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 1. Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 2. Industrial Technology Institute, Sri Lanka

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

3. Plant partRemedy for BarkJuice extracted is administrated to treat mouth sores, dysentry, dropsy (Edema ), Tonsillitis, Bronchitis SeedsFevers, diarrhoea and dysentery, intestinal worms, Antibilious, promote conception, making the muscles of vaginal tissue stronger and firmer after delivery FlowersSnake bites, Russell’s viper bites LeavesSkin disorders, Psoriasis, Nonspecific dermatitis, antitubercular RootsHemorrhage Walidda antidysenterica (Chopra et al; 1986, Frondozo et al; 2009 Shah et al; 2010)

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

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

8. Stirred at 30 rpm for an hour at RT Vacuum filtered (Celite filter) Rotary evaporated under the vacuum at 40 °C Further dried By exposing to air for overnight at RT Passing N 2 gas Transferred to pre-weighed glass bottles Stem BarkLeafFlower Plant extract preparation

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

10. Overnight incubation in a humidified CO 2 incubator Wells seeded with cells (2x10 5 ) Treated with different concentrations of plant extracts Cell culture and in vitro treatment 10,20,30,40,50,100,200, 400,800 µg/ml

11. Cytotoxicity assessment Trypan Blue 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. antidysenterica leaves extract WSE- W. antidysenterica stem bark extract WFE- W. antidysenterica flower 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 H 2 O 2 Negative Control (C-) Vehicle (DMSO)+ Culture media

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

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

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

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

21. Genotoxic potential of ELE, ESE and EFE Genotoxic potential 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 genotoxicity with respect to the % Tail DNA

23. ConclusionsConclusions Ethanol flower extract of Walidda antidysenterica was neither cytotoxic nor genotoxicity 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

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26. AcknowledgementAcknowledgement National Science Foundation, Sri Lanka for financial support,

27. Thank you