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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2023 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                 Research Article

Toxicity and anti-diabetic effectiveness of polymeric nanoparticles containing natural compounds

Trilochan Satapathy¹*, Abinash Satapathy², Neha Yadav², Ansuman Satapathy³, Kunal Chandrakar⁴, Shiv Kumar Bhardwaj¹

 1 Columbia Institute of Pharmacy Tekari, Near Vidhansabha, C.G., India

 2 College of Veterinary Science and Animal Husbandry, Anjora-491001, Durg, C.G., India

 3 Faculty of Pharmacy, Kalinga University, Kotni, Near Mantralaya Naya Raipur – 492101, Chhattisgarh, India

 4 University College of Pharmacy, CSVTU, Bhilai, Durg, C.G. India

 

 

INTRODUCTION

Diabetes Mellitus (DM) is considered as a metabolic disorder which is characterized by hyperglycemia and the incidence of the disease is high throughout the world. This disorder is common, chronic and serious believed to be due to resulting from insufficient or inefficient insulin or both. The prevalence of DM is rapidly increasing all over the globe at alarming rate. According to the data available with International Federation of Diabetes (IFD), 415 million adults over the world are presently suffering from diabetes, and this value will reach around 642 million by 2040.^1-2 As per the data published in 2018, the overall diabetes burden estimates for the 1·3 billion in population of India mask wide variations across the states of the country, many of which are comparable to large countries in terms of population.³ This disorder is related to high risk of several complications which is major incumbrance for patients, society, health care systems and economy of the country. The present treatments have limitations due to their side effects, particularly weight gain and hypoglycemia, or contraindications that limit their use.¹ Recent epidemiological data suggested that, the various types of anti-diabetic therapies on long term use can enhance the risk to establish pancreatic cancer. Among the various treatment options available, sulfonylurea, thiazolidinediones, and alpha-glucosidase inhibitors appear to have little or no effect for the establishment of pancreatic cancer where as some of the newer anti diabetic agents are burdened with some concerns in the respect of pancreatic cancer ^4-5. The DPP-IV inhibitor sitagliptine was seen to induce pancreatic ductal metaplasia, and a 4-fold increase in duct cell proliferation ⁶. Herbal based medicines are in practice since as of mankind and during many decades chemical evaluations as well as pharmacological screenings have been progressed using many plant extracts to establish their chemical composition as well as to confirm their indications in different health conditions. Various herbal drugs and herbal extracts, although they are showing great potential and better in-vitro findings but demonstrate little or no in-vivo actions which may be due to their poor lipid solubility or in appropriate molecular size or both. Hence, poor absorption that in turn ultimately resulting in poor bio-availability. ⁷ Curcumin (1, 7-bis-(4-hydroxy-3-methoxyphenyl)-1, 6-hepadiene, 3, 5,-dione) is one of the several components in the rhizome of turmeric and has been used over centuries in traditional remedies for the treatment of a variety of diseases, including inflammatory conditions.⁸ Numerous molecular targets for Curcumin have been identified over the years. Several pre-clinical and clinical studies indicated that, Curcumin is safe at very high concentrations. ⁹ In spite of its safety and effectiveness, Curcumin is not approved as a therapeutic agent till yet. The major barrier for the clinical use of Curcumin includes is poor oral bioavailability. However, this is mainly due to its poor aqueous solubility, increased intestinal metabolism and hepatic metabolism as well as rapid systemic clearance.^10-11 Now-a-days according to the advancement of technology, novel drug delivery systems paved the way towards the enhancement of bioavailability of many herbal drug delivery systems. These limitations may be overcome by formulating as nanoparticle-based drug delivery system. Many herbal compounds such as Piperine, quercetin, genistein, sinomenine, glycyrrhizin, naringin and nitrile glycoside have proved their capability to enhance the bio-availability when co-administered with other agents. ¹² Hence, the primary aim of this study was to develop Curcumin-Piperine nanoparticles by the suitable method and to evaluate the therapeutic effectiveness of prepared formulation in Streptozotocin induced diabetes model.

 MATERIALS AND METHODS

 Materials

Chitosan (Hi-media, Mumbai, India), Polycaprolactone and Streptozotocin (Sigma-Aldrich, Mumbai, India), Curcumin and Piperine (Sun pure Extract Pvt. Ltd., India) and all other chemicals used in this study were analytical reagent grade.

Preparation of polymeric nanoparticles^{13, 14, 15}

In this research, Curcumin/Piperine loaded Chitosan/Polycaprolactone (PCL) nanoparticles were developed by nano-co-precipitation method. Briefly, accurately weighed quantity of pure Curcumin/Piperine and polymer Chitosan and co-polymer PCL were co-dissolved into 10 ml of 90% acetic acid solution to form a homogeneous solution. This solution was then added drop wise under continuous stirring, into 50 ml of distilled water solution to obtain the Curcumin/Piperine loaded Chitosan/PCL nanoparticles. Finally, the obtained nanoparticles were centrifuged at 13,000 rpm/min for 30 min, discarding the supernatant, and re-suspended with 10 ml of distilled water solution for the further characterization and applications.

Spectral analysis (FT-IR) ¹⁶

FTIR stands for Fourier transform infrared which is considered as most useful method for identifying chemicals that are either organic or inorganic. It can be utilized to quantitate some components of an unknown mixture and for the analysis of solids, liquids, and gases. The term Fourier Transform Infrared Spectroscopy (FTIR) refers to a development in the manner in which the data is collected and converted from an interference pattern to a spectrum. It is a powerful tool for identifying types of chemical bonds in a molecule by producing an infrared absorption spectrum that is like a molecular "fingerprint". The wavelength of light absorbed is characteristic of the chemical bond as can be seen in this annotated spectrum. The basic principles of FTIR are that, the Molecular bonds vibrate at various frequencies depending on the elements and the type of bonds. For any given bond, there are several specific frequencies at which it can vibrate. In this research, FTIR spectra of pure Curcumin, Piperine, Polycaprolactone (PCL), Chitosan, and finally developed formulations (nanoparticles) were recorded on FTIR spectrometer (Nicolet, model Magna 550, USA). In brief, about 2-3mg of sample was mixed with dried potassium bromide (KBr) of equal weight and compressed under a hydraulic pressure of 600 kg to form a KBr disk. These disks containing samples were scanned from 400 to 4000 cm^₋1.

Morphological analysis of the Nano formulations by Scanning Electron Microscopy (SEM)

The prepared nanoparticles were subjected to morphological analysis. The surfaces were studied using Scanning Electron Microscope (JEOL.JSM-6480LV, Japan). The nanoparticles were mounted on metal grids using double sided tape and gold coated under vacuum and the image was taken.

Particle size measurement of different nanoparticle formulations

Particle size and poly dispersity index of formulated nanoparticles were measured by using a zeta sizer Nanoseries ((Malvern Instruments, Malvern, UK).

 Differential Scanning Colorimetry (DSC)

Differential Scanning Colorimetry (DSC) is a thermo dynamical process in which the difference in the amount of heat required to increase the temperature of sample and reference are measured as a function of temperature. The main aim of DSC is studying the phase transitions such as melting, Glass transitions or exothermic decompositions. These transitions involve energy changes or heat capacity changes that can be detected by DSC with great sensitivity. Differential Scanning Colorimetry (Mettler-Toledo, PerkinElmer, TA Instrument) was performed to characterize the physical state of Curcumin, Piperine, Polycaprolactone (PCL), Chitosan, and final formulation. About 5 mg of sample was weighed, crimped into an aluminum pan and analyzed at a scanning temperature range from 50 to 300°C at a heating rate of 10°C/min.

X-ray diffraction study (XRD)

X-ray diffraction analysis was employed to detect the crystallinity of the pure drug and the nanoparticle formulation, which was conducted using a Philips PW 3710 x-ray diffractometer (XRD) with a copper target and nickel filter (Philips Electronic Inst, Holland). Powders were mounted on aluminum stages with glass bottoms and smoothed to a level surface. The XRD pattern of each sample was measured from 10 to 50 degrees 2-theta using a step increment of 0.1 2-theta degrees and a dwell time of 1 second at each step . 

Determination of Drug Entrapment Efficiency

The percentage of drug entrapped in the different nanoparticle formulations was determined by weighing the accurately amount of different nanoparticle formulations and ultracentrifugation at 15000rpm for 45 min in an ultracentrifuge to separate the loaded drugs from the formulations. Then the supernatant was separated and analyzed by U V Visible spectrophotometer at 421nm and 342nm for Curcumin and Piperine respectively. The amount of drug entrapped was calculated by the formula given below.

DEE (%) = (Actual drug content/Theoretical drug content) x100

In-vitro Release Study

Curcumin nanoparticles, Piperine nanoparticles and nanoparticles containing both the drugs were prepared by nano co-precipitation method. In Vitro drug release from different nano particle formulations were performed by using membrane dialysis method. It is one of the most popular and versatile method to assess the drug release pattern of different nano sized dosage forms. At first the membrane was clamped in open glass tube for drug release. A 200ml of Phosphate buffer solution (PBS) with pH 7.4 and other with pH 3.4 were used as dissolution medium and taken in a receiver compartment. Before the release test, an accurately weighed quantity of the formulation dissolved in accurate quantity of the dissolution medium and placed in the glass tube maintained with temperature 37^oC at 100 rpm by magnetic stirrer and bead. A 5ml of aliquot was withdrawn from the receiver compartment at fixed time interval and maintained the sink condition with fresh medium immediately. Samples were analyzed by UV-Visible spectrophotometer at 421 and 342 for Curcumin and Piperine respectively. 

IN VIVO STUDY:

The developed formulations were then screened for In vivo pharmacological activity to evaluate their toxicity, therapeutic effectiveness against STZ induced diabetes using rat model. Experimental animals:

 To carry out the toxicity study, Wistar albino rats were used because rats are recommended rodent species for conducting acute toxicity studies as per OECD guideline and also sensitive for expression of toxic responses. All the experimental animals were obtained from Animal house facility, Columbia Institute of Pharmacy, Raipur, Chhattisgarh, India. Animals were maintained at standard environmental temperature and humidity, fed commercial pellet rat chow (M/s Hindustan lever foods, Bangalore, India) and water ad libitum. A 12:12h light and dark cycle (7am-7pm) was maintained throughout the experimental protocol. This study was conducted with prior permission to Institutional Animal Ethics Committee, Columbia Institute of Pharmacy, Raipur, Chhattisgarh, India, Regd. No (1321/po/ReBi/S/10/CPCSEA dated 22.10.2014).The animals were allowed to acclimatize to laboratory conditions two weeks prior to the experiments. 

 Grouping of animals:

In both the experiments, the animals were randomly allocated into different groups. The grouping of animals with their numberings and treatment schedule for toxicity study is depicted in Table-3 whereas for STZ induced diabetes model, the grouping and treatment details are presented in table-13. The preliminary experiment was conducted for evaluation of toxicity of developed formulations. In this experiment, ten healthy animals of controlled age and body weight in each group were used (Five males and five females) as per Organization of Economic Co-operation and Development (OECD-407) guideline. The therapeutic effectiveness of the developed formulations was investigated using STZ induced diabetes rat model. For this experiment six animals in each group were used. All the female animals used in these experiments were nulliparous and non-pregnant. The animals were housed five each, of same gender in polypropylene cages provided with bedding of husk. Each animal was fur markedwith picric acid. 

Selection of dose and route of administration: 

All the dosing was continued up to 28 days .The animals were observed daily for any toxic manifestation and mortality. Body weights and food consumption were measured at different time interval during the experimental period. The oral route was selected for administration of test substances because oral route is considered as a proposed therapeutic route of administration in human being. 

Repeated dose toxicity study (OECD-407, 2008)¹⁷

Repeated dose toxicity was performed using Wistar rats of either gender of controlled age and body weight as per OECD guideline-407.Animals were randomly allocated into five groups (Five male and five female in each group).All the animals received treatments after overnight fasting. The details of animal grouping, their numbers and treatment are described in table no-3 below.

Table 1:  Allocation of experimental animals into different groups for evaluation of toxicity of different nano formulations.

All treatments were administered orally suspended in 0.3 %CMC (vehicle)

RESULTS:

Composition and encapsulation efficiency of different nanoformulations 

The details of Composition i.e drug polymer ratio and encapsulation efficiency of different nanoformulations has been depicted in table no 2. The encapsulation Efficiency (EE %) was found to be 82%,71% and 67% for Curcumin nanoparticles, Piperine nanoparticles and for Cu+Pi dual drug loaded nanoparticles(NP).

Table-2. Composition and encapsulation efficiency of different nanoformulations.

 

 

 

Figure 1: FTIR spectra of of Pure Curcumin         Figure 2: FTIR spectra of Piperine

 

 

Figure 3: FTIR spectra of Polycaprolactone (PCL)                Figure 4: FTIR spectra of Chitosan

 

 

Figure 5: FTIR spectra of Final Formulation

 

Scanning Electron Micrographs of different Nanoparticle formulations

 

Figure 6: Scanning Electron Micrographs of Blank NP                                                Figure 7:   Scanning Electron Micrographs of Curcumin loaded NP

 

                    Figure 8: Scanning Electron Micrographs of Piperine loaded NP       Figure 9: Scanning Electron Micrographs of Cu+Pi NP

A Mean particle size and poly dispersity Index of different nano formulations. 

      

Figure 10:   Graph of mean particle size and                           Figure 11:   Graph of mean particle size and

poly dispersity index of Curcumin NPs                                      poly dispersity index of Blank NPs

 

Figure 12: Graph of mean particle size and poly                     Figure 13: Graph of mean particle size and poly

 Dispersity index of Cu+Pi Nanoparticles                                dispersity index of Piperine Nanoparticles

 

Figure 14: Differential scanning                                                                            Figure 15: Differential scanning 

Colorimetric spectra of Curcumin                                                                         Colorimetric spectra of Piperine

 

 

Figure 16: Differential scanning Colorimetric                                             Figure 17: Differential scanning 

spectra of Poly caprolactone (PCL)                                                                  Colorimetric spectra of Chitosan

 

Figure 18: Differential scanning Colorimetric spectra of Final Formulation (Cur+ PIP+ Chitosan/PCL)

 

Figure 19:  XRD of Curcumin                                               Figure 20: XRD of Piperine

 

Figure 21: XRD of Polycaprolactone                                            Figure 22: XRD of Chitosan

 

 

 

Table 3: Summary of Functional Observational Battery (FOB) Gender-MALE

DAY-AT TERMINATION

 

 

Table 4: Summary of Functional Observational Battery (FOB) Gender-FEMALE

DAY-AT TERMINATION

 

 

Table 5: Result of different nano formulations on hematological findings in Male Wistar rats for Twenty eight days

 

Figure 23: Effect of different nano formulations on hematological findings in Male rats for Twenty eight days

Table 6: Hematological findings of different nano formulations in Female Wistar rats repeated dose toxicity for twenty eight days

Data are represented as mean±SD (n =5), significantly different at *p<0.05, **p<0.01, ***p<0.001

Figure 24: Hematological findings of different nano formulations in Female Wistar rats repeated dose toxicity for twenty eight days.

Table 7: Effect of different formulations on Biochemistry of male rats repeated dose toxicity (Twenty eight days study)

Gender-Male

Groups	Total protein(g/dl)	BUN (Mg/dl)	ALT (IU/L)	AST  (IU/L)	γGT (U/L)	Cholesterol (mg/dl)	Triglycerides (mg/dl)	LDH (IU/L)	Bilirubin (mg%)	Albumin (g/dl)
Control	7.14±0.97	30.42±1.30	36.24±4.56	66.2±9.06	14.44±2.55	63.94±8.22	108.06±8.72	324.56±20.40	0.61±0.08	3.57±0.38
Blank nanoparticle	7.54±0.93	36.94±4.01	40.72±1.39	68.44±4.25	18.12±1.08	67.49±3.20	115.3±7.88	361.5±11.15***	0.70±0.03	4.29±0.63
Curcumin nanoparticles	7.42±0.72	35.08±4.07	39.72±1.65	67.22±4.28	17.4±1.29	67.6±2.70	113.6±9.07	357.2±12.51**	0.68±0.05	3.9±0.45
Piperine nanoparticles	7.18±0.61	35.02±4.32	39.56±1.90	67.4±4.39	16.88±1.54	67.64±2.57	114.12±9.80	359.2±9.20**	0.72±0.05	3.79±0.41
Curcumin+ Piperine nanoparticle	7.4±1.06	32.32±1.85	37.68±3.05	67.5±7.76	15.52±1.81	65.46±5.73	109.32±9.98	327.2±21.30	0.64±0.05	3.57±0.54

Data are represented as mean±SD (n =5), significantly different at *p<0.05, **p<0.01, ***p<0.001

Figure 25: Effect of different formulations on Biochemistry of male rats repeated dose toxicity (Twenty eight days study).

 

Table 8: Effect of different formulations on Biochemistry of female rats repeated dose toxicity (Twenty eight days study).

Data are represented as mean±SD (n =5), significantly different at *p<0.05, **p<0.01, ***p<0.001

Figure 26: Effect of different formulations on Biochemistry of female rats repeated dose toxicity (Twenty eight days study)

Table 9: Effect of different formulations on anti-diabetic activity against Streptozotocin induced diabetes in experimental animals (Rats)

Data are represented as mean±SD (n =5), significantly different at *p<0.05, **p<0.01, ***p<0.001

Figure 27: Effect of different formulations on Anti diabetic activity against Streptozotocin induced diabetes in experimental animals (Rats).

 

DISCUSSION AND CONCLUSION:

The FT-IR spectra of Curcumin, Piperine, PCL, Chitosan and final formulation were depicted in fig 1, fig 2,fig 3, fig 4 and fig 5 respectively. The result of FT-IR spectral analysis indicated that, in the nanoparticle formulation the major characteristic bands of both the drug and polymer are displayed without any significant spectral shift. This indicates that there was no potential interaction between the different components of the final formulation. From the SEM study, our observations indicated that, drugs like Curcumin and piperine both are compatible to polymer and co polymer used during preparation there by nanoparticles of appropriate size, shape and smooth surface have been obtained and there was no significant change in their surface topography. For final formulation the mean particle size fall in the range of 831.2 nm with polydispersity Index value is 0.702. The DSC thermo gram of Curcumin, Piperine, PCL, Chitosan and final formulation (Cur+ Pip+ PCL/Chitosan) have been represented in fig no.14, fig no.15, fig no.16, fig no.17 and fig no.18 respectively. The DSC curve of Curcumin showed a single endothermic peak being started at 172.82^oC and ended at 180.76^oC. Similarly the DSC curve of piperine also showed a single endothermic peak being started at 128.78^oC and ended with midpoint 123.29^oC. The DSC thermogram of PCL showed a broad endothermal peak being started at 83.11^oC and ended at 84.55^oC. The DSC thermogram of Chitosan showed a less intense endothermal peak being started at 54.35^oC and ended at midpoint 52.94^oC.  The DSC thermogram of final formulation showed two major endothermal peaks one being started at 71.33^oC and ended at midpoint 72.15^oC and other peak at about 114^oC -115^oC as well as one less intense peak at about 152^oC-154^oC.Here in the final formulation the sharp endothermic peak of pure Curcumin was shifted to a lower temperature. Such behavior suggests the minute or partial loss of drug crystallinity when the drug was incorporated into the nanoparticle formulation. Since there was no major shift in the glass transition of the polymer in drug loaded formulation, it can be concluded that there was no significant interaction occurring between the drug and polymer. The X-ray diffraction of Curcumin, piperine, Poly caprolactone (PCL), Chitosan have been carried out and are represented in fig.19, fig.20, fig.21, fig.22 respectively. The different formulations were also screened for various in vitro parameters before proceed to in-vivo activity. The 28 days repeated dose toxicity studies were carried out as per OECD guideline-407. The functional observational battery (FOB) was carried out to quantify their neurotoxic effects. In this experiment the FOB for group-I and group-V animals (comparison study) were carried out to evaluate the effects of developed nano formulations on alteration of neuro behavioral activities in both the gender of rat strain. The result of Functional Observational Battery (FOB), has been indicated in table no-3 and table no-4 for male gender and female gender respectively. The results of FOB in both the gender did reveal neither any abnormalities nor any mortality. The blood withdrawn from retro orbital plexus of different groups of animals and hematological as well as biochemical tests was performed. There were no major changes of in values of test parameters of control and test group of animals that indicates the final formulation did not exhibit any toxicity. The anti-diabetic potential of developed formulations was screened by STZ induced diabetes in rats model. The result is tabulated in Table no-9 and graphically represented in fig no-27. The result revealed that, the final formulation shows better diabetic control over other prepared formulations.

Conflicts of Interest: The authors declare no Conflict of Interest

Acknowledgement: Authors are thankful to Sun pure extract Pvt Ltd for providing Curcumin and Piperine as gift sample for this research work. The authors are also thankful to Principal and management of Columbia Institute of Pharmacy, Raipur, C.G., Principal, Faculty of Pharmacy, Kalinga University, Raipur and Dean, College of Veterinary Science and Animal Husbandry, Anjora-491001, Durg, C.G., India for providing necessary facilities to complete this research work.

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