1. IN VITRO ANTI
diABETIC
EXPERIMENTS

2. INTRODUCTION TO DIABETES
 Diabetes Mellitus is a chronic disease that
occurs when the pancreas does not produce
enough insulin or when the body cannot
effectively use it, resulting in hyperglycaemia.
 DM is a
chronic, progressive, systemic, metabolic
disorder

3. • Over 400 million of the world’s
population are suffering with
obesity .
• India is the major center for
diabetes victims sharing the
highest percentage of the world’s
obese people.

4. Diabetes is of 2
types –
Type -1 diabetes Type -2 diabetes
IDDM NIDDM
Juvenile onset of Hyperglycaemic
diabetes Hyperosmolar
Keto acidosis Non-ketotic
prone diabetes

5. Anti diabetic agents

6. INTRODUCTION TO INVITRO
EXPERIMENTTS
 Invitro experiments are the one, which
are done in specified lab
environments, there where the
environment is made simulated to that
of in the animal’s body.
 These are the basic experiments done
primarily before the invivo experiments.
 Here the systems similar to the body’s
tissues and organs, and the
environments similar to that of in the
animal’s body are created. I


7. The primary aim of these experiments is to save the animals
by avoiding the usage of them, if chance.
So, instead, various artificial systems resembling the
natural organs and tissues are used.
Some such examples are various cell lines and their
cultures, artificial contractile muscle systems, etc.,
So that the animals can be avoided from being inflicted to
any unnecessary pains.
And moreover the results will approximately be nearer to
that of the invivo experiments. This helps in assessing the
properties and characteristics of the drug primarily.

8. HYPOGLCEMIC ACTIVITY OF
ALKALOIDAL FRACTION OF
TINOSPORA CORDIFOLIA

9. Materials in experiments in vitro
Rat insulinoma RINm5F cells were obtained from National
Centre for Cell Sciences (NCCS, Pune, India).
Cell culture media, phosphate saline buffer (PBS), dimethyl sulfoxide
(DMSO), glutamine,sodium bicarbonate, glucose, HEPES,
sodium pyruvate, fetal bovine
serum, streptomycin, penicillin, and MTT (3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyl tetrazoliumbromide) were
procured from Hi Media (Mumbai, India). Dexamethasone
(DEX),STZ and 8-(4-chlorophenylthio) adenosine 3,5-cyclic
monophosphate sodium salt (pCPT-cAMP) were purchase from
Sigma–Aldrich Corporation (Bangalore, India).
Tolbutamide was obtained as a gift sample from Zydus Research
Centre (Ahmedabad, India).
Glucose assay kit purchased from Beackon Diagnostics
(Navsari, India) was used
Rat insulin ELISA kit was procured from Linco Research (MO, USA).

10. Cell culture and treatment for insulin secretion assay
RINm5F cells were cultured in 75 cm3 culture flasks and incubated at 37 ◦C
in an atmosphere of 5% CO2 and 95% air.
Cells were provided with liquid growth media of RPMI-1640 (with
glutamine, 2 mM; sodium bi carbonate, 1.5 g/l; glucose, 4.5 g/l;
HEPES, 10 mM; and sodium pyruvate, 1.0 mM) supplemented
with 10% fetal bovine serum, 100 IU/ml penicillin G sodium and
100 IU/ml streptomycin sulphate.
Cell confluency was measured by trypan blue dye exclusion method. Cells
were sub-cultured when 80% confluency was reached.
The insulin secreting assay was carried out according to the protocol
described by Bahekar et al. (2007).
Samples for testing were initially dissolved in an amount of DMSO
and further diluted with PBS (pH 7.2).
The final concentration of DMSO in the test system was not greater than
2% which has no effect on the growth of RINm5F cells.

11. Effect on insulin secretion after treatment with
AFTC and isolated compounds
After trypsinization, RINm5F cells were seeded at
concentration of 2×105 cells per well, in 96-well
plates.
The cells were grown overnight and allowed to
reach a 70–80% confluent state.
Culture medium was then replaced with 0.5 ml of
PBS (pH 7.2) followed by 40 min incubation in
fresh Krebs–Ringer Balanced Buffer(NaCl, 115
mM/l; KCl, 4.7 mM/l; CaCl2, 1.28 mM/l;
MgSO4·7H2O, 1.2 mM/l; KH2PO4, 1.2 mM/l;
NaHCO3, 10 mM/l; and HEPES, 25
mM/l), supplemented with glucose, 1.1 mM and
bovine serum albumin, 0.5% (pH 7.4).
The effect of increasing concentrations (5–80 g/ml) of
AFTC and isolated compounds were studied .

12. The buffer was replaced after 40 min and the
cells were incubated (37 C, 5% CO2) with
different concentrations of test and the
standard (tolbutamide10 M) compounds for
30 min both in the presence (16.7 mM) and
absence (0 mM) of glucose load.
The supernatant was collected and the insulin
produced was measured by enzyme-linked
immunosorbent assay using commercial rat
insulin ELISA kit.
The viability of the hepatocytes was
assessed by trypan blue dye exclusion
method.
The method of Tinstorm and Obrink (1989) was
adopted for preparation of primary culture of
rat hepatocytes (Tinstorm and Obrink, 1989).

13. The glucose production was measured by incubating the
culture in glucose free RPMI-1640 medium.
Rat hepatocytes were treated with 500 nM of
dexamethasone (DEX) and 0.1 mM of 8-(4-
chlorophenylthio) adenosine 3,5-cyclic
monophosphate sodium salt (pCPT-cAMP) in the
presence or absence of insulin (10 nM) or test samples
(5–80 g/ml) for 5 h at 37 ◦C.
Cells were incubated for an additional 3 h in glucose
production buffer (glucose-free Dulbecco’s modified
essential medium, pH 7.4, containing 20 mM sodium
lactate and 2 mM sodium pyruvate without phenol
red) with DEX/pCPT-cAMP in the presence or absence
of insulin or test samples.
At the end of this incubation, 0.5 ml of medium was taken
to measure the glucose concentration in the culture
medium using a glucose assay kit.

14. ANTIDIABEIC ACTIVITY
OF SOME PENTACYCLIC
ACID TRITERPENOIDS

15. In vitro PTP-1B assay
To test the ability of each compound to inhibit PTP-1B, aliquots of
stock solutions (dissolved in DMSO to prepare 20 mM) were diluted
with the assay buffer, containing 2.5 mM p-nitrophenyl
phosphate(pNPP), to obtain two different inhibitor concentrations.
Samples were incubated at 37 C, and the reactions were initiated by
adding an appropriate enzyme aliquot. Fig. 2 shows the preliminary
results.
This figure clearly shows that all compounds induced a significant
enzyme inhibition at both evaluated concentrations (10 and 50 mM),
and the inhibition effects were more pronounced at the highest
concentration.
In order to determine the IC50 value of each compound, the
enzyme activity was measured at a fixed substrate
concentration(corresponding to the Km) and varying inhibitor
concentrations.

16. IC50 values were calculated by fitting experimental
data in the following equation, using a non-
nonlinear fitting program (Fig Sys Biosoft, UK):
Y=Max-Min + Min
(1 + x/IC50)slope
where y=v/vo is the ratio between the measured
activity in the presence of the inhibitor (vi) and the
activity of the control without the inhibitor(vo). X is
the inhibitor concentration.

17. All compounds induced a PTPe1B enzymatic inhibition in
a concentration-dependent manner (Fig. 3AeD).
Moreover, RE-01 and RE-04 were the most potent
inhibitors. RE-01 and RE-0 acids have been reported as
inhibitors of PTPe1B with IC50 values of 3.28 and 3.02
mM, respectively [15,16].
These results are in agreement with our previous
observations (Fig. 3AeD).
Yet for RE-03 and RE-04 acids, there are no reports
concerning their PTPe1Binhibitory properties.
Also, the selectivity and type of inhibition for these
compounds have not been reported.
Therefore, the dilution method was used to determine the
ability of each compound to dissociate from PTPe1B
after binding.
The enzyme was incubated for 1 h at 37 C in the presence of
each inhibitor.

18. Then aliquots of enzyme mixture was withdrawn and diluted with
the assay buffer to determine the residual activity.
Fig. summarizes the results indicating that the recovery of enzyme
activity is almost complete in all cases, with the exception of RE-02,
suggesting that most compounds behave as reversible inhibitors.
We also determined the selectivity of each inhibitor towards
other none structurally related PTPases, such as the IF1, IF2 isoenzymes
of human LMW-PTP, the yeast LMW-PTP (LTP1) and human LAR.
The selectivity was measured at the corresponding experimental
IC50 value of each compound. Results are summarized in
Table 2.
All IC50 values for other PTPases were higher than the corresponding IC50
value for PTP-1B.
These results indicate that all compounds are selective towards PTPe1B with
respect to the other PTPases tested.

19. To determine the inhibition mechanism of each
compound, we studied the effects of increasing
inhibitors concentration on main kinetics
parameters, Km and Vmax (Figs. 5.8).
We deduced from the experimental results that the inhibition
mechanism of compounds RE-01, RE-02 and RE-03
(Figs. 5.7) can be referred to a linear mixed-type
inhibition model (Scheme 1), suggesting a possible
binding in a different cavity from the catalytic site.
This was further supported with molecular docking results
discussed later.
In contrast, the inhibition mechanism of RE-04 (Fig. 8) can
be referred to a simple linear non-competitive inhibition
model.
The Kid value for all compounds were calculated using the
appropriate equations and are presented in table 3.

25. Molecular docking of triterpene acids derivatives with
PTP-1B:
In order to gain insight into the putative binding mode of RE-01, RE-02, RE-
03 and RE-04 with PTPe1B, these compounds were docked with a
crystallographic structure of human PTP-1B.
The crystallographic structure was obtained from the Protein Data Bank
(PDB), accession code 1C83 [20].
This is the first study describing the docking of these triterpene derivatives
with PTP-1B.

26. E+S ES E+P
+ +
I I
EI + S ESI
SCHEME 1. LINEAR MIXED-TYPE INHIBITION OF EVALUATED COMPOUNDS

28. In addition to the PTP-1B catalytic site, Puius
et al. identified a second aryl phosphate
binding site in PTP-1B [21].
Fig. 9 shows the catalytic binding site (site A)
and the second binding site (siteB), as
proposed by Puius et al. [21].
The most important residues of site B are
Arg24, Arg254, and Glu262.
Other residues in this site are Tyr46, Asp48,
Val49, Ile219, and Met258 [21].
Before docking the triterpenic acids, the
docking protocol was validated by
predicting the binding mode of the
crystallographic

29. 6-(oxalyl-amino)-1H-indole-5-carboxilic acid,a
competitive inhibitorof PTPe1B [20].
Fig. 9 shows a comparison between binding mode of
the crystallographic ligand and the binding mode
predicted by AutoDock.
Fig. 9 clearly shows that AutoDock successfully
predicted the bindingmode of crystallographic
ligand with a root-mean square (RMS) deviation of
0.35 Å.
Predicted binding energies for the triterpenes are
summarized in Table 3.
The four compounds showed comparable energies
as calculated by AutoDock. Fig. 10 summarizes
the binding modes of the triterpenic acids
predicted by AutoDock.
According to the docking models, all four
compounds were predicted to bind into the
site B, thus sharing a very similar binding
mode(Fig. 10A and B).

30. Interestingly, no binding poses were found by AutoDock into the
catalytic binding site.
Fig. 10C depicts the optimized binding mode of RE-01, the most
active triterpene. In this binding model, the carboxylic group
of RE-01 at C28 forms an extensive hydrogen bond network
with Arg24, Arg254 and Gln262; the surface of these
residues is showed in red in Fig. 10B.
Other residues that form van der Waals contacts with the
triterpene are Tyr46, Asp48, Val49, Met258, and Gly259
(showed in green in Fig. 10B).
Fig. 10D shows the corresponding two-dimensional interaction
diagram of the optimized binding model generated with the
program Molecular Operating Environment (MOE) [22

31. Conclusions
We obtained four pentacyclic triterpenic acids from P. reichenbachianum
with significant in vivo antidiabetic activity on noninsulin dependent
diabetic rat model.
Moreover, the main mode of action of triterpenic acids was produced by
PTP-1B enzymatic inhibition with potent, reversible, selective, and linear
mixed-type inhibition model. According to the docking results, the
proposed binding mode of the triterpene derivatives in a second binding
site (site B) of PTP-1B suggests a new strategy to obtain compounds with
higher affinity and specificity. Arg254 and Gln262 are conserved among
all PTPases, though other residues such as Gln262, Met258 and Gly259
are less conserved.
Finally, pentacyclic triterpenic acids could be potential drugs for the
treatment of type 2 diabetes as insulin sensitizer [23].