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Rashini Yasara Baragama-arachchi 1 Dr Jagath Weerasena 1 Dr Shiroma Handunnetti 1

Cytotoxic and genotoxic potential of Walidda antidysenterica on human lymphocytes – A herb use in Sri Lankan traditional medicine. Rashini Yasara Baragama-arachchi 1 Dr Jagath Weerasena 1 Dr Shiroma Handunnetti 1 Dr Radhika Samarasekara 2.

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Rashini Yasara Baragama-arachchi 1 Dr Jagath Weerasena 1 Dr Shiroma Handunnetti 1

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  1. Cytotoxic and genotoxic potential of Waliddaantidysentericaon human lymphocytes – A herb use in Sri Lankan traditional medicine RashiniYasara Baragama-arachchi1 DrJagathWeerasena1 • DrShiromaHandunnetti1 • DrRadhikaSamarasekara2 1. Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 2. Industrial Technology Institute, Sri Lanka

  2. Waliddaantidysenterica • Family : Apocynaceae • Widely used in traditional medicine to • treat a broad spectrum of diseases • Bark - has anti-microbial, antidiarrheal, • antidontalgicand anti-inflammatory properties • The seeds are astringent, antidiarrheal and febrifuge • Leaves possess antioxidant and antibacterial properties (Wickramaratne et al; 2015)

  3. Waliddaantidysenterica (Chopra et al; 1986, Frondozo et al; 2009 Shah et al; 2010)

  4. Waliddaantidysenterica • Phytochemicals include alkaloids, flavonoids, sterols and quinine • More than 30 alkaloids have been isolated from W. antidysentericaand most were isolated from the stem bark (Ganapathy et al, 2009) • It has been reported that W. antidysentericacontain a potent genotoxic compound (pyrrolyzidine alkaloids) • (Arseculeratneet al, 1981) Pyrrolizidine alkaloid (Shah et al, 2010)

  5. Objectives of the study To investigate the in vitro cytotoxic and genotoxic potential of ethanol leaf, stem bark and flower extracts of W. antidysenterica

  6. Methodology

  7. Plant extract preparation Dust free leaves, flowers & stem bark were dried under the shade for 2 weeks Leaves Stem bark Flowers Kept at RT for three nights

  8. Plant extract preparation Rotary evaporated under the vacuum at 40 °C Vacuum filtered (Celite filter) Stirred at 30 rpm for an hour at RT • Further dried • By exposing to air for • overnight at RT • Passing N2 gas Stem Bark Leaf Flower Transferred to pre-weighed glass bottles

  9. Lymphocytes purification Dilute + + Blood layered over Histopaque Whole blood (1666 µl) PBS (1666 µl) Diluted blood Histopaque (1ml) 800 x g for 20 min at 4 °C without breaks

  10. Cell culture and in vitro treatment Overnight incubation in a humidified CO2 incubator Wells seeded with cells (2x105) Treated with different concentrations of plant extracts 10,20,30,40,50,100,200, 400,800 µg/ml

  11. Cytotoxicity assessment • TrypanBlue dye exclusion assay was carried out to check the viability after treatment • Concentrations, which retained >70% viability was selected for Comet assay

  12. Genotoxicity assessment by Comet assay Base slide preparation Embedding cells in LMPA Preparation of microgel slides Cell lysis Alkaline unwinding and electrophoresis Visualization and comet scoring Statistical analysis

  13. Comet scoring and statistical analysis • Assay was performed in triplicates for each concentration • 100 cells per each concentration were scored • “Casp 1.2.3b.1” image analysis software was used to assess the quantitative and qualitative extent of DNA damage in the cell • Results were analyzed using SPSS statistical software (version17.0) • The results were considered to be significantly different at P < 0.05

  14. Results & Discussion

  15. Cytotoxic potential of W. antidysenterica WLE- W. antidysentericaleaves extract WSE- W. antidysentericastem bark extract WFE- W. antidysentericaflower extract viability of lymphocytes (n=100) with treated concentrations of plant extracts. Results of 3 independent experiments

  16. Comets of control cells Positive Control (C+) 200 µM H2O2 Negative Control (C-) Vehicle (DMSO)+ Culture media

  17. Effect of different ELE concentrations on comet formation ELE 40 µg/ml 50 µg/ml 20 µg/ml 30 µg/ml 10 µg/ml

  18. Effect of different ESE concentrations on comet formation ESE 50 µg/ml 40 µg/ml 30 µg/ml 20 µg/ml 10 µg/ml

  19. Effect of different EFE concentrations on comet formation EFE 400 µg/ml 800 µg/ml 50 µg/ml 200 µg/ml 100 µg/ml

  20. Genotoxicpotential of ELE, ESE and EFE As detected by TM Dose dependency * TM : r = 0.921 ; p = 0.026 TM : r = 0.793 ; p = 0.110 TM : r = 0.952 ; p = 0.013

  21. Genotoxicpotential of ELE, ESE and EFE As detected by Tail DNA percentage (%) r = 0.928 ; p = 0.023 r = 0.899 ; p = 0.038 r = 0.995 ; p = 0.000 p < 0.05

  22. Summary of genotoxicitywith respect to the % Tail DNA

  23. Conclusions • Ethanol flower extract of Waliddaantidysentericawas neither cytotoxic norgenotoxicity compared to other plant parts • ESB showed moderate cytotoxicity while ELE showed the highest cytotoxic effect • High concentrations of leaves showed significant, dose-dependent genotoxicity where as stem barks showed moderate genotoxicity. • Presence of Pyrrolizidine alkaloids (e.g -: conessine, conessimine, iso-conessimine etc.) may account for the genotoxicity of leaves • Use of leaves to treat skin diseases can be justified with our study • However long term use is not recommended

  24. References • Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F et al. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. (2000) Mutation Research :463 ;111–172 • Arseculeratne SN, Gunathilaka AA and Panabokke RG. Studies on medicinal plants of Sri Lanka: Occurrence of Pyrrolizidine alkaloids and hepatotoxic properties in some traditional medicinal herbs. (1981) Journal of Ethnopharmacology: 4(2); 159-177 • Azqueta A, Gutzkow KB, Brunborg G and Collins AR. Towards a more reliable comet assay: Optimisingagarose concentration, unwinding time and electrophoresis conditions (2011) Mutation Research: 724; 41-45 • Chopra RN, Nayar SL, Chopra IC, Asolkar LV, Kakkar KK. Glossary of Indian medicinal plants ; [with] Supplement (1986)Council of Scientific & Industrial Research, Edition3 • Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. (2004b) Molecular Biotechnology: 26(3); 249-61 • Collins AR, OscoziAA,Brunborg G, Gaiva I, Giovannelli L, Kruszewski M et al. REVIEW:The comet assay: topical issues. (2008) Mutagenesis: 23 (3 ) ;143–151

  25. References • FrondozoSP,Villaflores OB, Paragas EM, Franzblau SG, Wang YH, Aguinaldo AM. (1970-2009) Phytochemical and antitubercular screening on the extracts of the aerial parts of white angel (wrightiaantidysenterica r. br.); isolation of metabolities from the chloroform leaf extract. UST College of Science Journal; University of Santo Tomas • Ganapathy PSS, Ramachandra YL, Sudeep HV, Bellamakondi PK, Achar KGS and Rai SP. Pharmacognostic and phytochemical evaluation of HolarrhenaantidysentericaWall. (2009) The Asian and Australasian Journal of Plant Science and Biotechnology: 3(1); 47-50 • Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P et al. Recommendations for conducting the in vivo alkaline Comet assay. (2003) Mutagenesis:18(1); 45–51 • Morley N, Rapp A, Dittmar H, Salter L, Gould D, Greulich KO et al. UVA-induced apoptosis studied by the new apo/necro-Comet-assay which distinguishes viable, apoptotic and necrotic cells. (2006) Mutagenesis: 21( 2 ); 105–114 • Nandhakumar S, Parasuraman S, Shanmugam M, Rao KR, Chand P and BhatBV. Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay). (2011) Journal of Pharmacology and Pharmacotherapeutics: 2(2); 107–111 • World Health Organization. Pyrrolizidinealkaloids,health and safety guide.IPCS International Programme on chemical safety (Health and safety guide N0.26)1989 • Wickramaratne MN, Gunatilake LP, Anuradha NGD, Godavillathanna AN, Perera MGN, and Nicholas I. Antioxidant Activity and Antibacterial Activity of Waliddaantidysenterica. 2015; Journal of Pharmacognosy and Phytochemistry:24(2);121-126

  26. Acknowledgement National Science Foundation, Sri Lanka for financial support • ,

  27. Thank you

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