Impact of Targeted Specific Antibiotic Deliveryfor Gut Microbiota Modulation on High-Fructose-FedRats

Prasant Kumar Jena & Shilpa Singh &

Bhumika Prajapati & G. Nareshkumar & Tejal Mehta &

Sriram Seshadri

Received: 26 August 2013 /Accepted: 3 February 2014# Springer Science+Business Media New York 2014

Abstract The objective of present investigation was to study the effect of gut microbiotaalteration by oral administration of targeted delivery of pH sensitive cefdinir microspheres tohigh-fructose-fed (HFD) rats. Rats were fed with a high-fructose diet with or without cefdinirmicrosphere administration for 30 days. The fecal microbiota community, oral glucosetolerance, the markers of liver injury, plasma and hepatic lipids profile, and histologicalevaluation were investigated. The levels of blood glucose, liver injury markers, lipid profilein plasma and liver, and fat tissue were significantly increased in high-fructose-fed rats.However, after pH-sensitive cefdinir microsphere administration, the elevation of these pa-rameters was significantly suppressed. Cef EL significantly lowered the increased AST(p<0.05) and ALT (p<0.001) levels in HFD group. There is a significant lower (p<0.01)AUCglucose level in Cef EL group than HFD group The histological changes in the liver and thesmall and large intestines were more profound in HFD group as compared to cefdinir-treatedHFD and control groups. Feeding of cefdinir microsphere sustained lactobacilli andbifidobacteria and significantly decreased (p<0.05) the number of Enterobacteriaceae inducedby HFD. Experimental evidences demonstrated that the effectiveness of pH-specific cefdinirmicrosphere on reducing insulin resistance and development of metabolic changes in high-

Appl Biochem BiotechnolDOI 10.1007/s12010-014-0772-y

Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-0772-y)contains supplementary material, which is available to authorized users.

P. K. Jena : S. Singh : B. Prajapati : S. Seshadri (*)Institute of Science, Nirma University, Sarkhej-Gandhinagar Highway, Chandlodia, Ahmedabad 382481Gujarat, Indiae-mail: sriram.seshadri@nirmauni.ac.in

Sriram. Seshadrie-mail: sriramsjpr@gmail.com

G. NareshkumarMolecular Microbiology and Biochemistry Laboratory, Department of Biochemistry, M. S. University ofBaroda, Vadodara, Gujarat, India

T. MehtaDepartment of Pharmaceutics, Institute of Pharmacy, Nirma University, Ahmedabad, Gujarat, India

fructose-fed rats and suggested that it may be a promising therapeutic agent in treating type 2diabetes. Intestinal-targeted antibiotic delivery needs to be further explored for its therapeuticapplications.

Keywords Cefdinir . Microspheres . Fructose . Gut microbiota . Diabetes . Inflammation

Introduction

Diabetes is the most common endocrine disorder, and the number of people withdiabetes in 2011 has reached a surprising 371 million. Around 4.8 million deaths arebecause of diabetes, and 300 million will be later added by 2025 [1]. Obesity, type 2diabetes, and hyperlipidemia often coexist and associate with significantly increasedmorbidity and mortality [2]. A significant increase in total refined carbohydrate intake,fructose has paralleled recent increase in incidence of obesity and diabetes. Metabo-lisms of sugars, particularly fructose, occurs mainly in the liver, and high-fructose fluxleads to enhanced hepatic triglyceride accumulation resulting in impaired glucose andlipid metabolism and increased proinflammatory cytokine expression [3].

Persistent fructose consumption shows to decrease insulin sensitivity, increasesinflammation, oxidative stress, and pancreatic islet dysfunction, and promotes dyslip-idemia, which may increase the risk for development of type 2 diabetes [4]. Type 2diabetes (T2D) processes by impaired glucose tolerance (IGT), where the metabolicand endocrine changes take place, which can effectively prevented or even delayedthrough lifestyle changes or drug treatment [5]. Because of the increasing incidence ofT2D, there is an extensive need to find new effective strategies for type 2 diabetesprevention and/or treatment, including the potential use of nutritional supplements.

The mammalian host colonizes by trillions of microbes, which inhabit the gastro-intestinal (GI) tract, predominantly in symbiotic relationship to their host [6]. Severallines of evidence suggest that dietary factors might profoundly influence gut micro-biota composition. Switching to a high-fat diet resulted in a reduction ofBacteroidetes, while the figures of Firmicutes and Proteobacteria had been increased[7]. Importantly, this was observed both in the presence and absence of obesity, whichclearly suggests that diet must be considered as a confounding factor affectingmicrobial composition. A change in the diet (i.e., from a low-calorie fat plantpolysaccharide to a high-fat sugar-rich diet) shifted the gut microbiota structure withina single day, along with the changes in major metabolic pathways in the microbiome.Community population and function of the microbiota can change due to variousways, including antibiotic treatment, inflammation, or changes in diet pattern [8].Antibiotic therapy not only target pathogen but also to commensal inhabitants ofthe human host. The degree of the impact on non-target microbial populationsdepends on the specific antibiotic drug used, its mode of action, and the degree ofresistance within the community [9].

Extended loss of the distinctive composition of gut microbiota links with several disorders,including inflammatory bowel diseases [10]. Changes in microbial composition have beenassociated with obesity and weight reduction; however, factors associated with these changesare not well defined [11]. The alterations in community population, whether chronic or acute,are accompanied by alteration in the microbiota’s collective genome, or microbiome, and thepatterns and specific metabolic capabilities [12]. Recently, it was observed that feeding mice ahigh-fat diet also caused an increased Firmicutes and reduced Bacteroidetes-type microbiota,

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as described before [13]. Therefore, the relevant variables, such as changes in the diet tochanges in the microbiome, are important in understanding how environmental factors andbehavior influence physiology of individual people.

Besides immune and inflammatory mechanisms, other pathways may involve, which arethe link between gut microbiota and metabolic syndrome. The microbiota produces enzymesthat degrade ingested polysaccharides, which promotes the absorption of various nutrients(especially carbohydrates) that result in increased liver lipogenesis, hyperinsulinemia, andhepatic insulin resistance. It has been demonstrated that high intake of cereal fiber associateswith reduced risk for T2D [14].

Many pharmaceutical dosage forms irritate the stomach due to their chemicalproperties. Others undergo biochemical changes in the gastric acid through the actionof various enzymes. Specific Eudragit acrylic polymers have been developed forperoral dosage forms with step-wise release of active ingredients in the digestivetract. The pharmaceutical principle of Eudragit coating is to solubilize in a specificenvironmental pH value [15].

Eudragit L100-55, composed of methacrylic acid and methyl methacrylate (1:2,Mw=approx. 135,000), was chosen as a pH-sensitive polymer owing to its uniquedissolution behavior above pH 5.5. Eudragit have been used as pH-sensitive polymersin various applications including enteric coating materials and drug delivery vehiclesand exhibited plastic deformation and significant speed sensitivity [16]. Eudragit incombinations with other polymers, such as hydroxypropyl methyl cellulose and talc,stabilized loaded drugs and provided a controlled release of them [17]. Microparticlesmade from Eudragit polymers utilize for protein drug delivery to the lower intestineafter oral administrations are based on the change of pH during the gastrointestinalpassage [18].

Cefdinir [8-(2-(2-amino-1,3-thiazol-4-yl)-1-hydroxy-2-nitroso-ethenyl]amino-4-ethenyl-7-oxo-2-thia-6-azabicyclo[4.2.0]oct-4-ene-5-carboxylicacid is a semisyntheticthird-generation broad-spectrum oral cephalosporin active against both gram-positiveand gram-negative bacteria and is widely used to treat acute chronic bronchitis,rhinosinusitis, and pharyngitis. It has only 21–25 % of oral bioavailability [19], whichis probably due to low aqueous solubility.

In trying to answer these problems, this investigation studied the effect of an excess fructoseleading pathological changes and activity levels within the liver defense system as well as gutmicrobiota. The alteration of gut flora by using Eudragit L100-55-coated cefdinir microspheres(Cef EL) which used to disrupt the microbiota in small intestine of male Wistar rat. This wouldhelp to understand better the role of gut microbiota in fructose-induced metabolic changes aswell as to develop appropriate strategies in the prevention and treatment of obesity and T2Dtriggered by unhealthy diets.

Materials and Methods

Starting Materials and Reagents

Eudragit L100-55 (average molecular weight approximately 320,000 g/mol) was provided byRohm GmbH and Co. KG (Germany). Cefdinir (cefdinir is an extended spectrum oral third-generation antimicrobial agent with a broad-spectrum activity against enteric gram-negativerods and has low permeability) was received as a gift from Macleods Pharmaceuticals Limited(Mumbai, India). All other reagents and solvents were of analytical grade.

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Preparation of Microspheres

The preparation of microspheres was either based on an oil/water emulsification—solventevaporation or solvent extraction method. The usually employed oil/water emulsificationprocess is given as a standard in the preparation of Cef EL. For all different techniques, afixed amount of polymer (100 mg) and drug (10 mg) were used. Accurately weighed antibioticcefdinir was taken in different drug polymer ratio (1:2) and was added in an 5-ml acetoneorganic phase (internal aqueous phase) having 10 % w/v Eudragit L100-55 (EL), and sonicatedin an ultrasonicator (JY92-11DN, Syclon, Japan) for 10 min. This solution was slowly injected(0.33 ml/min) into an external aqueous phase containing paraffin light liquid (100 ml) and wasemulsified containing the emulsifier, span 80 (2 %v/v). The system was stirred continuouslyusing a mechanical stirrer at 1,000 rpm and 37±0.5 °C for 5 h to form a uniform emulsion andallowed complete evaporation of the solvent to form microspheres. The paraffin was decantedoff; the microspheres were washed 3–4 times with petroleum ether (40–60 °C), collected byfiltration and finally dried at room temperature for 3 h [20].

In Vitro Release Profile Study

The transit time of a drug through the absorptive area of the gastrointestinal (GI) tract isbetween 9 and 12 h, whereas γ scintigraphy studies confirmed that short GI transit time frommouth to cecum is of 4 to 6 h Thus, assuming a maximum GI tract transit time of 12 h, aformulation in the cecum is expected to release its drug load within 6 h. Considering the same,in vitro drug release from all the batches of microspheres was studied for duration of 6 h. Forthe first 2 h, the drug release profile was performed in simulated gastric fluid (pH 1.2 with0.1 N HCl). Remaining 4 h of the release profile of cefdinir from Eudragit (L100-55)microspheres was evaluated in phosphate buffer (pH 5.5), which represents the pH of smallintestine. Microspheres equivalent to 1.5-mg drug/ml were transferred to the dissolution media(20 ml) which maintained at 37ºC±0.5ºC under stirring at 75 rpm. A 0.5 ml of samples waswithdrawn every regular time interval up to 6 h, and the withdrawn volume was replenishedimmediately by same volume of fresh phosphate buffer. Amount of drug released in thewithdrawn sample was estimated by measuring absorbance in a UV spectrophotometer at287 nm for cefdinir against a phosphate buffer (pH 5.5) as control blank.

Enteric Nature of Microspheres

This test was performed to determine whether the drug would be released in the acidicenvironment of the stomach (i.e., pH between 1 and 3). Cef EL equivalent to 1.5-mg drugwas transferred to 20 mL of 0.1 N HCl that was maintained at 37±0.5 °C under stirring at75 rpm. A 0.5 ml of samples was withdrawn every regular time interval up to 3 h, and thewithdrawn volume was replenished immediately by same volume of 0.1 N HCl. Amount ofdrug released in the withdrawn sample was estimated by measuring absorbance in a UVspectrophotometer at 287 nm against a 0.1 N HCl as blank.

Physicochemical Characterization of Cef EL

Scanning Electron Microscopy

The external and internal morphology of Cef EL was analyzed by scanning electron micros-copy (SEM). The microspheres were fixed on supports with carbon-glue and coated with gold

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using a gold sputter module in a high vacuum evaporator. Samples were then observed withthe SEM (LEO-1430VP, UK.) at 10 kV.

Particle Size Analysis

The particles was analyzed basis on the dynamic light scattering technique (DLS) by using aMastersizer (Malvern Instrument, Malvern, UK), Particle sizes are expressed as the weighedmean of the volume distribution. Each value resulted from a triplicate determination.

FT-IR Analysis

Fourier transform infrared (FT-IR) spectra of Cef EL were obtained on a PerkinElmer, GX-FT-IR spectrometer. Eudragit L100-55-containing microspheres (with or without cefdinir) wereprepared in KBr disks (2 mg sample in 200 mg KBr). The scanning range was 400–4,000 cm−1

and the resolution was 1 cm−1.

In Vitro Antimicrobial Profile Study

In vitro antimicrobial studies of Cef EL were performed as described earlier [21] withmodification. Cefdinir was dissolved in small amount of dimethylformamide andsuitably diluted with distilled water. Similarly, Cef EL and Eudragit polymer wasdissolved separately in distilled water and suitably diluted with the same to obtain theconcentration of 5 μg/mL. One milliliter overnight grown culture of Escherichia coliMTCC 443 and Lactobacillus casei MTCC1423 was inoculated in freshly preparedLuria-Bertani and MRS broths, respectively. Both the broth was adjusted to pH 5.5before addition of inoculum. After 2 h of incubation at 37 °C at shaking 150 rpm,prepared microsphere Cef EL (cefdinir concentration 5 μg/mL (MIC 0.1–0.5 μg/ml)was added to both the cultures and incubated at 37 °C with shaking at 150 rpm.Changes in optical density (600 nm) were recorded every hour for 7 h. Activities ofCef EL were compared with control cells treated with pure Eudragit L100-55 andcefdinir.

Animal Experiments

Eight to 10 week-old healthy male Wistar rats weighing 150–200 g were procuredfrom the Animal Research Facility, Torrent Research Center, Ahmedabad (India) underthe approval Institutional Animal Ethics Committee, protocol no. IS/BT/PhD11-12/1004 and were maintained at the animal house of Institute of Pharmacy (NirmaUniversity, India). The animals were acclimatized at temperature of 25±2 °C andrelative humidity of 50–60 % under 12/12 h light/dark conditions for 1 week beforeexperiments. Animals were assigned to a normal control group (CD; n=6) thatconsumed a standard diet and normal drinking water, a high-fructose-fed controlgroup (HFD; n =6) that consumed a standard diet having 65 % fructose with normaldrinking water, and cefdinir microsphere-treated group (HFD Cef EL, n=6) thatconsumed a high-fructose diet for 30 days except for the days before oral glucosetolerance tests (OGTTs) and blood collection. The composition of experimental dietsis presented in Table 1. Animals had free access to food and water with or withoutfructose. The food and water intakes were recorded daily by correction of spillage andbody weight was measured twice a week.

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Oral Glucose Tolerance Test

Oral glucose tolerance tests were performed between 8.0 and 10.0 h at weeklyintervals. The diets were removed from animal cages for 12 h before the administra-tion of an oral glucose load (2 g/kg of body weight) by orogastric gavage. Bloodsamples were collected from the tail vein at 0, 15, 30, 60, 90, and 120 min afterglucose administration. Glucose concentration was determined with an Accu-CheckAdvantage Blood Glucose Monitor (Roche Group, Indianapolis, IN, USA). Area underthe curve for glucose (AUCglucose) was determined using the trapezoidal rule.

Blood and Tissue Sample Collection

At the end of the experiments, the animals were sacrificed by a deep dose ofanesthesia. The blood samples were collected, processed for plasma and serum, andthen stored at −80 °C. Blood parameters including blood glucose, plasma triglycer-ides, total cholesterol and high-density lipoprotein cholesterol (HDL-C), liver patho-logical marker such as SGPT and SGOT from serum, hepatic triglycerides, andcholesterol were analyzed by enzymatic kit (Accucare diagnostics, India). The liverglycogen was determined as described earlier [22]. Body fats were measured while theliver, distal ileum, and proximal colon were washed with ice-cold saline and thenwere stored at −80 °C until used. A small portion of the liver, distal ileum, andproximal colon were excised from animals of each group, fixed with 10 %v/vformalin saline, and processed for standard histopathological procedures. Paraffin-embedded specimens were cut into 5 μm sections (Yorco Sales Pvt. Ltd., New Delhi)and stained with hematoxylin and eosin (H&E). The histopathological tissue sectionswere viewed and digitally photographed using a Cat-Cam 3.0MP Trinocular micro-scope with an attached digital 3XM picture camera (Catalyst Biotech, Mumbai, India).

Quantification of Fecal Bacteria

For quantitative determination of Enterobacteriaceae, lactobacilli, and bifidobacteria, 1-gcecal content mixed 0.85 % of NaCl solution and homogenized by vortexing for 10 min.Serial 10-fold dilutions were prepared and volumes of 100 μl of three appropriate dilutions

Table 1 Formulation of the diets

aFrom Central Drug House PvtLimited, IndiabFrom Ankur Foods, IndiacFrom M. P. Biosciences, UKdFrom Amrut Agrofoods, Mumbai

Ingredients (%) Controld High fructose High sucrose

Starch 65 0 0

Fructosea 0 65 0

Sucrosea 0 0 65

Caseina 20 20 20

Corn oilb 5 5 5

Wheat bran 5 5 5

Mineral mixc 3.5 3.5 3.5

Vitamin mixc 1 1 1

D-Methionine 0.3 0.3 0.3

Choline chloride 0.2 0.2 0.2

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were loaded onto the surface of plates in triplicate. EMB, MRS, and BS agars were used forgrowing Enterobacteriaceae, lactobacilli and bifidobacteria, respectively. All media wereobtained from Himedia (Mumbai, India). All these plates were incubated anaerobically at37 °C for 72 h. The number of colony counting for each microbiota species was expressed aslog CFU/g fecal content. Microscopic characteristics of all of the colonies were investigated byGram staining methods [23].

Statistical Analysis

All the values are expressed as mean±SD. Statistics was applied using GraphPad Prismsoftware version 5. One-way ANOVA followed by Tukey’s multiple comparison tests wasused to determine the statistical significance of the test groups with the control and within thetest groups. Differences were considered to be statistically significant when p<0.05.

Results

Preparation of Microspheres

Three different formulations containing cefdinir with Eudragit L100-55 of various drugs topolymer ratio were prepared by using emulsion solvent evaporation techniques. The propertiesof these formulations, process yield, and entrapment efficiency are listed (Table S2, supple-mentary data). From the results, it was observed that increased polymer ratio with respect todrug leads to enhance drug entrapment efficiency, so as the amount of drug remaining andavailable for encapsulation increased as the theoretical drug loading increased. However, thepolymer with lower concentration lowers the entrapment efficiency because loss of productduring the successive decantation, washing, and drying process. The entrapment efficiency ofcefdinir in microsphere formulation was found to be enhanced by increasing the drug topolymer ratio from lower to higher side, and maximum entrapment efficiency was observed atdrug to polymer ratio 1:1 (Table S2, supplementary data).

In Vitro Drug Release Study

To evaluate the pH-dependent release profiles of cefdinir microspheres, in vitro releasetests were performed up to 6 h in simulated gastric fluid (Fig. 1). In the dissolutionmedium at pH 1.2, nearly 5–8 % of the drug was released in 2 h. It was shown thatslow drug release behavior was observed for microspheres at pH 1.2. Since thepolymer is insoluble in the release media with pH 1.2, the microparticles were onlyslightly swollen and remained intact in this case. At a pH of 5.5, the polymerdissolved rapidly and the microspheres’ disintegration resulted in a faster drug releaserate compared with pH 1.2. As the concentration of polymer increases, release of drugwas found to be much slower because more amount of drug entrapped inside thepolymer matrix. Whereas more drug concentration, rapid release was observed for theabsorption on the surface of microsphere.

Scanning Electron Microscopy

The SEM images of Cef EL and Eudragit L100-55 are presented (Fig. 2). The unprocessedcefdinir particles (Fig. 2a) have needle-like crystals, and drastic change in the morphology and

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shape of particles was observed for both processed particles (Fig. 2c) and Eudragit polymer(Fig. 3b). Under SEM examination, Cef EL exhibited spherical morphology and smoothsurfaces as well as a monodispersed size distribution are shown (Fig. 2c). The microsphereparticle size distribution of Cef EL (cefdinir and Eudragit 100-55 drug polymer ratio 1:1) waspresented (Fig. S1 supplementary data).

FT-IR Study

The FT-IR spectra of intact cefdinir, Eudragit L100-55, and Eudragit L100-55-coatedcefdinir (Cef EL) was shown (Fig. 3). IR spectrum of cefdinir (Fig. 3a) is character-ized by principal absorption peaks at 2,928 cm−1 (O–H stretch COOH), 2,849 cm−1

(C–H stretch cyclic), 1,761 cm−1 (C═O), 1,678 cm−1 (C═C alkene), 1,620 cm−1 (C═Caromatic), 1,516 cm−1 (N–H bending), 1,391 cm−1 (C–N stretch), and 656 cm−1 (C–S). The spectrum of Eudragit L100-55 has a broad band characteristic of hydroxylgroups (O–H stretch vibration) in the range of 3,476–2,358 cm−1, characteristic bands

Fig. 1 Drug release profiles of Cef EL

Fig. 2 SEM image of a unprocessed crystalline cefdinir particles (×1,000), b Eudragit L100-55 and c cefdinircoated with Eudragit L00-55 polymer

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of methyl and methylene (C–H stretch vibration) at 2,976 cm−1 and 2,895 cm−1, and astrong band due to carbonyl groups (C–O stretch vibration) at 1,733 cm−1 and twobands due to ester linkages (C–O stretch vibration) at 1,368 and 1,266 cm−1 (Fig. 3b).FT-IR spectra of Cef EL polymer mixture shows prominent peaks at 3,427 cm−1 (O–H), 2,922 cm−1 and 2,852 cm−1 (C–H), and 1,730 cm−1 (H–O–H bending). All thepeaks of Cefdinir (CEF) completely disappeared with a shift of 1,781–1,767 cm−1.The FT-IR spectra of Cef EL (Fig. 3c) complex shows complete disappearance of theCEF peaks at 3,300, 2,976, 2,895, and at 652 cm−1 with strong decrease in peakintensity. This suggested that, CEF could form inclusion complex with Eudragit L100-55 in solid state. The microspheres of Cef EL did not show any new peaks, indicatingno chemical bond formation in the complexes. The significant differences in theobserved vibrational transitions and the bands in the spectrum of the crystalline formwere clearer and sharper than the bands of the amorphous forms. In addition, in IR-spectra, it is already known that significant differences between hydrate and anhydrateform were observed around 2,800–3,800 cm−1 [24]. Due to the O–H stretchingvibration of water molecules, the unprocessed cefdinir, which is in monohydrate form,had characteristic peaks observed at 1,164, 1,118, 1,620, 1,761 3,257, and3,357 cm−1, but not in processed particles.

In Vitro Antimicrobial Profile Study

The antimicrobial activity of Cef EL, cefdinir, and Eudragit L100-55 against gram-positive (L. casei MTCC1423) and gram-negative (E. coli MTCC443) species waschecked and results are summarized (Fig. 4). These studies revealed that Cef EL haveshown similar antimicrobial activity like CEF alone against L. casei while EudragitL100-55 alone have no properties of microbial inhibition as it shows similar growthpattern like L. casei (Fig. 4a) However, Cef EL inclusion polymer complex has shownsignificant microbial inhibition against both the microorganisms as like pure CEF inpH 5.5. However cefdinir known for its gram-negative antimicrobial agents, theeffects after Eudragit polymer coating also not reduced much as shown (Fig. 4b).The antimicrobial activity of Cef EL polymer and pure CEF alone have identicalinhibition properties against E. coli in pH 5.5 where as Eudragit alone had noinhibitory properties as grown with E. coli.

Fig. 3 FT-IR spectra of cefdinirand Eudragit L100-55 polymer(A) Eudragit L100-55; (B) cefdinircoated with Eudragit L100-55 and(C) cefdinir

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In Vivo Experiments

Body Weight, Food, and Water Intake

As shown (Table 2), the rats fed with a high-fructose diet (HFD) showed a significant increasein body weight (p<0.001); as compared to the normal control diet (CD) group at week 4. Itwas found that body weight was significantly reduced (p<0.001) when administered withcefdinir microsphere (Cef EL) as compared HFD-fed group. There are no significant changesobserved in food consumption among the groups while water intake was significantly higherin HFD group (p<0.01) than CD group but no significant changes observed in Cef EL groupin comparison with CD and/or HFD group.

Body weights of HFD groups were recorded significantly higher (p<0.001) than CD groupwhile Cef EL group has no significant changes than CD group animals after the 4-weekcoadministration period. But the Cef EL group animals had a significantly reduced (p<0.001)body weight than HFD animals. The weight of abdominal fat content was significantly higher(p<0.001) in the HFD group animals (12.8±0.96 g) than CD group (6.32±0.43 g) while CefEL (5.17±0.26 g) group were significantly different from CD group (p<0.05) and HFD group(p<0.001) animals.

Fig. 4 In vitro antimicrobial assay of Cef EL against a L. caseiMTCC 1423 and b E. coliMTCC443. Values arepresented as mean±SEM (n=3). Values with superscript letters are significantly compared with control(*p<0.05)

Table 2 Food intake and body weight of experimental rats fed with different experimental diets during 1-monthperiod

Parameters CD HFD HFD + Cef EL

Food intake (gm/day per rat) 19.93±3.2 16.58±2.6 17.84±2.3

Water intake (ml) 38±4.5 48±5.2b 42±4.2

Initial body weight (g) 170±12.30 165±11.50 165±10.6

Final body weight (g) 195±8.5 225±9.5 198±8.3

Body weight gain (g) 25±7.8 65±9.4c 33±4.6c

Body fat (g) 6.32±0.43 12.8±0.96c 5.17±0.26a, c

Values are presented as mean±SEM drawn from pooled spillage of three cages of the same group and calculatedas six animals per group. Values with different superscript letters are significantly different (a p>0.05; b p>0.01;c p>0.001)

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Oral Glucose Tolerance Test

The OGTTs value after the second week in the HFD group and the AUCglucose values weresignificantly higher (p<0.01) (46 %) than that of the CD group while Cef EL group weresignificantly 14 % lower (p<0.05) than that of HFD group (Fig. 5b). However, OGTTs wereimpaired after 4 weeks in the Cef EL animals, and the AUCglucose values were significantly(p<0.01) lower (34 %) than those of the HFD animals. After the fourth-week experimentalperiod, the AUCglucose was 150 % higher (p<0.001) in the HFD than the CD group, whereas inCef EL animals, it was 34 % lower, which is significant (p<0.01) than that of the HFD animalsyet significantly (p<0.001) 65 % higher than that of the CD animals (Fig. 5d).

Blood and Tissue Biochemical Analysis

Earlier studies indicated that higher triglyceride (TG) and cholesterol levels were the mainpredictors and causative factors for inducing insulin resistance in type 2 diabetes. In our currentresults, the fasting blood glucose levels in the HFD group were significantly higher (p<0.001)

Fig. 5 Effect of Cef EL on oral glucose tolerance test of HFD rats. a–d represents the OGTT and AUCglucose

values of animals after 15 and 30 days of intervals, respectively. Rats (6–8-week old, male, n=6/group) wereadministered with 2 g of glucose per kilogram of body weight after fasting for 12 h. The blood glucose levelswere measured from 0 to 120 min. Values are presented as mean±SD (n=3). Values with different letters aresignificantly different (p<0.05). AUCglucose area under the curve for glucose, HFD Cef EL, Cef EL and high-fructose-treated group, HFD high-fructose-fed group, CD control group

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than those in the CD group. However, Cef EL group was significantly lower (p<0.001) thanthat of HFD group (Table 3). After 4 weeks of fructose consumption, liver triglyceride level ofHFD was significantly increased (p<0.001) while liver cholesterol and hepatic glycogendecreased significantly (p<0.01 and p<0.05, respectively) than CD group (Table 4). In otherside, hepatic cholesterol level of Cef EL group was significantly reduced (p<0.001). Nosignificant differences were observed in liver glycogen between the CD and Cef EL groups.However, plasma total cholesterol, triacylglycerol, and LDL-C levels were significantly higher(p<0.001) in the HFD than the CD group, whereas these variables in the Cef EL group weresignificantly lower (p<0.001, p<0.05, and p<0.001, respectively) than those in the HFDgroup. However, HDL-C was significantly lower (p<0.05) in the HFD group than in the CDgroup, whereas no significant differences were observed between the HFD and Cef EL groups.Increased serum levels of AST and ALT were found in HFD group (Table 3). These resultsshowed that long-term high-fructose treatment induced damage to liver cells. However,coadministration of Cef EL significantly lowered the increased AST (p<0.05) and ALT(p<0.001) levels in HFD group.

Histological Analysis

Histopathological assessments were carried out to determine the possibility of pathogenicityinduced by a high-fructose diet in the liver and small and large intestines of rats. Thehistological examination of the liver showed a series of morphological alterations, notablyhepatic steatosis in fructose-fed diet group. Reduced tissue damage and liver steatosis wereobserved in Cef EL group rats. In control group, liver sections, normal hepatic cells (HC),central vein (CV), and kupffer cell (KC) were blurred observed due to staining (Fig. 6, series1A). High-fructose-fed rats showed prominent macro- and microvesicular steatosis and necro-sis around the portal triad as well as focal areas of the inflammatory cell infiltrate around theportal triad (Fig. 6, series 1B) compared to the normal histological appearance of the liver fromcontrol group (Fig. 6, series 1A). However, coadministration of Cef EL significantly reversedthe formation of hepatic steatosis induced by HFD group (Fig. 6, series 1C). Histologicalexamination of the intestinal segments revealed villi edema, lymphocytes infiltration, andgoblet cell hyperplasia in rats of HFD group (Fig. 6, series 2B) as compared to CD group(Fig. 6, series 2B) animals. However, Cef EL group shows recovery of mucosa, villus, cryptal,and repair of injury (Fig. 6, series 2C). The crypts of colon contained many goblet cells in HFD

Table 3 Biochemical analysis from blood

Parameters CD HFD HFD+Cef EL

Fasting blood glucose (mg/dl) 110.02±8.2 190.08±10.37b 119 .97±10.38B

Total cholesterol (mg/dl) 64. 02±5.63 95.85±4.36b 69.73±8.3B

Triglycerides (mg/dl) 74. 02±4.36 105.34±7.26b 77.65±9.4B

HDL-cholesterol (mg/dl) 29.5±7.52 23. 8±4.7 25.38±3.8

LDL-cholesterol (mg/dl) 56.75±4.2 77.64±2.8b 52. 64±5.4B

SGPT (ALT) IU/L 31±2.8 44.3±6.1b 36.5±3.2A

SGOT (AST) IU/L 28.2±1.5 46.3±2.2b 31.9±0.98a, B

Values are presented as mean±SEM drawn from pooled spillage of three cages of the same group andcalculated as six animals per group. Values with different superscript letters are significantly different i. e.,A p>0.05; B p>0.001 when compared with HFD group; a p>0.01; b p>0.001 when compared with CD group

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rats (Fig. 6, series 3B) while reduced histological parameters of colonic injury was observed inCef EL (Fig. 6, series 3C) as well as of control group (Fig. 6, series 3A).

Enumeration of Fecal Microbiota

Bifidobacteria and lactobacilli have been regarded as beneficial microflora species, whereassome species, Enterobacteriaceae for example, would be harmful as a consequence of theirmetabolic activities. Quantification of fecal bacteria of experimental animal was showed(Fig. 7). The number of bifidobacteria, lactobacilli, and Enterobacteriaceae in control groupwas regarded as 100 % for comparison. Our present results indicated that the number ofEnterobacteriaceae, lactobacilli, and bifidobacteria in HFD group were 146.56±27.28,

Table 4 Tissue biochemistry

Parameters CD HFD HFD + Cef EL

Liver Triglyceride (mg/gm) 10.46±1.76 22.39±3.89c 17.56±2.91b, A

Liver Cholesterol (mg/gm) 1.47±0.14 1.76±0.13b 1.57±0.08A

Liver Glycogen (mg/gm) 8.19±0.9 6.49±1.3a 7.91±0.72

Values are presented as mean±SEM drawn from pooled spillage of three cages of the same group and calculatedas six animals per group. Values with different superscript letters are significantly different, i.e., A p>0.05 whencompared with HFD group; a p>0.05; b p>0.01; c p>0.001 when compared with CD group

Fig. 6 Effects of Cef EL on HFD treated rats. At the end of the experimental period, the livers, distal ileum, andproximal colon from sacrificed rats were collected. a Control. b HFD group. c HFD Cef EL (in series 1: CVcentral vein,KC kupffer cell; in series 2: CP crypts, LP lamina propria, SM submucosa, AD adventitia; in series 3:CE crypt epithelium, GC goblet cell, CP crypts)

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63.08±1.24, and 94.33±4.04 %, respectively. These results revealed that significant decreaseof lactobacilli was observed in HFD group (p<0.01) and Cef EL group (p<0.05). After30 days of Cef EL dosing, the number of Enterobacteriaceae, lactobacilli, and bifidobacteriawere 59.04±3.4, 116.38±12.88, and 92.38±0.53 %, respectively. These results demonstratedthat administration of Cef EL significantly decreased (p<0.05) the number of Enterobacteri-aceae; however, there are no significant changes observed in lactobacilli and bifidobacteriawhen compared to those in HFD group.

Discussion

Modulation of the gut microbiota with antibiotics or pre- and probiotics improves themetabolic benefits of the host, in terms of glucose tolerance and inflammatory cytokine profile,although exposure to antibiotics in early life may actually predispose to obesity and metabolicdisease in later life. Since inflammatory and metabolic pathways are interconnected, themicrobial signals acting on either or both may suggest adjunctive therapeutic or preventivestrategies for obesity [25].

The findings show that Cef EL altered the gut microbiota in diet-induced diabetic ratsin distinct ways. In the present study, cefdinir was chosen for its ability to target thegram-negative component of the gut microbiota and its limited systemic impact. In thecurrent study, pH-sensitive cefdinir microsphere treatment of diet-induced diabetic ratresulted in a major alteration in the composition of the gut microbiota, whereby therespective proportions of the three dominant genera were altered dramatically with respectto each other, with a large reduction in Enterobacteriaceae population. However, theintestinal microflora across the subjects with type 2 diabetes was relatively enriched withgram-negative bacteria, belonging to the phyla Bacteroidetes and Proteobacteria. Thesealterations were related with a reduction in body weight gain and an improvement ininflammatory and metabolic health of the host. Earlier reports suggested that modulationof the microbiota by broad-spectrum antibiotics helps in reduction in metabolicendotoxemia in both high-fat-fed and ob/ob mice, with improvements in inflammation,glucose tolerance and hepatic steatosis [26, 27]. However, whether the effect on weightgain is persistent or is overcome by microbial community, adjustments are unclear, andmore studies in animal models and humans are required.

Fig. 7 In vivo quantification of fecal bacteria from experimental animal. Values are presented as mean±SEM(n=3). Values with superscript letters are significantly compared with control (*p<0.05)

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Previous study exposed that a higher weight gain and fat deposition were responsible forinsulin resistance [28]. In addition, a number of studies reported that a reduction of visceral fatin animal models and humans was associated with increased insulin sensitivity.

Our results shown in Table 2 demonstrated that the body weight and fat tissue weight inHFD group were significantly higher than those of control group. These results were in linewith previous reports [3]. Administration of Cef EL significantly suppressed the enhancingeffect of high-fructose load on body weight and fat tissue weight. These results implied thatreduction of body weight and fat tissue weight by Cef EL treatment may be responsible forimproving insulin sensitivity in HFD rats.

Impaired glucose tolerance test is an important pathological indicator for type 2 diabetes[29]. Our results from OGTT indicated that an increase in blood glucose level was observedafter high-fructose diet. Coadministration of Cef EL significantly decreased the blood glucoselevels enhanced by high-fructose feeding. These results established that feeding of Cef EL mayimprove glucose intolerance and prevent the development of hyperglycemia in high-fructose-fed diabetic rats.

As shown in our study, coadministration of Cef EL dramatically decreased the levels ofimportant components of metabolic syndrome, including blood glucose, plasma LDL, TG, andcholesterol enhanced by high-fructose treatment. In addition to plasma levels, the increasedhepatic levels of TG and cholesterol treated with high fructose were also found to besuppressed by oral administration of Cef EL. In present study, our data implicated thatadministration of Cef EL could improve insulin resistance via downregulation of both serumand hepatic lipid concentrations. Also, the serum levels of AST and ALT, markers of liverinjury, were increased in HFD rats; after treatment with Cef EL, downregulation of serum ASTand ALT levels in HFD-treated rats were noticed. These findings answered that Cef ELconsumption significantly reduced liver damage in HFD rats via enhancing hepatic antioxi-dants expressions [30].

In the Cef EL-treated HFD rats, the level of liver glycogen was markedly increased showsin this study. Improvement of hepatic insulin sensitivity leads to the suppression of hepaticglucose output and an increase in liver glycogen storage [22, 27]. Liver glycogen is often lowerin patients with type 2 diabetes, thus increment of liver glycogen storage is associated withincreased hepatic insulin sensitivity. Liver glycogen synthesis has not been assessed directly intype 2 diabetic patients; however, splanchnic glucose uptake is decreased after glucoseadministration [31]. In the present study, no significant difference noticed between CD andCef EL-treated HFD group. However, significant reduction in liver glycogen was observed inHFD group than in control group (Table 4).

The prepared Cef EL was found to be discrete, spherical, and uniform in size with a rangeof 230–280 μm. The SEM photograph indicates that the microspheres were smooth, spherical,and free flowing. From the result of FT-IR, it was clear that there was no interaction betweendrug and polymer used in the formulation. The drug release from Eudragit L100-55 polymershowed slow release at gastric pH but higher release obtained at intestinal pH. The Cef ELwith a coat consisting of Eudragit L100-55 exhibited desired antimicrobial properties in theex vivo release study against L. casei and E. coli with a pH of 5.5. The microsphere startedinhibiting the growth of L. casei and E. coli in different proportion after 2 h of drug loading.But as per spectrum of the activity, a higher gram-negative microorganism E. coli inhibitionwas observed as compared to gram-positive L. casei. Earlier report of ranitidine hydrochloridemicrospheres coated Eudragit E100 size was ranging from 247 to 286 μm [32] and size ofEudragit RS 100 microparticles containing 2-hydroxypropyl β-cyclodextrin and glutathionewas ranging from 150 to 350 μm for oral administration [33]. Above results are comparablewith the particle size of prepared Cef EL in the present investigation. There are no reports

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available till date for cefdinir coating with Eudragit L100-55 polymer. As per our results, thesize of Cef EL was suitable for oral administration following efficient targeting to the intestinalmicroflora within the range of pH 5.5.

Conclusion

In summary, gut microbiota modulation with cefdinir microsphere reversed the insulinresistance from male Wistar rat. It is possible that the presence of certain bacteria inthe gut might aggravate low grade systemic inflammation, which causes insulinresistance. In line with earlier studies, oral administration of pH sensitive cefdinirmicrosphere was significantly improved insulin resistance, glucose intolerance, triglyc-erides, and hepatic damages in rats fed with high-fructose diet, which not onlyprovided a solid theoretical basis regarding how exhibited its effectiveness on treatingdiabetes but also shed light on the alteration of selective gut microbiota as apromising therapeutic option for the management of type 2 diabetes. This furtherprovides a confirmation on the microbiota in metabolic dysfunctions, and a supportingevidence for modulating the microbiota as a prophylactic strategy using antimicrobialagents specifically targeting to intestinal delivery but specificity of action will beimportant. However, more work has to be done in order to prove that gut microbiotamodulation is a safe and effective therapeutic strategy in treating or managing type 2diabetes in humans.

Acknowledgments The authors are thankful to Nirma Education and Research Foundation (NERF),Ahmedabad for providing infrastructure and financial support. Authors are also thankful for the help andcooperation rendered by Dr. Sanjiv Acharya and Mr. Prerak Patel (Institute of Pharmacy, Nirma University).

Conflict of Interest The authors declare that there are no conflicts of interest.

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