evaluvation anti tumor activity of epipremnum aurium against dal cell lines

1. DEVAKI AMMA MEMORIAL COLLEGE OF PHARMACY,
Chelembra.
ANTI TUMOR ACTIVITY OF ETHANOLIC EXTRACT OF
“Epipremnum aureum Linn .” LEAVES AGAINST DALTONS LYMPHOMA ASCITES
INDUCED TUMOR IN MICE
Dissertation submitted to Kerala University of Health Sciences, Thrissur
in partial fulfillment of the requirements for the award of the Degree of
MASTER OF PHARMACY
in
PHARMACOLOGY
by
MUHAMED.K
(Reg. No - 152800056)
Under the guidance of
Mr.S.VENKATESH, M. Pharm.,
Assistant Professor
1

2. CONTENTS
 INTRODUCTION
 AIM AND PLAN OF WORK
 REVIEW OF LITERATURE
 MATERIAL AND METHOD
 RESULT AND DISCUSSION
 SUMMARY AND CONCLUTION
 REFERENCES
2

3. INTRODUCTION
3

4. CANCER
4

5. • Cancer is one of the most common diseases in
the world:
• 1 in 4 deaths are due to cancer
• Lung cancer is the most common cancer in men
• Breast cancer is the most common cancer in
women
• There are over 100 different forms of cancer
• Neoplasm- abnormal growth of cells
• Benign neoplasms are not cancerous
Encapsulated; Do not invade neighboring
tissue or spread
• Malignant neoplasms are cancerousNot
encapsulated; Readily invade neighboring
tissues May also detach and lodge in distant
places – metastasis
5

6. 2. AIM AND PLAN OF WORK
AIM
Evaluation anti tumor activity of Ethanolic leaf extract of Epipremnum
aureum (Linn.) Leaves against Daltons ascites lymphoma in Swiss albino
mice.
PLAN OF WORK
• To collect and authenticate Epipremnum aureum plant leaf
• To prepare ethanolic extract with leaves
• To carry out preliminary phytochemical screening
• To carry out antioxidant studies
• To carry out in-vivo and in-vitro anticancer studies
• To validate the results statistically
6

7. PLANT PROFILE
Kingdom : plantae
Botanical Name : Epipremnum aureum Linn
Family : Araceae
Sub Family : Monsteroideae
Commonly also known as Money Plant
Synonyms
Pothos aureus
Raphidophora aurea
Scindapsus aureus
7

8. PHYTOCHEMICAL
CONTENT
 The phytochemical components of the leaves and
aerial roots were screened for the presence of
alkaloids, saponin, glycosides, tannins, flavonoid,
and anthraquinones .
 E. aureum is a rich source of anti-oxidative
compounds such as bi-phenol, polyphenols and
tannins
8

9.  Flavanoids have a capacity to elevate the anti
oxidant levels and prevent the free radical
reactions. And also reported that Flavanoids have
a anticancer activity a specially in breast cancer.
 So the present study Epipremnum aureum linn
have high amount of Flavanoids.
 Till now, there is no anti tumor study was reported
on Leaves of Epipremnum aureum linn, for that
reason this present study chooses Antitumor
Status of Epipremnum aureum linn leaves against
Dalton’s Lymphoma Ascites-Induced Ascitic Tumor
in Mice.
9

10. TRADITIONAL USES
 Anti-oxidant property
 Anti microbial property
 Anti termite property
 Anti hyperlipedimic
 Anti inflammatory
 Anti viral
 Anti bacterial
 Neuroprotective
10

11. 3. REVIEW OF LITERATURE
Epipremnum aureum (L.).
SL
NO
AUTHOR SCIENTIFIC CLAIM
1 Nidhi Srivasatava et
al., (2011)
Study: Anti- Termite
2 Anjumshram et al.,
(2014)
Study: molecular and physiological
role.
3 Abhilash sing
madurya et al.,
(2015)
Study: cathode candidate in
microbial fuell cell
4 Ritha mehtha et al.,
(2013)
Study: Anti microbial activity.

12. 5 S. Hemalatha et
al., (2014)
Study: Anti oxidant activity
6 Sreemoy kandi das
et al., (2016)
Study: phytochemical
investigation and anti
oxidant screening
7 Arulpriya et al.,
(2011)
Study: pootential use of
E.A
8 Rita himanshu
mehta et al .,
(2016)
Study: pharmacognostic
and phytochemical study
of E.A
12

13. 4. MATERIAL AND METHOD
PLANT
Epipremnum aureum Linn
AUTHENTICATED BY
Dr. A. K. Pradeep,
Herbarium Curator,
Department of Botany,
University of Calicut (Specimen No. 148207)
EXTRACTED BY
Continuous Hot Extraction method, using Soxhlet
apparatus
SOLVENT USED
Ethanol Fig. No. 1
Soxhlet extraction
13

14. ANIMALS
 The study was approved by IAEC (Proposal No. DAMCOP/ IAEC/ 022)
 Animal : Swiss albino mice
 For acute toxicity: 5 (Female)
 Anticancer study: 30 (male)
 The animals are kept in separate cages have 6 animals.
 Maintain the environmental condition [ Temperature (22-26°c),
Relative humidity (60±10%), 12hr alternate light and dark cycle with
100% fresh air exchange in animals rooms and uninterrupted power
and water supply.
 Food: Should be fed palatable, non contaminated and nutritionally
adequate.
 Bedding: Absorbent, Free of toxic chemicals or personnel and of a
type not readily eaten by animals.
14

15. TOXICOLOGICAL STUDIES
Acute oral toxicity study: Limit test
• Swiss Albino mice / OECD Guidelines No. 425.
• Limit dose 2000 mg/Kg.b.wt. were given to 5 animals and compare with
control(1 animal).
• Fasted over night.
• Animals were observed for first four hours individually.
• Various parameters were observed for 14 days.
15

16. IN-VITRO ANTICANCER STUDY (MTT Assay)
• MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide]
• Taken up by the viable cells and reduced to formazan by
mitochondrial enzyme Succinate dehydrogenase
• The Optical Density (OD) of purple colored solution was
measured using a ELISA plate reader at 540 nm (λmax)
• The %inhibition was determined by using formula
PHARMACOLOGICAL SCREENING
% inhibition = 100 – Abs (sample)/Abs (control) × 100
16

17. TRYPAN BLUE DYE EXCLUSION ASSAY
• Trypan blue is a vital stain, leaves non-viable cells with a
distinctive blue colour when observed under microscope.
• Non viable cells do not have an intact and functional membrane
hence do take up the dye from their surroundings.
• % inhibition determined by using the formula
% inhibition = 100 – Abs (sample)/Abs (control) × 100
17

18. IN-VIVO ANTICANCER STUDY (Liquid Tumour Model) (DAL)
• Swiss Albino Mice ( Male)
• 5 groups each group contain 6 animals.
Treatment:
Group I: Normal (0.5% Sod. CMC suspension dose of 10
ml/kg.b.wt.)(oral)
Group II: DAL induced +
(0.5% Sod. CMC suspension dose of 10 ml/kg.b.wt.) (oral)
Group III: DAL induced +
Standard (5-Fluorouracil dose of 20 mg/kg.b.wt.)(oral)
Group IV: DAL induced +
EEEA dose of 200 mg/kg. b. wt.(oral)
Group V: DAL induced +
EEEAdose of 400 mg/kg. b. wt.(oral)
18

19.  Continued the treatment once daily for 14 days
 On 15th day, half of animals from each group are
anesthetized and blood was collected by retro-orbital
puncture.
 Haematological parameters like, Haemoglobin (Hb),
RBC, and WBC.
 The remaining animals in each of the groups are kept
to check the Mean Survival Time (MST) and % increase
in Life span of the tumour bearing hosts.
 Statistical analysis was performed using instat
software.
19

20. • Antioxidants are the substance that when present in low
concentrations compared to those of an oxidizable substrate
significantly delays or prevents oxidation of that substance.
Methods employed for the study
1) Nitric oxide free radical scavenging activity
2) DPPH ASSAY
IN-VITRO ANTIOXIDANT STUDY
20

21. 1) Nitric oxide free radical scavenging activity :
 Sodium nitroprusside in aqueous solution at physiological pH
spontaneously generates Nitrite oxide which interacts with
oxygen to produce Nitrite ions, which can be measured at
540nm by spectrophotometer in the presence of Griess
reagent.
 The nitric oxide radical scavenging activity was calculated as:
Where,
 Acontrol = absorbance of the blank control (containing all
reagents -
except the extract solution)
 Asample = absorbance of test sample.
S% = [(Acontrol – Asample) / Acontrol] × 100
21

22. 2) DPPH scavenging activity
 In DPPH transfer of an electron or hydrogen atom take
place. The reduction capacity of DPPH could be
determined by colour changes from purple to yellow
colour which can be measured at 517 nm.
 The percentage of inhibition was calculated as
S% = [(Acontrol – Asample) / Acontrol] × 100
22

23. EXTRACTION
The authenticated leaves of Epipremnum aureum (L.) were used for the
study.
The extraction was carried out and the percentage yield of extract is
shown in Table No 1 below.
Sl. No. Extract Percentage yield
1 Ethanolic extract of leaves 8.03% w/w
Table No. 1 Percentage yield of EEEA
4. RESULTS AND DISCUSSION
23

24. RESULTS
Phytochemical studies of Ethanolic extracts of leaves of
Epipremnum aureum Linn.
S.No.
Phytochemical
constituents
Epipremnum aureum linn
1. Carbohydrates + ve
2. Alkaloids + ve
3. Steroids & sterols + ve
4. Glycosides + ve
5. Saponins + ve
6. Flavonoids + ve
7.
Tannins & phenolic
compound
+ ve
8. Proteins & amino acids + ve
24

25. 25
•No mortality was observed at 2000 mg/kg for the extract.
•All the animals were found to be normal.
•No gross behavioural changes at the end of study period.
• From the study, 1/5
th and 1/10
th of 2000 mg/kg dose was selected for further
pharmacological screenings.
TOXICOLOGICAL STUDIES
Acute oral toxicity study: Limit test
Fig.3Weight variation of animals treated with EEEA in acute oral toxicity
study

26. Cell line Samples % inhibition
IC50
(µg/ml)
Concentration (µg/ml)
10 20 50 100 200
DAL cell line 5-FU 22.07 30.76 56.01 81.39 86.99 59.80
EEEA 9.87 15.25 22.89 40.01 58.24 158.31
Table No. 3 - % inhibition and IC50 value of in-vitro anticancer study(MTT assay)
26
(µg/ml)

27. Cell line Samples
% inhibition
IC50 (µg/ml)Concentration (µg/ml)
10 20 50 100 200
DAL cell line
5-FU 22.07 30.76 56.01 81.39 86.99 59.82
EEEA 10.1 15.06 23.03 40 58 158.89
Table No. 4 - % inhibition and IC50 value of in-vitro anticancer study (Trypan blue dye
exclusion assay)
EEEA shows cytotoxic effect in DAL cell line at 158.89 (µg/ml)
27

28. Sl.
No.
Concentration
(μg/ml)
Absorbance % Inhibition
(mean±SD)
IC 50
(μg/ml)
1 2 3
1 Control blank 1.4102 - -
Standard (Ascorbic acid)
1 12.5 1.0925 1.0971 1.1190 21.85±0.1452
48.65
2 25 0.8730 0.7989 0.7996 41.57±0.317
3 50 0.6193 0.6174 0.6084 69.47±0.088
4 100 0.3298 0.3421 0.3347 86.94±0.043
EEEA
1 50 0.6821 0.7215 0.7216 45.81±0.021
69.17
2 100 0.5241 0.5427 0.5241 58.02±0.032
3 150 0.3987 0.4521 0.4421 69.47±0.0888
4 200 0.1682 0.1621 0.1565 86.93±0.020
IN-VITRO ANTIOXIDANT ACTIVITY
Table No.15 : % inhibition and IC 50 values of Nitric oxide scavenging activity
28

29. Fig. No.21 : Concentration dependent % inhibition of Nitric oxide scavenging activity
• Based on the IC50 values of standard ascorbic acid and EEEA as shown in
Table no. 15, it is indicated that the leaf extract shows better nitric oxide
scavenging activity in a dose of 69.17(µg/ml)
• Nitric oxide free radical scavenging activity:
0
20
40
60
80
100
0 50 100 150 200 250
Ascorbic
acid
EEEA
y=0.571x+22.22
R2= 0.922
y= 0.2696x+31.35
R2 = 0.99
%Inhibition
Concentration (µg/ml)
29

30. Sl.
No.
Concentratio
n
(μg/ml)
Absorbance % Inhibition
(mean±SD)
IC 50
(μg/ml)
1 2 3
1 Control blank 1.4102 - -
Standard (Ascorbic acid)
1 12.5 1.0925 1.0971 1.1190 21.85±0.145
2
48.65
2 25 0.8730 0.7989 0.7996 41.57±0.317
3 50 0.6193 0.6174 0.6084 69.47±0.088
4 100 0.3298 0.3421 0.3347 86.94±0.043
EEEA
1 50 0.6112 0.6983 0.7241 45.81±0.021
69.17
2 100 0.5584 0.5453 0.5732 58.02±0.032
3 150 0.4231 0.4121 0.4309 69.47±0.088
4 200 0.1452 0.1654 0.1841 86.93±0.020
IN-VITRO ANTIOXIDANT ACTIVITY
Table No. 16 : % inhibition and IC 50 values of DPPH scavenging activity
30

31. Fig. No. 22 : Concentration dependent % inhibition of DPPH
• DPPH scavenging activity:
• Based on the IC50 values of standard ascorbic acid and EEEA as shown in
Table no. 16, it is indicated that the leaf extract shows better DPPH
scavenging activity in a dose of 69.17(µg/ml)
0
10
20
30
40
50
60
70
80
0 50 100 150 200
Ascorbic acid
EEEA
%Inhibition
y=0.604x+17.367
R2 =0.9442
31

32. WHITE BLOOD CELL COUNT
Parameter Normal control DAL control 5-FU 20mg/kg EEEA 200mg/kg EEEA 400mg/kg
WBC
16.12±0.086 38.77±0.037*** 19.92±0.018*** 24.30±0.151* 36.51±0.161***
RBC
8.85±0.062 3.583±0.038*** 8.035±0.07201*** 6.76±0.171** 7.06±0.140***
Hb 14.68±0.081 6.101±0.030*** 15.23±0.0410*** 12.77±0.0969** 8.745±0.0534**
R e d b lo o d c e ll c o u n t
G r o u p s
RBCcells
(10
3
/cu.mm)
N
o
rm
a
l
c
o
n
tro
lD
A
L
c
o
n
tro
l5
-fU
2
0
m
g
/k
g
E
E
E
A
2
0
0
m
g
/k
g
E
E
E
A
4
0
0
m
g
/k
g
0
2
4
6
8
1 0
N o rm a l co n tro l
D A L c o n tro l
5 -fU 2 0 m g /kg
E E E A 2 0 0 m g /kg
E E E A 4 0 0 m g /kg
Table No: 6 Effect on RBC, WBC, Hb count.
Fig. No.6, 7– Effect on white blood cell count, Red blood cell count
32
Values are mean±S.E.M; no. of mice in each group (n=6); *p<0.05, **p<0.01, ***p<0.001, – other groups compared with
DAL control

33. Hb
Fig. No.8 – Effect on Haemoglobin
• EEEA shows a significant decrease
in WBC cell count as compared to
DAL control
• EEEA shows significant increase in
RBC and Hb as compared to DAL
control
33

34. VIABLE END NON-VIABLE CELL COUNT
Parameter DAL control 5-FU 20mg/kg EEEA 200mg/kg EEEA 400mg/kg
Viable cells(x107cells
ml-1)
9.71±0.0175 8.81±0.059*** 4.54±0.0091** 4.0966±0.0578***
Non-viable cells
(x107cells ml-1)
0.815±0.0076 5.61±0.0493*** 4.66±0.035*** 3.6±0.011***
Table No: 8 Effect on Viable and Non- viable cell count
Fig. No.11 – Effect on Viable cell count Fig. No.12– Effect on Non-Viable cell count
EEEA Shows significant decreas in viable cell and increase in NON- viable cell
34

35. Parameter Normal control DAL control 5-FU 20mg/kg EEEA 200mg/kg EEEA 400mg/kg
Body
weight(g)
27.96±0.391 40.53±0.836
***
32.70±0.827
***
37.52±1.208
ns
35.46±0.678
***
Table No.10 – Effect on body weight
Fig. No.13 – Effect on body weight
35
BODY WEIGHT
Values are mean±S.E.M; no. of mice in each group (n=6); ***p<0.001, ns – other groups compared with DAL
control

36. TUMOR WEIGHT AND TUMOR VOLUME
Parameter DAL control 5-FU 20mg/kg EEEA 200mg/kg EEEA 400mg/kg
Tumour weight (g) 23.08±0.582 8.8±0.435*** 18.59±0.274** 15.7±0.1562***
Tumour volume (ml) 19.80±0.341 7.89±0.035*** 16.02±0.4599*** 16.93±0.7954***
T u m o u r v o lu m e
G r o u p s
Tumourvolume
(ml)
D
A
L
c
o
n
tro
l
5
-F
U
2
0
m
g
/k
gE
E
E
A
2
0
0
m
g
/k
gE
E
E
A
4
0
0
m
g
/k
g
0
5
1 0
1 5
2 0
2 5
D A L c o n tro l
5 -F U 2 0 m g /kg
E E E A 2 0 0 m g /kg
E E E A 4 0 0 m g /kg
Table No.7 – Effect on Tumour weight, Tumour volume
Fig. No.9 – Effect on Tumour volume Fig. No.10 – Effect on Tumour weight
EEEA shows significant decrease in tumor weight and tumor volume as compared to DAL
control 36

37. Parameter DAL control 5- FU
EEEA
200mg/kg
EEEA
400mg/kg
Mean Survival
Time
21 days 28 days 22 days 24 days
Percentage Life
Span
- 33.33% 8.62% 18.64%
Table No.9 – Effect on survival time of tumour bearing mice.
.
InDAL control, showed a significant change in MST and %LS.
From the results of haematological and other parameters,
5-FU showed its potent anti-tumour nature.
When compared to DAL control, EEEA showed a significant effect on these
parameters while the EEEA200mg/kg showed lesser effect than the EEEA400mg/kg
37

38. 5.SUMMARY AND CONCLUSION
• Continuous hot soxhlet extraction of authenticated plant material (Leaves)
Epipremnum aureum (L.)
• Phytochemical screening of EEEA
• Performed the in-vitro anticancer activity of EEEA against the DAL cell line by
MTT assay and Trypan blue dye exclusion assay. EEEA showed moderate
activity against the DAL cell line .
• Assessed the short term toxicity of EEEA.
• Performed the in-vivo anticancer activity of EEEA against the DAL cell lines
on mice. EEEA showed a significant activity against the DAL cell line. However
statistical data shows that EEEA have Anti tumor activity against DAL cell line
in some parameters.
38

39. • In conclusion, the present study investigated the anticancer activity of
EEEA against DAL cell line through in-vivo and in-vitro methods.
showed a significant effect towards the methods.
39

40. REFERENCE
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Huxley A, Griffiths M, Levy M, editors. The New Royal Horticultural Society Dictionary of
Gardening. London: Macmillan; 1994. p. 3353.
 Boyce P. Areview of epipremnum (Araceae). In: Cultivation. Aroideana 2004;27:205-11.
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chloroplast biogenesis. Plant Cell Environ 2007;30:350-65.
 Hung CY, Sun YH, Chen J, Darlington DE, Williams A, Burkey KO, et al. Identification of a
Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP,
differentiallyexpressed in variegated Epipremnum aureum‘GoldenPothos’ is achieved
through a unique method of comparative study using tissue regenerated plants. J Exp Bot
2010;61:1483-93.
 Weidner M, Martins R, Muller A, Simon J, Schmitz H. Uptake, transport and accumulation
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41. THANK YOU
41