EVALUATION OF ANTI-OXIDANT AND ANTI-TUMOUR ACTIVITIES OF CROTON LAEVIGATUS Vahl.

BY
DAWN V TOMY, B.Pharm.,
Reg.No: 26106393
1
RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013

Thesis Submitted to
The Tamilnadu Dr. M.G.R Medical University, Chennai In partial
fulfillment of the requirements For the award of the Degree of
MASTER OF PHARMACY IN PHARMACOLOGY

Submitted by
DAWN. V .TOMY, B. Pharm.,
Reg.No: 26106393
Dept. of Pharmacology
RVS College of Pharmaceutical Sciences, Sulur, Coimbatore.
2

Under the guidance of
Institutional Guide Industrial Guide
• Mrs. C. Maheswari, • Dr. T. D Babu, Ph.D.,
M.Pharm, (Ph.D)., • Asst. Professor, Dept. of
• Lecturer, Dept. of Pharmacology, Biochemistry,
• RVS College of Pharmaceutical • Amala Cancer Research Centre,
Sciences, Sulur, Coimbatore - 641 • Amala Nagar, Thrissur, Kerala -
402. 680 555.

3

INTRODUCTION
The nature has provided mankind the greatest
gift, ‘Medicinal Plants’. They play an important role in the
preservation of our health by curing infectious and
degenerative diseases. The medicinal properties of the plants
are due to the presence of bioactive compounds, produced
as by-products of primary metabolism. These compounds
protect the plants from infections
microorganisms, pests, various climatic stress conditions and
other hazards in the environment. They also contribute to
the development of new drugs. Hence, it has always been of
great interest to the scientists working on various disease 4
conditions. PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE
RVS COLLEGE OF Friday, March 15, 2013

The systematic screening of the plant, C. laevigatus Vahl.
The plant has been reported to be used in the traditional
Chinese medicine. Roots and leaves of the plant were
usually used by the Dai people (China) as a traditional drug
for treating fractures, malaria, stomach ache and wounds
received from falling. Almost 29 compounds were isolated
from the methanol extract of its leaves. Cytotoxicity of
diterpenoids isolated from C. laevigatus were initially
investigated on HeLa cell line by applying MTT method and
found modest cytotoxicity against HeLa cells (Zou et
al., 2010). No other pharmacological research has been
reported on this plant.
5

LITERATURE REVIEW
The C. laevigatus Vahl. belonging to the family Euphorbiaceae is
distributed mainly in India and China in the provinces of
Yunnan, Guangdong, and Hainan.
They are usually shrubs or trees, up to 1-5 m tall. Found as dense or
open forests; below 100-600 m. Roots and leaves of the plant are
usually used by the Dai people as a traditional drug for treating
fractures, malaria, stomach ache and wounds received from falling.
The chemical constituents of C. laevigatus were systematically
investigated with 29 compounds isolated from the methanol extract
of its leaves.
Cytotoxicity of diterpenoids isolated from C. laevigatus were initially
investigated on HeLa cell line by applying MTT methods. 6

AIM AND OBJECTIVES
•Phytochemical determination of biologically
active compounds from C. laevigatus Vahl.
•Determination of sub-acute toxicity of
methanolic extract of C. laevigatus.
•Analysis of cytotoxicity and anti-tumour
activities of C. laevigatus.
•Evaluation of anti-oxidant and anti-
inflammatory properties of C. laevigatus.
7

PLAN OF WORK

Sub-acute toxicity studies

8

PLANT PROFILE
Botanical Name: Croton laevigatus Vahl.
Common Names: Koteputol, Pongalam,
Somaraaji, Thomarayam.
Scientific Classification:
Kingdom - Plantae
Subkingdom - Angiosperms
Super division - Magnoliophyta
Division - Magnoliopsida
Class - Eudicots
Sub class - Rosids
Order - Malpighiales
Family - Euphorbiaceae
Subfamily - Crotonideae
Genus - Croton
Species - laevigatus 9

MATERIALS AND METHODS
1. Collection of plant sample
The leaves of C. laevigatus Vahl.
was collected from the Thrissur district
of Kerala. The plant was identified by Dr.
P. Sujanapal, Taxonomist, Kerala Forest
Research Institute (KFRI), Peechi.
The sample was dried in shade and
powdered using mixer grinder. 10

2. Preparation of extract
The powder was extracted
with methanol by maceration. The
extract was filtered through
Whatman #1 filter paper and the
filtrate was evaporated to
dryness, dissolved in Normal
Saline (NS) and used for biological
11
analysis.

3. Phytochemical screening
Qualitative phytochemical
screening of plant extracts was
done
The extract of the plant was
subjected to chemical tests for the
identification of various active
constituents
12

4. Determination of in vitro cytotoxicity
of leaves of C. laevigatus extracts (trypan blue
die exclusion method)

Tumour cell lines
•Daltons Lymphoma Ascites (DLA)
cells.
•Ehrlich Ascites tumour (EAC) cells.
•Jurkat cells.
% of cytotoxicity = Number of dead cells x 100
Number of total cells
13

5. In vitro anti-oxidant activities
The anti-oxidant properties of
the plant extract was analyzed by
determining the scavenging effects
of free radicals such as
superoxide, hydroxyl and DPPH
radical with in vitro assay systems.
14

DPPH radical scavenging assay:
Principle
In its radical form, DPPH has an absorption band at 515 nm, which
disappear on its reduction. Scavenging of DPPH radical by anti-oxidant compounds will
decrease the colour intensity, which can be compared with the control to get the
percent inhibition.
Reagents
Methanol and freshly prepared DPPH stored in brown bottle
Procedure
Different concentrations of extract of C. laevigatus were added to 0.375 ml
of freshly prepared DPPH solution in methanol. The volume was made up to 2 ml with
methanol. After 20 minutes, the absorbance was measured at 515 nm. The
percentage inhibition was calculated and concentration needed for 50% inhibition was
found out.
% of inhibition = OD of control - OD of test X 100
OD of control

15

5.2 Superoxide radical scavenging activity
Principle: The superoxide scavenging was determined by the Nitro Blue Tetrazolium
(NBT) reduction method, which depends on light induced super oxide radical generation
by riboflavin. The super oxide radical will reduce the NBT to a blue coloured formazan
complex, which is measurable at 560 nm.

Reagents: Phosphate buffer (0.06M, pH 7.8), EDTA (0.1M) containing 1.5 mg of sodium
cyanide per 100 ml, riboflavin (0.12 mM) stored cold in a dark bottle and NBT (1.5 mM)
stored cold.

Procedure: Different concentrations of extracts ranging from 100 to 1000 μg/ml were
added to the reaction mixture containing 3 μg NaCN in 0.1 M EDTA; NBT 50µg, 0.12 mM
riboflavin and 0.6 M phosphate buffer (pH 7.8) in a final volume of 3 ml. The tubes
containing the reaction mixture were continuously illuminated with incandescent lamp for
15 min. The Optical Density (OD) measurements were taken at 560 nm before and after
illumination. The effect of test material to inhibit superoxide generation was evaluated by
comparing the OD of control and treated samples.
% of inhibition = OD of control - OD of test X 100
OD of control
16

5.3 Hydroxyl radical scavenging activity
Principle: Hydroxyl radical scavenging was measured by studying the competition between
deoxyribose and the test compounds for hydroxyl radicals generated from the
Fe3+/ascorbate/EDTA/H2O2 system (Fenton reaction). The hydroxyl radical attacks epoxy
ribose, which eventually results in Thiobarbituric Acid Reacting Substances (TBARS)
formation, which is estimated using a spectrophotometer at 530 nm.

Reagents: Hydroxyl buffer (pH 7.4), H2O2 (1 mM) prepared freshly and stored in a brown
bottle, EDTA (0.1 mM), FeCl3 (0.1 mM) prepared freshly and stored in brown bottle, Ascorbic
acid (0.1 mM) freshly prepared, deoxy ribose (2.8 mM).

Procedure: The reaction system contained deoxyribose (2.8 mM), EDTA (0.1 mM), FeCl3 (0.1
mM), KH2PO4 KOH buffer (20 mM; pH 7.4) and from 100 to 1000 μg/ml of the test material
in a final volume of 1 ml. The reaction mixture was incubated for 37o C for 1 hour and is
estimated using a spectrophotometer at 530 nm.. The scavenging activity of hydroxyl radicals
was expressed as
% of inhibition = OD of control – OD of test X 100
OD of control
17

6. Acute toxicity study
Swiss albino mice were administered the extract of C.
laevigatus in doses of 500 and 1000 mg/kg by oral for 14 days
and the following parameters were determined to evaluate the
toxicity.
a) Mortality
b) Weight change
c) Food and water consumption
d) Hematological function
e) Liver Function
f) Renal Function
Adult Swiss albino mice of age approximately 6 to 8
weeks old with weights in the range of 25-30g were used for
experiment.
18

Parameters estimated

Liver Function Test (LFT) Method

1. Serum Gutamate Oxalo acetate Transaminase (SGOT) IFCC method
2. Serum Glutamate Pyruvate transaminase (SGPT) IFCC method
3. Alkaline phosphatase (ALP) PNPP hydrolysis Method/kinetic
4. Albumin BCG Dye Binding Method
5. Globulins Calculation
6. Total Protein Biuret method
7. Bilirubin Total Jendrassik-Diazotized Sulfanilic acid method

Renal Function Test (RFT)

1. Serum Urea Urease calorimetric kinetic method
2. Creatinine JAFFE-kinetic method
19

Hematological profile
Blood was analyzed to determine the variations in
hematological parameters like haemoglobin, RBC and
platelet using haematology analyzer (Model ABX-
Micro-S-60).
The total white blood cells (WBC) were measured
after diluting the blood in Turk’s fluid and counting them
using a haemocytometer under magnification by
microscope (10 X).
To measure the differential count, blood was
spread on a clean slide and stained with Leishman’s
stain. Various types of cells were counted using a
microscope under magnification (100 X). 20

7. Anti-inflammatory activities
a) Carrageenan induced acute inflammation.
Increase in paw thickness was measured using Vernier caliper
before and after carrageenan injection and thereafter at
first, second, third, fourth, fifth and twenty-fourth hours. The
increase in thickness as a measure of inflammatory edema can be
calculated by using the formula, Pt - Po where Po is initial
thickness and Pt is thickness at time‘t’
b) Formalin induced chronic inflammation.
The increase in thickness as a measure of inflammatory
oedema can be calculated by using the formula, Pt - Po where
Po is initial thickness and Pt is thickness at time‘t’.

21

8. Anti-tumour
a) Solid tumour: The tumour volume was
calculated using the formula V = 4/3 π r12 r2
where r1 is the minor radius and r2 is the major
radius.
b) Ascites tumour: The life span of animals
was calculated using the formula:
% Increase in life span (ILS) = (T-
C)/C 100, where T and C are mean survival of
treated and control mice. 22

Statistical analysis
The values are presented as mean SD.
Differences between group’s means were
estimated using a one way analysis of
variance followed by Tukey test, using
Graph Pad In stat Software. The results
were considered statistically significant when
P<0.05.

23

RESULTS
1. Phytochemical screening
The phytochemical screening of the C. laevigatus Vahl.
methanol extract showed the presence of alkaloids, carbohydrates,
proteins, steroids, glycosides and polyphenols (Tab. 1).
Table1: Results of the phytochemical screening
“+”Present” Phytochemicals Methanol extract “-“Absent
Alkaloids +
Carbohydrates +
Proteins +
Amino acids -
Steroids +
Glycosides +
Saponnins -
Polyphenols + 24

2. In vitro cytotoxicity
The C. laevigatus methanol extract showed marked cytotoxicity
activity against DLA, EAC and Jurkat cell lines. The concentration
required for 50% death (IC50) was found to be 37, 52 and 87µg/ml
respectively.
100
90
80
% of inhibition

70
60
50
40 IC50 = 37μg/ml
30
20
10
0
0 25 50 75 100 125 150
Concentration Of Drug In µg/ml
Figure 2: Cytotoxicity effect of C. laevigatus against DLA cell lines. 25

100
90
80
70
60
50
% of inhibition

40 IC50 = 52μg/ml
30
20
10
0
0 25 50 75 100 125 150

Figure 3: Cytotoxicity effect of C. laevigatus against EAC cell lines.
26

100
90
80
70
60
% of inhibition

50 IC50 = 87μg/ml
40
30
20
10
0
0 25 50 75 100 125 150

Figure 4: Cytotoxicity effect of C. laevigatus against Jurkat cell lines. 27

3. In vitro anti-oxidant activities
In vitro anti-oxidant activities of C. laevigatus methanol extract was evaluated
using DPPH, superoxide and hydroxyl radical scavenging assays. The results were
expressed in IC50 values.
3.1 DPPH radical scavenging activity
The reduction capability was determined by reduction in the absorbance at 515
nm. The stable free radical DPPH was effectively scavenged by C. laevigatus methanol
extract with IC50 value 130 μg/ml.
100
90
80
70
% of inhibition

60
50
40
30 IC50 = 130μg/ml
20
10
0
0 100 200 300 400 500 600 700 800 900 1000

28
Concentration of drug (µg/ml)
Figure 5: Effect of C. laevigatus crude extract in inhibiting DPPH radical.


3.2 Superoxide radical scavenging activity
The C. laevigatus methanol extract have significant scavenging of
superoxide radicals. The amount of extract needed for 50% inhibition was
found to be 110 μg/ml.

100
90
80
70
60
% of inhibition

50
40
30 IC50 = 110μg/ml
20
10
0
0 100 200 300 400 500 600 700 800 900 1000 1100

Figure 6: Effect of C. laevigatus crude extract in inhibiting superoxide radical scavenging 29

3.3 Hydroxyl radical scavenging activity
The concentration of the extract needed for 50% (IC50) inhibition of hydroxyl
radical was found to be 890 μg/ml.

100
90
80
70
60
50
% of inhibition

40
30
IC50 = 890μg/ml
20
10
0
0 100 200 300 400 500 600 700 800 900 1,000 1,100

Figure 7: Effect of C. laevigatus crude extract in inhibiting hydroxyl radical scavenging. 30

4. Acute toxicity study
Croton laevigatus methanol extract treatment caused no deaths in any
of the groups. Organ weight, food and water consumption, hemoglobin content and total
WBC count were found to be almost similar to that of control. Biochemical assays for liver
and kidney functions also revealed the safety of C. laevigatus methanol extract as an anti-
oxidant.
The administration of C. laevigatus methanolic extract did not produce
any mortality at doses of 500 and 1000 mg/kg of body weight for 14 days. This indicates
that the extract prepared from C. laevigatus, even at very high concentration was not lethal
to treated mice.
.
Table 2: Effect of C. laevigatus methanolic extract on mortality

Group Dose Mortality
(mg/kg of body weight)

C. laevigatus methanolic extract (500 mg/kg) 500 No

C. laevigatus methanolic extract (1000 mg/kg) 1000 No
31

Administration of extract at doses 500 and 1000mg/kg of body weight did not produce any
decrease in the body weight in Swiss Albino mice used in our study.

35

30

25
Body weight in grams

20

15
Normal

10 CL-500mg/kg

CL-750mg/kg
5

0
1 st 4 th 7 th 10 th
Days
Figure 8: Effect of C. laevigatus methanolic extract on body weight of animals. 32

Administration of C. laevigatus methanolic extract at doses 500 and 1000mg/kg did not
produce significant alteration of food and water consumption in treated groups.

18 Normal

16 CL-500mg/kg
Food consumption gms/cages (5 animals)

14 CL-750mg/kg

12

10

8

6

4

2

0
Days

Figure 9: Effect of C. laevigatus methanolic extract on food consumption of animals. 33

9
Normal
8
Water consumption ml/cages(5animals)

CL- 500mg/kg
7
CL-750mg/kg
6

5

4

3

2

1

0

34
Days
Figure 10: Effect of C. laevigatus methanolic extract on water consumption of animals.

Administration of C. laevigatus methanolic extract at doses of 500 and
1000 mg/kg of body weight did not produce any change in the weight
of organs such as liver, kidney, spleen, heart and lungs.

Table 3: Effect of C. laevigatus methanolic extract on organ weight. Values are mean ± SD
of 5 animals/group and expressed as the organ weight/100 g of body weight.

Group Liver (g) Kidney (g) Spleen (g) Heart (g) Lungs (g)

Normal 1.425±0.47 0.442 ± 0.21 0.103 ± 0.018 0.131±0.03 0.257±0.052

C. laevigatus 1.42±0.19 0.415±0.036 0.097 ±0.016 0.145±0.021 0.289±.05
methanolic extract
(500 mg/kg)

C. laevigatus 1.54±0.19 0.415±0.036 0.097± 0.02 0.151±0.017 0.305±0.071
methanolic extract
(1000 mg/kg) 35

Administration of C. laevigatus methanolic extract at doses of 500 and did not produce any
change in hepatic parameters such as SGOT, SGPT, bilirubin, albumin & globulin. This
indicates that C. laevigatus methanolic extract at high concentration did not produce any
hepatic damage

Table 4: Effect of C. laevigatus methanolic extract on Liver Function test. Values are mean ± SD of
5 animals/group.

Group SGOT (U/L) SGPT (U/L) Bilirubin Total protein Albumin Globulin
(mg/dL) (g/dL) (g/dL) (g/dL)

Normal 154.2± 29.63 88.42±22.15 0.2±0.11 6.28±0.5 2.82±0.53 2.1±0.24

C. laevigatus 149.5± 50.53 82.9±19.4 0.2±0.88 7.48±1.2 3.54±0.19 2.5±0.34
methanolic extract
(500 mg/kg)
C. laevigatus 162.2± 42.43 76.2±27.83 0.17±0.13 8.34±0.98 3.23±0.65 3.2±0.29
methanolic extract
(1000 mg/kg)
36

Administration of C. laevigatus methanolic extract at doses of 500 and 1000 mg/kg of body
weight for 14 days did not produce any change in the renal function as the creatinine level
was similar to that of normal. This indicates that C. laevigatus methanolic extract did not
produce any toxicity to kidney and other renal tissues at the concentrations studied here.

Table 5: Effect of C. laevigatus methanolic extract on Renal Function test. Values are mean
± SD of 5 animals/group.

Group Creatinine (mg/dL)

Normal 0.53 ± 0.09

C. laevigatus methanolic extract (500 mg/kg) 0.48 ± 0.2

C. laevigatus methanolic extract (1000 0.52 ± 0.17
mg/kg)
37

Administration of C. laevigatus methanolic extract for 14 days, at doses of 500 and 1000
mg/kg did not produce any change in hematological parameters in the animals such as
total, WBC, RBC, platelet, haemoglobin as well as the differential count. This again
indicates that C. laevigatus methanolic extract high doses were found to be non toxic to
hematological system.

Table 6: Effect of C. laevigatus methanolic extract on hematological parameters. Values are
mean ± SD of 5 animals/group.
Group Hb (g/dL) WBC (mm3) RBC Platelet (105/cmm)
(106/cmm)

Normal 12.37±0.697 9170±149.88 7.86±0.6 6.48±1.84

C. laevigatus methanolic extract (500 13.18 ± 1.09 8450±528.98 6.89±0.81 5.89±2.19
mg/kg)
C. laevigatus methanolic extract 13.64±0.687 7445±527.89 7.01±0.98 6.68±1.15
(1000 mg/kg)
38

Table 7: Effect of C. laevigatus methanolic extract on hematological parameters - on
differential counts. Values are mean ± SD of 5 animals/group.

Group Lymphocyte Eosinophils Basophils Neutrophils Monocytes
(mm3) (mm3) (mm3) (mm3) (mm3)

Normal 6450.4±324.52 256±40.83 227±52.27 1264±70.5 225.4±64.78

C. laevigatus 5974.8±412.25 196.2±40.31 204±44.2 1064±126.5 210±72.1
methanolic extract
(500 mg/kg)

C. laevigatus 5780.6±246.85 224±74.81 196±33.8 1664±39.1 273±19.4
methanolic extract
(1000 mg/kg)

39

5 Anti-inflammatory activity
5.1 Carrageenan induced acute inflammatory model
The sub-plantar injection of carrageenan into the mice hind paw elicited an
inflammation that was maximal at 3rd hour. The inflammatory response was significantly
reduced by the C. laevigatus methanol extract at doses of 500 and 750 mg/kg body weight
when compared to control with the percentage inhibition of 46.29 (p<0.01) and 50.74%
(p<0.001) respectively, 3rd hour following carrageenan injection.

Table.8: Effect of C. laevigatus methanol extract in carrageenan induced paw oedema. Values
are Mean ± SD; n=6 in each group (***) p<0.001, (**) p<0.01 as compared to control.
Groups Initial paw Paw thickness on 3rd Increase in paw % of
thickness hour thickness inhibition
Control 0.2185±0.0110 0.3647±0.0519 0.1462 ___
Diclofenac 0.2010±0.0189 0.2665±0.0192*** 0.0655 55.19
C. laevigatus methanolic 0.2082±0.0215 0.2867±0.0149** 0.0785 46.29
extract (500 mg/kg)
C. laevigatus methanolic 0.2035±0.0168 0.2755±0.0268*** 0.0720 50.74
extract (750 mg/kg) 40

5.2. Formalin induced chronic inflammatory model
In mice treated with C. laevigatus methanol extract showed reduction in paw oedema
induced by sub-plantar injection of formalin with percentage inhibition 25.78 (p<0.01) and
33.43% (p<0.001) respectively at the doses 500 and 750 mg/kg body weight, on the 3th
day.
Table 9: Effect of C. laevigatus methanol extract in formalin induced paw edema. Values are
Mean ± SD; n=6 in each group (***) p<0.001, (**) p<0.01 as compared to control.
Groups Initial paw Paw thickness on 3rd Increase in paw % of inhibition
thickness day thickness
Control 0.1936±0.0154 0.4000±0.0350 0.2064 ___
Diclofenac 0.1888±0.0184 0.3204±0.0144*** 0.1316 36.24
C. laevigatus 0.1898±0.0061 0.3430±0.0103** 0.1532 25.78
methanolic extract
(500 mg/kg)
C. laevigatus 0.1938±0.0147 0.3312±0.0189*** 0.1374 33.43
methanolic extract
(750 mg/kg)
41

6 Anti-tumour analysis
6.1 Effect of C. laevigatus methanol extract on solid tumour development
The animals injected with DLA cell lines alone showed marked increase in tumour volume
on the 30th day of inoculation in control group. The tumour volume in the mice treated with 500 and
750 mg/kg on the 30th day of inoculation decreased significantly by 58.35 and 62.02% respectively.
The tumour volume had decreased by 64.78% in cyclophosphamide (10 mg/kg) treated group.
0.40
Normal
0.35 Cyclophosphamide 10mg/kg
C. laevigatus 500 mg/kg
0.30
C. laevigatus 750 mg/kg
Tumor volume in cm3

0.25

0.20

0.15

0.10

0.05

0.00
0 3 6 9 12 15 18 21 24 27 30 33
Time intervels in Days
Figure 11: Effect of C. laevigatus methanol extract on solid tumour development. 42

6.2 Effect of C. laevigatus methanol extract on ascites tumour development
The animals of the tumour control group inoculated with EAC cells survived for
a period 16±1.095 days. The C. laevigatus methanol extract at the 500 and 750 mg/kg
body weight was found to be inhibiting the proliferation of EAC with the percentage
increase in life span by 60.44 and 84.38%. In cyclophosmide treated mice (10 mg/kg), the
percentage increase in life span was found to be 93.75%.

Table 10: Effect of C. laevigatus methanol extract on average life span of ascites tumour
bearing mice. Values are Mean ± SD; n=6 in each group (***) p<0.001, (**) p<0.01 as
compared to control.
Treatment Mean survival days % increase in life span

Control (Normal Saline) 16±1.095 ___

Cyclophosphamide 10 31±2.28*** 93.75
mg/kg

C. laevigatus 500 mg/kg 25.67±6.623** 60.44

C. laevigatus 750 mg/kg 29.50±5.505*** 84.38 43

DISCUSSION
•The plant C. laevigatus spread along India
to China have been in use as traditional anti-
inflammatory medicine it has never been
studied for its pharmacological activity
except for the diterpenoids which is found in
most of the Crotons and the less abundant
neocrotocembraneic acid.

44

•The extract showed cytotoxicity towards the DLA, EAC and
Jurkat cells with an IC50 value of 37, 52 and 87
µg/ml, respectively.

•The extract on anti-oxidant screening showed significant
activity with an IC50 value of 130, 110 and 890 µg/ml with
DPPH, superoxide and hydroxyl radical assay, respectively.

•The acute toxicity studies showed the safety profile of the
extract. The extract at doses of 500 and 1000 mg/kg did not
produce any mortality in mice; it also showed that the
haematological and non-haematological parameters were
similar to that of normal.
45

•It has been already reported that diterpenoids and
laevigatbenzoate isolated from C. laevigatus has shown
modest cytotoxicity towards HeLA cell lines (Zou et al., 2010).

•The cytotoxicity exhibited towards the DLA, EAC and Jurkat
cell lines in the present study may be due to the presence of
the same constituents.

•The polyphenols present in the C. laevigatus may responsible
the anti-oxidant property.

46

•The inflammation in the carageenan induced
inflammatory model has significantly reduced
in paw thickness on the 3rd hour which is
comparable to the standard diclofenac.

•The formalin induced chronic inflammation
has considerably reduced on the 3rd day
which is comparable to standard dicofenac.
47

•The anti-inflammatory activity may be due to the
present of the β-sitosterol which is a cholesterol
analogue (Zou et al., 2010).
•The steroids are reported to inhibit the
phospholipase A2 which prevent the synthesis of
inflammatory mediators.
•The lactonecembranoids belonging to the
neocrotcembraneic acid isolated from the C.
laevigatus is known to posses inhibitory action on
cAMP phosphodiesterase may also support the anti-
tumour activity.
48

•The lactonecembranoids which belongs to
the neocrotocembraneic acid previously
isolated from the stem bark of Croton
oblongifolius showed considerable inhibition
of cAMP Phosphodiesterase (Sophon et
al., 2002) which is also isolated from C.
laevigatus (Zou et al., 2010) may be
responsible for its significant anti-
inflammatory activity.
49

•The anti-tumour analysis was performed based on
the cytotoxicity activity observed in various
transformed cells.

•The anti-tumour studies showed that the extract
posses significant activity. It has increased the
percentage life span by 60.44 and 84.38% in 500
and 750 mg/kg extract treated mice.

•It has also reduced the tumour volume by 58.35
and 62.02% in 500 and 750 mg/kg extract treated
mice on the 30th day of DLA inoculation in mice.
50

•The acute toxicity studies have proved the safety
profile of C. laevigatus and all the parameters
assessed was similar to that of the untreated
normal. It also signifies the importance of the plant
C. laevigatus as potent anticancer drug.

•The anti-oxidant activity along with its anti-
inflammatory effect has established its safeness to
be used for further studies.

51

CONCLUSION
•A considerable increase in life span in ascites tumour
bearing mice and percentage decrease in the tumour
volume in the solid tumour model in a dose dependent
manner when compared with the standard drug is
evident for the anti-tumour activity of the plant C.
laevigatus Vahl.
•The toxicity study result showed very little changes
when compared with normal result along with the anti-
oxidant and anti-inflammatory activity has established
the bio-safety profile of the plant, supporting its use in
the future studies to determine its hidden potentials. 52

•The result of the anit-oxidant, anti-inflammatory
assays performed also supports the anti-tumour
activity.

•Further research has to be performed to evaluate
their pharmacological profile and to check the
efficiency of the plant C. laevigatus in controlling
various types of diseases.

53

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