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

1,845 views

Published on

MY THESIS

Published in: Health & Medicine
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
1,845
On SlideShare
0
From Embeds
0
Number of Embeds
2
Actions
Shares
0
Downloads
51
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide
  • My project title is evaluation of anti-oxidant and anti-tumour activities of croton laevigatusvahl.
  • The natures greatest gift to mankind are the medicinal plants. These compounds protect the plants from infections microorganisms, pests, various climatic stress conditions and other hazards in the environment.
  • MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • AIM & OBJECTIVEThe present investigation is focused on detecting therapeutics from the plant Croton laevigatusVahl.
  • PLAN OF WORK The selected plant C. laevigatusVahl. will be collected and authenticated. The leaves of the plant will be made free from dust particles, dried under shade and powdered coarsely. The powder will be extracted using methanol by overnight stirring (maceration). The extract will be subjected to preliminary phytochemical screening to determine the phytoconstituents present. Then in-vitro studies i.e. cytotoxicity and anti-oxidant studies will be carried out to decide whether to continue the study with the extract. If the methanol extract shows promising activity, the screening will be proceeded further for in-vivo anti-inflammatory and anti-tumour activity. The schematic representation of the plan work is shown in the Fig.1.
  • Distribution: India to China. In Kerala the plant is seen in Kasaragode, Malappuram, Thrissur, Eranakulam and Alappuzha.Habitat: Scrub forests; also in plains.Description: Usually shrubs or trees, up to 15 m tall; indumentum with dense, appressedstellate hairs; branches glabrescent, older branches silvery gray.Stems and leaves: Leaves clustered at stem apex; stipules subulate, 2-3 mm, caducous; petiole 1-5 cm; leaf blade elliptic or oblong-elliptic to oblanceolate, 7-25 × 3-9 cm, papery, abaxially with sparse stellate hairs, glaucous-gray when dried, adaxially glabrescent, base attenuate, base of midrib with abaxial sessile semiorbicular glands, margins subentire, usually with glands, bilateral apex acuminate.Flowers: Inflorescences terminally clustered, 10-30 cm, persistently hairy. Male flowers: sepals ca. 2 mm, densely and appressedstellate-hairy; petals oblong, ca. 2 mm, margins woolly; stamens 12-15. Female flowers: sepals as in male; petals small; ovary with densely appressedstellate hairs. Capsules obovoid or trigonous, ca. 10×8 mm, densely hairy. Flowering during October to December.Fruits: fruiting during December to March.Specimen sampled: leaf.Authentication: The plants were authenticated by Dr. P. Sujanapal, Taxonomist, Kerala Forest Research Institute (KFRI), Peechi.
  • The sample was dried in shade and powdered using mixer grinder.
  • The powder was extracted with HPLC grademethanol 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.
  • Alkaloids, Carbohydrates, Proteins, Amino acids, Steroids, Glycosides, Saponnins, Phenolic Compounds and Tannins.A) Tests for Alkaloids: 1. Dragendorff’s Test: To the extract, add 1ml of Dragendorff’s reagent. An orange red colour precipitate indicates the presence of alkaloids.2. Wagner’s Test: To the extract, add 1ml of Wagner’s reagent. Reddish brown colour precipitate indicates the presence of alkaloids.3. Mayer’s Test: To the extract, add 1ml of Mayer’s reagent. A dull white colour precipitate indicates the presence of alkaloids.4. Hager’s Test: To the extract, add 3ml of Hager’s reagent. Yellow colour precipitate indicates the presence of alkaloids.B) Tests for Carbohydrates:1. Molish Test: To the extract, 1ml of α-naphthol solution was added and conc. Sulphuric acid was added along the sides of the test tube. Purple or Reddish violet colour at the junction between the two liquids indicates the presence of carbohydrates.2. Fehling Test: To the extract, equal quantities of Fehling’s solution A & B were added. Upon heating gently, a brick red precipitate indicates the presence of carbohydrates.3. Benedict’s Test: To 5ml of Benedict’s reagent, 8 drops of solution under the test was added and mixed well. Then it was boiled vigorously for 2 minutes and cooled. Red precipitate indicates the presence of carbohydrates.C) Tests for Proteins:1. Biuret Test: To the extract, 1ml of 40% sodium hydroxide and 2 drops of 1% copper sulphate solution was added. A violet colour indicates the presence of proteins.2. Xanthoprotic Test: To the extract, 1ml of conc. Nitric acid was added. When a white precipitate was formed, it is boiled and cooled. Then 20% of sodium hydroxide or ammonia was added. Orange colour indicates the presence of aromatic amino acids.3. Lead Acetate Test: To the extract, 1ml of lead acetate solution was added. A white precipitate indicates the presence of proteins.D) Test for Amino acids: 1. Ninhydrin Test: 2 drops of freshly prepared 0.2% ninhydrine reagent was added to the extract and heated. Development of blue colour indicates the presence of proteins, peptides or amino acids.E) Tests for Steroids: 1. LibbermannBurchard Test: The extract was dissolved in 2ml chloroform in dry test tube. 10 drops of acetic anhydride and 2 drops of conc. Sulphuric acid were added. The solution becomes red, and then blue and finally bluish green in colour indicates the presence of steroids.2. Salkowaski Test: The extract was dissolved in chloroform and equal volume of Sulphuric acid was added to it. Bluish red to cherry red colour was observed in chloroform layer; whereas acid layer assumes marked green fluorescence indicates the presence of steroids.F) Test for Glycosides:1. Keller-killani Test: Test sample was dissolved in acetic acid containing trace of ferric chloride and transferred to the surface of conc. Sulphuric acid. At the junction, reddish brown colour was formed, which gradually becomes blue indicates the presence of cardiac glycosides.G) Test for Saponnins:1. Foam Test: About 1ml of extract is diluted separately with distilled water to 20ml and shaken in a graduated cylinder for 15 minutes. A 1cm layer of foam indicates the presence of saponnins.H) Test for Phenolic Compounds and Tannins: Small quantities of alcoholic and aqueous extracts in water were tested for the presence of phenolic compounds and tannins with dilute ferric chloride solution (5%), 1% solution of gelatine containing 10% sodium chloride, 10% lead acetate and bromine solutions.
  • 4.1 In vitro cytotoxicity assay by trypan blue dye exclusion methodPrinciple Viable cells exclude the dye while non-viable cells take up the dye and will appear in blue colour under magnification by a microscope.Materials requiredDaltons lymphoma ascites, Jurkat and Ehrlich ascites carcinoma cells maintained in Amala Cancer Research centre were used for the assay, Phosphate Buffer Saline (PBS pH 7.4), trypan blue, and haemocytometer.Procedure Short-term cytotoxicity activity of the C. laevigatus methanol extract was assayed by determining the percentage viability of DLA, EAC and Jurkat cells using the trypan blue dye exclusion technique (Moldeuset al., 1978). DLA and EAC cells were cultured in the peritoneal cavity of healthy albino mice weighing between 25 to 30g by injecting a suspension of cells (1x 106 cells/ml), intraperitoneally. The DLA and EAC cells were aspirated aseptically from the peritoneal cavity of the mice on day 15 and Jurkat cells were collected from the in vitro culture bottles (RPMI1640 medium with 10% Fetal Bovine Serum (FBS) and supplemented with antibiotics penicillin and streptomycin) and washed with PBS (pH 7.4) and centrifuged for 15 min at 1,500 rpm in a centrifuge. The pellet was re-suspended with PBS and the process was repeated three times. Finally, the cells were suspended in a known quantity of PBS and the cell count was adjusted to 1×106 cells/ml. The tumour cells (1x106 cells/ml) were incubated at 37°C for 3 hrs with different concentrations of drug (25-150 µg). After incubation, 0.1 ml of trypan blue was added. The cytotoxicity activity was determined by counting the number of dead cells to that of live cells using haemocytometer and substituting in the equation:  % of cytotoxicity = Number of dead cells x 100 Number of total cells
  • DPPH-1,1-diphenyl-2-picrylhydrazyl.5.1 DPPH radical scavenging assayPrinciple 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 100 OD of control5.2 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 colouredformazan 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 The scavenging activityof extracts on superoxide anion radicals was determined by light induced superoxide generation with riboflavin and subsequent reduction of nitro blue tetrazolium (NBT) (McCord and Fridovich, 1969). 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; 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 530 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 control5.3 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.ReagentsHydroxyl 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 scavenging activity of extracts on the hydroxyl radical (OH-) was measured by the Thiobarbituric Acid Reacting Substances (TBARS) method. The scavenging activity was measured by studying the competition between scavenging and test compounds for hydroxyl radical generated by the Fe3+/ascorbate/H2O2 system (Fenton reaction). The hydroxyl radical attacks deoxyribose eventually resulting in the formation of Thiobarbituric Acid Reacting Substances (Elizabath and Rao, 1990). The reaction system contained deoxyribose (2.8 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. The scavenging activity of hydroxyl radicals was expressed as % of inhibition = OD of control – OD of test X 100 OD of control
  • DPPH-1,1-diphenyl-2-picrylhydrazyl.5.1 DPPH radical scavenging assayPrinciple 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 100 OD of control5.2 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 colouredformazan 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 The scavenging activityof extracts on superoxide anion radicals was determined by light induced superoxide generation with riboflavin and subsequent reduction of nitro blue tetrazolium (NBT) (McCord and Fridovich, 1969). 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; 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 530 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 control5.3 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.ReagentsHydroxyl 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 scavenging activity of extracts on the hydroxyl radical (OH-) was measured by the Thiobarbituric Acid Reacting Substances (TBARS) method. The scavenging activity was measured by studying the competition between scavenging and test compounds for hydroxyl radical generated by the Fe3+/ascorbate/H2O2 system (Fenton reaction). The hydroxyl radical attacks deoxyribose eventually resulting in the formation of Thiobarbituric Acid Reacting Substances (Elizabath and Rao, 1990). The reaction system contained deoxyribose (2.8 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. The scavenging activity of hydroxyl radicals was expressed as % of inhibition = OD of control – OD of test X 100 OD of control
  • DPPH-1,1-diphenyl-2-picrylhydrazyl.5.1 DPPH radical scavenging assayPrinciple 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 100 OD of control5.2 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 colouredformazan 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 The scavenging activityof extracts on superoxide anion radicals was determined by light induced superoxide generation with riboflavin and subsequent reduction of nitro blue tetrazolium (NBT) (McCord and Fridovich, 1969). 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; 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 530 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 control5.3 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.ReagentsHydroxyl 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 scavenging activity of extracts on the hydroxyl radical (OH-) was measured by the Thiobarbituric Acid Reacting Substances (TBARS) method. The scavenging activity was measured by studying the competition between scavenging and test compounds for hydroxyl radical generated by the Fe3+/ascorbate/H2O2 system (Fenton reaction). The hydroxyl radical attacks deoxyribose eventually resulting in the formation of Thiobarbituric Acid Reacting Substances (Elizabath and Rao, 1990). The reaction system contained deoxyribose (2.8 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. The scavenging activity of hydroxyl radicals was expressed as % of inhibition = OD of control – OD of test X 100 OD of control
  • DPPH-1,1-diphenyl-2-picrylhydrazyl.5.1 DPPH radical scavenging assayPrinciple 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 100 OD of control5.2 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 colouredformazan 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 The scavenging activityof extracts on superoxide anion radicals was determined by light induced superoxide generation with riboflavin and subsequent reduction of nitro blue tetrazolium (NBT) (McCord and Fridovich, 1969). 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; 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 530 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 control5.3 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.ReagentsHydroxyl 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 scavenging activity of extracts on the hydroxyl radical (OH-) was measured by the Thiobarbituric Acid Reacting Substances (TBARS) method. The scavenging activity was measured by studying the competition between scavenging and test compounds for hydroxyl radical generated by the Fe3+/ascorbate/H2O2 system (Fenton reaction). The hydroxyl radical attacks deoxyribose eventually resulting in the formation of Thiobarbituric Acid Reacting Substances (Elizabath and Rao, 1990). The reaction system contained deoxyribose (2.8 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. The scavenging activity of hydroxyl radicals was expressed as % of inhibition = OD of control – OD of test X 100 OD of control
  • ProcedureMice were randomly divided into 3 groups. Each group consisted of 6 mice. The 1st group was kept as control and received 1 ml of vehicle normal saline every day. Group 2 and 3 received the drug at doses of 500 and 1000 mg/kg, respectively. Each dose was administered in such a way that all the animals received same volume (200 μl) of the drug. Drugs were administered orally in the prescribed dose (once daily) using oral needle and continued for 14 days. The animals were monitored, during this period for any type of clinical symptoms, mortality and any adverse reaction of the administered drug. Body weights were determined every 3 days. Animals were sacrificed on day 15th by ether anaesthesia; blood was collected by direct heart puncture method and taken in the EDTA and non-EDTA vials for assaying hematological parameters and serum biochemistry. All the organs were examined visibly for any type of abnormalities in the structure and selected organs like liver, kidney, heart, lungs and spleen were dissected out and washed thoroughly in ice-cold normal saline and weights were recorded.
  • 7.1 Carrageenan induced acute inflammation The anti-inflammatory activity was evaluated by the carrageenan-induced paw edema test in the mice. Four groups of 6 animals each male Swiss albino mice (25-30 g, 6-8 weeks old) were injected with 0.02 ml of 1% suspension of carrageenan in normal saline, sub-plantar to the right hind paw. First group with carrageenan alone served as positive control. The group second was administered with diclofenac (10 mg/kg body weight) intraperitoneally as standard reference drug. The remaining group received extract at the doses of 500 and 750 mg/kg of body weight orally, for 5 consecutive days and on fifth day drug was administered 1 hour prior to induction of inflammation by carrageenan injection. 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’. 6.7.2 Formalin induced chronic inflammationThe inflammation was induced by using formalin in Swiss albino mice. All the animals were injected with 0.02 ml of 1% solution of formalin sub-plantar into right hind paw. Paw volume was measured 1hr prior and for 6 days after formalin administration using a Vernier caliper. The methanol extract of C. laevigatus leaves was given at doses of 500 and 750 mg/kg of body weight orally 1 hr prior to formalin injection. Diclofenac (10 mg/kg) was used as standard reference drug. The control group received equivalent volume of the vehicle. 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’. 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.
  • 8 Anti-tumour activity of methanol extract of leaves C. laevigatus.For assessing the anti-tumour activity, the Dalton’s lymphoma Ascites (DLA) and Ehrlich’s Ascites Carcinoma (EAC) cells induced solid and ascites tumour were used respectively.8.1 Solid tumour The dose of the extract of C. laevigatus was evaluated by a toxicity study and safe doses of 500 and 750 mg/kg body weight were determined. Animals were grouped in to four (6 animals/group). Group 1 served as untreated control, group 2 received cyclophosphamide (10 mg/kg), group 3 received C. laevigatus methanol extract (500 mg/kg) and group 4 received C. laevigatus methanol extract (750 mg/kg). The drug treatment was stared next day after the induction of tumour and continued for 10 days. The tumour development on animals in each group was determined by measuring the diameter of tumour growth in two perpendicular planes using Vernier calipers every third day for 4 week. 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.8.2 Ascites tumourEhrlich’s Ascites Carcinoma cells were aspirated from peritoneal cavity of the tumour bearing mice and 0.1 ml containing 1x106 cells was injected intraperitoneally into the Swiss Albino mice. Injecting EAC cells into the intra peritoneal cavity of mice produced ascites tumour. Animals were grouped in to four groups (6 animals/group). Group 1 served as untreated control, group 2 received cyclophosphamide (10 mg/kg), group 3 received C. laevigatus methanol extract (500 mg/kg) and group 4 received C. laevigatus methanol extract (750 mg/kg). The drug treatment has stared next day after the induction of tumour and continued for 10 days. The animals were observed for the development of ascites tumour and death due to tumour burden was recorded for 30 consecutive days. 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.
  • The effect of an anti-oxidant on DPPH radical scavenging was thought to be due to their hydrogen donating ability. DPPH, a stable free radical accepts an electron/hydrogen to become a stable diamagnetic molecule.
  • Superoxide anion derived from riboflavin reaction reduces NBT and the decrease of absorbance at 560 nm with anti-oxidants indicates the consumption of superoxide anion in the reaction mixture.
  • The degradation of deoxyribose to TBARS by hydroxyl radicals generated from Fe3+/ascorbate/EDTA/H2O2 system markedly decreased by C. laevigatus methanol extract.
  • EVALUATION OF ANTI-OXIDANT AND ANTI-TUMOUR ACTIVITIES OF CROTON LAEVIGATUS Vahl.

    1. 1. BY DAWN V TOMY, B.Pharm., Reg.No: 26106393 1RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    2. 2. 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 PHARMACOLOGYSubmitted by DAWN. V .TOMY, B. Pharm., Reg.No: 26106393 Dept. of Pharmacology RVS College of Pharmaceutical Sciences, Sulur, Coimbatore. 2RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    3. 3. Under the guidance ofInstitutional 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. 3RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    4. 4. INTRODUCTIONThe nature has provided mankind the greatestgift, ‘Medicinal Plants’. They play an important role in thepreservation of our health by curing infectious anddegenerative diseases. The medicinal properties of the plantsare due to the presence of bioactive compounds, producedas by-products of primary metabolism. These compoundsprotect the plants from infectionsmicroorganisms, pests, various climatic stress conditions andother hazards in the environment. They also contribute tothe development of new drugs. Hence, it has always been ofgreat interest to the scientists working on various disease 4conditions. PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE RVS COLLEGE OF Friday, March 15, 2013
    5. 5. The systematic screening of the plant, C. laevigatus Vahl.The plant has been reported to be used in the traditionalChinese medicine. Roots and leaves of the plant wereusually used by the Dai people (China) as a traditional drugfor treating fractures, malaria, stomach ache and woundsreceived from falling. Almost 29 compounds were isolatedfrom the methanol extract of its leaves. Cytotoxicity ofditerpenoids isolated from C. laevigatus were initiallyinvestigated on HeLa cell line by applying MTT method andfound modest cytotoxicity against HeLa cells (Zou etal., 2010). No other pharmacological research has beenreported on this plant. 5 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    6. 6. 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. 6RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    7. 7. AIM AND OBJECTIVES•Phytochemical determination of biologicallyactive compounds from C. laevigatus Vahl.•Determination of sub-acute toxicity ofmethanolic extract of C. laevigatus.•Analysis of cytotoxicity and anti-tumouractivities of C. laevigatus.•Evaluation of anti-oxidant and anti-inflammatory properties of C. laevigatus. 7RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    8. 8. PLAN OF WORK Sub-acute toxicity studies 8RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    9. 9. 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 9 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    10. 10. MATERIALS AND METHODS1. Collection of plant sample The leaves of C. laevigatus Vahl.was collected from the Thrissur districtof Kerala. The plant was identified by Dr.P. Sujanapal, Taxonomist, Kerala ForestResearch Institute (KFRI), Peechi.The sample was dried in shade andpowdered using mixer grinder. 10RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    11. 11. 2. Preparation of extract The powder was extractedwith methanol by maceration. Theextract was filtered throughWhatman #1 filter paper and thefiltrate was evaporated todryness, dissolved in NormalSaline (NS) and used for biological 11analysis.RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    12. 12. 3. Phytochemical screeningQualitative phytochemicalscreening of plant extracts wasdone The extract of the plant wassubjected to chemical tests for theidentification of various activeconstituents 12RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    13. 13. 4. Determination of in vitro cytotoxicityof leaves of C. laevigatus extracts (trypan bluedie 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 13RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    14. 14. 5. In vitro anti-oxidant activities The anti-oxidant properties ofthe plant extract was analyzed bydetermining the scavenging effectsof free radicals such assuperoxide, hydroxyl and DPPHradical with in vitro assay systems. 14RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    15. 15. DPPH radical scavenging assay:Principle In its radical form, DPPH has an absorption band at 515 nm, whichdisappear on its reduction. Scavenging of DPPH radical by anti-oxidant compounds willdecrease the colour intensity, which can be compared with the control to get thepercent inhibition.Reagents Methanol and freshly prepared DPPH stored in brown bottleProcedure Different concentrations of extract of C. laevigatus were added to 0.375 mlof freshly prepared DPPH solution in methanol. The volume was made up to 2 ml withmethanol. After 20 minutes, the absorbance was measured at 515 nm. Thepercentage inhibition was calculated and concentration needed for 50% inhibition wasfound out. % of inhibition = OD of control - OD of test X 100 OD of control 15RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    16. 16. 5.2 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 generationby riboflavin. The super oxide radical will reduce the NBT to a blue coloured formazancomplex, which is measurable at 560 nm.Reagents: Phosphate buffer (0.06M, pH 7.8), EDTA (0.1M) containing 1.5 mg of sodiumcyanide 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 wereadded to the reaction mixture containing 3 μg NaCN in 0.1 M EDTA; NBT 50µg, 0.12 mMriboflavin and 0.6 M phosphate buffer (pH 7.8) in a final volume of 3 ml. The tubescontaining the reaction mixture were continuously illuminated with incandescent lamp for15 min. The Optical Density (OD) measurements were taken at 560 nm before and afterillumination. The effect of test material to inhibit superoxide generation was evaluated bycomparing the OD of control and treated samples. % of inhibition = OD of control - OD of test X 100 OD of control 16 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    17. 17. 5.3 Hydroxyl radical scavenging activityPrinciple: Hydroxyl radical scavenging was measured by studying the competition betweendeoxyribose and the test compounds for hydroxyl radicals generated from theFe3+/ascorbate/EDTA/H2O2 system (Fenton reaction). The hydroxyl radical attacks epoxyribose, 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 brownbottle, EDTA (0.1 mM), FeCl3 (0.1 mM) prepared freshly and stored in brown bottle, Ascorbicacid (0.1 mM) freshly prepared, deoxy ribose (2.8 mM).Procedure: The reaction system contained deoxyribose (2.8 mM), EDTA (0.1 mM), FeCl3 (0.1mM), KH2PO4 KOH buffer (20 mM; pH 7.4) and from 100 to 1000 μg/ml of the test materialin a final volume of 1 ml. The reaction mixture was incubated for 37o C for 1 hour and isestimated using a spectrophotometer at 530 nm.. The scavenging activity of hydroxyl radicalswas expressed as % of inhibition = OD of control – OD of test X 100 OD of control 17 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    18. 18. 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 daysand the following parameters were determined to evaluate thetoxicity.a) Mortalityb) Weight changec) Food and water consumptiond) Hematological functione) Liver Functionf) Renal Function Adult Swiss albino mice of age approximately 6 to 8weeks old with weights in the range of 25-30g were used forexperiment. 18RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    19. 19. Parameters estimatedLiver Function Test (LFT) Method1. 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 methodRenal Function Test (RFT)1. Serum Urea Urease calorimetric kinetic method2. Creatinine JAFFE-kinetic method 19 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    20. 20. Hematological profile Blood was analyzed to determine the variations inhematological parameters like haemoglobin, RBC andplatelet using haematology analyzer (Model ABX-Micro-S-60). The total white blood cells (WBC) were measuredafter diluting the blood in Turk’s fluid and counting themusing a haemocytometer under magnification bymicroscope (10 X). To measure the differential count, blood wasspread on a clean slide and stained with Leishman’sstain. Various types of cells were counted using amicroscope under magnification (100 X). 20 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    21. 21. 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’. 21RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    22. 22. 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 animalswas calculated using the formula: % Increase in life span (ILS) = (T-C)/C 100, where T and C are mean survival oftreated and control mice. 22RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    23. 23. Statistical analysis The values are presented as mean SD.Differences between group’s means wereestimated using a one way analysis ofvariance followed by Tukey test, usingGraph Pad In stat Software. The resultswere considered statistically significant whenP<0.05. 23RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    24. 24. 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 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    25. 25. 2. In vitro cytotoxicity The C. laevigatus methanol extract showed marked cytotoxicityactivity against DLA, EAC and Jurkat cell lines. The concentrationrequired for 50% death (IC50) was found to be 37, 52 and 87µg/mlrespectively. 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. 25RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    26. 26. 100 90 80 70 60 50 % of inhibition 40 IC50 = 52μg/ml 30 20 10 0 0 25 50 75 100 125 150 Concentration Of Drug In µg/ml Figure 3: Cytotoxicity effect of C. laevigatus against EAC cell lines. 26RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    27. 27. 100 90 80 70 60 % of inhibition 50 IC50 = 87μg/ml 40 30 20 10 0 0 25 50 75 100 125 150 Concentration Of Drug In µg/ml Figure 4: Cytotoxicity effect of C. laevigatus against Jurkat cell lines. 27RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    28. 28. 3. In vitro anti-oxidant activities In vitro anti-oxidant activities of C. laevigatus methanol extract was evaluatedusing DPPH, superoxide and hydroxyl radical scavenging assays. The results wereexpressed in IC50 values.3.1 DPPH radical scavenging activity The reduction capability was determined by reduction in the absorbance at 515nm. The stable free radical DPPH was effectively scavenged by C. laevigatus methanolextract 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.RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    29. 29. 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 Concentration of drug (µg/ml)Figure 6: Effect of C. laevigatus crude extract in inhibiting superoxide radical scavenging 29 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    30. 30. 3.3 Hydroxyl radical scavenging activity The concentration of the extract needed for 50% (IC50) inhibition of hydroxylradical 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 Concentration of drug (µg/ml)Figure 7: Effect of C. laevigatus crude extract in inhibiting hydroxyl radical scavenging. 30RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    31. 31. 4. Acute toxicity study Croton laevigatus methanol extract treatment caused no deaths in anyof the groups. Organ weight, food and water consumption, hemoglobin content and totalWBC count were found to be almost similar to that of control. Biochemical assays for liverand kidney functions also revealed the safety of C. laevigatus methanol extract as an anti-oxidant. The administration of C. laevigatus methanolic extract did not produceany mortality at doses of 500 and 1000 mg/kg of body weight for 14 days. This indicatesthat the extract prepared from C. laevigatus, even at very high concentration was not lethalto 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 31RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    32. 32. Administration of extract at doses 500 and 1000mg/kg of body weight did not produce anydecrease 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. 32RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    33. 33. Administration of C. laevigatus methanolic extract at doses 500 and 1000mg/kg did notproduce 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 1 st 4 th 7 th 10 th Days Figure 9: Effect of C. laevigatus methanolic extract on food consumption of animals. 33RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    34. 34. 9 Normal 8 Water consumption ml/cages(5animals) CL- 500mg/kg 7 CL-750mg/kg 6 5 4 3 2 1 0 1 st 4 th 7 th 10 th 34 DaysFigure 10: Effect of C. laevigatus methanolic extract on water consumption of animals.RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    35. 35. Administration of C. laevigatus methanolic extract at doses of 500 and1000 mg/kg of body weight did not produce any change in the weightof 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±0.052C. laevigatus 1.42±0.19 0.415±0.036 0.097 ±0.016 0.145±0.021 0.289±.05methanolic extract(500 mg/kg)C. laevigatus 1.54±0.19 0.415±0.036 0.097± 0.02 0.151±0.017 0.305±0.071methanolic extract(1000 mg/kg) 35RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    36. 36. 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 damageTable 4: Effect of C. laevigatus methanolic extract on Liver Function test. Values are mean ± SD of5 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±0.24C. laevigatus 149.5± 50.53 82.9±19.4 0.2±0.88 7.48±1.2 3.54±0.19 2.5±0.34methanolic 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±0.29methanolic extract(1000 mg/kg) 36 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    37. 37. Administration of C. laevigatus methanolic extract at doses of 500 and 1000 mg/kg of bodyweight for 14 days did not produce any change in the renal function as the creatinine levelwas similar to that of normal. This indicates that C. laevigatus methanolic extract did notproduce 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 ± 0.09C. laevigatus methanolic extract (500 mg/kg) 0.48 ± 0.2C. laevigatus methanolic extract (1000 0.52 ± 0.17mg/kg) 37RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    38. 38. Administration of C. laevigatus methanolic extract for 14 days, at doses of 500 and 1000mg/kg did not produce any change in hematological parameters in the animals such astotal, WBC, RBC, platelet, haemoglobin as well as the differential count. This againindicates that C. laevigatus methanolic extract high doses were found to be non toxic tohematological 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) 38RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    39. 39. 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±64.78C. laevigatus 5974.8±412.25 196.2±40.31 204±44.2 1064±126.5 210±72.1methanolic extract(500 mg/kg)C. laevigatus 5780.6±246.85 224±74.81 196±33.8 1664±39.1 273±19.4methanolic extract(1000 mg/kg) 39 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    40. 40. 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. Valuesare 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 inhibitionControl 0.2185±0.0110 0.3647±0.0519 0.1462 ___Diclofenac 0.2010±0.0189 0.2665±0.0192*** 0.0655 55.19C. laevigatus methanolic 0.2082±0.0215 0.2867±0.0149** 0.0785 46.29extract (500 mg/kg)C. laevigatus methanolic 0.2035±0.0168 0.2755±0.0268*** 0.0720 50.74extract (750 mg/kg) 40 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    41. 41. 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 areMean ± 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 thicknessControl 0.1936±0.0154 0.4000±0.0350 0.2064 ___Diclofenac 0.1888±0.0184 0.3204±0.0144*** 0.1316 36.24C. laevigatus 0.1898±0.0061 0.3430±0.0103** 0.1532 25.78methanolic extract(500 mg/kg)C. laevigatus 0.1938±0.0147 0.3312±0.0189*** 0.1374 33.43methanolic extract(750 mg/kg) 41 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    42. 42. 6 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 volumeon the 30th day of inoculation in control group. The tumour volume in the mice treated with 500 and750 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. 42RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    43. 43. 6.2 Effect of C. laevigatus methanol extract on ascites tumour development The animals of the tumour control group inoculated with EAC cells survived fora period 16±1.095 days. The C. laevigatus methanol extract at the 500 and 750 mg/kgbody weight was found to be inhibiting the proliferation of EAC with the percentageincrease in life span by 60.44 and 84.38%. In cyclophosmide treated mice (10 mg/kg), thepercentage increase in life span was found to be 93.75%.Table 10: Effect of C. laevigatus methanol extract on average life span of ascites tumourbearing mice. Values are Mean ± SD; n=6 in each group (***) p<0.001, (**) p<0.01 ascompared 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 43RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    44. 44. DISCUSSION•The plant C. laevigatus spread along Indiato China have been in use as traditional anti-inflammatory medicine it has never beenstudied for its pharmacological activityexcept for the diterpenoids which is found inmost of the Crotons and the less abundantneocrotocembraneic acid. 44RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    45. 45. •The extract showed cytotoxicity towards the DLA, EAC andJurkat cells with an IC50 value of 37, 52 and 87µg/ml, respectively.•The extract on anti-oxidant screening showed significantactivity with an IC50 value of 130, 110 and 890 µg/ml withDPPH, superoxide and hydroxyl radical assay, respectively.•The acute toxicity studies showed the safety profile of theextract. The extract at doses of 500 and 1000 mg/kg did notproduce any mortality in mice; it also showed that thehaematological and non-haematological parameters weresimilar to that of normal. 45RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    46. 46. •It has been already reported that diterpenoids andlaevigatbenzoate isolated from C. laevigatus has shownmodest cytotoxicity towards HeLA cell lines (Zou et al., 2010).•The cytotoxicity exhibited towards the DLA, EAC and Jurkatcell lines in the present study may be due to the presence ofthe same constituents.•The polyphenols present in the C. laevigatus may responsiblethe anti-oxidant property. 46RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    47. 47. •The inflammation in the carageenan inducedinflammatory model has significantly reducedin paw thickness on the 3rd hour which iscomparable to the standard diclofenac.•The formalin induced chronic inflammationhas considerably reduced on the 3rd daywhich is comparable to standard dicofenac. 47RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    48. 48. •The anti-inflammatory activity may be due to thepresent of the β-sitosterol which is a cholesterolanalogue (Zou et al., 2010).•The steroids are reported to inhibit thephospholipase A2 which prevent the synthesis ofinflammatory mediators.•The lactonecembranoids belonging to theneocrotcembraneic acid isolated from the C.laevigatus is known to posses inhibitory action oncAMP phosphodiesterase may also support the anti-tumour activity. 48RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    49. 49. •The lactonecembranoids which belongs tothe neocrotocembraneic acid previouslyisolated from the stem bark of Crotonoblongifolius showed considerable inhibitionof cAMP Phosphodiesterase (Sophon etal., 2002) which is also isolated from C.laevigatus (Zou et al., 2010) may beresponsible for its significant anti-inflammatory activity. 49RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    50. 50. •The anti-tumour analysis was performed based onthe cytotoxicity activity observed in varioustransformed cells.•The anti-tumour studies showed that the extractposses significant activity. It has increased thepercentage life span by 60.44 and 84.38% in 500and 750 mg/kg extract treated mice.•It has also reduced the tumour volume by 58.35and 62.02% in 500 and 750 mg/kg extract treatedmice on the 30th day of DLA inoculation in mice. 50RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    51. 51. •The acute toxicity studies have proved the safetyprofile of C. laevigatus and all the parametersassessed was similar to that of the untreatednormal. It also signifies the importance of the plantC. laevigatus as potent anticancer drug.•The anti-oxidant activity along with its anti-inflammatory effect has established its safeness tobe used for further studies. 51RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    52. 52. CONCLUSION•A considerable increase in life span in ascites tumourbearing mice and percentage decrease in the tumourvolume in the solid tumour model in a dose dependentmanner when compared with the standard drug isevident for the anti-tumour activity of the plant C.laevigatus Vahl.•The toxicity study result showed very little changeswhen compared with normal result along with the anti-oxidant and anti-inflammatory activity has establishedthe bio-safety profile of the plant, supporting its use inthe future studies to determine its hidden potentials. 52 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    53. 53. •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. 53RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    54. 54. BIBLIOGRAPHY• Abo KA, Ogunleye VO and Ashidi JS (1999). Antimicrobial potential of Spondias mombin, Croton zambesicus and Zygotritonia crocea. Phytotherapy Research, 13(6): 494-497.• Amrithpal S (2007). Herbal Medicine-Dream unresolved. Pharmacognosy reviews 1(2), 375-377.• Baderschneider B and Winterhalter P (2001). Journal of Agriculture and Food Chemistry 49: 2788.• Bisset NG (1991). Journal of Ethnopharmacolology, 32:71.• Block S, Stevigny C, Llabres G, deHoffman E, Adjakide V, DePauw-Gillet M and Quertin-Leclercq J (2002). Ent-Trachyloban-3β-ol, a new cytotoxic diterpene from Croton zambesicus. Plantae Medica, 68:647-648.• Choi EM and Hwang JK (2005). Effect of some medicinal plants on plasma anti- oxidant system and lipid levels in rats. Phytother Res 19:382-386.• Di Rosa M, Giroud JP and Willoughby DA (1971). Studies of the mediators of the acute inflammatory response induced in rats in different sites by carrageenin and turpentine. Journal of Patholgy 104, 15. 54RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    55. 55. • Elizabeth K and Rao MNA 1990. Oxygen radical scavenging activity of curcumin. International Journal of Pharmaceutics 58, 237-240.• Guo AZ, Hong WZ, Haji AA, Jun SY, Chao ZP and Zhong MZ (2011). Laevigatbenzoate from Croton laevigatus Vahl. Journal of Natural Medicine, 65:391-394.• Halliwell B and Gutteridge JMC (1995). Protection of oxygen radicals in Biological systems and free radicals in biology and medicine.• Ialenti A, Ianaro A, Moncada S and Di Rosa M (1995). Modulation of acute inflammation by endogenous nitric oxide. European Journal of Pharmacology 211, 177- 184.• Logani MK and Davis RE (1979). Lipid peroxidation in biologic effects and anti- oxidants. A Rev Lipids 15:485-93.• Mitchell RN and Cotran RS (2000). Robinsons Basic Pathology. 7th edition, 33-42.• Newman DJ et al. (1997). Journal of Natural Products, 60: 52.• Okokon JE and Nwafor PA (2009a). Antiplasmodial activity of root extract and fractions of Croton zambesicus. Journal of Ethnopharmacology, 121: 74-78.• Osbourn AE, Qi X, Townsend B and Qin B (2003). Dissecting plant secondary metabolism - constitutive chemical defences in cereals. New Phytol., 159, 101-108.• Samir M and Amrit PS (2007). A review of pharmacology of phytochemicals from indian medicinal plants. The Internet Journal of Alternative Medicine 5, 1. 55 RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    56. 56. •Santos PML, Schripsema J, Kuster, RM (2005). glycosylated flavonoids from Crotoncampestris 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 plantteas recommended as nutritional source for element supplementation. Acta Aliment34:161-167.•Website: Phytochemicals for cancer prevention.•Wong C, Li H, Cheng K and Chen F (2006). A systematic survey of anti-oxidantactivity of 30 Chinese medicinal plants using the ferric reducing anti-oxidant powerassay. Food Chemistry 97, 705–711.•Zhang L and Demain AL (2005). Natural products drug discovery and therapeuticmedicine. (ed) Springer.•Zheng W and Wang SY (2001). Anti-oxidant activity and phenolic compounds inselected herbs. J. Agric. Food Chem, 49:5165-5170.•Zou GA, Aisa HA, Zhang HW, Yang JS and Zou ZM (2012). Chemical composition ofCroton 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. 56RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013
    57. 57. 57RVS COLLEGE OF PHARMACEUTICAL SCIENCES, SULUR, COIMBATORE Friday, March 15, 2013

    ×