**In Vitro Characterization, Modelling, and Antioxidant Properties of Polyphenon-60 from Green Tea in Eudragit S100-2 Chitosan Microspheres**

**Eliana B. Souto 1,2,\*, Raquel da Ana 1, Selma B. Souto 3, Aleksandra Zieli ´nska 1, Conrado Marques 4,5,6, Luciana N. Andrade 7, Olaf K. Horba ´nczuk 8, Atanas G. Atanasov 9,10,11,12, Massimo Lucarini 13, Alessandra Durazzo 13, Amélia M. Silva 14,15, Ettore Novellino 16,\*, Antonello Santini 16,\* and Patricia Severino 4,5,6**


Received: 27 February 2020; Accepted: 30 March 2020; Published: 31 March 2020

**Abstract:** Eudragit S100-coated chitosan microspheres (S100Ch) are proposed as a new oral delivery system for green tea polyphenon-60 (PP60). PP60 is a mixture of polyphenolic compounds, known for its active role in decreasing oxidative stress and metabolic risk factors involved in diabetes and in other chronic diseases. Chitosan-PP60 microspheres prepared by an emulsion cross-linking method were coated with Eudragit S100 to ensure the release of PP60 in the terminal ileum. Different core–coat ratios of Eudragit and chitosan were tested. Optimized chitosan microspheres were obtained with a chitosan:PP60 ratio of 8:1 (Ch-PP608:1), rotation speed of 1500 rpm, and surfactant concentration of 1.0% (*m*/*v*) achieving a mean size of 7.16 μm. Their coating with the enteric polymer (S100Ch-PP60) increased the mean size significantly (51.4 μm). The in vitro modified-release of PP60

from S100Ch-PP60 was confirmed in simulated gastrointestinal conditions. Mathematical fitting models were used to characterize the release mechanism showing that both Ch-PP608:1 and S100Ch-PP60 fitted the Korsmeyers–Peppas model. The antioxidant activity of PP60 was kept in glutaraldehyde-crosslinked chitosan microspheres before and after their coating, showing an IC50 of 212.3 μg/mL and 154.4 μg/mL, respectively. The potential of chitosan microspheres for the delivery of catechins was illustrated, with limited risk of cytotoxicity as shown in Caco-2 cell lines using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The beneficial effects of green tea and its derivatives in the management of metabolic disorders can be exploited using mucoadhesive chitosan microspheres coated with enteric polymers for colonic delivery.

**Keywords:** green tea; epigallocatechin gallate; chitosan; microspheres; Eudragit; metabolic diseases

#### **1. Introduction**

Green tea is obtained from the fresh leaves of*Camellia sinensis,* a plant from the *Theaceae*family which has been used for centuries as a natural antioxidating beverage. Its polyphenolic constituents provide it with additional therapeutic benefits in the modulation of oxidative stress-induced cardiovascular diseases associated with diabetes, already confirmed in clinical trials [1–6]. Green tea extracts, rich in antioxidant polyphenols (e.g., epigallocatechin-3-gallate), have been reported for reducing lipid peroxidation, oxidized low-density lipoproteins (LDL), cholesterol levels, and anti-hypertensive effects, factors that are relevant to reduce cardio-metabolic disease risk [7]. A catechin extract from green tea, containing a mixture of the main active green tea polyphenols components, named polyphenon-60 [8], has recently drawn attention. Indeed, the role of green tea polyphenon-60 (PP60) in decreasing metabolic risk factors, oxidative stress, inflammation, and in the amelioration of cardiac apoptosis in experimentally induced diabetes has been described [9]. Among the naturally derived catechins (flavonoids composing the majority of soluble solids of green tea extracts), epigallocatechin gallate (EGCG) has already been proposed as active ingredient in polymeric nanoparticles for oral administration [10], and as lipid nanoparticles for ocular administration [11,12].

From a quick search using as keywords "epigallocatechin and nanoparticles" 376 publications appeared indexed in the Web of Science dated between 2000 and 2020, while "green tea and nanoparticles" resulted in the list of 780 publications. When associating epigallocatechin and dyslipidemia/dyslipidemia [13], only 33 works were listed as published over the last 20 years.

While the molecular mechanisms involved in the effect of catechins on the metabolism of lipids and sugars remains to be fully described, catechins are known to induce antioxidant enzymes, inhibit pro-oxidant enzymes and scavenge reactive oxygen species (ROS), and to chelate metals [14–16]. EGCG has been reported to improve insulin-resistance and metabolic profiles, as well as to reduce adipocyte area as a consequence of lipolytic action [17]. Anti-obesity effects, such as inhibition of fatty acid absorption and reduction in leptin levels were reported in a high-fat diet rat model combined with green tea extract administration [18]. Green tea and its polyphenols have been widely reported to exhibit positive effects against inflammation, cancer, aging, and others [11,19].

Casanova et al. [20] reviewed the effect of epigallocatechin gallate (EGCG) on oxidative stress and inflammation linked to the metabolic dysfunction of skeletal muscle in obesity and their underlying mechanisms and highlighted that in order to overcome the problem of EGCG instability and low bioavailability, future direction is the use of nanocarriers [20]. Chitosan is a biodegradable linear biopolyaminosaccharide obtained by alkaline deacetylation of chitin, with several advantages for oral drug delivery. It is a non-toxic, mucoadhesive natural polymer, with a high charge density, showing not only the capacity to improve dissolution of drugs, but also to improve the fat metabolism in the body [21,22]. Chitosan is being extensively used in the production of drug delivery systems (e.g., silica nanoparticles, microspheres) for oral administration [22–26]. The production of microspheres can be

achieved by the electrostatic interaction between the biopolyaminosaccharide and the low molecular counterions such as polyphosphates, sulphates, and cross-linking with glutaraldehyde producing a gel [27–29].

To be effective in the management of metabolic diseases, the oral administration route is of preference due to higher convenience and higher patient compliance. Polyphenon-60 contains pure catechins, mainly EGCG, and has been selected for the present work as the active ingredient to be loaded into chitosan microspheres coated with Eudragit S-100 for delayed release in the gut. The choice of the oral route is primarily due to it being non-invasive and appropriate for self-administration, which increases the success of the therapeutic outcome. However, the hydrophilic environment of the gastrointestinal tract (GIT) compromises the absorption of many sensitive drugs [22].

The aim of this work was the development of a delayed release oral formulation for PP60. Chitosan microspheres produced by the ionic cross-linking method was been loaded with PP60 and further coated with methacrylic anionic copolymers (Eudragit S-100) to obtain an enteric dosage form, capable of releasing the active in the ileum with improved bioavailability aiming the prevention of metabolic diseases.

#### **2. Materials and Methods**

#### *2.1. Materials*

Polyphenon-60 (PP60, yellow powder with a total cathecin content >60%), chitosan from shrimp shells (molecular weight 150 kDa, 95% deacetylated, low viscosity <200 mPa·s), glutaraldehyde, ascorbic acid, and Span 80 (sorbitan monooleate) were purchased from Sigma-Aldrich (Saint Louis, Missouri, USA). Eudragit S-100 (poly(methacylic acid-co-methyl methacrylate) 1:2) was received as a kind gift from Evonik (São Paulo, Brazil). Liquid paraffin was obtained from VWR Chemicals (Lisbon, Portugal). All other reagents (glacial acetic acid, monobasic potassium phosphate, sodium dihydrogen phosphate, sodium hydroxide, toluene, petroleum ether, acetone, ethanol, and methanol) were obtained from Reagente-5 (Porto, Portugal). For cell culture, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Minimum Essential Medium Eagle's (MEME), Trypsin-0.3% EDTA, phosphate buffered saline (PBS), L-glutamine, non-essential amino acids (NEAA), sodium dodecyl sulfate (SDS), dimethylformamide (DMF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Triton® X, and gentamicin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Caco-2 cell line was purchased from American Type Culture Collection (ATCC, Pensabio Biotecnologia, São Paulo, Brazil). The water used in all experiments was ultrapure, obtained from a MilliQ® Plus, Millipore® (Germany).

#### *2.2. Production of Chitosan Microspheres*

Chitosan microspheres were produced by emulsion cross-linking, at room temperature and following the method described by Jose et al. [26]. Briefly, PP60 was added to a solution of 2% (*m*/*v*) chitosan prepared in 1% (*w*/*v*) of glacial acetic acid aqueous solution. From the obtained solution, a volume of 3 mL was sampled and injected into 20 mL of oil phase of paraffin containing Span 80 with a syringe (No. 23) under mechanical stirring (Ultra-Turrax, T18, IKA, Staufen, Germany) for 30 min to form a w/o emulsion. A volume of 1.5 mL of toluene-saturated glutaraldehyde (8:1) was then added to the obtained emulsion, which was left to stabilize and to cross-link over a period of 5.5 hours. The obtained microspheres were centrifuged at 4000 rpm, the precipitate washed with petroleum ether and acetone and dried in a laboratory hot air oven (Binder Inc, Germany) at 50 ◦C. A total of 10 batches were produced by varying the processing parameters as shown in Table 1. For the first set of batches (Ch-PP602:1, Ch-PP604:1, Ch-PP608:1, and Ch-PP6010:1), the rotational speed was kept constant at 1500 rpm and the concentration of Span 80 in liquid paraffin was kept at 1% (*m*/*v*), and varying the chitosan:PP60 ratios. For the second set of batches (Speed10, Speed15, and Speed20), the chitosan:PP60 ratio was maintained at 4:1, the concentration of Span 80 in liquid paraffin was kept at 1% (m/v), and the rotational speed varied from 1000–2000 rpm. For the third set of batches (S800.5,

S801.0, and S801.5), the chitosan:PP60 ratio was maintained at 4:1, the rotational speed at 1500 rpm, while the concentration of surfactant (Span 80) in liquid paraffin ranged from 0.5%–1.5% (*m*/*v*).


**Table 1.** Variable parameters of chitosan microspheres produced by emulsion cross-linking method (PP60, polyphenon 60; rpm, rotations per minute; % (*w*/*v*), percentage weight per volume).

#### *2.3. Eudragit S-100 Coating of PP60-Loaded Chitosan Microspheres*

The coating of Ch-PP60 microspheres with Eudragit S-100 to obtain S100Ch-PP60 was done by emulsion-solvent evaporation technique, as described by Jose et al. [26]. Ch-PP60 microspheres were suspended in a 10% (*w*/*v*) of Eudragit S-100 in ethanol (2.5 mL) and then emulsified in light liquid paraffin (40 mL) containing 1.0% (*w*/*v*) Span 80. To form a stable emulsion, 2 mL of ethanol was added drop wise. The emulsion was kept for 3 h under mechanical stirrer (Ultra-Turrax, T18, IKA, Staufen, Germany) at 1000 rpm. The S100Ch-PP60 was collected, rinsed with petroleum ether, and dried in a laboratory hot air oven (Binder Inc., Germany) at 50 ◦C.

#### *2.4. Particle Size Analysis*

The particle size of Ch-PP60 (uncoated) and S100Ch-PP60 (coated) microspheres was measured in an optical Zeiss microscope (Oberkochen, Germany) fitted with a calibrated eyepiece micrometer under a magnification of 40×. The diameter of about 100 microspheres was measured randomly and the average size (*Dmean*) determined using the Edmondson's equation [26]:

$$D\_{\text{mean}} = \frac{\sum nd}{n} \tag{1}$$

where *n* is the number of counted microspheres and *d* is the mean size range.

#### *2.5. Yield of Production, Loading Capacity, and Encapsulation E*ffi*ciency*

The yield of production (YP%) was calculated based on the dry weight of microspheres, applying the following equation:

$$
\bar{Y}P^{\diamondsuit} = \frac{\mathcal{W}\_{\text{m}}}{\mathcal{W}\_{\text{PP60}} + \mathcal{W}\_{\text{c}}} \times 100\tag{2}
$$

where *Wm* is the mass of produced microspheres, and *WPP60* and *Wc* are the mass of PP60 and chitosan, respectively, initially taken for the production of the microspheres. For the determination of the loading capacity (LC%) and encapsulation efficiency (EE%), 10 mg of microspheres were weighted and triturated in a mortar and pestle with 20 mL methanol. The mixture was kept overnight for the extraction of the active from chitosan. After filtration and proper dilution with methanol, the absorbance was read in a UV spectrophotometer Shimadzu UV-1601 (Shimadzu Italy, Cornaredo, Italy) at 280 nm

against a calibration curve for the quantification of EGCG (WEGCG read <sup>λ</sup>280nm) [30]. The LC% and EE% were calculated using the following equations:

$$L\text{C\%} = \frac{W\_{\text{EGCG}} \text{ rand } \lambda 280 nm}{\mathcal{W}\_m} \times 100 \tag{3}$$

$$EE\% = \frac{W\_{\text{EGCG}} \cdot \text{rand } \lambda 280 nm}{\mathcal{W}m} \times 100\tag{4}$$

#### *2.6. In Vitro Release Assay*

The in vitro release of PP60 from Ch-PP60 (uncoated) and S100Ch-PP60 (coated) microspheres was evaluated in simulated gastrointestinal (GI) conditions using the United States Pharmacopoeia (USP) rotating paddle dissolution apparatus at 100 rpm and at 37 ± 0.5 ◦C, as described by Jose et al. [26]. Accurately weighed mass of microspheres, equivalent to 30 mg of PP60, was added to 450 mL of dissolution medium and GI conditions simulated over time by modifying the pH at pre-determined time intervals. From 0–2 h, the pH was kept at 1.2 by adding HCl (0.1 N). From 2–4 hours, 1.7 g of KH2PO4 and 2.225 g of Na2HPO4 · 2H2O were added to the medium and the pH adjusted to 4.5 with NaOH (1.0 M). From 4–12 h, NaOH (1.0 M) was added to adjust the pH to 7.4. Over the course of the assay, and at pre-determined time intervals up to 12 hours, a volume of 2 mL was withdrawn from the medium and replaced with fresh dissolution medium to ensure sink conditions over the entire experiment. Samples were analyzed by reading the absorbance in a spectrophotometer Shimadzu UV-1601 (Shimadzu Italy, Cornaredo, Italy) at 280 nm against a calibration curve for the quantification of EGCG [30]. The effect of the chitosan:PP60 ratio on the in vitro drug release was analyzed and the best ratio compared to the coated formulation. All measurements were done in triplicate. The in vitro drug release data of the coated S100Ch-PP60 formulation was fitted to four kinetic models i.e., zero order, first order, Higuchi, and Korsemeyer–Peppas models [25], selecting the most appropriate model based on the obtained *R2* values.

#### *2.7. Antioxidant Activity*

#### 2.7.1. DPPH Assay

The antioxidant activity of PP60 was measured when loaded into chitosan microspheres, and the effect of the enteric Eudragit S-100 coating (Ch-PP60 versus S100Ch-PP60) was compared. The assay evaluated the ability of the loaded PP60 to scavenge the stable DPPH• radical [31]. Briefly, microspheres (Ch-PP60; S100Ch-PP60) were dissolved in 0.1 mM DPPH methanolic solution. Then, a volume of 20 μL of sample was placed in the microplate wells to which 200 μL DPPH methanolic solution (0.1 mM) was added. Methanol was used as negative control and butylated hydroxytoluene (BHT, 0–6 μg/mL) was used as the positive control. The microplates were incubated at 25 ◦C for 30 min, and then read at 517 nm in a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc.). The percentage of the antioxidant activity (AA (%)) was calculated from the recorded optical densities (OD), using the following equation:

$$\text{AA}(\%) = \frac{\text{OD of negative control } - \text{OD of sample}}{\text{OD of negative control}} \times 100\tag{5}$$

The linear regression equation was obtained by plotting the concentration in the X-axis (μg/mL) against AA(%) in the Y-axis (% inhibition), from which the IC50 value could be calculated.

#### 2.7.2. In Vitro Caco-2 Cells Proliferation Assay

The MTT assay was used for the evaluation of the proliferative capacity of Caco-2 cells when treated with Ch-PP60 (uncoated) and S100Ch-PP60 (coated) microspheres [32]. Caco-2 cell lines were firstly seeded in 96-well microtiter plates (0.1×106 cells/mL; 100 <sup>μ</sup>L/well). After 24 h of incubation, serum DMEM was replaced with serum free DMEM. The next day, cells were treated with the microspheres. Solutions of Ch-PP60 and S100Ch-PP60 in dimethyl sulfoxide (DMSO 0.7%) at gradient concentrations (0.5, 2.5, 5, 10, and 15 μg/mL of microspheres) were prepared in serum free DMEM, added to each well and incubated for more 24 and 48 h at 37 ◦C in a 5% CO2 atmosphere. A solution of DMSO 1% was set as the negative control, whereas a doxorubicin solution (100 μg/mL) was set as the positive control. At the end of the incubation period, test solutions were removed. MTT solution (150 μL) at 0.5 mg/mL was added to each well and incubated in the dark for 4 h at 37 ◦C in a 5% CO2 atmosphere. The experiments were repeated three times, and quadruplicates were done for each condition in each assay. Cell viability was determined as the ability of viable cells to reduce the yellow dye MTT to the purple formazan. The obtained precipitate was dissolved in 150 μL DMSO and the absorbance was read at 595 nm using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc.). The results were expressed as percentage of cell viability in relation to the negative control as follows:

$$\text{Cell viability} \left[ \% \right] = \left[ \frac{Abs\_{Test}}{Abs\_{Negative\\_Control}} \times 100 \right] \tag{6}$$

#### *2.8. Statistical Analysis*

All measurements were performed in triplicate, and results expressed as the mean ± S.D. Statistical significance was established at *p* < 0.05 and was calculated using a one-way analysis of variance ANOVA followed by the Tukeys Test. Values of *p* < 0.05 were considered significant. All statistical analyses were carried out using the GraphPad program 5.0® (Intuitive Software for Science, San Diego, CA, USA).

#### **3. Results**

The microspheres produced by emulsion cross-linking between chitosan and glutaraldehyde to load PP60 were yellowish because of the natural color of the active ingredient. To select the best combination of chitosan and PP60, and the production parameters, the particle size (*Dmean*), yield of production (YP%), loading capacity (LC%), and encapsulation efficiency (EE%) were determined for the different batches (as shown in Table 1), and the results of the physicochemical characterization are given in Table 2.

**Table 2.** Particle size, percentage yield, percent drug content, and entrapment efficiency of uncoated and Eudragit coated chitosan microspheres. The results were subjected to one-way ANOVA (Tukeys Test. Data are presented as mean ± SD (standard deviation); *n* = 3.


Both Ch-PP608:1 and S100Ch-PP60 were tested for their release profile in simulated gastrointestinal fluids using USP dissolution test apparatus at 37 ± 0.5 ◦C (Figure 1). The release profile of epigallocatechin gallate (EGCG) from non-coated microspheres (Ch-PP608:1) and Eudragit S-100 coated microspheres (S100Ch-PP60) formulations were compared over the pH range from 1.2 (simulated gastric fluid) in acid buffer solution for 2 h, to pH 4.5 (simulated duodenum) for another 2 h, to pH 7.4 (simulated distal ileum and colon) for the remaining 20 h.

**Figure 1.** Cumulative percentage of epigallocatechin gallate (EGCG) release from non-coated microspheres (Ch-PP608:1, -) and Eudragit S-100 coated microspheres (S100Ch-PP60, ) in simulated gastrointestinal conditions. Error bars ± standard deviation (SD); *n* = 3.

Mathematical fitting models (Higuchi model, Korsmeyer–Peppas model, zero order, and first order) have been used to describe the recorded profiles from both tested batches and results are shown in Figures 2 and 3, respectively.

**Figure 2.** Mathematical fitting models (Higuchi model, Korsmeyer–Peppas model, zero order, and first order) of the cumulative percentage of EGCG release from non-coated microspheres (Ch-PP608:1) in simulated gastrointestinal conditions.

**Figure 3.** Mathematical fitting models (Higuchi model, Korsmeyer–Peppas model, zero order, and first order) of the cumulative percentage of EGCG release from Eudragit S-100 coated microspheres (S100Ch-PP60) in simulated gastrointestinal conditions.

The capacity of S100Ch-PP60 to neutralize reactive oxygen species (ROS) was evaluated using the DPPH scavenging assay, which was shown to be concentration dependent. The absorbance decay of the control test was compared with the recorded absorbance decay of Ch-PP608:1 versus S100Ch-PP60, resulting in the percentage scavenging of free radicals translated as the antioxidant activity (Table 3) [33]. Differences were shown to be statistically significant. For the positive control (BHT) 78.11% scavenging of DPPH radical was recorded at the highest tested concentration (6.0 μg/mL) [33,34]. For the Ch-PP608:1, the linear regression of *R*<sup>2</sup> = 0.9941 (y = 4.2743x – 1.45) was obtained and the IC50 calculated as 212.3 μg/mL, which confirms the cross-linking of chitosan with glutaraldehyde did not compromise the antioxidant activity of catechin. The coating with Eudragit (S100Ch-PP60) resulted in the linear regression of *R*<sup>2</sup> = 0.9895 (y = 3.1023x – 0.728) with the IC50 of 154.4 μg/mL.


**Table 3.** Percentage of scavenging of free radical DPPH (antioxidant activity; *AA (%)*) by Ch-PP608:1 and S100Ch-PP60. Values are mean ± SD (*n* = 3).

From the MTT assay (Figure 4), we can see that there was no significant difference in cell viability, over the concentration range tested, i.e., between 0.5 and 15 μg/mL. Despite the statistical (*p* < 0.05) significant reduction in cell viability observed in all tested concentrations and at both time-points (compared to the control), the cell viability remained above 70% at all tested concentrations for the non-coated microspheres, indicating limited risk of cytotoxic events. The Eudragit coating slightly reduced the cell viability. At the end of the 48 h, the reduction in cell viability was 35.41% and 40.63% for Ch-PP608:1 and S100Ch-PP60, respectively, at the highest tested concentration.

**Figure 4.** Evaluation of the cytotoxic activity of Ch-PP608:1 and S100Ch-PP60 in Caco-2 cell line using the MTT assay at 24 and 48 h. The data represent the mean values ± SD (*n* = 3).

#### **4. Discussion**

When varying the chitosan:PP60 ratio from 2:1 to 10:1, the mean diameter of microspheres increased from 5.57 μm to 7.83 μm (Table 2), which was an expected result as the increase of polymer concentration contributes to an increase the mean particle size. The higher encapsulation efficiency was obtained for the ratio 8:1 (Ch-PP608:1, 87.21 ± 0.33%) with a loading capacity of 7.72 ± 0.11%. Increasing the amount of chitosan also resulted in the increase of the yield of production up to 89.99 ± 0.70%. When increasing the speed rotation from 1000 rpm to 2000 rpm, the size decreased from 9.22 μm down to 6.97 μm. This is attributed to the improved distribution of small emulsion droplets within the aqueous phase, which are then stabilized with the surfactant molecules. The highest yield of production (92.27 ± 0.55%), loading capacity (11.32 ± 0.41%), and encapsulation efficiency (83.55 ± 0.81%) were achieved with 1% (*m*/*v*) of surfactant concentration, with this amount suitable to cover all new particle surfaces being formed upon emulsion cross-linking. When varying the speed, no significant changes were seen for the loading capacity as the chitosan:PP60 ratio remained 4:1 (Table 1). The best results (highest YP%, LC%, and EE%) were obtained with the 1.0% (*w*/*v*) of Span 80, resulting in microspheres with a mean diameter of 6.45 μm. The formulations produced with a chitosan:PP60 ratio of 8:1 (Ch-PP608:1) dispersed in 1% (*m*/*v*) Span 80 at 1500 rpm have been selected for further studies. The obtained microspheres (Ch-PP608:1) were coated with Eudragit S-100 (S100Ch-PP60) and showed a significant increase of the *Dmean* (51.4 μm). Eudragit S-100 is an anionic copolymer based on methacrylic acid and methyl methacrylate, both polymers contributing for the increase of the particles diameter which demonstrate that particles are coated with the enteric copolymer.

When comparing the release profile between Ch-PP608:1 and S100Ch-PP60 in simulated gastrointestinal conditions, the results depicted a delayed release of the active ingredient when coating the chitosan microspheres with the enteric polymer. Within the first 2 h, about 53.45 ± 0.28% of EGCG released from non-coated microspheres was quantified in the dissolution medium, whereas only ca. 2.24 ± 0.52% was released from the coated microspheres. The increase of the pH to 4.5 induced the further release up to 61.90 ± 1.59% and 5.51 ± 0.22% by the end of the 4th hour from non-coated and Eudragit S-100 coated microspheres, respectively. After 24 h of assay, the cumulative amount reached 88.56 ± 1.24% and 79.54 ± 0.52% when released from non-coated and Eudragit S-100 coated microspheres, respectively. These results also demonstrate that S100Ch-PP60 was effectively coated with polyacrylic polymer and this is able to ensure an enteric resistance of the microspheres until they reach the colon.

Comparing the *R2* values recorded for the different fitting models, the release plots of both profiles followed the Korsmeyer–Peppas model with the highest correlation coefficient values of 0.9779 (Ch-PP608:1) and 0.9680 (S100Ch-PP60). When coating the microspheres with Eudragit S-100, the release mechanism fitted to the Power Law profile which follows the equation *Mt*/*M*<sup>∞</sup> = *k t n*, where *Mt* is the cumulative amount of active released at time t, *M*<sup>∞</sup> is the cumulative amount of active released at infinite time, *k* is the Korsmeyers–Peppas constant that is governed by the physicochemical properties of the microspheres. The diffusional exponent *n* translates the release mechanism, i.e., if *n* = 0.5 the release follows the Fickian diffusion, and if 0.5 < *n* < 1.0 it follows a non-Fickian diffusion. A Case II transport is seen as *n* approaches 1.0 when the release independent of time and reaches zero-order release; a super Case II transport is followed when *n* > 1.0 [35]. Interestingly the Eudragit coating significantly changed the transport mechanism; for the non-coated microspheres the release followed the Fickian diffusion, which means that the active was released from the glutaraldehyde cross-linked chitosan microspheres by the usual molecular diffusion attributed to a chemical potential gradient. With the enteric coating, a super Case II was approached, in which the transport mechanism of the active from the microspheres is associated with the erosion of polymeric coating, as seen with the increase of the pH up to 7.4 (Figure 1). The second-best fitting model for the coated microspheres was the zero-order release. The obtained profiles seem to be appropriate to the proposed colonic delivery of PP60. Indeed, it is expected that the release of the active is kept at minimum through the transport of the microspheres through the stomach and small intestine before they reach colon. Eudragit S-100 is soluble at pH above 7; when reaching pH 7.4 the amount of active being released increased almost 40%. The presence of a modified release profile in both formulations could be confirmed, with a drug protective effect promoted by the enteric coating.

The antioxidant activity represents the first step for the evaluation of health benefits [36–38]. For an effective activity against metabolic diseases, the well-known antioxidant activity of green tea should be kept until it is released from the microspheres. The DPPH test demonstrated that the scavenging capacity of S100Ch-PP60 was dependent on the concentration, i.e., the higher the concentration the higher the scavenging activity. The coating of the microspheres with the polyacrylic polymer did not compromise the antioxidant activity of the loaded PP60, a property that can be further exploited for the treatment/prophylaxis of metabolic diseases.

Prior to any in vivo experiment, toxicological studies should be first performed in vitro, using cell models that mimic the body conditions, in order to minimize the number of animal studies and to have an idea of the cytotoxicity of the drug delivery system at an early stage. Although known to be biocompatible and biodegradable, glutaraldehyde-cross-linked chitosan microspheres should be characterized for their capacity to maintain the viability of cells in vitro. For oral delivery, the main barrier of drug absorption is the intestinal epithelium. The Caco-2 cell line is a human colon epithelial cancer cell line often used as a model to mimic the gastrointestinal conditions. While other cell lines are available for cyto/geno-toxicity assessment [39,40], this model seems to be the most realistic cell culture to test oral drug delivery systems. Cytotoxicity of drug delivery systems in the gut is frequently estimated by colorimetric methods in Caco-2 cells [27,41,42]. Among these methods, the most frequently used is the MTT assay. In the MTT assay, the mitochondrial function of the cells is tested. Only live cells will produce the enzymes capable to reduce the MTT reagent. The cytotoxicity of Ch-PP608:1 was checked in the Caco-2 cell line, in comparison to the Eudragit-coated microspheres (S100Ch-PP60). From the obtained results, cells remained viable when treated with both Ch-PP608:1 and S100Ch-PP60 over the tested concentration range over a period of 24 h or 48 h. The coating of the chitosan microspheres with the acrylic polymer reduced the cell viability down to approximately 60% at the highest tested concentration (15 μg/mL) after 48 h. The effect of size and concentration of

particles on cell viability is well-described in scientific literature, being also very much dependent on the type of cell lines [43]. Monolayer type adherent cells, i.e., cells that adhere onto the surface of the culture dish as happens with Caco-2, are more sensitive to the effect of size and concentration as more surface area is exposed. The smaller the size and the lower the concentration, the higher the cell uptake which in principle would induce higher cytotoxicity [44]. Our results show that cell viability was slightly compromised by the coating with the polyacrylic polymer attributed to the density of particles onto the surface of the cell's monolayer.

It is expected that S100Ch-PP60 microspheres can be further processed in foodstuff and in beverages to provide an alternative approach for the administration and delivery of phytochemicals with nutraceutical value. Indeed, micro/nano-nutraceuticals represent a useful tool in managing health conditions, particularly in patients not eligible for conventional therapy [45,46]. Follow up studies, use, and compliance [47–50], as well as communication strategies and assessment [51], should be applied also to nutraceuticals. This approach will allow the management of different health conditions, as happens with the metabolic syndrome [52,53], obesity, and dysmetabolism [54–58], which are often related to the food intake/dietary habits. Given its high levels of antioxidants and polyphenols, green tea is sometimes seen as the healthiest beverage on earth. It has recognized health benefits in metabolic syndrome, e.g., against fat gain, in preventing and managing type 2 diabetes, besides lowering the risk of cancer among others biological effects. Metabolic syndrome is a combination of risk factors ending up in chronic diseases, such as obesity, and is intimately related to oxidative stress and inflammation. As a prophylactic measure, antioxidants, such as those of green tea, can further be exploited in foodstuff as nutraceutical. The smart delivery of nutraceuticals [3,5,6,59–65], through their encapsulation in micro/nanoparticles, can offer an approach to increase their bioavailability. Besides, chitosan microspheres coated with an enteric polymer can be formulated in different food matrices for a modified release in the gut, offering the opportunity to enrich the nutraceutical value of food, supplements, and beverages recommended for the prevention and/or treatment of health conditions linked to dysmetabolism. Such micro/nano-based products should become part of an improved lifestyle as a prophylactic approach against metabolic disorders.

#### **5. Conclusions**

Polyphenon-60-loaded chitosan microspheres cross-linked with glutaraldehyde were successfully prepared. The microspheres were then coated with Eudragit and tested for their modified release in simulated gastrointestinal conditions. The delayed release of green tea was confirmed; both non-coated and Eudragit coated microspheres followed the Korsmeyers–Peppas release model, demonstrating capacity to retain the antioxidant activity of the active ingredient. The coated microspheres increased their size significantly, however without significantly compromising their biocompatibility with the intestinal epithelial Caco-2 cells. The potential for this formulation to deliver poorly water-soluble drugs, such as catechins, was illustrated and can be exploited for the management of metabolic diseases, exploiting the biological effects of green tea, as well as being applied to other matrices of vegetal origin and mucoadhesive chitosan microspheres.

**Author Contributions:** R.d.A., S.B.S., A.Z., C.M., L.N.A., O.K.H., A.G.A., M.L., and E.N. contributed to the methodology, formal analysis, investigation, resources, and data curation; production of non-coated and coated microspheres and the study of optimized parameters have been carried out by R.d.A., A.Z., C.M., and M.L.; physicochemical characterization of the microspheres and quantification of the catechin have been performed by A.D., E.N., and A.S.; the in vitro release assay and mathematical modelling have been done by E.B.S., S.B.S., L.K.H., and A.G.A.; the antioxidant activity and cell line studies have been carried out by L.N.A., P.S., and A.M.S.; writing of the original manuscript was contributed to by E.B.S., S.B.S., R.d.A., A.D., A.S., and P.S.; conceptualization, review, and editing of the manuscript, as well as project administration, supervision, and funding acquisition was contributed to by E.B.S., A.M.S., A.S., and P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Coordenação Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Sergipe (FAPITEC) CHAMADA MS/CNPq/FAPITEC/SE/SES N◦ 06/2018 – PROGRAMA DE PESQUISA PARA O SUS: GESTÃO COMPARTILHADA EM SAÚDE – PPSUS

SERGIPE 2017/2018, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was also financed through the projects M-ERA-NET/0004/2015-PAIRED, UIDB/04469/2020 (strategic fund) and PEst-OE/UID/AGR/04033/2019 (CITAB strategic fund), receiving support from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, and co-financed by FEDER, under the Partnership Agreement PT2020. The authors acknowledge the support of the research project: Nutraceutica come supporto nutrizionale nel paziente oncologico, CUP: B83D18000140007.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Synergistic E**ff**ect of Omega-3 Fatty Acids and Oral-Hypoglycemic Drug on Lipid Normalization through Modulation of Hepatic Gene Expression in High Fat Diet with Low Streptozotocin-Induced Diabetic Rats**

#### **Suresh Khadke 1, Pallavi Mandave 1, Aniket Kuvalekar 1, Vijaya Pandit 2, Manjiri Karandikar <sup>3</sup> and Nitin Mantri 4,\***


Received: 26 October 2020; Accepted: 23 November 2020; Published: 27 November 2020

**Abstract:** Type 2 diabetes mellitus, which an outcome of impaired insulin action and its secretion, is concomitantly associated with lipid abnormalities. The study was designed to evaluate the combinational effect of omega-3 fatty acids (flax and fish oil) and glibenclamide on abnormal lipid profiles, increased blood glucose, and impaired liver and kidney functions in a high fat diet with low streptozotocin (STZ)-induced diabetic rats, including its probable mechanism of action. The male Wistar rats (*n* = 48) were distributed into eight groups. All animal groups except the healthy received a high fat diet (HFD) for 90 days. Further, diabetes was developed by low dose STZ (35 mg/kg). Diabetic animals received, omega-3 fatty acids (500 mg/kg), along with glibenclamide (0.25 mg/kg). Both flax and fish oil intervention decreased (*p* ≤ 0.001) serum triglycerides and very low density lipoprotein and elevated (*p* ≤ 0.001) high density lipoprotein levels in diabetic rats. Total cholesterol and low-density lipoprotein level was decreased (*p* ≤ 0.001) in fish oil-treated rats. However, it remained unaffected in the flax oil treatment group. Both flax and fish oil intervention downregulate the expression of fatty acid metabolism genes, transcription factors (sterol regulatory element-binding proteins-1c and nuclear factor-κβ), and their regulatory genes i.e., acetyl-coA carboxylase alpha, fatty acid synthase, and tumor necrosis factors-α. The peroxisome proliferator-activated receptor gamma gene expression was upregulated (*p* ≤ 0.001) in the fish oil treatment group. Whereas, carnitine palmitoyltransferase 1 and fatty acid binding protein gene expression were upregulated (*p* ≤ 0.001) in both flax and fish oil intervention group.

**Keywords:** type 2 diabetes mellitus; glibenclamide; omega-3 fatty acids; high fat diet; transcription factors; streptozotocin

#### **1. Introduction**

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by an increase in blood glucose due to impaired insulin secretion and its action [1]. The consistent hyperglycemia, insulin resistance, and insulin deficiency contribute to lipid abnormalities in T2DM [2]. The lipid abnormality is an independent risk factor for cardiovascular disease (CVD) development and commonly found in T2DM individuals [3]. Diabetic dyslipidemia is significantly associated with mortality and morbidity due to cardiovascular complications [4]. It accounts for 80% of deaths in diabetic individuals due to CVD [5]. The hyperglycemia, along with lipid abnormalities, is a modifiable risk factor for CVD, and remains uncontrolled in T2DM individuals [4,6]. In spite of advancements in therapeutic strategies, there has been no significant decrease in the mortality related to CVD [7]. The majority of T2DM individuals failed to achieve all standard goals for lipid management, and, therefore, aggressive management strategies are required to lower lipid abnormalities in T2DM individuals [7,8].

Omega-3 fatty acids are a principle component of cell membranes, which serve several important physiological functions, including as signaling molecules, transporters, and modulators of gene expression [9,10]. Previous studies reported several pharmacological activities of omega-3 fatty acids such as anti-hyperlipidemic, anti-inflammatory, and vasodilatory effects [9,11,12]. They have benefited the management of numerous chronic diseases like diabetes, CVD, and autoimmune disorders [9,13,14]. Over a period of three decades, epidemiological studies also reported that omega-3 fatty acids dietary intake provides beneficial effects in cardiovascular diseases [15,16]. Although statin drug treatment lowered the CVD incidence and its associated mortality, increased triglyceride (TG) levels and residual CVD risk remains in diabetic dyslipidemic individuals despite a decrease in LDL levels [16,17]. Therefore, adjunctive therapy is needed to lower the CVD risk. This study was designed to examine the synergistic effect of omega-3 fatty acids and oral hypoglycemic drugs i.e., glibenclamide compared with glibenclamide alone and in combination with statin drug treatment.

Various animal models have been used to assess the pathogenesis of diabetes and its associated complications [18–20]. High fat diet with low dose streptozotocin induces insulin resistance, hyperglycemia, hyperinsulinemia, and hyperlipidemia, which are characteristics of T2DM [21,22]. With this background, we investigated the effect of omega-3 fatty acids i.e., flax and fish oil, along with glibenclamide, against diabetic dyslipidemia by using a high fat diet with low dose streptozotocin-induced diabetic rat model.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Flax oil capsules were procured from the Real World Nutritional Laboratory, Pune, India (Alvel-500). Fish oil capsules purchased from a local pharmacy (Merck Ltd., Pune, India) (Maxepa-500). Streptozotocin (STZ) (Sigma-AldrichSt. Louis, Missouri, USA), and glibenclamide tablets (Daonil-5 mg; Aventis Pharma, Pune, MH, India) were procured from a local pharmacy. The lard oil was purchased from the local market. The standard chow diet was procured from Nutrivet life sciences (Pune, MH, India).

#### *2.2. Animals*

Design of experiment, along with their procedures and techniques, was sanctioned by the Institutional Animal Ethics Committee (IAEC) of Bharati Vidyapeeth University, Pune, India. The study was approved through sanction number: (BVDUMC/2881/2016/001/001). Forty-eight male Wistar rats weighing (120–150 gm, 10 weeks old) which were received from Medical College of Bharati Vidyapeeth, Pune, India. The animals were kept in standard animal house conditions (temperature 22 ± 2 ◦C and 12:12 hr light and dark cycle condition with 55 ± 5% humidity, about 3 animals per cage). The high fat diet (HFD) composition is represented in Table 1.



After acclimatization, rats were randomly distributed into eight groups (*n* = 6) and treatment protocol as follows:

All groups of animals except healthy control received high fat diet (HFD) and water *ad libitum* during the experimental period. Group I (HC): healthy control; received standard chow diet for90 days; group II (HFDC): high fat diet control; group III (DC): diabetic control received low dose streptozotocin (35 mg/kg); group IV (GC): glibenclamide control treated with STZ and glibenclamide; group V (SC): statin control treated with STZ and statin; group VI (GSC): glibenclamide—statin control given glibenclamide and statin; group VII (flax oil): received flax oil and glibenclamide; group VIII (fish oil): received fish oil and glibenclamide.

The glibenclamide and statin was given at 0.25 mg/kg and 10 mg/kg body weight (b.w.)/day, p.o. respectively. All standard drug interventions were given after the development of stable hyperglycemia. The flax and fish oil were given daily at a dose 500 mg/kg body weight (b.w.), p.o. The flax and fish oil intervention was given throughout the experiment i.e., from 1st day to 90th day. However, after the confirmation of stable hyperglycemia, flax and fish oil interventions were continued with glibenclamide (0.25 mg/kg b.w./day, p.o.) till completion of the experiment.

#### *2.3. Experimental Design*

All animals were kept on a respective diet for 90 days. Intraperitoneal glucose tolerance test (IPGTT) was done on 51st day for the detection of glucose intolerance in animals. After confirmation of glucose intolerance, rats from different groups (III-VIII) were injected with a single dose of STZ (35 mg/kg body weight (b.w., i.p.) and wait for the development of stable hyperglycemia. The design of the experiment is demonstrated in Figure 1. The intake of food and water intake was recorded daily. At the end of the experiment, all animals were sacrificed. For various biochemical estimations, the blood was collected at 0 (before providing HFD), 52nd (before STZ induction) and 90th day (at end of the experiment). The different tissues like liver, kidney, pancreas, visceral adipose tissue near kidney, gastrocnemius muscle (hindlimb muscle), and heart were excised, snap-frozen immediately in liquid nitrogen and kept at −80 ◦C. The liver was used for gene expression studies. A small parts of the tissues (liver, pancreas, and kidney) were kept in neutral buffered formalin (10%) for the histopathological examination.

**Figure 1.** Design of the experiment. HFD: High fat diet, IPGTT: Intraperitoneal glucose tolerance test, STZ: streptozotocin; p.o. per os.

#### *2.4. Intraperitoneal Glucose Tolerance Test (IPGTT)*

After seven weeks of HFD, supplementation, all animal groups were fasted for 6 hrs. Initial blood glucose levels (0 min) were assessed. The glucose (2 gm/kg b.w.) solution was injected (i.p.) to all animals. Blood glucose levels were assessed using Accu-Chek monitor (Roche Diagnostics Pty. Ltd., Basel, Switzerland) at different time points (i.e., 0, 15, 30, 60, 90, and 120 min) from tail vein.

#### *2.5. Assessment of Insulin Resistance*

Insulin was estimated through rat-specific ELISA assay kits (Ray Biotech, GA, USA) after confirmation of glucose tolerance by IPGTT. The HOMA-IR (homeostasis model assessment of insulin resistance) was calculated as per the formula from Uma [23]:

HOMA-IR = Insulin (μU/mL) × glucose (mM)/22.5

#### *2.6. Biochemical Parameters*

The biochemical assessments were done by commercially available kits (Coral Clinical Systems, Goa, India). The glucose, lipid profile, liver, and kidney function markers were estimated from serum at 0 day (before providing HFD), 52nd (before STZ induction) and 90th day. Triglycerides (TGs), total cholesterol (TC), very low density lipoprotein (VLDL), low-density lipoprotein (LDL), and high density lipoprotein (HDL) were measured. In the liver function tests, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were also assessed. Urea and creatinine markers were estimated to assess the kidney function.

#### *2.7. Selection of Gene for Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis*

In this study, 3 transcription factors [Sterol Regulatory Element-Binding Protein-1c (SREBP-1c), Nuclear Factor-κβ (NFκβ) and Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ)] were selected, which regulates the expression of target genes, such as Fatty Acid Synthase (FASN), Acetyl-CoA Carboxylase Alpha (ACACA), Carnitine Palmitoyltransferase 1 (CPT1) and inflammatory marker [Tumor Necrosis Factor—Alpha (TNF-α)]. The Fatty Acid Binding Proteins (FABP) gene was also studied. KicqStart® Primers were procured from Sigma Aldrich (New York, USA). The selected genes and their primer sequences are depicted in Table 2.

#### *2.8. Assessment of Hepatic Gene Expression by qRT-PCR*

Total RNA was extracted from liver by TRIZOL method (Invitrogen, Carlsbad, CA, USA). The quality of RNA was assessed by agarose gel electrophoresis (BioRad, Hercules, CA, USA). The RNA quantification was achieved by ND-1000 UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). For qRT-PCR assessment, the isolated RNA (2 μg) was used to synthesize cDNA by using SuperScriptTM first strand synthesis kit (Invitrogen).

The Real-time PCR analysis was done by SYBr green assays (Applied Biosystems, Waltham, Massachusetts, CA, USA) on StepOne real-time PCR system (Applied Biosystems, Waltham, Massachusetts, CA, USA). The following qRT-PCR protocol was used: initial denaturation step was done at 95 ◦C for 10 min. This step was followed by the 40 cycles of denaturation (95 ◦C for 3 s); annealing (60 ◦C for 30 s) and extension (95 ◦C for 15 s). The final extension step was achieved at 60 ◦C for 15 s. Three biological replicates were analyzed from each group. The reaction was carried out in duplicate and the Ct (cycle threshold) values of all samples were normalized by using Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (endogenous housekeeping control).


**Table 2.** The selected genes and their primer sequences.

#### *2.9. Histological Examination*

All animals were sacrificed and different tissues (liver, pancreas, kidney, adipose tissue, muscle and heart) were collected for further analysis. The part of liver, pancreas, and kidney were fixed in buffered formalin solution (10%, pH 7). Then all tissues were embedded in paraffin for block preparation. The tissue sections were cut to 4 μm thickness and stained by Hematoxylin and Eosin. The slides were observed under the light microscope (EVOS™ FL Auto 2 Imaging System, Invitrogen, Carlsbad, CA, USA).

#### *2.10. Statistical Analysis*

The data are represented as Mean ± standard error (SE). Statistical analysis was carried out by using one-way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparison Test using GraphPad Instat (Version 5, GraphPad Software Inc., San Diego, CA, USA).

#### **3. Results**

#### *3.1. Assessment of Average Body Weight, Feed and Water Consumption*

The feed and water intake and body weight of experimental groups is represented in Table 3. The water and feed intake increased (*p* ≤ 0.001) in the diabetic rats (DC) as compared to the healthy control rats (HC). Initially, the average body weight of all animals was between 120–150 gm. The body weight was not statistically significant between diabetic and healthy rats. The flax oil-treated rats showed an increase (*p* ≤ 0.001) in feed consumption as compared to the diabetic rats. The omega-3 fatty acid intervention groups showed decreased (*p* ≤ 0.001) water intake as compared to the diabetic group. Whereas, body weight was significantly (*p* ≤ 0.001) elevated in flax and fish oil treatment groups as compared to the diabetic group.


**Table 3.** Average body weight, feed, and water intake of the experimental groups.

Results are denoted as Mean ± SE (standard error) (*n* = 6 for each group). \*\*\* *p* ≤ 0.001, when compared with the diabetic control group (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control.

#### *3.2. Estimation of Organ Weight*

Organ weight of all experimental animals are shown in Table 4. Diabetic rats showed increased liver, adipose tissue and muscle tissue weight (*p* < 0.001) increased as compared to the HC rats. Whereas, kidney (*p* < 0.001) and heart weight was decreased in the DC group as compared to the healthy group.

**Table 4.** Measurement organ weight (gm).


Results are recorded as Mean ± SE (*n* = 6 for each group). \*\* *p* ≤ 0.01 and \*\*\* *p* ≤ 0.001, when compared with the diabetic control group (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control.

The flax (*p* < 0.001) and fish (*p* < 0.01) oil intervention group showed decreased liver weight as compared to the diabetic group. Similarly, adipose tissue weight also decreased (*p* < 0.001) in flax and fish oil intervention groups as compared to DC group. Whereas, muscle weight was decreased in both flax and fish (*p* < 0.001) oil treatment groups as compared to DC group. The flax and fish oil intervention groups showed increase in kidney weight (*p* < 0.001) as compared to DC group. The heart weight was decreased in flax oil treatment group and elevated (*p* < 0.001) in fish oil treatment group as compared to the DC.

#### *3.3. Assessment of Biochemical Parameters at Zero Day*

Biochemical estimations of all experimental animals before providing HFD are shown in Table 5. Serum glucose, lipid profile (total cholesterol, triglycerides, VLDL, LDL and HDL) liver (SGOT and SGPT) and kidney function tests (creatinine and urea) were found not significantly different among all experimental groups before providing of respective diet.

#### *3.4. IPGTT and Area under the Curve (AUC) for the Experimental Groups*

Figure 2A,B depicts the glucose clearance and area under the curve (AUC) of IPGTT. The blood glucose levels at 0 and 120 min was elevated in all the experimental group as compared to the healthy group (Figure 2A). All experimental groups showed increase (*p* ≤ 0.001) in AUC as compared to the

healthy control group. The glucose clearance was not statistically significant between HFDC, flax, and fish oil intervention groups.


**Table 5.** Biochemical assessment before initiation of HFD.

Results are represented as Mean ± SE (*n* = 6 for each group and reactions were carried out in triplicates). All values for experimental groups were non-significantly different as compared with the healthy control group (Dunnett's Multiple Comparisons Test). Glu: Glucose, TC: Total cholesterol, TGs: Triglycerides, LDL: Low-density lipoprotein, VLDL: Very low-density lipoprotein, HDL: High-density lipoprotein, SGOT: Serum glutamic oxaloacetic transaminase, SGPT: Serum glutamic pyruvic transaminase.

**Figure 2.** IPGTT and area under the curve (AUC) for the experimental groups. (**A**) Variations in blood glucose levels during IPGTT; (**B**) Area under the curve (AUC) for IPGTT. Results are represented as Mean ± SE (*n* = 6 for each group). \*\*\* *p* ≤ 0.001, when compared with the HC animals (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control.

#### *3.5. Insulin Resistance*

Serum glucose and insulin levels are shown in Table 6. Serum glucose level was significantly elevated (*p* ≤ 0.001) in HFD fed rats (all groups except HC) as compared to healthy control rats. Flax and fish oil-treated rats showed non-significant difference in serum insulin level as compared to the healthy rats. HOMA-IR of all the experimental groups is represented in Figure 3. All animals from HFDC, DC and treatment groups of (GC, SC, GSC, flax and fish oil groups) rats showed a significantly increased (*p* ≤ 0.001) HOMA-IR as compared to the healthy control group. Prophylactically, omega-3 fatty acids significantly lowered lipid profile, liver function markers (SGOT and SGPT), and kidney function markers (creatinine and urea) (supplementary Figures S1 and S2).


**Table 6.** Serum glucose and insulin for HOMA-IR assessment.

Results are represented as Mean ± SE (*n* = 6 for each group). \* *p* ≤ 0.05 and \*\*\* *p* ≤ 0.001, when compared with the HC (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control, HOMA-IR: Homeostasis model assessment of insulin resistance.

**Figure 3.** HOMA-IR of different experimental groups Results are represented as Mean ± SE (*n* = 6 for each group). \*\*\* *p* ≤ 0.001, when compared with the HC (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control, HOMA-IR: Homeostasis model assessment of insulin resistance.

#### *3.6. Estimation of Biochemical Parameters*

#### 3.6.1. Fish Oil Treatment Significantly Lowered Serum Glucose

Figure 4 represents the serum glucose levels of all the experimental groups. Serum glucose level was significantly elevated (*p* ≤ 0.001) in diabetic rats as compared to healthy control rats. Fish oil-treated rats had significantly lower (*p* ≤ 0.001) serum glucose levels as compared to the diabetic rats.

#### 3.6.2. Fish Oil Treatment Lowered Abnormal Lipid Profile

Figure 5A–E depicts the serum lipid profile of all experimental groups. Diabetic animals showed significantly (*p* ≤ 0.001) increased serum TC, TGs, LDL, and VLDL levels as compared to the healthy control animals. The HDL level was significantly (*p* ≤ 0.001) decreased in diabetic animals as compared to healthy control animals. Flax and fish (*p* ≤ 0.001) oil treatment group showed decreased serum TC level as compared with diabetic group. Serum TG and VLDL levels were significantly (*p* ≤ 0.001) decreased in flax and fish oil-treated animals as compared to the diabetic animals. Serum LDL level was decreased (*p* ≤ 0.001) in fish oil-treated animals as compared to the diabetic animals. Flax and fish oil intervention elevated (*p* ≤ 0.001) serum HDL levels as compared to the DC group. Flax oil-treated animals showed significant (*p* ≤ 0.001) increase in TC, TGs, LDL, VLDL and HDL as compared to SC and GSC-treated animals. The serum TC level found to be comparable among SC, GSC and fish oil-treated groups. Serum LDL level increased in fish oil group as compared to SC and GSC group. Serum TGs, VLDL and HDL levels were significantly increased (*p* ≤ 0.001) in fish oil intervention group as compared to SC and GSC group. The fish oil treatment showed a significant decrease in abnormal lipid profile and increase serum HDL.

**Figure 4.** Fish oil intervention lowered serum glucose Results are represented as Mean ± SE (*n* = 6 for each group). \*\* *p* ≤ 0.01 and \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control.

**Figure 5.** Assessment of lipid profile from experimental groups. Results are represented as Mean ± SE (*n* = 6 for each group). \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). (**A**) Serum total cholesterol level, (**B**) Serum triglycerides level, (**C**) Serum low-density lipoprotein level, (**D**) Serum very low-density lipoprotein level, (**E**) Serum high-density lipoprotein level. HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control, LDL: Low-density lipoprotein, VLDL: Very low-density lipoprotein, HDL: High-density lipoprotein,.

#### 3.6.3. Flax and Fish Oil Interventions Decreases Level of Hepatic Enzymes

In diabetic rats, serum SGOT and SGPT levels were elevated (*p* ≤ 0.001) as compared to the healthy rats. Flax and fish oil-treated group showed significant decrease (*p* ≤ 0.001) in serum SGOT and SGPT level as compared to the diabetic group. The SGOT level was increased in flax (*p* ≤ 0.001) and fish oil intervention groups as compared to SC and GSC. Serum SGPT level decreased in both flax and fish oil groups as compared to SC and GSC. Serum SGOT and SGPT levels of all the experimental groups are represented in Figure 6A,B.

**Figure 6.** Flax and fish oil interventions lowered level of hepatic enzymes. Results are represented as Mean ± SE (*n* = 6 for each group). \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). (**A**) Serum glutamic oxaloacetic transaminase level (**B**) Serum glutamic pyruvic transaminase level. HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control, SGOT: Serum glutamic oxaloacetic transaminase, SGPT: Serum glutamic pyruvic transaminase.

3.6.4. Flax and Fish Oil Intervention Improved Kidney Function

Serum creatinine and urea levels of all experimental groups were depicted in Figure 7A,B. Diabetic animals showed significantly (*p* ≤ 0.001) increased serum creatinine and urea levels as compared to the healthy control animals. Flax oil intervention groups showed decreased serum creatinine and urea (*p* ≤ 0.001) levels as compared to the diabetic group. While fish oil intervention significantly (*p* ≤ 0.001) lowered both serum creatinine and urea levels as compared to the diabetic group. Serum creatinine and urea levels were non-significantly decreased in flax oil intervention groups as compared to SC and GSC. The serum creatinine level was decreased in fish oil intervention groups as compared to SC (*p* ≤ 0.001) and GSC. Serum urea was not significantly different among SC, GSC, and fish oil groups. Fish oil intervention effectively improved kidney function.

**Figure 7.** Flax and fish oil interventions improved kidney function. Results are represented as Mean ± SE (*n* = 6 for each group). \* *p* ≤ 0.05 and \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). (**A**) Serum creatinine level, (**B**) Serum urea level. HC: Healthy control, HFDC: High fat diet control, DC: Diabetes control, GC: Glibenclamide control, SC: Statin control, GSC: Glibenclamide statin control.

#### *3.7. Expression of Transcription Factors and Their Regulatory Genes*

In the present study, we have examined the effect of flax and fish oil along with glibenclamide against diabetic dyslipidemia. For gene expression studies, three transcription factors and five regulatory genes were examined. The expression profiles are shown in (Figures 8–10). qRT-PCR amplification efficiencies are depicted in Table 7.

**Figure 8.** Expression of transcription factors are modulated after intervention of flax and fish oil. Results are represented as Mean ± SE (*n* = 3 for each group). \* *p* ≤ 0.05 and \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). (**A**) Expression of sterol regulatory element-binding proteins-1c gene, (**B**) Expression of nuclear factor-κβ gene, (**C**) Expression of peroxisome proliferator-activated receptor gamma gene. HC: Healthy control, HFDC: High fat diet control, DC: Diabetic control, PPAR-γ: Peroxisome proliferator-activated receptor gamma, SREBP-1c: Sterol regulatory element-binding proteins-1c, NFκβ: Nuclear factor-κβ.

3.7.1. Flax and Fish Oil Interventions Modulates the Expression of Transcription Factors Resulting in Lipid Normalization

The expression of transcription factors is depicted in Figure 8A–C. In diabetic animals (DC), SREBP-1c expression was significantly (*p* ≤ 0.001) upregulated by ~1.98 and ~1.51-fold as compared to the HC and HFDC animals, respectively. Comparatively, flax and fish oil intervention groups showed significant downregulation by ~1.59 and ~2.84-fold as compared to the diabetic group.

NFκβ gene expression was significantly (*p* ≤ 0.001) upregulated in the diabetic animals by ~7.27 and ~2.27-fold as compared to the HC and HFDC animals, respectively. On the other hand, its expression was significantly (*p* ≤ 0.001) downregulated by ~1.59 and ~6.16-fold in flax and fish oil treatment groups as compared with the diabetic group.

**Figure 9.** Expression profiles of fatty acid metabolism genes. Results are denoted as Mean ± SE (*n* = 3 for each group). \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). (**A**) Expression of fatty acid synthase gene, (**B**) Expression of acetyl-CoA carboxylase alpha gene, (**C**) Expression of carnitine palmitoyl transferase 1 gene, (**D**) Expression of fatty-acid-binding proteins gene. HC: Healthy control, HFDC: High fat diet control, DC: Diabetic control, ACACA: Acetyl-CoA carboxylase alpha, FASN: Fatty acid synthase, CPT1: Carnitine palmitoyl transferase 1, FABP: Fatty-acid-binding proteins.

**Figure 10.** Expression of TNF-α in experimental groups. Results are represented as Mean ± SE (*n* = 3 for each group). \*\*\* *p* ≤ 0.001, when compared with the DC group (Dunnett's Multiple Comparisons Test). HC: Healthy control, HFDC: High fat diet control, DC: Diabetic control group-treated STZ and TNF-α: Tumor necrosis factor-alpha.


**Table 7.** The qRT-PCR efficiency for absolute mRNA quantification.

Efficiency is calculated from the slope of the curve as E = 10(−1/slope)<sup>−</sup>1.

Expression of PPAR-γ was significantly downregulated in diabetic animals by ~3.00 and ~2.15-fold, as compared to the HC (*p* ≤ 0.001) and HFDC (*p* ≤ 0.05) control animals. Flax oil-treated animals showed non-significant increased expression as compared to the diabetic group. While, fish oil-treated animals showed significant (*p* ≤ 0.001) upregulation expression by ~8.95, ~4.94, ~5.84, and ~4.04-fold as compared to the diabetic animals.

3.7.2. Flax and Fish oil Intervention Modulates the Expression Fatty Acid Metabolism Genes Which Results in Decreased Lipid Abnormality

Figure 9A–D represents the expression of lipid metabolism genes. Expression of FASN genes was significantly upregulated (*p* ≤ 0.001) in the diabetic animals by ~70.48 and ~3.47-fold as compared to HC and HFDC animals. Flax and fish oil-treated animals showed downregulation (*p* ≤ 0.001) by ~6.01 and ~4.04-fold as compared to the diabetic animals.

ACACA gene expression was significantly (*p* ≤ 0.001) upregulated in the diabetic rats by ~3.26-fold as compared to the healthy rats. Flax (*p* ≤ 0.001) and fish (*p* ≤ 0.05) oil-treated groups showed upregulation by ~2.12 and ~1.78-fold as compared to the diabetic group, respectively.

Expression of CPT1 genes was non-significantly downregulated in the diabetic rats by ~4.95 and ~4.63-fold as compared to the HC and HFDC rats. Flax and fish oil intervention groups showed significant upregulation (*p* ≤ 0.001) by ~14.17 and ~15.20-fold as compared to the diabetic group.

FABP gene expression was significantly downregulated (*p* ≤ 0.001) by ~8.87-fold in diabetic rats as compared to the healthy rat group. Both flax and fish oil intervention groups showed significant upregulation (*p* ≤ 0.001) by ~5.64 and ~10.51-fold as compared to the diabetic groups, respectively.

#### 3.7.3. Fish Oil Intervention Downregulates the Expression of TNF-α

In DC rats, TNF-α gene expression was upregulated (*p* ≤ 0.001) by ~12.31 and ~3.09-fold as compared to the healthy and high fat diet control rats, respectively. Comparatively, flax and fish (*p* ≤ 0.001) oil-treated groups showed downregulation by ~1.24 and ~3.28-fold as compared to the diabetic group, respectively. Figure 10 represents the inflammatory gene expression, TNF-α.

#### *3.8. Histological Examination of Liver, Pancreas, and Kidney from Experimental Animals*

Animals from experimental groups developed typical changes in liver, pancreas and kidney. Their histopathological examination is shown in Figures 11–13.

**Figure 11.** Histological examination of liver tissue (Scale bar 50 μm). Healthy. (**A**) Healthy control, (**B**) High fat diet control, (**C**) Diabetic control, (**D**) Glibenclamide control, (**E**) Statin control, (**F**) Glibenclamide statin control, (**G**) Flax oil (500 mg/kg), (**H**) Flax oil (500 mg/kg).

**Figure 12.** Histological examination of kidney (Scale bar 50 μm). (**A**) Healthy control, (**B**) High fat diet control, (**C**) Diabetic control, (**D**) Glibenclamide control, (**E**) Statin control, (**F**) Glibenclamide statin control, (**G**) Flax oil (500 mg/kg), (**H**) Flax oil (500 mg/kg).

**Figure 13.** Histological examination of pancreas (Scale bar 50 μm). (**A**) Healthy control, (**B**) High fat diet control, (**C**) Diabetic control, (**D**) Glibenclamide control, (**E**) Statin control, (**F**) Glibenclamide statin control, (**G**) Flax oil (500 mg/kg), (**H**) Flax oil (500 mg/kg).

#### 3.8.1. Histological Examination of Liver

Healthy animals showed normal architecture of hepatocytes (Figure 11A). High fat diet control group rats showed focal fatty changes in the liver (Figure 11B). Diabetic animals develop microvesicular fatty changes in the liver (Figure 11C). The flax oil intervention, along with glibenclamide showed focal fatty changes in the liver (Figure 11G). However, fish oil intervention, along with glibenclamide showed near-normal architecture of hepatocytes (Figure 11H).

#### 3.8.2. Histological Examination of Kidney

Healthy and high fat diet control group animals showed normal architecture of the kidney (Figure 12A,B). Diabetic rats showed tubules with vacuolated cells (Figure 12C). The animals receiving standard drugs (GC, SC and GSC) also showed tubules with vacuolated cells (Figure 12D,F). Flax and fish oil interventions along with the combination of glibenclamide showed tubules with vacuolated cells (Figure 12G,H).

#### 3.8.3. Histological Examination of Pancreas

Healthy and high fat diet control animals showed normal architecture of the pancreatic tissue (Figure 13A,B). Diabetic rats showed reduced number and size of islets of Langerhans and β cells (Figure 13C). Flax and fish oil intervention group showed reduced number and size of Langerhans and β cells (Figure 13G,H).

#### **4. Discussion**

The provision of HFD, along with a low dose of streptozotocin in rats, results in a condition that mimics the pathophysiology of type 2 diabetes (T2DM) in humans and is thus a suitable model for the practical investigations and testing of different natural compounds for the effective management of type 2 diabetes and its complications [20–22]. Despite advancement in the prevention and management strategies of diabetes and its associated complications in recent years, still, it has been growing alarmingly with the high rate of morbidity and mortality [24]. Therefore, aggressive management strategies for T2DM and its associated lipid abnormalities are highly recommended [4,22].

STZ-treated diabetic rats showed decreased body weights and elevated blood glucose, which are characteristic features of diabetes [18]. In the present study, a significant decrease in body weight and sustained hyperglycemia was observed in diabetic rats. The lipid abnormality is a very frequent impairment in type 2 diabetes patients [24]. For its management, they are commonly prescribed lipid-lowering drugs like statins. Therefore, one of the groups was given treatment with a normolipidemic drug just to evaluate the effect of the same drug on lipid abnormalities.

Several studies have reported the triglyceride-lowering effect of omega-3 fatty acids [9,25–27]. Some also studied cardioprotective effects of omega-3 fatty acids in animal models as well as in humans [28]. Flax oil, is a major source of alpha linolenic acid (ALA), and fish oil, predominantly contain eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the principle sources of omega-3 fatty acids [9]. Previous studies showed that, flax and fish oil treatment lowered abnormal lipid profile in diabetic rats [9]. Our results are in accordance with previous findings. Both flax and fish oil exhibit beneficial effects on hepatic cholesterol metabolism in HFD-fed animals [29].

Hendrich [30] reviewed the effect of omega-3 fatty acid in human clinical trials and concluded that its effects in T2DM were not well studied [9,30]. The management of metabolic disorders (T2DM and its associated complications) recommended combining lifestyle changes with pharmacological therapy [31]. In this regard, several studies reported, the beneficial effect of omega-3 fatty acids with different allopathic drugs (thiazolidinediones, pioglitazone, rosiglitazone, etc.) in HFD-fed mice results in the increased adiponectin secretion [31,32]. With this background, we have studied the effect of flax and fish oil intervention in combination with glibenclamide in HFD with low STZ-induced diabetic dyslipidemia. The present study helps to fill the gap and investigates the combinational effect of omega-3 fatty acids and oral hypoglycemic drug on lipid abnormalities through modulation of transcription factors and their regulatory genes.

Several studies reported that flax and fish oil intervention exhibited triglyceride-lowering effect in streptozotocin-induced diabetic rats [9,27,33,34]. However, the treatment did not show any effect on serum TC and LDL levels. In the present study, flax and fish oil along with glibenclamide treatment effectively lowered serum TC, triglycerides, VLDL, and LDL levels in diabetic rats. It has been previously reported that flax and fish oil intervention significantly increase HDL levels in STZ-induced diabetic rats [9,27,33]. In our study, a similar trend was observed. Thus, effective lowering of the abnormal lipid profile was observed in fish oil and glibenclamide combinational treatment. The overall mechanism of the action of flax and fish oil on hepatic gene expression is depicted in Figure 14.

SREBP is an important transcription factor that plays a crucial role in the regulation of fatty acid and cholesterol metabolism in the liver [35]. It consists of two isoforms i.e., SREBP-1a and SREBP-1c, which are expressed highly in the liver. Its overexpression was associated with elevated levels of cholesterol and triglycerides [36,37]. Earlier studies document that upregulated lipogenic gene expression, such as for fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACACA), was resulting in increased SREBP-1c expression, which leads to hepatic steatosis [38,39]. Hence, downregulation of SREBP-1c has a therapeutic value in the treatment of diabetic dyslipidemia [22,40,41]. The omega-3 fatty acid supplementation effectively lowered triglycerides level through downregulation of SREBP-1c gene expression [9,27,42]. Similarly, in our study, both flax and fish oil-treated animals showed downregulated SREBP-1c expression followed by a decrease in the expression of FASN and ACACA genes. These gene modulations may be one of the reasons for lowering the lipid abnormalities in flax and fish oil-treated rats.

CPT1 gene plays a major role in the uptake of fatty acids by the mitochondria for fatty acids β-oxidation [43]. In our study, the CPT1 expression was found to be increased in flax and fish oil-treated animals. This might be the probable reason for lowering serum triglyceride levels in diabetic rats.

Several studies reported that NF-κβ, a transcription factor (TF), plays a crucial role in insulin resistance and T2DM pathogenesis [22,41,44,45]. In diabetic conditions, upregulated NF-κβ expression leads to an increase inflammatory cytokine expression, e.g. tumor necrosis factor-α (TNF-α) [22,46,47]. In turn, it is associated with atherosclerotic lesions, lipolysis, and lipogenesis. Overall, this may result

in an increased risk of cardiovascular complications in T2DM individuals [46,47]. In the present study, both TNF-α expression and its transcription factor NF-κβ were found to be downregulated in flax and fish oil-treated groups.

**Figure 14.** Mechanism of action of flax and fish oil intervention. PPAR-γ: Peroxisome proliferatoractivated receptor gamma, SREBP-1c: Sterol regulatory element-binding proteins-1c, NFκβ: Nuclear factor-κβ, ACACA: Acetyl-CoA carboxylase alpha, FASN: Fatty acid synthase, CPT1: Carnitine palmitoyl transferase 1, FABP: Fatty-acid-binding proteins, TNF-α: Tumor necrosis factor-alpha, TCA: Tricarboxylic acid cycle, FFA: Free fatty acids, LCFA: Long chain fatty acids.

PPAR-γ, a transcription factor, is a member of the nuclear receptor family PPARs [48]. It plays an important role in carbohydrate and lipid homeostasis [48]. The activation of PPAR-γ stimulates β-oxidation of fatty acids and it results in lower serum triglyceride levels [49]. In the present study, flax and fish oil treatment upregulated the expression of PPAR-γ, and this may result in decreased serum triglyceride levels.

The FABP are members of a multigene family of cytoplasmic lipid transport proteins [50]. It is a potential target in the treatment of insulin resistance, lipid abnormalities, and atherosclerosis [50]. It facilitates fatty acid oxidation in the liver and may be beneficial for normalizing the hyperlipidemic condition [51]. Newberry et al. [52] reported that the L-FABP-null mice exhibit poor triglyceride accumulation in the liver, which leads to an increased serum triglyceride level [52]. A Wolfrum et al. [53] study shows that L-FABP acts as a gateway for the hypolipidemic drug and polyunsaturated fatty acids, which acts as a PPAR agonists [53]. Thus, upregulation of L-FABP expression would enhance the activation of PPAR through these agonists. In the present study, both flax and fish oil supplementation upregulated the expression of L-FABP in diabetic rats and this might be one of the reasons behind lowering serum triglyceride levels. FABPs are also associated with the docosahexaenoic acid (DHA) uptake and this might be the reason behind accelerating β-oxidation of fatty acids through higher activation of PPAR. This ultimately results in lowering serum triglyceride levels [54]. Our results are in accordance with the above findings [53,54].

#### **5. Conclusions**

The combinational treatment of glibenclamide and flax/fish oil intervention prophylactically against diabetic dyslipidemic rats exhibited potential effects on improving lipid abnormalities through modulating the expression of transcription factors (SREBP1-c, NF-kβ and PPAR-γ) and their regulatory genes i.e., ACACA, FASN, CPT1, FABP, and TNF-α. In the future, combination therapy of glibenclamide

and omega-3 fatty acid intervention at a therapeutic level is worth investigation in the diabetic dyslipidemic condition.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/12/3652/s1, Figure S1: Assessment of glucose and lipid profile before diabetes development, Figure S2: LFT and KFT before diabetes development.

**Author Contributions:** Conceptualization, A.K. and N.M.; Data curation and analysis, S.K.; Investigation, S.K.; Methodology, A.K., V.P., and N.M.; Performed the assays and acquisition of data, S.K. and P.M.; Project administration, A.K.; Supervision, A.K. and N.M.; Histological examination, M.K.; Experimental design, V.P.; Writing—original draft, S.K.; Writing—review & editing, A.K. and N.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors are grateful to Bharati Vidyapeeth Deemed University for the financial support of this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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