BIOLOGICAL SCREENING OF HERBAL DRUGS FOR ANTI-

CANCER ACTIVITYPRESENTED BY,

SHAFNA HUSSAINFIRST YEAR MPHARM

JAMIA SALAFIYA PHARMACY COLLEGE

BIOLOGICAL SCREENING OF HERBAL DRUGS FOR ANTI CANCER ACTIVITY

INTRODUCTION Cancer is uncontrolled division of cells. It has the ability to invade other tissues either by

direct growth into adjacent tissue , through invasion or by implantation by metastasis.

Metastasis is the stage in which cancer cells are transported through the bloodstream to sites far removed from the original site.

Treated with a combination of surgery ,chemo therapy & radio therapy.

Many non toxic herbs are used now a days thus reducing toxic effects of chemo & radio therapies.

HERBAL DRUGS AS ANTI CANCER AGENTS

Madagascar periwinkle ( Catharanthus roseus or Vinca roseus )

Taxus baccata :Arrest multiplication of cancerous cells by cross linking.

Curcumin :Inhibit the growth of cancer by preventing pdn of harmful eicosanoids.

Vinca alkaloids & podophyllin : Act by breaking the cytoskeletal organelle.

CYTOTOXICITY SCREENING

TWO MODELS ARE THERE :

INVITRO METHOD INVIVO METHOD

ADVANTAGE OF INVITRO OVER INVIVO

Can be screened about 85000compounds in a short term assay.

Provide information about the fun of specific organs &how a toxic agent can adversely affect them.

More specific Accurate Less time consuming

INVITRO METHODSTHREE CATEGORIES1.To indicate drug has altered cell viability.a) Trypan blue dye uptakeb) Lactate dehydrogenase (LDH) leakage.c) Neutral red dye retension.d) Propidium iodide or Flourescent marker attachment to double-stranded nucleic

acids.

2.To assess if the drug has an effect on cell growth & cell proliferation.e) [³H]thymidine uptake assay.f) Total DNA synthesis measured by [1251] Udr uptake.g) Cell cycle analysis with propidium iodide.3. To assess whether the toxicant has affected cellular

metabolic processes.h) MTT dye conversion by mitochondria.i) Alamar blue reduction by respiring cells.j) Fluorescent dye loss.

To indicate drug has altered cell viability

1.Trypan blue dye uptake: Simplest & cheapest method Qualitative & indicates only if a cell is activePRINCIPLE: Based on the ability of a cell with an intact plasma

membrane, to exclude trypan blue dye which is impermeable to healthy cells but can permeate through compromised membrane of dying cells.

Can be counted manually using a haemocytometer. Detects loss of memberane integrity during later

stages of cell death.

PROCEDURE:1. 20µl cells +20µl trypan blue dye in an Eppendroff tube.2. Mixture is stand for 5 min.3. Surface of heamocytometer is cleaned with 70%

alcohol & cover slip is placed.4. Each chamber is allowed to fill by capillary action.5. Total no: of stained cells & total no: of cells in the four 1

mm squares are counted.6. Cells which touches the top & left middle line are

included.7. The cells in the 4 corner squares in both chambers are

countedCells/ml =avg cell count per sq × dil. Fac × 10% Cell viability =[total viable cells (unstained cells) ÷ total

cell]×100Dil fac=[vol of sample+vol of diluent]÷vol of sample

To assess drug has an effect on cell growth

[³H] Thymidine uptake to cell growth: Relatively complex Sensitive procedure Requires use of radio-labelled chemicals. Used as an indication of cytotoxicity because

toxicants can decrease the rate of cell proliferation.

Used as an indicator of cell proliferation as [³H]thymidine incorporates to the nucleic acids of proliferating cells.

PROCEDURE:1. 1×10 cells are placed wells of a flat bottomed 24well micro titer plate.2. Allowed to settle for 24 hrs.3. Medium from the wells are replaced with 1ml serum free chemically

defined medium ,24-48 hrs before treating cells with test cpds.4. 50µl drug/vehicle control is added.5. 50µl/well thymidine soln is added for last 2hrs of incubation with the

test substance & incubated @ 37˚c.6. Reaction is stopped by placing on ice bath & cells are washed 3 times

with 1ml ice cold serum free medium7. Then carefully removed from the medium by vaccum aspiration.8. 1ml of ice cold trichloro acetic acid is added to each well.9. The plates are left on ice for 10 min to allow precipitation of DNA &

protein.10. 1ml 10%TCA is added & left ice for again 5min.11. Washed again with TCA.12. Then TCA is removed.13. Precipitated protein is solubilised with 0.5ml of 0.3N sod.hydroxide by

incubating for 30-60 min @ room temp.14. 250µl is transferred to a mini- scintillation vial, with 5ml scintillation

fluid and count.

To assess whether the toxicant has affected cellular metabolic processes MTT dye conversion by mitochondria:[3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazoliumbromide] A std colorimetric assay for measuring cellular proliferation. Quantitative & very sensitive test. Growth &death rates can be measured easily.

PRINCIPLE:Based on the ability of a mitochondrial dehydrogenase

enzyme present in viable cells to cleave the tetrazolium rings of the pale yellow MTT & form dark blue formazan crystals which are largely impermeable to cell memb thus resulting in its accumulation with in healthy cells.

PROCEDURE:o 500-10000 cells plated in 200µl media per well in a 96

well plate.o 8 wells are kept as blank.o Incubated (37˚C,5% carbon dioxide) overnight.o 2µl drug + DMSO is added to each well.o Placed on a shaking table @150rpm for 5min.o 20µl MTT soln is added to each well.o Incubated 2hr @ 37˚C.o 100µl of lyses buffer is added to each well.o Incubate overnight @ 37˚C.o Quantification done on a micro plate reader @

wavelength 600nm.% Cell viability= (absorbance of treated grp ÷

absorbance of control grp) × 100.

INVIVO METHODS Common models are rats. Models are ;A. Models of spontaneous & chemically induced

colorectal cancer (CRC).B. Models of chemically induced breast cancer.C. Hollow fibre assay.D. Xenograft tumour models.E. Orthotropic & metastasis tumour models.F. Autochthonous models.G. Genetically engineered cancer models.

Models of spontaneous & chemically induced colorectal cancer(CRC)

Frequently used carcinogens are dimethylhydrazine (DMH), its metabolite azoxy methane (AOM),MNNG,MNU.

71 Sprague-Dawley/Wistar rats (20mg/kg/s.c.)- 20 wks injected with DMH .

Barium enema is performed to see the size of tumour. Animals divided to 3 grps & subjected to 5-FU, 1(2-

tetra hydro furyl)-5 FU(FT) & mixture of UFT & FT. After 5 wks barium enema was repeated. Response was assessed on basis of tumour doubling

time. Response rate 5-FU, FT, UFT were 25%, 33%,36%.

Models of chemically induced breast cancer

1-methyl-1-nitrosourea (MNU) is the carcinogen used to induce adenocarcinomas in the rat΄s mammary glands.

It induced in female Sprague-Dawley rats by the i.p.administration of MNU (50mgMNU/kg bodywt) @21 days of age.

Divided into 4 grps.• Ad libitum fed.• Restriction of calorie intake 90%, 80%, 60% of ad libitum

intake.• Energy restriction (ER) reduces the ductal extension of the

mammary gland into fat pad.• Animal΄s breast density is predictive to its carcinogenic

response.• ER inhibits cell proliferation & induce apoptosis in

premalignant &malignant mammary gland lesions.

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