Evaluation of Antioxidant and Antitumour Activities of Croton

BY DAWN V TOMY, B.Pharm.,

Reg.No: 26106393

EVALUATION OF ANTI-OXIDANT AND ANTI-

TUMOUR ACTIVITIES OF CROTON LAEVIGATUS Vahl.

Saturday, April 8, 2023RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE

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DEPARTMENT OF PHARMACOLOGY

Thesis Submitted toThe Tamilnadu Dr. M.G.R Medical

University, Chennai In partial fulfillment of the requirements For

the award of the Degree ofMASTER OF PHARMACY IN PHARMACOLOGY

Submitted by

DAWN. V .TOMY, B. Pharm.,

Reg.No: 26106393

Dept. of Pharmacology

RVS College of Pharmaceutical Sciences, Sulur, Coimbatore.Saturday, April 8, 2023RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE

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Under the guidance ofInstitutional Guide

• Mrs. C. Maheswari, M.Pharm, (Ph.D).,

• Lecturer, Dept. of Pharmacology,• RVS College of Pharmaceutical

Sciences, Sulur, Coimbatore - 641 402.

Industrial Guide

• Dr. T. D Babu, Ph.D.,• Asst. Professor, Dept. of

Biochemistry,• Amala Cancer Research Centre,• Amala Nagar, Thrissur, Kerala -

680 555.


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INTRODUCTIONThe 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 conditions.


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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.


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LITERATURE REVIEWThe 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.


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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.


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PLAN OF WORK

Sub-acute toxicity studies


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PLANT PROFILEBotanical 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

http://eol.org/pages/282/entries/34425377/overview

http://eol.org/pages/283/entries/34425378/overview


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MATERIALS AND METHODS1. 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.


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2. Preparation of extractThe 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 analysis.


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3. Phytochemical screeningQualitative phytochemical screening of plant extracts was done

The extract of the plant was subjected to chemical tests for the identification of various active constituents


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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


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5. In vitro anti-oxidant activitiesThe 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.


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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 bottleProcedure

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 100OD of control


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DEPARTMENT OF PHARMACOLOGY5.2 Superoxide radical scavenging activityPrinciple: 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 100OD of control


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DEPARTMENT OF PHARMACOLOGY5.3 Hydroxyl radical scavenging activityPrinciple: 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 100OD of control


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6. Acute toxicity studySwiss 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) Mortalityb) Weight changec) Food and water consumptiond) Hematological functione) Liver Functionf) 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.


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DEPARTMENT OF PHARMACOLOGYParameters estimated

Liver Function Test (LFT) Method

1. Serum Gutamate Oxalo acetate Transaminase (SGOT) IFCC method2. Serum Glutamate Pyruvate transaminase (SGPT) IFCC method3. Alkaline phosphatase (ALP) PNPP hydrolysis Method/kinetic4. Albumin BCG Dye Binding Method5. Globulins Calculation6. Total Protein Biuret method7. Bilirubin Total Jendrassik-Diazotized Sulfanilic acid method

Renal Function Test (RFT)

1. Serum Urea Urease calorimetric kinetic method2. Creatinine JAFFE-kinetic method


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Hematological profileBlood 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).


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7. Anti-inflammatory activitiesa) 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’.


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8. Anti-tumoura) 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.


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Statistical analysisThe 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.


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RESULTS

Phytochemicals Methanol extract

Alkaloids +

Carbohydrates +

Proteins +

Amino acids -

Steroids +

Glycosides +

Saponnins -

Polyphenols +

1. Phytochemical screeningThe 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” “-“Absent


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DEPARTMENT OF PHARMACOLOGY2. 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.

0 25 50 75 100 125 1500

102030405060708090

100

Concentration Of Drug In µg/ml

% o

f in

hib

itio

n

IC50 = 37μg/ml

Figure 2: Cytotoxicity effect of C. laevigatus against DLA cell lines.


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0 25 50 75 100 125 1500

10

20

30

40

50

60

70

80

90

100%

of

inh

ibit

ion


IC50 = 52μg/ml

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


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0 25 50 75 100 125 1500

10

20

30

40

50

60

70

80

90

100

IC50 = 87μg/ml


% o

f in

hib

itio

n

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


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DEPARTMENT OF PHARMACOLOGY3. 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 activityThe 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.

0 100 200 300 400 500 600 700 800 900 10000

102030405060708090

100

Concentration of drug (µg/ml)

% o

f in

hib

itio

n

IC50 = 130μg/ml

Figure 5: Effect of C. laevigatus crude extract in inhibiting DPPH radical.


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DEPARTMENT OF PHARMACOLOGY3.2 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.

0 100 200 300 400 500 600 700 800 900 1000 11000

10

20

30

40

50

60

70

80

90

100


% o

f in

hib

itio

n

IC50 = 110μg/ml

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


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DEPARTMENT OF PHARMACOLOGY3.3 Hydroxyl radical scavenging activity

The concentration of the extract needed for 50% (IC50) inhibition of hydroxyl

radical was found to be 890 μg/ml.

0 100 200 300 400 500 600 700 800 900 1,000 1,1000

10

20

30

40

50

60

70

80

90

100


% o

f in

hib

itio

n

IC50 = 890μg/ml

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


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Group Dose

(mg/kg of body weight)

Mortality

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

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

4. Acute toxicity studyCroton 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


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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.

1 st 4 th 7 th 10 th0

5

10

15

20

25

30

35

Normal

CL-500mg/kg

CL-750mg/kg

Days

Bod

y w

eigh

t in

gra

ms

Figure 8: Effect of C. laevigatus methanolic extract on body weight of animals.


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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.

1 st 4 th 7 th 10 th0

2

4

6

8

10

12

14

16

18Normal

CL-500mg/kg

CL-750mg/kg

Days

Foo

d c

onsu

mp

tion

gm

s/ca

ges

(5 a

nim

als)

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


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1 st 4 th 7 th 10 th0

1

2

3

4

5

6

7

8

9

Normal

CL- 500mg/kg

CL-750mg/kg

Wat

er c

onsu

mp

tion

ml/c

ages

(5an

imal

s)

Days

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


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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

methanolic extract

(500 mg/kg)

1.42±0.19 0.415±0.036 0.097 ±0.016 0.145±0.021 0.289±.05

C. laevigatus

methanolic extract

(1000 mg/kg)

1.54±0.19 0.415±0.036 0.097± 0.02 0.151±0.017 0.305±0.071

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.


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DEPARTMENT OF PHARMACOLOGYAdministration 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

Group SGOT (U/L) SGPT (U/L) Bilirubin

(mg/dL)

Total protein

(g/dL)

Albumin

(g/dL)

Globulin

(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

methanolic extract

(500 mg/kg)

149.5± 50.53 82.9±19.4 0.2±0.88 7.48±1.2 3.54±0.19 2.5±0.34

C. laevigatus

methanolic extract

(1000 mg/kg)

162.2± 42.43 76.2±27.83 0.17±0.13 8.34±0.98 3.23±0.65 3.2±0.29

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

of 5 animals/group.


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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

mg/kg)

0.52 ± 0.17

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.


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Group Hb (g/dL) WBC (mm3) RBC

(106/cmm)

Platelet (105/cmm)

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

C. laevigatus methanolic extract

(500 mg/kg)

13.18 ± 1.09 8450±528.98 6.89±0.81 5.89±2.19

C. laevigatus methanolic extract

(1000 mg/kg)

13.64±0.687 7445±527.89 7.01±0.98 6.68±1.15

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.


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Group Lymphocyte

(mm3)

Eosinophils

(mm3)

Basophils

(mm3)

Neutrophils

(mm3)

Monocytes

(mm3)

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

C. laevigatus

methanolic extract

(500 mg/kg)

5974.8±412.25 196.2±40.31 204±44.2 1064±126.5 210±72.1

C. laevigatus

methanolic extract

(1000 mg/kg)

5780.6±246.85 224±74.81 196±33.8 1664±39.1 273±19.4

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


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Groups Initial paw

thickness

Paw thickness on 3rd

hour

Increase in paw

thickness

% of

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

extract (500 mg/kg)

0.2082±0.0215 0.2867±0.0149** 0.0785 46.29

C. laevigatus methanolic

extract (750 mg/kg)

0.2035±0.0168 0.2755±0.0268*** 0.0720 50.74

5 Anti-inflammatory activity5.1 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.


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Groups Initial paw

thickness

Paw thickness on 3rd

day

Increase in paw

thickness

% of inhibition

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

methanolic extract

(500 mg/kg)

0.1898±0.0061 0.3430±0.0103** 0.1532 25.78

C. laevigatus

methanolic extract

(750 mg/kg)

0.1938±0.0147 0.3312±0.0189*** 0.1374 33.43

5.2. Formalin induced chronic inflammatory modelIn 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.


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DEPARTMENT OF PHARMACOLOGY6 Anti-tumour analysis6.1 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 3 6 9 12 15 18 21 24 27 30 330.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40Normal

Cyclophosphamide 10mg/kg

C. laevigatus 500 mg/kg

C. laevigatus 750 mg/kg

Tu

mor

vol

um

e in

cm

3

Time intervels in Days

Figure 11: Effect of C. laevigatus methanol extract on solid tumour development.


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Treatment Mean survival days % increase in life span

Control (Normal Saline) 16±1.095 ___

Cyclophosphamide 10

mg/kg

31±2.28*** 93.75

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

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

6.2 Effect of C. laevigatus methanol extract on ascites tumour developmentThe 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.


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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.


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•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.


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DEPARTMENT OF PHARMACOLOGY• 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.


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• 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.


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• 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.


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• 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.


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•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.


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•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.


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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.


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• 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.


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DEPARTMENT OF PHARMACOLOGY•Santos PML, Schripsema J, Kuster, RM (2005). glycosylated flavonoids from Croton campestris St. Hill.(Euphorbiaceae). Rev. Bras. Farmacog., 15:321-325.•Sneader W (2005). Drug Discovery: a History.•Szentmihalyi K, Taba G, Lado C, Fodor J, Then M, Szoke E, (2005). Medicinal plant teas recommended as nutritional source for element supplementation. Acta Aliment 34:161-167.•Website: Phytochemicals for cancer prevention.•Wong C, Li H, Cheng K and Chen F (2006). A systematic survey of anti-oxidant activity of 30 Chinese medicinal plants using the ferric reducing anti-oxidant power assay. Food Chemistry 97, 705–711.•Zhang L and Demain AL (2005). Natural products drug discovery and therapeutic medicine. (ed) Springer.•Zheng W and Wang SY (2001). Anti-oxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem, 49:5165-5170.•Zou GA, Aisa HA, Zhang HW, Yang JS and Zou ZM (2012). Chemical composition of Croton laevigatus. Chemistry of Natural Compounds 47, 6.•Zou GA, Ding G, Su ZH, Yang JS, Zhang HW, Peng CZ, Aisa HA and Zou ZM (2010). Lactonecembranoids from Croton laevigatus. Journal of Natural Products 73, 792-795.

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Evaluation of Antioxidant and Antitumour Activities of Croton

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