in groups of 6 rats each, and received orally treatments of 75 mg.kg–1.day–1 of NaCl (Salt solution), 330 mg.kg–1.day–1of vitamin C (Vitamin C), 830 mg.kg–1.day–1 extract of each species (Extract) and 830 mg.kg–1.day–1 of each extract plus 330 mg.kg–1.day–1 of vitamin C (Extract plus vitamin C) for 14 days.
After 14 days the rats were sacrificed in CO2 chamber. Total blood was collected by cardiac puncture and transferred to tubes containing heparin. An aliquot was centrifuged to separate the plasma (5,000rpm, 15min). The samples of blood and plasma were frozen at‑20°C and later used to perform, respectively, the comet assay and carbonyl proteins dosage.
The genotoxicity of Maytenus extracts was evaluated by the alkaline comet
assay as described by Singh et al. (1988)Singh, NP., McCoy, MT., Tice, RR. and Schneider, EL., 1988. A simple technique
for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research,
vol. 175, no. 1, p. 184-191. http://dx.doi.org/10.1016/0014-4827(88)90265-0.
PMid:3345800
http://dx.doi.org/10.1016/0014-4827(88)9...
, with minor
modifications. A 5mL aliquot of previously collected blood were mixed with 100 μL of 0.75%
low-melting point agarose, and immediately spread onto a glass microscope slide pre-coated with
1% normal melting point agarose. Agarose was allowed to set at 4°C for 5min. To remove cell
proteins, slides were incubated with ice-cold lyses solution (2.5M NaCl, 10mM Tris, 100mM EDTA,
1% Triton X-100, and 10% DMSO, pH 10.0) at 4°C for at least 1h. To promote DNA unwinding and the
exposure of alkali-labile sites, slides were placed on a horizontal electrophoresis unit,
covered with fresh buffer (300mM NaOH, 1mM EDTA, pH 13.0) for 20min at 4°C. Electrophoresis was
performed in alkaline conditions (pH ~ 13) for 15min at 25V and 300mA. After electrophoresis,
slides were neutralized (0.4M Tris, pH 7.5), washed in bi-distilled water, stained with silver
solution (0.1% ammonium nitrate; 0.1% de silver nitrate; 0.25% tungstosilicic acid; 0.15%
formaldehyde), dried overnight and analyzed by optical microscopy. In order to prevent
additional DNA damage, the slides preparation was made in a dark room.
For each animal were prepared two independently slides, being evaluated 100 random cells in each one. Cells were visually scored according to tail length into five classes, from 0 (undamaged, without a tail) to 4 (maximum damage, comets with no heads). A damage index (DI) was assigned to each comet according to its class. The damage index is based on the length of migration and on the amount of DNA in the tail, and it is considered a sensitive DNA measurement. Damage index, can range from 0 (completely undamaged 100 cells×0) to 400 (with maximum damage 100 cells×4).
Evaluation of oxidative damage in plasmatic proteins was based on detection of carbonyl groups
by reaction with 2,4-dinitrophenylhydrazine (DNPH). Previously collected plasma samples were
mixed with acetic glacial acid (0.2mL), incubated on the ice (5min), centrifuged (10,000rpm,
5min) and the supernatant was discarded. The remained precipitate received 1mL of DNPH (10mM)
and was incubated at 37°C for 60min. A blank was made using HCl (2M) instead DNPH. Following the
samples was mixed in vortex (30s), centrifuged (10,000rpm, 5min) and precipitated proteins
dissolved in 6M guanidine. The latter procedure was repeated three times; in the last, the
supernatant phase was collected and subjected to carbonyl content determination by measure of
absorbance at 370nm (Levine et al., 1990Levine, RL., Garland, D., Oliver, CN., Amici, A., Climent, I., Lenz, AG., Ahn,
BW., Shaltiel, S. and Stadtman, ER., 1990. Determination of carbonyl content in oxidatively
modified proteins. Methods in Enzymology, vol. 186, p. 464-478.
http://dx.doi.org/10.1016/0076-6879(90)86141-H. PMid:1978225
http://dx.doi.org/10.1016/0076-6879(90)8...
) and total
protein quantification by Bradford method (Bradford,
1976Bradford, MM., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding. Analytical
Biochemistry, vol. 72, no. 1-2, p. 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3.
PMid:942051
http://dx.doi.org/10.1016/0003-2697(76)9...
).
The results were analyzed statistically using one-way ANOVA plus Tukey´s post-test, with p values < 0.05 accepted as significant.
3 Results and Discussion
In these work the in vitro antioxidant activity and the in vivo genotoxicity of
Maytenus dasyclada was compared to the reference species M.
aquifolium and M. ilicifolia. As elucidated by DPPH method,
M. dasyclada as well the two other Maytenus spp.
investigated, showed in vitro antioxidant activity (Table 1), but M. ilicifolia was clearly more efficient as
radical scavenger since its IC50 is dozens of times smaller. The DPPH result obtained
to M. ilicifolia is similar to previously published data (Pessuto et al., 2009Pessuto, MB., Costa, IC., Souza, AB., Nicoli, FM., Mello, JCP., Petereit, F. and
Luftmann, H., 2009. Antioxidant activity of extracts and condensed tannins from leaves of
.Maytenus ilicifolia Mart. ex ReissQuimica Nova, vol. 32, no. 2, p. 412-416.
http://dx.doi.org/10.1590/S0100-40422009000200027.
http://dx.doi.org/10.1590/S0100-40422009...
). In these sense, is possible conclude that M.
dasyclada have antioxidant capacity comparable to the M.aquifolium
which, in its turn, acts as scavenger of several oxidants, including O2.–,
NO.and HCOl (Vellosa et al., 2007Vellosa, JCR., Barbosa, VF., Khalil, NM., Santos, VAFFM., Furlan, M., Brunetti,
IL. and Oliveira, OMMF., 2007. Profile of action over free radicals and reactive oxygen
species. Maytenus aquifoliumBrazilian Journal of Pharmaceutical Sciences, vol.
43, no. 3, p. 447-453.).
Total phenols content was also similar in M. dasyclada and M. aquifolium, both species showing values that are approximately half of M. ilicifolia (Table 1). Using another methodology, Pereira et al. (2005)Pereira, AMS., Januário, AH., Queiroz, MEE., Biondo, R. and França, SC., 2005. Evaluation of Maytenus aquifolium Mart. and Mart. chemotypes for tannins, total phenols and triterpenes. Maytenus ilicifoliaRevista Brasileira de Plantas Medicinais, vol. 8, no. 1, p. 13-17. has demonstrated that the yields of phenolic compounds in M. ilicifolia can indeed be until three fold higher than in M. aquifolium (14 to 20% and 4.23 to 7.53%, respectively). No previous data were found in the literature about total polyphenols content in M. dasyclada.
Is interesting to note that in the three species, DPPH IC50 is inversely correlated
with phenolic results and this correlation shown a good linear regression (Pearson correlation
of –0.932) (Figure 1), suggesting the importance of
phenolic compounds to antioxidant proprieties of the genus. A similar correlation had previously
been demonstrated to ethanolic and aqueous extracts of M. ilicifolia using DPPH
and phosphomolybdenum assay; being that the antioxidant potential of ethanolic extract was
better than aqueous extract and was also proportionally increased by the presence of
hydroxylated phenolic groups in the fraction (Pessuto et al.,
2009Pessuto, MB., Costa, IC., Souza, AB., Nicoli, FM., Mello, JCP., Petereit, F. and
Luftmann, H., 2009. Antioxidant activity of extracts and condensed tannins from leaves of
.Maytenus ilicifolia Mart. ex ReissQuimica Nova, vol. 32, no. 2, p. 412-416.
http://dx.doi.org/10.1590/S0100-40422009000200027.
http://dx.doi.org/10.1590/S0100-40422009...
).
Correlation between phenolic content and antioxidant activity in three Maytenus spp. (A) M. ilicifolia, (B) M. aquifolium and (C) M. dasyclada.
Antioxidant-rich fractions (e.g. polyphenols) from plant extracts have been used in
experimental models to prove their inhibitory effect on induced ulceration (Vilegas et al., 1999Vilegas, W., Sanommiya, M., Rastrelli, L. and Pizza, C., 1999. Isolation and
structure elucidation of two new flavonoid glycosides from the infusion of maytenus aquifolium
leaves. Evaluation of the antiulcer activity of the infusion. Journal of Agricultural and Food
Chemistry, vol. 47, no. 2, p. 403-406. http://dx.doi.org/10.1021/jf980114i.
PMid:10563907
http://dx.doi.org/10.1021/jf980114i...
). The protective effect of rutin, a
widespread flavonol glycoside, against ethanol induced gastric lesions as well as the inhibition
of gastric H+, K+ – ATPase by catechins have already been assessed (La Casa et al., 2000La Casa, C., Villegas, I., Alarcón de la Lastra, C., Motilva, V. and Martín
Calero, MJ., 2000. Evidence for protective and antioxidant properties of rutin, a natural
flavone, against ethanol induced gastric lesions. Journal of Ethnopharmacology, vol. 71, no.
1-2, p. 45-53. http://dx.doi.org/10.1016/S0378-8741(99)00174-9. PMid:10904145
http://dx.doi.org/10.1016/S0378-8741(99)...
). The presence of polyphenols and the
antioxidant activity on Maytenus spp. can contribute for the efficiency of its
infusion as an antiulcer agent and legitimate its medicinal use (Corsino et al., 2003Corsino, J., Silva, DHS., Zanoni, MVB., Bolzani, VS., França, SC., Pereira, AM.
and Furlan, M., 2003. Antioxidant flavan-3-ols and flavonol glycosides from Maytenus
aquifolium. Phytotherapy research : PTR, vol. 17, no. 8, p. 913-916.
http://dx.doi.org/10.1002/ptr.1249. PMid:13680823
http://dx.doi.org/10.1002/ptr.1249...
). Besides the best characterized M.
ilicifolia, other species of the same genus can became, with greater or lesser
intensity, pharmacological alternatives.
To ensure their secure use, is important know the biological effects of the plants in
vivo. In this work the genotoxicity of the Maytenus extracts and
their ability to oxidize proteins was investigated by the comet assay and carbonyl proteins
measure, respectively. The alkaline comet assay is able to detect DNA strand breaks produced by
ROS or other toxic substances; it is a sensible and low cost test widely used in the analysis of
genotoxicity (Collins et al., 2008Collins, AR., Oscoz, AA., Brunborg, G., Gaivão, I., Giovannelli, L., Kruszewski,
M., Smith, CC. and Stetina, R., 2008. The comet assay: topical issues. Mutagenesis, vol. 23,
no. 3, p. 143-151. http://dx.doi.org/10.1093/mutage/gem051.PMid:18283046
http://dx.doi.org/10.1093/mutage/gem051...
). Considering that
the maximum DNA damage index (DI) that can be achieved in the comet assay is 400, the results
showed that the three Maytenus spp. have induced a low and similar level of
genotoxicity, which was partially recovered by a co-treatment with vitamin C (Figure 2). In relation to the carbonyl content, M.
dasyclada and M. aquifolium extracts have also a similar effect,
promoting the in vivo protein oxidation (Figure 3). Again,
this effect could be reverted by combination with vitamin C.
Genotoxicity of the three Maytenus species in rats leukocytes. Animals were treated with salt solution (75mg.kg–1.day–1), vitamin C (330mg.kg–1.day–1), extract (830mg.kg–1.day–1) or extract (830mg.kg–1.day–1) plus vitamin C (330 mg.kg–1.day–1) for 14 days. Statistically significant differences between the treatments are indicated by different letters using one-way ANOVA plus Tukey’s post test.
Oxidative damage produced by three Maytenus species in plasmatic proteins of rats. Animals were treated with salt solution (75 mg.kg–1.day–1), vitamin C (330 mg.kg-1.day-1), extract (830 mg.kg–1.day–1) or extract (830 mg.kg–1.day–1) plus vitamin C (330 mg.kg–1.day-1) for 14 days. Statistically significant differences between the treatments are indicated by different letters using one-way ANOVA plus Tukey’s post test.
Although the extracts studied had in vitro antioxidant activity, the reduction of DNA damages
by vitamin C is an indicative that their slight genotoxic effect in vivo can be mediated by an
oxidative mechanism. This observation is in agreement with the pro-oxidant action upon proteins
(Figures 2 and 3).
M. ilicifolia does not cause significant change upon carbonyl content, which
can be related with its high content of phenolic compounds comparative to the two other analyzed
species (Table 1). The in vivo antioxidant properties of
M. ilicifolia were previously demonstrated (Melo et al., 2001Melo, SF., Soares, SF., Costa, RF., Silva, CR., Oliveira, MB., Bezerra, RJ.,
Caldeira-de-Araújo, A. and Bernardo-Filho, M., 2001. Effect of the Cymbopogon citratus,
Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride
oxidative damage in Escherichia coli. Mutation Research, vol. 496, no. 1-2, p. 33-38.
http://dx.doi.org/10.1016/S1383-5718(01)00216-9.PMid:11551478
http://dx.doi.org/10.1016/S1383-5718(01)...
) and seems be important to the protection of gastric lesions in rats
(Baggio et al., 2007Baggio, CH., Freitas, CS., Otofuji, GM., Cipriani, TR., Souza, LM., Sassaki,
GL., Iacomini, M., Marques, MC. and Mesia-Vela, S., 2007. Flavonoid-rich fraction of Maytenus
ilicifolia Mart. ex. Reiss protects the gastric mucosa of rodents through inhibition of both
H+,K+ -ATPase activity and formation of nitric oxide. Journal of Ethnopharmacology, vol. 113,
no. 3, p. 433-440. http://dx.doi.org/10.1016/j.jep.2007.06.015. PMid:17706386
http://dx.doi.org/10.1016/j.jep.2007.06....
).
Despite the use of Maytenus ilicifolia as medicinal plant, there are few
works that have investigated their genotoxic effects. Camparoto
et al. (2002)Camparoto, ML., Teixeira, RO., Mantovani, MS. and Vicentini, VEP., 2002. Effects
of Mart. and . Maytenus ilicifoliaBauhinia candicans Benth
infusions on onion root-tip and rat bone-marrow cellsGenetics and Molecular Biology, vol. 25,
no. 1, p. 85-89. http://dx.doi.org/10.1590/S1415-47572002000100016.
http://dx.doi.org/10.1590/S1415-47572002...
showed that infusions of M. ilicifolia no produce
alterations on the cell cycle and chromosomes structure of onion root and bone-marrow cells,
while Vargas et al. (1991)Vargas, VMF., Guidobono, RR. and Henriques, JAP., 1991. Genotoxicity of plant
extracts. Memorias do Instituto Oswaldo Cruz, vol. 86, no. 2, supplement 2, p. 67-70.
http://dx.doi.org/10.1590/S0074-02761991000600017. PMid:1842016
http://dx.doi.org/10.1590/S0074-02761991...
demonstrated that the plant
is not mutagenic in the AMES test using Salmonella typhimurium as biological
model. The clinical toxicology of the M. ilicifolia has also been investigated
in humans and the plant does not altered biochemical e hematologic parameters in the subjects
(Tabach et al., 2002Tabach, R., Carlini, E. and Moura, YG., 2002. Um novo extrato de Mart. ex Reiss.
(Toxicologia clínica – Fase I) Avaliação em seres humanos. Maytenus
ilicifoliaRevista Racine, vol. 71, p. 38-43.).
Particularly, the comet assay was not previously employed with all Maytenus
spp. target of the present study in vivo, only in vitro
experiments (Schwanzet al., 2013). Although the three species have presented a slight increase
in DI, the obtained values are comparable to that seemed in other medicinal plants. Treatment of
rats with Calendula officinalis during the same time employed in this study
results in similar comet levels (DI 34-72 for C. officinalis versus 46-52 for
Maytenus spp.); and even so C. officinalis have an
antigenotoxic effect that repair the DNA damage caused by methyl methanesulfonate (Leffa et al., 2012Leffa, DD., Rosa, R., Munhoz, BP., Mello, AAM., Mandelli, FD., Amaral, PA.,
Rossatto, AE. and Andrade, VM., 2012. Genotoxic and antigenotoxic properties of .
Calendula officinalis extracts in mice treated with methyl
methanesulfonateAdvances in Life Sciences, vol. 2, no. 2, p. 21-28.
http://dx.doi.org/10.5923/j.als.20120202.05.
http://dx.doi.org/10.5923/j.als.20120202...
). Extracts of Baccharis
trimera, showing DI value in the same range, have also protective action against DNA
breaks induced by hydrogen peroxide (Rodrigues et al.,
2009Rodrigues, CR., Dias, JH., de Mello, RN., Richter, MF., Picada, JN. and Ferraz,
AB., 2009. Genotoxic and antigenotoxic properties of Baccharis trimera in mice. Journal of
Ethnopharmacology, vol. 125, no. 1, p. 97-101. http://dx.doi.org/10.1016/j.jep.2009.06.006.
PMid:19539021
http://dx.doi.org/10.1016/j.jep.2009.06....
). Schwanz et al. (2013)Schwanz, M., Dresch, RR., Manfredini, V. and Henriques, AT., 2013. Antioxidative
effects of Mart. (Celastraceae). Maytenus dasycladaInternational Journal of
Pharma and Bio Sciences, vol. 4, no. 3, p. 957-969. evaluating the
in vitro antioxidative effects of different extracts of Maytenus
dasyclada, observed reduction in the DI compared to the control, with 100 and10μg/mL,
regard less of the fraction of the extract (ethyl-acetate or ethanolic).
4 Conclusions
The aforementioned data permit to conclude that therapeutic application of M. ilicifolia is secure in male Wistar rats. However, some cautions relative to dosage and/or time of use as well take into account some particularities of the subjects are necessary. In this work we demonstrated that M. dasyclada as antioxidant activity in vitro, and that its genotoxic and pro-oxidant effects in vivo are comparable to the M. ilicifolia and M. aquifolium.
Acknowledgements
The authors thanks to CNPq, CAPES, FAPERGS and SCIT-RS to financial support.
References
- BAGATINI, MD., SILVA, ACF. and TEDESCO, SB., 2007. The use of test as a bioindicator of genotoxicity of medicinal plants infusions. Allium cepaBrazilian Journal of Pharmacognosy, vol. 17, p. 444-447.
- Baggio, CH., Freitas, CS., Otofuji, GM., Cipriani, TR., Souza, LM., Sassaki, GL., Iacomini, M., Marques, MC. and Mesia-Vela, S., 2007. Flavonoid-rich fraction of Maytenus ilicifolia Mart. ex. Reiss protects the gastric mucosa of rodents through inhibition of both H+,K+ -ATPase activity and formation of nitric oxide. Journal of Ethnopharmacology, vol. 113, no. 3, p. 433-440. http://dx.doi.org/10.1016/j.jep.2007.06.015. PMid:17706386
» http://dx.doi.org/10.1016/j.jep.2007.06.015 - Bradford, MM., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, vol. 72, no. 1-2, p. 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3. PMid:942051
» http://dx.doi.org/10.1016/0003-2697(76)90527-3 - Brasil. Agência Nacional de Vigilância Sanitária – ANVISA,2011.Formulário de Fitoterápicos da Farmacopeia Brasileira. Brasília: ANVISA.
- Camparoto, ML., Teixeira, RO., Mantovani, MS. and Vicentini, VEP., 2002. Effects of Mart. and . Maytenus ilicifoliaBauhinia candicans Benth infusions on onion root-tip and rat bone-marrow cellsGenetics and Molecular Biology, vol. 25, no. 1, p. 85-89. http://dx.doi.org/10.1590/S1415-47572002000100016.
» http://dx.doi.org/10.1590/S1415-47572002000100016 - Collins, AR., Oscoz, AA., Brunborg, G., Gaivão, I., Giovannelli, L., Kruszewski, M., Smith, CC. and Stetina, R., 2008. The comet assay: topical issues. Mutagenesis, vol. 23, no. 3, p. 143-151. http://dx.doi.org/10.1093/mutage/gem051.PMid:18283046
» http://dx.doi.org/10.1093/mutage/gem051 - Corsino, J., Silva, DHS., Zanoni, MVB., Bolzani, VS., França, SC., Pereira, AM. and Furlan, M., 2003. Antioxidant flavan-3-ols and flavonol glycosides from Maytenus aquifolium. Phytotherapy research : PTR, vol. 17, no. 8, p. 913-916. http://dx.doi.org/10.1002/ptr.1249. PMid:13680823
» http://dx.doi.org/10.1002/ptr.1249 - Galvão, EL., Silva, DCF., Silva, JO., MOREIRA, AVB. and SOUSA, EMBD., 2008. Evaluation of the antioxidant potential and sub-critical extraction of linseed oil. Ciência e Tecnologia de Alimentos, vol. 28, no. 3, p. 551-557.
- Glasl, H., 1983. ZurPhotometrie in der drogenstandardisierung -3. Gehaltsbestimmung von gerbstoffdrogen. Deutsche Apotheker-Zeitung, vol. 123, p. 1979-1987.
- Halliwell, B. and Gutteridge, JMC., 2007. Free Radicals in biology and medicine. 4th ed. New York: Oxford University Press Inc.
- La Casa, C., Villegas, I., Alarcón de la Lastra, C., Motilva, V. and Martín Calero, MJ., 2000. Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions. Journal of Ethnopharmacology, vol. 71, no. 1-2, p. 45-53. http://dx.doi.org/10.1016/S0378-8741(99)00174-9. PMid:10904145
» http://dx.doi.org/10.1016/S0378-8741(99)00174-9 - Leffa, DD., Rosa, R., Munhoz, BP., Mello, AAM., Mandelli, FD., Amaral, PA., Rossatto, AE. and Andrade, VM., 2012. Genotoxic and antigenotoxic properties of . Calendula officinalis extracts in mice treated with methyl methanesulfonateAdvances in Life Sciences, vol. 2, no. 2, p. 21-28. http://dx.doi.org/10.5923/j.als.20120202.05.
» http://dx.doi.org/10.5923/j.als.20120202.05 - Levine, RL., Garland, D., Oliver, CN., Amici, A., Climent, I., Lenz, AG., Ahn, BW., Shaltiel, S. and Stadtman, ER., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology, vol. 186, p. 464-478. http://dx.doi.org/10.1016/0076-6879(90)86141-H. PMid:1978225
» http://dx.doi.org/10.1016/0076-6879(90)86141-H - Mariot, MP. and Barbieri, RL., 2007. Metabólitos secundários e propriedades medicinais da espinheira-santa (Maytenus ilicifolia Mart. ex Reiss. e Mart.). Maytenus aquifoliumRevista Brasileira de Plantas Medicinais, vol. 9, no. 3, p. 89-99.
- Melo, SF., Soares, SF., Costa, RF., Silva, CR., Oliveira, MB., Bezerra, RJ., Caldeira-de-Araújo, A. and Bernardo-Filho, M., 2001. Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli. Mutation Research, vol. 496, no. 1-2, p. 33-38. http://dx.doi.org/10.1016/S1383-5718(01)00216-9.PMid:11551478
» http://dx.doi.org/10.1016/S1383-5718(01)00216-9 - Miranda, ALP. and FRAGA, CAM., 2006. Atividade sequestradora de radical livre determinação do potencial antioxidante de substâncias bioativas. In MONGE, A. and GANELLIN, CR. (Eds.). Practical Studies for Medicinal Chemistry. IUPAC. p. 1-14.Available from: <http://www.iupac.org/fileadmin/user_upload/publications/cd/Medicinal-Chemistry/Practica-I-5.pdf>. Access in: 10 Mar. 2015.
» http://www.iupac.org/fileadmin/user_upload/publications/cd/Medicinal-Chemistry/Practica-I-5.pdf - Mossi, AJ., Mazutti, M., Paroul, N., Corazza, ML., Dariva, C., Cansian, RL. and OLIVEIRA, JV., 2009. Chemical variation of tannins and triterpenes in Brazilian populations of Maytenus ilicifolia Mart. Ex Reiss. Brazilian Journal of Biology, vol. 69, no. 2, p. 339-345. http://dx.doi.org/10.1590/S1519-69842009000200015.
» http://dx.doi.org/10.1590/S1519-69842009000200015 - Pereira, AMS., Januário, AH., Queiroz, MEE., Biondo, R. and França, SC., 2005. Evaluation of Maytenus aquifolium Mart. and Mart. chemotypes for tannins, total phenols and triterpenes. Maytenus ilicifoliaRevista Brasileira de Plantas Medicinais, vol. 8, no. 1, p. 13-17.
- Pessuto, MB., Costa, IC., Souza, AB., Nicoli, FM., Mello, JCP., Petereit, F. and Luftmann, H., 2009. Antioxidant activity of extracts and condensed tannins from leaves of .Maytenus ilicifolia Mart. ex ReissQuimica Nova, vol. 32, no. 2, p. 412-416. http://dx.doi.org/10.1590/S0100-40422009000200027.
» http://dx.doi.org/10.1590/S0100-40422009000200027 - Rodrigues, CR., Dias, JH., de Mello, RN., Richter, MF., Picada, JN. and Ferraz, AB., 2009. Genotoxic and antigenotoxic properties of Baccharis trimera in mice. Journal of Ethnopharmacology, vol. 125, no. 1, p. 97-101. http://dx.doi.org/10.1016/j.jep.2009.06.006. PMid:19539021
» http://dx.doi.org/10.1016/j.jep.2009.06.006 - Santos-Oliveira, R., Coulaud-Cunha, S. and Colaço, W., 2009. Review of Mart. Ex Reissek, Celastraceae. Contribution to the studies of pharmacological properties. Maytenus ilicifoliaBrazilian Journal of Pharmacognosy, vol. 19, no. 2b, p. 650-659.
- Schwanz, M., Dresch, RR., Manfredini, V. and Henriques, AT., 2013. Antioxidative effects of Mart. (Celastraceae). Maytenus dasycladaInternational Journal of Pharma and Bio Sciences, vol. 4, no. 3, p. 957-969.
- Singh, NP., McCoy, MT., Tice, RR. and Schneider, EL., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research, vol. 175, no. 1, p. 184-191. http://dx.doi.org/10.1016/0014-4827(88)90265-0. PMid:3345800
» http://dx.doi.org/10.1016/0014-4827(88)90265-0 - Soares, M., Welter, L., Gonzaga, L., Lima, A., Macini-Filho, J. and Fett, R., 2008. Evaluation of antioxidant activity and identification of phenolic acids present in the pomace of Gala variety apples. Ciência e Tecnologia deAlimentos, vol. 28, no. 3, p. 727-732.
- Souza-Formigoni, ML., Oliveira, MG., Monteiro, MG., Silveira-Filho, NG., Braz, S. and Carlini, EA., 1991. Antiulcerogenic effects of two Maytenus species in laboratory animals. Journal of Ethnopharmacology, vol. 34, no. 1, p. 21-27. http://dx.doi.org/10.1016/0378-8741(91)90185-G. PMid:1753784
» http://dx.doi.org/10.1016/0378-8741(91)90185-G - Tabach, R., Carlini, E. and Moura, YG., 2002. Um novo extrato de Mart. ex Reiss. (Toxicologia clínica – Fase I) Avaliação em seres humanos. Maytenus ilicifoliaRevista Racine, vol. 71, p. 38-43.
- Vargas, VMF., Guidobono, RR. and Henriques, JAP., 1991. Genotoxicity of plant extracts. Memorias do Instituto Oswaldo Cruz, vol. 86, no. 2, supplement 2, p. 67-70. http://dx.doi.org/10.1590/S0074-02761991000600017. PMid:1842016
» http://dx.doi.org/10.1590/S0074-02761991000600017 - Vellosa, JCR., Barbosa, VF., Khalil, NM., Santos, VAFFM., Furlan, M., Brunetti, IL. and Oliveira, OMMF., 2007. Profile of action over free radicals and reactive oxygen species. Maytenus aquifoliumBrazilian Journal of Pharmaceutical Sciences, vol. 43, no. 3, p. 447-453.
- Vilegas, W., Sanommiya, M., Rastrelli, L. and Pizza, C., 1999. Isolation and structure elucidation of two new flavonoid glycosides from the infusion of maytenus aquifolium leaves. Evaluation of the antiulcer activity of the infusion. Journal of Agricultural and Food Chemistry, vol. 47, no. 2, p. 403-406. http://dx.doi.org/10.1021/jf980114i. PMid:10563907
» http://dx.doi.org/10.1021/jf980114i
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(With 3 figures)
Publication Dates
-
Publication in this collection
May 2015
History
-
Received
10 Oct 2013 -
Accepted
04 Dec 2013