In-vitro antidiabetic activity of methanolic extract of leaves and fruits of Pouteria campechiana

Introduction and Aim: Herbal medicine have been used to treat several ailments since decades. Pouteria campechiana (Kunth) Baehni belongs to the family Sapotaceae which is widely found around the world. In folk medicine, various parts of P. campechiana is used to treat various illness. Inhibition of alpha-amylase and alphaglucosidase enzymes can be an important strategy in management of postprandial blood glucose level in non-insulin dependent diabetic patient. Hence, present study focused to evaluate the in vitro antidiabetic activity of leaf and fruit methanolic extract of Pouteria campechiana (Kunth) Baehni. Materials and Methods: Methanolic extract of P. campechiana leaf (PCL) and fruit (PCF) was screened by biochemical assay such as α-amylase inhibition activity by CNPG3 method (2-chloro-p-nitrophenyl-α-Dmaltotrioside) and αglucosidase inhibition activity and in vitro cellular assay such as glucose uptake assay in 3T3L1 cell line. Results: Methanolic extract of P. campechiana leaf and fruit showed inhibition of α-Amylase and α-Glucosidase enzymes. The methanolic extract of P. campechiana leaf and fruit at varying concentrations (μg/ml), did not exhibit cytotoxicity against 3T3-L1 cell line after 24 hours of incubation. The test compounds PCL and PCF induced the uptake of 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino) 2-Deoxyglucose (2-NBDG) in 3T3L1 cells. PCF and PCL both showed almost similar activity of standard drug, Metformin at higher concentrations. Conclusion: Based on the results, it can be concluded that methanolic leaf and fruit extract of P. campechiana possess antidiabetic activity.


INTRODUCTION
iabetes mellitus (DM) is a metabolic disorder characterized by a chronic hyperglycemia with dysregulation of carbohydrates, fats, and protein metabolism resulting from defective secretion and function of insulin (1). Diabetes mellitus is also characterized by a reduced reaction of insulinsensitive peripheral tissues and a marked decrease in glucose uptake and metabolism in response to insulin. Rapid development and progression of Type 2 DM has been evolved from past few decades. Its main cause is progressively reduced insulin secretion by pancreatic β-cells of pre-existing insulin resistance in skeletal muscle, liver and adipose tissue (2).
Inhibition of the -amylase should reduce the unfavourable high postprandial blood glucose peak in diabetics. Intestinal α-glucosidase inhibitors are reported to be powerful therapeutic agents in carbohydrate metabolic disorders, especially diabetes mellitus and obesity (6). To control post-prandial hyperglycemia with non-insulin-dependent diabetes mellitus (NIDDM), biguanides such as metformin are used for the enhancement of glucose uptake by peripheral cells stimulation of basal glucose transport and metabolism in muscle and fat cells would explain increased glucose utilization. Therefore, cellular assays are used to determine the mechanism of action of natural or synthetic compounds from isolated rat diaphragms, as well as isolated and cultured rat 3T3 adipocytes. Hence, it is highly desirable to find new antidiabetic agents from natural resources that stimulate glucose uptake by peripheral tissues such as adipose tissue or muscle cells (7). In this study, we evaluated the antidiabetic properties of P. campechiana leaf and fruit extract in vitro using the 3T3-L1 as a model system.

Preparation of plant extract
The extraction was carried out by the continuous hot percolation method in a Soxhlet apparatus using methanol. The methanolic extract obtained were concentrated to dryness using the rotary vacuum evaporator, dried and stored in airtight bottles in a refrigerator at 4°C for further use.

α -Amylase inhibitory assay
The assay for inhibition of α-amylase was carried out as described by Kumar et al.,(8) with slight modification. The enzyme solution was prepared by mixing α-amylase (3.246 mg) in 100 ml of 40 mM phosphate buffer (pH 6.9). A positive control was prepared by dissolving acarbose in phosphate buffer and diluted to get a various concentration using phosphate buffer (pH 6.9). P. campechiana leaf and fruit extract was dissolved in phosphate buffer to give final concentrations of 37.5, 75, 150, 300, 600 and 1200 µg/ml. The acarbose and plant extract were mixed with 2-Chloro-4-Nitrophenol α-D-Maltotrioside (CNPG3) and incubated for 8 min at 37°C. Reaction was arrested by heating on boiling water bath for 2 min and cooled. The absorbance was read at 405 nm. A control reaction was prepared without the plant extract/acarbose and measured in a similar manner.

α -Glucosidase inhibitory assay
The α-glucosidase inhibitory activity was measured as described by Dayananda et al., (9). Reaction mixture consisted of crude enzyme solution (50 µl), sucrose (37 mM, 500 µl) in 80 mM potassium phosphate buffer (pH 7.0), and the test sample (1 mg/ml) in dimethyl sulfoxide (DMSO) and diluted to get various concentrations 3.125, 6.25, 12.5, 25, 50 and 100 µg/ml. After incubation for 20 min at 37°C, the reaction was stopped by heating on boiling water bath for 2 min. The amount of liberated glucose was measured by the glucose oxidase method.

In vitro cytotoxicity assay
The Cell viability of 3T3-L1 cells was determined using 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) assay (10). Cytotoxicity assay is a colorimetric assay to determine the noncytotoxic concentration of P. campechiana leaf (PCL) and fruit (PCF) after 24 h exposure period based on the conversion of MTT to formazan crystals by the lactate dehydrogenase present in live cells. 3T3-L1 cells (200 μl cell suspension) were seeded in a 96-well plate at required cell density (20,000 cells per well), without the test agent. Cells were allowed to grow for about 24 h. Cells were then treated with different concentrations 10, 20, 40, 80, 160, 320 µg/ml of leaf and fruit extract. Then the plates were incubated for 24 h at 37°C in a 5% CO2 atmosphere. After the incubation period, plates were taken out from the incubator and spent media was removed. MTT reagent to a final concentration of 0.5 mg/ml of total volume was added and further incubated for 3 h at 37°C. After incubation, MTT reagent was taken out and formazan crystals were dissolved with 100 μl of DMSO, solubilisation solution. Absorbance was measured at 570 nm on ELISA reader. Percentage viability was determined using the formula: Mean OD (Test) at 570nm Percentage of viability = × 100 Mean OD (untreated cells) at 570nm Glucose uptake assay 3T3-L1 cell line were cultured in 6-well plate at a density of 2 x 10 5 cells/2 ml and incubated in a CO2 incubator overnight at 37°C. After 24 h, spent medium was removed through aspiration and cells were treated with extract (40 μg/ml and 320 μg/ml) and controlmetformin (100 uM). The extract and control were prepared of 2-NBDG and the cells were incubated for 2 h. After 2 h of incubation, cells were washed with PBS. For adherent cells, PBS was removed and 200 μl trypsin was added and incubated at 37°C for 3-4 min. 2 ml of culture medium was added and the cells were harvested directly into in 12 x 75 mm tubes. Tubes were centrifuged and pellet was re-suspended in 0. 5 ml of PBS. Cellular uptake of 2-NBDG was analysed with a flow cytometer (11).

Statistical analysis
All experiments were performed in triplicates and results were expressed as mean percentage inhibition ± standard deviation (SD) (n=3). IC50 values in enzyme inhibition assays were determined using linear regression graph (concentration versus percentage enzyme inhibition). All statistical analyses and IC50 values determination were carried out in GraphPad Prism (version 3.1) software (San Diego, CA).

Inhibition of α-amylase activity
Percentage inhibition of -amylase activity by P. campechiana leaf and fruit extract was estimated with acarbose as positive control. The PCL and PCF extract showed 10.31 ± 1.06% and 11.49 ± 2.35% inhibition of α-amylase activity at 37.5 µg/ml and 66.93 ± 1.16% and 56.75 ± 1.47% inhibition at 1200 µg/ml concentration respectively. The IC50 value of PCL and PCF extract was found to be 516.4 µg/ml and 927.4 µg/ml. The standard drug acarbose exhibited 12.14 ± 0.53% inhibition of α-amylase activity at 0.3125 µg/ml and 71.30 ± 1.30% inhibition at 10µg/ml concentration. The IC50 value for acarbose was found to be 3.449 µg/ml. The percentage inhibition of -amylase activity by the extract and drug at different concentrations represented in Figure 1.

Inhibition of α-glucosidase activity
Percentage inhibition of -glucosidase activity by P. campechiana leaf and fruit extract was estimated with acarbose as positive control. Methanolic extract of PCL and PCF showed significant inhibition of α-Glucosidase activity. The lowest concentration of the leaf and fruit extract showed 9.24 ± 1.70 % and 10.23 ± 1.93% inhibition respectively and the highest concentration showed 58.67 ± 1.01% and 67.75 ± 1.35% of inhibition in α-Glucosidase activity respectively. Acarbose, the positive control drug, showed a percent inhibition, in α-Glucosidase activity, of 13.45 ± 0.87% at 1 µg/ml and 76.38 ± 0.50% at 32 µg/ml. The IC50 values of acarbose, PCL and PCF extract were found to be 6.11 µg/ml, 62.68 µg/ml and 45.03 µg/ml, respectively, as shown in Figure 2. and PCF extract (10 µg/ml) showed 99.72 ± 0.84% and 99.48 ± 0.15% viability respectively, and the highest concentration (320 µg/ml) showed 102.21 ± 0.37% and 101.79 ± 0.46% of viability, respectively after 24 h of exposure. These results indicated that methanolic extract of PCL and PCF is not toxic to mammalian cells even at higher concentrations and could be used to analyze other parameters of in vitro antidiabetic studies.

DISCUSSION
Adipocyte differentiation inhibitors may be effective in preventing various diseased conditions such as obesity, atherosclerosis, diabetes and other associated complications. Under the appropriate conditions, 3T3-L1 cells are known to differentiate into adipocytes and used as a model for adipose cells, which are one of the major sites of lipid and glucose metabolism (12). One of the approaches in tackling DM has been the use of agents that inhibit the action of enzymes involved in the absorption and metabolism of carbohydrates. The major enzymes involved in the absorption of glucose from the gut include the pancreatic -amylase and -glucosidase enzymes (13). Inhibition of these enzymes regulate the postprandial blood sugar level in Type 2 DM patients by reducing the absorption of sugars from gut. Existent antidiabetic drugs that specifically inhibit the activity of enzymes include acarbose, voglibose and miglitol. However, usage of these drugs can have adverse effects such as flatulence and abdominal bloating. Natural compounds from ethnomedicinal plants that do not have such effects are candidates of interest in the treatment of Type 2 diabetes. Such medicinal plants play a vital role in herbal medicine, particularly in treating ailments like diabetes (14).
The decrease in glucose uptake in type 2 diabetes is due to the insulin resistance developed by the cells. A fluorescent deoxyglucose analog, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino (-2-Deoxy glucose)) (2-NBDG) used as a probe for the detection of glucose taken up by cultured 3T3 L1 cells. It is transported by both SGLTs and GLUTs and has allowed the study of the effects of drugs targeting glucose uptake and glycolysis (15). In this study, experiments on cellular uptake of glucose revealed that P. campechiana has the capability to induce glucose utilization by 3T3L1 cells. After incubation of cells with 40 μg/ml and 320 μg/ml of PCL and PCF extract for 2 h at 37°C, 2-NBDG uptake was observed in 11.91%, 62.21%, 11.57% and 58% of cells respectively, whereas it was observed in 74.73% of cells in the presence of 100 µM of the standard drug, metformin and increased when compared to cell control as indicated in figure 6.
The present study evaluated the antidiabetic properties of P. campechiana and results indicate that the methanolic extract of PCL and PCF effectively inhibits α-amylase and α-glucosidase activities. These inhibitory effects were estimated with acarbose as the standard drug. Furthermore, P. campechiana had no cytotoxic effect and we have proven the anti-diabetic activity of methanolic extract of P.campechiana in an in vitro cell-based model. However, further studies need to be carried out to understand the exact mechanism of action.

CONCLUSION
This novel study reports the antidiabetic properties of P. campechiana leaf and fruit as evidenced by biochemical assays and in vitro cellular assay. Methanolic extract of PCL and PCF extract showed no inherent cytotoxicity in 3T3-L1 cell lines tested, with IC50 values > 100 µg/ml. PCL and PCF extract effectively inhibited the activity of the enzymes α-amylase and α-glucosidase, enhanced glucose uptake in 3T3-L1 cells. These results were comparable to the action of acarbose and metformin. This suggests that P. campechiana leaf and fruit has potential antidiabetic properties and may serve as a source of lead molecules possessing potential uses in the treatment of DM. However, further studies to identify and characterize these bioactive constituents must be performed.