Alcoholic turmeric extract simultaneously

activating murine lymphocytes and inducing

apoptosis of Ehlrich ascitic carcinoma cells

International Immunopharmacology 5 (2005) 1574– 1581

Impact factor: 2.157
Ashim Kumar Chakravarty*, Hadida Yasmin
North Bengal University, Siliguri – 734430, West Bengal, India

Presented by Inder Makhija

Dept of pharmacognosy
M. Pharm I
 Abstract
 Ethanolic Turmeric Extract (ETE) has been found to play
diabolically opposite role on murine lymphocytes and on
Ehlrich Ascitic Carcinoma Cells (EACC).
 Turmeric stimulates the lymphocytes as studied through
 In vitro viability,
 Blastogenesis and
 3H-TdR incorporation.
 Turmeric induces apoptosis, studied by monitoring the cell
topography of EACC under Scanning Electron Microscopy
(SEM) after treating with ETE for a period of 16 hours
 SEM revealed the formation of cytoplasmic blebs and
plasma membrane disintegration of tumor cells with ETE,
suggesting turmeric to be initiating apoptosis of tumor cells
 Introduction
 The rhizomes of Curcuma longa Linn. (turmeric) is widely
used in different food preparations and external application
in India for its medicinal value [1] .
 Curcumin : Major pigment and phenol compound, Possess
 Antiinflammatory[2,3]
 Antioxidant properties [4–6]
 Inhibitory against chemical induced carcinoma in several
experimental models [7–9]
 Materials and methods
1. Animals : Inbred adult Swiss mice
2. Chemicals: (i) Purchased from SIGMA,
USA
 Ficoll (type 400)  PBS

 RPMI 1640 medium  Toluene

 HEPES buffer  POPOP

 Nystatin  PPO

(ii) Bhaba Atomic Research Centre 3

H-thymidine

(iii) Robin’s Scientific Corporation, USA Nylon wool

3. Tumor induction: Chittaranjan National Cancer Ehlrich ascitic carcinoma

Research Institute cell line
4. Ethanol turmeric extract (ETE) preparation
 10 g of fresh rhizomes crushed to a paste with mortar and
pestle
 Add 10 ml of absolute alcohol, kept in a refrigerator at 4˚C
 After 12 h filtered through Whatman filter paper 1
 Refiltered through Millipore filter paper
 Final solution obtained was stored at 4˚C for further use. One
ml of ETE contain 0.435 mg dry weight of turmeric.

5. Cell suspension
 Spleen and lymph nodes collected aseptically, cells were
dissociated in PBS, pH 7.2 separately, layered on Ficoll
Hypaque gradient , spun at 3000 rpm for 30 min. The
Lymphocytes were finally washed twice with PBS.
6. Separation of B and T cells
 Cells were separated by nylon wool fiber column technique
from the lymphocyte suspension
 Nylon wool adherent cells were eluted out with an excess
amount of cold RPMI by vigorous agitation of the wool and
then re-suspended in fresh medium. T and B cells were counted
with the help of haemocytometer.

7. Culture medium For in vitro culture, lymphocyte suspensions

were aliquot separately in round bottom glass culture tube at a
conc. of 1*106 cells in 2 ml of RPMI {supplemented with
glutamine, HEPES buffer, 200 mg NaHCO3/ 100 ml, 100 U of
penicillin/ml, 100 mg/ml streptomycin, 50 mg/ml nystatin and
10% heat inactivated goat serum}.
8. Cell viability assay
 Viability of splenocytes in each culture tube was counted at 8,
16, 24, 48 and 72 h with a 25 µl dose of ETE.
 % of viable cells were counted in a haemocytometer in the
presence of trypan blue, considering the number of viable cells
at the beginning as a hundred.

8. Measure of blastogenesis
 Blastogenic activity of turmeric was compared with that of
5 µg/ml ConA, a polyclonal activator.
 The % of blast cells was counted by a haemocytometer in the
presence of trypan blue. Cells with a diameter over 6 µm and
ranging more than 10 µm were considered as blast.
10. Measure of DNA synthesis
1. Proliferation of lymphocytes was determined by
measuring the rate of 3H-thymidine (3H-TdR)
incorporation in vitro.

2. 200 µl of cell suspension was aliquot in each well of a 96-

well micro-culture plate. In each of the experimental wells 25
µl ETE and in control, an equivalent amount of alcohol was
added.

3. After 24, 48 and 72 h of incubation at 37 ˚C in humidified

atmosphere containing 5% CO2 in air, the cells were labeled
with 1 µCi of 3H-thymidine (specific activity—15.8 Ci/mM) per
well.
4.Washed with cold PBS and precipitated with cold 10%
trichloroacetic acid (TCA). The TCA precipitate was then filtered
onto glass fiber filters; washed with methanol and dried.

5. The discs from filter paper containing individual samples were

placed in standard scintillation vials (Beckman, USA). At the time
of radioactivity count, 5 ml of scintillation fluid (6 g PPO, 0.5 g
POPOP/1 t of toluene) was added into each vial. Radioactivity
was counted in β-scintillation counter (LS 1800 BECKMAN,
USA).
 Results
1. In vitro viability of murine lymphocytes
2. In vitro blastogenic response of lymphoid cell
types in the presence of ETE

Fig. 2. Histogram showing blastogenic response of lymphocytes from spleen treated

with ETE, alcohol and ConA;
A) B lymphocytes, B) T lymphocytes.
Fig. 3. Histogram showing blastogenic response of lymphocytes from lymph node
treated with ETE, alcohol and ConA;
A) B lymphocytes, B) T lymphocytes.
3.
H-TdR incorporation in lymphocytes with
3

ETE treatment

Fig. 4. Pattern of incorporation of 3H-thymidine by lymphocytes

treated for different hours with ETE;
A) lymphocytes from spleen,
B) lymphocytes from lymph node.
4. Scanning electron microscopic study

Fig. 5. Scanning electron micrographs of murine lymphocytes from

spleen.
A) After 16 h of turmeric treatment, lymphocytes showing no
significant changes on its surface topography and seem to be healthy.
B) Control, lymphocytes at 16 h.
A) Tumor cells prior to turmeric treatment, B) After 10 min of turmeric treatment, cells
showing ruffles distributed evenly all over showing formation of cytoplasmic blebs
the surface

C) After 4 h of turmeric treatment, D) After 8 h of treatment, cytoplasmic blebs,

cytoplasmic blebs were broader and became numerous, cell volume tends
conspicuous to decrease

Fig. 6. Scanning electron micrographs of Ehlrich ascitic carcinoma cells

E) After 16 h of turmeric F) Control, after 16 h of culture
treatment, loss of cellular tumor cells remain unchanged
organization with disintegration
of plasma membrane

Fig. 6. Scanning electron micrographs of Ehlrich

ascitic carcinoma cells
 Discussion
1) The foregoing study was an effort to prove that ethanol
extract of turmeric stimulates the lymphocyte blastogenisis
and induces apoptosis in cancer cells

a) Viability: % of viable lymphocytes cultured with ETE was

much higher than the alcohol treated lymphocyte.

b) Blastogenic response: ETE provides blastogenic stimulus

for T cells but less extent from ConA.

c) DNA synthesis: 3H-TdR incorporation by the lymphocytes

treated with ETE also show the peak at 24 h.
2) Study shows pronounced cytoplasmic blebing and plasma
membrane disintegration leading to apoptosis of Ehlrich
ascitic carcinoma cells following adminstration of ETE.

PROPOSED MECHANISMS
 Due to prominent downregulation of Bcl-2 & upregulation of Bax
expressions ( Kim et al. ).
 Activation of caspase-3 and by generation of RO intermediates
( Khar et al. ).
 Inhibit the expression of several protooncogenes, such as c-jun, c-
fos and c-myc in mouse skin and transcription factors such as NF-
Kb ( Kakar et al.& Singh S et al. ).

HENCE MOLECULAR MECHANISM OF ACTION OF CURCUMIN

SHOWS TWO WAY EFFICACY ON TWO TYPES OF CELLS
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