- Email: [email protected]
Role of Syzygium cumini seed extract in the chemoprevention of in vivo genomic damage and oxidative stress
Journal of Ethnopharmacology 134 (2011) 329–333
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Role of Syzygium cumini seed extract in the chemoprevention of in vivo genomic damage and oxidative stress Renganathan Arun a , M. Velayutham Dass Prakash a , Suresh K. Abraham b , Kumpati Premkumar a,∗ a b
Department of Biomedical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India
a r t i c l e
i n f o
Article history: Received 11 March 2010 Received in revised form 21 November 2010 Accepted 14 December 2010 Available online 21 December 2010 Keywords: Syzygium cumini Urethane DMBA Antioxidant Micronucleus Chromosomal aberration Oxidative stress
a b s t r a c t Ethanopharmacological relevance: The seeds of Syzygium cumini, Skeels (Jamun) are extensively used in India for treatment of diabetes and other ailments. Aim of the study: The aim of this work was to assess the role of Jamun seed extract (JSE) as a chemoprotective agent against in vivo oxidative stress and genomic damage. Materials and methods: Experiments were carried out to evaluate in vitro protective effects of JSE against hydroxyl radical induced damage in pBR322 DNA, and in vivo genomic damage and oxidative stress in mice which received JSE orally for 5 days before exposure to genotoxic carcinogens urethane (URE) and 7,12-dimethyl benz(a)anthracene (DMBA). Results: Aqueous and ethanolic extracts of JSE showed significant protective effects against hydroxyl radical induced strand breaks in pBR322 DNA. The in vivo experiments with aqueous JSE showed significant protective effects against chromosomal damage induced by the genotoxic carcinogens URE and DMBA. Biochemical assays registered significant inhibition of hepatic lipid peroxidation and increase in GSH level and activity of GST, SOD and CAT. Conclusion: Our findings suggest that JSE can possibly play an important role as a chemopreventive agent against oxidative stress and genomic damage. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The present study was initiated with the main aim of evaluating the possible antigenotoxic effects of the medicinal seed extract obtained from Syzygium cumini, Skeels (Synonym: Eugenia jambolana Lam.; Family Myrtaceae). In India, the deep purple colored, oblong edible fruit with a large centrally located seed is commonly known as Jamun. The fruit pulp and seed extract of Jamun have a long history of medicinal use. Extensive laboratory investigations carried out during the last three decades have furnished substantial information on the antidiabetic properties of this tropical fruit (Prince et al., 2003). Besides, there are reports on the antioxidant (Banerjee et al., 2005; Benherlal and Arumughan, 2007), antiinflammatory (Chaudhuri et al., 1990), antipyretic (Ghosh et al., 1985), anti-allergic (De Brito et al., 2007), anti-bacterial (Bhuiyan
Abbreviations: JSE, Jamun seed extract; URE, urethane; DMBA, 7,12-dimethyl benz(a)anthracene; LPO, lipid peroxidation; GSH, reduced glutathione; GST, glutathione S-transferase; SOD, superoxide dismutase; CAT, catalase; SD, standard deviation; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; MnPCE, micronucleated PCE. ∗ Corresponding author. Tel.: +91 8056589893; fax: +91 0431 2407045. E-mail address: [email protected] (K. Premkumar). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.12.014
et al., 1996), gastro-protective (Chaturvedi et al., 2007) and radioprotective properties of Jamun seed extract (JSE) (Jagetia and Baliga, 2002). In view of the paucity of information on the antigenotoxic effects of this antioxidant-rich medicinal fruit (Veigas et al., 2007; Reynertson et al., 2008), we initiated the present study to evaluate the possible protective effects of aqueous JSE against in vivo genomic damage and oxidative stress induced by the genotoxic carcinogen urethane (URE) and 7,12-dimethylbenz(a)anthracene (DMBA). Prior to this, in vitro tests were carried out with JSE to evaluate the free radical induced strand breaks in pBR322 DNA. 2. Materials and methods 2.1. Chemicals Genotoxic chemicals and stains used for the present study were obtained from Sigma Chemicals (USA). All the other chemicals were purchased from Merck (India) and Qualigens (India). 2.2. Plant materials Syzygium cumini (Jamun) seeds were collected freshly during June–August from a location in Tiruchirappalli (Tamil Nadu, India).
330
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
The seed coat was removed after partial dryness and shade dried again for 3 days. The dried seeds were coarsely ground the powder was stored in a cool and dry place at room temperature. The plant was identified by Dr. S. John Britto SJ, Director, RAPINET Herbarium & Centre for Molecular Systematics, St. Joseph College Campus, Tiruchirappalli and a voucher specimen No. RHT. 68473 has been deposited in the herbarium. 2.3. Sample preparation 100 g of seed powder was extracted with different solvents (ethanol, acetone, ethyl acetate and aqueous) in material to solvent ratio of 1:2 (w/v) at ambient temperature (25–30 ◦ C) under continuous stirring for 5 h and repeated three times. After each extraction, the residue was filtered through muslin cloth and the filtrate was pooled and stored at 4 ◦ C until further use. The clear filtrate was concentrated in a rotary evaporator at low temperature (<40 ◦ C) under vacuum. The concentrated extracts were made to dry in oven at 60 ◦ C and stored at −20 ◦ C till further analysis. 2.4. Qualitative phytochemical screening of extracts Chemical tests were carried out using the different extracts from plants and/or the powdered specimens, using standard procedures to identify the constituents as described by Sofowora (1993), Trease and Evans (1989) and Harborne (1973). 2.5. Determination of total phenolic and flavonoid content of extract Total phenolic and flavonoid content of Jamun seed extracts (JSEs) was determined colorimetrically by the method of McDonald et al. (2001) and Chang et al. (2002), respectively. 2.6. Determination of free radical scavenging activity and reducing power of JSE The DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity and reducing power of JSEs was determined by the method of Koleva et al. (2002) and Oyaizu (1986).
Fig. 1. Protective effects of ethanol and aqueous JSE on hydroxyl radical (OH• − ) induced pBR322 DNA strand breaks. (A) Lane 1: DNA + potassium phosphate buffer (PPB; 20 mM), Lane 2: DNA + Fentons reagent (FR), Lane 3: DNA + EtOH JSE (250 g/ml) + FR, Lane 4: DNA + EtOH JSE (500 g/ml) + FR, Lane 5: DNA + Aq. JSE (250 g/ml) + FR, Lane 6: DNA + Aq. JSE (500 g/ml) + FR, Lane 7: DNA + EtOH JSE (500 g/ml), Lane 8: DNA + Aq. JSE (500 g/ml). (B) Total protective percentage of ethanol and aqueous JSE (250 and 500 g/ml) on hydroxyl radical (OH• − ) induced plasmid DNA strand breakage. Data are presented as mean ± SD of three experiments.
2.9. Animals All the experiments were carried out using 10–12 weeks old male and female Swiss albino mice maintained in the University animal house on standard mouse pellets and water ad libitum in accordance with CPCSEA, India guidelines. Approval for this work was obtained from the University animal ethical committee. 2.10. In vivo assays for determining genomic damage
2.7. DNA nicking assay DNA cleavage assay was performed using supercoiled pBR322 DNA (Bangalore Genei Ltd., Bangalore, India) by the method of Lee et al. (2002). 2.8. HPTLC determination of flavonoids and phenolic acids Preliminary qualitative phytochemical screening of JSE tested for the presence of flavonoids and phenols. The HPTLC quantification was performed at Indian Institute of Crop Processing Technology (IICPT), Ministry of Food Processing Industries, Govt. of India, Tanjavur, Tamil Nadu, India with the standardized protocol. Briefly, aqueous extract of Jamun seed and mixed standards (gallic acid, rutin, ferulic acid, caffeic acid and quercetin) were dissolved in HPLC grade methanol and applied to pre washed silica gel 60 F254 HP-TLC plates (10 cm × 10 cm, silica-gel thickness 2 mm, Merck) with an automator applicator (Linomat IV; CAMAG). The samples were then separated (migration distance 75 mm) using HPLC grade solvents. Then the plates were scanned in CAMAG TLC scanner and the peaks were recorded at a wavelength of 366 nm. The Rf values and concentration of separated compounds were determined with WINCATS planar chromatography Manager Software.
2.10.1. Mouse bone marrow micronucleus test Genotoxic effects were evaluated in the mouse bone marrow micronucleus test according to Schmid (1975). 2500 polychromatic erythrocytes (PCEs) were scored per animal per slide to determine the frequency of micronucleated polychromatic erythrocytes (Mn PCEs). All the slides were scored by the same observer. 2.10.2. Mouse bone marrow chromosome aberration test The in vivo chromosomal aberration analysis was performed according to the protocol of Kilian et al. (1977). One hundred-well spread and uniformly stained metaphases, having 40 ± 1 chromosomes, per animal and 600 metaphases per dose were observed for the presence of chromosomal aberrations. 2.11. Biochemical assays Hepatic reduced glutathione (GSH) level and glutathione Stransferase (GST) activity was determined by the method of Moron et al. (1979) and Habig et al. (1974). Activities of superoxide oxide (SOD) and catalase (CAT) were assayed by the method of Marklund and Marklund (1974) and Sinha (1971), respectively. Lipid peroxidation (LPO) in liver was estimated colorimetrically by the method
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
331
Fig. 2. Effects of aqueous JSE on urethane induced oxidative stress in liver. Effects of aqueous JSE on (A) lipid peroxidation (LPO) expressed as nmol of MDA formed per mg protein. (B) Superoxide dismutase (SOD) activity expressed as 50% inhibition of pyrogallol auto-oxidation/min/mg protein. (C) Catalase (CAT) activity expressed as moles of H2 O2 consumed/min/mg protein. (D) Glutathione S-transferase (GST) activity expressed as nM of CDNB conjugated min−1 mg−1 protein. (E) Reduced glutathione (GSH) levels expressed as g of GSH formed mg−1 protein. All values are expressed in mean ± SD with six mice in each group. ### p < 0.001; ## p < 0.01 compared with normal control group; ***p < 0.001; **p < 0.01compared with URE and aqueous JSE treated groups.
of Ohkawa et al. (1979). The total protein was determined by the method of Bradford (1976). 2.12. Statistical analysis All data were expressed as means ± SD of number of experiments (n = 6). The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 11.5 (SPSS, Cary, NC, USA) and the individual comparison were obtained by Duncan’s Multiple Range Test (DMRT). A value of p < 0.05 was considered to indicate a significant difference between groups. 3. Results Preliminary phytochemical screening of aqueous, ethanol, acetone and ethyl acetate extracts of Jamun seed have shown positive results for carbohydrates, phytosterol, phenols, tannins, phlobatannins and flavonoids. Terpenoids were detected in the
ethanol, acetone and ethyl acetate extracts. The total phenolic content in the aqueous (168.33 ± 7.64), ethanol (471.67 ± 29.3), acetone (230 ± 25) and ethyl acetate (375 ± 40.93) extracts were determined with respect to gallic acid equivalent (mg/g of extract). The flavonoids content in the aqueous (65.31 ± 1.77), ethanol (114.88 ± 5.36), acetone (103.86 ± 3.67) and ethyl acetate (138.26 ± 6.58) extracts were determined with respect to quercetin equivalent (mg/g of extract). The DPPH radical scavenging activity of different JSEs at 5, 10, 25, 50 and 100 g/ml was less when compared to gallic acid. However, with 100 g/ml of different JSEs, the antioxidant activity was similar to that of gallic acid. The IC50 values for antioxidant activity of gallic acid, aqueous, ethanol, acetone and ethyl acetate extracts are 25.33, 25.02, 24.53, 24.29 and 24.42 g/ml, respectively. The reductive potential of different JSEs at concentrations 20, 40, 60, 80, 100 g/ml were approximately one-third of that observed for the positive control ascorbic acid. The IC50 values for reductive potential of ascorbic acid, aqueous, ethanol, acetone and ethyl acetate
332
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
Fig. 3. Effects of aqueous JSE on URE or DMBA induced micronucleated polychromatic erythrocytes (MnPCEs) in bone marrow cells of mice. All bars are expressed in mean ± SD of total number of MnPCEs per 2500 PCEs with six mice in each group. (a) ### p < 0.001; compared with normal and JSE alone groups; (b) ***p < 0.001; compared with urethane and aqueous JSE treated groups; (c) ***p < 0.001; compared with DMBA and aqueous JSE treated groups.
extracts are 40.10, 60.10, 59.10, 59.10 and 59.95 g/ml, respectively. The IC50 values obtained for ascorbic acid was lower when compared to that for the different JSEs. From Fig. 1, it is evident that the presence of aqueous and ethanol JSEs along with plasmid pBR322 DNA during Fenton’s reaction protected DNA from OH• radical induced strand breaks. At a concentration of 500 g/ml, the aqueous and ethanol extract protected the DNA from OH• radicals to the extent of 90% and 99%, respectively. The results of the biochemical assays for determining the hepatic LPO, GSH level and activity of SOD, CAT and GST in the control and treated mice have been presented in Fig. 2. Increase in LPO level and a concomitant decrease in GSH, GST SOD and CAT were observed in mice treated with the genotoxin URE alone. Administration of three doses of aqueous JSE to the experimental animals for five consecutive days before exposure to URE showed a reduction in LPO and a dose-related increase in GSH, GST, SOD and CAT. Fig. 3 illustrates the results of the in vivo assays for evaluating genomic damage. It shows that pre-treatment of mice with three doses (500, 1000 and 1500 mg/kg b.w.) of aqueous JSE for five consecutive days resulted in significant reductions in the frequencies of micronuclei induced by the genotoxic carcinogen either URE or DMBA. This protective effect was not always dose-related. A similar trend was observed when the frequencies of chromosome aberrations were evaluated in the metaphase cells of mice pretreated with three doses of aqueous JSE and challenged with URE (Fig. 4). The incidence of aberrations/cell was significantly reduced when compared to that of URE alone. Treatment of mice with the combination of URE/DMBA with JSEs did not show any significant change in the percentage of PCEs. There was no significant increase in genotoxicity following pre-treatment of mice for 5 days with the highest test dose (1500 mg/kg b.w) of aqueous JSE.
Fig. 4. Effects of aqueous JSE on urethane induce chromosomal aberrations in bone marrow cells of mice. All bars are expressed in mean ± SD of total chromosomal aberrations per cell with six mice in each group. ### p < 0.001 compared with normal control group; ***p < 0.001 compared with URE and aqueous JSE treated groups.
The results from our present study have demonstrated for the first time that aqueous JSE can exert significant in vivo antigenotoxic effects against the indirectly acting carcinogens URE and DMBA, and protect against in vitro DNA damage induced by free radicals. DNA damage resulting from free radical induced oxidative stress can lead to gene mutation and chromosome aberrations. In this study, we observed significant reductions in chromosome aberrations in metaphase cells and micronuclei in polychromatic erythrocytes formed as a result of chromosomal damage. Many phytochemicals with antioxidant activity can either minimize or prevent this potentially harmful genomic damage mediated by oxidative stress. From the biochemical assays carried out with the liver samples of these animals, there is evidence for the role of JSE pre-treatment in inhibiting LPO and enhancing the level of GSH and activity of the antioxidant enzymes GST, SOD and CAT. These results suggest that the antioxidant property of JSE is responsible for the observed chemopreventive effect. URE, the genotoxin used in our study is found in trace amounts in fermented foods and beverages and it is not genotoxic or carcinogenic per se (Kemper et al., 1995). However, vinyl carbamate epoxide is believed to be the metabolite responsible for its genotoxic and carcinogenic effects. Conjugation of vinyl carbamate epoxide with GSH can lead to detoxification and this process is catalyzed by GST (Kemper et al., 1995). From our results it is evident that pre-treatment of mice with JSE has increased the level of GSH and activity of GST. These results indicate that the inducible GST defense system has possibly played an important role in reducing the reactive form of URE which can cause DNA lesions. A search for the bioactive constituents of JSE would lead to several potential candidates. HPTLC analysis of aqueous JSE used in the present study showed the presence of rutin, quercetin, gallic acid, ferulic acid and caffeic acid (data not shown). When a complex mixture of bioactive phytochemicals is used for evaluating antigenotoxic effects, the results would largely depend on the synergistic/additive interaction between the above chemopreventive phytochemicals (Liu, 2004).
4. Discussion 5. Conclusion Today, a wide range of antioxidant phytochemicals present in food stuffs and beverages are known to exert chemopreventive effects. This prompted us to investigate whether the traditional phytomedicine JSE which has shown antioxidant activity can protect against DNA damage induced by environmental genotoxins/carcinogens.
In conclusion, our present study has demonstrated for the first time that oral administration of the phytomedicine aqueous JSE to mice can enhance the activity of the antioxidant defense and lead to protective effects against genomic damage induced by the carcinogens DMBA and URE.
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
Acknowledgements The authors thank Bharathidasan University, Tiruchirappalli, Tamil Nadu, India for providing the Financial Support to Dr. K. Premkumar as a startup fund. The authors would like to thank Mr. S. Kumaravel, Senior Scientist and Mr. R. Paranthaman, Chemist, IICPT, Thanjavur, Tamil Nadu, India for the technical help rendered for HPTLC analysis. References Banerjee, A., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of S. cumini fruit. Food Chemistry 90, 727–733. Benherlal, P.S., Arumughan, C., 2007. Chemical composition and in vitro antioxidant studies of S. cumini fruit. Journal of the Science of Food and Agriculture 87, 2560–2569. Bhuiyan, M.A., Mia, M.Y., Rashid, M.A., 1996. Antibacterial principles of the seeds of Eugenia jambolana. Bangladesh Journal of Botany 25, 239–241. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Chang, C.C., Yang, M.H., Wen, H.M., Chern, J.C., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis. 10, 178–182. Chaturvedi, A., Kumar, M.M., Bhawani, G., Chaturvedi, H., Kumar, M., Goel, R.K., 2007. Effect of ethanolic extract of Eugenia jambolana seeds on gastric ulceration and secretion in rats. Indian Journal of Physiology Pharmacology 51, 131–140. Chaudhuri, A.K.N., Pal, S., Gomes, A., Bhattacharya, S., 1990. Anti-inflammatory and related actions of S. cumini seed extract. Phytotherapy Research 4, 5–10. De Brito, E.S., de Araújo, M.C.P., Alves, R.E., Carkeet, C., Clevidence, B.A., Novotny, J.A., 2007. Anthocyanins presented in selected fruits: acerola, jambolão, jussara and guajiru. Journal of Agricultural and Food Chemistry 55, 9389–9394. Ghosh, K., Chakraborty, D., Chatterjee, G.K., Chaudhuri, A.K.N., Pal, M., 1985. Studies on anti-inflammatory and antipyretic activities of S. cumini Linn, seeds. International Research Communications System: Medical Science Biochemistry 13, 340–341. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130–7139. Harborne, J.B., 1973. Phytochemical Methods: A Guide to Modern Technique of Plant Analysis. Chapman and Hall, London. Jagetia, G.C., Baliga, M.S., 2002. S. cumini (Jamun) reduces the radiation-induced DNA damage in the cultured human peripheral blood lymphocytes: a preliminary study. Toxicology Letters 7, 19–25.
333
Kemper, R.A., Myers, S.R., Hurst, H.E., 1995. Detoxification of vinyl carbamate epoxide by glutathione: evidence for participation of glutathione S-transferases in metabolism of ethyl carbamate. Toxicology Applied Pharmacology 135, 110–118. Kilian, D.J., Moreland, F.M., Benge, M.C., Legator, M.S., Whorton, E.B., 1977. A collaborative study to measure interlaboratory variation within the in vivo bone marrow metaphase procedure. In: Kibey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.), Handbook of Mutagenicity Test Procedures. Elsevier/North-Holland, Amesterdam, pp. 243–260. Koleva, I.I., Van Beek, T., Linssen, J.P.H., de Groot, A., Evstatieva, L.N., 2002. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis 13, 8–17. Lee, J.C., Kim, H.R., Kim, J., Jang, Y.S., 2002. Antioxidant property of an ethanol extract of the stem of Opuntia ficus-indica var. Saboten. Journal of Agricultural Food Chemistry 50, 6490–6496. Liu, R.H., 2004. Potential synergy of phytochemicals in cancer prevention. Mechanism of action. Journal of Nutrition 134, 3479–3485. McDonald, S., Prenzler, P.D., Autolovich, M., Robards, K., 2001. Phenolic content and antioxidant activity of olive extracts. Food Chemistry. 73, 73–84. Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry 47, 389–394. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 582, 67–78. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Oyaizu, M., 1986. Studies on products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition. Japanese Journal of Nutrition. 44, 307–315. Prince, S.M., Kamalakkannan, N., Menon, V.P., 2003. S. cumini seed extracts reduce tissue damage in diabetic rat brain. Journal of Ethnopharmacology 84, 205– 209. Reynertson, K.A., Yang, H., Jiang, B., Baile, M.J., Kennelly, E.J., 2008. Quantitative analysis of antiradical phenolic constituents from fourteen edible Myrtaceae fruits. Food Chemistry 109, 883–890. Schmid, W., 1975. The micronucleus test. Mutation Research 31, 9–15. Sinha, K., 1971. Colorimetric assay of catalase. Analytical Biochemistry 47, 389–394. Sofowora, L.A., 1993. Medicinal plants and traditional medicine in Africa. Spectrum Books Ltd. Ibaban., 55–71. Trease, G. E., Evans, W. C., 1989. Trease and Evans Pharmacognosy: A Physician’s Guide to Herbal Medicine. 13, Bailliere Tindall, London. Veigas, J.M., Narayan, M.S., Laxman, P.M., Neelwarne, B., 2007. Chemical nature, stability and bioefficacies of anthocyanins from fruit peel of S. cumini Skeels. Food Chemistry 105, 619–627.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Role of Syzygium cumini seed extract in the chemoprevention of in vivo genomic damage and oxidative stress Renganathan Arun a , M. Velayutham Dass Prakash a , Suresh K. Abraham b , Kumpati Premkumar a,∗ a b
Department of Biomedical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India
a r t i c l e
i n f o
Article history: Received 11 March 2010 Received in revised form 21 November 2010 Accepted 14 December 2010 Available online 21 December 2010 Keywords: Syzygium cumini Urethane DMBA Antioxidant Micronucleus Chromosomal aberration Oxidative stress
a b s t r a c t Ethanopharmacological relevance: The seeds of Syzygium cumini, Skeels (Jamun) are extensively used in India for treatment of diabetes and other ailments. Aim of the study: The aim of this work was to assess the role of Jamun seed extract (JSE) as a chemoprotective agent against in vivo oxidative stress and genomic damage. Materials and methods: Experiments were carried out to evaluate in vitro protective effects of JSE against hydroxyl radical induced damage in pBR322 DNA, and in vivo genomic damage and oxidative stress in mice which received JSE orally for 5 days before exposure to genotoxic carcinogens urethane (URE) and 7,12-dimethyl benz(a)anthracene (DMBA). Results: Aqueous and ethanolic extracts of JSE showed significant protective effects against hydroxyl radical induced strand breaks in pBR322 DNA. The in vivo experiments with aqueous JSE showed significant protective effects against chromosomal damage induced by the genotoxic carcinogens URE and DMBA. Biochemical assays registered significant inhibition of hepatic lipid peroxidation and increase in GSH level and activity of GST, SOD and CAT. Conclusion: Our findings suggest that JSE can possibly play an important role as a chemopreventive agent against oxidative stress and genomic damage. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The present study was initiated with the main aim of evaluating the possible antigenotoxic effects of the medicinal seed extract obtained from Syzygium cumini, Skeels (Synonym: Eugenia jambolana Lam.; Family Myrtaceae). In India, the deep purple colored, oblong edible fruit with a large centrally located seed is commonly known as Jamun. The fruit pulp and seed extract of Jamun have a long history of medicinal use. Extensive laboratory investigations carried out during the last three decades have furnished substantial information on the antidiabetic properties of this tropical fruit (Prince et al., 2003). Besides, there are reports on the antioxidant (Banerjee et al., 2005; Benherlal and Arumughan, 2007), antiinflammatory (Chaudhuri et al., 1990), antipyretic (Ghosh et al., 1985), anti-allergic (De Brito et al., 2007), anti-bacterial (Bhuiyan
Abbreviations: JSE, Jamun seed extract; URE, urethane; DMBA, 7,12-dimethyl benz(a)anthracene; LPO, lipid peroxidation; GSH, reduced glutathione; GST, glutathione S-transferase; SOD, superoxide dismutase; CAT, catalase; SD, standard deviation; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; MnPCE, micronucleated PCE. ∗ Corresponding author. Tel.: +91 8056589893; fax: +91 0431 2407045. E-mail address: [email protected] (K. Premkumar). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.12.014
et al., 1996), gastro-protective (Chaturvedi et al., 2007) and radioprotective properties of Jamun seed extract (JSE) (Jagetia and Baliga, 2002). In view of the paucity of information on the antigenotoxic effects of this antioxidant-rich medicinal fruit (Veigas et al., 2007; Reynertson et al., 2008), we initiated the present study to evaluate the possible protective effects of aqueous JSE against in vivo genomic damage and oxidative stress induced by the genotoxic carcinogen urethane (URE) and 7,12-dimethylbenz(a)anthracene (DMBA). Prior to this, in vitro tests were carried out with JSE to evaluate the free radical induced strand breaks in pBR322 DNA. 2. Materials and methods 2.1. Chemicals Genotoxic chemicals and stains used for the present study were obtained from Sigma Chemicals (USA). All the other chemicals were purchased from Merck (India) and Qualigens (India). 2.2. Plant materials Syzygium cumini (Jamun) seeds were collected freshly during June–August from a location in Tiruchirappalli (Tamil Nadu, India).
330
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
The seed coat was removed after partial dryness and shade dried again for 3 days. The dried seeds were coarsely ground the powder was stored in a cool and dry place at room temperature. The plant was identified by Dr. S. John Britto SJ, Director, RAPINET Herbarium & Centre for Molecular Systematics, St. Joseph College Campus, Tiruchirappalli and a voucher specimen No. RHT. 68473 has been deposited in the herbarium. 2.3. Sample preparation 100 g of seed powder was extracted with different solvents (ethanol, acetone, ethyl acetate and aqueous) in material to solvent ratio of 1:2 (w/v) at ambient temperature (25–30 ◦ C) under continuous stirring for 5 h and repeated three times. After each extraction, the residue was filtered through muslin cloth and the filtrate was pooled and stored at 4 ◦ C until further use. The clear filtrate was concentrated in a rotary evaporator at low temperature (<40 ◦ C) under vacuum. The concentrated extracts were made to dry in oven at 60 ◦ C and stored at −20 ◦ C till further analysis. 2.4. Qualitative phytochemical screening of extracts Chemical tests were carried out using the different extracts from plants and/or the powdered specimens, using standard procedures to identify the constituents as described by Sofowora (1993), Trease and Evans (1989) and Harborne (1973). 2.5. Determination of total phenolic and flavonoid content of extract Total phenolic and flavonoid content of Jamun seed extracts (JSEs) was determined colorimetrically by the method of McDonald et al. (2001) and Chang et al. (2002), respectively. 2.6. Determination of free radical scavenging activity and reducing power of JSE The DPPH (1,1-diphenyl-2-picryl hydrazyl) radical scavenging activity and reducing power of JSEs was determined by the method of Koleva et al. (2002) and Oyaizu (1986).
Fig. 1. Protective effects of ethanol and aqueous JSE on hydroxyl radical (OH• − ) induced pBR322 DNA strand breaks. (A) Lane 1: DNA + potassium phosphate buffer (PPB; 20 mM), Lane 2: DNA + Fentons reagent (FR), Lane 3: DNA + EtOH JSE (250 g/ml) + FR, Lane 4: DNA + EtOH JSE (500 g/ml) + FR, Lane 5: DNA + Aq. JSE (250 g/ml) + FR, Lane 6: DNA + Aq. JSE (500 g/ml) + FR, Lane 7: DNA + EtOH JSE (500 g/ml), Lane 8: DNA + Aq. JSE (500 g/ml). (B) Total protective percentage of ethanol and aqueous JSE (250 and 500 g/ml) on hydroxyl radical (OH• − ) induced plasmid DNA strand breakage. Data are presented as mean ± SD of three experiments.
2.9. Animals All the experiments were carried out using 10–12 weeks old male and female Swiss albino mice maintained in the University animal house on standard mouse pellets and water ad libitum in accordance with CPCSEA, India guidelines. Approval for this work was obtained from the University animal ethical committee. 2.10. In vivo assays for determining genomic damage
2.7. DNA nicking assay DNA cleavage assay was performed using supercoiled pBR322 DNA (Bangalore Genei Ltd., Bangalore, India) by the method of Lee et al. (2002). 2.8. HPTLC determination of flavonoids and phenolic acids Preliminary qualitative phytochemical screening of JSE tested for the presence of flavonoids and phenols. The HPTLC quantification was performed at Indian Institute of Crop Processing Technology (IICPT), Ministry of Food Processing Industries, Govt. of India, Tanjavur, Tamil Nadu, India with the standardized protocol. Briefly, aqueous extract of Jamun seed and mixed standards (gallic acid, rutin, ferulic acid, caffeic acid and quercetin) were dissolved in HPLC grade methanol and applied to pre washed silica gel 60 F254 HP-TLC plates (10 cm × 10 cm, silica-gel thickness 2 mm, Merck) with an automator applicator (Linomat IV; CAMAG). The samples were then separated (migration distance 75 mm) using HPLC grade solvents. Then the plates were scanned in CAMAG TLC scanner and the peaks were recorded at a wavelength of 366 nm. The Rf values and concentration of separated compounds were determined with WINCATS planar chromatography Manager Software.
2.10.1. Mouse bone marrow micronucleus test Genotoxic effects were evaluated in the mouse bone marrow micronucleus test according to Schmid (1975). 2500 polychromatic erythrocytes (PCEs) were scored per animal per slide to determine the frequency of micronucleated polychromatic erythrocytes (Mn PCEs). All the slides were scored by the same observer. 2.10.2. Mouse bone marrow chromosome aberration test The in vivo chromosomal aberration analysis was performed according to the protocol of Kilian et al. (1977). One hundred-well spread and uniformly stained metaphases, having 40 ± 1 chromosomes, per animal and 600 metaphases per dose were observed for the presence of chromosomal aberrations. 2.11. Biochemical assays Hepatic reduced glutathione (GSH) level and glutathione Stransferase (GST) activity was determined by the method of Moron et al. (1979) and Habig et al. (1974). Activities of superoxide oxide (SOD) and catalase (CAT) were assayed by the method of Marklund and Marklund (1974) and Sinha (1971), respectively. Lipid peroxidation (LPO) in liver was estimated colorimetrically by the method
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
331
Fig. 2. Effects of aqueous JSE on urethane induced oxidative stress in liver. Effects of aqueous JSE on (A) lipid peroxidation (LPO) expressed as nmol of MDA formed per mg protein. (B) Superoxide dismutase (SOD) activity expressed as 50% inhibition of pyrogallol auto-oxidation/min/mg protein. (C) Catalase (CAT) activity expressed as moles of H2 O2 consumed/min/mg protein. (D) Glutathione S-transferase (GST) activity expressed as nM of CDNB conjugated min−1 mg−1 protein. (E) Reduced glutathione (GSH) levels expressed as g of GSH formed mg−1 protein. All values are expressed in mean ± SD with six mice in each group. ### p < 0.001; ## p < 0.01 compared with normal control group; ***p < 0.001; **p < 0.01compared with URE and aqueous JSE treated groups.
of Ohkawa et al. (1979). The total protein was determined by the method of Bradford (1976). 2.12. Statistical analysis All data were expressed as means ± SD of number of experiments (n = 6). The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 11.5 (SPSS, Cary, NC, USA) and the individual comparison were obtained by Duncan’s Multiple Range Test (DMRT). A value of p < 0.05 was considered to indicate a significant difference between groups. 3. Results Preliminary phytochemical screening of aqueous, ethanol, acetone and ethyl acetate extracts of Jamun seed have shown positive results for carbohydrates, phytosterol, phenols, tannins, phlobatannins and flavonoids. Terpenoids were detected in the
ethanol, acetone and ethyl acetate extracts. The total phenolic content in the aqueous (168.33 ± 7.64), ethanol (471.67 ± 29.3), acetone (230 ± 25) and ethyl acetate (375 ± 40.93) extracts were determined with respect to gallic acid equivalent (mg/g of extract). The flavonoids content in the aqueous (65.31 ± 1.77), ethanol (114.88 ± 5.36), acetone (103.86 ± 3.67) and ethyl acetate (138.26 ± 6.58) extracts were determined with respect to quercetin equivalent (mg/g of extract). The DPPH radical scavenging activity of different JSEs at 5, 10, 25, 50 and 100 g/ml was less when compared to gallic acid. However, with 100 g/ml of different JSEs, the antioxidant activity was similar to that of gallic acid. The IC50 values for antioxidant activity of gallic acid, aqueous, ethanol, acetone and ethyl acetate extracts are 25.33, 25.02, 24.53, 24.29 and 24.42 g/ml, respectively. The reductive potential of different JSEs at concentrations 20, 40, 60, 80, 100 g/ml were approximately one-third of that observed for the positive control ascorbic acid. The IC50 values for reductive potential of ascorbic acid, aqueous, ethanol, acetone and ethyl acetate
332
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
Fig. 3. Effects of aqueous JSE on URE or DMBA induced micronucleated polychromatic erythrocytes (MnPCEs) in bone marrow cells of mice. All bars are expressed in mean ± SD of total number of MnPCEs per 2500 PCEs with six mice in each group. (a) ### p < 0.001; compared with normal and JSE alone groups; (b) ***p < 0.001; compared with urethane and aqueous JSE treated groups; (c) ***p < 0.001; compared with DMBA and aqueous JSE treated groups.
extracts are 40.10, 60.10, 59.10, 59.10 and 59.95 g/ml, respectively. The IC50 values obtained for ascorbic acid was lower when compared to that for the different JSEs. From Fig. 1, it is evident that the presence of aqueous and ethanol JSEs along with plasmid pBR322 DNA during Fenton’s reaction protected DNA from OH• radical induced strand breaks. At a concentration of 500 g/ml, the aqueous and ethanol extract protected the DNA from OH• radicals to the extent of 90% and 99%, respectively. The results of the biochemical assays for determining the hepatic LPO, GSH level and activity of SOD, CAT and GST in the control and treated mice have been presented in Fig. 2. Increase in LPO level and a concomitant decrease in GSH, GST SOD and CAT were observed in mice treated with the genotoxin URE alone. Administration of three doses of aqueous JSE to the experimental animals for five consecutive days before exposure to URE showed a reduction in LPO and a dose-related increase in GSH, GST, SOD and CAT. Fig. 3 illustrates the results of the in vivo assays for evaluating genomic damage. It shows that pre-treatment of mice with three doses (500, 1000 and 1500 mg/kg b.w.) of aqueous JSE for five consecutive days resulted in significant reductions in the frequencies of micronuclei induced by the genotoxic carcinogen either URE or DMBA. This protective effect was not always dose-related. A similar trend was observed when the frequencies of chromosome aberrations were evaluated in the metaphase cells of mice pretreated with three doses of aqueous JSE and challenged with URE (Fig. 4). The incidence of aberrations/cell was significantly reduced when compared to that of URE alone. Treatment of mice with the combination of URE/DMBA with JSEs did not show any significant change in the percentage of PCEs. There was no significant increase in genotoxicity following pre-treatment of mice for 5 days with the highest test dose (1500 mg/kg b.w) of aqueous JSE.
Fig. 4. Effects of aqueous JSE on urethane induce chromosomal aberrations in bone marrow cells of mice. All bars are expressed in mean ± SD of total chromosomal aberrations per cell with six mice in each group. ### p < 0.001 compared with normal control group; ***p < 0.001 compared with URE and aqueous JSE treated groups.
The results from our present study have demonstrated for the first time that aqueous JSE can exert significant in vivo antigenotoxic effects against the indirectly acting carcinogens URE and DMBA, and protect against in vitro DNA damage induced by free radicals. DNA damage resulting from free radical induced oxidative stress can lead to gene mutation and chromosome aberrations. In this study, we observed significant reductions in chromosome aberrations in metaphase cells and micronuclei in polychromatic erythrocytes formed as a result of chromosomal damage. Many phytochemicals with antioxidant activity can either minimize or prevent this potentially harmful genomic damage mediated by oxidative stress. From the biochemical assays carried out with the liver samples of these animals, there is evidence for the role of JSE pre-treatment in inhibiting LPO and enhancing the level of GSH and activity of the antioxidant enzymes GST, SOD and CAT. These results suggest that the antioxidant property of JSE is responsible for the observed chemopreventive effect. URE, the genotoxin used in our study is found in trace amounts in fermented foods and beverages and it is not genotoxic or carcinogenic per se (Kemper et al., 1995). However, vinyl carbamate epoxide is believed to be the metabolite responsible for its genotoxic and carcinogenic effects. Conjugation of vinyl carbamate epoxide with GSH can lead to detoxification and this process is catalyzed by GST (Kemper et al., 1995). From our results it is evident that pre-treatment of mice with JSE has increased the level of GSH and activity of GST. These results indicate that the inducible GST defense system has possibly played an important role in reducing the reactive form of URE which can cause DNA lesions. A search for the bioactive constituents of JSE would lead to several potential candidates. HPTLC analysis of aqueous JSE used in the present study showed the presence of rutin, quercetin, gallic acid, ferulic acid and caffeic acid (data not shown). When a complex mixture of bioactive phytochemicals is used for evaluating antigenotoxic effects, the results would largely depend on the synergistic/additive interaction between the above chemopreventive phytochemicals (Liu, 2004).
4. Discussion 5. Conclusion Today, a wide range of antioxidant phytochemicals present in food stuffs and beverages are known to exert chemopreventive effects. This prompted us to investigate whether the traditional phytomedicine JSE which has shown antioxidant activity can protect against DNA damage induced by environmental genotoxins/carcinogens.
In conclusion, our present study has demonstrated for the first time that oral administration of the phytomedicine aqueous JSE to mice can enhance the activity of the antioxidant defense and lead to protective effects against genomic damage induced by the carcinogens DMBA and URE.
R. Arun et al. / Journal of Ethnopharmacology 134 (2011) 329–333
Acknowledgements The authors thank Bharathidasan University, Tiruchirappalli, Tamil Nadu, India for providing the Financial Support to Dr. K. Premkumar as a startup fund. The authors would like to thank Mr. S. Kumaravel, Senior Scientist and Mr. R. Paranthaman, Chemist, IICPT, Thanjavur, Tamil Nadu, India for the technical help rendered for HPTLC analysis. References Banerjee, A., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of S. cumini fruit. Food Chemistry 90, 727–733. Benherlal, P.S., Arumughan, C., 2007. Chemical composition and in vitro antioxidant studies of S. cumini fruit. Journal of the Science of Food and Agriculture 87, 2560–2569. Bhuiyan, M.A., Mia, M.Y., Rashid, M.A., 1996. Antibacterial principles of the seeds of Eugenia jambolana. Bangladesh Journal of Botany 25, 239–241. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Chang, C.C., Yang, M.H., Wen, H.M., Chern, J.C., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis. 10, 178–182. Chaturvedi, A., Kumar, M.M., Bhawani, G., Chaturvedi, H., Kumar, M., Goel, R.K., 2007. Effect of ethanolic extract of Eugenia jambolana seeds on gastric ulceration and secretion in rats. Indian Journal of Physiology Pharmacology 51, 131–140. Chaudhuri, A.K.N., Pal, S., Gomes, A., Bhattacharya, S., 1990. Anti-inflammatory and related actions of S. cumini seed extract. Phytotherapy Research 4, 5–10. De Brito, E.S., de Araújo, M.C.P., Alves, R.E., Carkeet, C., Clevidence, B.A., Novotny, J.A., 2007. Anthocyanins presented in selected fruits: acerola, jambolão, jussara and guajiru. Journal of Agricultural and Food Chemistry 55, 9389–9394. Ghosh, K., Chakraborty, D., Chatterjee, G.K., Chaudhuri, A.K.N., Pal, M., 1985. Studies on anti-inflammatory and antipyretic activities of S. cumini Linn, seeds. International Research Communications System: Medical Science Biochemistry 13, 340–341. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130–7139. Harborne, J.B., 1973. Phytochemical Methods: A Guide to Modern Technique of Plant Analysis. Chapman and Hall, London. Jagetia, G.C., Baliga, M.S., 2002. S. cumini (Jamun) reduces the radiation-induced DNA damage in the cultured human peripheral blood lymphocytes: a preliminary study. Toxicology Letters 7, 19–25.
333
Kemper, R.A., Myers, S.R., Hurst, H.E., 1995. Detoxification of vinyl carbamate epoxide by glutathione: evidence for participation of glutathione S-transferases in metabolism of ethyl carbamate. Toxicology Applied Pharmacology 135, 110–118. Kilian, D.J., Moreland, F.M., Benge, M.C., Legator, M.S., Whorton, E.B., 1977. A collaborative study to measure interlaboratory variation within the in vivo bone marrow metaphase procedure. In: Kibey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.), Handbook of Mutagenicity Test Procedures. Elsevier/North-Holland, Amesterdam, pp. 243–260. Koleva, I.I., Van Beek, T., Linssen, J.P.H., de Groot, A., Evstatieva, L.N., 2002. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis 13, 8–17. Lee, J.C., Kim, H.R., Kim, J., Jang, Y.S., 2002. Antioxidant property of an ethanol extract of the stem of Opuntia ficus-indica var. Saboten. Journal of Agricultural Food Chemistry 50, 6490–6496. Liu, R.H., 2004. Potential synergy of phytochemicals in cancer prevention. Mechanism of action. Journal of Nutrition 134, 3479–3485. McDonald, S., Prenzler, P.D., Autolovich, M., Robards, K., 2001. Phenolic content and antioxidant activity of olive extracts. Food Chemistry. 73, 73–84. Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry 47, 389–394. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 582, 67–78. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Oyaizu, M., 1986. Studies on products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition. Japanese Journal of Nutrition. 44, 307–315. Prince, S.M., Kamalakkannan, N., Menon, V.P., 2003. S. cumini seed extracts reduce tissue damage in diabetic rat brain. Journal of Ethnopharmacology 84, 205– 209. Reynertson, K.A., Yang, H., Jiang, B., Baile, M.J., Kennelly, E.J., 2008. Quantitative analysis of antiradical phenolic constituents from fourteen edible Myrtaceae fruits. Food Chemistry 109, 883–890. Schmid, W., 1975. The micronucleus test. Mutation Research 31, 9–15. Sinha, K., 1971. Colorimetric assay of catalase. Analytical Biochemistry 47, 389–394. Sofowora, L.A., 1993. Medicinal plants and traditional medicine in Africa. Spectrum Books Ltd. Ibaban., 55–71. Trease, G. E., Evans, W. C., 1989. Trease and Evans Pharmacognosy: A Physician’s Guide to Herbal Medicine. 13, Bailliere Tindall, London. Veigas, J.M., Narayan, M.S., Laxman, P.M., Neelwarne, B., 2007. Chemical nature, stability and bioefficacies of anthocyanins from fruit peel of S. cumini Skeels. Food Chemistry 105, 619–627.