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BRIEF RESUME OF THE INTENDED WORK
Need for the study:
Cancer is the second leading cause of death in the world1. Throughout history and across the world, the plant kingdom has provided a variety of medicines for cancer treatment, currently over 60٪ of the drugs are derived in one or other way from natural source including plant, marine organism and micro-organism. There are worldwide efforts to discover anticancer agents from plants2.
Cancer is a disease characterized by uncontrolled multiplication and spread of abnormal forms of body’s own cells. It is one of the major causes of death in the developed countries. One in three people will be diagnosed with cancer during their life time and in 2001, 270,000 new cases were reported in the UK with lung and bowel cancer comprising the large category closely followed by breast and prostate cancer1. Heart diseases have declined by 45%, but increasing cancer death still is continued since 1997 according to the data base of the National Cancer Institute Survival Epidemiology and End {NCISEE}3.
Remarked differences can be found in incidence of cancers depending upon genetic and environmental factors. The carcinogenicity by UV rays and chemical agents are the major causes for cancer4. Cigarette smoking is probably the most significant single factor that contributes to the development of cancer5. Cancer can be treated with chemotherapeutic agents along with radiation and surgery. The chemotherapeutic agents provide a temporary improve of symptoms and signs of cancers and most of the chemotherapeutic agents are given in combination with the radiation therapy and surgery, but patient has to consume for the long duration and these drugs are more toxic and economically burden to the patients6.
Several molecules isolated from various marine organisms (algae, fungi, invertebrates and vertebrates) are currently under study at an advanced stage of clinical trials, either directly or in the form of structural modification. Some of them have already been marketed as drugs7. The novel anti-tumor agent trabectedin and aplidine are under phase III clinical trials8,9. With the above the background main goal of the present study is to explore the anticancer activity of Matricari recutita. an in vitro and in vivo evaluation in EAC bearing mice.
6.2 REVIEW OF LITERATURE:
Plant Profile:
Title of the plant : Matricaria recutita Linn.
Family : Asteraceae (Compositae)
Synonyms : Matricaria, German Chamomile, Sweet False Chamomile’s.
Habitat : German Chamomile is annual herb originally from Europe which has to the wild and now naturalized in almost every continent. It can now be found growing along fence rows and in sunny open field from Southern Canada to Northern U.S. West to Minnesota.
Chemical constituents:
The plant contains Terpenoids like α-Bisabolol, α-Bisabolol oxide A and B, Chamazulene, Sesquiterpenes. Flavonoids like Apigenin, Luteolin, Quercetin, Coumarins, Umbelliferone and others are Anthemic acid, Choline, Tannin and Polysaccharides.
Medicinal uses :
Matricaria recutita Linn. has been used medicinally for thousands of years and is widely used in Europe. It is a popular treatment for numerous ailments including sleep disorders, anxiety, digestion, intestinal conditions, skin infections, inflammations (including eczema), wound healing, infantile colic, teething pains and diaper rash. In the USA, Chamomile is the used externally for wounds, ulcers, eczema, gout, neuralgia, rheumatic pain, hemorrhoids and leg ulcers etc 10.
Pharmacological actions :
Matricaria recutita is the potentially used as an antioxidant 11, neuroprotective12, diabetic complications13, cardiac effects 14, antimicrobial activity 15, anti proliferative and apoptic activity16. Cardiovascular effects as increase atrial rate, relaxed thoracic aorta17, hemodynamic effects and anti ulcer activity18. Chamomile’s main active constituents are apigenin and bisabolol. Apigenin have cardio protective activity 19, 20.
6.3 Objectives of the study:
The main goal of the present study is to evaluate the anticancer activity of Matricaria recutita. species with the following objectives:
In vitro evaluation of anticancer activity of the extract of Matricaria recutita. species.
In vivo evaluation of anti cancer activity of Matricaria recutita extracts in EAC and DAL Tumour cells bearing mice.
Effect of the Matricaria recutita. extracts on physiological and haematological parameters in EAC EAC and DAL Tumour cells bearing mice.
The effect of the Matricaria recutita. extracts on biochemical and histopathological parameter changes in EAC and DAL Tumour cells bearing mice.
METHODS AND MATERIALS:
7.1. Source of data: All the data will be collected from the experimental animal model and the animals will be subdivided into following groups and the dose of extract of Matricaria recutita.. was selected on the basis of acute toxicity test as per OECD guidelines.
I Anticancer activity of Matricaria recutita. against Ehrlich Ascites Carcinoma bearing Balb/c mice
a) Group I : Normal/positive control (n=8)
b) Group II : Tumor control (n=8)
c) Group III : 5-Flurouracil (n=8)
d) Group IV : Low dose of methanol extract (n=8)
e) Group V : Moderate dose of methanol extract (n=8)
f) Group VI : High dose of methanol extract (n=8)
II Anticancer activity of Matricaria recutita. against Dalton’s lymphoma Ascites (DLA) bearing Balb/c mice
a) Group I : Normal/positive control (n=8)
b) Group II : Tumor control (n=8)
c) Group III : 5-Flurouracil (n=8)
d) Group IV : Low dose of methanol extract (n=8)
e) Group V : Moderate dose of methanol extract (n=8)
f) Group VI : High dose of methanol extract (n=8)
7.2 Materials :
Animals : Female Balb/c mice
Chemicals : All the chemicals of AR grade
Instruments : Haemocytometer, Binocular microscope, Centrifuge machine etc.
7.3 Methods:
Preparation of Plant extract:-
The plant will be authenticated and collected from NBRI, Lucknow in ideal conditions will be air dried under the shade and powdered to a fine texture of uniform size by passing through the sieve # 44. The powder collected will be extracted with polar and non polar solvent and the extract obtained will be used for the present study.
In vitro studies: Tryphan Blue Exclusion Method (Cell viability test)21:
This is one of the methods to assess cytotoxicity of anticancer compounds. It comes under preliminary screening of anticancer compounds. This test is based on the principle that the living cell membrane has the ability to prevent the entry of dye. Hence, they remain unstained and can be easily distinguished from dead cells, which take the dye.
Method: For EAC
The Ascitic fluid was withdrawn from animals which were induced with EAC cell lines. Ascitic fluid of 0.1 ml was aspirated and it was diluted with 2ml of phosphate buffered saline (PBS). To this equal volume of different extracts were mixed and this mixture was incubated for 3hrs at 370C. Then the mixture was added to 0.5ml of tryphan blue and mixed thoroughly. The diluted suspension was charged into haemocytometer. The viable cells (unstained) were counted in a WBC chamber under a microscope and the mean numbers of cells in four chambers were calculated as follows
Total number of cells = Mean number of cells Dilution factor (2) ×10 4
Method: For DLA22
Cell lines:
Dalton s Lymphoma Ascites (DLA) tumour cells were obtained through the courtesy of Amala Cancer Research Centre, Thrissur, Kerala, India.
In vitro cytotoxic assay
DLA was maintained by serial transplantation from mice to mice .The ascetic fluid of the DLA was drawn out from the donor mice carrying tumour for 7-9 days.the freashly drawn ascetic fluid from the peritoneal cavity was washed thrice with phosphate buffer saline (PBS,pH7.4) and diluted in PBS to a concentration of 106 cells/ml.One million cells were incubated with various concentrations of the herbal extract() in a total volume of 1ml for 3 hours at 370C. 0.1% carboxy methyl cellulose (CMC) was used as control.After incubation the viability of the cells was determined by the tryphan blue exclusion method23
Effect of ethanolic herbal extract residues on ascites tumour bearing animals
BALB/c mice (20-25 g) were obtained from the Breeding section, Amala Cancer Research
Centre, Thrissur. They were kept in groups of six in well ventilated cages in air controlled room, fed with normal mice chow (Sai Feeds, Bangalore, India) and water adlibitum. All animal experiments were conducted after permission from the Institutional Ethical Committee. DLA cells were aspirated from the peritoneal cavity and washed three times with PBS. Ascites tumour cells were then inoculated to three groups (6 animals / groups) of BALB / c mice by injecting one million cells in to their peritoneal cavity. After 24 h of tumour inoculation two different dosages of herbal extract residues were given and continued for ten consecutive days.
Group I : Untreated control for DLA
Group II : DLA cells + 10 mg/kg b.wt of Matricaria recutita herbal extract residue in 0.1% CMC (ip) – low dosage
Group III : DLA cells +50 mg/kg b.wt of Matricaria recutita herbal extract residue in 0.1% CMC (ip) – high
The death pattern of animals due to tumour burden was noted every day and the percentage of increase in life span was calculated using the formula, T-C/C X 100 where ‘T’ and ‘C’ are the number of days the treatedand control animals survived respectively24 .
Haematological parameters
On the 15th day post tumour inoculation, blood was collected in heparinzed tubes from the respective animals by tail vein and the haematological parameters such as white blood cell count, red blood cell count and percentage of haemoglobin were determined by following the standard procedures .24
Tumour cell count
0.1 ml of the ascitic fluid was aseptically withdrawn on the 15th day post tumour inoculation using a 1 ml syringe and diluted with 0.9 ml of PBS (pH 7.4) to adjust the cell count to 1x106 cells. From the stock 0.1 ml was taken and mixed with 0.8 ml of PBS and then incubated for 3 hours at 37 0C. 0.1 ml of tryphan blue was then added to this Then a drop of the resulting solution was loaded on the heamocytometer and the number of tumour cells randomly in every 100 cells was counted 24.
Estimation of SGPT and SGOT
On 15th day post tumour inoculation, blood from the tail vein of treated and control mice was withdrawn individually into test tubes which were then centrifuged at low speed for about 10-15 min to collect the serum for enzyme analysis following the method 25.
In vivo Anticancer activity of extracts:
Induction of Ehrlich Ascites Carcinoma:
The Ascitic carcinoma bearing mice (donor) was taken 15 days after tumor transplantation. The Ascitic fluid was drawn using an 18-gauge needle into sterile syringe. A small amount was tested for microbial contamination. Tumor viability was determined by Trypan blue exclusion test and cells were counted using haemocytometer. The Ascitic fluid was suitably diluted in normal saline to get a concentration of 106 cells/ml of the tumor cell suspension. This was injected by intraperitonial route to obtain ascitic tumor. The mice were weighed on the day of tumor inoculation and then for three each days. Treatment was started 24 hours after tumor inoculation. 5-Flurouracil (Standard) and extracts were administered till the 9th day by intraperitonial route26.
Induction of DLA27:
DLA was maintained by serial transplantation from mice to mice .The ascetic fluid of the DLA was drawn out from the donor mice carrying tumour for 7-9 days.the freashly drawn ascetic fluid from the peritoneal cavity was washed thrice with phosphate buffer saline (PBS,pH7.4) and diluted in PBS to a concentration of 106 cells/ml. Ascites tumour cells were then inoculated to three groups (6 animals / groups) of BALB / c mice by injecting one million cells in to their peritoneal cavity. After 24 h of tumour inoculation two different dosages of herbal extract residues were given and continued for ten consecutive days.
A]. Physical parameters :
Percentage increase in body weight as compared to day-0.
Median survival time (MST).
Percentage increase in life span (%ILS) 28.
Mean survival time (MEST).
Cell viability test (% survivors of malignant cells in ascitic fluid) 29.
B]. Haematological parameters30.
Total WBC, DLC, RBC and Hemoglobin content.
C] Biochemical parameters :
After the collection of the blood samples the mice were sacrificed and their liver was excised, rinsed in ice-cold normal saline followed by cold 0.15 mol/lit Tris-Hcl buffer (pH 7.4), blotted dry and weighed. The homogenate 10% w/v was prepared in 0.15 mol/lit Tris-Hcl buffer and a portion of it was utilized for the measurement of LPO31 and other portion of the same after precipitating with TCA was used for the estimation of glutathione. The remaining homogenate was centrifuged at 1500 rpm for the estimation of SOD, catalase and protein content32.
Statistical analysis:
FOR EAC:
All the data were expressed in mean + SEM. The significance of differences in mean between control and treated animals for different parameters was determined by using one way ANOVA followed by Dunnett’s multiple comparison (Post-hoc) test. Percentage tumor inhibition can be calculated by Chi-Square test.
FOR DLA:
Results were expressed as mean + standard deviation and Student’s ʻt’ test was used.
7.4 Does the study require any investigation or to be conducted on patients or other humans/animal? If so please describe briefly
Yes, the above study requires to be carried out in mice. The effects of preparation on cancer will be studied by considering physiological, pathological and biochemical parameters using animal models.
7.5 Has ethical clearance been obtained from your institution for performing various tests on animals?
Yes, the study is cleared from institutional animal ethics committee. copy is enclosed with protocol.
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