2.1 Chemicals and reagents

All the chemicals used in this study was obtained from Himedia, India. Blood glucose level was analysed using Accu-Chek® Active Blood Glucose Meter. Real-time PCR analysis was performed using KAPA SYBR® FAST qPCR Master Mix (2X). Rat IgA ELISA kit was obtained from eBioscience, Germany. Primary and secondary antibodies for western blotting obtained from Calbiochem, Germany and Santa Cruz Biotechnologies, USA.

2.2 Plant material collection, authentication and sequential extraction

Leaves of J. gossypifolia was collected from east coast region of Chennai, Tamil Nadu, India. The collected material was endorsed by Dr P. Jayaraman, Plant Anatomy Research Institute, Chennai, Tamil Nadu, India (Authentication: No: PARC/2015/3142). The leaves of J. gossypifolia were dried and powdered (50 g). To increase the order of polarity (hexane, dichloromethane, ethyl acetate and methanol), the powder was subjected to sequential extraction using Soxhlet devices with different solvents. The obtained filtrate was subjected to rotary evaporation and obtained dried extract (Anandharajan et al., 2006).

2.3 Estimation of phytochemical components

All the four leaves extracts (n-hexane, Dichloromethane, Ethyl acetate and Methanol extracts) of J. gossypifolia were subjected to preliminary phytochemical testing that was performed to analyse the presence of flavonoids using colorimetric aluminium chloride method (Chang et al., 2002). Total phenolic content was determined by Folin-Ciocalteau method (McDonald et al., 2001; Silva et al., 2006) and determination of Tannins was performed according to Porter et al. (1985).

2.4 HPLC analysis of ME

HPLC (Agilent Technologies, 1200 infinity series, Germany) analysis was carried out in crude ME and tannic acid was used as a standard (Beasley et al., 1977; Kanaujia et al., 2010). The following protocol was used for the HPLC analysis (Mohammadi et al., 2014): Mobile phase-Water: Acetonitrile: Acetic acid [89:10:1], Flow rate – 0.8 ml/min, Injection sample – 10 µL, Sample concentration – 1 mg/mL and UV–VIS reading – 280 nm.

2.5 Experimental animals

The in vivo study was conducted with the consent from the animal ethical committee of the Institute (Ethical Clearance No. 090/835/IAEC-2014). Wistar male rats aged between 6 and 8 weeks old and weighed 180–200 g was used in the study. Thirty animals have been recruited from the University of Veterinary Sciences of Tamil Nadu, Chennai, India, transported to the animal house in the Institute and acclimatized for a week. All animals fed with sterilized water and pellet diet ad libitum during the acclimatization period. The rats were kept with sterile paddy husk as bedding in sterile polypropylene cages; the temperature and light and dark condition was maintained as 25 °C and 12L/12D.

2.6 Induction of diabetes

Diabetes was instigated by intraperitoneal administration in male wistar rats with the combination of Nicotinamide (NIC) (110 mg/kg) dissolved in saline and Streptozotocin (STZ) (single dose of 50 mg/kg b.w.) dissolved in 0.01 M freshly prepared citrate buffer (pH 4.5); STZ was injected after 15 min from the injection of Nicotinamide (Masiello et al., 1998).

2.7 Animal grouping and drug administration

Thirty rats (20 diabetic induced rats and 10 normal rats as control) divided into 6 groups; each consists 5 rats. Regular diet was provided to the control groups and high fat diet (commercially available) was supplied to the diabetic induced rats. The diet composition of normal and diabetic induced rats mentioned in Table 1.

A small cut was made in the tail and blood was drawn to measure the blood glucose level using glucometer, (Accu-check, Germany). On day 3 and 7 of induction, the rats pronounced with hyperglycemia (FBG 250 mg/dl) which further selected and studied as follows:

• Group 1: Control rats (0.5% CMC); n = 5.

• Group 2: Control rats treated with ME (100 mg/kg b.w.); n = 5.

• Group 3: Diabetic rats (STZ + NIC); n = 5.

• Group 4: Diabetic rats (STZ + NIC) treated with ME (50 mg/kg b.w.); n = 5.

• Group 5: Diabetic rats (STZ + NIC) treated with ME (100 mg/kg b.w.); n = 5.

• Group 6: Diabetic rats (STZ + NIC) treated with metformin (500 mg/kg b.w.); n = 5.

Body weight and blood glucose level was periodically checked on day 0, 14, 21, 28 and 35. Blood from the internal canthus of the eye was obtained by retro-orbitally capillary tubes. Glucometer was used to assess the blood glucose levels. The serum was centrifuged 2000 rpm for 2 min and stored at −20 °C for further study.

2.8 Intraperitoneal glucose tolerance test (IPGTT)

At the end of the experimental phase, the rats were fasted overnight and injected with glucose (2 g/kg) intraperitoneally. Then, blood was drawn from the tail vein at 0, 15, 30, 60, 90 and 120 min and the glucose level was measured using Accu-check glucometer (Andrikopoulos et al., 2008).

2.9 Sacrification of animals and organ collection

At the end of the experimental phase (day 35), the animals were deprived of food and sacrificed. For the blood glucose assessment, blood was collected in a tube containing EDTA; and for insulin level measurement, the plasma was isolated from the blood by centrifugation. Pancreas and skeleton muscles are dissected out washed in ice-cold saline and stored in 10% formalin for further studies.

2.10 Estimation of insulin level and HOMA index

The obtained blood samples were used to determine the plasma insulin level using Rat IgA ELISA kit. Further, to quantify the insulin resistance, homeostatic model assessment (HOMA) index was calculated as follows (Mohammadi et al., 2014). $$\mathit{HOMA}\text{-}R=\left[\mathit{fasting}insulin\left(\mu U/mL\right)Xfastingglu\cos e\left(\mathit{mmol}/L\right)\right]/22.5$$

2.11 Histopathological examination

Thin sectioning of the pancreas (10 µm) was sectioned using microtome (Leica Bio system RM 2125 RTS, Germany) and further processed and stained with 0.1% haematoxylin followed by eosin (Abdollahi et al., 2011; Kumar et al., 2013). The section were observed under microscope at 40× magnification.

2.12 Isolation of RNA and real-time RT-PCR analysis

Muscle tissue was homogenized using trizol reagent and obtained the total RNA as explained earlier (Arockiaraj et al., 2011, 2012a; Kumaresan et al., 2014). The isolated RNA was converted to cDNA as reported (Arockiaraj et al., 2012b, 2013; Chaurasia et al., 2016a,b; Chomczynski and Sacchi, 1987). The obtained cDNA was used as template and subjected to qPCR analysis (Bhatt et al., 2014; Kumaresan et al., 2015). Real-time PCR investigation was achieved by KAPA SYBR® FAST qPCR Master Mix (2X) in a total volume of 20 µL with specific primers as mentioned in Table 2. GAPDH served as internal control gene. Initial denaturation carried out at 95 °C for 5 min, trailed at 95 °C for 1 min for denaturation. The annealing temperature maintained at 1 min at 64 °C for IRTK; and 62 °C for IRS-1, PI3K and 63 °C for GSK-3β, GS and GLUT 4. Extension was accomplished at 72 °C for 1 min. This entire process repeated for 40 cycles, trailed by a final extension at 72 °C for 10 min and hold at 4 °C. The ΔΔct value was calculated after normalizing with GAPDH and the fold change was estimated (Arasu et al., 2014; Chaurasia et al., 2016a,b; Palanisamy et al., 2015).

2.13 Preparation of plasma membrane fractions and total cell lysate from skeletal muscle

Plasma membrane fractions (PM) were obtained as described earlier (Sujatha et al., 2010). Skeletal muscle tissues were homogenized with Glass-Teflon potter homogenizer using 2 ml of the ice-cold buffer (pH 7.4) that contained 250 mM/L Sucrose, 5 mM NaN3, 20 mM HEPES, 200 µM/L PMSF, 1 µM/L Pepstatin, 1 µM/L Aprotinin and 2 µM/L EGTA. Cell lysate was obtained using Dounce homogenizer (20 S, 0.5 cycles, 10 pulses; 2 min each and lag time of 1 min for each pulse). The total cell lysate homogenate was centrifuged in12000 RPM at 4 °C for 20 min. The cell homogenates was further centrifuged in 12,000 RPM for 5 min at 4 °C in order to remove cell debris. The resulting supernatant was again centrifuged in 15,000 RPM for 40 min at 4 °C; and the obtained pellets were resuspended in buffer to constitute the Plasma Membrane (PM) fraction.

Total cell lysate was prepared as described earlier (Kanaujia et al., 2010). In a summary, homogenized tissue washed with ice-cold buffer contained 50 mM Hepes, 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 1 mM sodium orthovanate, 50 mM NaF, 10 µg/ml Aprotinin, 10 µg/ml Leupeptin and 1% Triton X 100 (pH: 7.4). Cell lysate obtained was homogenized using Dounce homogenizer (20 S, 0.5 cycles, 10 pulses; 2 min each and lag time of 1 min for each pulse). The Total cell lysate homogenate was centrifuged at 4 °C 12,000 rpm for 20 min.

Bradford method was used to determine the protein contents; the protein was subjected to 10% of SDS-PAGE and transferred onto a membrane with nitrocellulose. The membrane is blocked at 4 °C overnight in 5% skimmed milk powder and immunoblotted with the respective antibody. PM fractions were used to analyse the GLUT 4 translocation, whereas whole cell lysate was used to detect the proteins including IR-β and GSK-3β.

2.14 Western blot analysis

The immunoblot was probed with the respective antibodies (GLUT 4, IRβ and GSK3β) for 2 h. Upon washing, the membrane was incubated with HRP antibody; and the immunosignals were detected using luminol/H₂O₂ reagent and visualized under chemiDoc XRS+ imaging system (Biorad, USA). Quantification of the protein expression was performed in densitometric scanning using Image Studio Lite (ver. 5.2).

2.15 Statistical analysis

All the data were shown in this study are mean of three replicates ± standard deviation (SD). Data were subjected to one-way variance analysis (ANOVA) followed by Duncan’s multiple range test (DMRT) using statistical package of social science (SPSS, ver. 17.0). The significance level considered at p < 0.05.

3.1 Phytochemical screening

It is imperative to note that the majority of the phytochemical constituent reported in literature are not about compounds isolation, rather on the phytochemical screening of the significant classes through qualitative methods. Phytochemical screening of n-hexane, Dichloromethane, Ethyl acetate and ME of J. gossypifolia showed the presence of phytochemical constitution such as tannin, flavonoid and phenolic compounds. Particularly, ME have shown significant (p < 0.05) increase in these phytochemical constituents than the other extracts as shown in Table 3. Presence of flavonoids, phenols and tannins in plants are found to be possessed antidiabetic activity (Kumari and Jain, 2012; Tiwari and Rao, 2002). Similarly, phytochemical screening of the ME of Melastoma malabathricum indicated the presence of terpenoids, flavonoids, phenolic compound, tannins, Saponins and triterpenes; however, flavonoid and phenolic substances are present in significant amount. Altogether, ME of M. malabathricum showed potential antidiabetic activity as reported by Kumar et al. (2013).

3.2 HPLC analysis of ME

HPLC analysis of J. gossypifolia crude ME presented in Fig. 1(a and b). Totally 10 phenolic compounds were identified in ME to the retention time using tannic acid as standard (Fig. 1b). Maximum peak (37.77%) in the ME was observed due to the presence of Tannic acid followed by Rutin (11.41%) and Resorcinol (9.78%) as represented in Table 4. Among phenolic compounds present in ME, tannic acid availability is significantly abundant. Souza et al. (2008), investigated the presence of condensed tannins in Brazilian folk medicine Maytenus ilicifolia using HPLC analysis which contains epigallocatechin, epicatechin and epiafzelechin that supports our data.

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Fig. 1

(a) HPLC analysis of standard Tannic acid, (b) HPLC analysis of J. gossypifolia ME.

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3.3 Effect of ME on body weight in STZ-NIC induced diabetic rats

STZ-NIC induction led to significant (p < 0.05) body weight loss (from 201.6 ± 2.5 g to 124 ± 1.7 g) compared to the controls. This proves that STZ-NIC had induced diabetes in rats, which is characterized by severe loss in the bodyweight. The treated groups (50 and 100 mg/kg b.w.) showed significant improvement in body weight from 199.6 ± 4.7 g (day 0) to 201 ± 3.1 g (day 35) and 199 ± 2.6 g (day 0 to 201.6 ± 2.5 g (day 35 compared to diabetic group, respectively. Metformin (a diabetic drug) treatment also showed weight gain from 198.2 ± 2.6 g to 205.6 ± 2.5 g (Table 5). STZ is transported into β-cells via the glucose transporter GLUT2 and causes DNA damage and the partial protective action of β cells in NA is due to the inhibition of PARP-1 activity. NA inhibits this enzyme, preventing depletion of NAD⁺ and ATP in cells exposed to STZ. The reduction in body weight noticed in the diabetic group due to the effect of protein squandering, because of inaccessibility of carbohydrate for use as a vital source. Similar findings were observed in type 2 diabetic induced (by streptozotocin-nicotinamide) rats, which was treated by Alangium lamarckii Thwaites leaves (AAL) alcoholic extract. Further, the reports showed a decrease in body weight from 174.16 ± 8.07 g (day 0) to 131.16 ± 7.12 g (day 14 in diabetic group; and in AAL treated group (250 and 500 mg/kg), it was 178.83 ± 4.72 g (day 0) to 142.66 ± 4.92 g (day 14) and 171.50 ± 3.51 g (day 0) to 157.83 ± 3.32 g (day 14) (Kumar et al., 2011).

3.4 Effect of ME on blood glucose level in STZ-NIC induced diabetic rats

During the entire study, fasting blood glucose level was validated; oral administration of 50 and 100 mg/kg ME showed a significant anti-hyperglycemic effect. Reduction of blood glucose level from before treatment 416.8 ± 3.9 mg/dL (day 0 to after treatment 142.6 ± 3.5 mg/dL (day 35); and 413.4 ± 3.8 mg/dL (day 0) to 120.8 ± 3.3 mg/dL (day 35) was observed upon treatment with J. gossypifolia ME at the concentration of 50 and 100 mg/kg b.w., respectively; however, maximum reduction was observed after treatment. Metformin exhibited 108.8 ± 3.4 mg/dL reduction in blood glucose level at the end of the study as compared to the diabetic group. J. gossypifolia ME treated groups (100 mg/kg) displayed a maximum lowering effect of glucose in diabetic rats compared to low dose of ME (50 mg/kg). The data substantiates the anti-hyperglycemic effect of ME as comparable with the positive control (Table 6). The hyperglycemia was elevated in the blood glucose because of the decreased secretion of insulin. The prolonged blood glucose level after intraperitoneal administration of STZ infers the hyperglycemic condition; and oral organization of 50 and 100 mg/kg of ME for 35 days essentially decreased the fasting blood glucose level in the diabetic rats, which demonstrated the antidiabetic activity of J. gossypifolia ME. Similarly, maximum reduction of blood glucose level was observed in oral administration of ethanolic extract of Helianthus annuus L., seed on day 21 i.e. 175.24 ± 4.12 mg/dL with 250 mg/kg and 124.65 ± 2.64 mg/dL with 500 mg/kg (Saini and Sharma, 2013).

3.5 Effect of ME in IPGTT

At the conclusion of the treatment cycle with vehicle and insulin, glucose tolerance was tested by IPGTT (2 g/kg. i.p). Fig. 2 shows that nearly all animals are hyperglycemic at 30 min. This hyperglycemia exacerbated by the i.p glucose load; and after 150 min it did not return to the baseline, which is suggesting the glucose intolerance in diabetic rats. Blood glucose reduction was observed in rats treated with ME (50 and 100 mg/kg) it is revealed a decrease in blood glucose levels of 121.5 ± 2.5 mg/dL (50 mg/kg b.w.) and 118.8 ± 2.1 mg/dL (100 mg/kg b.w.). This is due to the interference of intestinal absorption or by the potentiation of insulin secretion and in addition, by increasing the use of glucose levels in muscles as demonstrated by Fuhlendorff et al. (1998). Equally, oral administration of Codariocalyx motorius treatment (200 mg/kg b.w.) was also significantly increased the glucose tolerance after glucose administration from 30 to 120 min (Uma et al., 2014).

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Fig. 2

Effect of J. gossypifolia ME dose 50 (LD) and 100 mg/kg b.w (HD) on intra peritoneal glucose tolerance test. Values are given as means ± SD (n = 5) from three independent experiments in triplicate. Statistical evaluation done by one-way ANOVA followed by Duncan’s multiple range test (DMRT). *p < 0.05 as compared with control group, #p < 0.05 as compared with Diabetic group.

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3.6 Histopathology analysis of pancreas

Control and drug control rats (Fig. 3a and b) showed the presence of normal pancreatic β cells in islets of Langerhans on endocrine portion. Diabetic rats (STZ + NIC) as shown in Fig. 3c displayed a dilated and degranulated islet cells with damage and reduction in size of pancreatic β cells. Diabetic rats treated with ME (Fig. 3d and e) 50 and 100 mg/kg, re-established the necrosis and fibrotic changes; and increased the pancreatic β cells in islets of Langerhans. In other hands, Metformin-treated diabetic rats (500 mg/kg b.w.) showed a normal pancreatic islet cells (Fig. 3f). J. gossypifolia ME increased the number of insulin positive cells in the pancreas and either prevented or improved the β-cells of the islets of Langerhans with accords reported in C. motorius (Uma et al., 2014).

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Fig. 3

Histopathology of pancreas in STZ-NIC induced diabetic rats after 35 days of treatment. (a and b) Control and drug control (ME 100 mg/kg b.w.) rat indicates the normal pancreas, (C) Diabetic control-degranulated, (d) Diabetic + ME (LD – 50 mg/kg b.w.) and (e) Diabetic + ME (HD – 100 mg/kg b.w.) – showing granulated pancreatic islets, (f) Diabetic + Metformin – granulated and normal pancreas appearance.

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3.7 Effect of ME on serum insulin level

The control and the experimental rats displayed serum insulin levels (Fig. 4). A significant increase (p < 0.05) in serum insulin level was observed in the oral administration of 50 and 100 mg/kg b.w. up to 13.01 ± 0.41 µU/ml and 14.13 ± 0.52 µU/ml, respectively as compared to the insulin level dropped in diabetic rats (5.20 ± 0.14 ml). The decrease in the blood glucose level due to the oral administration of ME for 35 days increased the plasma insulin level, this may be due to the phyto-constituents present in the plant extract, which influenced the insulin secretion and protected the active β-cells that yielded insulin and reduced the glucotoxicity. Similarly, Ficus amplissimaIn smith. bark extract in STZ induced diabetic rats showed a decrease in serum insulin level; and administration of extract in three different doses (50, 100 and 150 mg/kg b.w.) caused significant increase in the serum insulin level by 11.81 ± 0.38 µU/ml, 15.77 ± 0.42 µU/ml and 15.87 ± 0.52 µU/ml, respectively (Arunachalam and Parimelazhagan, 2013).

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Fig. 4

Effect of ME on level of serum insulin on STZ-NIC induced diabetic rats. Metformin used as a standard drug. Data are given as means ± SD (n = 5) from three independent experiments in triplicates. Statistical evaluation done by one-way ANOVA followed by Duncan’s multiple range test (DMRT). *p < 0.05 as compared with control group, #p < 0.05 as compared with Diabetic group.

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3.8 HOMA-IR index

The HOMA-IR index in ME treated groups (50 and 100 mg/kg b.w) displayed a decreased peripheral resistance to insulin of 4.5 ± 1.5 (50 mg/kg b.w.) and 4.2 ± 1.2 (100 mg/kg b.w.), respectively as compared to the diabetic group (6.7 ± 2.1) (Fig. 5). Insulin plays an important role in maintaining the normal level of blood glucose, which was confirmed by the HOMA-IR results that the insulin resistance developed in the experimental groups. Metformin treated rats showed an index of 4.3 ± 1.6, which is indicating that, the oral treatment of ME improved insulin resistance on par with the standard drug. Previous studies by Wang et al. (2013), demonstrated that the HOMA-IR value of Swertia macrosperma extracts treated diabetic rats were extremely less (ethanolic extract 250 and 500 mg/kg b.w., showed 5.72 ± 0.59 and 5.61 ± 0.78 and n-butanol 250 and 500 mg/kg b.w., showed 6.64 ± 1.07 and 5.25 ± 0.57, respectively) than the diabetic rats 10.19 ± 0.72.

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Fig. 5

HOMA-IR index on STZ-NIC induced diabetic rats. Metformin used as a standard drug. Data are given as means ± SD (n = 5) from three independent experiments. Statistical evaluation done by one-way ANOVA followed by Duncan’s multiple range test (DMRT). *p < 0.05 as compared with control group, #p < 0.05 as compared with Diabetic group.

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3.9 Effect of ME treated diabetic rats on the gene expression in muscle tissues

Administration of STZ-NIC induced experimental diabetes in the rats was causing damages in the pancreatic β-cells and leads to a reduction in the cellular response to insulin resistance. Insulin-signalling pathway impairment leads to insulin-stimulated glucose uptake failure in targeted tissues such as muscle and fat. This is because of the insulin is incapable of stimulating a GLUT 4 translocation from intracellular pool to PM in muscle cells (Epstein et al., 1999). To examine the mechanism underlying the effects of ME on insulin signalling pathway, IRTK, IRS-1, PI3K, GLUT-4, GSK-3β and GS gene expression were analysed. The results showed that 50 and 100 mg/kg b.w ME treatment to the experimental rats significantly (p < 0.05) increased the expression of IRTK (2.3 and 2.9 fold), IRS1 (2.4 and 2.7 fold) PI3K (2.2 and 2.4 fold), GSK-3β (0.4 and 0.3 fold), GS (2.3 and 2.5 fold) and GLUT 4 (2.5 and 2.9 fold) (Fig. 6a–f).

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Fig. 6

Effect of ME treated diabetic rats on the gene expression in muscle tissues on IRTK (a), IRS1 (b), PI3K (c), GSK-3β (d), GS (e) and GLUT 4 (f) mRNA expression level using quantitative (real time) PCR. Data expressed as mean ± S.D of three independent experiments in triplicates. Statistical evaluation done by one-way ANOVA followed by Duncan’s multiple range test (DMRT). *, P < 0.05 as compared with control, #p < 0.05 as compared with Diabetic group.

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These results suggested that ME induced insulin to bind to its receptor of tyrosine kinase; the insulin receptor substrate (IRS-1) is leading to tyrosine phosphorylation and the activation of phosphatidyl inositide 3-kinase (PI3 K). AKT/PKB (Protein Kinase B) and PKC (Protein Kinase C) are the downstream targets of PI3K; the activation/phosphorylation of which causes GLUT 4 translocation to the plasma membrane and improves glycogen synthase enzyme by inhibiting the glycogen synthase kinase-3β (GSK-3β). Sujatha et al. (2010), stated that both crude and (3β)-STIGMAST-5-EN-3-OL can increase the expression of mRNA in IRTK, IRS1and PI3K; and eventually the downstream kinases are also activated. Our results are corroborate with the earlier findings in activating the expression of the key targets of insulin signalling pathway.

3.10 Effect of ME on IRβ and GSK-3β protein expression

Insulin receptor originates from the member of receptor tyrosine kinase family, which comprises the receptors for insulin, insulin like growth factors and several other growth factors (Petruzzelli et al., 1982). Effect of ME on insulin receptor β protein in muscle samples are analysed using blot (Fig. 7a). Diabetic rats showed 0.4 fold decrease in IRβ protein, whereas ME treated group (50 and 100 mg/kg b.w.) showed 2.4 and 2.7 fold increase in IRβ protein levels, respectively (Fig. 7b). The oral treatment of ME remarkably reduced the level of lipids in the diabetic induced rats, henceforth increased level of insulin receptor was observed. (Satyanarayana et al., 2015), reported that Azadirachta indica, being a effective antioxidant and anti-hyperlipidemic agent was able to increase the protein expression of IR by reducing the lipid level in type 2 diabetic rats equivalent to that of metformin.

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Fig. 7

(a) Effect of ME treated diabetic rats on IRβ and GSK-3β protein expression. Lane 1 denotes control, Lane 2 drug control (ME 100 mg/kg b.w.), Lane 3 indicates diabetic, Lane 4 and 5 indicates ME (LD 50 and HD 100 mg/kg b.w.) respectively. IRβ and GSK-3β protein expressions shown. β-Actin served as loading control. (b) Densitometric analysis of IRβ and GSK3β protein expression upon treatment with ME (50 and 100 mg/kg b.w.). Data expressed as mean ± S.D of three independent experiments in triplicates. Statistical evaluation done by one-way ANOVA followed by Duncan’s multiple range test (DMRT). *P < 0.05 as compared with control, #p < 0.05 as compared with Diabetic group.

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GSK-3β inhibition stimulate an increase in glycogen synthesis and insulin sensitivity. Activation of GSK-3β contributes to a reduction in glycogen synthesis and decreased insulin sensitivity (Coghlan et al., 2000; Liberman and Eldar-Finkelman, 2005). Western blot analysis revealed that the ME groups (50 and 100 mg/kg b.w.) showed 2.2 and 2.4 fold increase in GSK-3β (serine 9 phosphorylation) protein level, respectively as compared with diabetic group (0.4 fold decrease in the expression level) (Fig. 8a and b). Previous studies have shown that the inhibition of GSK3, oral glucose disposal could be improved by increasing liver glycogen synthesis in a Zucker diabetic fatty (falfa) rats (Cline et al., 2002). A selective GSK3 inhibitor upregulated the intracellular glycogen level and increased GLUT 4 expression in the skeletal muscle of ob/ob diabetic mice which was well correlated with our data (Kaidanovich-beilin and Eldar-finkelman, 2006).

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Fig. 8

(a) GLUT 4 protein expression by ME treated diabetic rats. Lane 1 denotes control, Lane 2 drug control (ME 100 mg/kg b.w.), Lane 3 indicates diabetic, Lane 4 and 5 indicates ME LD 50 and HD 100 mg/kg b.w. respectively. GLUT 4 protein expressions were shown. β-Actin served as a loading control. (b) Densitometric analysis of translocation of GLUT 4 using PM fractions. The fold increase of GLUT 4 upon treatment with ME quantified arbitrarily. Data expressed as mean ± S.D of three independent experiments in triplicates. Statistical evaluation done by one-way ANOVA followed by Duncan’s multiple range test (DMRT). *P < 0.05 as compared with control, #p < 0.05 as compared with Diabetic group.

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3.11 Effect of ME on GLUT 4 protein expression in PM fractions

Skeletal muscles highly express GLUT 4, which is responsible for the insulin stimulated glucose uptake. Effect of GLUT 4 protein expression in plasma membrane fraction detected in the muscle of Albino wistar rats. Western blot revealed that there was no significance difference in the GLUT 4 protein expression in the control group and the extract treated non-diabetic control group. Diabetic rats, showed a decreased GLUT 4 protein expression level (0.4 fold). The 50 and 100 mg/kg b.w. of ME treated rats showed 2.2 and 2.4 fold increase in GLUT 4 protein levels, respectively (Fig. 8a and b)). The oral administration of ME to the experimental groups improved the insulin sensitivity by activating the insulin signalling cascades, which reduced the lipid accumulation and controlled obesity contributing to the restoration of GLUT 4 expression. Similarly, GLUT 4 expression in plasma membrane measured in soleus muscle of STZ-diabetic mice. Treatment with Gardeniae Fructus significantly increased GLUT 4 expression, 1.4 time-greater than that of H₂O- treated control group (Yu et al., 2013).