Introduction to medicinal plants.

Medicinal plant Melia azedarach L.

Immunomodulation and immunomodulators

In- vitro & in-vivo tests for evaluation of

immunomodulators

MTT assay

Thesis work plan

India has a wealth of medicinal plants most of which have been traditionally used in Ayurveda, Unani & Sidhaa.

There are about 6000-7000 medicinal plant species(NMPB)in India, with concentrated spots in the region of Eastern Himalayas, Western Ghats and Andaman & Nicobar Island.

India is the largest producer of medicinal herbs and is called the botanical garden of the world.

25% of modern pharmaceutical drugs contain plant ingredients.

herbals are relatively inexpensive

need for alternative treatments for drug-

resistant pathogens

expanding contact and growing respect for

foreign cultures, including alternative systems

of medicine

High commercial value of medicinal plants.

phenolics 45%

alkaloids 18%

terpenoids & steroids27%

others 10%

Ref:International journal of herbal medicine

Common name:chinaberry, White cedar,

bakain, syringa, ghora neem, umbrella tree etc.

native to Southeast Asia and northern

Australia

deciduous tree growing to 50 ft. in height and

2 ft. in diameter.

Plant Parts Used : Leaf, flower, root, young

branches, fruit and bark.

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Magnoliopsida

Order: Sapindales

Family: Meliaceae

Genus: Melia

Species: azedarac

Medicinal activity Targeted organism or disease

Extracts Basic Reference

Larvicidal & anti- oviposition

Pediculus humanus capitis Leaf & Fruit Carolina et al, 2008, , Carolina et al. 2004, Cori a et al 2008,

Pediculicidal & Ovicidal

Pediculus humanus capitis Fruit Caprinella et al, 2003-2007

Antifeedant Pseudaletia unipunctataTrachiaplusia niSpodoptera eridania

Seed Seed Fruit

Akhtar et al 2008 Carpinella et al., 2002

Medicinal activity Targeted organism or disease

Extracts Basic Reference

Rodenticidal Albino rat Seed Leaves

Roop et al 2005, Keshri et al 2003, Keshri et al, 2004

Fungicidal Aspergillus flavusDiaporthe phseolorumSchlertina sclertiorum

Seed Carpinella et al, 1999-2003-2005

Antibacterial Proteus mirabilis Shigella flexeneriStaphyloccocus aureusBacillus subtilis

Leaf Abdul Viqar et al., 2008

Anti helminthal Haemonchus contortus Seed, Leaf Maciel, et al, 2006

Medicinal activity Targeted organism or disease Extracts Basic Reference

Antiviral Foot and mouth Disease Herpes simplex,(VSV) , (HSV)(VSV), (HSV1) Ocular Herpes simplex

Leaf Leaf Leaf Leaf

Wachsman et al., 2008 Wachsman et al., 1982 Alche, L. et al. 2002 Pifarri, et al. 2002

Antiprotozoal Trichomonas vaginalis Seed, Leaf

Lee et al., 2007

Anti hatchability Erias vitella Seed Gajmer et al, 2001

Immunomodulation is the the alteration of immune response

which may increase or decrease the immune responsiveness.

The immune response is how our body recognizes and

defends itself against bacteria, viruses, and substances that

appear foreign and harmful

An immunomodulators may be defined as a substance,

biological of synthetic, which can stimulate, suppress or

modulate any of the components of the immune system

including both innate and adaptive arms of the immune

response

2 types of immunomodulator

1. immunostimulators, are substances (drugs and nutrients)

that stimulate the immune system by inducing activation or

increasing activity of any of its components.

2. An immunosuppressant is any agent that weakens the

immune system.

1) In vitro

a) lymphocyte proliferation assay

Quantitates the response of T cells to a variety of stimuli

including mitogens, alloantigens and specific recall antigens.

b) Jerne’s plaque assay

an assay that enumerates individual antibody-forming

cells

c) IL-1 and TNF – alfa production by

macrophages in response to

lipopolysaccharides

d) quantitation of the expression of cell

activation markers on cell surface viz.

CD25,CD69,CD86 by fluorescent activated

cell sorting(FACS).

e) augmentation or inhibition of NK cell activity

in absense or presense of IFN- gama.

IN- VIVO:

a) estimation of specific antibody profile and titer to specific response.

b) Delayed Type Hypersensitivity (DTH) response to specific antigen

c) spleen foci (colony formation) assay for immunostimulant activity

d) Induction/progression of autoimmune disease in

experimental animal models for immunosuppresant

activity.

e) Prevention of allograft or xenograft rejection.

f) Evaluation in rodent malaria and japanese

encephalitis virus models for immunostimulant

activity.

MTT assay is a laboratory test and a standard colorimetric assay (an assay

which measures changes in color) for measuring cellular proliferation (cell

growth).

It can also be used to determine cytotoxicity of potential medicinal agents

and other toxic materials.

Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide, a tetrazole) is reduced to purple formazan in the mitochondria of

living cells.

A solubilization solution (usually either dimethyl sulfoxide, an acidified

ethanol solution, or a solution of the detergent sodium dodecyl sulfate in

dilute hydrochloric acid) is added to dissolve the insoluble purple formazan

product into a colored solution.

Fig: A microtiter plate after anMTT assay increasing amount ofcells resulted in increased purplecolor

Establishment of chicken lymphocyte cell line

culture

In – vitro test for evaluation of immunomodulator

Expression analysis of cell cytokines

Making of aqueous/methanol extract from M.

azedarach leaf.

chicken lymphocyte cell line culture

determination of max. non cytotoxic dose of

leaf extract by MTT assay

performing lymphocyte proliferation assay at max.

non cytotoxic dose

expression analysis of cell cytokines (IL-2,IL-6)

• Scientific validation of this plant as an immunomodulator.