BIOASSAY SCREENING OF TRADITIONALLY USED MEDICINAL PLANTS OF KASHMIR VALLEY AND ISOLATION OF ANTIPROLIFERATIVE & APOPTOSIS INDUCING COMPOUNDS.

By:

ShariQ Ahmad M.Sc BiochemistryUniversity of Kashmir

IntroductionCancer is the leading cause of mortality, strikes more

than one- third of the world’s population & is cause of more than 20% of all deaths

India results about 5,80,000 cancer related deaths every year

In males oral, lung, & stomach and in females cervical, breast & oral cancers are prone.

Plant materials have been used for the treatment of malignant diseases for centuries.

Plant derived medicines are better tolerated by patients & have less side effects.

An effective tool for the treatment of ovarian and breast cancer is the best example to bench mark the importance of medicinal plants for curing cancer.

MIRACLE DRUG Pain fever reducer Blood Thinner against strokes Acts against Cancer Acts against Alzheimer

Origin of proposal

Since the mortality rates of people with cancer are so high , the discovery of new drugs is a priority.

A plethora of new chemical structures with diverse biological activates can be found through natural products research.

Approximately half of the drugs that are in clinical use today can be traced back to either a natural product source or they are based on a natural product template

Plants under study

Lavatera cashmeriana

Delphinium cashmerianum

Rhododendron campanulatum.

REASONS FOR THE SELECTION OF PLANTS

Depending upon the information gathered from traditional healers.

Their usage at folklore claims.

These plants contain the free radical scavenging properties.

Review of the Literature

Lavatera cashmerian-protease inhibitors have been found in vitro to strongly inhibit trypsin,chymotrypsin and elastase that contribute to tumor invasion & metastasis.

S.Rakhshanda etal. (Asian Pacific J Cancer Prev, 14

(6), 3975-3978) University of

Kashmir.

Studies conducted 0n a thia traditional medicine ,found that a fraction from Methanolic extract of Delphinium cashmeriana , has anti proliferative effect on SRKB3 and MDA-MB435 human breast cancer cells.

(Pudoham et al.,2007)

Plant Monograph

Common Name: sazposh/sazmoolFamily: MalvaceaeHabitat : Forest clearings, meadows, shrubberies etc.

Distribution : Himalaya : Kashmir, Gilgat Botanical features : Perennial herb, Stem upto 2 m tall, Leaves 3-5 lobed.

Medicinal  uses :in throat problems, The herb is given as a mild laxative,The roots are collected in large quantities and sold as crude drug in market.

Available at:Jammu and Kashmir Medicinal Plants Introduction CentreIst street, Nambalbal, Pampore PPR J&K 192121PO Box 667 GPO Srinagar SGR JK 190001

Family: Ranunculaceae Common name: Kashmiri Larkspur

Plant Classification: perennial herb, Minimum Height: 25 cm Maximum Height: 30 cm Distribution: Asia, the Western Himalayas, Kashmir and Tibet, ranges from 3000 to 4800 metres.

Medicinal Uses: Different parts especially root is used as medicine by the tribal people.

Plant Monograph

Delphinium cashmerinum

Available at:Jammu and Kashmir Medicinal Plants Introduction CentreIst street, Nambalbal, Pampore PPR J&K 192121PO Box 667 GPO Srinagar SGR JK 190001

Plant Monograph

Rhododendron campanulatum

Family: Ericaceae

Plant Classification: evergreen shrub, Minimum Height: 25 cm Maximum Height: 30 cm Distribution: found on open slopes in the alpine zone at elevations between 2,800 and 4,400 meters.

Medicinal Uses: leaves are used as a snuff in the treatment of colds and headaches that affect only one side of the head. juice of the leaves is also used in the treatment of chronic rheumatism, sciatica and syphilis.

Specific Objectives

isolation and characterization of the bio-active molecule(s).

To investigate the anticancer activity of isolated compounds against murine tumour models.

To study the measurement of plasma membrane destabilization & detection of changes in mitochondrial membrane potential.

DNA CELL CYCLE ANALYSIS THROUGH CELL FLOW CYTOMETRY

INVIVO STUDIES IN MURINE CANCER MODELS

MEASUREMENT OF MEMBRANE DESTABLISATION DURING APOPTOSIS

CYTOTOXIC ASSAY

CELL SURVIVAL ASSAY

PREPARATION OF PLANT EXTRACT

Methodology

Cell survival assay

Carried out as described by( Sun et al,. in 1997)

Cancer cell lines will be allowed to grow on tissue culture in presence of test material

The cell growth will be measured on ELISA reader after staining with SRB dye

Cytotoxic assay

Cytotoxic assay based on MTT will be done

In mitochondria of viable and glycolytically active cells MTT is reduced to insoluble, purple formazan

The formazan product can be solubilized and OD can be measured which reflects number of cells present at the end of assay period

Measurement of membrane destabilization

This event can be detected by flow cytometry using Annexin-v which is antibody against PS

The fluorochrome( isothiocynate, biotin)attached to antibody gives signal that can be detected

DNA cell cycle analysis through cell flow cytometry

Cytostatic drugs can inhibit the progress of cell cycle in G1 phase & induce apoptosis.

DNA content changes as the cell progresses through G1-S-G2 phases.

It can be analyzed through flow cytometery using fluorescent dye (propidium iodide)

Invivo studies in murine Cancer modelsEAC cells will be maintained in the peritoneal cavity of swiss albino mice will be collected from an animal having 8-10 days old ascitic tumour by aspirating ascitic fluid.

Mice will be selected for the experiment & will be injected EAC cells.

On day 1 the animals will be randomized & divided into different groups.

The test material prepared in solution form will be given both orally & intraperitoneally

All the animals will be sacrificed & peritoneal fluid will be collected & its volume will be determined in each animal.

%tumor inhibition : Av. No. of cells in control group _ Av No of cells in test group ------------------------------------------------------------------------------ x 100

Av. No. of cells in the control group

Importance of the proposed studyNumerous Drugs which are currently in use for cancer chemotherapy exhibit:

Cell toxicity genotoxic effects Carcinogenic effects Teratogenic effects

These side effects limit the use of chemotherapeutic agents despite their high efficacy in treating target malignant cells

Therefore the search for using plant extracts that are both effective & non toxic has been increased.

Budget particularsS.No

Budget (in Rs) TOTAL

Head1st Year 2nd Year 3rd Year

Total

1. Salaries/Wages 2,40,000@20,000p.m.

2,40,000@20,000p.m.

2,40,000@20,000p.m.

7.20 Lac

2.Consumables

5.0 Lac 4.0 Lac 3.0 Lac 12.0 Lac

3.Travel (Within India)

0.3 Lac 0.3 Lac 0.3 Lac 0.90 Lac

4 Contingency (Including Research Publication charges) 1.0 Lac 1.0 Lac 1.0 Lac 3.0 Lac

5Equipments and Chemicals Nil Nil Nil Nil

6Overhead Costs (@20% of project cost)

1.54 Lac

1.54 Lac 1.54 Lac 4.62 Lac

Total cost: Rs 23.10 Lac Overhead Costs (@20% of project cost: Rs. 4.62 Grand total: 27.72 Lac

Cancer affects everyone, and its up to all of us to support the important research that can one day

make a much sought –after cure a reality.

Angie Harmon