INVITRO STUDIES ON THE EFFECT OF TERMINALIA ARJUNA IN ADIPOCYTE 3T3 - L1 CELL LINES

 

MATERIALS AND METHODS:
Sample Collection:
Matured leaves wear collected in the Ghandhi Krushi Vignana Kendram (GKVK) Bangalore. The plant samples were collected and identified by an agriculturist in the GKVK. The samples were allowed to dry at room temperature under a shade. The dry samples were then crushed in fine powder and stored in tightly sealed polyethylene bags.

Extraction procedure: Plant leaves were washed thoroughly with distilled Water.The leaves were dried under shade at room temperature. The dried leaves of Terminalia Arjuna were finely grinded using electrical grinder and stored in air tight containers for further use. A total of 250 g of the pulverized plant material was extracted for 4 days in Methanol. The extracts were then filtered through Whatman’s No. 1 filter paper and then condensed to dryness using rotary evaporator. The thick extracted mass was then dried at room temperature (Hemant et al., 2002)

1. Phytochemical analysis:
Phytochemical analysis of Terminalia Arjuna extracts were done using the protocols described by; segelman et al for the following (Segelman et al., 1969).
Test for Sterols  - Liebermann Burchard reaction
Tests for Alkaloids  -  Mayer’s and Wagner’s test
Tests for Tannins  - Ferric chloride reagent test
Tests for Saponins  - Foam test
Tests for Phenols  - Ferric chloride reagent test
Tests for Flavonoids - Sodium hydroxide test
Test for Terpenoids - Salkowiski test
Test for Carbohydrate - Benedict’s test
Test for protein - Biuret test

2. Hydroxyl radical scavenging assay:
Principle:
Hydroxyl radicals, generated by the Fenton type reaction system (Fe+3 + EDTA / H2O2 / Ascorbic acid), are known to damage deoxyribose and form TBA reactive chromogen, which forms a pink colour measured spectrophotometrically at 532 nm.
Procedure:
The deoxyribose assay is performed as described by Halliwell et al. with minor changes. 1ml of reaction volume have 5.6 mM deoxyribose, 2.8 mM H2O2 , 40 µM FeCl3 , 100 
µM EDTA, and varying concentrations of the sample in 2.5 mM phosphate buffer, pH 7.4. Initiation of the reaction is by the addition of 0.1 mM Ascorbic acid. The mixture is then incubated for 90 minutes at 37˚C. After incubation 1ml of TBA (0.7% in 0.05 N KOH) and 1ml of 2.5% TCA. The mixture is heated at 100˚C for 8 minutes, cooled and the pink colour formed is measured spectrophotometrically at 532 nm. Controls are to be run, which are devoid of test samples.  Quercitin is used as the reference standard (Olatunde Farombi et al., 2002).

3. HPLC analysis of Quercetin:
Plant Extraction:
10gms plant powder was extracted with 50ml Methanol at 50oC for 4 hours. The Methanolic extracts were filtered through Whatmann No. 1 filter paper and filtrate was evaporated to dryness. Methanolic extract (10mg/ml) was used for HPLC analysis.
Quercetin Standard  : 100ug/ml prepared in Methanol
HPLC Condition:
Instrument :  Shimadzhu LC- Prominence 20AT
Column  :  C18 column 250 mm x 4.6 mm, 5u particle
Mobile Phase   : Linear
A :  HPLC grade Acetonitrile (60%)
B : HPLC grade Water (40%)
Flow Rate  :  1.0 ml/min
Injection volume : 10ul

Quantification of Quercetin in plant extracts
Concentration of Standard injected :  100
µg/ml
Sample concentration :  10mg/ml
Formula used for quantification of quercetin in plant extract
Quercetin (Mg/g) = Sample area / Standard area x Standard concentration injected x Dilution factor.

4. Cytotoxicity studies using 3T3-L1 cell line by MTT assay:
3T3-L1cell line was obtained from American Type Culture Collection (ATCC) (Rockville, MD USA) (ATCC Number-CL-173). The steps and procedure for cell culture, Thawing, Revival and Propagation of Cells were followed as described by Kangas, 1984.
Procedure
The collected cells should reach about 70-80% confluency. Check the viability of the cells and centrifuge it.Take about 50,000 cells / well and seed it in 96 well plates and incubate for 24 hrs at 370C, 5% CO2 incubator. Add plant samples which is to be tested from 0-320μg/ml (2 fold variation) concentration in RPMIwithout FBS & are incubated for 24 hr. Add 100μl/well of the MTT (5 mg/10ml of MTT in 1X PBS) to incubated plant samples to the respective wells and incubated for 3to 4 hours.Discard the MTT reagent by pipetting without disturbing cells and add 100 µl of DMSO to rapidly solubilize the formazan. Measure the Absorbance at 590 nm.
Calculating Inhibition
% Inhibition = 100 – (OD of sample/OD of Control) x 100

RESULTS:
1. Phytochemical analysis

S.No

Tests

Observation

Inference

1

Froth formation test

Formation of stable froths was observed.

Presence of Saponins was confirmed.

2

Mayer’s and Wagner’s test

A brown colour Precipitates was observed.

Presence of Alkaloid was confirmed.

3

Ferric Chloride test

Dark green colour was not developed.

Absence of Tannin was confirmed.

4

Liebermann-Burchard test

Formation of bluish green colour was observed.

Presence of Steroid was confirmed.

5

Sodium hydroxide test

Change from yellow colour to colorless was observed.

Presence of Flavonoid was confirmed.

6

Ferric chloride test

Violet colour was not developed.

Absence of Phenol was confirmed.

7

Salkowski test

Reddish brown coloration was observed.

Presence of Terpenoid was confirmed.

8

Benedict’s test

Formation of an orange red precipitate was observed.

Presence of reduceing sugar was confirmed.

9

Biuret test

Formation of pink colour in the extract layer was found.

Presence of protein was confirmed.

Table – 1: Phytochemical Analysis Terminalia Arjuna leaves extracts

2. Hydroxyl radical scavenging assay:

Table – 2: Hydroxyl Radical Scavenging Assay

Hydroxyl Radical Scavenging Assay

Figure – 1: Hydroxyl Radical Scavenging Assay

3. HPLC analysis of Quercetin and Terminalia arjuna


S. No.


Retention. Time [min]


Area [mV.s]


Height [mV]


Area [%]


Height [%]


W05 [min]


1


1.933


373.177


22.881


20.5


14.0


0.20


2


3.107


92.433


3.352


5.1


2.0


0.49


3


3.487


1296.195


133.916


71.3


81.6


0.14


4


4.207


55.054


3.869


3.0


2.4


0.22


 


Total


1816.859


164.018


100.0


100.0


 

Table – 3: HPLC analysis of Standard Quercetin

Figure – 2: HPLC analysis of standard Quercetin


S. No.


Retention. Time [min]


Area [mV.s]


Height [mV]


Area [%]


Height [%]


W05 [min]


1


1.547


229.640


11.970


44.3


45.3


0.27


2


2.220


101.600


4.922


19.6


18.6


0.28


3


3.323


138.804


7.220


26.8


27.3


0.28


4


3.873


12.358


0.706


2.4


2.7


0.30


5


5.613


36.406


1.612


7.0


6.1


0.36


 


Total


518.808


26.429


100.0


100.0


 

Table – 4: HPLC analysis of Quercetin content in Terminalia arjuna

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