EVALUATION OF CYTOTOXIC EFFECT OF PHYTOCONSTITUENTS OF TINOSPORA

CORDIFOLIA USING 3T3L1 FIBROBLAST CELL LINE

PRESENTED BYPRIYANKA CHAURASIA

THE STUDY…….

prevalence of genetic and stress related diseases is on increasing trend such as diabetes.

Tinospora cordifolia - antidiabetic activity.

3T3L1 cell line - pivotal in understanding - basic cellular mechanisms associated with diabetes, obesity and related disorders.

present study - to evaluate the cytotoxic potential of the Tinospora cordifolia and its isolated fractions in 3T3L1 cells using an MTT assay.

INTRODUCTION

Diabetes mellitus - global health crisis - attained a pandemic form.

important to control diabetes and its complications - alleviate the human suffering.

Prior to investigating the plants for their anti-diabetic activity, it is first essential to evaluate their cytotoxic potential.

Evaluating in vitro cytotoxic potential - provide important preliminary data - help select plant extracts with potential anti-diabetic potential for future work. 

Tinospora cordifolia used for the treatment of diabetes.

Oral administration of an aqueous T. cordifolia root extract (TCRE) to alloxan diabetic rats caused a significant reduction in blood glucose and brain lipids, increase in body weight, total haemoglobin & hepatic hexokinase.

lowers hepatic glucose-6-phosphatase and serum acid phosphatase, alkaline phosphatase, and lactate dehydrogenase in diabetic rats.

hypoglycaemic and hypolipidaemic effect.

AIMS AND OBJECTIVES

Aim: To evaluate the cytotoxic potential of Tinospora cordifolia & its isolated fractions using 3T3L1 Fibroblast cell line.

Fractions of T. cordifolia: isolated fractions viz., Water, Butanol, Undissolved

and Ethyl acetate fractions using MTT assay.

TYPE 2 DIABETIC COMPLICATIONS

1. HEART AND BLOOD VESSEL DISEASE :- Increases the risk of various cardiovascular problems. risk of stroke and death rate is more.2. NEUROPATHY(NERVE DAMAGE) :- Nerves damage - problems with nausea, vomiting,

diarrhoea or constipation.3. NEPHROPATHY(KIDNEY DAMAGE) :- Severe damage - kidney failure or irreversible end-stage

kidney disease, requiring dialysis or a kidney transplant.4. EYE DAMAGE :- Retina damage – blindness – condition termed as

Diabetic Retinopathy Serious vision conditions – cataracts & glaucoma

3T3L1 CELL LINE

3T3-L1 is a cell line derived from 3T3- Swiss albino mouse cells that is used in biological research on adipose tissue.

Source – Adipose tissue

3T3-L1 cells have a fibroblast-like morphology and cells differentiate into adipocyte phenotype.

3T3L1 cells of adipocytes increases the synthesis and accumulation of triglycerides.

3T3 cell line established by two scientists, George Todaro and Howard Green in 1962.

3T3L1 CELLS

Fibroblast phenotype Adipocyte phenotype

OTHER CELL LINES THAT CAN BE USED FOR IN VITRO STUDIES ON DIABETES

CELL LINE

CELL ORIGIN SPECIES ADVANTAGES

DISADVANTAGES

MIN 6 INSULINOMA MOUSE EXPRESS GLUCOKINASE & GLUT 2

TREATMENT WITH NICOTINAMIDE MAKES THEM RESPONSIVE TO GLUCOSE

RINm INSULINOMA RAT INSULIN LEVEL DECREASES. SECRETES SOMATOSTATIN.NOT RESPOND TO GLUCOSE

INS 1 INSULINOMA RAT RESPONDS TO GLUCOSE. HIGH INSULIN CONTENT

REQUIRE MERCAPTO- ETHANOL IN CULTURE MEDIUM

KNOWN ANTI-DIABETIC DRUGS

Modern Drug:

Sulphonylureas, Pioglitazone, Metformin, Thiazolidinediones

etc.

Pioglitazone is known insulin sensitizer.

Ayurveda drugs:

Phyllanthus emblica, Allium sativum, Aloe vera, Tinospora

cordifolia.

Tinospora cordifolia is widely used for its antidibetic properties.

TINOSPORA CORDIFOLIA

Tinospora cordifolia (Guduchi) is a large, glabrous, deciduous climbing shrub belonging to the family Menispermaceae.

Chemical constituents from this shrub belong to different classes - alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds and polysaccharides.

medicinal properties - anti-diabetic, anti-periodic, anti-spasmodic, anti-inflammatory, anti-arthritic, anti-oxidant, anti-allergic, anti-stress, anti-leprotic, anti-malarial, hepatoprotective, immunomodulatory and anti-neoplastic

PHARMACOLOGICAL ACTION

aqueous extract of guduchi stem has shown the presence of arabinogalactan that showed immunological activity.

stem is used in dyspepsia, fevers and urinary diseases.

reduces oxidative stress and lowers blood sugar level in body.

used in leprosy, diabetes, cardiac debility, jaundice and general weakness.

used in inflammations, gout and skin diseases.

CYTOTOXIC EFFECT

Guduchi is very well established immunomodulatory agent and proven antioxidant herb.

Dichloromethane extract is the most promising one as far as cytotoxic effect is concerned. These preliminary studies were done to establish the cytotoxic effect of guduchi at higher doses.

The anti tumor activity of TC may be due to decreased lipid peroxidation, glutathione-S-transferase activity or due to the release of lactate dehydrogenase.

OTHER CYTOTOXICITY ASSAYS

1. LDH assay: The LDH assay give satisfactory responses by using cell membrane damaging agents like triton X-100; e.g. sodium azide which inhibits the respiratory chain.

2. Neutral red assay: This assay is less sensitive (excitoxic model) & cannot recommend it for the use in ion channel studies. Measures the cell death.

3. ATP based assay:It is the most sensitive assay. The disadvantage of this method is the luminescence-readout, which could be influenced by quenching side effects in the samples.

OUTLINE OF PROJECT

SOLUBILITY

VIABILITY

D/W DMSO

MTT ASSAY

TC

FRACTIONS OF TC

Water- soluble in 100% D/W.

Butanol- soluble in 100% D/W.

Undissolved- soluble in 100% D/W.

Ethyl acetate- soluble in 100% DMSO when kept overnight.

PRINCIPLE OF MTT ASSAY

This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.

The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product.

Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.

RESULTS

Aqueous extract of

Tinospora cordifolia:Water fraction of

Tinospora cordifolia:

Control cells

1 5 10 500

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Abs

orba

nce

at 5

70 n

m

Control cells

1 5 10 500

0.2

0.4

0.6

0.8

1

Abs

orba

nce

at 5

70 n

m

Butanol fraction of

Tinospora cordifolia: Undissolved fraction

of Tinospora cordifolia:

Control cells

1 5 10 500

0.2

0.4

0.6

0.8

1

Abs

orba

nce

at 5

70 n

m

Control cells

1 5 10 500

0.2

0.4

0.6

0.8

1

Abs

orba

nce

at 5

70 n

m

Ethyl acetate fraction of Tinospora cordifolia

Control cells 1 5 10 500

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Absorb

ance a

t 570 n

m

DISSCUSSION

Aqueous extract of Tinospora cordifolia and its 5 fractions viz., Water, Butanol, undissolved and Ethyl acetate fraction were studied over a concentration range of 1 to 50 µg/ml.

aqueous extract of Tinospora cordifolia, Water, Butanol and Undissolved fraction - not show a significant increase in the absorbance values for a time period of 24 hours.

ethyl acetate fraction - a dose dependent decrease in the absorbance as compared to untreated cells.

necessary to study EAF at lower concentrations below 5 μg/ml to evaluate its cytotoxic potential.

CONCLUSION

The present study was conducted to evaluate the cytotoxic potential of aqueous extract of Tinospora cordifolia and its isolated fraction.

observed - aqueous extract, Water, Butanol and Undissolved fraction - not affect the viability of the 3T3L1 cells at the concentrations studied - no cytotoxic effect.

Ethyl acetate fraction of Tinospora cordifolia - a concentration dependent effect on the viability of the 3T3L1 cells - cytotoxic effect.

All the fractions except ethyl acetate can be further evaluated for their anti diabetic potential

REFERENCES

Colagiuri R. Diabetes: A pandemic, a development issue or both? Expert Rev CardiovascTher 2010;8:305-9.

Grover JK, Yadav S, Vats V; Medicinal plants of India with anti-diabetic potential, JEthnopharmacol; 2002 Jun;81(1):81-100.

Gogte, V.M.,Ayurvedic Pharmacology & Therapeutics Uses of Medicinal plants (Dravyagunavigyan).BharatiyaVidyaBhavan publication;Part II: Medicinal plants. In Ramakrishnan S (Ed), 2000.p.514-515

Stanely M, Prince P, Menon VP, Gunasekaran G.,Hypolipidaemic action of Tinosporacordifolia roots in alloxan diabetic rats. J Ethnopharmacol; 1999; 64:53-7.

Stanely M, Prince O, Menon VP.,Hypoglycaemic and other related actions of Tinosporacordifolia roots in alloxan-induced diabetic rats, J Ethnopharmacol; 1999; 70: 9-15.

Gupta SS, Varma SCL, Garg VP, Rai M, Antidiabeticeffect of Tinosporacordifolia on fasting blood sugar level, glucose tolerence and adrenaline induced hyperglycemia. Indian J. Exp. Biol; 1976; 55: 733–45.

Green H., Kehinde O., Sublines of mouse 3T3 cells that accumulate lipid. Cell 1:113–116, (1973)

Green H., Meuth M., An established preadipose cell line and its differentiation inculture Cell 3:127–133,(1974)

Da Poian AT, El-Bacha T and Luz MRMP. Nutrient utilization in humans: metabolism pathways. Nature Education 2010;3(9):11.

Blin C, Panserat S, Médale F, Gomes E, Brèque J, Kaushik S and Krishnamoorthy R. Teleost liver hexokinase- and glucokinase-like enzymes: partial cDNA cloning and phylogenetic studies in rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata). Fish Physiology and Biochemistry 1999;21(2):93-102.