Pharmacological evaluation of embelin - chitosan nanoparticles as an antidiabetic agent.

Embelin has been reported to possess variety of pharmacological activities such as androgenic antagonists, antiangiogenic, antibacterial, anticancer, anticonvulsant, antidiabetic, antidepressant, antihelmintic, antifertility, antihyperlipidemic, anti-inflammatory, antimalarial, antimitotic, antiobesity and antioxidant properties. The current research work aimed to study the hypoglycemic effect of embelin-chitosan nanoparticles (ECNPs) diabetic rats provoked by streptozotocin (STZ). Embelin nanoparticles (ENPs) were created by combining chitosan, a natural biopolymer, and glutaric acid, a new cross-linker. STZ 50 mg/kg was given intravenously into Sprague-Dawley rats weighing 250-300 g (75-90 days) to induce experimental diabetes. The antidiabetic efficacy of orally administered ECNPs in diabetic rats developed by STZ was investigated, as well as histological examination. When compared to diabetic control rats, ECNPs (25 mg/kg body weight and 50 mg kg body weight) and standard glibenclamide (10 mg/kg body weight) treated rodents exhibited a remarkable drop in glucose contents. Furthermore, histological research showed that ECNPs-treated rats were harmless up to amount of 25 mg/kg bwt. Thus current investigation reveals that ECNPs have antidiabetic potential and may be beneficial in treating hyperglycemia in people.

Introduction

Diabetes mellitus, also known as diabetes, is a metabolic illness that produces high amounts of blood sugar. It is categorized into Type I (due to-cell destruction, generally resulting in absolute insulin shortage) and Type II (due to-cell destruction, usually resulting in absolute insulin deficit) (because of a growing insulin secretory malfunction in the context of insulin resistance).[1] By the end of 2030, the global and regional diabetes population is predicted to grow at an alarming rate of 10.2% (578 million).[2] Diabetes Type 2 is a chronic metabolic condition that is rapidly becoming a primary cause of morbidity and mortality. The majority of the existing oral dose forms for diabetes have various and sometimes life-threatening side effects; hence, treating diabetes without these side effects is a significant issue.[3]

As a result, numerous researchers are working in traditional and complementary medicine to cure diabetes. Interestingly, 400 traditional plants have shown to possess antidiabetic activity so far.[4] However, not all herbal treatments have scientific evidence to support their usefulness in treating diabetes.[5] Embelia ribes is a well-known and thoroughly explored herbal plant for a variety of pharmacological actions. Embelin is a natural benzoquinone (2, 5-dihydroxy-3-undecyl-p-benzoquinone) that was discovered to be a major ingredient of Embelia ribes and has been linked to a variety of pharmacological properties.[67] Embelin has been demonstrated to be nontoxic and capable of rapidly reducing and stabilizing metal ions to nanoparticles. These embelin-stabilized nanoparticles were found to be biocompatible with human blood and cells.[8] The purpose of this study is to see how embelin-chitosan nanoparticles (ECNPs) affect plasma glucose levels and histopathological alterations in streptozotocin (STZ)-induced diabetic mice.

Materials and Methods

Animal groupings/experimental animals

For the current in vivo animal investigation, 150–200 g of female Sprague-Dawley rats were obtained from the SRMC central animal house in Chennai and maintained in regular laboratory settings (temperature 24°C ± 2° C; humidity 45 ± 10%) with a 12 h day and night cycle, respectively. All experimental rats were fed with standard laboratory diet and had unrestricted access to drinking water. Then, assessment of antidiabetic activity was carried out with the agreement of the (SRM-IEC) and in consent with the institutional ethical instruction for the safeguard of animals at SRMC, Chennai (IAEC/XXXVII/SRU/342/2014).

Isolation of embelin

Embelin was extracted from Embelia ribes berries and described using Radhakrishnan's techniques (2012).[9]

Chemicals

Chemicals such as chitosan, glibenclamide, glutaric acid, sodium azide, sodium carboxy methylcellulose (CMC), and streptozotocin (STZ) were procured from Sigma-Aldrich, USA. Similarly biochemical reagents for determination of plasma glucose, creatinine, and urea were procured fromRadiant Specialty Diagnostics (Accurex Biomedical Reagents), Nungambakkam, Chennai.

Preparation of embelin-chitosan nanoparticles

ECNPs were synthesized and characterized in accordance with previous reports.[1011]

In vivo experimental design

The rats were sorted into five batches of 6 (female) rats each group. Group I rats were healthy and served as standard controls, with 0.5% CMC functioning as a vehicle. Rats from Groups II, III, IV, and V were given a single dose intraperitoneal injection of STZ (STZ - 50 mg/kg body weight) and served as untreated diabetes controls (0.5% CMC), diabetic glibenclamide, diabetic ECNPs (minimum dose - 25 mg/kg/day), and diabetic ECNPs (maximum dose - 50 mg/kg/day). The experiment was conducted 2 days after STZ injection in diabetic rats with glycosuria and hyperglycemia (blood glucose values of 200–300 mg/ml). The rats were starved overnight before blood was drawn from the tail vein. Groups III, IV, and V received glibenclamide (10 mg/kg/day), ECNPs (25 mg/kg/day), and ECNPs (50 mg/kg/day) orally. After 2 weeks, the rats were slaughtered and blood samples were drawn from the heart. Plasma glucose levels were measured at 0, 7, and 14 days after an overnight fast. On the 14th day, the 24 h urine was collected by placing rats in individual metabolic cages and utilizing sodium azide as a preservative. Following drug administration, the levels of plasma and urine glucose, creatinine, and urea were measured in urine.

Histopathological examination

Following the experimental period, the collected organs (pancreas, liver, and kidney) were fixed in 10% buffered neutral formalin solution for 48 h before being processed using the standard inclusion into paraffin technique. The preparations were then stained with hematoxylin-eosin (H and E) and histopathologically evaluated under a microscope, while the remaining sections were frozen at 80°C.

Biochemical analysis

Accurex kits (Radiant Specialty Diagnostics, Nungambakkam, Chennai) were used to estimate blood glucose levels as well as biochemical parameters in urine such as glucose, creatinine, and urea levels.

Statistical analysis

A one-way variance analysis was performed using SPSS version 3.5 (IBM SPSS Statistics., Chicago, USA). For group comparisons, a post hoc Tukey's test was used. With P = 0.001, values were considered statistically significant.

Results

Extraction and chemical characterization of embelin (active principle of Embelia ribes)

Embelin was extracted from Embelia ribes berries and chemically analyzed utilizing a range of tools in this study (thin-layer chromatography, ultraviolet, Fourier transform infrared, differential scanning calorimetry, thermogravimetry, and nuclear magnetic resonance). The current study, however, did not demonstrate the findings.

Physicochemical characterization of NPS

Embelin nanoparticles (ENPs) were created in this study using the natural biopolymer chitosan and the new cross-linker glutaric acid. The prepared ECNPs were discovered to be of a discrete, free-flowing nature. As shown in Table 1, approximately three batches of ECNPs were prepared. The percentage yields of ECNPs ranged from 75.23 to 78.45 ± 1.0106 % [Table 1], while the encapsulation efficiency of ECNPs ranged from 71.64 to 75.50 ± 1.1908 % [Table 1].

Table 1

Composition, yield, and entrapment efficiency of embelin loaded chitosan nanoparticles

Batch code	Drug: Carrier ratio	Percentage yield	Entrapment efficiency
B1	1:2	75.23	72.31
B2	1:2	78.45	75.50
B3	1:2	75.65	71.64

The relevance of the in vitro drug release evaluation results has shown the product quality and performance of the offered drug.

Embelin release investigations in the current study show that the release was uniform, and the amount of drug released at the end of 6 h was judged to be acceptable and sustainable [Table 2 and Figure 1]. Further, transmission electron microscopy analysis shows that a well dispensability of ECNPs pointing outward with size up to 100 nm was observed as shown in Figure 2.

Table 2

In vitro release profiles of embelin loaded chitosan nanoparticles

Time (h)	Percentage cumulative release of nanoparticles
	B1	B2	B3	Average
0	0	0	0	0
1	29.76	25.92	27.65	27.77
2	36.45	28.32	33.56	32.77
3	47.65	34.56	41.35	41.18
4	54.76	44.54	47.68	48.99
5	60.23	51.23	56.32	55.92
6	62.34	60.42	61.54	61.43

Figure 1

Cumulative percentage release of embelin nanoparticles

Figure 2

TEM image of embelin nanoparticles

Antidiabetic activity screening in normal and streptozotocin-induced diabetic rats

STZ-induced diabetic rats were used to test the antidiabetic activity of ECNPs. The data obtained on the fasting blood sugar level of normal and diabetic rats of antidiabetic effects of ECNPs are depicted in Tables 3 and 4. Moreover, in our study, there was a significant reduction of plasma and urine glucose level was observed in all the treatment groups compared to standard [Tables 3 and 4]. There is no statistically significant different among the groups at the 0th day and 7th day except standard treated rats.

Table 3

Estimation of plasma glucose level

Group	Batch code	Plasma glucose level (mg/dl)
		0 day	7th days	14th days
1	Normal control (0.5% CMC)	79.5±5.21	74.25±4.20	71.47±2.61
2	Diabetic control (0.5% CMC)	341±46.63	346.75±16.16	272.91±4.21
3	Standard (10 mg per kg body weight/day)	353.62±54.93	179.98±12.85*	142.16±12.06*
4	Low dose of ENPs (25 mg per kg body weight/day)	409.25±39.93	282.59±31.46	148.58±20.78*
5	High dose of ENPs (50 mg per kg body weight/day)	434.75±25.90	301.40±25.97	154.83±17.44*

*P<0.001 compared to diabetic control (Group-II) (one-way ANOVA followed by a post hoc test). Values are expressed as mean±SEM (n=4). SEM=Standard error of the mean, CMC=Carboxy methylcellulose, ENPs=Embelin nanoparticles

Table 4

Estimation of urine glucose, urea, and creatinine level on 14th day

Group	Batch code	Urine glucose, urea, and creatinine level
		Glucose	Urea	Creatinine
1	Normal control (0.5% CMC)	1.3425±0.0698	53.9375±5.3768	0.0375±0.0010
2	Diabetic control (0.5% CMC)	13.4775±0.3047	21.3119±0.4205	0.0331±0.0012
3	Standard (10 mg per kg body weight/day)	10.635±0.3781*	40.7638±4.8314*	0.0525±0.0053*
4	Low dose of ENPs (25 mg per kg body weight/day)	10.9625±0.5467*	29.595±4.4470	0.0588±0.0016*
5	High dose of ENPs (50 mg per kg body weight/day)	7.585±0.3508*	33.0431±6.5820	0.065±0.0022

*P<0.001 compared to diabetic control group (Group II) (one-way ANOVA by a Dunnett’s statistics method). Values are mentioned as mean±SEM, where (n=4). SEM=Standard error of the mean, CMC=Carboxy methylcellulose, ENPs=Embelin nanoparticles

However, a statistically significant reduction in the measurement of plasma glucose of the standard and treatment groups compared to Group II was revealed at the 14th day (diabetic control animals). Glibenclamide demonstrated a slower onset of hypoglycemic action and showed a sustained decrease in blood sugar levels. ECNPs at doses of 25 and 50 mg/kg body weight decreased blood sugar levels significantly and were found to be more effective. The treatment groups exhibited dose-dependent effects. Diabetes, as depicted/demonstrated in Table 3, resulted in a significant initial increase in fasting blood glucose levels across the board.

Histopathological studies

For this purpose the obtained liver, kidney and pancreas were fixed in 10% buffered neutral formalin solution for 48 hours and then included in paraffin by the usual technique. It was processed by the usual technique of inclusion into paraffin. The preparations were then stained with Haematoxylin-Eosin and they were examined under microscope for histopathological evaluation [Figures 3-5], whereas the remaining sections were frozen at −80°C.

Figure 3

Histopathological change in kidney of embelin nanoparticles on Sprague-Dawley rats

Figure 5

Histopathological change in pancreas of embelin nanoparticles on Sprague-Dawley rats

Histopathological change in liver of embelin nanoparticles on Sprague-Dawley rats

Discussion

Embelin from Embelia ribes has numerous applications in Indian medicine. ENPs were created in this study using the natural biopolymer chitosan and the new cross-linker glutaric acid. Similarly, in our prior investigation, we reported ECNPs that were cross-linked with glutaraldehyde.[11] At the end of the 6th h, the in vitro cumulative percent drug release was reported to be 61.43%. As a result, the current study's findings were consistent with previous reports.[11] The doctoral thesis report includes a chemical characteristic study of manufactured ECNPs.[12] Embelin has been shown to have antidiabetic properties in both alloxan and STZ-induced diabetic rats. STZ-induced hyperglycemia, on the other hand, has been regarded as a helpful experimental paradigm for studying the activity of hypoglycemic medications.[12] Similarly, embelin has been shown in silico to decrease human pancreatic alpha-amylase and human aldose reductase activity.[1314] According to the findings, ECNPs have strong antidiabetic effect in both normal and STZ-induced hyperglycemic rats. Diabetes mellitus was produced by STZ injection, most likely due to elimination of the beta cells of the pancreatic islets of Langerhans. Oral administration (per os) of the ECNPs at doses of 25 and 50 mg/kg body weight exhibited no remarkable change in behavior, showing that the ECNPs are not harmful in tested animals at the observable conditions. Oral delivery of ECNPs resulted in no toxicity, mortality, or behavioral abnormalities for up to 14 days.[15] The standard showed the greatest reduction, followed by ECNPs (25 mg/kg), hence the current study results were consistent with previous publications.[1516] Similarly, the current study reveals that ECNPs have antidiabetic potential and may be beneficial in treating hyperglycemia in people. Both serum creatinine and urea levels were excessively high, indicating compromised kidney function.[17] Histopathological examination also reveals that ECNPs are safe at a dose of 25 mg/kg body weight. The current study's findings were consistent with previous publications.[17] Long-term research of ECNPs is thus required to establish the specific mechanism of action to develop it as a commercially accessible powerful antidiabetic medicine to protect body organs from diabetic alterations.

Conclusion

Embelin from Embelia ribes has numerous applications in Indian medicine. Oral administration (per os) of the ECNPs at doses of 25 and 50 mg/kg body weight results in no remarkable changes in behavior, showing that the ECNPs are not hazardous in tested animals at the observable conditions. In conclusion, the findings indicated that ECNPs had antidiabetic potential and may be beneficial in treating hyperglycemia in people.

Financial support and sponsorship

This research work was partially supported by a Young Faculty Research Grant (C. No. 36/Dean/2012) provided by the Sri Ramachandra University, Porur, Chennai.

Conflicts of interest

There are no conflicts of interest.