CY TOTOXIt:lTya'r TO TUMOUR CELLS AND CEL.LS lPI1 CULrrTJIltE (IN VITRO)

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3.1. INTRODUCTION

Traditional and folk medicine have long used plant products for

therapeutic purposes. But it is only since 1959 that a concerted

systematic effort has been made to screen crude plant extracts for

their inhibitory activity against animal tumour systems. In the

Indian system of medicine many of the plant products are used for the

treatment of various diseases including cancer (285).

In this study we have investigated the cytotoxic effect of

Saraca asoca bark, flower, and leaves extract to various tumour cells

in vitro and also the growth inhibitory effect of these extracts to - - cultured cell S.

3.2. Materials and Methods:

DLA, EAC, S-180 and P-388 tumour cells were obtained from

tumour bearing mice as described in 2.1.4. Radioactive isotopes,

trypan blue and culture medium was obtained from sources mentioned in

Chapter-2.

3.2.1. Differential extractions:

Preliminary experiment was carried out by extracting Saraca

asoca bark, flower and leaves, powder with petroleum - ether,

choroform, 90% acetone, Ethylacetate, Ethanol (95x1, methanol and

water were used. The extract was evaporated under reduced pressure

and active pfinciple was isolated as stated in 2.2.2. This was used

for cytotoxicity studies.

3.2.2. Determination of cytotoxicity to tumour cells:

The cytotoxic effect of Saraca asoca bark,flower and leaves

extract was evaluated using murine tumour cells like DLA, EAC, S-180

and P-388 (2.2.5). Brief1 y 1 X 106 cells were incubated with

various drug concentrations at 37'~ for 3 hour. Cell viability was

ascertained by the trypan blue exclusion method.

3.2.3. Determination of cytotoxicity to isolated Iymphocytes:

Lymphocytes were is01 ated from the peripheral blood of heal thy

normal persons and patients with leukaemia (acute lymphocytic

leukaemia and chronic myeloid leukaemia) by the Ficol Hypaque Method.

(273) Chronic myelogenous leukemia (CML) and Acute Lymphoblastic

Leukaemia (ALL) cells were obtained from a 52 and a 9 year old

patient admitted in our oncology department. The blood was obtained

from these patients before the commencement of any kind of treatment.

The is01 ated l ymphocyte/ml PBS a1 ong with various extract

concentrations was incubated at 37'~ for 3 hour and the viability of

lymphocytes was determined by trypan blue exclusion method.

3.2.4. Cytotoxicity of extracts in in vitro cultures:

5x10~ K-562 cell S were added to each of the culture bottles

containing 10 m1 RPMI medium, different concentrations of extracts

were then added. Controls were maintained without the drug. All the

culture bottles were incubated at 37'~ in 5% CO2 and the inhibition

in the growth of cells was assessed as detailed in (2.2.4.1)

4 5x10 CH0,KB or Vero cells were added to tissue culture bottles

containing 10 m1 Eagle's minimum essential medium and incubated for

24 hour at 37'~. Drugs at different concentration was added after 24

hours and the bottles incubated for seven days (37'~) After

incubation, the cells were harvested and the growth inhibitory effets

ascertained (2.2.4.1)

3.2.5. Determination of intracellul ar enzyme l eve1 of S-180

tumour cel l S in cul ture :

lxlo7 Sarcoma-180 tumour cell S were cultured in 10 m1 RPMI

medium containing 10% calf serum at 3 7 O ~ in CO2 for 48 hours in the

presence of various concentrations of Saraca asoca bark and flower

extract and assay was carried out as described in 2.2.13.4

Glutathione reductase (GSH-R) was determined from the tumour

cells and glutathione S-transferase (GSH-S) levels using 2,4 -

dinitro - chl or benzene as substrate. Cell suspensions (2x107/ml )

were sonicated on ice (90s) using a sonicator and the resultant

sonicated rn'ixture was spun at 1500 rev/min for 6 min. The protein

content in the supernatent was also determined (283).

3.2.6. Inhibition of the synthesis of macromolecules:

The effect of Saraca asoca bark, flowers and leaves extracts on

DNA synthesis was studied using radio labelled precursors by the

method developed by Pavlik et a1 (1903). Briefly 3(H) thymidine was

added (2p ci/ml) to the tubes containing one million tumour cells/ml

and incubated with various drug concentrations in a total of 2 m1

MEM(+10% FCS) for four hour at 37'~. The precipitated DNA was

dissolved and radio activity measured.

Tritiated uridine and leucine were used to study the effect of

Saraca asoca bark, flowers and leaves extracts on the synthesis of

RNA and protein respectively. Details of the assay procedure are

given in 2.2.7 and 2.2.8.

3 . 3 RESULTS

The extraction of Saraca W, bark,flowers and leaves with

solvents of different polarity and their cytotoxic study indicated

maximum extraction at the active principle with 95% methanol in case

of flowers, Ethyl acetate in Bark and 90% acetone as in leaves

(Tab1 e-20) ,-

3 . 3 . 1 . Cytotoxicity of Saraca asoca active principle.

In vitro studies indicated that the active principle of ethyl - - acetate extract of Saraca asoca bark, methanolic extract of flower

which furthur purified as stated in 2.2.2 and was highly active

against .bDCA, S-180, and P-388 tumour cells. The concentrations

of the "Ashoka" bark needed to produce 50% cytotoxicity were 16

yg/ml, 23 pg/ml, 47 pg/ml and 55 pg/ml respectively for DLA, 5-180,

EAC, P-388 tumour cells Table(21). In the case of flower extract the

corresponding figures were 24pg,37pg/ml and 52pg as in the case of

DLA, S-180, and P-388,Table(22). Extracts of leaves required higher

concentrations 65 and 73yg/ml to produce 50% cytotoxic effect

Table(23). While the flower and leaves extract were insensitive to

Ehrl ich ascites carcinoma cel l S.

3.3.2. Effect of extracts on lyrnphocytes:

It was found that normal lymphocytes were insensitive to drug

while lymphocytes from leukaemic patients were acted upon hy bark

and flower extract. 50% cytotoxicity was obtained at a concentration

of 11 and 22pg to ALL and CML in the case of flower extract (Fig 4)

and it was 9 pg and 16 pg in the case of bark extract (Fig 5)

Table 21: in v i t ro cytotoxicity of the act ive pr inciple of Saraca asoca ba rk -- extracts on tumour cell l i nes

Percentage of Live cells

=onckntration of ................................................................................. extract ( ~ g ) DLA S-180 EAC cells P-388 cells

.............................................................................................................. 80 ND ND 10.25+1.37 - 13.6 - +1.79

Values a re average of mean - + SD from 4 separate studies

N.D. - Not determined

N . A . - No activity

DLA - Dalton's 1yrnphQha a s d t e s ..

5-180 - Sarcoma - 180

DAC - Dalton's lymphoma ascites Carcinoma

Amount of extract needed for 50% Cytotoxicity was determined from graph by plotting concentration

against percent of l ive cells.

Table 22: -- i n vitro cytotoxidty of the active principle of 'Ashoka' (Saraca asoca) flower

extract on tumour cell l ines

Percentage of l ive cells Concentratiori of .......................................................................................

extracts ( ~ g ) DLA S-180 E . A C P 388

Values are average of mean + S.D. from 4 separate determination5

N D - Not determined

DLA - Dalton's lymphoma ascites

EAC - Ehrlich a s d t e s carcinoma

Amount of extract needed for 50% cytotoxicity was determined from graph by plotting

concentration against percent of live cells.

Table 23: i n v i t ro cytotoxicity of t h e active principle of Saraca asoca -- leaves extracts on tumour cell l ines

Concentrdtion of Percentage of l ive cells

extract ......................................................................................... (p) DLA S-80 EAC P 388

.............................................................................................................. 9 0 12.42+1.32 - 16.81+1.13 - NA N A

Values a r e average of mean - + S.D. from 4 separa te studies

N . A . - No activity

D L A - Dalton's lym??iama : ascites

EAC - Ehrlich a s d t e s carcinoma

S-180 - Sarcoma 180

Amount of extract needed for 50% cytotoxicity w a s determined from graph by plotting concentration

against percent of live cel ls .

Concentrat ion o f drug (pg/ml I

Fig. 4 : I n vi t ro Cytotoxic effects of Saraca asoca flower extract to -- P

normal lymphocytes and lymphocytes from leukaemic patients

( ) Chronic myelogenous leukemia

( Acute lymphoblastic leukemia

n 1 Normal lymphocytes.

Concentration o f drug ( p g / m l )

Fig. 5: I n vi t ro Cytotoxic effects of Saraca asoca ba rk extract to

normal lymphocytes and lymphocytes from leukaemic patient*

) Chronic myelogenous leukemia.

-m Acute lymphoblastic leukernia.

( 0 ) Normal lymphocytes.

3 . 3 . 3 . Inhibitory effect of active principle of Saraca asoca

on the growth of K-562 c e l l s in culture.

The active principle of Saraca asoca bark and flower inhibited

the growth of K-562 cells in culture. The percentage of cell survival

0.5 ~ ~ / m l and 1.5 pg/ml active principle of bark after 24 hour of

treatment was found to be 16% and 11%. The corresponding figures

were 36% and 22% after 72 hour. While in presence of flower extract

the percentage of cell survival was 31% and 17% (day-l) and 52% and

39% (day-3) at a concentration of 3 and 41~glml respectievely (Table-

24)

3 . 3 . 4 . Effect of Saraca aeoca (Aehoka) on the growth of K B , CH0

and vero cell S in culture:

The growth of KB, CH0 and Vern cells in culture was inhibited

by active principle of Saraca asoca bark and flower extracts. The

concentration required to produce 50% growth inhibition in the case

of Ashoka bark, flower and leaves were found to be 0.6 yg/ml, 1.3

pg/ml and 2.4 )lg/mI respectively when cultured with KB cell S (Fig.6).

Vero cells were found to be more sensitive to bark and flower

extracts in culture (fig.7,8). The 50% growth inhibition to vero

cells waso.16pg and 0.34 pg of Ashoka bark and flower respectively.

While vero cells were insensitive to leaves extracts.

0.5 1.0 1.5 2 .0 2.5

Concentration o f drug ( ~ g l r n l 1

Fig. 6: Effect of Saraca asoca active principle on KB cells i n

culture.

Different concentrations of Saraca asoca bark or flower or -- - leaf extract were added to t issue culture bottles, 24 h after

cell inoculation. After incubation for 6 more d-lys cells

were detached and viable cells counted. Cell . surv iva l

was 100% i n the control (without drug)

(U) Saraca 3 bark

(W) Saraca asoca flower. -- (0-0) Saraca leaf .

Concentration of drug ( p g/ml )

Fig. 7 : Effect of Saraca asoca active principle on Vero cells i n -- - culture. Different concentrations of Saraca asoca bark or

flower or leaf extract were added to t issue culture bottles

24 h a f te r cell inoculation. After incubation for 6 more

days cells were detached and viable cells counted. Cell

surv iva l was 100% i n control bottles (without d r u g ) .

(M) Saraca asoca bark .

(U) Saraca asoca flower.

(0-0) Saraca asoca leaf.

Fig. 8: Effect of Saraca active principle on CH0 cells i n cu l ture .

Different centrations of Saraca asoca b a r k or flower or leaf extract were added to t issue culture bottles 24 h a f te r cell inoculation. After incubation for 6 more days cells were detached and viable cells counted. Cell surv iva l was 100% i n control (without drug)

(U) Saraca asoca Dark.

( 0 - 4 -- Saraca -. asoca flclwer.

(M) Saraca .- asocc l ea f .

3.3.5 Effect of Ashoka extracts on the intracellular glutathione

l eve1 s of S-180 tumour cell s . 3.

The studies on the effect of the purified compound from the

bark and flower extract of ashoka on the intracellular glutathione

levels of S-180 tumour cells indicated a concentration dependent

increase in the levels of intracellular reduced glutathione (GSH)

glutathione reductase (GSH-R) and glutathione S-transferase (GSH-S).

The elevation (P < 0.001) in GSH was almost 4-fold and 3-fold in bark

and flower respectively whereas GSH-R and GSH-S were increased by

near1 y 2-fold in the presence of 1.0 ,ug/ml of ashoka bark and 1.5

fold in the presence of 1.0 pg/ml of flower as compared to control s

(Table-25).

3.3.6. Effect of active principle of Saraca asoca on the

synthesis of macro m01 ecul es: -

The effect of Saraca asoca bark, flower and leaves active --

principle on DNA synthesis was studied by incorporating tritiated

thymidine into Dalton's lymphoma ascites cells. The results showed a

concentration dependent decrease in thymidine incorporation into

cellular DNA, as 50% inhibition in incorporation was achieved at a

concentration of 4.7~glrn1, 6.5 pg/ml and 17 pg/ml as in the case of

bark, flower and leaves extracts respectively (Table 26,27,28).

Table 25: Effect of Saraca asoca bark and flower extract on the in t race l lu la r glutathione a n d glutathione related enzyme

activity i n Sarcoma-180 tumour cells ( in vitro) ..............................................................................................................

Saraca Saraca Saraca -- Saraca asoca asoca asoca asoca - -

Control Bark Bark Flower Flower 0.25 1.0 0.25 1.0 pg/ml pg/ml p s / m l pg/ml ..............................................................................................................

Reduced glutathione l .82+0.61 - (GSHI -

- 6 (nmol/lO cells)

Glutathione 18.58+1.71 - 23.41+0.99 - 38.22+4.0*** - 20.36+1.76 - 32.26+4.65 reductase (GSH-R) (nmol NADPH oxidised/min/mg protein)

Glutathione S-transferase (GSH-S) (nmol CDNB conjugated/min/ mg protein) ..............................................................................................................

Values represented a r e mean + SD from three separate experiments i n duplicate significantly - different from control.

Table 26 : Effect of Saraca asoca ba rk extracts on the incorporation of t r i t ia ted thymidine

into the cellulose DNA of Dalton's lymphoma ascites cells

Concentration of the drug CPM + mean S.D. Percent incorporation - Percent of inhibition

pg/ml - + mean S.D. + mean S.D. -

50% I . C . (inhibition in incorporation) was obtained a t a concentration of 4.7 pg/ml 10 m1

cell suspension ( 2 x 1 0 ~ cells/rnl MEM) was incubated with and without (control) the drug and

0.5 micro curi m 1 3 ( ~ . ~ d r ) at 37Oc for 4 hours. Values a r e CPM + mean S.D. from 4 observations. I -

Table: 27.

Effect of Saraca asoca flowers on the DNA bio-synthesis of DLA tumour cells (in m -- ..................................................................... Concentration CPM - + Mean S.D. Percent Percent of the drug incorporation inhibition (pg/ml) Mean+ - Mean - + S.D. .....................................................................

50% IC (Inhibition in incorporation was obtained at a concentration

of 6 . 5 ) ~ g / m l . Values are CPM+Mean - S.D. from 4 observations.

Table: 28.

Effect of Saraca asoca leaves extracts on the incorporation of tritiated t-nemo the cellulose DNA of Dal ton's l ymphoma ascites cell S. ..................................................................... Concentration CPM - + Mean S.D. Percent Percent of of the drug incorpration inhibition (pg/ml) - + mean S.D. - + means S.D. .....................................................................

50% I.C. (Inhibition in incorporation) was obtained at a

concent ration of 17 pglml . 10 m1 cell suspension ( 2 x 1 0 ~ cell s/ml MEM) was incubated with anc

without (control ) the drug and 0.5 micro c u r i / i l 3 (H.Tdr) at 37O(

for 4 hrs.

Values are CPM - + mean S.D. from 4 observations

Saraca asoca a c t i v e pr inci -ples of ba rk , f lower and l eaves

f a i l e d t o i n h i b i t t h e i n c o r p o r a t i o n a t Ur id ine and l e u c i n i n t o

c e l l u l a r RNA "and p r o t e i n r e spec t ive1 y ( F i g . 9 , 1 0 )

3 . 4 . DISCUSSION:

Saraca asoca i s widely used i n Ayurvedic Medicines ( 1 8 5 ) . But

very l i t t l e has been r epo r t ed on i t s a n t i c a n c e r a c t i v i t y . I t i s

c l e a r l y e s t a b l i s h e d from t h e above s t u d i e s t h a t t h e p l a n t Saraca

asoca con ta in c y t o t o x i c p r i n c i p l e s . The d i f f e r e n t i a l e x t r a c t i o n

procedure adopted i n d i c a t e d maximum e x t r a c t i o n of t h e a c t i v e

component from bark wi th e thy l a c e t a t e , from f lowers 95% methanol and

from l eaves 90% ace tone . I t was no t i ced t h a t a comparatively

increased a c t i v i t y i n t h e a c t i v e p r i n c i p l e of bark than o t h e r p a r t of

t h e p l a n t .

I n a d d i t i o n t o DLA, a c t i v i t y was t e s t e d wi th P-388, Eh r l i ch

a s c i t e s carcinoma and S-180 grown i n t h e p e r i t o n i a l c a v i t y of mice.

Grea t e r a c t i v i t y was observed i n DLA system probably due t o t h e

s p e c i f i c n a t u r e of t h e drug. E h r l i c h a s c i t e s tumour c e l l s were l e s s

s e n s i t i v e t o e i t h e r of t h e e x t r a c t s . Another p o i n t of i n t e r e s t was

t h a t t h e e x t r a c t s were i n s e n s i t i v e t o normal lymphocytes while

lymphocytes from leukaemia p a t i e n t s were ac t ed upon by t h e bs rk and

f lower e x t r a c t . Acute lymphoblas t ic leukaemia ( A L L ) c e l l s were more

s e n s i t i v e t o t h e acc ive p r i n c i p l e a s compared t o ch ron ic myelogenous

leukaemia c e l l s ( C M L ) . The p r e f e r e n t i a l a c t i o n no t having any e f f e c t

Fig. 9: Effect of active principle of Saraca on 3H-Uridine incorporation into cel lular macromolecules. 3FI-Uridine ( 2 U d / m l ) were incubated with different

loo

C .-O 80 .d

g P

6 0 - C .-

% A O - - C Y Y 2 2 0 -

.. concentrations of active principle for 4 h a t 37Oc. After precipitation, radioactivity was measured in a Liquid scinti l lat ion counter.

-4 ,. W = - 8 ,. - - -

I I I

0 5 10 l5 2 0 2 5

(W ) Saraca asoca bark

on cent rat ion of drug [pg/ rnl 1

( W 3 Saraca -- asoca flower.

(U) Saracs asoc'3 leaf. -- --

Concentration of drug ( pg/ml 1

Fig. 10: Effect of active principle of Saraca asoca on 3 -1eucine H incorporation into cel lular macromolecules. 3 -1eucine H ( 2 u ci'./ml) were incubated with different concentrations of

active principle for 4 h at 37Oc; a f t e r precipitation, radioactivity measured i n a l iquid scinti l lat ion counter.

( W ) Saraca W bark .

(M ) Saraca 5 flower.

( ) Saraca leaf

on normal lymphocytes clearly shows the added benefit of the drug in

case it is used as a mode of treatment in future.

The growth inhibitory effects and cytotoxicity of the extracts

was further evaluated using K-562, KB, CH0 and Vero cells. Active

principles of bark and flower was found to inhibit the growth of

above tumour cell S. CH0 and Vero cells were found to be the most

sensitive towards active principles. The enhancement of reduced

glutathione (GSH), glutathione reductase (GSH-R) and g1utathione-S-

transferase (GSH-S) levels in cultured tumour cells may be attributed

to the cytotoxic potentials of Saraca asoca bark and flower extracts.

Results of thymidine incorporation studies indicated that DNA

synthesis was inhibited by active principles of Saraca -- asoca

bark,flower and leaves extracts in a dose dependent manner. The

concentration required to produce 50% inhibition of thymidine

incorporation was lower than that required for 50% cytotoxicity.

Therefore it seems possible that the mechanism of action of the drug

is the inhibition of DNA synthesis. On the other hand, the active

principles failed to inhibit the incorporation of Uridine and leucine

into cellular RNA and protein respectively. Thus active principles

(bark, flower, leaves) may not have any effect on RNA or protein

biosynthesis.

The exact nature of the active components responsible for

cytotoxicity is not known now. Chemical analysis of Saraca asoca

bark active principle yielded (-)-Epicatechin, Proanthocyanidin B2

and a new Proanthocyanidin. The extract of flower showed the

presence of (-)-epicatechin, leucopelargonidin and dihydro

chalcone. Whether the cytotoxic action is due to these

compounds alone or in combination with' other compounds remains

to be investigated.