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The plant material consists of dried powdered bark of Hymenodictyon excelsum Roxb. belongs to the family Rubiaceae and dried powdered seeds of Apium graveolense Linn. belonging to the family Apiaceae.
Stem bark of Hymenodictyon excelsum Roxb. was collected from Thrissure district, Kerala, India and seeds of Apium graveolense Linn. collected from Institute of horticulture, Ooty, Tamilnadu district, India during the month of June 2010. The plant material was identified and authenticated by Mr. G. V. S Murthy, Joint Director, Scientist, C-I/C, Botanical survey of India, Tamil Nadu Agricultural University Campus, Coimbatore bearing the reference number BSI/SRC/5/23/10-11/Tech-657 and BSI/SRC/5/23/10-11/Tech - 730 respectively.
The fresh bark of Hymenodictyon excelsum Roxb. was collected. The collected bark was dried in shade under room temperature, powdered mechanically and sieved through No. 20 mesh sieve. The finely powdered bark was kept in airtight container until the time of use. The Fresh stem bark (1OOg) was exhaustively extracted with 95% MeOH (250 ml) using Soxhlet apparatus. It was filtered and the filtrate condensed under reduced pressure. The extract was concentrated to dryness under controlled temperature between 40-500C. The percentage yield of Hymenodictyon excelsum Roxb. bark (HE) was 8.55%w/w.
4.5. Extraction procedure for Apium graveolense Linn.
The fresh seeds of Apium graveolense Linn. was collected, dried in shade under room temperature, powdered mechanically and sieved through no. 40 mesh sieve. Powdered dried seeds (250g) were extracted in round-bottomed flask with 1000 ml petroleum ether (60-800C) and then repeatedly with methanol using a Soxhlet apparatus. Then, methanol extract was recovered and evaporated to dryness by distillation under reduced pressure in rotatory evaporator. The percentage yield of Apium graveolense Linn. seeds were 10.68%w/w.
4.6. Phytochemical screening
Chemical test were carried out for the bark of Hymenodictyon excelsum Roxb. and seeds of Apium graveolense Linn. for the presence of phytochemical constituents (Trease and Evans, 2002; Kokate, 1994).
4.6.1. Test for alkaloids
A small portion of the extract was stirred with few drops of dilute HCl and filtered.
To the filtrate, Mayer's reagent was added and a cream precipitate indicates the presence of alkaloids.
To the filtrate, Dragendroff's reagent (potassium bismuth iodide solution) was added and an orange brown precipitate indicates the presence of alkaloids.
4.6.2. Test for Glycosides
A portion of extract was mixed with few drops of Fehling's solution A and B and heated gently. A brick red precipitate indicates the presence of glycosides.
4.6.3. Test for flavonoids
To a portion of the extract, concentrated H2SO4 was added. A yellow colouration observed indicate the presence of flavonoids. The yellow colouration vanished on standing.
Few drops of 1% AlCl3 solution was added to a portion of extract. A yellow coloration indicates the occurrence of flavonoids.
A portion of the extract was heated with 10ml of ethyl acetate over a steam bath for 3min. the mixture was filtered and 4ml of the filtrate was shaken with 1ml of dilute ammonia solution. A yellow coloration indicates a positive test for flavonoids.
4.6.4. Test for tannins and phenolics
To the solution of the extract, added a few drops of 0.1% ferric chloride and observed for brownish green or a blue-black coloration.
4.6.5. Test for terpenoids
About 5ml of the extract was treated with 2ml of chloroform and 3ml concentrated H2SO4 was carefully added to form layer. A reddish brown coloration of the interface formed shows the presence of terpenoids.
4.6.6. Test for saponin
About 10ml of the extract was mixed with 5ml of distilled water and shaken vigorously for a stable persistent froth. Frothing then mixed with 3 drops of olive oil and shaken vigorously and then observed for the formation of an emulsion.
4.7. CYTOTOXICITY SCREENING
4.7.1. Cell Lines
Dalton's lymphoma ascites (DLA) cells were used for short term in vitro cytotoxicity experiments. This cell lines were maintained as ascites tumors in swiss albino mice. The cells were aspirated, washed thrice in normal saline counted using a haemocytometer and cell suspension of 1million cells/ml was prepared. One ml of this suspension was injected into peritoneal cavity of swiss albino mice. Mouse L929 cell lines (Lung fibroblast) were used for long term in vitro cytotoxicity experiments.
4.7.2. Short term in vitro cytotoxicity assay by trypan blue dye exclusion technique:-
Any compound, which is cytotoxic to cells, inhibits the cell proliferation and kills the cells. Trypan blue has the ability to penetrate in to the dead cells and give it blue color. This method gives an exact number of dead and viable cells (Kuttan et al., 1985).
Phosphate buffer saline( PBS)
Cells were aspirated from the peritoneal cavity of tumor bearing mice and it was washed three times using PBS. The viability of cells were checked using trypan blue (cell viability should be above 98%). The cell suspension was added to tubes containing various concentrations of the test compounds and the volume was made up to 1ml using phosphate buffered saline (PBS). Control tubes containing only cell suspension. These assay mixtures was incubated for 3h at 370C and then 1ml of trypan blue was added after incubation and the number of dead cell was counted using a haemocytometer. The percentage cytotoxicity was calculated using the formula (Shrivastava and Ganesh, 2010).
% cytotoxicity = [ ] x 100
4.7.3. Long term in vitro cytotoxicity by MTT assay
The ability of the cells to survive a toxic insult is the basis of most cytotoxic assays. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium. The assay depends both on the mitochondrial activity per cell and number of cells present. The cleavage of 3-(4, 5 dimethyl thiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) to a blue formazan derivative by living cells is clearly a very effective principle on which the assay is based.
The principle involved is the cleavage of tetrazolium salt, 3-(4, 5 dimethyl thiazole-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) in to a blue colored product (formazan) by mitochondrial enzyme succinate dehydrogenase. The amount of cells was found to be proportional to the extent of formazan production by the cells used (Ramnath et al., 2009).
L929 cell line
Microtitre plate (96 well)
Microplate reader (ELISA READER, Bio-rad)
Cells were seeded in a 96-well flat-bottom plate (5000 cells/well) and permitted to adhere for 24h at 370 with 5% CO2 atmosphere. Different drug concentration was added and incubated further for 48hrs. Before 4h of the completion of incubation, 20µl of MTT (5mg/ml) was added. Dead cell percentage was determined using an ELISA plate reader set to record absorbance at 570nm. The percentage growth inhibition was calculated using the formula given below (Hajighasemi and Mirshafiey, 2010).
% Growth inhibition =100-[ ] x 100
4.8. Apoptosis detection assay
Apoptois is the mechanism by which the cell undergo programmed cell death. It is a physiological program characterized by chromatin condensaion, membrane blebbing, cell shrinkage and DNA fragmentation (Sharma et al., 2007).
4.8.1. DNA ladder analysis
Benzyl pencillin and Ambistyrin
Saturated phenol (in NTE buffer)
Sodium Dodecyl Sulphate
Protein kinase K
Prepare DMEM medium (912mg/100ml) containing benzyl pencillin 1mg/litre, Ambistyrine (1mg/litre) adjust the pH to 7.0with sodium bicarbonate (colour deep yellowish red : when pink colour comes do not use the medium). To the medium add 2 X 106 DLA cells (suspended in minimal volume), 2µl trypsin and different drug concentration and then make the final volume to 2ml. incubate at 370C for various hours ( from 1-24hrs). After incubation centrifuge the cells at 10,000 rpm for 10mins, discard the medium and wash the cells for 2 times in NTE buffer. Suspend the cells in 2ml NTE buffer and 2% trypsin (100µg/ml) and add 20% SDS (25µl/ml) and proteinase K (100µg/ml). Incubate the cells at 370C for overnight. Add 1ml NTE buffer saturated phenol and 1ml chloroform and shake the vial 12 times (turning up and down slowly) centrifuge at 10,000 rpm for 10 min (2 phases are seen) and transfer the upper portion to another vial and add 1ml chloroform, repeat this for 4 times. To this RNase was added and incubated at 350C for 2hrs. Centrifuge the vial at 10,000rpm for 10mins and decant the solvent and take the pellet (DNA) and dissolve in TAE buffer. Then the dissolved DNA is subjected to horizontal electrophoresis (Lin et al., 2009).
4.8.2. Morphological studies
To detect the morphological changes during apoptosis, 5x106 DLA cells were incubated in DMEM at 370C in the presence of 5% CO2 for 48 hrs. After incubation, the cells were washed twice with PBS, centrifuged and the cell pellets were smeared on a clean glass slide. The slides were fixed in methanol and stained with hematoxylin-eosin method. The slides were observed at 100X for the changes (Zahri et al., 2009).
4.9. ANTICOAGULANT SCREENING
4.9.1. Prothrombin time (PT) assay
Tissue thromboplastin, in the presence of calcium ions, is an activator which initiates the extrinsic pathway of coagulation. When a mixture of tissue thromboplastin and calcium ions is added to normal anticoagulated plasma, the clotting mechanism is initiated leading to formation of a fibrin clot. If a deficiency exists within the extrinsic pathway, the time required for clot formation will be prolonged depending on the severity of the deficiency. The PT measures effects on the exogenous pathway of coagulation.
Plasma was acquired by centrifuging citrated blood for 15 min at 1500 - g. Thromboplastin-calcium reagent was reconstituted with distilled water according to the manufacturer's instructions. It is then prewarmed by placing it in a water bath at 370C for at least 10 min before commencement of the test. One hundred micro liters of plasma is placed in a test tube and incubated in the water bath for 180s. For the controls, 100µl of prewarmed PBS, followed by 200µl of the prewarmed thromboplastin-calcium reagent was rapidly pipette into the plasma while simultaneously starting a timer. The test tube was then gently twisted back and forth, until a clot formed, at which time the timer stopped and the clotting time recorded. For the tests, 100µl of the prewarmed sample solution in PBS was mixed with the plasma, just before adding the thromboplastin-calcium reagent (Manicam et al., 2010).
4.9.2. Activated partial thromboplastin time (APTT) assay
APTT is used for the estimation of coagulation factors such as VIII, IX, XI, XII and prekallikrein in the intrinsic blood coagulation pathway.
100µl of alexin (partial thromboplastin with activator, and calcium chloride (0.02M) were prewarmed to 370C) was added to 100µl of incubated (180s) plasma, and the contents were mixed rapidly. The mixture was then incubated for another 180s, after which 100µl of PBS, then 100µl of the prewarmed calcium chloride solution was added while simultaneously starting a timer. The tube was then permitted to remain in the water bath while gently tilting the test tube every 5s. At the end of 20s, the test tube was then removed from the water bath, wiped clean with dry gauze, and gently tilted back and forth until a clot was seen and the time recorded (Zhou et al., 2009).