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

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)

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)

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

Objectives of the study

 

To investigate the in vitro cytotoxic and genotoxic potential of ethanol leaf, stem

bark and flower extracts of W. antidysenterica

Methodology

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

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

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

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

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

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

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

Results & Discussion

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

Comets of control cells

Positive Control (C+) 200 µM H2O2

Negative Control (C-) Vehicle (DMSO)+ Culture media

Effect of different ELE concentrations on comet formation

ELE

50 µg/ml

30 µg/ml 20 µg/ml 10 µg/ml

40 µg/ml

Effect of different ESE concentrations on comet formation

ESE

30 µg/ml

50 µg/ml

20 µg/ml

40 µg/ml

10 µg/ml

Effect of different EFE concentrations on comet formation

EFE

200 µg/ml 100 µg/ml 50 µg/ml

800 µg/ml 400 µg/ml

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

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

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

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

References

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References

Acknowledgement

National Science Foundation, Sri Lanka for financial support

• ,

Thank you