Summer Report

“IN VITRO CYTOTOXICITY OF NATURAL PRODUCTS AGAINST HUMAN CANCER CELL LINES”

UNDER THE GUIDANCE OF

Dr.A.K.SAXENA, SCIENTIST F Department Of Pharmacology INDIAN INSTITUTE OF INTEGRATIVE MEDICINE (CSIR),

BY NIKHIL DEEP SINGH B.Tech (2nd Year) IIT KANPUR® 1

#INTRODUCTION CANCER: Cancer (medical term: malignant neoplasm) is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancers differentiate them from benign tumors, which are selflimited, and do not invade or metastasize. Most cancers arise from stem cells or early progenitor cells. Stem cells exist at specific locations within a given tissue (niches) and are permanently resident at this location. They have a proliferative potential that exceeds an individual’s life time ( Renetan and Potten ., 2001).

CAUSES: Abnormalities in the genetic material of the transformed cells are the main cause. (Kinzler, Kenneth W.; Vogelstein, Bert 2002.) This may be due to the effects of carcinogens, such as tobacco smoke, benzene, radiation, chemicals, or viruses e.g.; HPV. Other cancer-promoting genetic abnormalities may be randomly acquired through errors in DNA replication, or are inherited, and thus present in all cells from birth. The heritability of cancers is usually affected by complex interactions between carcinogens and the host's genome.

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GENES RESPONSIBLE

When proto-oncogene are mutated they become oncogene. Often a whole series of these genes become activated into oncogenes (Dollinger., 1997), giving the cells new properties, such as hyperactive growth and division, protection against programmed cell death (Apoptosis), loss of respect for normal tissue boundaries, and the ability to become established in diverse tissue environments. It’s apparent that epigenetic abnormalities in the expression of these genes also play an important role in carcinogenesis (Weinsten., 2000).

Tumor-suppressor genes. (p-53, p-21) are then inactivated in cancer cells, resulting in the loss of normal functions in those cells, such as accurate DNA replication, control over the cell cycle, orientation and adhesion within tissues, and interaction with protective cells of the immune system.

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TYPES AND TREATMENT: Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor.Examples of general categories includes: 

Carcinoma: Malignant tumors derived from epithelial cells. This group includes the common forms of breast, prostate, lung and colon cancer.



Sarcoma: Malignant tumors derived from connective tissue, or mesenchymal cells.



Lymphoma and leukemia: Malignancies derived from hematopoietic (bloodforming) cells



Germ cell tumor: Tumors derived from totipotent cells. In adults most often found in the testicle and ovary; in fetuses, babies, and young children most often found on the body midline, particularly at the tip of the tailbone; in horses most often found at the poll (base of the skull).

The four most deadly cancers are lung, stomach, liver, and colorectal (Parkin et al., 2001). More than 35% of cancer cases in men are related to the oral cavity, larynx and pharynx (all tobacco related), and about 40% and 30% of cancer cases in women are cervical and breast cancer, respectively, in India. (Rao et al., 2002). Leukemia and lymphoma are malignant tumors of hematopoetic cells of the bone marrow and account for 9% of cancer. Benign cell is a hyperplasia or non malignant lesion that is often premalignant (Doll and Pelo., 1981).

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There are different types of therapies available Surgery e.g.; biopsy, Cryosurgery – (Extreme cold to kill cancer cells), Radiation Therapy (use of ionizing radiation to kill cancer cells and shrink tumors ), Cancer Vaccines, Herbal Therapy (A recent survey lists over 1400 genera of herbs that have a history of use in cancer treatments. (Hartwell, 1967, 1971), Hormonal Therapy (providing or blocking certain hormones), Gene Therapy, Immunotherapy (diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the tumor), Chemotherapy

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#REVIEW OF LITERATURE: Cancer drug is a major transition from the previous pre genomic cytotoxic era to the new post genomic targeted era ( Workman et al., 2001 ; Guillemard et al., 2004; Sawyers et al 2004; Segota et al., 2004; Pesgram et al 2005) . Some e.g. of currently marketed targeted drugs for cancer therapeutics are shown in this table. Trade Name Trastuzumab (Herceptin )

Company

Mechanism

Genentech

Humanized

monoclonal

antibody against HER 2 Imatinib (Gleevec)

Novartis

Small molecule Inhibitor of Bcr-Abl and C-kit tyrosine kinases

Gefitinib (Iressa)

Astrazeneca

Small molecule Tyrosine kinase inhibitor of EGFR

Cetuximab (Erbitux)

Bevacizumab (Avastin)

Imclone/Bristol-Myers

Chimeric

squibb

antibody against EGFR

Genetech

monoclonal

Humanized monoclonal antibody against vascular endothelial growth factor (VEGF)

Bortezomib (Velcade)

Millennium

co- Small

developed

molecule

with Proteasome inhibitor

Johnson

-

Johnson

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and

DEVELOPMENT OF ANTICANCER DRUGS FROM NATURAL SOURCES: • Paclitaxel:

1. A Chemical discovered from the pacific Yew tree Taxus brevifolia in 1971 (Wani et al.,1971) 2. Effective drug for the treatment of breast and ovarian cancers. 3. It binds specifically to the beta-tubulin subunit of microtubules (Schiff et al., 1979; Horwitz 2004) • Camptothecin:

1. Alkaloid first found in Camptotheca acuminata. 2. Activity in colorectal, ovarian, and small cell lung cancer (Takimoto and Arbuck., 2001). 3. Enhances binding of topo-isomerase I to DNA, thus promoting DNA strand breaks. (Cragg and Newman. 2004) 7

• Mitomycin C:

1. Antibiotic isolated from Streptococcus caespitosus 2. Mitomycin contains an aziridine group and a quinone group in its structure, as well as a mitosane ring, and each of these participates in the alkylation reactions with DNA. (Verweij et al... 2001) • Doxorubicin ( Adiriamycin ):

1. Produced by the fungus Streptococcus peucetius var . caesius 2. Have been used

primarily in the acute

leukemias , whereas

doxorubicin displays broader activity against human neoplasm, including a variety of solid tumors

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#IN VITRO STUDIES: SOME OF THE COMMON CANCER CELL LINES. CANCER TYPE

CELL LINES

BREAST

MCF-7

CERVIX

HeLa, SiHa

CNS

IMR-32, , SK-N-SH, SNB-78,SF-295

COLON

Colo-205, HCT-15, HT-29, SW-620, 502713

LEUKEMIA

K-562, MOLT-4

LIVER

Hep-2

LUNG

A-549, HOP-62, H226

PROSTATE

DU-145, PC-3

ORAL

KB

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Materials and Methodology

• Chemicals Required: Growth medium (RPMI), Fetal calf serum, Trypsin , PBS , Tryphan blue, Ethanol, Penicillin , Streptomycin , Gentamycin, DMSO , Trichloroacetic acid,

Distilled

water,

Sodium

Hydroxide,

Tris-EDTA

Buffer,m

Sulphorhodamine , Tris buffer, Acetic acid , Sodium bicarbonate, Mitomycin C ,Palcitaxel (taxol), 5 Fluorouracil , Hydrochloric acid

,

Isopropanol , Tris-Acetate-EDTA Buffer.

• Apparatus: Tissue culture flasks, Incomplete growth medium (RPMI), 6-well flat bottomed culture plates Micropipettes 1.5 ml and 0.5 ml eppendorf centrifuge tubes 15ml centrifuge tubes 50 ml centrifuge tubes, 96-Well cell culture plates ,Sterile centrifuge tubes, Cryo vials, Glass bottles to store media etc. Glass pipettes Syringe

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Reagents:  Prepare RPMI-1640 with 2 mM glutamine medium or MEM in double distilled water. PH is adjusted to 7.2. Add penicillin (100 units/ ml dissolved in PBS) and sterilize by filtering through 0.2 µm filters in sterile laminar flow. Store the media in refrigerator (2-8 o C).  Complete growth medium: Contains 10 % FCS and 1% Penicillin. The amount of FCS may vary depending upon the requirements of cell lines used. Freezing Medium for cryopreservation contains 20 % FCS and 10 % DMSO in growth medium (RPMI or MEM).

 Phosphate Buffer Saline (PBS): Dissolve 9.6 gm/lt. in distilled water. PBS is used to prepare solutions of Penicillin and Trypsin EDTA.

 Penicillin Solution: Dissolve 100 units/ml or 625 mg/ ml in PBS. Penicillin is mixed in RPMI Medium to avoid contamination.

 Gentamycin Solution: Dissolve 50 mg/ ml in PBS. Gentamycin is added in the medium used in the preparation of dilutions of the test sample to avoid contamination because of plant extracts.

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 Trypsin EDTA: 0.05% Trypsin and 0.02% EDTA (disodium salt) are dissolved in PBS. Trypsin EDTA is used to detach the cells while sub-culturing and splitting the cell line.

 TCA: 50% (w/v) TCA solution is prepared in double distilled water. TCA is used for fixing the culture cells before washing.

 Acetic acid: Prepare 1% in distilled water. Acetic acid is used to prepare solution of SRB dye crystals and to remove unbound dye from cells after staining.  SRB Dye: Dissolve 0.4% in 1% acetic acid. SRB Dye is used to stain the basic proteins of cancer cells fixed by 50% TCA.

 Tris buffer: 10 mM (pH 10.5). Tris buffer is used to dissolve protein bound dye.

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INSTRUMENTS:

Laminar Air Flow

Microscope

Elisa Reader

Hemocytometer

Cryovial 13

• Hemocytometer (designed for the counting of blood cells and other cells),Cryo-containers(for storing cells),

Water

bath(for

incubation),

Filtration assembly, Deep freezer, Mechanical shaker, Centrifuge, Autoclave, ELISA Reader , Liquid Nitrogen Cylinder, CO2 Gas Cylinder, CO2 Incubator

• Handling of cell lines on arrival: The cells are first grown in complete RPMI growth medium then incubated in CO2 incubator (37oc, 5% CO2, 90% R.H) and observed under microscope, for apparent contamination and proper growth. After monolayer formation cells are harvested and then stored in Liquid N2 at -196oc.

• Revival and seeding of cells: The desired cryovial is removed from liquid nitrogen cylinder and thawed. Nearly 10 ml of complete growth medium is poured into the tissue culture flask. The cells are added to tissue culture flask. Mixed properly and incubated at 37oC, 5% CO2 atmosphere and 90% R.H.

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STAGES OF CELL GROWTH DURING CULTURING

 Attachment Stage: Within 24 hours of incubation after seeding or trypisinization, the cells get attached to the base of tissue culture flask.  Sub-confluent Stage: It is a stage of rapid growth of cells. In this stage, some space remains in between the growing cells. The cells are in log phase of their growth and are used for experimental purposes.  Confluent Stage: The medium turns turbid as nutrients are utilized & cell debris and metabolic wastes accumulate in the medium. When the cells are observed under microscope, they are seen to form a complete monolayer.

Sub-culturing of cell lines: Sub-culturing of cell lines Is done by trypsinisation to maint ain the cells in log phase of growth. The cells are detached when they reach subconfluent stage and to make single cell suspension for experimental purposes. The trypsinisation of a cell line should not be done more than 5 times as the cells lose their potential to grow.

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 Preparation of test material:

 Stock solution (depending upon required conc.) DMSO is used for dissolving 95 % MeOH / EtOH extracts, 50% aqueous DMSO for 50% Aqueous MeOH/EtOH extracts and distilled water for hot water extracts. The working solutions of test samples are prepared in the complete growth medium. Gentamycin (50 mg/ml) is added to check the microbial contamination in growth medium.  Positive control is essential for comparison of the activity of the test material with the already established drugs against cancer e.g.; a) 5-fluorouracil: It is a pro-drug undergoing a series of biotransformation reactions to ribosyl and deoxyriboxyl nucleotide metabolites. One of these metabolites is 5-fluoro-2-deoxyuridine-5monophosphate (FdUMP) which gets incorporated in to RNA where it interferes with r-RNA and m-RNA translation.

b) Mitomycin-C: It is an antibiotic isolated from Streptomyces caespitosus. It is an alkylating agent that undergoes metabolic activation through an enzyme-mediated reduction, to generate an alkylating agent that cross-links DNA.

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Blank (negative) Control: Blank control is essential to find the OD of medium and thus that can be reduced from total OD to get the OD of the test sample. Gentamycin media and test sample solution are added but cell suspension is not added.

Determination of Cytotoxicity: In a single 96 well plate, the Cytotoxicity of sample can be determined on 2 cell lines at a time. The number of plates depends on the number of test samples

*96 Well Plate

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• Preparation of cell suspension for the assay: Skehan et al., (1990) • Grow the desired human cancer cell line in tissue culture flasks at 370 C, in an atmosphere of 5% CO2 and 90% relative humidity in complete growth medium to obtain enough number of cells. • Select the flask with sub-confluent stage of growth. • Harvest the cells by treating the cells with Trypsin- EDTA. • Count the number of cells/ ml of suspension with the help of haemocytometer. • Adjust the cell density to 10,000 cells /100 ml (or the standard density depending upon cell line) in the cell suspension. • Add 100 µl of cell suspension to each well of 96 well plates with the help of a handy-step. • Incubate the plates at 370 C, in an atmosphere of 5% CO2 and 90% relative humidity for 24 hours. • After 24 hours the 100 µl of working solution of each test material is added to the wells of 96 well plates. • The wells 1 to 4 and 9 to 12 of first row in each plate is control growth (CG i.e. blank) i.e. having the cell suspension and medium. The growth of the cells in these wells is maximum as no test sample is added.

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• The second row of one plate have 100 µ/well positive control (PC) i.e. having 5-FU or Mitomycin C etc. The growth of these wells is less, as it is the known inhibitor of cells.

Addition of test material:

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• If different concentrations of the same test sample are used, they should be in the successive wells in the same plate. • 100 ml of working solution of the test material is added to rest of the wells excluding the first row of each plate and where the negative control (1%gentamycin medium) has been added. • Incubate the plates for 48 hours at 37oC, in an atmosphere of 5% CO2 and 90% relative humidity. • Determine the cell growth after 48 hours of adding sample • To stop the reaction, gently add 50 µl of chilled 50% TCA (trichloroaceticacid ) to each well of the plate, making final concentration of 10%.

• Incubate the plates at 4C for one hour to fix the cells attached to bottom of the wells.

• Wash the plates 5-6 times with distilled water.

• Plates are air-dried.

• Add 100 µl of SRB dye(0.4%in1%acetic acid) to each well of the plate and leave the plates at room temperature for 30 minutes

• Wash the plates with 1% acetic acid after 30 minutes.

• Plates are again air-dried.

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• 100 µl of Tris buffer (10.5M) added to each well.

• Shake the plates gently for 10- 15 minutes on a mechanical shaker. • Record the optical density with ELISA reader at 540nm wavelength and maintain the data

• Cytotoxicity

assay

by

Sulforhodamine

B

Dye:

*.figure depicting the absorbance phenomenon of Elisa reader where hѵ is the energy of the photon coming in and hѵ’ the energy of photon that reflects back after absorbance by the cells having Sulphorhodamine B bound to them. 21

• SRB assay is a rapid, sensitive and inexpensive method for measuring the cytotoxic potential of test substances, based on the cellular protein content of adhered suspension cultures in 96 well plate. This method is suitable for ordinary laboratory purposes and for large-scale applications like high throughput in vitro screening in anticancer drug development.

• Principle of assay: SRB is a bright pink aminoxanthene dye with 2 sulphonic groups. Under mild acidic conditions, Sulphorhodamine B binds to the protein’s basic amino acid residues in TCA fixed cells to provide a sensitive index and cellular protein content. The anticancer activity is determined by the cytotoxic potential of the test material using human cancer cell line, which is allowed to grow on tissue culture plate in the presence of test material

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PROCEDURE Seeded human cancer cell lines (96 well plates)  Added test samples  Stopped cell growth  Stopped reaction with 50% w/v TCA  Kept at 4*C for 1hr. and washed with distilled water and left the plates to dry @ room temp.  Added SRB into plates and kept for 30 minutes.  Washed plates with 1% acetic acid, left the plates to dry@ room temp.  Added tris buffer to dissolve bound dye  Read plates on ELISA READER at 540 nm 23

#5.RESULTS AND DISCCUSIONS: RESULT -1 IN VITRO CYTOTOXICITY AGAINST HUMAN CANCER CELL LINES CELL LINE TYPE CCL Codes 10809 10810 10811 10812 10813 10814 10815 10816 10817 10818 10819 10820 10821 10822 10823 10824 10825 10826 10827 10828 10829 10830 10831 10832 10833 10834 10835 10836 10837 10838 10839 10687 10688 10689

Inst. Codes RBH 1762 P13 A001 RBH 1762 P13 A002 RBH 1969 P01 A001 RBH 1982 P01 A001 RBH 1982 P01 A002 RBH 2298 P13 A001 RBH 2307 P04 A001 RBH 2310 P03 A003 RBH 2321 P04 A002 RBH 2325 P14 A002 RBH 2467 P02 A003 RBH 2467 P09 A001 RBH 2476 P01 A002 RBH 2470 P13 A001 RBH 2470 P13 A002 RBH 2470 P13 A003 RBH 2485 P09 A001 RBH 2566 P09 A001 RBH 2575 P13 A001 RBH 2575 P13 A002 IHB 1422 P02 A001 IHB 1422 P02 A002 IHB 1422 P02 A003 IHB 1423 P02 A001 IHB 1423 P02 A002 IHB 1423 P02 A003 IHB 1423 P03 A001 IHB 1423 P03 A002 IHB 1423 P13 A001 IHB 1423 P13 A002 IHB 1423 P13 A003 RJO 1888 P13 A001 RJO 1888 P13 A001 RJO 1888 P13 A001 5-Fu Paclitaxel Adriamycin Mitomycin-C

Colon

Lung

Liver

Conc 502713 A-549 Hep-2 (μg/ml) 100 24 35 45 100 20 32 41 87 84 100 63 100 46 30 44 100 23 12 29 71 100 37 41 100 15 0 21 100 4 60 20 75 100 65 54 100 23 41 22 100 28 48 30 100 61 52 57 100 48 60 42 100 26 19 22 100 7 0 13 100 27 13 33 100 37 10 50 100 23 15 33 97 91 100 43 94 86 100 31 100 41 0 28 100 15 20 8 100 51 51 12 93 100 49 36 91 100 40 31 93 87 100 54 97 100 44 63 92 100 26 32 73 100 40 42 80 100 18 40 83 76 100 49 83 100 39 41 100 50 39 35 100 32 1 12 2X10-5M 53 -5 1X10 M 63 1X10-6M -5 1X10 M 69 24

Neuroblastoma

IMR-32 0 15 65 24 1 18 0 23 38 31 19 28 29 15 16 6 27 19 56 41 0 3 24 50 29 99 64 38 22 23 48 80 28 41 72 -

DISCUSSION: Remarks: Stock solution (20 mg/ml) was prepared in DMSO, 50%DMSO and water and further dilution was carried out with medium. In vitro cytotoxicity against 4 human cancer cell lines was determined using SRB assay. Criteria of activity: >70% growth inhibition at 100 µg/ml against three cell lines Discussion: All the samples have been evaluated at 100 µg/ml and one sample (IHB 1423 P02 A003) shows such growth inhibition.

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RESULT 2: Cell line type CCL CODES 10926 10927 10928 10929 10930 10931 10932 10933 10934 10935 10943 10944 10945 10946 10947 10948 10949 10950 10951 10952 10953 10954 10955 10956 10957 10958 10959 10960 10961 10962 10963 10964 10965 10460 10461 10462 10463 10464

INST. CODES RLB 1241 P05 A001 RLB 2401 P02 A001 RLB 2401 P02 A002 RLB 2401 P02 A003 RLB 2424 P14 A001 RLB 2424 P14 A002 RLB 2424 P14 A003 RLB 2425 P14 A001 RLB 2425 P14 A002 RLB 2425 P14 A003 IHB 1423 P01 A001 IHB 1423 P01 A002 IHB 1423 P01 A003 IHB 1423 P03 A003 IHB 1423 P04 A001 IHB 1423 P04 A002 IHB 1423 P04 A003 IHB 1423 P14 A001 IHB 1423 P14 A002 IHB 1423 P14 A003 IHB 1424 P13 A001 MAP 2601 P13 A001 MAP 2601 P13 A002 MAP 2601 P13 A003 MAP 2603 P02 A001 MAP 2603 P02 A002 MAP 2603 P02 A003 MAP 2603 P03 A001 MAP 2603 P03 A002 MAP 2603 P03 A003 MAP 2609 P13 A001 MAP 2609 P13 A002 MAP 2609 P13 A003 RJO 1932 P13 A001 RJO 1932 P13 A002 RJO 1932 P13 A003 RJO 2351 P13 A003 RJO 2360 P13 A001 5-FU

Conc. 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 100ug/ml 1X10-6M 26

Colon Lung Liver Prostrate 502713 A-549 Hep-2 PC-3 GROWTH INHIBITION% 59 17 60 60 19 2 49 41 0 2 25 17 0 14 12 13 64 42 59 62 62 32 56 56 0 12 0 0 0 9 6 9 0 4 5 22 0 13 0 16 17 66 79 80 17 2 25 9 43 90 95 93 68 33 68 77 38 91 94 86 32 83 90 73 19 77 91 79 43 89 95 83 18 82 91 75 14 94 99 94 4 42 88 70 61 17 57 34 0 16 47 13 54 95 98 95 60 35 44 49 18 27 14 29 34 40 89 72 13 24 27 35 35 26 30 27 3 9 10 7 17 14 37 12 16 20 25 13 15 0 0 28 47 6 37 36 14 2 23 12 68 20 50 75 0 30 0 14 11 21 39 39 44 31 24 33

PACLI MITO-C

1X10-5M 1X10-5M

64 49

60 23

88 69

48 29

DISCUSSION: Remarks : Stock solution (20 mg/ml)was prepared in DMSO,50%DMSO and water and further dilution was carried out with medium. In vitro cytotoxicity against 4 human cancer cell lines was determined using SRB assay. Criteria of activity: >70% growth inhibition at 100 µg/ml against three cell lines Discussion : All the samples have been evaluated at 100 µg/ml and eight(IHB 1423 P01 A003, IHB 1423 P14 A002, IHB 1423 P14 A003, IHB 1423 P04 A001, IHB 1423 P04 A003, IHB 1423 P14 A001, IHB 1423 P04 A002, MAP 2601 P13 A003) samples show such

growth inhibition.

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CALCULATIONS AND DISCUSSIONS: The viability and growth in the presence of test material is calculated as:

OD Change = Mean of OD of Test sample – Mean of OD of Blank

% Growth in presence of the Control = 100 / OD change in presence of control

% Growth in the presence of Test sample = (% Growth of in presence of control) X (OD change in presence of test sample) % Inhibition by the Test sample = 100 - % Growth in the presence of Test sample

Growth in presence of test material =

------------------------------------------------------- X 100 Growth in absence of test material

T / C value for 50% growth inhibition for each test material is calculated from its three concentrations

Criteria for activity: The test material showing >70% activity at 100ug/ml is considered to be active

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