IN VITRO ANTI diABETIC EXPERIMENTS

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

• 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.

Diabetes is of 2 types – Type -1 diabetes IDDM Juvenile onset of diabetes Keto acidosis prone diabetes

Type -2 diabetesNIDDMHyperglycaemic Hyperosmolar Non-ketotic

Anti diabetic agents

IINTRODUCTION 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.

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.

HYPOGLCEMIC ACTIVITY OF ALKALOIDAL FRACTION OF TINOSPORA CORDIFOLIA

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).

Cell culture and treatment for insulin secretion assayRINm5F 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.

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.

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).

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.

ANTIDIABEIC ACTIVITY OF SOME

PENTACYCLIC ACID TRITERPENOIDS

In vitro PTP-1B assay

To test the ability of each compound to inhibit PTP-1B, aliquots ofstock solutions (dissolved in DMSO to prepare 20 mM) were dilutedwith 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, theenzyme activity was measured at a fixed substrate concentration(corresponding to the Km) and varying inhibitor concentrations.

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.

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.

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.

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.

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.

E + S ES E + P + + I I

EI + S ESI SCHEME 1. LINEAR MIXED-TYPE INHIBITION OF EVALUATED COMPOUNDS

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

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).

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

ConclusionsWe 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].