evaluation of cytotoxic effect of phytoconstituents of tinospora (new)
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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
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