Comparative Study of Free Radical Scavenging and Lipid Peroxidation Inhibitio...
Result and Discussion_Dabur
1. A Project Report on
″Evaluation Of Antidiabetic Potential Of
Syzygium jambolanum″
By:
Sonali Uttam
Amity Institute Of Biotechnology
B.Tech-Biotechnology
Section-D
BTB/11/4042
Industry Guides: Internal Guide:
Kalpana Meena Dr.Ritu Verma Dr. Madhumita P. Ghosh
Senior Research Scientist,
Cell Biology,
Dabur Research Foundation
Deputy Manager,
Cell Biology,
Dabur Research
Foundation
Assistant Professor,
Amity Institute Of
Biotechnology,
Amity University
2. RESULT AND DISCUSSION
Microscopic examination of preadipocytes and mature adipocytes:
(a) (b)
(a) The morphology of preadipocytes was fibroblast like. They were cultured until confluent state. (b) On
differentiation the mature adipocytes appear round and yellowish and start to accumulate lipid droplets in
their cytoplasm. The signet ring appearance of adipocytes is clearly seen in the cells indicated with red
arrows.
Oil Red O staining:
The accumulation of the lipid droplets was visualized by staining them with oil red O stain.
4. The preadipocytes were incubated in different concentrations (10%-0.001%) of aqueous extracts
of Jamun pulps and seeds separately. Serum free DMEM media was taken as control. The cell
viability in the serum free media was regarded as 100% and viability of cells incubated with the
SA and PA extracts was calculated with respect to serum free media.
Concentrations corresponding to absorbance values equal to 70% or lower than the control cells
are regarded as toxic. Conversely, a higher absorbance rate indicates an increase in proliferation.
In the MTT assay the absorbance values for all the concentrations of Jamun Pulp Aqueous
extract are higher than the control and hence are proliferative. Therefore all the doses of Jamun
Pulp Aqueous crude extract ranging from 10% to 0.001% are safe.
Incubation with Jamun Seed Aqueous Extract also produced absorbance values greater than the
control. However the cell viability corresponding to 10% of Jamun Seed Aqueous extract was
equal to 71% of that of the control. This indicates that Jamun Seed extract at a concentration
higher than 10% of the crude extract may be toxic.
The 10% of the aqueous crude extract of Jamun seed was noticeably colored. This could also
interfere with the growth of the cells.
Lipogenesis Assay and Oil Red O Staining:
5.
6. In order to investigate the potential of S.jambolanum aqueous pulp and seed extracts on
lipogenesis, differentiation media was supplemented with various concentrations of the extracts
(7%-0.001%). The rate of preadipocyte differentiation/ lipogenesis was assessed by staining the
lipid droplets with Oil Red O dye and quantified spectrophotometrically at 510nm.
Aqueous pulp(PA) extract at the concentration of 5% showed maximum lipogenesis, which is
35.68% greater than the positive control (differentiation media without any extract, and
containing 10µg/ml insulin, 0.5µM Dexamethasone, 0.5mM IBMX) followed by 1% PA extract
which showed 8.93% increase with respect to the control.
Aqueous seed (SA) extract showed maximum lipogenesis at the concentrations of 1%, 0.05%
and 0.01% which are 39.72%, 37.36% and 39.32% respectively, higher than the control. SA
extract at the concentration of 0.005% increased lipogenesis by 27.85%.
It is clearly observed that the seed aqueous extract was more efficient in enhancing lipogenesis.
Higher concentrations of the extracts, 7% SA, 5% SA and 7% PA did not exert any enhancement
of lipogenesis. The lower dose range 0.05% PA, 0.01% PA and 0.001% PA, the lipogenic effect
of the extract was attenuated significantly. 0.005% PA extract showed only a 28.88% decrease
in lipogenesis with respect to the control. A reasonable explanation for this deviation is the
8. In the α-amylase inhibitory assay, the enzyme α-amylase was preincubated with various
concentrations of the aqueous crude extracts of both Jamun pulp and seeds for 15mins at 27ºC.
Following which the substrate 1% starch was added and incubated for 30min at 27ºC. 1N HCl
was used to stop the reaction and iodine solution was added for color development that results
from iodine binding to starch polymers. Absorbance was measured at 540nm. The absorbance
corresponds to the amount of starch present in the test tube. In the test tube labeled as Enzyme
control, where no extract (potential inhibitor) was present the Enzyme showed breakdown of
starch added initially. In the presence of either Quercetin or the extracts, the enzyme is inhibited
to some extent and the breakdown of starch is reduced.
The standard quercetin (20mM) showed 63.14% inhibition. The 100% crude aqueous extract of
Jamun seed showed 63.28% inhibition which is as high as the standard. The Jamun seed aqueous
extract with concentrations increasing from 0.1% to 100% showed a dose dependent increase in
inhibition.
In case of Jamun pulp aqueous extract, maximum inhibition of 40.16% was observed at the
concentration of 70% PA extract. The percentage inhibition decreased with decreasing
concentration of the PA extract, up to 10%.
