cytotoxic and genotoxic potential of walidda antidysenterica on human lymphocytes – a herb use in...
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Cytotoxic and genotoxic potential of Walidda
antidysenterica on human lymphocytes – A herb use in
Sri Lankan traditional medicine
Rashini Yasara Baragama-arachchi1
Dr Jagath Weerasena1
Dr Shiroma Handunnetti 1
Dr Radhika Samarasekara2
1. Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 2. Industrial Technology Institute, Sri Lanka
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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)
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Plant part
Remedy for
Bark Juice extracted is administrated to treat mouth sores, dysentry, dropsy (Edema ), Tonsillitis, Bronchitis
Seeds Fevers, diarrhoea and dysentery, intestinal worms, Antibilious, promote conception, making the muscles of vaginal tissue stronger and firmer after delivery
Flowers Snake bites, Russell’s viper bites
Leaves Skin disorders, Psoriasis, Nonspecific dermatitis , antitubercular
Roots Hemorrhage
Walidda antidysenterica
(Chopra et al; 1986, Frondozo et al; 2009 Shah et al; 2010)
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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
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Objectives of the study
To investigate the in vitro cytotoxic and genotoxic potential of ethanol leaf, stem
bark and flower extracts of W. antidysenterica
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Methodology
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Leaves
Kept at RT for three nights
Stem bark Flowers
Dust free leaves, flowers & stem bark were dried under the shade for 2 weeks
Plant extract preparation
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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 N2 gas
Transferred to pre-weighed glass bottles
Stem Bark Leaf Flower
Plant extract preparation
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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
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Overnight incubation in a humidified CO2 incubator
Wells seeded with cells (2x105)
Treated with different concentrations of plant extracts
Cell culture and in vitro treatment
10,20,30,40,50,100,200,400,800 µg/ml
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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
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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
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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
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Results & Discussion
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Cytotoxic potential of W.
antidysenterica
0 10 20 30 40 50 100 200 400 8000
20
40
60
80
100
120
WLE
WSE
WFE
Concentration (µg/ml)
Per
cen
tage
via
bil
ity
(%)
WLE- W. antidysenterica leaves extractWSE- W. antidysenterica stem bark extractWFE- W. antidysenterica flower extract
viability of lymphocytes (n=100) with treated concentrations of plant extracts. Results of 3 independent experiments
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Comets of control cells
Positive Control (C+) 200 µM H2O2
Negative Control (C-) Vehicle (DMSO)+ Culture media
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Effect of different ELE concentrations on comet formation
ELE
50 µg/ml
30 µg/ml 20 µg/ml 10 µg/ml
40 µg/ml
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Effect of different ESE concentrations on comet formation
ESE
30 µg/ml
50 µg/ml
20 µg/ml
40 µg/ml
10 µg/ml
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Effect of different EFE concentrations on comet formation
EFE
200 µg/ml 100 µg/ml 50 µg/ml
800 µg/ml 400 µg/ml
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C (-) C (+) 10 20 30 40 500
20
40
60
80
100
120
TM
Concentration (µg/ml)
Arb
rita
ry U
nit
s
Genotoxic potential of ELE, ESE and EFE
As detected by TM
C (-) C (+) 10 20 30 40 500
20
40
60
80
100
120
TM
Concentration (µg/ml)
Arb
rita
ry U
nit
s
C (-) C (+) 50 100 200 400 8000
20
40
60
80
100
120
TM
Concentration (µg/ml)
Arb
rita
ry U
nit
TM : r = 0.921 ; p = 0.026
TM : r = 0.793 ; p = 0.110
TM : r = 0.952 ; p = 0.013
* Dose dependency
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C (-) C (+) 10 20 30 40 500
10
20
30
40
50
Concentration (µg/ml)
Tail
DN
A %
C (-) C (+) 10 20 30 40 500
10
20
30
40
50
% Tail DNA
Concentration (µg/ml)
Tail
DN
A (
%)
C (-) C (+) 50 100 200 400 8000
10
20
30
40
50% Tail DNA
Concentration (µg/ml)
Tail
DN
A (
%)
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
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ELEESE
EFE
0
5
10
15
20
25
30
10
20
30
40
50
100
200
400
800
10
20
30
40
50
100
200
400
800
Tai
l DN
A p
erce
nta
ge (
%)
Concentration (µg/ml)
Summary of genotoxicity with respect to the % Tail DNA
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Conclusions
• 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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References
1. 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
2. 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
3. Azqueta A, Gutzkow KB, Brunborg G and Collins AR. Towards a more reliable comet assay: Optimising agarose concentration, unwinding time and electrophoresis conditions (2011) Mutation Research: 724; 41-45
4. Chopra RN, Nayar SL, Chopra IC, Asolkar LV, Kakkar KK. Glossary of Indian medicinal plants ; [with] Supplement (1986)Council of Scientific & Industrial Research, Edition3
5. Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. (2004b) Molecular Biotechnology: 26(3); 249-61
6. Collins AR, Oscozi AA,Brunborg G, Gaiva I, Giovannelli L, Kruszewski M et al. REVIEW:The comet assay: topical issues. (2008) Mutagenesis: 23 (3 ) ;143–151
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7. Frondozo SP,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 (wrightia antidysenterica r. br.); isolation of metabolities from the chloroform leaf extract. UST College of Science Journal; University of Santo Tomas
8. Ganapathy PSS, Ramachandra YL, Sudeep HV, Bellamakondi PK, Achar KGS and Rai SP. Pharmacognostic and phytochemical evaluation of Holarrhena antidysenterica Wall. (2009) The Asian and Australasian Journal of Plant Science and Biotechnology: 3(1); 47-50
9. 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
10. 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
11. 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
12. World Health Organization. Pyrrolizidine alkaloids,health and safety guide.IPCS International Programme on chemical safety (Health and safety guide N0.26)1989
13. Wickramaratne MN, Gunatilake LP, Anuradha NGD, Godavillathanna AN, Perera MGN, and Nicholas I. Antioxidant Activity and Antibacterial Activity of Walidda antidysenterica. 2015; Journal of Pharmacognosy and Phytochemistry :24(2);121-126
References
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Acknowledgement
National Science Foundation, Sri Lanka for financial support
• ,
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Thank you