Nano‐encapsulated Tinospora cordifolia (Willd.) using poly (D, L‐lactide) nanoparticles educe effective control in streptozotocin‐induced type 2 diabetic rats

Abstract

The therapeutics for type 2 diabetes mellitus has emerged in the current century towards nanomedicine incorporated with plant active compounds. In this study, Tinospora cordifolia loaded poly (D, L‐lactide) (PLA) nanoparticles (NPs) were evaluated in vivo for their anti‐hyperglycemic potency towards streptozotocin‐induced type 2 diabetic rats. T. cordifolia loaded PLA NPs were synthesised by the double solvent evaporation method using PLA polymer. The NPs were then characterised and administrated orally for 28 successive days to streptozotocin‐induced diabetic rats. The PLA NPs had significant anti‐diabetic effects which were equal to the existing anti‐diabetic drug glibenclamide. The antidiabetic activity is due to the synergism of compounds present in stem extract of the plant which reduced the side effects and anti‐diabetic.

1 Introduction

Diabetes mellitus is metabolic syndrome interpreted by hyperglycemia due to deformity in insulin secretion or less binding efficiency of insulin. It is classified into type 1, type 2 and gestational diabetes. Type 1 is an autoimmune disorder due to the disruption of insulin‐generating beta cells of islets of Langerhans in the pancreas. Type 2 is a chronic condition in which the insulin becomes resistant or lack of insulin secretion [1]. Gestational diabetes occurs during pregnancy due to hormonal changes, which increase blood sugar level [2]. Currently, diabetes supervision is carried out with insulin therapy and a hypoglycemic drug which causes adverse side effects that lead to various organ failures like nephropathy, retinopathy, neurological disorder and cardiovascular alignments [3]. So, as an alternative remedy a large number of medicinal plants, widely recognised as an important source of drugs, have been used to treat diabetes because these natural products have lesser toxic effects. It is also estimated that about 80% of diabetics around the world population presently depending upon herbal medicine for their successful treatments [4]. Tinospora cordifolia (Willd.) Miers ex Hook.f.&Thoms is a prodigious, barren, ephemeral climbing shrub correlate to the family menispermaceae [5]. The stem and root of T. cordifolia are predominantly used in ayurvedic and ancestry medicine for restorative, anti‐inflammatory, antiarthritic and antiallergic effects [6, 7]. The stem extract of T. cordifolia is extensively used in a folk system of medicine for treating diabetes [8]. This plant is rich in alkaloids, phenols, glycosides, aliphatic compounds and natural polymers like mucilages and gums [9]. The water‐dispersible alkaloids like berberine, palmatine, tinosporine and isocolumbine are present in this plant, which acts as a glucosidase inhibitor [10, 11]. Nanoparticle (NP) could act as a useful carrier for a range of biomolecules such as protein, enzymes, amino acids and drugs. They bind effectively and have enhanced stability with low toxicity [12].

In this study, polymers are used as the entrapment ingredients for the release of the compound, which acts as pro‐drug or drug carriers. Numerous biodegradable polymeric NPs have been broadly used for delivery of molecules and drugs [13, 14, 15]. The proficiency of polymeric NPs acts as a carrier of drugs to target sites by reducing the adverse side effects [16]. Diverse biodegradable polymers such as poly (D, L‐lactide) (PLA), poly‐lactic‐co‐glycolic acid (PLGA), poly‐ɛ‐caprolactone, gelatine, chitosan and gelatine poly (alkyl cyanoacrylates) have been notably used for targeted delivery of drugs associated with diabetes, cancer, asthma, malaria and other malignant diseases [17, 18, 19, 20, 21]. PLA and PLGA are mostly used in the synthesis of NPs and approved in a parental application by the FDA [22]. Among many biodegradable polymers, PLA has been substantially used for encapsulation of therapeutic agents relevant to its high biodegradability, low toxicity, biocompatibility, bioavailability and prolonged drug release [23, 24, 25, 26].

The hypothesis behind this study was to analyse the efficiency of nano‐encapsulated total cholesterol (TC)‐loaded PLA NPs and its antidiabetic activity on streptozotocin (STZ)‐induced type 2 diabetic Wistar rats.

2 Materials and methods

2.1 Materials

PLA and polyvinyl alcohol (PVA) was procured from Sigma Aldrich, USA. STZ, phenazinemethosulphate, dichloromethane (DCM), nitrobluetetrazolium, N‐butanol, potassium dichromate, trichloroacetic acid, pyridine was purchased from Himedia Laboratories Ltd, rat insulin enzyme‐linked immunosorbent assay (ELISA) kit (RayBio®, USA) and other chemicals used were of analytical grade.

2.2 Synthesis of TC‐loaded PLA NPs

TC‐loaded PLA NPs were synthesised using a double solvent evaporation method and based on the optimisation of full factorial design. PLA and lyophilised TC stem extract were sonicated together at 40% amplitude in DCM solution for 30 s at room temperature to form a primary emulsion, followed by addition of PVA solution and sonicated to form a secondary emulsion and the organic solvent DCM present in the emulsion was evaporated by stirring for 3 h. After stirring the solution was centrifuged at 16,500 rpm for 10 min at 10°C and washed thrice with distilled water. The resulting NPs were lyophilised using lyophiliser (Lark Innovative Fine Technology, India) and stored at 2–8°C till further use. The synthesis and characterisation of NPs such as Fourier transform infrared (FT‐IR), liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS/MS), X‐ray diffraction (XRD) and high‐resolution scanning electron microscopy was already reported in our earlier study [27].

2.3 In vitro haemolysis assay

The haemolytic activity was performed to study the compatibility of TC‐loaded PLA NPs in red blood cells (RBCs). The blood sample was collected from a healthy rat. Alsever's solution (5 ml) which is used as an anticoagulant was added to the blood sample (5 ml) and the sample was centrifuged at 1500 rpm for 10 min. The pellet was washed using 5 ml of phosphate buffer saline (PBS). RBC solution was prepared by adding 5 ml of PBS to the washed pellet. Different concentration (7.8, 15.6, 31.2, 62.5, 125, 250, 500 and 1000 µg/ml) of synthesised TC‐loaded PLA NPs was prepared in 1 ml of PBS. 0.5 ml of RBC solution was added to different concentration of synthesised NPs and the reaction mixture was incubated for 30 min at 37°C. After incubation, the samples were centrifuged at 1500 rpm for 5 min and the supernatant obtained was estimated for absorbance at 540 nm. 0.1% of Triton‐X 100 solution as the positive control (PC) and RBCs without treatment as the negative control. Graphs are plotted using the percentage haemolysis at Y ‐axis and the concentration of the sample in X ‐axis [28].

2.4 Cell viability

The toxicity of the TC‐loaded PLA NPs was assayed in RIN5F cell lines by (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐tetrazolium bromide) (MTT) assay. Cell lines were obtained from National Centre for Cell Sciences (NCCS), Pune and cells were maintained in dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 50 μg/ml CO₂ at 37°C is recommended. Cells concentration of (1 × 10⁵ /well) were plated in 24‐well plates and incubated at 37°C with 5% CO₂ condition. At confluence stage, different concentrations (7.8, 15.6, 31.2, 62.5, 125, 250, 500 and 1000 µg/ml) of the synthesised TC‐loaded PLA NPs samples were added and incubated for 24 h. After the incubation period, the sample was removed from the well and washed with phosphate‐buffered saline (pH 7.4) or DMEM without serum. In total, 100 μl/well of 0.5% MTT was added and incubated for 4 h. After incubation, 1 ml of DMSO was added in all the wells and the absorbance was measured at 570 nm in ultraviolet‐spectrophotometer using DMSO as the blank. Graphs are plotted using the percentage of cell viability at Y ‐axis and the concentration of the sample in X ‐axis.

2.5 Experimental animals

Female Wistar albino rats (Rattus norvegics) of 8–10 weeks old, weighing between 150 and 200 g were chosen for the study. They were procured from the animal house, Vellore Institute of Technology, Vellore. The animal experimental procedures were followed as per the consent and approval from the Institutional animal ethical committee (VIT/IAEC/11th October 10th/No.38). Animals were treated according to the prescribed guidelines by the Committee for the Purpose of Supervision and Control of Experiments on Animals (CPSCEA), Government of India, Chennai, Tamil Nadu and IAEC. Animals were housed in standard laboratory conditions of 27°C and subjected to 12‐h day/night cycle. They were served with pelleted feed and water ad libtium.

2.5.1 Acute toxicity study

Toxicity studies were carried out by adapting OECD guidelines‐423 [29]. In this study female rats were divided into three groups consisting of three rats in each group to study the toxicity profile. The acute toxicity of TC‐loaded PLA NPs was studied by preparing three different concentrations at a dose with two minimal and one higher dose range (Group A‐5 mg, Group B‐10 mg and Group C‐50 mg/kg b.wt.) and administered orally. Prior to conducting the experiment animals fasted for 24 h and dose was administered. The animal was observed during the test period for 14 days.

2.5.2 Induction of type 2 diabetes

The rats were supplied with 10% fructose in drinking water for two weeks to make partial dysfunction of beta cells of islets [30]. After induction of fructose, the rats fasted overnight. STZ which was prepared freshly in 0.1 M citrate buffer (pH 4.5) and the calculated amount (40 mg/kg b.wt.) of the solution was injected intraperitoneally to overnight fasted rats [31]. Blood glucose levels in the rats were determined and rats with blood glucose ranging over 250 mg/dL were used for the study.

2.5.3 Experimental design

Experimental rats were divided into five groups with each group consisting of six rats and the treatment was as follows:

• Group I: Normal control (non‐diabetic rats treated with citrate buffer alone).

• Group II: PC (diabetic rats treated with glibenclamide 0.1 mg/kg b.wt.).

• Group III: Negative control (STZ‐induced diabetic rats).

• Group IV: Diabetic rats treated with TC‐loaded PLA‐NPs 5 mg/kg b.wt.

• Group V: Diabetic rats treated with TC‐loaded PLA‐NPs 10 mg/kg b.wt.

The TC‐loaded PLA NPs and glibenclamide were administered orally through oral gavage no 18, 2 inches in length and ball diameter is 2.25 mm with a curved shape. The body weight and blood glucose levels were examined in all the groups at 1, 7, 14, 21 and 28 days. The GOD‐POD was determined using Glucose Test kit (Arkray®) as per manufactures protocol and the absorbance of quinone imine is measured at 505 nm and which is directly proportional to glucose concentration in the sample. At the end of the experimental period, the 12 h fasted animals were sacrificed under anaesthesia and blood samples were collected, the serum was separated by centrifugation (2000g for 20 min) and stored at −20°C for biochemical assays. The liver and pancreas tissues were removed from the sacrificed rats, washed in ice‐cold saline to remove the bloodstain and stored at −80°C for further assay.

2.5.4 Biochemical parameters

Biochemical parameters were evaluated in the blood serum and liver homogenate. The serum profile markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), TC, high‐density lipoprotein (HDL), low‐density lipoprotein (LDL) and triglycerides (TGs) are estimated by using the commercial kit (Arkray®) as per the manufacturer's protocol. The liver homogenate was prepared using homogenisation buffer containing 0.25 M sucrose, 1 mM EDTA, 10 mM HEPES‐NaOH (pH 7.4) and centrifuged at 4°C for 20 min at 9000 rpm [32]. The resulting supernatant was used to determine the liver profile using spectrophotometric methods such as catalase (CAT) [33], superoxide dismutase (SOD) [34], glutathione peroxidase (GPx) [35] and Lipid peroxidation (LPx) [36].

2.5.5 Quantitative determination of rat insulin by ELISA

The rat serum was diluted using assay diluents and was subjected to ELISA analysis using the rat insulin ELISA kit (RayBio®) as per the manufacturer's protocol.

2.5.6 Histopathological examinations

Liver and pancreas tissues were fixed in 10% formalin buffer. Following the fixation, tissues were sliced using an automated tissue processor and embedded in wax. Tissue sections were cut to a thickness of 5 μm using a Leica microtome and stained by hematoxylin and eosin staining. The slides thus obtained were subjected to histopathological analysis.

2.6 Statistical analysis

All data are represented as mean ± standard error mean (SEM) analysed using GraphPad Prism® 7 software (GraphPad Software, Inc. La Jolla, CA, USA). The groups were compared by one‐way analysis of variance (ANOVA) with Tukey test.

3 Results and discussion

The synthesis and characterisation of TC loaded PLA NPs were already reported in our previous study. The polymeric NPs were used as drug delivery molecules due to the properties such as biodegradability, biocompatibility, high stability and encapsulation efficiency and sustained release effect [37, 38]. The TC stem extract was prepared and polymeric TC‐loaded PLA NPs were synthesised using a solvent evaporation method. The FT‐IR analysis of the extract showed a major curve corresponds to an alcohol group which is further confirmed by liquid chromatography mass spectrometry (LC‐MS/MS) analysis. The insulin receptor undergoes the conformational changes by activating the tyrosine kinase β subunit and induces glucose uptake in cells [39]. The FT‐IR spectrum of synthesised TC‐loaded PLA NPs confirms the encapsulation of polymer with TC extract and revealed the presence of alkaloids. The XRD pattern predicted the amorphous structure of synthesised NPs and the zeta potential value of the NPs was −28.0 mV, implying the good stability of the nanoformulation. The high resolution scanning electron microscopy (HR‐SEM) image confirmed the spherical nature of NPs and the size was predicted between 134.4 and 236.8 nm. The size and shape of NPs directly influenced their cellular uptake, which was accompanied in the cell by endocytosis process [40]. TC release from PLA NPs through the dialysis membrane displayed a controlled release up to 20 h and release kinetic constant showed good correlation with Higuchi kinetics. A drug release study proved that the nanoformulations have a sustained release profile compared to their pure forms due to the encapsulation of the drug moiety in the polymer matrix [41].

The haemolysis activity of TC‐loaded PLA NPs was estimated and the haemolytic percentage was determined using the formula

$$\text{\%}\mathrm{Hemolysis}=\frac{\mathrm{O}{\mathrm{D}}_{\mathrm{Sample}}-\mathrm{O}{\mathrm{D}}_{\mathrm{Saline}}}{\mathrm{O}{\mathrm{D}}_{\mathrm{PC}}-\mathrm{O}{\mathrm{D}}_{\mathrm{Saline}}}\times 100$$ (1)

TC‐loaded PLA showed good haemocompatibility profile, with the lowest activity (0.89% at 7.8 µg/ml and 5.05% at 1000 µg/ml) considering non‐toxic and safe to use as a drug (Fig. 1 a).

Fig. 1.

In vitro analysis of TC loaded PLA NPs

(a) Percentage haemolysis of TC‐loaded PLA NPs,

(b) Cell viability of TC‐loaded PLA NPs

The percentage of cell viability was calculated using the following formula

$$\text{\%}\mathrm{Cell}\mathrm{viability}=\frac{\mathrm{A}570\mathrm{of}\mathrm{treated}\mathrm{cells}}{\mathrm{A}570\mathrm{of}\mathrm{control}\mathrm{cells}}\times 100$$ (2)

Graphs are plotted using the percentage of cell viability at Y ‐axis and the concentration of the sample in X ‐axis. Cell control and sample control are included in each assay to compare the full cell viability assessment. The cell viability was 50.75% at 500 µg/ml concentration of TC‐loaded PLA NPs. (Fig. 1 b). From the previously reported studies, T. cordifolia stem extract at 100 µg/ml concentration shows a higher cell viability percentage [42].

Animals were observed during the experimental acute toxicity study period and parameters such as skin and fur condition were normal. No breathing abnormalities and eye dullness was nil. Body weight was noted at the beginning, 7th and 14th day of the study period, increase in body weight was observed in all three groups (Fig. 2 a). No lethality was observed which manifests that TC‐loaded PLA NPs was non‐toxic and safe. At the end of the study period, the animals were sacrificed under anaesthesia. Liver tissue was dissected and subjected to histological examinations (Fig. 2 b). Histopathological examination of Group A rat liver showed normal architecture with central veins and portal vessels. Group B showed liver with preserved architecture and mild portal congestion was observed, no evidence of inflammation and necrosis. In Group C liver parenchyma with preserved architecture, dilated and congested portal vessels. Based on the acute toxicity study dosage were selected for the treatment group.

Fig. 2.

Acute toxicity study

(a) Effects of body weight in rats,

(b) Histological examination of rat liver. Tissues were stained with haematoxylin and eosin (100×)

In an in vivo anti‐diabetic study, reduction in the body weight was observed in diabetic rats might be the result of degradation of structural proteins and fats due to deficiency of carbohydrate for the energy metabolism [43], while a decrease in serum insulin level was due to the destruction of the pancreatic β cells [44]. Fig. 3 a interprets the variations in the final body weight upon administration with TC‐loaded PLA NPs. Treatment of rats with glibenclamide and TC‐loaded PLA NPs increased in body weight of diabetic rats. Change in body weight was minimal in both glibenclamide and TC‐loaded PLA NPs treated rats. However, the body weight of the negative control (Group III) STZ‐induced diabetic rats reduced at the final week of the treatment period.

Fig. 3.

In vivo antidiabetic study

(a) Effects of body weight in rats,

(b) Effects of fasting blood glucose level

Fasting blood glucose level was measured in normal and experimental groups at the initial day, 7th day, 14th, 21st and 28th day of treatment. STZ administration in diabetic rats (Group III) exhibited an increase in the blood glucose level when compared to Group I and Group II. TC‐loaded PLA NPs treated rats showed an increase in the blood glucose level when compared with normal control (Group I), but when compared with the negative control (Group III) a reduction in blood glucose level was observed.

A decrease in blood glucose level in TC‐loaded PLA NPs treated rats is almost similar to glibenclamide‐treated rats, signified the hypoglycemic potential of TC‐loaded PLA NPs (Fig. 3 b). The treatment of TC‐loaded PLA NPs (5 and 10 mg/kg. b.wt.) in diabetic rats for 28 days significantly increased insulin level and decreased the blood glucose levels might have, either by stimulating the beta cells to secrete insulin or by regeneration of pancreatic β cells, which was supported by histopathological investigations.

The results of in vivo study proved that the TC‐loaded PLA NPs were able to decrease blood sugar level equivalent to the glibenclamide an anti‐diabetic drug.

Usually, the oxidative stress due to hyperglycemia increases through the overproduction of ROS, which disturbs the antioxidant defence systems of the cells. The antioxidant enzymes CAT, SOD, GPx and LPx levels in the liver of different experimental groups were shown in (Table 1). Compared with normal (Group I) and PC (Group II), the level of MDA (P  < 0.001) significantly increased in STZ‐induced diabetic rats (Group III), whereas the activities of CAT (P  < 0.001) and GPx (P  < 0.001) was decreased in Group III rats. As shown in the data, Group II PC rats showed a significant increase (P  < 0.001) in CAT and SOD level compared to Group III. Treatment of TC‐loaded PLA NPs led to significant increases (P  < 0.01) in CAT, SOD, GPx level and diminution in LPx level was observed in comparison with Group III diabetic rats. These results represented that glibenclamide and TC‐loaded PLA NPs had an apparent capacity to regulate antioxidant enzymes level or to decrease damage in the liver caused due to diabetes. (Table 1). The antioxidant activity of alkaloids presents in the TC‐loaded PLA NPs has attributed to the increased levels of antioxidant enzymes CAT and GPx in TC‐loaded PLA NPs in treated rats.

Table 1.

Enzymatic activity

Parameters	Group I	Group II	Group III	Group IV	Group V
CAT (µmoles of H2 O2 consumed/min/mg protein)	458.85 ± 10.7	436.30 ± 9.1	130.74 ± 5.85a*** b***	425.89 ± 5.91 a*c***	441.88 ± 3.28 c***
SOD (nmoles of enz/g tissue)	161.5 ± 3.15	150 ± 7.23	99.22 ± 6.66 a*** b***	146.29 ± 2.88 c***	152.17 ± 2.57 c***
GPx (U/mg protein)	14.45 ± 0.44	12.77 ± 1.2	7.33 ± 0.69 a*** b**	12.28 ± 0.44 c**	14.21 ± 0.76 c***
LPx (nmoles of MDA formed/mg tissue wt)	0.087 ± 0.001	0.095 ± 0.002	0.178 ± 0.012 a*** b***	0.099 ± 0.004 c***	0.091 ± 0.002 c***

Statistical analysis – ANOVA, followed by Tukey's comparison test.

a* indicates the comparison with Group I, b* indicates the comparison with Group II and c* indicates the comparison with Group III.

Values are expressed as mean ±SEM, n  = 6, *p  < 0.05, **p  < 0.01 and ***p  < 0.001.

Lipid metabolism plays an important role in the occurrence and development of diabetes. Over‐accumulation of lipids results in insulin resistance, islet β‐cell damage and eventual development of diabetes. Anti‐diabetic effect of TC‐loaded PLA NPs was observed through the determination of lipid profile and the levels of AST and ALT in the serum. It was observed that STZ‐induced rats (Group III) exhibited a significant increase in the levels of TC (P  < 0.001) and TG (P  < 0.001) when compared to normal control and PC groups. However, a significant decrease in the level of HDL (p  < 0.001) was observed in Group III diabetic rats when compared to normal control (Group I). Administration of TC‐loaded PLA NPs brought back the levels of AST, ALT, total cholesterol and HDL to near normal values, signified the hypolipidemic activity of TC‐loaded PLA NPs (Table 2).

Table 2.

Serum lipid profile analysis

Parameters	Group I	Group II	Group III	Group IV	Group V
AST (IU/L)	41.10 ± 2.74	46.85 ± 2.62	89.43 ± 3.15 a*** b***	48.47 ± 4.01 c***	43.16 ± 2.54 c**
ALT (IU/L)	39.92 ± 1.48	56.72 ± 5.81 a**	74.10 ± 2.95 a*** b**	51.27 ± 2.11 c***	42.72 ± 1.94 b*c***
total cholesterol (mg/dl)	154.16 ± 3.57	150.52 ± 2.5	254.68 ± 3.93 a*** b***	159.37 ± 2.21 c***	149.47 ± 2.40 c***
HDL (mg/dl)	93.76 ± 1.42	92.06 ± 2.13	37.56 ± 2.88 a*** b***	88.05 ± 2.87 c***	90.15 ± 2.78 c***
LDL (mg/dl)	38.13 ± 4.60	35.25 ± 1.42	173.85 ± 3.42 a*** b***	47.79 ± 3.28 c***	36.47 ± 4.66 c***
TGs (mg/dl)	111.3 ± 2.65	116.01 ± 0.74	216.34 ± 6.08 a*** b***	117.57 ± 5.34 c****	114.24 ± 2.24 c***

Statistical analysis – ANOVA followed by Tukey's comparison test.

a* indicates comparison with Group I, b* indicates comparison with Group II and c* indicates comparison with Group III.

Values are expressed as mean ±SEM, n  = 6, *p  < 0.05, **p  < 0.01 and ***p  < 0.001.

Decreased insulin secretion increases lipolysis, which ultimately causes elevated serum lipid levels, which was observed in group III diabetic rats. However, the administration of TC‐loaded PLA NPs significantly lowered TC, TG levels and increased HDL levels compared with the diabetic control group. This result suggests that TC‐loaded PLA NPs could efficiently adjust serum lipids in diabetic patients, and it might also reduce the risk of developing cardiovascular disease.

The changes in the level of serum insulin in the experimental animals were shown in (Fig. 4). The serum insulin level was significantly decreased in Group III rats (1.381 ± 0.007 µIU/ml) when compared to Group I rats (2.119 ± 0.035 µIU/ml). However, a marked increase in the level of insulin was observed in glibenclamide treated Group II rats (1.787 ± 0.040 µIU/ml) in comparison with Group III. A similar result was also observed in Group IV rats treated with TC‐loaded PLA NPs (5 mg/kg b.wt.) compared to Group II. The insulin level of Group V rats (1.803 ± 0.043 µIU/ml) increased significantly (P  < 0.001) when compared to Group III rats. Thus, the results depict the increase in insulin concentration after administration of TC‐loaded PLA NPs.

Fig. 4.

Quantitative determination of rat insulin concentration by ELISA

Histopathological examination of Group I rats liver showed the normal architecture with central veins, portal vessels and hepatocytes. (Fig. 5 a). The PC group treated with glibenclamide showed congested central veins and portal vessels. The hepatocytes appear normal and minimal Kupffer cell hyperplasia was seen Group II. In the negative control, STZ–induced rats liver sections were examined with dilated central veins and portal vessels showing chronic inflammatory cell infiltrates. The hepatic sinusoids showed congestion with Kupffer cell hyperplasia. The liver section of rats treated with TC‐loaded PLA NPs 5 mg/kg b.wt. (Group IV) was noted with dilated and congested central veins and portal vessels. Rats treated with TC‐loaded PLA NPs 10 mg/kg b.wt. (Group V) exhibited architecture with normal hepatocytes.

Fig. 5.

Histopathological examinations

(a) Histological examination of rat liver. Tissues were stained with haematoxylin and eosin (100×),

(b) Histological examination of rat pancreas.

Tissues were stained with haematoxylin and eosin (100×)

In a normal control Group I rats, pancreas exocrine glands exhibited normal morphology and islet of Langerhans morphology appear normal. (Fig. 5 b). The PC Group II treated with glibenclamide observed with a decreased number of the islet of Langerhans. In Group III STZ‐induced diabetic rat pancreas showed a great extent of, vacuolisation and islets of Langerhans is not seen. Reduced numbers of pancreatic islets were observed in Group IV rats treated with TC‐loaded PLA NPs 5 mg/kg b.wt pancreas tissue section shows normal morphology. Group V rats treated with TC‐loaded PLA NPs 10 mg/kg b.wt. pancreas shows normal morphology, increased number of the islet of Langerhans when compared to Group III.

4 Conclusion

The present study is evident that TC‐loaded PLA NPs exhibits anti‐diabetic effect in STZ‐induced type 2 diabetic rats as well as reduces ameliorates diabetes‐associated hyperlipidemic complications by improving carbohydrate metabolism and providing antioxidant potential due to the presence of phytocompounds, which act alone or in the synergism of compounds for its therapeutic effect.