Synergistic effect of Gentiana lutea L. on methyl methanesulfonate genotoxicity in the Drosophila wing spot test

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Synergistic effect of Gentiana lutea L. on methyl methanesulfonate genotoxicity in the Drosophila wing spot test
  Ethnopharmacological communication Synergistic effect of   Gentiana lutea  L. on methyl methanesulfonategenotoxicity in the  Drosophila  wing spot test Aleksandra Patenkovic´  a, n , Marina Stamenkovic´-Radak a,b , Dragana Nikolic´  b , Tamara Markovic´  b ,Marko An X elkovic´  a,b,c a Institute for Biological Research ‘‘Siniˇ sa Stankovic ´’’, Department of Genetics of Populations and Ecogenotoxicology, University of Belgrade, Bul. despota Stefana 142,11060 Belgrade, Serbia b Faculty of Biology, University of Belgrade, Studentski trg 3, 11000 Belgrade, Serbia c Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade, Serbia a r t i c l e i n f o  Article history: Received 9 November 2012Received in revised form11 January 2013Accepted 19 January 2013Available online 4 February 2013 Keywords:Gentiana lutea SMART wing testCo-genotoxicity Drosophila a b s t r a c t Ethnopharmacological relevance: Gentiana lutea  L., the yellow gentian, is herb known for its pharma-cological properties, with a long tradition of use for the treatment of a variety of diseases including theuse as a remedy for digestion, also in food products and in bitter beverages. The aim of the presentstudy is to evaluate, for the first time, genotoxicity of gentian alone, and its antigenotoxicity againstmethyl methanesulfonate (MMS). Materials and methods:  The water infusion of the underground part of gentian were evaluated  in vivo using the  Drosophila  wing spot test, at the dose commonly used in traditional medicine.For antigenotoxic study two types of treatment with gentian and MMS were performed: chronicco-treatment, as well as post-treatment with gentian after acute exposure with MMS. Results:  Water infusion of gentian alone did not exhibit genotoxicity. The results of co- and post-treatment experiments with gentian show that gentian enhanced the frequency of mutant clones overthe values obtained with MMS alone, instead of reducing the genotoxicity of MMS, for 22.64% and27.13% respectively. Conclusions:  This result suggests a synergism of gentian with MMS, and indicates that water infusion of gentian used in traditional medicine may have particular effects with regard to genotoxicity indicatingcareful use. &  2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Gentiana lutea  L. ( Gentianaceae ) isanherbaceousperennialplantnative to the mountains of central and southern Europe, with a longtradition of use in the treatment of gastrointestinal disorders. Theunderground part of this herb is one of the most important bitterdrugs widely used as a decoction or tea, as digestive aid. The gentianroot and rhizomes are also used as an ingredient in the liquor andspirit products, in food, cosmetics and anti-smoking products. Itsextracts show no toxicity and are generally-well tolerated. It hasbeen reported that gentian root increases gastric secretion, and ithas secretolytic, choleretic, anti-ulcer and gastroprotective, antimi-crobial, fungitoxic, free radical scavenging and antioxidant effects(EMEA, 2009). Gentian contains many physiologically importantconstituents that include iridoids, bitter constituents (gentiopicro-side, amarogentine, swertiamarin and sweroside), xantones(gentisin, isogentisin), triterpenoid derivatives, and essential oils(Aberham et al., 2007). The most common secoiridoid glycosidesfound in  Gentiana lutea  and close-related species have a variety of biological effects such as anti-inflammatory, antitumor and hepato-protective (Ghisalberti, 1998), wound-healing (Ozt ¨urk et al., 2006),antifungal and antibacterial (ˇ Siler et al., 2010), as well as antioxidantactivities ( Jaishree and Badami, 2010). Xanthones and xanthoneglycosides from  Gentiana lutea  are potent inhibitors of monoamineoxidase  in vitro  (Haraguchi et al., 2004), further, it was shown thatxanthone isogentisin from  Gentiana lutea  possesses protectiveeffects against endothelial damage caused by cigarette smoking(Schmieder et al., 2007).For lot of herbs and phytochemicals which are used inethnopharmacology or as food ingredients, have been reportedto act as dietary mutagens or/and antimutagens, or showco-mutagenicity, co-carcinogenicity. However, herbs and herbalproducts are increasingly used for treatments of various diseases,as complementary or alternative medicines, although in manycases, there is poor scientific evidence for their therapeuticefficacy and their potential risk to health (Elgorashi et al., 2002; Contents lists available at SciVerse ScienceDirectjournal homepage:  Journal of Ethnopharmacology 0378-8741/$-see front matter  &  2013 Elsevier Ireland Ltd. All rights reserved. n Corresponding author. Tel.:  þ 381 11 20 78 334; fax:  þ 381 11 27 61 433. E-mail address: (A. Patenkovic´). Journal of Ethnopharmacology 146 (2013) 632–636  Verschaeve and Van Staden, 2008). Thus, investigation of geno-toxicity and antigenotoxicity of traditionally used plants is valu-able and important. The extensive data of   Drosophila melanogaster  genetics combined with long experimental experience has madethis species of unique usefulness in mutation research and genetictoxicology. Also,  Drosophila melanogaster   has extensive genetichomology to mammals. The  Drosophila  wing spot test (Graf et al.,1984) is somatic mutation and recombination test (SMART). It isan efficient, sensitive,  in vivo  method for quantification of recom-binogenic and mutagenic potential of chemical and physicalagents, or complex mixtures, for genotoxic and antigenotoxicactivity (Lehmann et al., 2000; Idaomar et al., 2002). 2. Material and methods  2.1. Chemicals All treatments were carried out with the same sample of dried,fragmented, underground part of   Gentiana lutea  (plant materialwas collected on Mountain Suvobor, 44 1  9 0 32.07 00 N and20 1 10 0 10.81 00 E and was obtained from the Department of PlantPhysiology, Institute for Biological Research, Belgrade). Infusion of  Gentiana lutea  was prepared with 5 g dried roots dissolved in200 ml of distilled boiling water, with a prolonged extractionof 30 min. A 3 mM methyl methanesulfonate (MMS) [CAS no. 66-27-3, Aldrich, USA] was used as a positive control.  2.2. Wing spot test  Wing spot test detects several genetic endpoints. Geneticchanges induced in somatic cells of the wings imaginal discs leadto the formation of mutant clones on the wing blade. Single spots( mwh  or  flr  ) can result from mutational events, chromosomeaberrations or mitotic recombination (crossing over between thetwo marker genes). Twin spots ( mwh  and  flr  ) are producedexclusively by mitotic recombination.The experimental procedure was carried out as described byGraf et al. (1984). Two  Drosophila melanogaster   strains for thestandard (ST) cross were used: virgin females from the  multiplewing hairs  ( mwh ) strain:  y 1 ;  mwh 1  j v 1 , and males from the  flare strain:  y I ,  Dp (1 ; 3) sc   J  4  y þ  flr  1 / TM  1 . All treatments were done at25 1 7 1  1 C, 60% relative humidity, and with the 72 h-old larvae.For the genotoxicity studies, treatments were applied:acute — larvae were fed with gentian infusion for 4 h andchronic — larvae were fed with gentian infusion, until completionof their larval life,   48 h.Two separate treatments were applied for the antigenotoxicitystudies: co-treatment and post-treatment. For the co-treatmentlarvae were fed for 4 h with the test agents (gentian, MMS andcombination of gentian with MMS), and then larvae were trans-ferred on standard medium. For the post-treatment larvae werefirst treated with MMS or water, for 4 h. After that, larvae had thesecond treatment: 44 h with or without gentian in the standardmedium.The emerged flies were collected and wings from flies weremounted on slides. Both surfaces of the wings were analyzedunder an optical microscope (at magnification 400  ) for thepresence of spots according to standard procedures (Graf et al.,1984).  2.3. Data analysis For statistical evaluation of the genotoxic effect, the frequen-cies of spots per wing flies treated with  Gentiana lutea  werecompared with data from the corresponding negative controls,whereas the results from flies treated with  Gentiana lutea  plusMMS were compared with data from the corresponding negativeand positive controls. The data were evaluated using Kastenbaumand Bowman’s conditional binomial test, according to themultiple-decision procedure described by Frei and W¨urgler(1988).Because of the weak expression of the  flr   marker in smallclones and its lethality in large clones of mutant cells, only the mwh  clones are used to calculate the clone formation frequenciesper 10 5 cells, based on the number of wings analyzed, the numberof   mwh  clones and the number of cells scored in each wing(24,400) (Frei and W¨urgler, 1988).The percentage of inhibition or enhancement, by gentianinfusion was calculated as [(MMS alone — MMS plus gentian)/MMS alone]/100. 3. Results and discussion The activity of   Gentiana lutea  has been extensively studiedusing both  in vivo  and  in vitro  systems, but as far as we knowthere are no data on its genotoxic and antimutagenic effect in vivo .Frequencies of mutant spots in the trans-heterozygotemarkers flies ( mwh /  flr  ) fed on gentian water infusion or onMMS, in acute and chronic treatment given in Table 1, show thatgentian alone does not modify the spontaneous frequencies of spots, and has no genotoxicity effect. The negative (distilledwater) and positive (MMS) controls gave total spots/fly  Table 1 Summary of results obtained in the  Drosophila  wing spot test. Acute (4 h) and chronic feeding (chr) 3-day-old larvae of the standard (ST)  Drosophila melanogaster   cross withwater infusion of   Gentiana lutea . Marker-heterozygous flies ( mwh /  flr  ) were evaluated.  Treatment  N   SSS  m ¼ 2 LSS  m ¼ 5 Twin spots  m ¼ 5 Total spots  m ¼ 2 TMC  M mwh  c.s.c. CIF n fr D n fr D n fr D n fr D Water control 46 5 0.11 2 0.04 0 0 7 0.15 7 2.14 0.62Gentian 4 h 38 5 0.13  i  1 0.03  i  0 0  i  6 0.16  i  5 1.6 0.54 [  0.08]Gentian chr. 42 1 0.02    1 0.02  i  0 0  i  2 0.05    1 1 0.10 [  0.53]MMS 4 h 34 11 0.32  þ  8 0.24  þ  2 0.06  i  21 0.62  þ  17 2.95 2.05 [1.43]chr (chronic treatment),  N   (number of wings),  n  (number of spots),  fr   (frequency).SSS (small single spots, 1–2 cells), LSS (large single spots,  4 2 cells), TMC (total  mwh  clones). M mwh  c. s. c. (mean  mwh  clone size class) calculated  mwh  clones from  mwh  single spots and the  mwh  parts of twin spots. D  statistical diagnosis according to Frei and W¨urgler (1988): þ (positive),  (negative), i (inconclusive),  w  (weakly positive). m  (multiplication factor) Kastenbaum–Bowman tests, significance level  a ¼ b ¼ 0.05.CIF (clone induction frequency/10 5 cells/cell division) values in square brackets are induction frequencies corrected for spontaneous incidence estimated from negativecontrol.  A. Patenkovic ´ et al. / Journal of Ethnopharmacology 146 (2013) 632–636   633  frequencies within the values reported in the literature for same in vivo  assay.On the other hand, analyzing the effect of water infusion of gentian against direct-acting mutagen, MMS, the synergisticactivity of gentian is recorded in co- and post-treatment(Table 2). In fact, gentian produced an increase in MMS-inducedgenotoxicity, reflected through increase of the frequencies of largesingle, twin and total spots in marker flies. These results showgentian co-genotoxicity by enhancing the frequency of mutantclones over the values obtained with MMS alone, for 22.64% in co-treatment and 27.13% in post-treatment. Both treatments withgentian shows lower incidence of small single spots suggesting aslight inhibitory effect of gentian for this category of spots, inboth, marker and balancer flies. However, this is not interpretedas a real protective effect of gentian since this decrease is limitedonly to the category of small single spots and no reflected in thefrequency of total spots in marker fly. The results obtained showthat gentian had co-recombinogenic rather than co-mutagenicactivity.In this test it is possible to separate mutational events fromrecombinational events. The difference in  mwh  clone frequenciesbetween marker-heterozygous and balancer-heterozygous wingsis a direct measure of the proportion of recombination (Frei et al.,1992). We found that the induced spots were mainly due tomitotic recombination (Table 2). Results show slightly enhance-ment of MMS mitotic recombinogenicity in the presence of gentian, also suggesting that gentian has co-recombinogenicactivity. Although homologous mitotic recombination is animportant process for DNA repair, it has been viewed as a processhaving mainly negative consequences, especially because of itsrole in the loss of heterozygosity in carcinogenesis and otherpathologies such as atherosclerosis, certain types of coronaryheart diseases, autoimmune defects and diabetes.The size distribution of the  mwh  clones is analyzed with eachcompound. As can be seen from the mean  mwh  clone size class(Table 2) the average obtained clone size is different for allcompounds, and the largest  mwh  clones are obtained by gentianwith MMS under both treatments. There was an increase in largespots containing 17–32, 33–64 and 65–128 cells in the descen-dants of larvae treated with both gentian infusion and MMS(Fig. 1). Analyzed distribution of large spots was seen in thedescendants of larvae treated only with MMS, but in lowernumber. This finding indicates that co- and post- treatment withgentian and MMS increases the genotoxicity of MMS, allows theemergence of a greater number of large mutant spots, showingthe synergistic activity of gentian. There are no published articleson gentian co-genotoxicity, and it was only showed in earlierstudy, the mutagenicity of xanthones extracted from gentian root(isogentisin and gentisin) in the Ames test (Morimoto et al., 1983;Matsushima et al., 1985).A similar synergistic effect have been observed by Lehmannet al. (2000), using SMART assay, for tannic acid when adminis-tered simultaneously with either MMS or nitrogen mustard. Kayaet al. (2002) show, in the same test, that ascorbic acid acts as a co-  Table 2 Summary of results obtained in the  Drosophila  wing spot test in the marker-heterozygous (MH) and balanser-heterozygous (BH) progeny of the standard (ST) cross in co-and post-treatment with water infusion of gentian ( Gentiana lutea ) and methyl methanesulfonate (MMS). MMS  Gentian N   SSS  m ¼ 2 LSS  m ¼ 5 twin spots  m ¼ 5 total spots  m ¼ 2 TMC  M mwh  c.s.c. CIF  R   ( % )  E   ( % ) n fr D n fr D n fr D n fr D Co-treatment  a b a b a b a bmwh  /  flr  0 0 72 5 0.07 5 0.07 0 0 10 0.14 10 2.7 0.570 4 h 34 6 0.18  i  2 0.06    0 0  i  8 0.24  i  7 1.43 0.84 [0.27]  79.76 4 h 0 30 6 0.2  i  6 0.2  i  2 0.07  i  14 0.47  þ  13 2.92 1.78 [1.21]  42.31 Gentian þ MMS 18 2 0.11  i i  6 0.33  þ  i  2 0.11  þ  i  10 0.56  þ  i  9 4.67 2.05 [1.48]  66.67 22.64 mwh  / TM1 0 0 28 1 0.04 0 0 1 0.04 1 1 0.150 4 h 24 0 0  i  1 0.04  i  1 0.04  i  1 4 0.17 [0.02]4 h 0 28 4 0.14  i  3 0.11  i  7 0.25  þ  7 2.86 1.02 [0.88] Gentian þ MMS 12 1 0.08  i i  1 0.08  i i  2 0.17  i i  2 3 0.68 [0.54] Post-treatment mwh  /  flr  0 0 98 13 0.13 2 0.02 0 0 15 0.15 15 1.87 0.630 44 h 32 1 0.03    0 0  i  0 0  i  1 0.03  i  1 1 0.13 [  0.50]4 h 0 70 23 0.33  þ  16 0.23  þ  1 0.01  i  40 0.57  þ  37 2.65 2.17 [1.54]  80.43 4 h 44 h 92 20 0.22  i    35 0.38  þ   9 0.1  þ þ  64 0.7  þ   58 4.05 2.58 [1.96]  89.66 27.13 mwh  / TM1 0 0 22 1 0.05 0 0 1 0.05 1 1 0.190 44 h 68 3 0.04  i  2 0.03  i  5 0.07  i  3 1.33 0.18 [  0.01]4 h 0 58 5 0.09  i  1 0.02  i  6 0.10  þ  6 1.67 0.42 [0.24]4 h 44 h 46 0 0  i    3 0.07  i i  3 0.07  þ  i  3 6 0.27 [0.08] N   (number of wings),  n  (number of spots), fr (frequency).SSS (small single spots, 1–2 cells), LSS (large single spots,  4 2 cells), TMC (total  mwh  clones). M mwh  c.s.c. (mean  mwh  clone size class) calculated  mwh  clones from  mwh  single spots and the  mwh  parts of twin spots. D  statistical diagnosis according to Frei and W¨urgler (1988):  þ  (positive),   (negative),  i  (inconclusive),  w  (weakly positive). m  (multiplication factor)Kastenbaum–Bowman tests, significance level  a ¼ b ¼ 0.05. a  Statistical diagnoses for comparison with negative control. b  Statistical diagnoses for comparison with MMS alone.CIF (clone induction frequency/10 5 cells/cell division) values in square brackets are induction frequencies corrected for spontaneous incidence estimated from negativecontrol.Marker-trans-heterozygous flies ( mwh /  flr  ) and balancer-heterozygous flies ( mwh / TM1 ) were evaluated. Only  mwh  single spots can be observed in  mwh /TM 1  heterozygotesas the balancer chromosome TM 1  does not carry the  flr   mutation. R  (Recombination),  E   (Enhancement).  A. Patenkovic ´ et al. / Journal of Ethnopharmacology 146 (2013) 632–636  634  mutagen by inducing significant increases in CoCl 2  genotoxicpotential. Using the same test, de Rezende et al. (2011) haverecently shown that (-)-cubebin, a lignan extracted from seeds of pepper plant ( Piper cubeba  L.), at higher concentrations, potentiatethe genotoxic effects of doxorubicin.A probable explanation for results from present study comesfrom experiments using  Salmonella typhimurium  strains, whenMatsushima et al. (1985) found that derivatives of xanthone witholigo-hydroxy- or methoxy-substitutions were mutagenic andthat the position of the hydroxyl or methoxyl group influencedthe mutagenicity. With tri-hydroxy-xanthones (gentisein), monoreplacement of one hydroxyl group by a methoxyl group atposition 7 of gentisein increased the mutagenicity (isogentisin),and at position 3 of gentisein, decreased mutagenicity (gentisin).MMS, as direct acting alkylating agent, could modify xanthonesfrom gentian, and increase the mutagenicity. Moreover,Schmieder et al. (2007) showed that isogentisin alone caused amild form of intracellular oxidative stress and enhanced intracel-lular oxidative stress by H 2 O 2 , even they showed that isogentisinhave protective activity against cigarette smoke extract- andH 2 O 2 - induced cell death. They suggested that isogentisin doesnot interfere with the damaging activity of both agents, but ratheractivates cellular repair functions. This is important becauseability of MMS to induce excision-repairable lesions has beendemonstrated in a wide range of organisms, including  Escherichiacoli , yeast,  Drosophila  and mammalian cells.The mechanisms by which  Gentiana lutea  increases MMS-induced genotoxicity were not examined here, but consideringall these observations, these mechanisms probably are complexand possibly involving the interactions with excision repairmechanisms and the modulation of active constituents of gentian.Although homologous recombination causes rearrangements of DNA that can promote cancer, little is known about the ability of  Gentiana lutea  to enhanced recombination or modulates DNArepair mechanisms. 4. Conclusions The results showed that water infusion of   Gentiana lutea  is notgenotoxic in somatic cells of   Drosophila melanogaster  , at theconcentration of 25 mg/ml in both chronic and acute treatments.Also, results whit co- and post-treatments, show that gentianenhanced the frequency of mutant clones over the valuesobtained with MMS alone, for 22.64% and 27.13%, respectively,suggesting a co-recombinogenic activity and synergism of gentianwith MMS.Our results indicate that water infusion of gentian can inter-fere  in vivo  with genotoxic agents, and it raises some questionswith regard to safety. Due to the many positive features andword-wide use of gentian, it is obvious that more in depthinvestigations are necessary for better characterize the propertiesof   Gentiana lutea .  Acknowledgments This work was supported by the Ministry of Education, Scienceand Technological Development, Republic of Serbia, Grantno.173012. Thanks to colleagues Zorana Kurbalija Novicˇ ic´ andPredrag Kalajdzˇ ic´, for critical reading and language improvement.Herbal material was provided with courtesy of Prof. Dr DragoljubGrubiˇ sic´ (Department of Plant Physiology, Institute for BiologicalResearch, Belgrade). References Aberham, A., Schwaiger, S., Stuppner, H., Ganzera, M., 2007. Quantitative analysisof iridoids, secoiridoids, xanthones and xanthone glycosides in  Gentiana lutea L. roots by RP-HPLC and LC–MS. Journal of Pharmaceutical and BiomedicalAnalysis 45, 437– Rezende, A.A.A., e Silva, M.L.A., Tavares, D.C., Cunha, W.R., Rezende, K.C.S.,Bastos, J.K., Lehmann, M., de Andrade, H.H.R., Guterres, Z.R., Silva, L.P., Spano´,M.A., 2011. The effect of the dibenzylbutyrolactolic lignan (-)-cubebin ondoxorubicin mutagenicity and recombinogenicity in wing somatic cells of  Drosophila melanogaster  . 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Envir-onmental Mutagenesis 6, 153–188.Ghisalberti, E.L., 1998. Biological and pharmacological activity of naturallyoccurring iridoids and secoiridoids. Phytomedicine 5, 147–163.Haraguchi, H., Tanaka, Y., Kabbash, A., Fujioka, T., Ishizu., T., Yagi, A., 2004.Monoamine oxidase inhibitors from  Gentiana lutea . Phytochemistry 65,2255–2260.Idaomar, M., El Hamss, R., Bakkali, F., Mezzouga, N., Zhiri, A., Baudoux, D.,Mun˜oz-Serrano, A., Liemans, V., Alonso-Moraga, A., 2002. Genotoxicity andantigenotoxicity of some essential oils evaluated by wing spot test of  Drosophila melanogaster  . Mutation Research 513, 61–68. Jaishree, V., Badami, S., 2010. Antioxidant and hepatoprotective effect of swertia-marin from  Enicostemma axillare  against d-galactosamine induced acute liverdamage in rats. Journal of Ethnopharmacology 130, 103–106.Kaya, B., Creus, A., Vela´zquez, A., Yaniko˘glu, A., Marcos, R., 2002. Genotoxicity ismodulated by ascorbic acid; studies using the wing spot test in  Drosophila .Mutation Research 520, 93–101. water control4h gentian4h MMSco-treatment    S   i  n  g   l  e     m    w      h   s  p  o   t  s  p  e  r  w   i  n  g water control 44h gentian4h MMS post-treatment    S   i  n  g   l  e     m    w      h   s  p  o   t  s  p  e  r  w   i  n  g Spot size classSpot size class Fig. 1.  Spot size distribution of single  mwh  spots and single spot inductionfrequencies, recorder on marker-heterozygous (MH) wings after (a) co- and(b) post- treatments with water infusion of   Gentiana lutea  and MMS. Spot sizeclasses: (1): one cell; (2): two cells; (3): three to four cells; (4): five to eight cells;(5): 9–16 cells; (6): 17–32 cells; (7): 33–64; (8): more than 64 cells.  A. Patenkovic ´ et al. / Journal of Ethnopharmacology 146 (2013) 632–636   635  Lehmann, M., Graf, U., Reguly, M.L., Andrade, H.H.R., 2000. 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